1 Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand
2 Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Correspondence
Skorn Mongkolsuk
skorn{at}tubtim.cri.or.th
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Center for Vectors and Vector-Borne Diseases, Mahidol University, Bangkok 10400, Thailand.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The identity of key regulators mediating the expression of bacterial catalases can be inferred from observations in Escherichia coli and Salmonella typhimurium showing that the synthesis of catalase-peroxidase (hydroperoxidase I encoded by katG) is regulated by OxyR whereas expression of the monofunctional catalase (hydroperoxidase II encoded by katE) is under the regulation of a stationary-phase-specific S (Storz & Altuvia, 1994
). OxyR is a peroxide sensor and global transcriptional regulator of the peroxide stress response (Toledano et al., 1994
; Zheng et al., 1998
). The precise role of OxyR in the regulation of catalases seems to vary in different bacteria, particularly in non-enteric species. The expression of monofunctional catalase, encoded by katB, in Pseudomonas aeruginosa is activated by OxyR (Ochsner et al., 2000
), whereas expression of the Neisseria gonorrhoeae catalase, encoded by kat, is presumably repressed by OxyR (Tseng et al., 2003
). Streptomyces coelicolor produces multiple catalase isozymes, none of which is regulated by OxyR (Hahn et al., 2002
).
Xanthomonas campestris is an important bacterial phytopathogen. Treatment of X. campestris pv. phaseoli with sublethal concentrations of H2O2 or a superoxide-generating agent (menadione) induced elevated levels of total catalase activity during both exponential and stationary phase (Vattanaviboon & Mongkolsuk, 2000). The level of total catalase activity correlates with the ability to resist H2O2 toxicity (Fuangthong & Mongkolsuk, 1997
; Mongkolsuk et al., 1997a
). X. campestris pv. phaseoli produces two detectable isozymes of monofunctional catalase, denoted KatA and KatE, that are encoded by katA and katE, respectively. KatA is the major catalase produced during all phases of growth, while KatE is detected only as cells enter the stationary phase or under nutrient-starved conditions (Vattanaviboon & Mongkolsuk, 2000
). More recently, the X. campestris pv. phaseoli katA gene, encoding the major catalase, was cloned and characterized (Chauvatcharin et al., 2003
). Its putative amino acid sequence is highly homologous (87 % identity) to the clade I catalase from Pseudomonas syringae, CatF, whose crystal structure has been solved (Carpena et al., 2003
). In this paper, we report expression analysis and demonstrate the physiological importance of katA in protection against H2O2 toxicity. The involvement of ankA, encoding an ankyrin homologue, in the H2O2 resistance of X. campestris pv. phaseoli is also demonstrated.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nucleic acid manipulations.
All nucleic acid manipulations were performed using standard molecular biology techniques (Sambrook et al., 1989) or according to the manufacturers' recommendations. The labelling of DNA probes with [
-32P]dCTP was performed using a DNA labelling bead (Amersham Pharmacia Biotech). Southern and Northern blot analyses were performed as previously described (Mongkolsuk et al., 1997b
).
Catalase activity gels and assays.
Cell lysate preparation and catalase activity gel staining were performed as previously described (Vattanaviboon & Mongkolsuk, 2000). Bacterial cells were lysed in 50 mM sodium phosphate buffer, pH 7·0, containing 1 mM PMSF by brief sonication followed by centrifugation at 10 000 g for 10 min. Supernatants were used for catalase activity gels and catalase isozymes were visualized on native PAGE gels as previously described (Vattanaviboon & Mongkolsuk, 2000
). Catalase activity appeared as colourless bands against a dark brown background. The catalase assay was carried out spectrophotometrically, according to the method of Beers & Sizer (1952)
. One unit of catalase was defined as the amount of enzyme required to decompose 1·0 µmol H2O2 at 25 °C at pH 7·0.
Cloning of full-length ankA.
The full-length ankA gene in pKat29 (Chauvatcharin et al., 2003) was amplified using the oligonucleotide primers BT176 and BT177 (see Table 1
). The 620 bp PCR product was cloned into pGemT-easy and subsequently subcloned into the broad-host-range plasmid pBBR1MCS-5 (Kovach et al., 1995
) to generate the ankA overexpression plasmid, pAnkA (see Table 1
).
|
Gel mobility shift assay.
