Zentralbl. Mikrobiol. 143 (1988),591-594

VEB Gustav Fischer Verlag lena

[Department of Botany, Ranchi University, India]

Short Communication

The Free Amino Acid Composition During Growth

of the Xanthomonas campestris pv. malvacearum (SMITH) DOWSON

K. Lal, B. B. Lal, and M. PRASAD

With 1 Figure

Summary

12 amino acids were found to be present in the developing culture of Xanthomonas campestris pv. malvacearum. These were I-leucine/isoleucine, ~-phenylalanine, proline, dl-threonine, dl-alanine, glycine, tryptophan, glutamine. serine, homoserine, glutamic and aspartic acids. Out of these, only 6 were found to be present in the bacterial culture filtrate. The composition and pool size of intra- and extracellular amino acids varied considerably during the growth of the pathogen. Glutamic acid and alanine which form a part of the cell wall component were present in a ratio of2: I in the cell extract.

Zusammenfassung

1m Zellextrakt einer sich entwickelnden Kultur von Xanthomonas campestris pv. malvacearum wurden 12 Aminosauren gefunden: L-Leucinllsoleucin, ~-Phenylalanin, Prolin. DL-Threonin, DL-Alanin, Glycin, Tryptophan, Glutamin, Serin, Homoserin, Glutamin- und Asparaginsaure. 1m bakteriellen Kulturfiltrat wurden nur 6 von ihnen gefunden. Zusammensetzung und Menge der intra- und extrazellularen Aminosauren waren wahrend des Wachstums sehr unterschiedlich. Ala und Glu, die einen Teil der Zellwandkomponente bilden. waren im Zellextrakt im Verhaltnis 2: I vorhanden.

Variations in amino acids, depending on the age of the culture, occur in several bacterial cultures, as reported by LANG and RING (1976), PRASAD and PANDEY (1977), and PRASAD (1979). BIEBER and ZIMMERMANN (1971) found quantitative and qualitative fluctuations in different bacterial types. The variation may also occur in different nutritional environment (HOARE 1955). POWELL and STRANGE (1953) noticed biochemical changes in the ratio of amino compounds during germination of bacterial spores of Bacillus megatherium and B. subtilis. However, the number of amino acids remained the same in cell extract and culture filtrate of Corynebacterium diphtheriae (WORK 1949). In contrast to this, in Xanthomonas citri pronounced qualitative and quantitative differences were observed by PRASAD (1979). Therefore it is proposed here to present the changes that occur in amino acids, both qualitative and quantitative, during the growth phase of Xanthomonas campestris pv. ma[vacearum, the incitant of the angular leaf spot disease of cotton.

Materials and Methods

The pathogen, Xanthomonas campestris pv. malvacearum (SMITH) DAWSON, was isolated from infected cotton leaves. The culture was grown on solid medium (PDA) and DYE's liquid medium simultaneously for analysing amino acids in bacterial cells as well as in the culture filtrate. The investigation was carried out for a grown period of 12 to 96 h at an interval of 12 h each. Chromatographic analysis and detection of the amino acids was carried out as adopted earlier by PRASAD and CHAUDHARY (1973) and PRASAD and PAN DE)' (1977).

592 K. LAL et al.

_ AMINO ACIO PRESENT IN CULTURE FILTRATE

c::::J AMINO ACID PRESENT IN BACTERIAL CELL EXTRACT

r=u¢: TItCKNESS OF THE LINE DENOTES THE AMOUNT OF •• ANIIO ACIDS ASSESSED ARBITRARilY

HOMOSERINE

HOMOSERINE

ASPARTIC ACID

ASPARTIC ACID

GLUTAMIC ACID

~--------------~=:J GLUTAMIC ACID

SERINE

GLUTAMINE

~ _________________ ~ __ ~ TRYPTOPHANE

GLYCINE

THREONINE

THREONINE

DL - ALANINE

DL -ALANINE

~ ________________ '--:=J __ ---l PROLINE

'---_____________________________ ----Jl ,B-PHENY L ALANINE - L -LEUCINE /ISOLEUCINE

L-LEUCINE/ISOLEUCINE I I , , I

12 24 36 48 60 72 84 96 GROWTH IN HOURS

Abb. 1. The free amino acid composition during growth of the Xanthomonas campestris pv. malvacearum (SMITH) DoWSON.

Results

The results are presented in Fig. 1. As evident from our investigation, 12 amino acids, namely I-leucine/isoleucine, B-phenylalanine, proline, dl-alanine, threonine, glycine, tryptophan, glut­amine, serine, homoserine, glutamic and aspartic acids were detected in varying amounts in the cell extract. Out of these only I-leucine/isoleucine, dl-alanine, threonine, homoserine, glutamic and aspartic acids were found to be present in its culture filtrate.

