CHAPTER 1

INTRODUCTION TO MICROBIAL . PIGMENTS

Pigments and Microbes

A number of different kinds of pigments are produced by

microorganisms. The pigments vary in color from red, yellow,

orange, purple etc. These pigments have been subjected to a lot of

research not only for their colorful nature, but also because they

play important roles in photosynthesis, photoprotection,

pathogenesis etc. Yet other pigments are interesting because their

functional role is unclear. One of this latter class of pigments, the

xanthomonadins, is the topic of this thesis. This chapter is a review

of the structure and function of the xanthomonadins and two other

groups of microbial pigments, the carotenoids and melanins. The

emphasis on these pigments is due to the possibility that they

might be involved in photoprotection.

Xanthomonas

The genus Xanthomonas is comprised almost entirely of plant

pathogens. The affected plants include representatives from

practically all major groups of higher plants. Xanthomonads are

gram-negative, aerobic, rod-shaped bacteria with a single polar

flagellum. A characteristic feature of this genus is the production

of copious amounts .. of extracellular polysaccharide. Another -.:..-----~--·-·- ··--·- -o•- --· - ---- ·-----·--·--

distinguishing feature is that most xanthomonads produce yellow

pigments called xanthomonadins which are distinct from the yellow

1

2 (carotenoid) pigments produced by Erwinia, Pseudomonas,

Flavobacterium and Corynebacterium strains (see Carotenoids). The

characteristic presence of xanthomonadin in members of the genus

Xanthomonas has led them to be used as chemo-taxonomic and

diagnostic markers (Starr, 1981 ). A few non-pigmented

. xanthomonads have been reported viz. Xanthomonas campestris

pathovar (pv.) manihotis (causal agent of bacterial blight in

Cassava; Dye and Lelliott, 1974).

Xanthomonadin Pigment

Xanthomonadin (from Xanthomonas juglandis, a walnut pathogen)

was reported to be localized at the cytoplasmic membrane by

Stephens and Starr (1963). The membrane-bound nature of the

pigment was determined based on the kinetics of release of the

pigment from cells of X. juglandis upon sonic ·treatment. It was

observed that the ·cells subjected to sonic or ballistic

disintegration released xanthomonadin at a rate identical to that of

a membrane component like reduced nicotinamide adenine

dinucleotide (NADH) oxidase. The structure of a xanthomonadin,

isolated from X. juglandis, was solved by Andrews et a/. (1976)

using heavy atom Patterson and Fourier crystallographic methods

(Figure 1 . 1 ). Xanthomonadins were then deduced as brominated aryl

polyene pigments. The presence of bromine in this molecule is

unique since xanthomonads are terrestrial microorganisms which as

a group rarely incorporate bromine in organic molecules. Very little

information is available about the biosynthesis of this pigment. It

has been postulated that the aromatic ring may be derived from the

B r

OMe

Figure 1.1: Structure of Xanthomonadin I

0

c~ ~e '\OCH2CH

I Me

Xanthomonadins are membrane bound, brominated aryl polyene

pigments that are characteristic of the genus Xanthomonas. The

structure of xanthomonadin I was described from X. juglandis (a

walnut pathogen; Andrews et a/. 1976).

3

Figure 1.2: Colonies of Xanthomonas oryzae pv. oryzae

The above photograph shows yellow colonies of Xanthomonas oryzae

pv. oryzae as seen under a stereo microscope. Colonies were

photographed after 4 days of incubation on Peptone Sucrose agar

medium (see Materials and Methods in Chapter 2) at 28°C.