32P-labelled DNA fragments were prepared by PCR using the oligonucleotide primers BT151 and BT150 (see Table 1) and pKat29 (Chauvatcharin et al., 2003
) as the template to generate a 254 bp fragment spanning the katA promoter region. Gel mobility shift reactions were performed by adding 3 fmol labelled probe in 25 µl reaction buffer [20 mM Tris pH 7·0, 50 mM KCl, 1 mM EDTA, 5 %, v/v, glycerol, 50 µg BSA ml1, 5 µg calf thymus DNA ml1, 0·5 mg poly(dI/dC) ml1]; 400 ng purified OxyR (Loprasert et al., 2000
) was added and the reaction was incubated at 25 °C for 15 min. ProteinDNA complexes were separated by electrophoresis on 6 % non-denaturing polyacrylamide gel in 0·5x Tris/borate/EDTA buffer (TBE) at 4 °C.
RT-PCR of katAankA mRNA.
Reverse transcription (RT) of katAankA mRNA was performed to confirm the bicistronic transcriptional organization of these genes. Total RNA was isolated from X. campestris pv. phaseoli cultures using the hot acid/phenol method (Mongkolsuk et al., 2002). Purified RNA was treated with 10 U RNase-free DNase I for 30 min to remove contaminating DNA. Primer BT149 (located within ankA; see Table 1
) was mixed with 10 µg RNA, and 200 U cloned Moloney murine leukaemia virus (MMLV) reverse transcriptase (Promega) was added. The mixture was incubated at 42 °C for 60 min. Five microlitres of the mixture was added to a PCR reaction containing primers BT149 and BT148 (located in katA; see Table 1
). PCR was performed for 35 cycles under the following conditions: denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 30 s. The PCR products were analysed by agarose gel electrophoresis.
Primer extension.
Total RNA was isolated from uninduced and menadione-induced X. campestris pv. phaseoli cultures. Primer extension experiments were performed using 32P-labelled oligonucleotide primer BT150 (see Table 1), 5 µg total RNA and 200 U superscript II MMLV reverse transcriptase (Promega). Extension products were sized on sequencing gels next to dideoxy sequencing ladders generated using a PCR sequencing kit with labelled BT150 primer and pKat29 plasmid as the template (Chauvatcharin et al., 2003
).
Determination of oxidant resistance.
Analysis of the killing effects of various reagents on X. campestris pv. campestris strains was performed using inhibition zone assays as described by Mongkolsuk et al. (1998a). Briefly, overnight cultures were subcultured as 5 % inocula into fresh SB broth and incubated at 28 °C with shaking. One millilitre of exponential-phase cells (4 h, culture OD600
0·5) was mixed with 10 ml molten top agar (SB containing 0·7 % agar) held at 50 °C, and overlaid onto SB plates (14 cm diameter Petri dishes containing 40 ml SB agar). The plates were left at room temperature for 15 min to let the top agar solidify. Sterile 6 mm diameter paper discs soaked with 5 µl H2O2 (0·5 M), tBOOH (0·5 M) or menadione (1·0 M) were placed on top of the cell lawn and the diameters of the inhibition zones were measured after 24 h incubation at 28 °C.
Determination of adaptive and cross-protective resistance to H2O2.
The induced adaptive or cross-protective resistance to H2O2 killing was measured by adding H2O2 or menadione (100 µM), respectively, to exponential-phase cultures of X. campestris pv. campestris strains prior to treatment with lethal concentrations of H2O2 (10, 20, 30 mM) for 30 min. After treatment, cells were removed and washed once with fresh SB medium and cell survival was determined by plating appropriate dilutions on SB agar plates. Colonies were counted after 48 h incubation at 28 °C. The surviving fraction was defined as the number of c.f.u. recovered after treatment divided by the number of c.f.u. prior to treatment. Three independent experiments were performed in each case and representative data are shown.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The transcriptional organization of katA and ankA was determined by Northern blots. The results showed that a katA probe hybridized to two mRNA bands at 1·6 kb and 2·2 kb that corresponded to the predicted sizes of monocistronic katA and bicistronic katAankA mRNAs respectively (Fig. 1a). Northern blot analysis using an ankA-specific probe detected a single positively hybridizing band at 2·2 kb (Fig. 1a
), suggesting that ankA is co-transcribed with katA. This was confirmed by RT-PCR experiments that were performed using two specific primers, one located near the 3' end of the katA coding region and another located near the 5' end of ankA. RT-PCR using an RNA sample prepared from an uninduced Xp culture gave rise to a 550 bp product that corresponded to the expected size of a product derived from a katAankA mRNA template (Fig. 1b
). This 550 bp band was absent when the RNA sample was first treated with RNaseA, indicating that the product was not the result of priming to contaminating DNA (Fig. 1b
). Thus, katA and ankA are transcribed as a bicistronic mRNA of 2·2 kb. Densitometer analyses of katA Northern blots indicated that the monocistronic katA mRNA made up 85 % of the katA-encoding mRNA while 15 % consisted of the katAankA bicistronic message (Fig. 1b
). Examination of the DNA sequence in the katAankA intergenic region shows the presence of an inverted repeat sequence spanning 32 bp followed by a run of three T residues (Fig. 1c
). The stemloop structure resembles a typical rho-independent transcription terminator, suggesting that the major 1·5 kb katA monocistronic transcripts are the result of rho-independent transcription termination at this site. Moreover, the minor 2·2 kb katAankA bicistronic mRNA is likely to result from transcriptional readthrough at this site. The mechanism responsible for the antitermination at the katA terminator is not known. It could be mediated by a regulator, as with AmiR in the amidase operon (Wilson et al., 1996
). Alternatively, readthrough may be the result of the intrinsic efficiency of the terminator itself (Weisberg & Gottesman, 1999
), such that it permits 15 % readthrough transcription into ankA.
