Diversity of Microbial World Madam Noorulnajwa Diyana Yaacob PPK BIOPROSES April/May 2013

Diversity of Microbial World

Madam Noorulnajwa Diyana Yaacob

PPK BIOPROSES

April/May 2013

Course content ProkaryotesArchaeaBacteria Eukaryotes (microbial Protists)FungiAlgaeProtozoa Viruses

Introduction

Taxonomy is the science of the classification of organisms, with the goal of showing relationships among organisms.

Taxonomy also provides a means of identifying organisms.

consists of three separate but interrelated parts

classification – arrangement of organisms into groups (taxa; s., taxon)

nomenclature – assignment of names to taxa identification – determination of taxon to which an

isolate belongs

How would you classify?Types of classification: 1. natural2. polyphasic phenetic phylogenetic genotype

for reproduction or display.

Natural Classification arranges organisms into groups whose

members share many characteristics first such classification in 18th century

developed by Linnaeus based on anatomical characteristics

this approach to classification does not necessarily provide information on evolutionary relatedness

Polyphasic Taxonomy

used to determine the genus and species of a newly discovered prokaryote

incorporates information from genetic, phenotypic, and phylogenetic analysis

Phenetic Classification

groups organisms together based on mutual similarity of phenotypes (is the composite of an organism's observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior)

can reveal evolutionary relationships, but not dependent on phylogenetic analysis i.e., doesn’t weigh characters

best systems compare as many attributes as possible

Phylogenetic Classification also called phyletic classification

systems phylogeny

evolutionary development of a species usually based on direct comparison of

genetic material and gene productsWoese and Fox proposed using small

subunit (SSU) rRNA nucleotide sequences to assess evolutionary relatedness of organisms

Genotypic Classification

comparison of genetic (inherited instructions it carries within its genetic code) similarity between organisms individual genes or whole genomes can be

compared70% homologous belong to the same

species

Taxonomic Ranks - 1 microbes are placed in hierarchical

taxonomic levels with each level or rank sharing a common set of specific features

highest rank is domain Bacteria and Archaea – microbes only Eukarya – microbes and macroorganisms

within domain phylum, class, order, family, genus, species

epithet, some microbes have subspecies

Species definition

collection of strains that share many stable properties and differ significantly from other groups of strains

also suggested as a definition of speciescollection of organisms that share the same

sequences in their core housekeeping genes

Strains

descended from a single, pure microbial culture

vary from each other in many waysbiovars – differ biochemically and

physiologicallymorphovars – differ morphologicallyserovars – differ in antigenic properties

Type Strain

usually one of first strains of a species studied

often most fully characterized not necessarily most representative

member of species

well-defined group of one or more strains clearly separate from other genera often disagreement among taxonomists

about the assignment of a specific species to a genus

Binomial System of Nomenclature

devised by Carl von Linné (Carolus Linnaeus) each organism has two names

genus name – italicized and capitalized (e.g., Escherichia)

species epithet – italicized but not capitalized (e.g., coli)

can be abbreviated after first use (e.g., E. coli) a new species cannot be recognized until it has

been published in the International Journal of Systematic and Evolutionary Microbiology

Techniques for Determining Microbial Taxonomy and Phylogeny

classical characteristicsmorphologicalphysiologicalbiochemicalecologicalgenetic

Ecological Characteristics

life-cycle patterns symbiotic relationships ability to cause disease habitat preferences growth requirements

Molecular Approaches

extremely important because almost no fossil record was left by microbes

allows for the collection of a large and accurate data set from many organisms

phylogenetic inferences based on these provide the best analysis of microbial evolution currently available

Molecular Characteristics

nucleic acid base composition nucleic acid hybridization nucleic acid sequencing genomic fingerprinting amino acid sequencing

Nucleic Acid Base Composition

G + C contentMol% G + C =

(G + C/G + C + A + T)100usually determined from melting

temperature (Tm)variation within a genus usually <10%

Figure 17.2

Nucleic Acid Hybridization DNA-DNA hybridization

measure of sequence homology common procedure

bind nonradioactive DNA to nitrocellulose filter

incubate filter with radioactive single-stranded DNA

measure amount of radioactive DNA attached to filter

Table 17.4

Nucleic Acid Sequencing

Small subunit rRNAs (SSU rRNAs)sequences of 16S and 18S rRNA most

powerful and direct method for inferring microbial phylogenies and making taxonomic assignments at genus level

