Fol ia Microbiol. 26, 120-- 123 (1981)

Surface Layers of Xanthomonas malvacearum, the Cause of Bacterial Blight of Cotton J.P. VERMA a and H. FOR~A~EKb

aDivision of Plant Pathology, Indian Agricultural l~eaearch Institute, New Dethi.110 012, India, and bBotanisches Institut dcr Universitd.t Mi'tnchen, 2)-8000 M~tnchen-19, ~ ( ~

J~eceived November 3, 1979

ABSTRACT. Mure ins were i so la ted f rom two s t r a in s o f Xanthomonas malvacearum, a p h y t o p a t h o g e n i c b a c t e r i u m caus ing bacter ia l b l igh t of co t ton . T he p u r i t y o f m u r e i n was 70- - 95 % a n d t h e a m i n o ac id a n d a m i n o s u g a r c o m p o n e n t s (g lu tamic acid, a lanine , meso-diaminopimelic acid, m u r a m i c ac id a n d g lueosamine) w e r e p r e s e n t a t t he mo la r ra t io o f 1 : 1.9 : 1 : 1.12 : 0.85. T h e b a c t e r i u m secre ted a copious a m o u n t of s l ime wh ich m a s k e d i t s sur face s t r uc t u r e . T he s l ime was c o m p o s e d of dense ly i n t e r w o v e n n e t w o r k o f f i l amen tous ma te r i a l o r ig ina t ing f r o m t h e cell sur face a n d e x t e n d e d in to the m e d i u m w i t h o u t a n y discern- able b o u n d a r y . The s l ime was sec re ted t h r o u g h surface layers pores b y force, g iv ing t he effect of a s p r a y or jet . Sl ime a lso p l ayed a role in c h a i n f o r m a t i o n o f bacter ia l cells.

Phytopathogenic bacteria, in general, are Gram-negative. One of the reasons (Verma and Singh 1976a) why Gram-positive bacteria are poor potential pathogens may be due to the fact that plants contain toxic chemicals (natural products, known and unknown) which readily damage the mureins of these bacteria, because they are generally exposed, and any damage to murein causes the loss of shape and rigidity and ultimate death of the bacterial cell. In Gram-negative bacteria, on the other hand, the mureins are well protected by ]ipopolysaccharides and lipoproteins, and, therefore, the plant products are unable to penetrate this sheath to reach the murein layer, resulting in the uninhibited multiplication of these bacteria, which reach critical concentrations to cause disease symptoms.

Most of the Gram-negative bacteria belong r the directly cross-linked, meso- diaminopimelic acid (m-Dap) containing mureins belonging to the Group-A, sub- group-1 and variation y i.e. A-1-y (Schleifer and Kandler 1972). Among the phyCo- pathogenic bacteria some serious attention has been given only to the Gram-positive genus Corynebacterium. C. insidiosum and C. michiganensis have a modified type B-2-~r of murein, i.e. linkage between positions 2 and 4 of two peptide units and an interpeptide bridge containing a D-diamino acid, with L-2,4-diaminobutyrio acid (L-Dab) in position 3 (see Schleifer and Kandler 1972). C. tritici also contains Dap (Perkins 1965) while C. poinsettiae, C. berne and C. flaccumfaciens D-ornithine (Perkins and Cummins 1964). A soluble extract was prepared from C. insidiosum and C. poin- settiae which contain enzymes necessary for the biosynthesis of certain murein pre- cursors (Wyke and Perkins 1975). Some of the Gram-negative plant pathogens like Agrobacterium tumcfaciens (Mannase and Corps 1967; Kandler and Zehender 1956) and X. malvacearum (Verma and Singh 1971) were shown to contain m-Dap.

1981 SURFACE LAYERS OF X. malvacearura t2|

TABLE I. Amino acid and sugar content of isolatmd mureins of two races of X. malvacearura

Amino acid and sugar a Total murein Race Quantity NHa components

number ' Glu Ala Dap Mur GlcNH~ ~o

32 tLmol/mg dry mass 0.98 1.94 1.04 1.10 0.88 0.24 95

molar ratio 1.0 1.97 1.05 1.12 0.89 0.24 --

2 y.mol/mg dry mass 0.74 1.38 0.74 0.83 0.61 0.25 70

molar ratio 1.00 1.86 1.00 1.12 0.82 0.34 --

Traces of Asp, Thr, Ser, Gly, Val, Lou, Tyr, Pho wore also present as contaminants.

Cotton plants contain, on their surface, a large number of bacteria as a part of their phylloplane flora (Verma etal. 1978), of which a few species belonging to Flare- bacterium, Aeromonas and t~seudomonas on preinoculation protected the leaves from infection by a challenge dose (after 8--24 h) of X. malvacearum (Verma and Singh 1976b). These bacteria might be producing certain chemicals or inducing the for- mation/increase of certain plant products (Verma et al. 1978), which might inhibit the biosynthesis of mureins or surface structures of X. malvacearum, which is com- pletely unknown as far as its cell wall composition is concerned. In the present communication we report only on the surface structures including the composition of murein of X. malvacearum.

M A T E R I A L S A N D M E T H O D S

Strains. Race 32 and race 2 of Xanthomonas malvaeearum (E.F. Smith) Dowson, the causative agent of bacterial blight of cotton (Gossypium hirsutum L.) were described earlier (Verma and Singh 1974, 1976b).