|
|
Analysis of OxyR regulation of the katA promoter
A more detailed characterization of OxyR-regulated expression of katA was performed. First, the transcriptional start site of the katAankA operon was mapped. The results of primer extension analyses using total RNA samples prepared from uninduced and menadione-induced cultures showed a single extension product of 78 bases, indicating that katA transcription is initiated at the C residue located 21 nucleotides upstream of the katA translational start codon (Fig. 3). The proposed
70-type RNA polymerase consensus binding sequence for a X. campestris promoter consists of the 35 element, TTGTNN, separated by 16 to 24 nucleotides from the 10 element, T/AATNAA/T (Katzen et al., 1996
). Examination of the sequence upstream of the transcriptional start site revealed the presence of two sequence motifs, TTCTCA (34 to 29) and GATGAT (11 to 6), that are separated by 17 bp, and that closely matched the 35 and 10 consensus promoter sequences, respectively (Fig. 3
). A significant amount of katA primer extension products was detected in the absence of inducer (Fig. 3
, uninduced sample) indicating that the gene is constitutively transcribed. This is consistent with other observations (Fig. 2
) indicating that even in the absence of oxidant inducers, katA is highly expressed during exponential-phase growth. The amount of primer extension products in the menadione-treated sample was fivefold higher than that in the uninduced sample (Fig. 3
). This reinforced the Northern blot hybridization results and confirmed that menadione induction of KatA activity is the result of increased katA transcript levels.
|
In order to conclusively demonstrate the direct participation of OxyR in the activation of X. campestris pv. phaseoli katA transcription, purified Xp OxyR and a 254 bp DNA fragment spanning the katA promoter, including the putative OxyR binding site, were used in mobility shift assays. The results of these assays demonstrated that OxyR bound to the katA promoter (Fig. 4). OxyR binding was inhibited by the addition of excess unlabelled probe fragment (Fig. 4
, UP), but not by the addition of excess nonspecific competitor DNA (pBBR1MCS-5) (Fig. 4
, UD), indicating that binding was specific for the katA promoter. Moreover, the unrelated oxidant-sensing transcription repressor, OhrR (Mongkolsuk et al., 2002
), did not bind to the katA promoter, indicating that katA expression is not under direct OhrR control (Fig. 4
, OhrR). These data, coupled with the in vivo results, support the hypothesis that katA is directly regulated by OxyR.
