1.1. Introduction - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/35251/8/08...consequently to the other habitats throughout the world, would make the estimated 3 PUjaSalt-{/a

1.1. Introduction

PujaSaluja Ph D. TJ..sis, IMTECH Challdigarh

Chapter J: Introduction and Review o/LiteruhJre

The biodiversity of yeasts has not been as much appreciated as that of several other

microbes . The reasoning (although erroneous) behind this lack of attention has been

that they play only a minor role in the biosphere. All this has been despite of their well

known and wide-ranging roles in a) fermentation, b) as established model organisms

(Saccharomyces cere visiae , Schizosaccharomyces pombe), c) as pathogens (Candida

albicans, Cryptococcus neoformans), d) heterologous expression systems (Hansenula

polymorpha, Pichia pastoris), e) as probiotics, f) for production of industrially

important compounds; g) vitamins, h) carotenoids, i) lipids, j) organic acids and the list

continues on.

Despite the realized and/or unrealized biopotential of yeasts, the rate of species

discovery of yeasts has not increased significantly over the past 50 years . There has not

even a proper estimation of number of yeast species yet. However, Fell et al. (2000)

suggested that the presently described 800 species constitute only 1 % (may be even less

than) that existing in nature. Based on Fell's estimation, the number of yeast species

should be in the proximity of 1 lakh. The development of rapid molecular tools in late

nineties helped in the discovery of new yeast species to some extent, inspite of these

developments only about 1000 yeast species were described till 2005 (Fig. 1.1).

Number of Yeast Species Since 1952

1200 .-------------------------------------~__.

1000 +------------------------------------II) G/

1800 +-----------------------------~----II)

OJ ~800 +-------------~~---->-'0 ~ 400 +---------=-------

..0 E ~

z 200 +------

o

Year

• Number of Described Yeast Species

Fig. 1.1. Histogram showing the number of yeast species discovered and characterized since 1952. (modified from Daniel et aI. , 2006).

2

PujaSaluja Ph. D. Thesis, IMTECHChandigarh

Chapter 1: Introduction and Review o/Literature

Yeasts are explored from both natural (terrestrial and aquatic) and man-made

habitats (food and beverages), Although yeasts have been explored from variety of

habitats, the diversity of yeasts from natural habitats is mostly appreciated from plant

parts (flowers, fruits, phyllosphere, bark etc.), insects and soil among terrestrial habitats

and from fresh and marine water in aquatic habitats. Even though yeasts are not

considered as ubiquitous as bacteria, they were found even in the extreme environments

of Antarctica (Shivaji & Prasad, 2009; Vishniac & Hempfling, 1979a). The occurrence

of yeasts have also been marked in hydrothermal vent, (Gadanho & Sampaio, 2005),

hypersaline environment (Butinar et ai., 2005), even in the extreme acidic

environments of Iberian pyrite belt (Gadanho et ai., 2006). Yeasts are supposed to be

key players in the Yeast-Flower-Insect ecosystems, phyllosphere ecosystems and

extreme environments. In Yeast-Flower-Insect ecosystems and phyllosphere

ecosystems, they are suggested to play their roles as symbionts, as competitors or

antagonists (Rosa et aI., 2003; Buck & Burpee, 2002). In the extreme environments,

yeasts may hold keys to nutrient cycling, metal detoxification, and these psychrophillic

yeasts may serve as producers of industrially important compounds (de Garcia et aI.,

2007; Gadanho & Sampaio, 2005; Gadanho et ai., 2006).

Phaff et al. (1978) expressed optimism that, " we are convinced that imaginative

research by yeast ecologists will lead to the discovery of many additional interesting

associations and the isolation of novel yeast species". This optimism has become reality

when beetle gut was identified as a diverse source of yeasts by Suh et al. (2005a). A

limited sampling of beetles of only 27 families from a limited geographical region

yielded 650 isolates of which about 200 were putative new species on the basis of

DIID2 domain sequencing. This provided an indication of how dramatically limited our

knowledge is about these organisms and the extent to which they have been ignored.

The authors predicted on the basis of their statistical analysis that every .one of the

beetle species potentially corresponds to one new yeast species. If this were true, then

by studying the Erotylidae group alone (estimated number of species is about 4500) in

the limited geographical region explored by the authors, would increase the number of

yeast species four to five times of what are currently known. Further extension of this

prediction to other group of beetles, bees, flies and other insect species and

consequently to the other habitats throughout the world, would make the estimated

3

PUjaSalt-{/a Ph. D. Thesis, IMTECH Chondigarh


number of yeast species to be extremely high. Such studies would make the number of

currently recognized species almost negligible.

Traditionally, the generIc description of yeasts is performed by usmg the

information gathered from morphology, vegetative biology, sexual state, shape(s) of the

sexual spores and physiological tests. Species are usually differentiated by

physiological tests, in particular through their utilization of carbon and nitrogen

sources. It is usually necessary to perform nearly 90 tests to obtain reliable biochemical

identification of yeasts at the species level, and it takes at least 2-4 weeks to obtain the

final results (Barnett et aI., 2000). As an alternative approach, DNA reassociation

experiments are performed between closely related species to confirm their relatedness

(Martini & Kurtzman, 1985). Over the past few years, the development and use of

molecular techniques have provided new dimensions to the knowledge on

characterization and identification of medically (Sugita et al., 2000) and industrially

important yeasts (Petersen et aI., 2001). The molecular techniques include use of

species-specific PCR primers (Hierro et aI., 2004), analysis of Restriction Fragment

Length Polymorphism (RFLPs), randomly-amplified polymorphic DNA and micro

satellite fingerprinting (Howell et aI., 2004). The development of these techniques has

provided us with several new approaches for rapid identification of yeasts at species

level which have become indispensible tools for understanding the biodiversity of

yeasts.

Direct comparison of genomic DNA sequences is the best means of determining

phylogenetic relationship between two different microbes. The ribosomal RNA (rRNA)

genes have been found to be the best molecular chronometers, because a) they are

universal, b) evolve at approximately the same rate, and c) behave like a single copy

region. Comparative sequence analysis of rRNA sequences reveals stretches of highly

conserved, semi-conserved and other regions with considerable amount of variability.

The coding regions are highly conserved but show enough sequence variability to allow

global-level classification. The Internal Transcribed Spacer region (ITS) and D1ID2

variable domain of 26S rRNA have proved very useful in measurements of close

genealogical relationships (Kurtzman, 1993b; Scorzetti et al., 2002). The nucleotide

divergence of more than 1 % in the combined sequence analysis of ITS region and

D1ID2 domain is generally accepted for describing a novel yeast species.

4

PujaSaluja Ph. D. Thesis, IMTECH Chandigarh


A great majority of known yeast species comes from Western Europe, Japan

and North America. Larger parts of Africa, Antarctica, Asia, Australia and America are

mainly virgin territories in this respect (Boekhout, 2005). This indicates that a lot of

natural habitats of yeasts have yet to be investigated. Consequently, we can only

assume that many additional species await discovery. Because yeasts are widely used in

traditional and modem biotechnology, the exploration for new species should lead to

additional novel technologies. On the other side correct identification, naming and

placing of yeasts in their proper evolutionary frameworks is also essential for their

exploration and utilization in various fields and also for determining the intellectual

property rights. Surprisingly, despite of rich tradition of mycology in India over several

decades, yeast diversity in India is largely unexplored except for a few studies recently

(Bhadra et aI., 2008; Rao et ai., 2008).

Majority of the yeasts are mesophilic in nature, capable of growing at 25 to

30°C. The capability of a yeast species to grow at higher temperatures than those of

mesophilic yeasts is defined as thermophily. The demarcation of mesophilic,

thermotolerant and thermophilic yeasts is not very clear but, the yeasts which can grow

upto 37°C are regarded as mesophilic yeasts and those which can grow optimally above

37°C are regarded as thermotolerant or thermophilic yeasts. However, the mechanisms

for thermophily are not well studied. It becomes important to know the basis of high

temperature growth. As reviewed from the literature, it is well accepted that several

mechanisms are responsible for high temperature growth in some thermophilic bacteria

and archaea. Among those thermostability of proteins, presence of compatible

molecules like trehalose, cDPG, DGP and derivatives of myo-inositol phosphate

(Arguelles, 2000; Santos & da Costa, 2002) are already known. Some of the most

important parameters in thermal adaptation are changes in membrane fluidity (Arthur &

Watson, 1976); post-translational modifications (Olsen & Thomsen, 1991), presence of

some unique genes like reverse gyrase (porterre et ai., 2000; Takami et ai., 2004). The

molecular basis of thermophily in yeasts can be summarized as a composite of genes,

proteins, protein modifications and metabolites.

As yeast diversity is quite unexplored in India, we initiated this work to

examIne the diversity of yeasts from soil and flowers from two high temperature

5

PujaSaJuja Ph. D. Thesis, IMTECHChandigarh

Chapter}: Introduction andReview o/Literature

regions of Indian state of Rajasthan and coal-belt of Khammam district in Andhra

Pradesh, where temperatures in summers range between 40°C - 50°C. The reason

behind selecting such regions is that yeast diversity from high temperature regions was

not much explored. We felt that it would be interesting to explore yeasts from these

regions as well as to get insight to look at the phenomenon of high temperature growth

in yeasts, The following objectives were proposed for the current study.

1) Isolation of yeasts from high temperature regions ofIndia.

2) Molecular characterization of yeast.

3) Comprehensive analysis of interesting isolates by polyphasic approach.

4) To examine the basis of thermo tolerance in yeast.

During the course of our studies, we have isolated and characterized about 215

isolates of yeasts. More than 30 species turned out to be potentially novel yeast species

and among them 17 were characterized more extensively by polyphasic approach and

proved to be novel species. Three new species have been described as; Cryptococcus

rajasthanensis, Debaryomyces singareniensis, Candida ruelliae (Saluja & Prasad,

2007a; Saluja & Prasad, 2007b; Saluja & Prasad, 2008). Interestingly, our exploration

of high temperature regions did not yield any novel thermophilic yeasts.

Further to examme the basis of thermophily in yeast, a thermophlic yeast

Hansenula polymorpha (Pichia angusta) has been used as a model organism. We

designed a genetic screen to look for gene(s) that could confer thermophilic traits upon

a non-thermophilic yeast. The details of our experiments and the pertinent literature are

discussed in Chapter-7.

6

1.2. Review of Literature

1.2.1. History of Yeasts

PujaSaluja Ph. D. Thesis, IMTECH Chand;garh

Chapler 1: lnrroduclion and Review o/Lilerature

The word "yeast" comes from old English words gist, or gest which mean foam or froth

and from the Indo-European root jes-, meaning boil, foam, or bubble. Yeasts are

probably one of the earliest domesticated organisms. Even prior to 7000 B. c., beer was

being produced in Sumeria. Archaeologists digging in Egyptian ruins found early

grinding stones and baking chambers for yeasted bread, as well as drawings of 4,000

year-old bakeries and breweries. It is interesting that yeasts were being used far back in

history as industrial organisms, without people actually realizing them to be yeasts (or

even living organisms). It has been recently reported through chemical analyses of

ancient organics and preserves in pottery jars from Northern China that a mixed

fermented beverage of rice, honey, and fruit was being produced as far back as 9,000

years ago. It was approximately the same time that barley beer and grape wine were

beginning to be made in the Middle East (McGovern et ai., 2004).

Yeasts were first observed as globular structures rather than living organisms by

Antonie van Leeuwenhoek under microscope in 1680. It took until 1857 when Louis

Pasteur, a French microbiologist, first proved in his paper -Memoire sur la

fermentation alcoolique- that yeasts were the organisms responsible for alcoholic

fermentation (Barnett et ai., 2000). Yeasts have been classified under the kingdom

Fungi and are defined as a) organisms which survive as unicellular forms for at least a

small part of their life cycle and b) whose sexual states are not enclosed in perfect

fruiting bodies like that of fungi. Sexually reproducing yeasts are called perfect or

teleomorphic yeasts and the ones whose sexual state could not be determined are

known as asexual, anamorphic or imperfect yeasts.

1.2.2. Yeast Taxonomy Taxonomy is the science of classification, identification and nomenclature of

orgamsms. Classification implies the grouping of organisms into taxa, according to

actual similarities, presumed ancestral relationship or both. Identification requires the

comparison of unnamed species with similar, named species. Finally the nomenclature

is the naming of taxa as per rules of nomenclature codes (Mayr, 1942).

7

1.2.3. Classification and Nomenclature of Yeasts

PujaSaJuja Ph. D. Thesis, IMTECH Chandigarh


Yeasts and Fungi are classified using the rules of International Code of Botanical

Nomenclature. The most recent version of this code was adopted at the Seventeenth

International Botanical Congress at Vienna, Austria in July 2005. The official version

of the Code has been published as "International Code of Botanical Nomenclature

(Vienna Code)" (McNeill et ai., 2005). We are here describing briefly the Botanical

Code as it applies to Yeasts. The rules of this code are similar for describing genera,

families and orders to as of describing a new species. Some of the important rules

concerned with species description are described below.