L-leucine/isoleucine showed a greater fluctuation during 12 to 84 h of growth phase. Its amount increased between 36 to 60 h and, after showing a drop again, came to the same level between 72 to 84 h. B-phenylalanine and dl-alanine did not show any variation in their amounts, being present in fairly good proportion during the entire growth phase. Glutamic acid showed an increase in 48-h-old culture and thereafter disappeared. Aspartic acid, on the other hand, diminished in 36 h and again

The Free Amino Acid Composition 593

increased in 84-h-old culture. This amino acid was, however, continously present throughout the growth phase, of the bacterium. Homoserine, showing a drop 72 h, ceased completely after 84 h of growth. Glutamine, an amid, could also be associated with the initiation of growth phase, being present in 12-h-old culture and completely lost in 84 h of growth phase. Proline and tryptophane appeared during the log-phase of growth, and their amount declined sharply in 96-h-old culture.

Of the 6 amino acids found in bacterial culture filtrate, interestingly I-leucine/isoleucine could be initially detected in 12-h-old culture and lost abruptly and could not be detected in 24-h-old culture. It appears that active extrusion of I-leucine/isoleucine in bacterial culture filtrate was remotely connected with the initiation of growth of this pathogen. Threonine, aspartic acid, and homoserine were detected after 24 h onward.

Discussion

PRASAD and PANDEY (1977) detected 16 amino acids during active growth phase of Xanthomonas campestris. Of these common amino acids, found in X. campestris pv. malva­cearum and X. campestris. were I-leucine/isoleucine, B-phenylalanine, serine, dl-alanine, threo­nine, proline, and glutamic and aspartic acids. In our experiment all these amino acids were consistently present right from the beginning of the growth and were active during its log-phase. WACEK and SEQUEIRA (1973), while analysing the peptidoglycan component of Pseudomonas solanacearum. discovered the presence of glutamic acid and alanine in a ratio of 1 : 1. This was, however, present in a ratio of 2: 1 in the cell extract of X. campestris pv. malvacearum.

In contrast to the finding by WORK (1949) for Corynebacterium diphtheriae. there were both qualitative and quantitative differences in the amino acid constituents of the bacterial cell extract and culture filtrate of X. campestris pv. ma/vacearum. LANG and RING (1976) had detected substantial variations in the size and composition of the pool of free intracellular amino acids during the growth cycle of Streptomyces hydrogenans. Such tendency towards general decline in the size and composition of amino acid pool during static phase of growth attaining lowest values at the end of this phase was discernible in X. campestris pv. malvacearum culture, too.

Active excretion of I-leucine/isoleucine in culture filtrate within 12 h of growth appeared to be remotely responsible for initiation of growth in this pathogen. The log-phase was maintained by the intense activities of B-phenylalanine, dl-alanine, glycine, serine, glutamic and aspartic acids in the bacterial cell. On the other hand. threonine, aspartic acid and homoserine due to their rapid depletion in the culture filtrate helped good growth during active period. Proline and tryptophan were products of lag-phase of growth. Similarly, dl-alanine and glutamic acid due to their continued presence in culture filtrate during later part of growth had a bearing on its stagnent phase.

Acknowledgement

The senior author extends thanks to the University Grants Commission. New Delhi. for awarding some grants for this research work.

References

BIEBER. H., ZIMMERMAN, W.: Die Aminosauren. Zusammensetzung einiger aerober und anaerober Bakterien. Zbl. Bakl. I 217 (1971). 47-62.

CUMMINS. C. S., HARRIS, H.: The chemical composition of the cell wall in some Gram-positive bacteria and its possible value as a taxonomic character. J. Gen. Microbiol. 14 (1956),583-600.

HOARE, D. S.: The amino acid composition of Sarcina [mea grown on different media. J. Gen. Microbiol. 12 (1955),534-539.

LANG, H. W., RING, K.: Regulation of amino acid transport in growing cells of 5rreptomyces hydrogenans. I. Modulation of transport capacity and amino acid pool composition during the growth cycle. Arch. Mikrobiol. 109 (1976), 227-235.

594 K. LAL et al.

PRASAD, M.: The free amino acid pool composition during growth of the culture of Xanthomonas citri (HASSE) DOWSON. Zbl. Bakt. II. Abt. 134 (1979),692-696. CHAUDHARY, S. K.: Variations in amino acids and sugars and their effect on growth and sporulation in Fusarium oxysporum f. udum. Phytopathol. Z. 78 (1973), 147-151. PANDEY, P. K.: Variations in amino acids and sugars in the growing culture of Xanthomonas campestris (PAMMEL) DOWSON, a pathogen of blackvein of cabbage. Symp. Physiology of Microorganism (1977). New Delhi 1977, 303-308.

POWELL, J. F., STRANGE, R. E.: Biochemical changes occuring during the germination of bacterial spores. Biochem. J. 54 (1953),205-209.

W ACEK, T. J., SEQUEIRA, L.: The peptoglycan of Pseudomonas solanacearum: Chemical composition and biological activity in relation to hypersensitive reaction in tobacco. Physiol. PI. Path. 3 (1973),363-369.

WORK, E.: Chromatographic investigations of amino acids from microorganisms. I. The amino acids of Corynebacte­rium diphteriae. Biochem. Biophys. Acta 3 (1949), 400-411.

Authors addresses:

Dr. B. B. LAL and Dr. rer. nat. M. PRASAD, Department of Botany, Ranchi University, Ranchi 834008, India; K. LAL, Department of Botany, Bihar University. Muzaffarpur 842001, India.