|
|
katA contributes to H2O2-induced adaptive protection and menadione-induced cross-protection against H2O2 killing treatments
Physiological adaptation to stresses is an important response for bacterial survival under stressful conditions. The process often involves complex alteration in the expression pattern of genes involved in stress protection and repair of stress-induced damage. The oxidative stress-induced physiological adaptation and cross-protection responses are widely distributed in both Gram-negative and Gram-positive bacteria. We have reported the presence of H2O2-induced physiological adaptive and menadione-induced cross-protective responses to lethal concentrations of H2O2 in Xp (Mongkolsuk et al., 1998b). The H2O2-induced adaptive response is completely abolished in an oxyR mutant while the menadione-induced cross-protective response is only partially lost (Mongkolsuk et al., 1998b
). The role of OxyR-regulated katA expression in these responses was evaluated. Xp20 and its parental strain Xp were grown to exponential phase before being induced with either H2O2 or menadione (100 µM) for 30 min. The induced cultures were then treated with lethal concentrations of H2O2 for 30 min and the percentage survival relative to an untreated control culture was determined. The results show that Xp20 had significantly impaired H2O2-induced adaptive and menadione-induced cross-protection responses against H2O2 relative to the parental strain (Fig. 6
). In the parental strain Xp, pretreatment with H2O2 or menadione induced 100-fold and 1000-fold protection, respectively, against subsequent H2O2 killing treatments (Fig. 6a
). In the katA mutant, Xp20, the levels of induced protection against H2O2 killing decreased to 10-fold and 100-fold after induction with H2O2 or menadione, respectively (Fig. 6b
). This indicated that katA has a general role in the protection against killing by H2O2 in both uninduced cells and those induced by oxidants. Furthermore, even though uninduced and oxidant-induced Xp20 cells were 100-fold more sensitive to H2O2 killing than the parental strain Xp, strain Xp20 still retained the ability to mount a partial H2O2-induced adaptive response. This observation, combined with the fact that inactivation of oxyR in X. campestris pv. phaseoli has been shown to completely abolish the H2O2-induced adaptive response (Mongkolsuk et al., 1998b
), implies that OxyR-regulated genes other than katA must contribute to the H2O2-induced adaptive response in Xp20. In E. coli, the alkylhydroperoxidase AhpC has an essential role in scavenging H2O2 during normal growth (Seaver & Imlay, 2001
) and it has been shown that ahpC is highly induced by both H2O2 and menadione (Loprasert et al., 2000
). Thus, it is likely that induction of ahpC by oxidants contributed to both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris pv. phaseoli. The mechanism of menadione-induced cross-protection against H2O2 is more complex. The fact that OxyR was only partially responsible for the process (Mongkolsuk et al., 1998b
) suggests that, in addition to katA and ahpC, there must be other genes, independent of OxyR regulation, that are involved. The OxyR-independent menadione induction of these protective systems is currently being investigated.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 33893402.
Beers, R. F. & Sizer, I. W. (1952). A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195, 133135.
Bendtsen, J. D., Nielsen, H., von Heijne, G. & Brunak, S. (2004). Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340, 783795.[CrossRef][Medline]
Carpena, X., Soriano, M., Klotz, M. G., Duckworth, H. W., Donald, L. J., Melik-Adamyan, W., Fita, I. & Loewen, P. C. (2003). Structure of the clade 1 catalase, CatF of Pseudomonas syringae, at 1·8 Å resolution. Proteins 50, 423436.[CrossRef][Medline]
Chauvatcharin, N., Vattanaviboon, P., Switala, J., Loewen, P. C. & Mongkolsuk, S. (2003). Cloning and characterization of katA, encoding the major monofunctional catalase from Xanthomonas campestris pv. phaseoli, and characterization of the encoded catalase KatA. Curr Microbiol 46, 8387.[CrossRef][Medline]
Claros, M. G. & von Heijne, G. (1994). TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10, 685686.[Medline]
Farr, S. B. & Kogoma, T. (1991). Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol Rev 55, 561585.[Medline]
Fuangthong, M. & Mongkolsuk, S. (1997). Isolation and characterization of a multiple peroxide resistant mutant from Xanthomonas campestris pv. phaseoli. FEMS Microbiol Lett 152, 189194.[CrossRef][Medline]
Hahn, J. S., Oh, S. Y. & Roe, J. H. (2002). Role of OxyR as a peroxide-sensing positive regulator in Streptomyces coelicolor A3(2). J Bacteriol 184, 52145222.
Howell, M. L., Alsabbagh, E., Ma, J. F. & 10 other authors (2000). AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide. J Bacteriol 182, 45454556.
Katzen, F., Becker, A., Zorreguieta, A., Puhler, A. & Ielpi, L. (1996). Promoter analysis of the Xanthomonas campestris pv. campestris gum operon directing biosynthesis of the xanthan polysaccharide. J Bacteriol 178, 43134318.
Klotz, M. G., Klassen, G. R. & Loewen, P. C. (1997). Phylogenetic relationships among prokaryotic and eukaryotic catalases. Mol Biol Evol 14, 951958.[Abstract]
Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M. 2nd & Peterson, K. M. (1995). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175176.[CrossRef][Medline]
Loprasert, S., Fuangthong, M., Whangsuk, W., Atichartpongkul, S. & Mongkolsuk, S. (2000). Molecular and physiological analysis of an OxyR-regulated ahpC promoter in Xanthomonas campestris pv. phaseoli. Mol Microbiol 37, 15041514.[CrossRef][Medline]
Mongkolsuk, S., Loprasert, S., Vattanaviboon, P., Chanvanichayachai, C., Chamnongpol, S. & Supsamran, N. (1996). Heterologous growth phase- and temperature-dependent expression and H2O2 toxicity protection of a superoxide-inducible monofunctional catalase gene from Xanthomonas oryzae pv. oryzae. J Bacteriol 178, 35783584.