1.2.4. Description of Species or Taxa

The publication of a new species will be valid only if a) it provides the description of

essential characteristics and as well as diagnosis that distinguishes the taxon from the

previously described taxa, b) the description and name of taxa must be given in Latin

(since January, 1993), c) the species should be published in recognized journal. Non

compliance with any of the above criteria will invalidate the newly described species

and termed as "nomen invalidum". The other important recommendation is that the

type material known as Holotype should be deposited in a publicly accessible

herbarium or culture collection. The Holotype refers to the isolate on which the

description of particular taxa is based. With the amendment of the 1994 code (Greuter

et ai., 1994), lyophilized cultures are also accepted as valid type material (Holotype),

and the living cultures derived from it are considered as ex typo i.e. from the type.

1.2.5. Early Development in Yeast Taxonomy

The most important unit of yeast taxonomy is the species. The classical definition of

biological species was given by Mayr (1942). It states, "Species are groups of actually

or potentially interbreeding natural populations, which are reproductively isolated from

other such groups". The above definition is frequently referred to as the biological

species concept. This definition although seemingly adequate for species delineation in

general but was not completely applicable to yeasts for the following reasons.

a) Most of the described species of yeasts are anamorphic.

b) Many yeasts species are homothallic (self-fertile).

8


Chapter 1: Introduction and Review o/Lilerature

1.2.5.1. Species Delineation Based upon Phenotypic Characteristics

Although the genus Saccharomyces was introduced in 1837, but the concept of yeast as

species emerged only after the introduction of pure culture techniques by Hansen

during his studies on brewery yeasts (Hansen, 1888). The criteria employed by Hansen

for differentiation of yeast isolates were a) cellular and ascospore morphology, b)

optimal temperature of growth and c) fermentation ability. Hansen designated the

isolates to different species based upon these phenotypic characteristics. For his work

he is regarded as the founder of the phenotypic characterization as a means for species

delineation in yeasts. Several investigators have used a combination of morphological

(colony and cell morphology) as well as physiological characteristics for species

delineation in yeasts. Historically (between 1920s-1940s), the physiological

characteristics that were utilized involved the ability of yeasts species to ferment and/or

assimilate certain sugars, and assimilation of ethanol, sulphate, asparagine, urea,

peptone and nitrate (Kreger-Van Rij, 1987). Apart from these, the standard description

also included characteristics of the novel species' life cycle. The difference(s) in one

(mostly) or more characteristics of a novel strain with another known strain formed the

basis for describing a new species. Strength of phenotype-based approach lies in

identification of unique trait(s) that can be associated with a taxon (or higher levels)

with confidence and should not be subject to variability with time, space and laboratory

conditions. With the isolation of more and more species over time, the utility of this

less number of phenotypic characteristics for designating new species became limited.

In the 1950' s, the spectrum of phenotype-based tests was further extended by

Wickerham (1952) and today approximately 90 tests are performed routinely. These

tests include fermentation and assimilation of carbon compounds, assimilation of

nitrogenous compounds, resistance to cycloheximide and temperature requirements for

growth. In addition, wherever possible the sexual structures and sexual cycles were also

being taken into account.

1.2.5.2. Phenotype-based Classification of Yeasts

The classification of yeasts above the species level was also based upon a limited set of

phenotypic characteristics which include mode of vegetative reproduction, sexual

reproduction, physiological and biochemical characteristics as described for species

delineation. In addition, ultra structural details and coenzyme Q analysis were also

9



implicated for taxonomic groupmg. Electron mIcroscopy revealed the differences

between ascomycetous and basidiomycetous yeasts and that became the basis of first

level of dichotomy in classification. Ascomycetous yeasts have electron-transparent

cell wall and thin electron-dense outer layers, whereas basidiomycetous yeasts have

lamellate and electron-dense layers (Kreger-van Rij & Veenhuis, 1971).The cell walls

of these two groups also react differently to a chemical known as Diazonium blue B

(DBB). The basidiomycetous yeasts give red color with this reagent while

ascomycetous yeasts do not give any reaction (Hagler & Ahearn, 1981; Simmons &

Ahearn, 1987; Van Der Walt & Hopsu-Havu, 1976). These differences were useful in

the grouping of imperfect yeasts to ascomycetous or basidiomycetous yeasts.

Other methods like coenzyme Q analysis provide resolution only upto genus

level for the most part. The coenzyme Q or ubiquinones are components of respiratory

chain. In this system, variations are found as the number of isoprene units per molecule

and the number varies from Q-5 to Q-lO among yeasts (Yamada et aI., 1976a; Yamada,

et aI., 1976b; Yamada et aI., 1981). For example, the coenzyme Q-9 was reported in the

genus Dekkera and Debaryomyces and Q-6 in Saccharomyces and Arixozyma. A

combination of CoQ has been observed among the member of the genus Pichia. As

more than one genus can share similar type of Co-Q system this can not be used

inclusively for grouping of taxa.

The phenotype-based classification of yeasts as per Kreger-Van Rij (1987) has

been briefly described below. According to this classification yeasts were divided into

three categories.

a) The ascosporogenous yeasts,

b) The basidiosporogenous yeasts

c) The imperfect yeasts, based upon their mode of reproduction.

a) The ascosporogenous yeasts were classified under hemiascomycetous yeasts that

lack ascocarps (fruiting bodies in which ascopsores are formed) and ascogenous hyphae

(Ainsworth, 1973). Saccharomycetaceae and the Spermophthoraceae are the two

families belonging to the order Endomycetales under Hemiascomycetes. The two

families differ in shape of ascospores which are needle shaped in Spermopthoraceae

10


Chapter 1: Introduction and Review a/Literature

and have different shapes in Sachharomycetaceae. Further classification of lower ranks

was based upon the following characteristics.

i) Type of budding (monopolar, bipolar or multilateral budding)

ii) Shape of ascospores

iii) Ability to ferment

iv) Type of Coenzyme Q

b) The basidiomyceteous yeasts are the haploid phase in the life cycle of most

heterothallic basidiomycetes, where they originate from the germinated basidiospore

(Kreger-Van Rij, 1987). The basidiomycetous yeasts were classified further mainly on

the basis of following features.

I) Formation of true mycelium with or without clamp connection.

II) Presence of ball is to spore.

III) Fermentation ability

IV) Presence of chlamydospore and type of coenzyme Q.

The basidiomyceteous yeasts were divided into three groups.

i) Teliospore-forming yeasts

ii) Filobasidiaceae with a yeast phase

iii) Unclassified genus Sterigmatosoridium

c) The fungi imperfecti comprised the anamorphic yeasts for which the sexual state

could not be determined either because of the laboratory conditions (non-natural

habitat) or because of the isolation of only one mating type. Several species of this

group have close resemblance to other perfect species but lack sexual state. These

yeasts were separated into different families and genera on the basis of vegetative

reproduction, presence or absence of mycelium, pigmentation, fermentation ability,

nitrate assimilation, formation of asexual structure like ballistoconidia and blastospore

(Kreger-Van Rij, 1987).

1.2.5.3. Limitations of Phenotype-based Classification

The yeast classification and species delineation solely based upon morphological,

physiological and biochemical characteristics soon became inadequate. The results

were frequently found to be inconsistent, time consuming and labour intensive.

11

PtljaSaluja Ph. D. Thesis, IMTECH Chandigarh

Chapter J: Introduction and Review o/Literature

Outcome of many of these tests is highly dependent upon the media, purity of

chemicals, and methods used to conduct the tests. Some tests were found to be variable

even within strains of a species. For example, Ditlevsen in 1944 (as cited in van der

Walt, 1987) had found a strain of Saccharomyces italicus to be heterozygous for short

and long cells. With the discovery of newer species, the resolution of several

characteristics which were earlier thought to be decisive in delineation of species

became rather inadequate. A compelling example is shape of ascospores, which formed

the basis for separation of families Spermophothoraceae and Saccharomycetaceae,

became questionable with the introduction of Pichia ohmeri (Kodamaea ohmeri) which

produced both spherical and hat shaped ascospores (Kurtzman, 1998b). The above

example and many others like it indicated that the classification and species delineation

solely based upon such a limited set of characteristics was artificial.

1.2.6. Yeast Taxonomy Based upon Molecular Methods

1.2.6.1. Assessment of G+C Content

The transition phase from phenotypic characters to molecular taxonomy started with

the development of methods for assessment of DNA base composition. The G+C

content of the 800 yeast species, ranged between 27-70 mol%. In general, for the

ascomycetous yeasts, the G+C content was about 27-50% while in basidiomycetous

yeasts, it was 50-70 %. The G+C content measurement was also dependent upon the

method used for the assessment. It was suggested that strains differing by more than 1-

l.5% through thermal denaturation method (Phaff et al., 1985; Price et al., 1978) or 2-

2.5% using buoyant density method would potentially represent different species

(Meyer, 1979). But the G+C measurement can not be used as a sole criterion for

species delineation because it provides no information about the DNA sequence.

Therefore, many different species may share the same G+C content while being very

different in other respects.

1.2.6.2. DNA-DNA Hybridization

The DNA complementarity was so far the only technique that provided resolution at the

species level and could be applied to anamorphic species as well. This technique

provided a quantitative means for assessment of the relatedness of genetic materials

among species and strains. The commonly used methods include spectrophotometric

12

PujaSaluja Ph. D. Th"is, IMTECHChandigarh


analysis and membrane-bound reassociation techniques using radioisotopes or other

markers (Kurtzman, 1993a). Results of DNA complementation are expressed and

interpreted as percent relatedness. The correlation between the biological species

concept and DNA relatedness has been examined using genetic crosses utilizing both

homothallic and heterothallic species. Percent relatedness of 65-70% was found in

agreement for conspecific species in both homothallic and heterothallic backgrounds. A

DNA relatedness value of less than 20% showed distant relationship between the two

strains. Test subjects showing such values were often found to be new species. An

intermediate value of 35-60% suggested that the two strains being compared belonged

to different varieties of the same species.

The taxonomic interpretation of DNA hybridization values between 20-35% are

often difficult to interpret as exemplified below. Two heterothallic species Pichia

amylophila and P. mississipiensis (Kurtzman et aI., 1980b) showed 25% DNA

relatedness, the crosses between them showed abundant mating but limited ascus

formation where ascospores were not observed. Similar results were obtained upon

crosses between Pichia americana and P. bimundalis with 21 % DNA relatedness

(Kurtzman, 2006). In Issatchenkia scutulata and its variety exigua exhibited 25% DNA

relatedness but showed ascospore formation similar to intravarietal crosses. The

ascospore viability was only 5% and sibmating (mating among siblings) of the progeny

showed 17% viability, but the back cross to the parents resulted in low ascosporulation

and viability (Kurtzman et ai., 1980a). Thus the tests indicated that these were two

separate species. Homothallic species also gave similar results and can be defined in a

similar way. The above results suggested that although mating between homothallic

and heterothallic species could occur over a wide range of DNA relatedness values but

the highly fertile ascospores or conspecificity was observed only in cases showing 70%

or higher DNA relatedness.

Although the technique of DNA-DNA hybridization proved to be highly

conclusive but was not suitable for routine analysis. First, this technique is very

cumbersome and requires that DNA from a single species be hybridized to all the

related species followed by reciprocal hybridizations. Second, the information gathered

from this technique is limited to being applicable at the sister-species level.

13

1.2.6.3. Sequence-based Yeast Taxonomy

PujaSaluja Ph D. Tlresis, IMTECHChandigarh

Chapter 1: lntroduction and Review of Literature

The resolution of all the methods described above became limited with the increase in

the number of identified species and with the advent of new technologies. Sequence

based identification has revolutionized the taxonomy of yeasts. By using DNA

sequences, the phylogenetic relationships among the species can be easily established

and the sequence results are almost always consistent and not dependent upon space,

time and the method used. Such advantages have made it the method of choice. The

other major benefit of this technique is that the teleomorphic and anamorphic

relationship can be suspected by construction of phylogenetic trees. This reduces the

redundancy in nomenclature by combining the synonyms together.

Important Considerations for Selection of the Molecular Choronometer

The selection of a gene to be used for inferring the phylogeny is very crucial and

challenging task. A gene which can be used as a choronometer should fulfill the

following criteria (Kurtzman, 1994a; Valente et ai., 1999).

a) The gene must be present in all the organisms of interest. Thus, the genes which are

central (universal) to cellular functions are usually of choice. Examples include genes

whose products function in replication, transcription and/or translation - the processes

constituting the "Central Dogma" of molecular biology.

b) The gene of interest should not be subj ect to lateral transfer. The gene history may

not faithfully represent the history of the organism when a gene is capable of being

laterally transferred. When a gene performs a central function, an organism is unlikely

to acquire a copy by lateral transfer, since the organism must already have a functional

copy to be alive.

c) The gene must be large enough and contain appropriate level of conserved and

diverged regions so as to allow inference of its phylogeny at different taxonomic levels.