4

5 shikimate pathway and the polyene chain from the polyketide~

pathway (Jenkins and Starr, 1982a). }

Recently, a 23 Kb gene cluster required for xanthomonadin

biosynthesis in Xanthomonas campestris pv. campestris (a pathogen

of crucifers like cabbage, radish etc.) was cloned by Poplawsky et

a I. 1993. Complementation analysis suggests that this region

includes seven complementation groups. RFLP and dot blot analysis,

using the xanthomonadin clone as a probe, suggested that these

genes are present in all xanthomonads irrespective of whether they

produce pigment or not. Sequences homologous to the xanthomonadin

gene cluster were not detected in non-xanthomonads and therefore

these genes could be used as diagnostic probes for the

identification of xanthomonads. A subsequent report indicated that

one of the genes (pig B) in this cluster enco_des~a_di.ftusible_factor

required for both _ptg.r:n_e_nt_ a.nd __ extcacelh,Jlac_Roly_~accharide

production (PoQiawsky and Chun, 1997). ---·--· .· -------~

Pigment deficient mutants of many Xanthomonads viz. X

campestris pv. campestris and Xanthomonas oryzae pv. oryzae

(causal agent of bacterial blight of rice) are fully proficient for

virulence when wound inoculated onto their respective hosts

suggesting that pigment is not essential for virulence of the

pathogen (Poplawsky et a/. 1993; Tsuchiya et a/. 1982; Durgapal,

1996). A pigment deficient mutant of X. juglandis was observed to

be more sensitive to photobiological damage when compared to the

pigmented wild type strain suggesting that xanthomonadins might '

serve to protect the bacterium from photodamage (Jenkins and

Starr, 1982b).

Xanthomonas oryzae pathovar oryzae

This organism is the subject of study in this thesis. It causes a

serious disease of rice called bacteri.al leaf blight. The symptoms

are characterized by yellowing ·and drying which begin at the tips

of rice leaves and spread down either one or both sides of the leaf

or through the mid vein (Figure 1.3). The pathogen gains entry into

the rice leaf either through wounds or natural openings called

'hydathodes' that are present on the edges of the rice leaf. The

bacterium multipt"ies inside the xylem vessels and travels through

them to more distant parts of the leaf and occasionally to other

parts of the plant. It also exits from the leaf in the form of ooze

droplets that collect on the surface of the leaf. Dispersal of the

pathogen Is achieved when these ooze droplets are splashed onto

neighboring uninfected plants by wind or rain. Dispersal of the

pathogen from one infected leaf to other leaves on the same adult

rice plant might also occur through ooze (Sridhar Dharmapuri and

Ramesh Sonti, unpublished data). During the off-season, the

pathogen is presumed to survive either on seeds, dried leaves and

stubble or on neighboring alternate host plants (like weeds) till the

next season (Alvarez et al. 1989; Figure 1.4 ).

The objective of this study is to determine the role of the

xanthomonadin pigment in the life cycle of X oryzae pv. oryzae. One

of the questions addressed is whether the pigment is required for

virulence following epiphytic entry of the pathogen into the plant

i.e. entry through the hydathodes after residence on the leaf surface. I

This is more akin to the natural mode of entry into the plant an"' is

6

A B

Figure 1.3: Disease symptoms of bacterial leaf blight of

rice

Xanthomonas oryzae pv. oryzae is a vascular pathogen that traverses

down the xylem vessels of the leaf. A, a healthy rice leaf. B, leaf

blight symptoms caused by Xanthomonas oryzae pv. oryzae. Infection

symptoms begin at the leaf tips and advance through the veins/mid

rib of the infected leaves.

7

Off season

Wild rices Stubble Seed (?)

.._Entry • Bacterial ooze

Plant to plant spread

leaf to leaf

Figure 1.4: Life cycle of Xanthomonas oryzae pv. oryzae

The pathogen exits from infected leaves in the form of bacterial ooze. Wind and rain

contribute to splash dispersal of the pathogen onto neighboring uninfected plants.

During the off season the pathogen is presumed to survive in the straw, stubble or on

alternate host plants. It has also been suggested that the pathogen might survive the off

season in seeds.

8

9 likely to result in exposure of the pathogen to air and light. These

are conditions under which pigment may enhance viability of the

pathogen (by providing photoprotection). Pigment deficient mutants

that were isolated after either EMS mutagenesis or marker

exchange of transposon insertions obtained in cloned pigment

biosynthetic genes were used in this study. Reporter gene fusions to

putative pigment promoters were obtained and their expression was

studied; specifically to determine whether these genes are

expressed when the pathogen is present within the rice plant.

During the process of cloning the X. oryzae pv. oryzae genes involved

in pigment biosynthesis, a number of methods for conducting

molecular genetics on an Indian isolate of X. oryzae pv. oryzae were

standardized and these are detailed in the following chapter.