Comparative Analysis of 16S rRNA Sequences oligonucleotide signature sequences found

short conserved sequences specific for a phylogenetically defined group of organisms

either complete or, more often, specific rRNA fragments can be compared

when comparing rRNA sequences between 2 organisms, their relatedness is represented by percent sequence homology 70% is cutoff value for species definition

Figure 17.3

Genomic Fingerprinting used for microbial classification and

determination of phylogenetic relationships

requires analysis of genes that evolve more quickly than rRNA encoding genesmultilocus sequence analysis (MSLA)

the sequencing and comparison of 5 to 7 housekeeping genes is done to prevent misleading results from analysis of one gene

multilocus sequence typing (MLST) – discriminates among strains

Restriction Fragment Length Polymorphism (RFLP) uses restriction enzymes to recognize

specific nucleotide sequencescleavage patterns are compared

ribotyping similarity between rRNA genes is determined

by RFLP rather than sequencing

Figure 17.4

Single Nucleotide Polymorphism (SNP)

Looks at single nucleotide changes, or polymorphisms, in specific genes

16S rRNA focuses on one specific gene Regions targeted because they are

normally conserved, so single changes in a base pair reveal evolutionary change

Figure 17.5

Amino Acid Sequencing amino acid sequences reflect mRNA

sequence and therefore of the gene which encodes that protein

amino acid sequencing of proteins such as cytochromes, histones, and heat-shock proteins has provided relevant taxonomic and phylogenetic information

cannot be used for all proteins because sequences of proteins with different functions often change at different rates

Figure 17.6

The prokaryotes:

Domain Archaea

Domain Bacteria

1923,David Bergey (prof of bacteriology) published a classification of bacteria for identification of bacterial (and archaea) species.

Bergey’s Manual categorizes bacteria into taxa based on rRNA sequences.

Bergey’s Manual lists identifying characteristics such as Gram stain reaction, cellular morphology, oxygen requirements, and nutritional properties.

Bergey’s Manual of Systematic Bacteriology

The Archaea

Archaea

Scientist identified archaea as a distinct type of prokaryotes based on its unique rRNA sequence

Reproduce by : binary fusion, budding or fragmentation

Cells shape : cocci, bacilli, spiral, lobed, cuboidal etc

Not causing disease to humans/animals Cell wall contain proteins, glycoproteins,

lipoproteins, polysaccharides

Archaeal Cell Surfaces cell envelopes

varied S layers attached to plasma membrane

pseudomurein (peptidoglycan-like polymer) complex polysaccharides, proteins, or

glycoproteins found in some other species only Ignicoccus has outer membrane

flagella closely resemble bacterial type IV pili

Archaeal Membrane Lipids

differ from Bacteria and Eukarya in having branched chain hydrocarbons attached to glycerol by ether linkages

polar phospholipids, sulfolipids, glycolipids, and unique lipids are also found in archaeal membranes

Archaeal Lipids and Membranes

Bacteria/Eukaryotes

fatty acids attached to glycerol by ester linkages

Archaea branched chain

hydrocarbons attached to glycerol by ether linkages

some have diglycerol tetraethers

Figure 18.4

Archaeal Taxonomy

two phyla based on Bergey’s ManualEuryarchaeotaCrenarchaeota

16S rRNA and SSU rRNA analysis also shows Group I are ThaumarchaeotaGroup II are Korachaeota

Figure 18.1

Very high/low temp/pH, concentrated salts or completely anoxic (extreme environments)

Archae are either gram +ve or gram –ve Classified into two phylum :

1) Crenarchaeota

2) Euryarchaeota – 5 major physiologic groups (the metanogens, the halobacteria, the thermoplasms, extremely thermophilic S°-reducers and sulfate-reducing)