Divergent sequences have a tendency to become randomized, and therefore impose a

limit to which the divergence of sequences can be accurately inferred. If the sequences

are too conserved then there may be little or no change between the evolutionary

branches of interest, and it will not be possible to infer close (genus or species level)

relati onshi ps.

14

PujaSaluja Ph D. Thesis, IMTECH ChandiglITh


1.2.6.4. Ribosomal RNA Gene (Ribosomal DNA)-based Phylogeny

The rRNA genes attracted interest almost universally to infer the phylogeny as they

accomplish all the criteria of the molecular chronometer mentioned above. The regions

of rRNA gene were used to infer phylogeny from higher taxonomic levels such as

kingdom, orders and consequently to the level of species in all domains of life (Chaw et

ai., 1997; Field et ai., 1988; Gouy & Li, 1989; Hori et ai., 1985; Leclerc et ai., 1994).

Organisation of Ribosomal RNA gene cluster (rRNA genes or rDNA)

In Saccahromyces cerevisiae, the rRNA genes (rDNA) are arranged in a tandem array

of 140-200 rDNA units comprising of small subunit (SSUI18S), internal transcribed

spacer region-l (ITSl), S.8S, internal transcribed spacer region-2 (ITS2), large sub unit

(LSU/26S) rRNA, intergenic spacer region-l (IGSl), SS rRNA and internal

transcribed spacer region-2 (lGS2) (Fig. 1.2). Three coding genes of the cluster SSU

(I8S), S.8S and LSU (26S) rRNA are transcribed in one direction and the SS rRNA is

transcribed in the opposite direction (Philippsen et ai., 1978). Comparative sequence

analysis of rRNA gene sequences revealed some stretches of highly conserved, semi

conserved and other regions with considerable amount of variability. The coding

regions are highly conserved but show enough sequence variability to allow

classification upto species level. The non-coding regions (spacer regions) are less

subject to evolutionary constraints due to functiomilloss, hence show lot of variation in

the sequences. These are useful for inferring the phylogeny of closely related species.

)

<i-

RS1 RS2 1GS1 1652 RS1 RS2 1651 1652

1 1 H ! 1 H ! -1 HH HH

185 5.85 265 55 185 5.85 265 55

Unit-l Unit-2

Fig. 1.2. Schematic organization of the ribosomal RNA gene cluster in Saccharomyces cerevisiae.

Regions of Ribosomal RNA gene cluster used to infer phylogeny

Use of5S rRNA gene (5S rDNA)

The SS rRNA was the first gene to be used to infer phylogeny because of its conserved

nature and short length (Ca.120 nucleotide). Walker and Doolittle (I982 and 1983)

IS

PujaSaluja Ph. D. Thesis, IMTECH ChaIJdigarh


used 5S rRNA sequences to assign the phylogeny of basidiomycetous fungi and yeasts

(including anamorphs). They correlated the phenotype- and sequence-based

information and separated basidiomycetes in five clusters. Phylogeny of ascomycetous

yeasts was also studied similarly on the basis of 5S rRNA sequences (Walker, 1985).

The sequence of 5S rRNA region was used to establish broad phylogentic relationships

but its rather small size soon became limiting in resolving the relationships.

Use of small subunit (SSU) and large subunit (LSD) rRNA gene sequences

Other regions explored for studying phylogeny of yeasts were small subunit rRNA gene

(SSU rRNA or SSU rDNA) (Ca. 1800 nucleotides) and large subunit rRNA gene (LSU

rRNA gene or LSU rDNA) (Ca. 3200 nucleotides). These regions are larger than the 5S

rRNA therefore better resolution can be expected. Comparisons of sequences from

wide variety of organisms suggested that these regions comprised of various conserved

and variable domains having different rates of nucleotide substitutions. Because of

these characteristics, these regions have been regarded as a collection of chronometers,

with each region offering a different glimpse of the evolutionary history of an organism

(W oese, 1987). It was suggested that the partial sequence analysis of rRNAs could

display the same phylogeny as the complete sequence and thus promoted partial

sequence analysis as a rapid and economical method for ascertaining phylogenetic

relationships (Mc Carroll et al., 1983; Lane et al., 1985).

Use ofSSU rRNA gene (I8S rRNA gene or 18S rDNA)

The SSU rRNA gene is about 1800 nucleotides long in S. cerevisiae. The SSU rRNA

variability map of S. cerevisiae is shown in the Fig. 1.3. The color coding represents the

extent of nucleotide variability among different organisms. In this map, 5 variability

classes were identified which ranged from very conserved (blue) to highly variable

(red). Positions that are identical in all organisms taken into account are indicated in

purple. Positions which are present in less than 25% of these organisms or for which

the alignment was unreliable are indicated in grey in the map. This map is available for

download and is distributed for free by its author at the ribosomal RNA database

(http://bioinformatics. psb. ugent. be/webtool s/rRNA).

16

saccharomyces cerevisiae SSUrRNA variability map

0 0

0' c::J. 0' c· C' e <2"'-

PujaSal'l/a Ph D. Thesis, IMTECHChandigarh


Fig. 1.3. Variability map of SSU rRNA of Saccharomyces cerevisiae. The different colors represent the extent of variability and range from conserved (purple) to highly variab I e (red) (http://bioinformatics. psb. ugent. be/webtools/rRN N).

Petersen and Kurtzman (1991) selected four regions SSU rRNA gene to assess

their resolution in deliniating yeasts. The four regions selected of SSU rRNA (18S-566,

18S-901, 18S-1137 and 18S-1627) were found to be much too conserved to be useful in

delineation of individual species. However, the region 18S-1627 showed some

variability to differentiate among genera.

The complete sequencing of 18S rRNA gene was used to resolve the

phylogenetic relationships among the members of the genera Brettanomyces,

Debaryomyces, Dekkera, Kluyveromyces (Cai et al., 1996) and Saccharomyces (James

et aI., 1997). The teleomorph-anamorph relationship was established between

Brettanomyces (anamorph) and Dekkera (teleomorph), as members of these genera

formed a monophyletic group. The genera Saccharomyces (James et al., 1997) and

Kluyveromyces were suggested to be polyphyletic (Cai et al., 1996). In addition to this,

full sequencing of 18S region was used to infer phylogeny at higher taxa viz order, class

17



and family levels in fungi (Swann & Taylor, 1995; Swann & Taylor, 1993), and for

classifying yeasts and fungi of basidiomycetes (Sugiyama & Suh, 1993).

Use ofDI1D2 Domain ofLSU rRNA gene (DI1D2 Domain ofLSU rDNA) sequencing

The D1 and D2 domains are present towards the 5' end of LSU rRNA gene and

comprise of about 600 bases. The secondary structure showing the D11D2 domains is

depicted in the Fig. 1.4. The primer NL1 starts ahead of D1 region and primer NL4 is

beyond the D2 region.

Peterson and Kurtzman (1991) used the D2 regIOn (also known as the D2

domain) of LSU rRNA gene to assess its resolution at species level. The sequencing of

D2 region was found to be highly variable and it allowed separation of closely-related

species. Although D2 region was found to be much too variable to establish phylogeny

among distantly related species, several studies used the D2 sequences successfully to

differentiate among genera in ascomycetous as well as basidiomycetous yeasts (Fell &

Kurtzman, 1990; Fell & Statzell-Tallman, 1992; Fell et al., 1992; Walker, 1985;

Yamada et ai., 1994). Kurtzman and Robnett (1997) were the first to sequence the

D 11D2 domains of almost all the clinically important yeasts. They established

phylogenetic relationships upto species levels and also constructed the phylogenetic

trees to study anamorph-teleomorph relationships. Their results were verified and

correlated with nuclear DNA complementarity for closely-related representative species

included in the study. They further expanded the work to infer phylogeny of all the

ascomycetous yeasts and the database they generated has become an indispensible tool

for species identification (Kurtzman & Robnett, 1998).

18

.. ,

PujaSaJuja Ph D. Tlresis, IMTECH Chandigarh


Fig. 1.4. Schematic representation of the secondary structure of D 1 and D2 domains of LSU rRNA (modified from Inacio et aI., 2003).

On the basis of D IID2 domain sequences, it was suggested for ascomycetous yeast,

strains showing a difference of more than 6 nucleotides (1 %) would represent different

species whereas, conspecific strains usually exhibit 1-3 nucleotide divergence.

However, there are some exceptions like Clavispora lusitaniae, in which up to 5%

variation was observed in the DIID2 domain sequence among different strains

(Lachance et ai., 2003b). Fell et aI., (2000) sequenced DIID2 domains of

basidiomycetous yeasts thereby extending the database to encompass almost all the

yeasts known at that time.

Use of spacer regions

The other regions which gained interest in resolving phylogenetic relationships were

the spacer regions: internal transcribed spacer region (ITS) and intergenic spacer region

(IGS.). Although DIID2 variable domain was very useful in differentiating species of

yeasts, several studies have shown that the resolution of ITS region in general was

better than D IID2 region, and researchers started using this region to provide additional

information in support of differentiating species (Fell et al., 2002). The ITS region

comprises of two spacer regions viz ITSI and ITS2 which are separated by 5.8S rRNA

19



between them as shown in the Fig. 1.5. The length of these variable regions may vary

from 300-1300bp among different yeasts.

l8S

ITSln~2 Fig. 1.5. Schematic representation of a unit of r DNA to show Internal transcribed spacer region ITS 1 and ITS2 (modified from Nazar, 2004).

Sequencing of ITS from all known basidiomycetous yeasts was performed by

Fell et al. (2002). Their analysis suggested that combined sequence analysis (D11D2

and ITS) was more appropriate for species delineation of basidiomycetous yeasts as

their D11D2 region tends to be quite conserved (showed 2-3 base differences among

species of some groups) (Fell et ai., 2002). The ITS sequencing was suggested as a

rapid and accurate method for differentiation and identification of pathogenic yeasts

Peterson et ai, 2001; Leaw et aI., 2006; Wang et aI., 2007). ITS sequencing was also

performed to study the diversity of yeasts from arbuscular mycorrhizal roots (Renker et

ai., 2004), marine yeasts (Nagahama et ai., 2001), and yeasts from food (Foschino et

ai., 2004). Belloch et ai. (2002) used ITS sequence analysis along with electrophoretic

karyotyping and phenotypic characteristics for characterization of 39 strains belonging

to four species in the genus Kiuyveromyces. The ITS sequences sometimes could

resolve the relationship even below the species level. For example, five varieties of

Williopsis saturns containing identical 18S rDNA sequences, were resolved by ITS

sequences (James et ai., 1998). ITS sequence was found to be more useful than D11D2

sequence analysis for species delineation in the genus Taphrina (Rodrigues & Fonseca,

2003). Cadez et al. (2003) used both the ITS and D11D2 domain sequences to describe

four new species in the genus Hanseniaspora. Fifteen strains of Cryptococcus were

reclassified by combined sequence analyses of 18S, D11D2 and ITS regions and as a

result, three new combinations of yeasts viz Cr. aureus, Cr. carnescens and Cr. peneaus

were proposed (Takashima et ai., 2003).

Some studies on basidomycetous yeasts suggested that boundary for species

delineation using ITS sequence as 1% nucleotide divergence (Sugita et ai., 1999;

20



Takashima & Nakase, 2000). In contrast Bai et al. (2001a and 2001b) found

approximately 2% divergence among some conspecific strains. Fell et al. (2000) found

that some yeasts species whose relationships could not be resolved by DIID2 sequence

could be resolved well using ITS sequences. On the contrary, Bai et al. (2002) reported

that species could have similar ITS and quite divergent DIID2 sequences. For example,

S strains of Sporobolomyces phaffi differed from S. ruberrimus by 3 nucleotides in ITS

sequences but exhibited 18-19 nucleotide differences in D IID2 domain sequences. It

can be concluded on the basis of above discussion that analysis based upon more than

one sequence provides more reliable information and is preferred for phylogenetic

analysis. Therefore a combination of ITS and D IID2 sequences is recommended for

obtaining more reliable conclusions.

The other spacer region used in phylogeny is IGS which is highly divergent

when compared to ITS. The IGS region was used for generating RFLP patterns for

phylogenetic analysis of the species in Saccharomyces group (Montrocher et al., 1998).