Carotenoid Pigments

Carotenoids are isoprenoids containing a characteristic polyene

chain of conjugated double bonds and are located in chromatophores

(vesicular, tubular or lamellar membranous structures) which are

continuous with the cytoplasmic membrane. The two groups of

carotenoids known are the 'carotenes' or hydrocarbons and their

oxygenated derivatives called 'xanthophylls'. Unlike xanthomonadins

that are found exclusively in xanthomonads, carotenoids are present

not only in bacteria but also in photosynthetic algae, plants and

some fungi. About 600 carotenoids are known to occur naturally

with most of these being considerably similar to each other in

structure. Carotenoids usually contain 40 carbon atoms (C40) with

10 acyclic 'lycopene' (Figure 1.5A) considered as the basic compound

from which most, if not all, carotenoids are derived (Goodwin,

1973). The presence of carotenoids in bacteria and fungi is

discussed here.

Carotenoids. in photosynthetic bacteria

Approximately 60 different carotenoids have been identified

from photosynthetic bacteria and these have been tabulated by

Goodwin {1973). Photosynthetic bacteria are divided into two

classes i.e. the purple photosynthetic bacteria and the green

photosynthetic bacteria. All purple photosynthetic bacteria contain

acyclic carotenoids e.g. Spirilloxanthin (Figure 1 .58). In contrast,

the green photosynthetic bacteria are characterized by the presence

of aromatic carotenoids e.g. chlorobactene (Figure 1.5C). In

photosynthetic bacteria, two possible functions attributed to

carotenoids are that they act as accessory pigments in

photosynthesis and also function to provide protection against

photodamage.

The first study that provided evidence for photoprotection by

carotenoids was with a mutant of the photosynthetic purple

bacterium Rhodopseudomonas spheroides (Griffiths et a/. 1 955). It

was observed that the mutant strain (designated as 'blue-green')

accumulated a colorless carotenoid precursor called phytoene.

Bacteriochlorophyll was synthesized in the mutants but to lower

amounts when compared to the wild type. The growth of this mutant

strain proceeded normally under anaerobic conditions. However,

under aerobic conditions, the cells of 'blue-green' were rapidly

I I

A•

MeO

B_. eO

c ...

Figure 1 .5 A-C: Structures of lycopene (A), spirilloxanthin

(B) and chlorobactene (C).

Around 600 carotenoid structures have been reported. Most

carotenoids are derived from lycopene. An example of an acyclic

carotenoid i.e. spirilloxanthin and a cyclic carotenoid like

chlorobactene are shown here.

12 killed due to exposure to light and oxygen (in this case

bacteriochlorophyll acts as the photosensitizer). It was proposed

that the mechanism by which photoprotection occurs is that in the

presence of excess quanta, chlorophyll gets exCited to its triplet

state and chlorophyll in its triplet state could mediate abnormal

photosensitized reactions. Under these conditions, the carotenoid

pigments serve to quench the triplet state of chlorophyll. This

hypothesis is supported by the fact that carotenoid pigments can

interact with the triplet state of chlorophyll (Fujimori and

Livingston, 1957). It was also observed that carotenoids have the

ability to quench singlet oxygen (1 02) and that the ability of a

carotenoid to quench singlet oxygen in vitro is dependent on the

number of conjugated double bonds. The efficiency of

photoprotection by carotenoids was studied by a number of groups

and it was concluded that the least unsaturated carotenoid that

shows a protective effect was neurosporene with only 9 conjugated

double bonds and that with increased desaturation there was a

concomitant increase in photoprotection (Mathews-Roth et a/.

1974).