Phylum Crenarchaeota most are extremely thermophilic

hyperthermophiles (hydrothermal vents) most are strict anaerobes some are acidophiles many are sulfur-dependent

for some, used as electron acceptor in anaerobic respiration

for some, used as electron source

Figure 18.9

Figure 18.10

Crenarchaeota… include organotrophs and lithotrophs

(sulfur-oxidizing and hydrogen-oxidizing)

contains 25 genera two best studied are Sulfolobus and

Thermoproteus

Genus Thermoproteus long thin rod, bent or branched

cell walls composed of glycoprotein thermoacidophiles

70–97 °C pH 2.5–6.5

anaerobic metabolism lithotrophic on sulfur and hydrogen organotrophic on sugars, amino acids, alcohols,

and organic acids using elemental sulfur as electron acceptor

autotrophic using CO or CO2 as carbon source

Genus Sulfolobus irregularly lobed, spherical shaped

cell walls contain lipoproteins and carbohydrates

thermoacidophiles70–80°CpH 2–3

metabolism lithotrophic on sulfur using oxygen (usually)

or ferric iron as electron acceptororganotrophic on sugars and amino acids

Phylum Euryarchaeota consists of many classes, orders, and

families often divided informally into five major

groupsmethanogenshalobacteria thermoplasmsextremely thermophilic S0-metabolizerssulfate-reducers

The Methanogens - strict anaerobes- obtain energy by converting CO2, H2, methanol to methane or methane & CO2- eg. Methanobacterium, Methanococcus

- methanogenesis* last step in the degradation of organic compounds

*occurs in anaerobic environments e.g., animal rumens e.g., anaerobic sludge digesters e.g., within anaerobic protozoa

Ecological and Practical Importance of Methanogens important in wastewater treatment can produce significant amounts of

methane can be used as clean burning fuel and energy

source is greenhouse gas and may contribute to

global warming can oxidize iron

contributes significantly to corrosion of iron pipes

The Halobacteria- extreme halophiles- aerobic chemoorganotrophs (use organic compound as energy sources)- dependent on high salt content- cell wall dependent on NaCl, they disintegrated when [NaCl] < 1.5M- dead sea

Strategies to Cope with Osmotic Stress increase cytoplasmic osmolarity

use compatible solutes (small organics) “salt-in” approach

use antiporters and symporters to increase concentration of KCl and NaCl to level of external environment

acidic amino acids in proteins

The Thermoplasms- lack cell walls - but plasma membrane strengthen by diglycerol tetraether, lipopolysaccharides, and glycoproteins- grow best at 55-59°C, pH1-2- eg. Thermoplasma

Extremely Thermophillic So-Reducers- strictly anaerobic- can reduce sulfur to sulfide- grow best at 88-100°C- motile by flagella- eg. Thermococcus

Sulfate-reducing

- irregular garm –ve coccoid cellscell walls consist of glycoprotein subunits

- extremely thermophilicoptimum 83°C isolated from marine hydrothermal vents

- obtain their energy by oxidizing organic compounds or H2 while reducing sulfates to sulfides. In a sense, they "breathe" sulfate rather than oxygen

- eg. Archaeoglobus

Bacteria

Domain Bacteria

Bacteria are essential to life on Earth.

We should realize that without bacteria, much of life as we know it would not be possible.

In fact, all organisms made up of eukaryotic cells probably evolved from bacterialike organisms, which were some of the earlist forms of life.

The Proteobacteria Largest group of bacteria. More than 500 genera gram-negative, some motile using flagella Most are facultative/obligate anaerobes Share common 16s rRNA sequence 5 distinct classes of proteobacteria (α,β, ε, ɣ,δ) :

- Alphaproteobacteria - Betaproteobacteria- Gammaproteobacteria- Deltaproteobacteria- Epsilonproteobacteria

Figure 20.1

Alphaproteobacteria Gram -ve Most are oligotrophic (capable of growing at low nutrient

levels) Example of alphaproteobacteria ;

1) Most purple nonsulfur phototrophs are in this group (use light energy and CO2 and do not produce O2)

2) Nitrifying bacteria e.g. Nitrobacter (oxidize NH3 to NO3 by a process called nitrification)3) Pathogenic bacteria eg. Rickettsia (typhus), Brucella (brucellosis), Ehrlichia (ehlichiosis)4) Beneficial bacteria eg. Acetobacter and Caulobacter (synthesize acetic acid); Agrobacterium (used in genetic recombination in plants)

Purple Nonsulfur Bacteria with one exception (genus

Rhodocyclus) all are a-proteobacteria metabolically flexible

normally grow anaerobically as anoxygenic photoorganoheterotrophs

possess bacteriochlorophylls a or b in photosystems located in membranes that are continuous with plasma membrane

some can oxidize sulfide, but not elemental sulfur, to sulfate

Purple Nonsulfur Bacteria…

Rhodospirillum industrial importanceproduces H2 novel biodegradable plasticoxidize carbon monoxide to carbon dioxide

morphologically diversemost motile by polar flagella

found in mud and water of lakes and ponds with abundant organic matter and low sulfide levels; some marine species

Nitrifying Bacteria

very diverse chemolithoautotrophsnitrification – gain electrons from oxidation

of ammonium to nitrate or nitrite nitrite further oxidized to nitrate

Nitrification ammonia nitrite nitrate conversion of ammonia to nitrate by

action of two generae.g., Nitrosomonas – ammonia to nitritee.g., Nitrobacter – nitrite to nitrate

fate of nitrateeasily used by plants lost from soil through leaching or

denitrification

Betaproteobacteria Gram –ve oligotrophic (capable of growing at low nutrient

levels) Differ with alphaproteobacteria in rRNA sequence Example of betaproteobacteria :

1) nitrifying bacteria eg. Nitrosomonas

2) pathogenic species, Neisseria (gonorrhea), Bordetella (whooping cough)

3) Thiobacillus (ecologically important), Zoogloea (sewage treatment)

Gammaproteobacteria – largest class purple sulfur bacteria – obligate anaerobes that

oxidize hydrogen sulfide to sulfur intracellular pathogens (Legionella, Coxiella), methane oxidizers (Methylococcus), facultative anaerobes that utilize glycolysis and

the pentose phosphate pathway (Escherichia coli),

pseudomonads –aerobes that catabolize carbohydrates (Pseudomonas, and Azomonas)

Deltaproteobacteria Sulfate reducing microbes Eg. Desulfovibrio

(important in the sulfur cycle) Myxobacteria – gram negative, soil-dwelling

bacteria , dormant myxospores; common worldwide in the soils having decaying plant material or dung

Epsilonproteobacteria Gram-negative rods, vibrios, or spiral Include important human pathogens Eg. Campylobacter (causes blood poisoning)

The Gram Positive Bacteria

In Bergey’s Manual, gram-positive bacteria (able to form endospore) are divided into those that have :- low G + C ratio (base pair in genome below 50%)- high G + C ratio

Low G + C gram-positive bacteria include 3 groups clostridia, mycoplasms, Gram-positive Bacilli and Cocci

High G + C gram-positive bacteria include mycobacteria, corynebacteria, and actinomycetes.

Clostridia Eg. Clostridium – anaerobic, form

endospores, rod shape, gram +ve pathogenic bacteria causing gangrene,

tetanus, botulism, and diarrhea

Mycoplasmas Facultative or obligate anaerobes lack cell walls Gram +ve (previously under gram negative

category until nucleic acid sequences proved similarity with gram positive organisms)

When culture on agar, form ‘fried egg’ appearance bcoz cell in the center of the colony grow into the agar while those around the spread outward

Usually associated with pneumonia and urinary tract infections

Fried egg appearance

Gram positive Bacilli and Cocci Eg. Bacillus – form endospores, flagella

(B.licheniformis synthesis antibiotic. B.anthracis cause anthrax)

Eg. Lactobacillus – nonsporing rods, nonmotile, produce lactic acid as fermentation product. Mostly found in human mouth, intestinal tract, stomach. Protect body from pathogens

Streptococcus – nonmotile, cocci associated in pairs and chain. Cause pneumonia, scarlet fever

StreptococcusBacillus

High G+C gram-positive bacteria Include Corynebacterium, Mycobacterium and

Actinomycetes that have a G+C ratio > 50% in the phylum Actinobacteria, which have species with rod-shaped cells

Corynebacterium store phosphates in metachromatic granules. C. diptheria causes diphtheria

Mycobacterium cause tuberculosis and leporosy. It has unique resistant cell walls containing mycolic acids. Hence, acid fast stain (for penetrating waxy cell walls) is used for its identification

Actinomycetes resemble fungi as they produce spores and form filaments; important genera: Actinomyces found in human mouths; Nocardia useful in degradation of pollutants; and Streptomyces produces antibiotics

THE EUKARYOTES :FUNGI, ALGAE,

PROTOZOA

FUNGI Organisms in kingdom fungi include molds,

mushrooms, yeasts Fungi are aerobic or facultatively anaerobic

(yeast), chemoheterotrophs, spore-bearing, lack chlorophyll

Most fungi are decomposers, and a few are parasites of plants and animals

Some fungi – cause disease (mycoses) Some fungi – essential to many industries

(bread, wine, cheese, soy sauce)

Characteristics of Fungi Body/vegetative struc. of fungi – Thallus Thalli of yeast – small, globular, single cell Thalli of mold – large, composed of long,

branched, threadlike filaments of cell called hyphae that form mycelium

Hyphae - septate- Aseptate (coenocytic)

Fungi grow best in the dark, moist habitats

Acquire nutrients by absorption. Secrete enzyme to break large organic mol. Into simple mol.

Reproduction of fungi – sexual & asexual

Asexual reproduction

Several ways :

1) Transverse fission - Parent cell undergo mitosis, divide into daughter cell by formation of new cell wall

2) Budding – after mitosis, one daughter nucleus is sequestered in a small bleb that is isolated from parent cell by formation of cell wall

3) Asexual spore formation - filamentous fungi produce asexual spores through mitosis and subsequent cell division.several types of asexual spores :1) Sporangiospores form inside a sac called sporangium2) Chlamydospores form with a thickened cell wall inside hyphae3) Conidiospores (conidia) produced at the tip or side of hyphae, not within sac4) Blastospores produced from vegetative mother (hyphae) cell by budding5) Arthrospores hyphae that fragment into individual spores

sporangiospores

conidiospores

Chlamydiospores

Blastospores

Arthrospores

chlamydospores

sporangiospores

conidiospores

4) Sexual reproduction in fungi

Fungal mating type designated as + and –.

4 basic steps :

1) Haploid (n) cells from + and – thallus fuse, form dikaryon (cell with both +&- nuclei)

2) pair of nuclei within a dikaryon fuse to form one diploid (2n) nucleus

3) meiosis of the diploid restores the haploid state

4) haploid nuclei partitioned into + and - spores

Classification of fungi

1) Zygomycota Coenocytic molds – zygomycetes produce sporangiospores (asexual) and

zygospores (sexual) e.g. Black bread mold Rhizopus nigricans

usually reproduce asexually by spores that develop at the tips of aerial hyphae

sexual reproduction occurs when environmental conditions are not favorable requires compatible opposite mating types hormone production causes hyphae to produce

gametes gametes fuse, forming a zygote zygote becomes zygospore

Figure 24.4

2) Ascomycota Septate hyphae Form ascospores within sac-like structure

call asci (sexual) Form conidiospores in asexual

reproduction Eg Penicillium

Ascomycota ascomycetes or sac fungi

found in freshwater, marine, and terrestrial habitats

red, brown, and blue-green molds cause food spoilage

some are human and plant pathogenssome yeasts and truffles are ediblesome used as research tools

Figure 24.6

Ascomycota Yeast Life Cycle alternates between haploid and diploid

in nutrient rich, mitosis and budding occurs at non-scarred regions

stops after entire mother cell is scarred

nutrient poor, meiosis and haploid ascus containing ascospores formed

haploid cells of opposite mating types fuse tightly regulated by pheromones

Figure 24.7

3) Basidiomycota septate hyphae produce basidiospores (sexual), some produce

conidiospores (asexual) Eg mushrooms, puffballs, stinkhorns

Figure 24.12

Human Impact Basidiomycota decomposers edible and non-edible mushrooms

toxins are poisons and hallucinogenic pathogens of humans, other animals,

and plantse.g., Cryptococcus neoformans –

cryptococcosissystemic infection, primarily of lungs

and central nervous system

Protozoa

Eukaryotic, unicellular and lack of cell wall Motile (cilia, flagella, pseudopodia) Grow in moist habitats Some are in group of Planktonic (floating free in

lakes, ocean and form the basis of aquatic food chain)

Some protozoa can produce a cyst that provides protection during adverse environmental conditions

Asexuall reproduction by binary fission, schizogony/multiple fission

Sexually reproduction by conjugation

1)Nucleus undergoes mitosis2)Cytoplasm divides by cytokinesis

Schizogony/multiple fission

Classification of protozoa

Grouping based on locomotive structure do not reflect genetic relationship.

7 taxa of protozoa : alveolates, cercozoa, radiolaria, amoebozoa, eglenozoa, diplomonads, and parabasalids

Alveolates Have small membrane cavities called

alveoli beneath cell surface. 3 groups : ciliates (have cilia),

apicomplexans (pathogen to animal), dinoflagellates (have flagella)

Cercozoa Unicellular, called amoeba Move & feed by pseudopodia Have snail-like shells of calcium carbonate

Radiolaria Amoeba that have ornate shells composed of

silica

Amoebozoa Have lobe-shaped pseudopodia, no shell Eg Acanthamoeba, Naegleria

Eglenozoa move by means of flagella and lack sexual

reproduction; they include Trypanosoma

Diplomonad Lack mitocondria, golgi bodie Have 2 nuclei and multiple flagella

Giardia

Simple eukaryotic, phototrophic organisms, like plants

Carry out photosynthesis using chlorophyll Most live in aquatic environments

Characteristic of Algae

Unicellular or simple multicellular (thalli) Thallus of seaweed (large marine algae)

are complex, with holdfast (attached to rock), stemlike stipes and leaflike blades

Algae reproduce sexual and asexual (fragmentation & cell division)

Classification of algae Classify according to their structure and

pigment :

- red algae

- brown algae – cell wall composed of cellulosa & alginic acid

- green algae

- diatoms – silica cell wall composed of two halves called frustules that fit together like petri dishes

DIATOMS

frustule

VIRUSES

Virus Classification

classification based on numerous characteristicsnucleic acid typepresence or absence of envelopcapsid symmetrydimensions of viron and capsid

Figure 25.2

Characteristics of Viruses Viral disease – SARS, AIDS, influenza, herpes, common

cold Viruses – miniscule, infectious agent with simple acellular

organization and pattern of reproduction Viruses can exist – extracellular or intracellular Virion (complete virus particle) consist of :

- nucleocapsid (composed of 1 or more DNA or RNA, held within capsid)- in some viruses – envelope (phospholipid membrane)

Capsid - build by few types of protein = protomer - 3 types – helical, icosahedral, complex

symmetry

Virus structure

Different types of virus

Helical Helical Icosahedral Complex symmetry

Types of capsids

Virus size range 10-1000nm Most virus infect only particular host’s cells

Eg. HIV only infect T lymphocytes (a type of white blood cell)

Some viruses infect many kinds of cells in many different hosts

Eg. Rabies can infect most mammals Viruses are obligatory intracellular parasites.

They multiply by using the host cell’s synthesizing machinery to cause the synthesis of specialized elements that can transfer the viral nucleic acid to other cells

Viral replication

Virus cannot reproduce themselves bcoz:- have no genes for all enzyme needed for replication- have no ribosomes for protein synthesis

Viruses dependent of host’s organelles and enzymes to replicate

Virus replication – Lytic replication

Lytic replication of bacteriophage Consist of 5 stages – attachment, entry,

synthesis, assembly, release

1) Attachment – structure responsible for attachment to host = tail fiber. Attachment is dependent on chemical attraction and precise fit between T4 tail and protein receptor on E.coli cell wall

2) Entry – T4 release lysozyme to weaken peptidoglycan of E.coli cell wall. T4 inject genome into E.coli, leaving T4 coat outside.

3-4) Synthesis – viral enzyme degrade the bacterial DNA. E.coli start synthesis new viruses. T4 DNA is transcribed, producing mRNA which is translated to T4 protein (component of tail and head, lysozyme)

5) Assembly – T4 components are assemble in spontaneous manner to form mature virion

6) Release – newly assembled virions are released from the cell as lysozyme completes its work on the cell wall

Lytic replication takes about 25min can produce 100-200 new virions each cycle

Diversity of Microbial World Madam Noorulnajwa Diyana Yaacob PPK BIOPROSES April/May 2013

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