The difference in IGS length and sequence (upto 20%) was reported even among the

varieties of Cr. neoformans (Diaz et aI., 2000). The limitations in use of IGS are the

presence of homopolymeric regions and repetitive sequences which make its

sequencing rather problematic. Fell et al. (2007) used ITS and IGS sequences to reveal

the phylogenetic relationship between the species of Xanthophyllomyces which was not

resolved by DIID2 sequences. The level of differentiation that can be achieved from

different parts of rRNA gene cluster has been presented in Table 1.1.

Table 1.1. Taxonomic resolution of sequences of different parts of rRNA gene cluster.

Molecule Approx Higher Family Genus Species Sub Strains or region length category species

or variety

SSrDNA 120 bp + + + S.8S rDNA 158 bp + + + 18S rDNA 1800 bp + + + DIID2 600bp + + + + domain ITS 300-1300 + + + + +

bp IGS 2.4-4.1 + + + + + +

Kb

>,q' 5.{,2- 'T\-\- t6477 e 9eyC$ 81 (! Ubrary~' bL- ~ il 21 ~. ~

l#-

1.2.7. Modern Species Definition in Yeasts

PujaSaJuja Ph. D. Thesis, lMTECHChandigarh

Chapter}; Introduction andReview of Litera hire

The development in tools and techniques helped to generate the phylogenetic concept

of species delineation as an alternative of biological species concept According to this,

"species is defined as a monophyletic group composed of the smallest diagnosable

cluster of organisms within which there is a parental pattern of ancestory and discent"

(Cracraft, 1983).

The above definition can be put in a simplistic form as follows. In a system of

classification, all the taxonomic categories should be monophyletic from an

evolutionary point of view. So, the classification whether based upon the phenotypic

characters or the molecular methods relies on a set of characters which results in

monophyly.

The modern classification system of ascomycetous and basidiomycetous yeasts

as described in "The Yeasts: A Taxonomic Study" (Boekhout et al., 1998a; Kurtzman,

1998a) and its relation with rRNA-gene phylogeny of ascomycetous and

basidomycetous yeasts is presented in brief below.

1.2.8. Current System of Yeast Classification

1.2.8.1. Classification of Ascomycetous Yeasts

Historically, ascomycetes were divided into two taxonomic classes or subclasses called

Hemiascomycetes and Euascomycetes. Several studies on the phylogeny of the

ascomycetous yeasts (Hausner et al., 1992; Hendriks et aI., 1992; Kurtzman, 1993b;

Kurtzman, 1994a; Kurtzman & Robnett, 1994b; Nishida & Sugiyama, 1994; Walker,

1985), utilized different parts of rRNA gene sequences. On the basis of those studies,

Kurtzman (1998a) suggested the following three major lineages in ascomycetous

yeasts.

a) Hemiasocomycetes (Order Saccharomycetales) includes budding yeasts and

yeast-like taxa such as Ascoidea and Cephaloascus. In this group asci are not

formed in or on the fruiting bodies.

22


Chapter J: Introduction and Review of Literature

b) Euascomycetes is a sister group to the Hemiascomycetes and represents

the'filamentous' species, some of which are dimorphic. Here; the asci of nearly

all species form within or upon the fruiting bodies (Oosporidium).

c) Archiascomycetes represents a phylogenetically broad assemblage of yeast-like

taxa basal to the previous groups and comprised of the genera

Schizosaccharomyces, Saitoella, Protomyces, Taphrina and Pneumocystis.

The currently accepted classification is based upon the combination of phenotypic

and molecular methods. But this classification may be revised in future as the existence

of several orders, families and genera became doubtful on the basis ofDI1D2 sequence

analysis of ascomycetos yeasts by Kurtzman & Robnett (1998). For example, the

DIID2 sequence analysis showed that the family Lipomycetaceae to be monophyletic

and statistically well-supported (bootstrap = 98%). However, the seven presently

included teleomorphic genera were suggested to be paraphyletic, suggesting that ascus

morphology, ascospore ornamentation, and composition of coenzyme Q were

unreliable phylogenetic parameters. Similarly, the genera Nadsonia, Wickerhamia,

Hanseniaspora and Saccharomycodes all showed bipolar budding which would suggest

a close relationship among them. However, this character did not prove to be of

evolutionary significance on the basis of DIID2 sequences as the members of these

genera turned out to belong to entirely separate clades. Several such contradictory

results highlighted the poor resolution of phenotypic characters in inferring the

underlying phylogeny. The phylogenetic analysis of ascomycetous yeasts based upon

DIID2 alone was not sufficient to resolve basal lineages.

Kurtzman & Robnett (1998) suggested that additional DNA sequences (multi gene

sequence analysis) should be used to draw any genetic boundaries. They used the

combined sequence analysis of rRNA genes (18S, 26S and ITS), translation elongation

Factor Ia (EF-I a), actin-I, RNA polymerase II and mitochondrial-encoded gene

(SSUr DNA, COX II) to resolve the phylogeny of Saccharomyces species complex

(Kurtzman, 2003; Kurtzman & Robnett, 2003). This analysis identified 11 well

supported clades in the family Saccharomycetaceae and resulted in proposal of 5 new

genera and reassigned several species among currently accepted genera. This work was

further expanded to resolve phylogenetic relationship of Trichomonascus,

23

PujaSaJt{ja Ph. D. Thesis, IMTECH Chand;garh

Chapter]: Introduction and Review o/Literature

Wickerhamiella and Zygoascus yeast clades (Kurtzman & Robnett, 2007) and of

polyphyletic genus Pichia (Kurtzman et ai., 2008),

1.2.8.2. Classification of Basidiomycetous Yeasts

The ultra structure and molecular analysis based upon SSU rDNA, D1ID2 and ITS

sequence analyses of basidiomycetous yeasts suggested the occurrence of

basidiomycetous yeasts in three main phylogenetic classes of Basidiomycota namely

the Hymenomycetes, Urediomycetes and Ustilaginomycetes (McLaughlin et ai., 1990;

Swann & Taylor,1993; Swann & Taylor, 1995),

a) Hymenomycetous yeasts produce dolipore septa, and cell walls contain glucose,

mannose, and xylose (Fell et al., 2000). In these yeasts, inositol is usually

assimilated and starch-like compounds are also produced.

b) Uredinomycetous yeasts produce spores in which cell wall is attenuated towards

the central pore, Cell wall may contain mannose, glucose, fucose or rhamnose

but not xylose (Fell et ai., 2000). These yeasts donot produce starch-like

compounds and are unable to utilize inositol.

c) Ustilaginomycetous yeasts have microspore-like septa with or without an

inflated margin and which differ from simple pores because they do not have

tapering cell walls and probably lack a true pore (Bauer et al., 1989; Boekhout,

et al., 1998a; Boekhout et ai., 1992; McLaughlin et ai., 1995; O'Donnell &

McLaughlin, 1984), In these yeasts, glucose, galactose, and mannose are

present while xylose is absent in the cell wall, Inositol mayor may not be

present and strach-like compounds are not produced,

Phylogenetic analysis of basidiomycetous yeasts based upon DIID2 domain of

LSU rDNA was performed by Fell et al. (2000) and in combination with ITS was

performed by Scorzetii et al. (2002), In these studies several monophyletc clades were

identified in all the three classes and species boundaries were discussed, Members of

the class Uredinomycetes were distributed into Microbotryum, Sporidiobolus,

Agaricostilbum and Erythrobasidium clades, In the class Hymentomycetes;

Trichosporonales ord, nov., Filobasidiales and Cystofilobasidiales clades were

identified, Ustilaginales, Microstromatales and Malasseziales clades found their place

in the class Ustilaginomycetes. Members of some genera were distributed among more

24

PujoSaluja Ph. D. Thesis, IMTECH Chandigarh


than one clade and are thus polyphyletic. The Genera Cryptococcus, Rhodotorula and

Sporobolomyces are polyphyletic. In contrast, other genera, like Buliera,

Cystofilobasidium, Feliomyces, Filobasidielia, Filobasidium, Kondoa,

Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces

are monophyletic.

Although these yeasts have been separated into many clades but the

phylogenetic trees based upon the ITS and DIID2 sequences do not adequately resolve

the relationships among taxa as most of the clades exhibit low boot strap values. This

may be due to the insufficient data as current estimates suggested that only about 1%

(or less) of the total number of basidiomycetous yeast species have been identified (Fell

et al., 2000). With more research it is very likely that there will be more pieces to fill

the vacant places in this puzzle. It is also highly possible that with the discovery of

more species several of the currently drawn phylogenetic trees would undergo

rearrangements that could reflect their more accurate placing and the resulting

bootstrap values would improve.

-1.2.9. Modern Approaches for Species Delineation in Yeasts The identification of species based on phenotypic characteristics is frequently

insufficient and moreover can lead to errors in identification of species. Therefore the

standard fermentation and assimilation tests can not be used solely to unambiguously

define a new species. The results from such tests may however be used to

diagnostically recognize some genetically defined species or groups of species. These

tests provide general information about the physiology of the strain and its metabolic

properties. In addition, these tests are of immense value to biologists, ecologists and

others that need to know about the physiological properties of the species/strains under

investigation. DNA sequence on the other hand provides no information regarding

biology of the species. Therefore the descriptions of any new species can not solely rely

on nucleotide data. As a result, the current approach in yeast taxonomy follows a

combination of phenotypic as well as molecular characterization of yeasts for species

delineation. This approach is regarded as the polyphasic approach which afso includes

the study of sexual structures wherever possible.

25

1.2.10. Yeast Diversity

PujaSa/uja Ph. D. Thesis, lMTECH Chandigarh


Yeasts are not as ubiquitous as bacteria but have been isolated from a wide variety of

natural habitats. Yeasts are strictly chemo-organotrophic as they can not perform

photosynthesis. As a result they require fixed organic forms of carbon for growth. The

yeasts are known to utilize quite diverse compounds which include polyols, simple

sugars, aliphatic alcohols, hydrocarbons, various heterocyclic and polymeric

compounds, organic and fatty acids. The diversity of yeasts in a particular niche is

determined by its nutritional selectivity. Owing to that some species of yeasts which are

nutritionally heterogeneous have been isolated from many different habitats as a result

species of the genera e.g. Debaryomyces and Cryptococcus are considered as

cosmopolitan yeasts. The high surface/volume ratio of yeasts helps in rapid nutrient

absorption. The yeasts can grow over a wide range of pH values, but cannot grow at

very high temperatures. They generally grow in the range of 25°C -37°C (Ueno et al.,

2001). The maximum temperature of growth of yeasts is found to be 52°C. Yeast have

been explored from many diverse terrestrial as well as aquatic habitats. Terrestrial

habitats mostly include plants, animals and soil. Aquatic habitats include fresh-water

and marine habitats and study of estuaries as well. Study of fermentative and

pathogenic yeasts diversity is another point of focus in this field. Study of yeast

diversity is a broad wide-ranging topic, therefore we can not cover all of it, and instead

we have focused and described some of its important and pertinent aspects related to

our work.

1.2.10.1. Yeast Diversity from Flowers and Insects

Many insects are known to possess eukaryotic symbionts m their digestive tract.

Although this symbiotic relationship is not well understood but some studies have

indicated that certain microorganisms might play important roles for their hosts

including detoxifying food material and providing essential nutrients (Dowd, 1989;

Dowd, 1991; Vega & Dowd, 2005). Yeasts have been isolated from flowers and guts of

associated insects, examples include guts of basidiocarp-feeding beetles and several

other insects. Although the symbiotic relationship has been suspected but the

underlying mechanism is not understood. Clues about the unresolved question of

symbiotic relationship between the members of these ecosystems will help us

understand better the role of yeasts in ecosystem management.

26

Puja Saluja Ph. D. Thesis, IMTECH Chandigarh

Chapter 1: Introduction and Review of Literature

Three new yeast species were isolated during studies of yeasts associated with

ephemeral flowers in Brazil, Australia and Hawaii. Among them Kodamaea

nitidulidarum and Candida restingae were isolated from cactus flowers and associated

nitulid beetles in sand dune ecosystems (restinga) of Southeastern Brazil. Kodamaea

anthophila were isolated from Hibiscus and morning glory flowers (Ipomoea spp.) and

from associated nitulid beetles and Drosophila hibiscus in Australia (Rosa et al., 1999).

Since, these yeasts species were not isolated from any other habitat therefore; these

yeasts have been suggested to be the persistent members of yeast-flower-insect

community. Lachance and co-workers (2001c) studied the yeast communities of

ephemeral flowers and associated insects in the Neotropical, Nearctic and Australian

bio-geographic regions. They investigated the occurrence of yeasts from the flowers of

the families Malvaceae, Convolvulaceae and Cactaceae, and insects including beetles,

flies and bees, which regularly visit these flowers. They found some interesting

correlations between the yeast-flower-insect relationship. Australian community was

dominated by the insect Aethina concolor and the yeast species Kodamaea anthophila

where as the other regions dominated by Conotelus mexicanus and in turn by yeasts

species of Metschnikowia. Interestingly, in Hawaii region where both of these insects

are found, the yeast community also typically represents both geographic regions. This

specificity was further confirmed by the analysis of yeast diversity from other plants

which were not visited by insects. Thus the authors concluded that these yeasts were

the part of a specific insect-flower ecosystem.

Another comprehensive study of yeast-insect-morning glory system of Kipuka

Puaulu, Hawaii was performed by Lachance et al. (2003a). On the basis of their study,

they proposed that Metschnikowia hawaiiensis was confined to Hawaii as it has not

been isolated anywhere else after its first description by Lachance et al. (1990). They

concluded thatM hawaiiensis and Candida kipukae were specific symbionts ofNitulid

B (an unidentified beetle) and that other species like Candida ipomoeae and

Metschnikowia lochheadii are associated with Conotelus mexicanus (Beetle). This

study raised several interesting questions: what are the factors maintaining this type of

specificity? What type of interaction may be occurring there with the other species?

Another interesting distribution was found of ten putative new species of genus

Starmerella, which was specifically isolated from bees during the study by Lachance et

27

---- ---------~---------


Chapter J: Introduction and Review a/Literature

al, (2000) and Rosa et al. (2003). Several species of the genus Starmerella (probable

anamorphs of Starmerella) viz Candida cleridarum, Candida tilneyi, Candida powellii

(Lachance et al., 2001a), Candida riodocensis, and Candida cellae (pimentel et aI.,

2005) have been described recently from such habitats. A mutualistic relationship

among species of yeasts of Starmerella complex and associated bees has been

suggested for Candida magnoliae, Starmerella bombicola and Candida batistae (Rosa

et aI., 2003). The yeasts appeared to be viable as high cell count was found in honey

and pollen provisions of these bees. The authors suggested that yeasts may be involved

in increasing the nutritional quality of these resources as they are known to provide

nutrients in many insects (Lachance et al., 1990; Morais et aI., 1994; Starmer, 1981).

Although some of these species like Candida etchellsii, have also been isolated from

other habitats and some are known to be spoilage yeasts in sugary substances but the

authors suggested that these yeasts were vectored by these bees which are mostly

attracted by sugary substances. The other genus which was suggested to be a part of

such systems is Wickerhamiella and the five species currently known in this genus have

been isolated from yeast-insect-flower ecosystem (Lachance et aI" 1998).

Recently in a breakthrough research, beetles that feed on basidiocarps were

recognized as a hyper diverse habitat, and a total of 650 yeasts were isolated from

beetles of 27 families. Of the 650 yeast species, nearly 200 species were suspected to

be putative new species on the basis of their D11D2 domain sequences (Suh et al.,

2005a). This study provided an important insight into an unrealized truth that the

number of yeast species is grossly underestimated. The studies on insects revealed that

these yeast are often well separated as unique clades. For example, during the study of

yeasts from baisdiocarp-feeding beetle, more than 30% yeasts from 1000 guts of

beetles were clustered in clades near Candida tazawaensis and Candida krusii (Nguyen

et al., 2006; Suh et aI., 2006). The other unique clades were related to Candida

mesenterica, Candida membranaefaciens, Pichia guilliermondii and Geotrichum

species (Suh & Blackwell, 2004; Suh & Blackwell, 2005; Suh et al., 2005b; Suh &

Blackwell, 2006). In another study, the yeasts Spathaspora passalidarum gen nov. sp.

nov. and its sister taxa Candida jeffriesii sp. nov were isolated and proposed from two

wood-ingesting beetles of the families Passalidae and Tenebrionidae (Nguyen et aI.,

2006). Although the yeasts isolated from insects are distantly related but showed

similar physiological profiles and also shared the ability to ferment xylose, a major

28

PujaSaluja Ph D. Thesis, IMTECH Chandigarh


component of hemicellulose and lignocellulosic mass (Nguyen et aI., 2006; Suh et aI.,

2003). The fermentation capability of these yeasts provided a clue about the symbiotic

relationship between yeasts and wood-ingesting beetles.

Another system which is studied in relation to insects is the 'cactus-yeast

insects' community. Several yeast species have been isolated from cactus nacrosis.

Although not unique, Drosophila is the most important and perhaps the most

predictable vector in this system. The core species of cactus-yeast communities are

Pichia cactophila, Sporopachydermia cereana and Candida sonorensis, which were

specifically reported from yeast communities of stems of many columnar cacti or

opuntia-cladode rots but were not isolated from decaying cactus-fruit tissues and tree

fluxes. Other yeast species that are frequently reported from cactus-rot pockets are

members of the Starmera 'amethionina complex' (St. amethionina var. amethionina,

var. pachycereana and St. caribaea), Candida ingens-like species (Dipodascus starmeri

and close relatives), Clavispora opuntiae and Myxozyma mucilagina (Starmer et aI.,

2003). The most interesting feature of these communities is the species specificity and

consistency in terms of space and time. During the study of yeast community of

Stenocereus gummosus from five distinct localities in Baja California, Norte, Mexico,

over the span of 15 years, Latham (1998) found that diversity was quite constant in

different regions studied, different location of the region, different plants within the

location and with in the plant necrosis. On the basis of rRNA gene analysis the

polyphyletic origin of these species was reported which can be further correlated to

some of the unique aspects of cactus chemistry, survival in the extreme environments,

vector association and interactions among the cacti, yeasts and insects. Although the

underlying mechanisms of these interaction would be a subject matter for

comprehensive research in future but some clues can already be gained from the studies

in such ecosystems. It has been suggested that the community as a whole rather than a

specific yeast of a cactus plant stimulate sexual behavior in insects, helps in larval

development and contribute to the size and fecundity of the adult insect (Starmer, &

Aberdeen, 1990). This helps in maintaining the vector-host specificity. On the other

hand maintenance of specific yeast communities on cacti has been suggested to be at

least partially because of the special functional traits that these communities confer

upon their hosts. It was found that C. sonorensis could utilize methanol as a source of

carbon and therefore would be specific to those environments where methanol is

29

PujaSaltlja Ph. D. ThEsis, IMTECH Chandigarh


produced by plant cell-wall breakdown and it's the only species which can also ferment

glucose as well. S. cereana complex species can utilize inositol, a number of unusual

alcohols, aldehydes and ketones that are mostly produced in rotting cacti. This specific

utilization patterns might be responsible for maintaining its specificity in such

environments (Ganter, 1989).

1.2.10.2. Diversity of Phylloplane Yeasts

The external surface of leaves serves as a habitat for many microbes including yeasts.

This habitat is known as phyllosphere or phylloplane (Last & Price, 1969). The most

common yeasts of phylloplane are of basidiomycetous affinity and belong to the genus

Sporobolomyces, Rhodotorula (collectively referred to as the pink yeasts) and

Cryptococcus (white yeasts) (Inacio et ai., 2002; Phaff & Starmer, 1987). In addition,

members of the genera Bullera and Tilletiopsis are commonly isolated from leaves and

are thought to be especially adapted to leaf environment due to the production of

forcibly ejected ballistoconidia (Lindow & Brandl, 2003). Interestingly, the

ascomycetous yeasts were also isolated in equal proportions during studies of

phylloplane yeasts from Slovakia (Slavikova et al., 2007). Many new speCIes of

phyllolplane yeasts of genera Sporidiobolus (Wang & Bai, 2004), Dioszegia (Wang et

al., 2008), and Cryptococcus have been described recently (Inacio et al., 2005). Very

little is known about the ecology of phylloplane yeasts, and it has been suggested that

several factors including leaves and their surfaces determine the nature of yeast

community. Availability and the type(s) of nutrients available on the leaf surface are

considered as one of the important factor in determining the type of yeast community.

The availability of nutrients depends upon plant species, age of the leaf, and growth

conditions (Mercier & Lindow, 2000). The nutrients of the phylloplane, at least partly,

originate from the leaves themselves. Molecules leaching out of the plant leaves

include a variety of organic and inorganic compounds, such as sugars, organic acids,

amino acids, methanol and various salts (Slavikova et al., 2007). Other factors of the

leaf-surface environment to which the microbes needs to adapt, are thought to be

exposure of leaf surface to rapidly fluctuating temperature, relative humidity, and more

prominently the large fluxes of UV radiation from sun (Lindow & Brandl, 2003). Plant

surface represents a diverse habitat of microbes in general. So the occurrence of a

particular type of microbe(s) on a surface also depends upon their interactions with the

other microbes. In such habitats, yeasts are active as competitors for nutrients,

30

PtljaSaluja Ph. D. Thesis, IMTECHChandigarh


antagonists or symbiotic associates, or as the victims of the behaviour of their

neighbours (Do Carmo-Sousa, 1969).

1.2.10.3. Diversity of Soil Yeasts

Yeasts were very frequently explored from soil in the 20th century (Atlas et aI., 1973;

Bouthilet, 1951; Danielson & Jurgensen, 1973). The species which were frequently

isolated from soil in the 20th century belonged to the genera Cryptococcus,

Debaryomyces, Kluveromyces, Lipomyces and, Pichia etc. (phaff & Starmer, 1987).

The estimated number of yeasts species from soil ranges between a hundred to

few thousand cells/gram of soil (Latham, 1998). Although not always true, but in

general the diversity of basidiomycetous yeasts dominate over ascomycetous yeasts in

soil (poliakova et aI., 2001; Slavikova & Vadkertiova, 2000). The frequent occurrence

of basidiomycetous yeasts of genus Cryptococcus, Rhodosporidium and Lipomyces

from soil was correlated with the presence of capsule due to which they can survive

better in pure nutrient conditions and during desiccation (Slavikova & Vadkertiova,

2000). But there may be additional reasons as well because some basidiomycetous

yeasts like Cryptococus spp. are also known to survive in oligotrophic conditions as in

oligotrophic-ocean waters and glacial melt water in Argentina (de Garcia et al., 2007;

Nagahama et aI., 2001). Most of the soil yeasts lack fermentation ability and are

therefore dependent upon the aerobic respiration and thought to occur mostly at the

depths of 5-15 cm. Although there are very limited exploratory studies of yeasts from

soil but a few studies have examined their diversity from several regions in different

parts of the world including Antarctic soils. In a very interesting study, viable yeasts

(upto 9000 CFUs) were reported from the permafrost soils of Siberia with an estimated

age of 3 million years (Dmitriev, 1997). Thus, despite the complexity of the

organization of a eukaryotic cell, eukaryotic microorganisms can be found to be equally

resistant under the conditions of Siberian permafrost soils as prokaryotes. The overall

diversity patterns were the same as the members of Cryptococcus, Rhodotorula, and

Sporobolomyces predominate there as well. Similarly, yeasts have been reported from

the extreme environments of Antarctic soil. In such extreme environments where first

colonizer are thought to be heterotrophic microbes rather than photosynthetic

organisms (Tscherko et al., 2003), yeasts are speculated to play key roles in nutritional

31


Chapter 1: introduction and Review o/Literature

cycling and as members of food webs (Gadanho & Sampaio, 2005; Gadanho et aI.,

2006; Nagahama et aI., 2001).

The studies on soil yeasts based solely upon phenotypic characteristics (Mok et

aI., 1984; Slavikova & Vadkertiova, 2000; Slavikova & Vadkertiova, 2003; Vishniac,

1996) cannot be as informative and comparable to the current molecular studies

(Connell et al., 2008; Wuczkowski & Prillinger, 2004). The reason as explained earlier

is that the identification based solely upon phenotypic characters is often not reliable

and therefore as a result several species which were deposited in the culture collections

are being re-identified and reclassified based upon modern methods (Fonseca et aI.,

2000; Inacio & Fonseca, 2004; Takashima et al., 2003). A study on the diversity of soil

and litter yeasts from alluvial forest national park based upon molecular methods was

performed by Wuczkowski and Prillinger (2004). In their study, 136 yeast strains, with

36 different sequences belonging to 16 genera were identified. The dominating yeasts

from the soil and litter were related to Cryptococcus, Sporobolomyces and

Trichosporon. Study of yeast species from rhizosphere and non-rhizosphere soils of

Panax ginseng (a medicinal plant) cultivation field was performed recently (Hong et

al., 2006). Among the 34 isolated yeasts, 3 were ascomycetous and 14 were

basidiomycetous yeasts. Among the basidiomycetous yeasts, one was Uredinomycetous

yeast, Rhodotorula sloojiae and 12 were hymenomycetous yeasts of which, except one

isolate, all others were of different species of the genus Cryptococcus. Cryptococcus

podzolicus was originally isolated from forest soil in Siberia ( Fell & Statzell -Tallman,

1998a) and afterwards from rhizosphere soil in Korea. Cr. watticus which was isolated

from soil of Antarctica (Guffogg et aI., 2004), was also from rhizosphere soil of

Chinese balloon flower and apple tree (Hong et aI., 2002) and also from soil of Panax

ginseng field (Hong et aI., 2006). This type of species which have mainly been isolated

from soil samples provides clues towards understanding the ecology of such yeasts. As

the studies on the diversity of soil yeasts are rather limited throughout the world, a

comprehensive view cannot be drawn. Understanding the several different aspects of

their ecology in this habitat would require many more studies. Although biodiversity of

soil yeasts has been suggested to play only a minor role in soil but the occurrence of

specific types of yeasts in a limited survey of 3 millions years old soil and from

extreme environments of Antarctica strongly argue that they might be performing some

as yet unknown but important roles in those soils.

32

1.2.10.4. Diversity of Fermentative Yeasts



The foremost and oldest application of yeasts utilizes their fermentation ability to

produce ethanol, alcoholic beverages and fermented foods. The most popular yeasts

responsible for fermentation are the strains of Saccharomyces viz strains of S.

cerevisiae, S. exiguus and S. rosei. These include bakers yeast, wine yeasts (including

special flocculent strains for the production of champagne and film-forming strains for

the production of flor sherry), sake yeast, top and bottom fermenting brewing yeasts

and distiller stains used for alcohol production (Demain et aI., 1998). There is

continuing research on the role of Saccharomyces cerevisiae in beer, wine and bread

fermentations but with time research has expanded into exploring the role(s) of yeasts

in other products as well (Fleet, 2007). Several non-Saccharomyces yeasts in addition

to S. cerevisiae are attracting interests for many different applications that include

adding flavor, aroma, or improving the texture during cheese maturation (Addis et ai.,

2001), preparation of sausages (Cocolin et aI., 2006; Gardini et ai., 2001) and varieties

of sour dough (Fleet, 2007). Some of these non-conventional yeasts are: Debaryomyces

hansenii, Yarrowia lipoiytica, Kiuyveromyces maraxianus, Saccharomyces exiguus,

Candida milleri, C. humilis, C. krusei (Issatchenkia orientalis), Pichia anamoia, and P.

membranifaciens, Yarrowia lipoiytica is generating considerable research interest as it

showed lipolytic and proteolytic activities, both of which can be exploited for

biotechnological applications (Gardini et aI., 2001). Species of some other genera such

as Saccharomyces, Hanseniaspora, Candida, and Pichia contribute to generating the

precursors of chocolate flavor by fermentation of coca beans (Ardhana & Fleet, 2003;

Nielsen et aI., 2005).

Some yeasts can also have some detrimental effects on food and beverages.

These yeasts are known as spoilage yeasts and their examples include Candida stellata,

Pichia jermentans, and Metschnikowia puicherrima for fruit spoilage, species of

Brettanomyces for wine, beer and soft drink spoilage and certain strains of

Debaryomyces hansenii for meat products spoilage (Fleet, 2007). The yeasts of the

Zygosaccharomyces genus have had a long history as spoilage yeasts within the food

industry, The ability of these yeasts to function in food spoilage is due to their ability to

grow in the presence of high sucrose, ethanol, acetic acid, sorbic acid, benzoic acid, and

sulfur dioxide concentrations.

33

1.2.10.5. Pathogenic Yeasts

PujaSaluja Ph. D. Thesis, IMTECH Chand;garh

Chapter J: Introduction and Review a/Literature

Yeasts are considered to be human friendly organisms, but some species of yeasts are

known as human pathogens. Two of the well-known pathogens are: Cryptococcus

neojormans and Candida albicans. C. albicans is considered as commensal because its

host-free occurrence is rare. It is found in the oral, gastrointestinal, or urinogenital

tracts in human and other warm-blooded animals. C. albicans can cause vulvovaginitis,

dermatitis, cystitis, fever, myosistis, hepatic dysfunction, and mental confusion.

Depending upon the nature of infection which may be superficial, invasive or deep, one

or more type of abnormalities can be seen. Different strains of C. albicans show

differences in virulence but parasitism depends upon the physical status of human host.

A high frequency of oral Candidiasis is reported from AIDS patients (phelan et aI.,

1987).

Pathogenic yeasts responsible for candidiasis in probable descending order of

virulence for humans are: Candida albicans, Candida tropicalis, Candida stellatoidea,

Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii,

Candida viswanathii, Candida lusitaniae and Rhodotorula mucilaginosa. Candida

glabrata is the second most common Candida pathogen after C. albicans, causing

infections of the urogenital tract, and of the bloodstream (Candidemia) (Stoyan &

Carbon, 2004). During a survey of C. albicans and non C. albicans species of patients

with candidaemia at hospital of Heraklion, Greece, 46% had candidaemia due to C.

albicans and 54% due to non-albicans species (25%, C. glabrata; 40%, C. tropicalis;

26%, C. parapsilosis; 3%, C. lusitaniae; 4%, C. kruset; and, 3%, C. guilliermondii)

(Samonis et aI., 2008). Similarly, dominance of non C. albicans species has been

observed in Brazil (Colombo et al., 1999). Candida dubliniensis an another emerging

pathogen was described in 1995, which was misidentified earlier as C. albicans. This

yeast is capable of causing oropharyngeal, vaginal and bloodstream infections in human

immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome

(AIDS) patients (Sullivan et al., 1995). However, C. dubliniensis is rarely reported in

the oral microtlora of normal healthy individuals as compared to Candida albicans

(Sullivan et aI., 2004).

34



Cryptococcus neoformans is not usually commensal and is known to cause

Cryptococcosis in people. The cells of this yeast are surrounded by a rigid

polysaccharide capsule, which helps to prevent them from being recognized and

engulfed by white blood cells in the human body. Two varieties of Cryptococcus that

are known as pathogens are: Cr. neoformans var. neoformans and Cr. neoformans var.

gatti, Serotypes of Cr. neoformans var. neoformans (A, D, AD) are found in soil rich in

pigeon droppings. Cr. neoformans var. gatti serotype B has been associated with

flowerings of Eucalyptus camaldulensis. Cryptococus primarily infects the lungs. It can

produce pneumococcal-type pneumonia and can cause fatal meningoencephalitis In

untreated or immuno-compromised individuals (Hull & Heitman, 2002).

1.2.10.6. Diversity of Aquatic Yeasts

Aquatic yeasts occupy fresh water, estuary and marine habitats. In spite of recent

descriptions of new species from aquatic environments, updated information on the

ecology of aquatic yeasts is scarce. The fresh water and estuaries are difficult to be

classified as specific habitats as their microbiology is highly affected by the

surrounding fauna and flora, soil rain off and/or effluents of human sources. It was

found that the predominant yeasts from fresh waters were usually ubiquitous or were

associated with pollution (as cited by Lachance & Starmer, 1998). A very interesting

study on freshwater yeasts showed that the yeast community structure in the Patagonian

freshwater yeasts depends upon their ability to produce photoprotective compounds,

their tolerance to UV exposure and their success in colonizing habitats highly exposed

to UV radiations (Libkind et al., 2006). The yeast diversity of estuary of Tagus river,

Portugal has been studied recently by culturable and unculturable methods of yeast

identification. The dominant populations in the culturable class belonged to

Debaryomyces hansenii, Rhodotorula mucilaginosa, Oyptococcus longus, and in the

unculturable class to a basidiomycetous yeast phylogenetically close to Cr. longus

(Gadanho & Sampaio, 2004; Lachance, 1998). It was only recently that yeasts were

isolated from extreme aquatic habitats like glacial melt water rivers in Patagonia,

Argentina (de Garcia et ai., 2007; Libkind et al., 2003) and extreme acidic environment

of the Iberian Pyrite belt (Gadanho et al., 2006). Marine yeasts are considered as

versatile organisms for biodegradation. Marine yeasts are suggested to play an

important role in plant substrates decomposition, nutrient cycling and biodegradation of

oil compounds (Kutty & Philip, 2008). Yeasts were isolated from different sources in

35

PujaSaJuja Ph D. Thesis, IMTECH Chandigarh

Chapter J: Introduction and Review o/Literature

manne water viz seawater, manne deposits, seaweeds, fish, marine mammals and

seabirds. The number of yeasts varies from 10-1000 cells/litre of water near shores and

10 or fewer cells in deep sea and places with low organic contents. The number of

yeasts decreases with increasing depth, Aerobic forms are abundant in non-polluted

water while polluted water contains mostly the fermentative yeasts. Marine habitats

have been explored all over the world including India. Yeasts have been isolated from

Pacific ocean (Yamasato et aI., 1974), Indian ocean (Fell, 1967; Godinho et al., 1978),

Indo-Pacific ocean (Fell, 1976), North Sea (Ahearn & Crow, 1980) and Atlantic ocean

(Fell, 1970). The yeasts isolated from marine habitat frequently belong to the genera

Candida, Cryptococcus, Debaryomyces, Rhodotorula and Torulopsis. Ascomycetous

yeasts are more abundant than basidiomycetous yeasts from weed algae and animals.

These yeasts are usually of the genera Candida, Metschnikowia, Saccharomyces,

Pichia, and Debaryomyces (Kutty & Philip, 2008). There are also some recent reports

of yeasts from aquatic habitats from deep sea environment of Pacific ocean (Nagahama

et aI., 2001), from animals and sediment of deep sea floor of Suruga Bay (Nagahama et

al., 2003a; Nagahama et aI., 2003b) and from mid-atlantic ridge hydrothermal fields

near the Azores Archipelago (Gadanho & Sampaio, 2005).

1.2.11. Biotechnological Applications of Yeasts

1.2.11.1. Model Organisms

Yeasts such as the budding yeast, S. cerevisiae, and the fission yeast, S. pombe, are

among the most widely used eukaryotic model organisms for genetics, cellular and

molecular biology. Some of the properties that make yeasts particularly suitable for

biological studies include rapid growth, dispersed cells, the ease of replica plating,

mutant isolation, a well-defined genetic system, and most important, a highly versatile

DNA transformation system. Being nonpathogenic, these yeasts can be handled with

little precautions. S. cerevisiae was the first eukaryote with a fully sequenced genome

(Goffeau et aI., 1996). Fission yeast genome sequence followed a few years later

(Wood et aI., 2002). Over 20% of human disease genes have been reported to have

counterparts in yeast as revealed from the genome sequencing. This is largely because

the cell cycle in a yeast cell is very similar to the cell cycle in humans, and therefore the

basic cellular mechanics of DNA replication, recombination, cell division and

metabolism are comparable. Interestingly, many proteins important in human biology

36

PujaSa/uja Ph. D. Thesis, lMTECH Chandigarh

Chapter 1: lntroduction and Review o/Literature

were first discovered by studying their homologues in yeast; these proteins include cell

cycle proteins, signaling proteins, and protein-processing enzymes.

In addition, yeasts have proved to be extremely valuable tools for studies of

other organisms including the use of the two-hybrid screening system for the general

detection of protein-protein interactions and the use of Y ACs for cloning large

fragments of DNA. Finally, availability of synthetic genetic arrays, single gene

knockout libraries, ease of generating conditional knockouts and carrying out over

expression screens have made these yeasts instrumental in generating a plethora of

scientific knowledge (Ostergaard et al., 2000).

1.2.11.2. Bioethanol Production

World is facing an energy crisis because of increasing energy demands due to

continuous development in science and technology and on the other side continuous

fear of exhausting the present oil and petroleum reserves. The bioethanol production is

attracting considerable interest across the world as a biofuel which can provide an

alternative to the presently used non-renewable sources. Saccharomyces cerevisiae has

been the leading microbe capable ofbioethanol production since centuries and no other

organism has ever come close to posing a challenge. Throughout the world researchers

are trying to increase the bioethanol production by genetic modifications and metabolic

engineering of known yeasts, and by isolating new yeasts which may have the promise

for higher ethanol production. Today the leading countries for bioethanol production

are Brazil (17 x 106 t) and the USA (15.1 x 106 t) (Branduardi et al., 2008).

1.2.11.3. Food and Beverage Production

The role of yeasts specifically, the S. cerevisiae strains for baking and production of

alcoholic beverages is among the oldest applications of yeasts which are still mostly

dependent upon yeasts. Besides that the roles of conventional yeasts, non

Saccharomyces yeasts such as Debaryomyces hansenii, Yarrowia lipolytica, Pichia

anomala, Candida mil/eri, Candida humilis, Hanseniaspora spp. have been expanded

to include production of many food ingredients and additives (Fleet; 2007).

1.2.11.4. Production of Heterologous Proteins

37

PujaSaiuja Ph. D. Thesis, IMTECH Chandigarh


Yeasts' utilization for heterologous gene expression is of considerable interest for the

production of pharmaceutical proteins of therapeutic value (interferons, interleukins

and insulin, etc,) and commercial interest (industrial enzymes) (Gellissen, 2000).

Yeasts are advantageous than other expression systems as they do not contain toxic

cell-wall pyrogens (endotoxins) like prokaryotes and devoid of oncogenic or viral

DNAs of mammalian cells. As eukaryotes, yeasts are also capable of performing post

translational processing and modifications (disulphide bond formation, proteolytic

maturation of pro-hormones, N- and O-linked glycosylation etc.) on expressed

polypeptides, which may be essential for the functionality of many proteins

(Branduardi et ai., 2004). Several non-conventional yeasts viz Kluyveromyces lactis,

Yarrowia lipolytica, and two methylotrophs-Pichia pastoris and Hansenula

polymorpha have been utilized for the production of heterologous proteins (Gellissen et

al., 2005; Hull & Heitman, 2002).

1.2.11.5. Probiotics

Yeasts such as Saccharomyces boulardii are being used as probiotic supplements, to

maintain and restore the natural flora in the large and the small gastrointestinal tracts. S.

boulardii has been shown to reduce the symptoms of acute diarrhea in children

(Kurugol & Koturoglu, 2005), prevent reinfection of Clostridium difficile (McFarland,

1994); reduce the incidence of antibiotic diarrhoea (McFarland et ai., 1995), traveler's

diarrhoea (Scarpignato & Rampal, 1995) and mY/AIDS-associated diarrhoea (Saint

Marc et al., 1995). Other yeasts which can have potential as probiotics are suggested as

D. hansenii, Yarrowia lipolytica, Pichia jarinosa, Pichia anomala (Fleet & Bali a,

2006).

1.2.11.6. Production of Enzymes and Other Industrially Important Compounds

Yeasts have been explored for the production of many industrially important enzymes.

The foremost important among them are the production of Lipase (Pandey et ai., 1999)

and Uricase (Chen et ai., 2008). The lipase produced by Candida rugosa is fast

becoming one of the most frequently used enzyme industrially. This is because of its

use in a variety of processes due to its high activity, both in hydrolysis as well as

synthesis (Redondo et ai., 1995). The other lipase which is of equal interest is of

Pseudozyma antarctica lipase that exhibited unique property of being thermostable at

90°C, inspite of its production from a psychrophilic organisms. This lipase has

38

PujaSaltga Ph. D. Thesis, IMTECH Chandigarh

Chapter 1: Introduction and Review o!Literahire

widespread application in food, pharmaceutical, agricultural, cosmetics and chemical

industries (Shivaji & Prasad, 2009), Uricase enzyme (used for the treatment of gout)

produced by Candida utilis was found to be less immunogenic when compared to that

produced by other fungi (Chen et al., 2008). In addition, yeasts are being utilized for

production of numerous industrially important compounds, all of which are difficult to

be listed here. Some of the important among them are a) citric acid, b) vitamins, c)

capsular polysaccharides, d) carotenoids, e) lipids and f) glycolipids etc.

1.2.11.7. Yeasts as Biocontrol Agents

Biocontrol provides a non-hazardous means for effectively controlling post-harvest

diseases, and producing safe foods with high quality. Cryptococcus laurentii has been

showed to have antagonistic activity against many post-harvest pathogens (Roberts,

1990), Aureobasidium pullulans has been reported to exhibit antagonistic activity

against Rhizopus sp. in strawberries (Lima et al., 1997); Rhodotorula glutinis has been

shown to be effective against Penicillium expansum in post-harvest apples (Cal vente et

aI., 1999) and Candida oleophila and Pichia anomala work against major post-harvest

diseases of citrus fruits (Lahlali et aI., 2004). The above-cited are only a small number

of examples in the growing list utilization of yeasts in biocontrol measures.

1.2.11.8. Bioremediation

Yeasts have been identified as potential targets for bioremediation. The yeast, Yarrowia

lipolytica, is known to degrade palm-oil mill effluent, TNT (an explosive) (Jain et aI.,

2004) and other hydrocarbons such as alkanes, fatty acids, and oils (Fickers et al.,

2005). Pichia guilliermondii and other yeasts have been explored recently for

bioremediation of different metals (Ksheminska et aI., 2003).

1.2.12. Methods for Studying Yeast Diversity

1.2.12.1. Methods of Sample Collection and Processing

Sampling methods and sample processing depend upon the objective of the study. A

very general consideration is that samples need to be collected aseptically. All the

equipment and accessories being used should be made sterile by an appropriate

procedure, The method of sterilization depends upon the sample type. For example,

aseptic sampling of flowers, fruits and insects often involves sterile bags/containers,

39



sterile spatulas, needles, cotton swabs, pipettes, hand sterilization and use of gloves etc.

For clinical samples, sterilization is often performed by swabbing the area with alcohol,

iodine or 2-propanal to remove surface contaminants and bacteria (Boundy-Mills,

2006). Rogers et al. (2004) have described several methods for the surface sterilization

of ice glaciers viz: exposure to bleach, ethanol, UV radiation and use of acid, base or

hydrogen peroxide.

Samples can be collected and processed in several ways. Nectar samples can be

collected with a sterile capillary pipette and can be plated as such on agar medium

(Herzberg et ai., 2002). Similarly, samples such as tree exudates and insect frass can

also be directly plated. To study the yeasts of external surfaces of flowers like corolla

or phyllosphere yeasts, the samples are usually pressed on the surface of the agar media

(Brysch-Herzberg & Lachance, 2004). In some cases the processing of sample is not

that simple, for example the yeast inocula used in brewing industry become stratified

(ASBC, 2003) therefore multiple samples from multiple sections of yeast cake or slurry

are recommended for use as inocula. When the distribution of an organism is suspected

to be non-uniform, homogenization of the sample is recommended to get the proper

representation of the community. Incomplete and/or improper homogenization and

dilution can results in erroneous enumeration of cell density as discussed by Fleet

(1999) .

Some samples where number of yeasts are known to be less as in aquatic

habitats (Kutty & Philip, 2008) and in soil (Wuczkowski & Prillinger, 2004), samples

can be concentrated by direct centrifugation followed by resuspension or else

concentration of samples can also be achieved by using membrane filters (Gadanho et

ai., 2003). Samples with higher concentration of yeasts such as those from flowers,

insects, fruits and plant leaves need to be diluted to obtain countable colonies on the

plate. Aliquots of each dilution are plated on appropriate media, and the number of

colonies is used to calculate the concentration of yeasts in the original sample,

expressed as CFU/ml.

Several different dilutants are recommended for enumeration of yeasts.

Examples include saline solutions used by brewers (0.85%) (ASBC, 2003 ; Mian et ai.,

40

PujaSaluja Ph. D. ThEsis, IMTECH Chandigarh

Chapter J: lntroduction and Review o/Literature

1997); and milk or Butterfields phosphate-buffered water for dairy products (Boundy

Mills, 2006).

1.2.12.2. Isolation of Yeasts

Yeasts are isolated from both aquatic and terrestrial habitats. The number of yeasts per

gram of specimen is usually much lower than of bacteria and fungi which are

considered as more adaptive than yeasts. So during course of isolation growth of other

microbes should be suppressed in order to get colonies of yeasts.

Inhibition of Bacteria and Molds

The use of acidified media is suggested for retarding or suppression of bacterial growth.

The pH is maintained between 3.5-5.0 by using HCI or phosphoric acid. Alternatively,

antibiotics such as chloramphenicol, streptomycin, ampicillin, penicillin or cocktail of

these are used for yeast isolation (Martin, 1950; Saluja & Prasad, 2007b). Propionic

acid or calcium propionate decrease mold growth significantly, but also inhibit some

aerobic yeasts (Buhagiar & Barnett, 1971). Rose Bengal (Martin, 1950), oxgall (Miller

and Webb, 1954), eugenol, dichloran (Bell & Crawford, 1967), or oligomycin can be

added to inhibit rapidly spreading molds. The Rose Bengal Agar and/or

chloramphenicol agar has been used in some of the recent studies (Lachance et aI.,

2001b; Rodrigues et aI., 2001; Rosa et aI., 2003).

Media for Isolation of Yeasts

When yeasts are present in high numbers, they can be isolated on solidified media

either by using acidified media or antibiotics. The acidifying compound is added after

sterilization and cooling of media to 45°C as agar is hydrolyzed at low pH. Although

bacteria can be successfully inhibited at low pH but growth of some yeasts like

Schizosaccharomyces is also inhibited at that pH.

The liquid media can be used for the enrichment of yeasts if the yeast number is

less in the sample. Yarrow (1998) suggested that shaking of flasks with liquid media

would restrict the growth of molds, and in such a setting bacterial growth can be

inhibited by acidifying or addition of antibiotics to the media. Shaking inhibits the

sporulation in molds and they aggregate in pellets. The yeasts can then be separated

from molds by allowing the mold pellet to settle down and then streaking the

41

PtdaSa!uja Ph. D. Thesis, IMTECH Chandigarh


suspension of yeasts on to agar in petri plates, or by removing the pellet by aseptically

filtering the liquid culture through a loose plug of sterile glass wool.

1.2.12.3. Morphological characterization

Characteristics of Vegetative cells

This includes the study of colony and cell morphology as described by Yarrow (1998),

production of filaments, modes of asexual reproduction. Details of study of cell and

colony morphology are given in detail in Chapter-2, Some of the yeasts form chains of

cells where the bud fails to detach and these cells convert into pseudohyphae. The

pseudohyhae are very common in the genus Pseudozyma. Pseudohyphae may be

rudimentary or may develop into elongated cells where each may produce blastospore

in regular or characteristic manner, Some of the yeasts also produce true septate

hyphae.

Modes of Asexual Reproduction:

The asexual reproduction includes formation of buds, by fission or conidia formation

on short stalks. The other mode of asexual reproduction is the formation of arthospore,

endospore and chlamydospore or ballistospore.

Vegetative Bud Formation: This includes study of mode of bud formation and

arrangement of successive buds if present. Budding cells may be monopolar,

bipolar or multipolar. Acropetal budding is the formation of successive buds in a

chain with the youngest at the apex while opposite is the case in basipetal budding.

Arthoconidia Formation: Hyphae of some cells break up or disarticulate to form

one-celled arthospore or arthoconidia. They are arranged in a zig-zag fashion on

solid media.

Fission: Reproduction by fission IS characteristics of the genus

Schizosaccharomyces in which cells form an inward septum which bisect the long

axis of the cell.

42



Endospore Formation: Endospores are vegetative cells which are formed within

discrete cells and hyphae. Endospores are formed in the genus Trichosporon,

Candida, Cryptococcus, Oosporidium and Cystojilobasidium.

Chlamydospore Formation: These are thick-walled spores which may be terminal or

intercalary. They have been reported in C. albicans and Metschnikowia and rarely

from Trichosporon and Cryptococcus.

Ballistospore Formation: Ballistospores are specialized spores that form on

sterigmata which produced from vegetative cells. These spores are discharged into

the air by droplet mechanism.

Study of Sexual Reproduction

Many yeasts reproduce both asexually and sexually resulting in alteration of their

generations. A yeast that forms either asci or basidia is referred to as a perfect yeast or

in a perfect state. A yeast that does not form either asci or basidia is referred as an

imperfect yeast or in an imperfect state. Perfect state is also known as teleomorphic

state and the imperfect state is known as the anamorphic state. A yeast exhibiting the

combined states is termed as holomorph. The teleomorphic and anamorphic states of

the same yeasts have different names, for example Pichia jadinii (teleomorph) and

Candida utilis (anamorph). This study of sexual reproduction constitutes the mode of

karyogamy, plasmogamy, formation of sexual structures such as ascus or basidium,

formation of ascospores in ascomycetous yeasts and basidiospore formation in

basidiomycetous yeasts. Shape and number of ascospores is another interesting

characteristic of some genera, for example they are long and clavate in Metschnikowia

while sac-like in Lipomyces.

1.2.12.4. Physiological and Biochemical Characterization

Physiological and biochemical characterization involves a battery of about 90 tests

including assimilation and fermentation of several different carbon compounds, growth

and utilization of nitrogen sources, requirements for vitamins, growth at various

temperatures and media with high sugar content or sodium chloride, hydrolysis of urea,

and resistance to antibiotics. These tests are performed in liquid or solid media. For

fermentation tests, the Durham tube method is preferred and is performed in liquid

43



media containing 2% solution of different carbon sources. The liquid-based tests are

performed by inoculating the culture in the media containing the appropriate amount of

the carbon source under investigation that has been added to the basal medium. The

growth is compared to a positive as well as a negative control. The results of these tests

are interpreted as positive, negative, weak or delayed. The details for these tests are

provided in the Materials and Methods section of this dissertation.

For solid media, the tube method, replica plating and auxanogram are utilized.

In the tube and the replica plating methods the compounds are present in the media.

Results are read by comparing the growth of the strain under question with a negative

control consisting of the basal medium without the added compound. In the

auxanogram method, yeast cells are seeded in basal agar medium containing various

compounds at different points at the periphery. Results are read by examining the plates

for the formation of opaque zones at the points where the compound was added.

1.2.12.5. Molecular Methods to Study Yeast Diversity

Molecular methods include DNA-based techniques which are more rapid and accurate

than the traditional phenotypic methods. Several molecular methods are being used in

combination to study yeast diversity and for characterization of yeasts. These methods

include Random Amplified Polymorphic DNA (RAPD), microsatellite fingerprinting,

Restriction Fragment Length Polymorphism (RFLP), electophoretic karyotyping and

sequencing of rRNA and other genes. Some culture-independent methods include use

of Temperature Gradient Gel Electrophoresis (TGGE), Denaturing Gradient Gel

Electrophoresis (DGGE) microarray, flow cytometry, single-cell conformation

polymorphism and cloning. Some of these methods are relatively recent and are being

used for specific purposes mostly in combination with sequencing. A brief description

of some of these methods is provided in the following sub sections.

Randomly Amplified Polymorphic DNA (RAPD)

RAPD is a fingerprinting method based upon a single random primers of 10 bases

(Williams et aI., 1990). RAPD allows the separation of yeasts mostly at species level

and sometimes at strain level as well. It has been widely used to study yeasts diversity

in several different habitats. Balerias-Couto et al. (1994 and 1995) used RAPD to

differentiate spoilage and food-borne yeasts. They could assess variability among S.

44

PujaSaJ'!ia Ph. D. Thesis, IMTECH Chandigarh


cerevisiae and Zygosaccharomyces species by RAPD. The brine yeasts which were not

identified accurately by phenotypic methods could be discriminated by RAPD

(Prillinger et ai., 1999). RAPD was used to discriminate yeasts in dairy products

(Vasdinyei & Deak, 2003), sourdoughs of Italian sweet-baked products (Foschino et

aI., 2004) and brewery yeasts (Barszczewski & Robak, 2004). Although this method

seems to be useful in some specific cases, but it suffers from certain limitations. The

RAPD methods are not always reproducible among or even within the laboratories.

Conflicting results have been obtained by RAPD and sequence analysis during study of

yeasts from flowers (Herzberg et ai., 2002). The authors concluded that numerous

variables make RAPD PCR analysis unreliable, at least as a means of identifying yeasts

in ecological studies.

Simple Sequence Repeat Fingerprinting or Microsatellite Fingerprinting

Simple sequence repeats (SSRs), or microsatellites, are genetic loci where one or a few

bases are tandemly repeated for varying numbers of times (Katti et ai., 2001). These

repetitions occur primarily due to slipped-strand impairing and subsequent error(s)

during DNA replication, repair, or recombination (Levinson & Gutman, 1987). These

loci can mutate further by insertions or deletions of one or a few repeat units, and the

mutation rates generally increase with an increase in the length of repeat tracks (Wierdl

et aI., 1997). Eukaryotic genomes frequently contain several interspersed

microsatellites. Microsatellite loci show extensive length polymorphism, and hence

they are widely used in DNA fingerprinting and diversity studies. There may be several

locations in the genome where these loci may be present in such a way that they are not

too far away from each other and their directions converge such that a single PCR

primer could anneal at both of the loci and generate a unique PCR product. Several

such pairs of loci together can generate a pattern that is frequently referred to as the

Microsatellite fingerprint. PCR fingerprinting with the microsatellite primers (GTG)s,

(GACA)4, and the M13 sequence GAGGTGGCGGTTCT has been used for yeast

identification (Lavallee et aI., 1994; Lieckfeldt et ai., 1993).

In a study of diversity of S. cerevisiae strains from wine and beer, microsatellite

fingerprinting was used in combination with RAPD. The fingerprinting primer (GTG)s

was found to be more discriminatory as 16 stains were separated into 6 groups (a total

of 6 unique patterns) in comparison to the primer (GAC)s, which separated the 16

strains into only two different groups (Baleiras Couto et aI., 1996). Gallego et al.

(2005) compared SSR, RAPD, and AFLP (Amplified Fragment Length Polymorphism)

45

PujaSaluja Ph. D. Thesis. IMTECH Chandigarh

Chapter1: Introduction andReview ofLilerarure

markers for the genetic analysis of yeast strains of Saccharomyces cerevisiae isolated

from wineries. They used six microsatellite loci and appropriate primers needed for

their specific amplification. They considered SSR as reliable, fast, easy and highly

discriminatory method for identification and characterization of S. cerevisiae strains.

Microsatellite fingerprinting was also used to study the diversity of yeasts in marine

samples collected from Portugal (Gadanho et aI., 2003). In that study, a total of 31

MSP-PCR classes were formed, 8 for the pigmented yeasts and 23 for the non

pigmented yeasts. Similarly, microsatellite fingerprinting was successfully utilized to

study plant-associated yeasts in Brazil (Inacio et al., 2008), during polyphasic

taxonomy of the genus Rhodotorula (Gadanho & Sampaio, 2002), and for

differentiation of strains of Hanseniaspora uvarum isolated in the Finger Lakes

wineries in New York (Bujdos6 etal., 2001).

Restriction Fragment Length Polymorphism (RFLP)

RFLP is also a widely used fingerprinting technique in which different regions of

rRNA gene and mitochondrial DNA have been utilized for fingerprinting. The mt-DNA

RFLP has been employed for examining the authencity of S. cerevisiae commercial

strains of wine (F ernandez-Espinar et aI., 2001), for typing of D. hansenii strains from

dairy products (Petersen et aI., 2001). The ITS RFLP has been used successfully in

many studies for differentiation of S. cerevisiae strains isolated from wine (Guillamon

et al., 1998), wine and beer contaminants from a brewery (Hansen & Jakobsen, 2001).

The RFLP was also used to characterize non-Saccharomyces yeasts isolated from food

and beverages (Esteve-Zarzoso et aI., 2001; Fernandez et aI., 2000). However, during

characterization of yeasts from various food products, ITS-RFLP using the restriction

enzymes MspI and HaeIII, same patterns were obtained for D. hansenii and

Meschnikowia pulcherrima and several other yeasts (Senses-Ergul et aI., 2006).

Similarly, ITS RFLP was found to be ineffective in differentiating yeasts from dry

cured meat products (Andrade et aI., 2006), for strain typing of D. hansenii even after

using 9 different restriction enzymes (Petersen et aI., 2001) and even to discriminate,

different Debaryomyces species (Martorell et aI., 2005). In contrast, IGS-RFLP was used

successfully to differentiate among species and varieties of the members of the genus

Debaryomyces (Quiros et aI., 2006). Similarly, IGS-RFLP was used to discriminate D.

hansenii from other species isolated from intermediate moisture foods (Romero et aI.,

2005).

46

Electrophoretic Karyotyping

PujaSaluja Ph. D. Thesis, lMTECH Chandigarh


Karyotyping is the characterization of the number, size and form of the chromosome in

an organism, The electrophoretic karyotyping is performed by running the DNA sample

from the cells through an electric field such that chromosome-sized molecules get

separated according to their size. There are several electrophoretic systems available to

perform karyotyping and some of the widely used systems are: Pulse-Field Gel

Electrophoresis (PFGE), Orthogonal-Field Alteration Gel Electrophoresis (OF AGE),

Contour-clamped Homogeneous Electric Field Gel Electrophoresis (CHEF), and Field

Inversions Gel electrophoresis. Karyotyping using PFGE has been used to investigate

brewing contaminants (Jespersen et al., 2000), yeasts from dairy products (Hansen &

Jakobsen, 2001) and for wine yeasts (Fernandez et aI., 2000) as well.

Other Molecular Methods

There is ongoing research to develop new rapid and reliable tools/methodology to study

diversity and to help facilitate correct identification of yeasts. This becomes especially

valuable from industrial point of view. In addition, rapid identification is crucial from

clinical standpoint for identification of pathogenic yeasts. Some of the recently

developed methods includes Quantitative PCR, microarrays, TGGE, DGGE, Single

Strand Conformation Polymorphism (SSCP), and flow cytometry etc.

TGGE, DGGE and Cloning: TGGE refers to Temperature Gradient Gel

Electrophoresis and DGGE stands for Denaturating Gradient Gel Electrophoresis.

Both of these are electrophretic techniques which differ only in the nature of

gradient which is a denaturant in case of DGGE and Temperature in case of TGGE.

The double stranded DNAs are melted while passing through the gradient and

accordingly the mobility of DNA changes. As the DNA melting is sequence

dependent these techniques provide the separation of different sequences (of

different isolates of microorganisms under study) present in a community of DNAs

without the need for culturing the organisms.

Both of these are culture-independent methods which have been used to

assess genetic diversity in yeast populations from samples such as sourdough

(Meroth et al., 2003), during fermentation of coffee (Masoud et aI., 2004), wine

(Cocolin et aI., 2000; Cocolin & Mills, 2003), cocoa (Nielsen et aI., 2005), and

47

--------------------------------------



from slurry reactor systems (EI-LatifHesham et aI., 2006). PCR-DGGE was found

to be less sensitive than cl1lture-based approach for determining the yeast ecology

of grapes and could not reliably detect species present at populations less than 104

CFU/g. However, this method detected a greater diversity of species than traditional

agar plating (Prakitchaiwattana et al., 2004). During a survey of yeasts from estuary

of the Tagus river, Portugal, the number of species detected after enrichment was

higher than the number of taxa found using TGGE. Thus the authors suggested that

some yeast populations were present in densities below the detection threshold of

the method (Gadanho & Sampaio, 2004). Molnar et al. (2008) studied the yeast

biodiversity in the guts of several pests on maize by using culture-dependent and -

independent approaches; DGGE and cloning. Several genera could not be detected

through the culture-independent approaches. In addition, the most frequently

detected genera were the same as those found in classical isolation. Interestingly,

several fungi were also were detected along with yeasts, Yeasts diversity from

forest and grassland soil from Germany, could not be studied by unculturable

approach as only few yeasts were detected out of 102 clones sequenced. This study

implies, that yeast biomass in soil is smaller than other fungal groups (Kurth, 2008).

SSCP: In this method single-stranded PCR amplified DNAs are separated by

Polyacrylamide Gel Electrophoresis (pAGE) on the basis of small differences in

their secondary structures (generated by the DNA sequence). This method also

shares the limitation of DGGE and TGGE methods but is advantageous from them

as it does not require GC clamp like DGGE and TGGE. SSCP has been used to

detect polymorphism in clinical isolates of C. albicans (Graser et aI., 1996).

Flow cytometry: Flow cytometry has been used to detect as little as one yeast cell in

the background of 106 brewer's yeast cells (Jespersen et aI., 1993) and for clinical

isolates (Diaz & Fell, 2004; Page & Kurtzman, 2005). The major advantage of this

method is that multiple species can be identified from multiple samples by using a

mUltiplex assay.

Quantitative real time polymerase chain reaction (Q-PCR/qPCR): Q-PCR also

known as Real Time PCR is a technique based on the polymerase chain reaction,

which is used to amplify and simultaneously quantify a targeted DNA molecule. It

48

PujaSalr.ya Ph. D. Thesis, IMTECH Chandigarh


enables both detection and quantification (as absolute number of copies or relative

amount when normalized to DNA input or additional normalizing genes) of a

specific sequence in a DNA sample.

Q-PCR has been used to detect specific organisms in an environmental sample.

A very sensitive and accurate Q-PCR method has been developed for the detection

of six pathogenic Candida species in drinking water (Brinkman et al., 2003) and for

detection of spoilage yeast D. bruxellensis from wine based on specific D IID2

primers (Phister & Mills, 2003).

Microarray: Microarrays provide a new level of specificity to DNA-DNA

hybridization studies. A single array can contain several thousand specific DNA

sequences which can be used to detect a rather large number of target cells in a

given sample. These target sequences can be species-specific or function-specific

(to get functional diversity), Microarrays have been successfully used to detect

major microbial groups (EI Fantroussi et aI., 2003). Recently a sensitive microarray

has been developed to detect 10 most common pathogenic Candida species and

Aspergillus spp. by using specific ITS probes (Leinberger et al., 2005).

Some of the DNA-based methods have limitations and potential bias at

various steps including cell lysis, DNA extraction and purification. The efficiency

with which cells or mycelia are lysed can vary within and among microbial groups.

DNA and RNA extraction methods that result in DNA shearing, such as bead

beating, can also lead to biases (von Wintzingerode et aI., 1997). The use of some

of these methods is only restricted to studies of certain specific habitats of yeasts,

With time, these methods will probably be improved to overcome some of their

shortcomings and more widespread use of these methods will hopefully open new

horizons in the studies of yeast diversity.

49

Documents

1.1. Introduction - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/35251/8/08...consequently to the other habitats throughout the world, would make the estimated 3 PUjaSalt-{/a