Carotenoids in non-photosynthetic bacteria

A number of unique C30, C45 and C50 carotenoids are found in

non-photosynthetic bacteria. Photoprotection offered by

carotenoids in non-photosynthetic bacteria was first demonstrated

by Mathews and Sistrom ( 1 9 59) in Sarcina /utea. They observed that

a carotene-less mutant of S. lutea was rapidly killed upon exposure

to light, air and a photosensitizer like toluidine blue whereas the

13 pigmented strain was unaffected. A photoprotective effect offered

by carotenoids was also observed in Corynebacterium poinsettiae

where unlike the pigmented strain, the colorless mutant succumbed

rapidly to light and air in the, presence of an exogenous

photosensitizer (Kunizawa and Stanier, 1958). T~is photosensitive

effect on the mutant strain was not seen in an atmosphere of

nitrogen. Similar experiments without the use of an exogenous

photosensitizer were performed with Mycobacterium marinum. This

bacterium produces carotenoids only in the presence of light;

therefore, dark-grown pigmentless bacteria were compared with

light grown pigmented bacteria for survival under bright light in the

presence of air. The results showed that the non-pigmented bacteria

succumbed more rapidly to the photodamage than the pigmented

cells (Wright and Rilling, 1963).

Fungal Carotenoids

Many fungal species are characterized by the presence of

carotenoid pigments. Both carotenes and xanthophylls have been

reported. They can be found among Phycomycetes, Ascomycetes,

Basidiomycetes and Myxomycetes. Two fungi, Sporidiobolus

johnsonii and Dacryopinax spathularia, showed an increase in

sensitivity to light if the pigment was absent indicating a role for

the carotenoid pigment in photoprotection (Margalith, 1992).

Melanin Pigments

The black pigments often observed in microbes are considered to

be melanin or melanin-like pigments. Melanins are brownish or

14 black pigments obtained by the oxidation of aromatic amino acids,

principally tyrosine, accompanied by polymerization of the

resulting derivatives. Melanins are produced by a number of bacteria

for e.g. the marine bacterium Shewanella colwanella; Proteus

mirabilis as well as Rhizobium spp. Some Ps_eudomonas aeruginosa

strains were shown to produce melanin-like pigments under

specific growth conditions (Margalith, 1992). A number of other

microbes like Serratia marcescens and Streptomycetes have been

shown to produce melanin or melanin-like pigments in the presence

of tyrosine (Trias et a/. 1989; Margalith, 1992). A few Xanthomonas

spp. have also been observed to produce a brown diffusible pigment

e.g. Xanthomonas ampe/ina (causes canker disease on grapevine) and

Xanthomonas phaseoli pv. fuscans (which causes a disease on bean)

{Dye and Lelliott, 1974}. However, it is not known whether this

brown pigment is melanin or a melanin-like pigment.

In fungi, melanin production is observed in structures such as

hyphae, conidia etc. (Margalith, 1992). A number of phytopathogenic

fungi produce melanin (Margalith, 1992; Bell and Wheeler, 1986).

One such interesting example is Magnaporthe grisea which causes

the 'rice blast disease'. The fungus penetrates the host cell wall by

increasing the hydrostatic pressure on its hyphae. Howard and

Ferrari ( 1989) showed that melanins in the cell walls of

appressoria, or swollen hyphal tips, of this pathogen imparts

differential permeability leading to a high internal solute

concentration. This creates a turgor pressure which facilitates the

penetration into the host tissue. Melanin deficient mutants of

Magnaporthe grisea were observed to be non-pathogenic on rice

15 (Chumley and Valent, 1990). Melanin biosynthetic inhibitors like

'tricyclazole' have been useful in prevention of penetration of

appressoria into host tissues and are· therefore being used to

control the rice blast fungus (Woloshuk et · a/. 1983; Margalith,

1992). The human pathogenic fungus, Cryptococcus neoformans,

produces melanin and a polysaccharide coat. Both these phenotypes

have been associated with virulence of the pathogen (Nosanchuk and

Casadevall, 1997).

In several fungi, melanins have been shown to provide protection

to fungal spores against ultraviolet and solar radiation (Margalith,

1992). Survival of conidia and sclerotia over prolonged periods is

correlated with the occurrence of melanins or melanin-like

pigments. It was observed that melanized sclerotia or cell walls

are resistant to attack by microorganisms unlike non-melanized

portions of the two. Melanins have also been proposed to trap free

radicals (hydroxyl radical, OH0) and protect cells from oxidizing

conditions (Goodchild et a/. 1981; Margalith, 1992)

The genes responsible for melanin biosynthesis· have been

described in Streptomyces lividans, Streptomyces antibioticus and

from a number of Rhizobium species. Melanin biosynthetic genes

from Rhizobium are reported to be present on a plasmid (Cubo et a/.

1988). Melanin may be required for detoxification of polyphenolic

compounds which may accumulate in senescing nodules of legumes

(Margalith, 1992).

A great many other pigments, not discussed in this chapter, are

produced by microbes for e.g. bacteriochlorophyll produced by

photosyn,thetic bacteria, phenazine antibiotic pigments produced by

16 certain Pseudomonas aeruginosa strains, riboflavins produced by

fungi, bacterial indigo produced by Pseudomonas indoloxidans to

name a few. The function of some of these pigments is well

established while that of others is largely unknown. The pigments

whose functions are unknown are likely to be more than just

secondary metabolites. Their physiological functions may become

more apparent with further studies.

17 References

Alvarez, A. M., Teng, P. S., and Benedict, A. A. 1989. Methods for

epidemiological research on bacterial blight of rice. In: Bacterial

blight of rice, International Rice Research Institute, P. 0. Box 933,

Manila, the Philippines. pp 99-11 0.

Andrews, A. G., Jenkins, C. L., Starr, M. P., and Hope, H. 1976.

Structure of xanthomonadin I, a novel dibrominated aryl-polyene

pigment produced by the bacterium Xanthomonas juglandis.

Tetrahedron Letters 45: 4023-4024.

Bell, A. A., and Wheeler, M. H. 1986. Biosynthesis and functions of

fungal melanins. Ann. Rev. Phytopathol. 24: 411-451.

Cerda-Olmedo, E. 1985. Carotene mutants of Phycomyces. Methods in

Enzymol. 11 0: 220-243.

Chumley, F. G., and Valent, B. 1990. Genetic analysis of melanin

deficient, nonpathogenic mutants of Magnaporthe grisea. Mol. Plant

Microbe Interact. 3: 135-143.

Cubo, M. T., Buendia-Ciaveria, A. M., Beringer, J. E., and Ruiz-Sainz, J.

E. 1988. Melanin production by Rhizobium strains. Appl. Environ.

Microbial. 54: 181 2-1 81 7.

18 Durgapal, J. C. 1996. Albino mutation in rice bacterial blight

pathogen. Curr. Science 70: 15.

Dye, D. W., and Lelliott, R. A. 1974. Xanthomonas In: Bergey's Manual

of Determinate Bacteriology (Edited by Buchanan, R. E., and Gibbons,

N. E.) The Williams and Wilkins Company, Baltimore. U.S.A. pp 243-

253.

Fujimori, E., and Livingston, R. 1957. Interactions of chlorophyll in

its triplet state with oxygen, carotene, etc. Nature 180: 1036-1038.

Gantotti, B. V., and Beer, S. V. 1982. Plasmid borne determinants of

pigmentation and thiamine prototrophy in Erwinia herbicola. J.

Bacteriol. 1 51: 1627-1629.

Goodchild, N. T., Kwock, L., and Lin, P. S. 1981. Melanin, a possible

cellular superoxide scavenger. In: Oxygen and Oxy-radicals in

Chemistry and Biology (Edited by Rodgers, M.A. J., and Powers, E. L.)

Academic Press, London. pp 645-648.

Goodwin, T. W. 1973. Microbial carotenoids In: Handbook of

Microbiology (Edited by Laskin, I. A., and Lechevalier, H. A.) CRC

Press, Cleveland, Ohio, U. S. A. Vol 3, pp 75-83.

Griffiths, M., Sistrom, W. R., Cohen-Bazire, G., and Stanier, R. Y.

1 9 55. Function of carotenoids in photosynthesis. Nature 1 7 6: 1 211-

1214.

19

Howard, R. J., and Ferrari, M. A. 1989. Role of melanin in

appresorium function. Exp. Mycol. 13: 403-418.

Jenkins, C. L., and Starr, M. P. 1982a. The pigment of Xanthomonas

populi is a nonbrominated ary!-heptaene belonging to xanthomonadin

pigment group II. Curr. Microbial. 7:195-1,98.

Jenkins, C. L., and Starr, M. P. 1982b. The brominated aryl polyene

(xanthomonadin) pigments of X. juglandis protect against

photobiological damage. Curr. Microbial. 7: 323-326.

Johnson, E. A., and Schroeder, W. A. 1995. Microbial carotenoids In:

·Advances in Biochemical Engineering I Biotechnology (Edited by

Fiechter, A.) Springer-Verlag, Berlin. Vol 53: 120-178.

Kunizawa, R., and Stanier, R. Y. 1958. Studies on the role of

carotenoid pigments in a chemoheterotrophic bacterium

Corynebacterium poinsettiae. Arch. Microbial. 31: 146-1 56.

Margalith, P. Z. 1992. Pigment Microbiology. Chapman & Hall, London.

pp 1-157.

Mathews, M. M., and Sistrom, W. R. 1959. Function of carotenoid

pigments in non-photosynthetic bacteria. Nature 184: 1892-1893.

I'"

20 Mathews-Roth, M. M., Wilson, T., Fujimori, E., and Krinsky, N. I. 1974.

Carotenoid chromophore length and protection against

photosensitization. Photochem. and Photobiol. 22: 59-61.

Nosanchuk, J. D., and. Casadevall, A. 1997. Cellular change of

Cryptococcus neoformans : contributions from the capsular

polysaccharide, melanin and monoclonal antibody binding. Infect.

lmmun. 65: 1836-1841.

Poplawsky, A. R., Kawalek, M. D., and Schadd, N. W. 1993. A

xanthomonadin-encoding gene cluster for the Identification of

pathovars of Xanthomonas campestris. Mol. Plant-Microbe Interact.

6: 545-552.

Poplawsky, A. R., and Chun, W. 1997. pigS determines a diffusible

factor needed for extracellular polysaccharide slime and

xanthomonadin production in Xanthomonas campestris pv.

campestris. J. Bacteriol. 179: 439-444.

Starr, M. P. 1981. The genus Xanthomonas. In: The Prokaryotes.

(Edited by Starr, M. P ., Stolp, H., Truper, G. H., Balows, A. and

Schlegel, G. H.), Springer-Verlag, Berlin. Vol. 1, pp 742-763.

Stephens, W. L., and Starr, M. P. 1963. Localization of carotenoid

pigment in the cytoplasmic membrane of Xanthomonas juglandis. J.

Bacteriol. 86: 1070-1074.

21 Spurgeon, S. L., and Porter, J. W. 1980. Carotenoids In: The

Biochemistry of Plants (Edited by Stumpf P. K., and Conn. E. E.).

Academic Press Inc. N. Y. Vol4, pp 419-483.

Tabata, K., Ishida, S., Nakahara, T., and Hoshino, T. 1994. A

carotenogenic gene cluster exists on a large plasmid in Thermus

thermophilus. FEBS letters 341: 251-255.

Trias, J., Vinas, M., Guinea, J., and Loren, J. G. 1989. Brown pigment

in Serratia marcescens cultures associated with tyrosine

metabolism. Can. J. Microbial. 35: 1 037-1 042.

Tsuchiya, K., Mew, T. W., and Wakimoto, S. 1982. Bacterial and

pathological characteristics of wild types and induced mutants of

Xanthomonas campestris pv. oryzae. Phytopathology 72: 43-46.

Woloshuk, P. M., Sisler, M. D., and Vigil, E. L. 1983. Action of the

antipenetrant, tricyclazole, on appresoria of Pyricularia oryzae.

Physiol. Plant Pathol. 23: 245-259.

Wright, L., and Rilling, H. C. 1963. The function of carotenoids in a

photochromogenic bacterium. Photochem. and Photobiol. 2: 339-342.

Yo, K. Y., Lai, E. M., Lee, L. Y., Lin, T. P., Hung, C. H., Chen, C. L., Chang,

Y. S., and Liu, S. T. 1994. Analysis of the gene cluster encoding

carotenoid biosynthesis in Erwinia herbicola Eho13. Microbiology

140: 331-339.

22 CHAPTER II

DEVELOPMENT OF METHODOLOGY FOR CONDUCTING MOLECULAR

GENETIC STUDIES ON AN INDIAN ISOLATE OF XANTHOMONAS

ORYZAE PATHOVAR ORYZAE

Abstract

Xanthomonas oryzae pv. oryzae causes one of the serious

diseases of rice. This chapter describes the standardization of

methods for conducting molecular genetic studies on our laboratory

wild type strain of X. oryzae pv. oryzae, which belongs to the

dominant pathotype of this pathogen in India. Conditions for gene

transfer by electroporation as well as by conjugation with donor E.

coli cells were standardized. A genomic library of this strain, with

an average insert size of 30 Kb, was constructed in the broad host

range cosmid vector pUFR034. Restriction digestion and Southern

hybridization of two randomly selected clones from the library

suggests that cloned DNA is not rearranged when reintroduced into X

oryzae pv. oryzae. The results obtained from colony hybridizations,

using previously' identified sequences from a Philippine isolate of X

oryzae pv. oryzae as probes, indicate that the genomic library

constructed is an adequate representation of the X. oryzae pv .. oryzae

genome. Conditions were also standardized for performing

transposon mutagenesis of cloned X. oryzae pv. oryzae DNA in E. coli

using a mini-TnSgus transposon and marker exchange of these

insertions into the X. oryzae pv. oryzae chromosome.

23 Introduction

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial

leaf blight, a serious disease of rice (Starr, 1981 ). To date most of

the research on this pathogen has been conducted using Philippine

isolates. Owing to quarantine reasons, these strains could not be

used in this study. This chapter describes the standardization of

some methods for conducting molecular genetic studies on our

laboratory wild type strain (called BXO 1 ) of X. oryzae pv. oryzae.

This strain belongs to the dominant pathotype of X. oryzae pv. oryzae

in India (Reddy and Reddy, 1989; Yashitola et a/. 1997). It was

provided to us by Dr. A. P. K. Reddy of the Directorate of Rice

Research, Hyderabad where it was isolated from an infected leaf

sample collected at Chinsuria, West Bengal.

Conditions for gene transfer into BXO 1 by conjugation with donor

E. coli strains as well as by electroporation were standardized. A

genomic library of X. oryzae pv. oryzae was constructed in the broad

host range cosmid vector pUFR034 (DeFeyter et a/. 1990) using

Esherichia coli DHSa, as the host. Plasm ids were isolated from ten

random clones of the genomic library, to estimate the average insert

size. In order to confirm that the library included a complete

representation of the X. oryzae pv. oryzae genome, two previously

identified clones from a Philippine isolate of X. oryzae pv. oryzae

(Kelemu and Leach, 1990; Hopkins et a/. 1992) were used as probes

in colony hybridizations.

A previous report on Xanthomonas campestris pv. malvacearum (a

cotton pathogen) indicated that DNA cloned in E. coli can be

subjected to substantial rearrangement when reintroduced back into

24 X. campestris pv. malvacearum (DeFeyter et a/. 1990). If this were

the case in X. oryzae pv. oryzae, it would interfere with subsequent

genetic analysis of the cloned DNA. Therefore, two randomly

selected clones from the genomic library were mobilized into X

oryzae pv. oryzae. The plasmids were isolated from the

transconjugants and subjected to Southern analysis to assess their

structure in X. oryzae pv. oryzae.

In addition, procedures were standardized to obtain transposon

insertions into cloned X. oryzae pv. oryzae DNA using a mini Tn5

transposon harboring the E. coli gus gene (Wilson et a/. 1995). The

conditions for marker exchange of these Tn5 insertions into the X

oryzae pv. oryzae chromosome were also standardized. This mini Tn5

transposon functions as a promoter-probe transposon where f\­

glucuronidase activity is observed only if the gus gene is driven by

endogenous bacterial promoters. Since both X. oryzae pv. oryzae and

r.ice possess minimal endogenous f\-glucuronidase activity, the use

of the mini:-TnSgus transposon would facilitate studies on X. oryzae

pv. oryzae gene expression in planta.

Materials and Methods

Bacterial strains, media etc.

All bacterial strains used are listed in Table 2.1. BX01 is the

laboratory wild type strain that belongs to pathotype I, the dominant

pathotype of X. oryzae pv. oryzae in India (Reddy and Reddy, 1989;

Yashitola et a/. 1997). All X. oryzae pv. oryzae strains used here

were grown on Peptone-Sucrose (PS) medium (Tsuchiya et a/. 1982)