Culture media. The bacteria were cultured on a pep tone-sucrose medium (in g, peptone 5, sucrose 10, Na2HPO4.2H20, FeSO4 0.5, Ca(X03)~ 0.5, cycloheximide 0.05, water 1 L). The bacteria were harvested during late exponential phase for the preparation of mureins as well as for electron microscopy.

Isolation of mureins. Essentially the methods described earlier (Verma and Martin 1967) were used. This included extraction in hot sodium dodecyl sulphate and trypsin t reatment . Amino acids were assayed on a Beckman-Amino Analyser model 120-C.

Electron microscopy. 2 ~/a potassium phosphotungstate or 1--2 ~7 o u rany l (2~) acetate were used for staining (Martin etal. 1968). The preparations were examined under a Siemens Elmiskop 101.

RESULTS

Cell morphology and secretion of slime

The cells of X. malvacearum, both races 32 and 2, were rod-shaped and occurred in pairs or short chains (Plate 1A). The chains were held together by secreted slime, the secretion of which appeared to be in the form of spray or ooze (Plate 1B) a n d

122 J . P . VERMA and H. FORMANEK Vol. 26

a balooning effect, at one or several places, was quite evident in certain cases (Plate 1C). In others, the fibrous nature of slime strands was clear (Plate 1B, D). The surface structure was masked with slime, but in a few cases it appeared to be the usual reticulate type with channels (valleys or grooves) and raised structures (lobes or hills) as evident by negatively stained preparations (Plate 1D). Finer structures were not visible. However, certain bleb-like structures were present on the surface, which appeared to be secreted into the medium.

Composition of murein Electron microscopy revealed the presence of a sacculus possessing the shape and

size of the original cells. The negatively stained preparations (Plate 1E) showed no structural details but were so thin that the presence of small holes could not be excluded. Both the strains contained Glu, Mur, Ala, Dap and GleNH2 at the molar ratio of 1 : 1 . 1 2 : 1 . 9 : 1 : 0 . 8 5 (Table I) and the mureins were 70--95 ~ pure. Traces of Asp, Thr, Gly, Val, Leu, Tyr and Phe were present as contaminants.

D I S C U S S I O N

A few groups of bacteria are now known which do not have typical murein as a component of their cell wall (see Kandler and KSnig 1978; Schleifer and Kandler 1972). However, the cell envelope of X. malvacearum, a phytopathogenic, Gram- negative bacterium causing bacterial blight of cotton, contains a murein of the usual type i.e. A-I-~,, as found in Gram-negative bacteria (Schleifer and Kandler 1972; Verma 1970), although muramic acid was present in slightly higher amounts, i.e. Glu : Mur ---- 1 : 1.2. This might be characteristic for this species or for bacteria pathogenic on leaves of plants. More work, however, is needed to confirm this. I t must, however, be mentioned that it was relatively easy to prepare the mureins from this species as is evident from the high purity obtained in certain cases, i.e. up to 95 ~/o. The preparations were also very clean under the electron microscope. Although no ultra.thin sections were made, the thinness of the mureins suggests to conclude that these represent monolayers. The monolayer murein A-I-~, has been considered to be the most evolved p.rocaryote (Schleifer and Kandler 1972). One might conclude that phyCopathogenic bacteria may represen'~ one of the most evolved groups in bacteria.

The secretion of polysaccharide in a jet or spray fashion is quite interesting in X. malvacearum, the phytopathogenic slime-secreting bacterium, suggesting the presence of minute holes on the surface layers. These holes probably open when the pressure is exerted by the slime, which, according to certain indications, accumu- late in the underlying layers. The formation of long or short chains of cells due to end-to-end adherence through polysaceharide is also noteworthy. The polysaccharide contains glucose, mannose and glucuronic acid in the proportion of 38--42 and 5 - 1 4 ~/o, respectively; unknown materials accounted for 0 .1-13 ~o of the poly- saccharide (Ghowdhury and Verma, unpublished). The ejection of slime in a jet fashion may help the bacterial pathogen in forcing its way through the intercellular spaces of the host. Polysaccharide component of the slime created an extend period of water soaking at a concentration of 6 mg/mL or higher (Chowdhury and Verma, unpublished), which is favourable for the multiplication and spread of X. malva- cearum.

Thanks are due to ProL Dr. O. Kandler and Dr. W. Hannnes (Univ. of Miinehcn, Botanisehes Ins t i tu t ) and Dr. R.P. Singh (IARI, New Delhi) for thei r critical discussion; to Mrs. E. Hagner and Miss R. GSbbert

1981 SURFACE LAYERS OF X. malvaccarum |2~

(Botanisches Inst.) for technical help; to Mr. Liedl (Botanisches Inst.) and Mr. D.S. Rawat (IARI) for photographs; and to DAAD (Deutscher Akademischer Austausehdienst) to support a s tudy-turn- information t r ip of one of us (J.P.V.) to Munich during the tenure of which the main pa r t of the work was done.

R E F E R E N C E S

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MA~Ass R.J . , CORPS W.A.: Chemical composition of cell envelopes from Agrobacteriura tumefaciens, aa~. J.Microbiol. 41,243 (1967).

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The plate will be found a t the end of the issue.