Mongkolsuk, S., Vattanaviboon, P. & Praitaun, W. (1997a). Induced adaptive and cross-protection responses against oxidative stress killing in a bacterial phytopathogen, Xanthomonas oryzae pv. oryzae. FEMS Microbiol Lett 146, 217221.[CrossRef]
Mongkolsuk, S., Loprasert, S., Whangsuk, W., Fuangthong, M. & Atichartpongkun, S. (1997b). Characterization of transcription organization and analysis of unique expression patterns of an alkyl hydroperoxide reductase C gene (ahpC) and the peroxide regulator operon ahpF-oxyR-orfX from Xanthomonas campestris pv. phaseoli. J Bacteriol 179, 39503955.
Mongkolsuk, S., Praituan, W., Loprasert, S., Fuangthong, M. & Chamnongpol, S. (1998a). Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with a novel pattern of oxidative stress regulation from Xanthomonas campestris pv. phaseoli. J Bacteriol 180, 26362643.
Mongkolsuk, S., Sukchawalit, R., Loprasert, S., Praituan, W. & Upaichit, A. (1998b). Construction and physiological analysis of a Xanthomonas mutant to examine the role of the oxyR gene in oxidant-induced protection against peroxide killing. J Bacteriol 180, 39883991.
Mongkolsuk, S., Whangsuk, W., Vattanaviboon, P., Loprasert, S. & Fuangthong, M. (2000). A Xanthomonas alkyl hydroperoxide reductase subunit C (ahpC) mutant showed an altered peroxide stress response and complex regulation of the compensatory response of peroxide detoxification enzymes. J Bacteriol 182, 68456849.
Mongkolsuk, S., Panmanee, W., Atichartpongkul, S., Vattanaviboon, P., Whangsuk, W., Fuangthong, M., Eiamphungporn, W., Sukchawalit, R. & Utamapongchai, S. (2002). The repressor for an organic peroxide-inducible operon is uniquely regulated at multiple levels. Mol Microbiol 44, 793802.[CrossRef][Medline]
Ochsner, U. A., Vasil, M. L., Alsabbagh, E., Parvatiyar, K. & Hassett, D. J. (2000). Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J Bacteriol 182, 45334544.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Seaver, L. C. & Imlay, J. A. (2001). Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli. J Bacteriol 183, 71827189.
Storz, G. & Altuvia, S. (1994). OxyR regulon. Methods Enzymol 234, 217223.[Medline]
Toledano, M. B., Kullik, I., Trinh, F., Baird, P. T., Schneider, T. D. & Storz, G. (1994). Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection. Cell 78, 897909.[Medline]
Tseng, H. J., McEwan, A. G., Apicella, M. A. & Jennings, M. P. (2003). OxyR acts as a repressor of catalase expression in Neisseria gonorrhoeae. Infect Immun 71, 550556.
Vattanaviboon, P. & Mongkolsuk, S. (2000). Expression analysis and characterization of the mutant of a growth-phase- and starvation-regulated monofunctional catalase gene from Xanthomonas campestris pv. phaseoli. Gene 241, 259265.[CrossRef][Medline]
Vattanaviboon, P., Sriprang, R. & Mongkolsuk, S. (2001). Catalase has a novel protective role against electrophile killing of Xanthomonas. Microbiology 147, 491498.[Medline]
Visick, K. L. & Ruby, E. G. (1998). The periplasmic, group III catalase of Vibrio fischeri is required for normal symbiotic competence and is induced both by oxidative stress and by approach to stationary phase. J Bacteriol 180, 20872092.
Weisberg, R. A. & Gottesman, M. E. (1999). Processive antitermination. J Bacteriol 181, 359367.
Wilson, S. A., Wachira, S. J., Norman, R. A., Pearl, L. H. & Drew, R. E. (1996). Transcription antitermination regulation of the Pseudomonas aeruginosa amidase operon. EMBO J 15, 59075916.[Abstract]
Xu, X. Q., Li, L. P. & Pan, S. Q. (2001). Feedback regulation of an Agrobacterium catalase gene katA involved in Agrobacterium-plant interaction. Mol Microbiol 42, 645657.[CrossRef][Medline]
Zheng, M., Aslund, F. & Storz, G. (1998). Activation of the OxyR transcription factor by reversible disulfide bond formation. Science 279, 17181721.
Received 1 September 2004;
revised 22 October 2004;
accepted 5 November 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |