| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Laboratório de Microbiologia, Centro de Neurociências e Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal
2 Lehrstuhl für Mikrobiologie und Archaeenzentrum, Universität Regensburg, D-93053 Regensburg, Germany
3 Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal
4 Departamento de Bioquímica, Universidade de Coimbra, 3001-401 Coimbra, Portugal
Correspondence
André Antunes
aantunes{at}cnc.uc.pt
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Polar lipid profiles of Halorhabdus utahensis and strain SARL4BT, separated by mono-dimensional TLC, are shown in a supplementary figure available with the online version of this paper.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). All of the known deep-sea brines are associated with tectonic activity, though found in regions around the world that represent entirely different geodynamic environments, namely divergent and convergent plate boundary settings (Red Sea and Mediterranean Sea, respectively) and the salt tectonics of the Gulf of Mexico (Degens & Ross, 1969; Scientific Staff of Cruise Bannock 1984–12, 1985; Wiesenburg et al., 1985).
The first deep-sea brines to be discovered were in the Red Sea, but only a few microbiological studies have been performed on these brines and, consequently, only a few micro-organisms (all of which are bacteria) have been isolated from these unusual biotopes (Antunes et al., 2003, 2007; Eder et al., 2001; Fiala et al., 1990; Trüper, 1969). Nonetheless, data from phylogenetic and biochemical studies have shown that a diverse archaeal population exists in these deep-sea brines (Eder et al., 1999, 2001, 2002; Michaelis et al., 1990).
New samples for microbiological studies were retrieved from the northern-most brine-filled depths of the Red Sea during Cruise 52/3 of RV Meteor in 2002 (Antunes, 2003). Strain SARL4BT was isolated from the brine–sediment interface of the Shaban Deep as the result of a subsequent microbial diversity assessment study that relied on phylogenetic targeting of members of the Archaea. A phylogenetic analysis based on 16S rRNA gene sequencing revealed a close relationship between strain SARL4BT and Halorhabdus utahensis, the sole recognized species of the genus Halorhabdus. We propose that, on the basis of physiological, biochemical and phylogenetic evidence, isolate SARL4BT represents a novel species of the genus Halorhabdus.
Strain SARL4BT was isolated from a sample of the brine–sediment interface of the eastern basin of the Shaban Deep in the Red Sea (station no. 133-1; 2 ° 13.9' N 3 ° 21.3' E) taken at a depth of 1447 m, a pH of 6.0, a salinity of 24.4 % and an in situ temperature of 24.1 °C. Serum flasks (120 ml) were filled with 20 ml anaerobic HBM liquid medium (Halobacteria medium; DSMZ medium 372) containing the following (l–1): 5.0 g yeast extract, 5.0 g Casamino acids, 1.0 g sodium glutamate, 2.0 g KCl, 3.0 g sodium citrate, 20.0 g MgSO4 . 7H2O, 200.0 g NaCl, 36.0 mg FeCl2 . 4H2O and 0.36 mg MnCl2 . 4H2O. The liquid medium was dispensed, stoppered and sealed inside an anaerobic tent and the gas phase exchanged with N2 (300 kPa) prior to autoclaving. The medium was inoculated with 0.2 ml of this sample and then incubated at 22 °C until turbidity was observed (after approx. 1 month). Cultures were screened with fluorescence in situ hybridization for the presence of members of the Archaea by following the protocol of Antón et al. (1999) and using a mixture of universal, archaea-specific, fluorescently labelled oligonucleotide probes (Moissl et al., 2003). A pure archaeal culture was later obtained using visually controlled separation of the desired cell type from a freshly grown mixed culture, achieved with a laser-microscope procedure (Huber et al., 1995, 2000).
Cells were routinely grown in anaerobic HBM liquid medium containing 27.5 % (w/v) NaCl and at a pH of 7.5. HBM was prepared as a minimal medium by omitting the yeast extract and the Casamino acids. HBM solid medium was obtained by the addition of 1.5 % (w/v) agar before autoclaving. The type strain of Hrd. utahensis (DSM 12940T) was used for comparative purposes.
Cell morphology and motility were examined using phase-contrast microscopy. The presence of cytochrome oxidase and catalase was determined as described by Smibert & Krieg (1981). The Gram reaction, flagellation and the presence or absence of endospores and poly-β-hydroxybutyrate were examined by using the staining procedures described by Smibert & Krieg (1981), after desalting using the method of Dussault (1955). For the poly-β-hydroxybutyrate-production test, cells were grown in HBM minimal medium supplemented with NH4Cl at 0.005 % (w/v) and maltose at 0.5 or 1 % (w/v).
The temperature, pH and salinity ranges for growth of strain SARL4BT were tested in both HBM and HRM (Hrd. utahensis medium; DSMZ medium 927; Wainø et al., 2000) anaerobic liquid media. The temperature range for growth (between 10 and 60 °C) was determined. Growth in the presence of NaCl was tested at concentrations ranging from 5 to 30 % (w/v). The pH range for growth was determined by using 50 mM MES, HEPES, TAPS, CAPSO and CAPS between pH 5.5 and 10.5. The requirement for Mg2+ was tested under optimal growth conditions, by supplementing HBM anaerobic liquid medium with 0–25 % (w/v) MgSO4 . 7H2O. The influence of oxygen on growth was tested in HBM liquid medium with a headspace containing O2 at 0–15 % (v/v) or in medium reduced with 0.5 g Na2S l–1 and prepared as described by Balch et al. (1979).
Enzymic and biochemical characterization tests were performed using the API 20NE system and API ZYM test strips (bioMérieux), as recommended by the manufacturer but with the salinity adjusted to 26 % NaCl (w/v) and with incubation at 42 °C for up to 9 days (API 20NE) or 6 h (API ZYM). Nitrate reduction was tested using the method described by Smibert & Krieg (1981). Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities were determined in minimal medium supplemented with 0.05 % (w/v) NH4Cl, 0.1 % (w/v) yeast extract and 0.5 or 1 % (w/v) arginine, lysine or ornithine. pH values were monitored throughout growth, the results being considered positive when an increase in pH was detected. The Voges–Proskauer reaction was performed in minimal HBM medium supplemented with 0.05 % (w/v) NH4Cl and 0.5 % (w/v) maltose. At the end of the growth period, the reaction was tested as described elsewhere (Smibert & Krieg, 1981). Methyl red test reactions were determined by measuring the final pH of the medium described above and were considered positive if the pH was 
4.2.
Anaerobic growth in the presence of S0, Na2SO4, Na2S2O3 and KNO3 (0.5 %, w/v) was tested in HBM and in HBM minimal medium supplemented with 0.5 % (w/v) maltose, and in both media with MgSO4 . 7H2O having been replaced by an equimolar amount of MgCl2 . 6H2O. Hydrogen sulphide was detected using lead(II) acetate paper (Merck).
Single-carbon-source assimilation was tested for 20 days at 42 °C in minimal HBM medium supplemented with 0.05 % (w/v) NH4Cl and the substrate at 0.2, 0.5 or 0.7 % (w/v). Gas production from sugars was confirmed by measuring the pressure inside the anaerobic flasks at the end of the growth period. Sensitivity to antimicrobial agents was tested by the addition of filter-sterilized antibiotics (25 and 50 µg ml–1) to HBM anaerobic liquid medium (Oren et al., 1997).
Polar lipids and diether core lipids were extracted and analysed by using TLC as described previously (Ross & Grant, 1985). Lipoquinones were extracted from freeze-dried cells and purified by using TLC according to the method of Tindall (1989).
DNA for the determination of the G+C content was isolated as described by Nielsen et al. (1995). The G+C content of the DNA was determined by using HPLC according to the method of Mesbah et al. (1989). The extraction of genomic DNA, PCR amplification of the 16S rRNA gene and sequencing of the purified PCR products were carried out as described previously (Dyall-Smith, 2001; Eder et al., 2001). The novel 16S rRNA gene sequence was integrated into an alignment of approximately 30 000 full and partial 16S rRNA sequences available in public databases using the corresponding automated tools of the ARB software package (Ludwig et al., 2004). The resulting alignment was checked manually. The neighbour-joining phylogenetic tree was constructed using the methods available in the ARB software package. The phylogenetic distance was determined by distance matrix analyses (Jukes & Cantor correction) also included in the ARB software package.
DNA–DNA hybridization was carried out at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). DNA was isolated using a French pressure cell (Thermo Spectronic) and purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) with the modifications described by Huß et al. (1983) using a Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with an in situ temperature probe (Varian).
16S rRNA gene sequence analysis indicated that isolate SARL4BT was a member of the family Halobacteriaceae. Phylogenetic analysis showed that Hrd. utahensis, the sole species of the genus Halorhabdus, is the closest relative of isolate SARL4BT, having a sequence similarity value of 99.3 % (Fig. 1). The sequence similarity values for SARL4BT with respect to all other recognized members of the family Halobacteriaceae were found to be below 91.0 %. The mean levels of DNA–DNA hybridization between strain SARL4BT and Hrd. utahensis were 41.2 %. This value is well below the threshold value of 70 % DNA–DNA relatedness generally accepted for the definition of a novel species (Wayne et al., 1987).
|
The results of the physiological and chemotaxonomic characterization are given in Table 1 and in the species description. The polar lipid profile of strain SARL4BT was found to be similar to that of Hrd. utahensis, having phosphatidylglycerol, methylated phosphatidylglycerophosphate, triglycosyl diether and sulfated diglycosyl diether as the major lipids, while phosphatidylglycerosulfate was absent (see Supplementary Fig. S1 in IJSEM Online). The antibiotic-sensitivity profiles of the novel strain and Hrd. utahensis were also largely similar.
|
). One of the most striking differences was the absence of pigmentation in strain SARL4BT; perhaps pigmentation was lost during evolution in a light-deprived environment. Another significant difference concerns the ability of strain SARL4BT to utilize some complex substrates for growth, such as yeast extract and starch. The inability of Hrd. utahensis to use these substrates was referred to as being unique within the family Halobacteriaceae and was suggested as a distinguishing feature (Wainø et al., 2000). This characteristic trait was subsequently observed in the phylogenetically related genus Halosimplex (Vreeland et al., 2002). Isolate SARL4BT was able to grow under strictly anaerobic, microaerophilic and aerobic conditions. However, this organism, unlike Hrd. utahensis, exhibited extremely poor aerobic growth, having a clear preference for anaerobic conditions. Such a preference, never previously reported for members of this family, clearly distinguishes strain SARL4BT not only from Hrd. utahensis, but also from all the other currently described haloarchaea.
Strain SARL4BT represents the first described archaeon isolated from a deep-sea hypersaline anoxic basin. Interestingly, recent phylogenetic studies performed on the deep-sea hypersaline anoxic basins of the eastern Mediterranean Sea have revealed a high percentage of archaeal clone sequences similar to that of Hrd. utahensis (van der Wielen et al., 2005). The presence of members of the genus Halorhabdus in at least two deep-sea, anoxic brine pools located in different geographical locations suggests a high degree of evolutionary success in their adaptation to this type of extreme biotope.
The physiological and biochemical characteristics of isolate SARL4BT were very distinctive, despite the phylogenetic proximity to Hrd. utahensis.
Our results show that strain SARL4BT is sufficiently different from the single species currently recognized as belonging to the genus Halorhabdus to warrant the classification of this isolate as a novel species of the genus, for which we propose the name Halorhabdus tiamatea. In view of some notable differences with respect to the type species of the genus, we also propose the emendation of the description of the genus Halorhabdus so as to accommodate this novel species.
Emended description of the genus Halorhabdus
The main characteristics of the genus Halorhabdus are as previously described (Wainø et al., 2000). In addition, strains of some species are non-flagellated, non-motile, produce non-pigmented colonies and show a clear preference for anaerobic conditions. The DNA G+C content ranges from 61.7 to 64.0 mol%. The type species of the genus is Halorhabdus utahensis.
Description of Halorhabdus tiamatea sp. nov.
Halorhabdus tiamatea (ti.a.ma.te'a. N.L. fem. adj. tiamatea belonging to, or related to, Tiamat, the ancient Mesopotamian goddess of ‘the primal abyss’ and salty water).
Gram-negative, pleomorphic cells ranging from rods to coccoid or irregular forms (0.5–1x1–8 µm). Flagella are not observed. Colonies on HBM agar are round, non-pigmented and 2–3 mm in diameter. Extremely halophilic: growth occurs at NaCl concentrations between 10 and 30 % (w/v), with optimum growth occurring at approximately 27 % (w/v) NaCl. Growth occurs with MgSO4 . 7H2O at 0–25 % (w/v). The temperature range for growth is between 15 and 55 °C (optimal temperature, 45 °C) and the pH range is between approximately 5.5 and 8.5 (optimal pH, 6.5–7.0). Grows under anaerobic and microaerophilic conditions; shows extremely poor aerobic growth. Produces acid from maltose and yeast extract. Oxidase-negative and catalase-positive. Methyl red test is positive and Voges–Proskauer test is negative. Indole is not produced. Poly-β-hydroxybutyrate is produced. Gelatin and aesculin are hydrolysed; starch is weakly hydrolysed. Casein and Tween 80 are not hydrolysed. Maltose and yeast extract are used for growth. Glucose, xylose, fructose, galactose, and proline are weakly assimilated. Amylose, L-arabinose, lactose, sucrose, sodium acetate, sodium citrate, sodium formate, sodium glucuronate, sodium lactate, sodium pyruvate, N-acetylglucosamine, alanine, betaine, lysine, phenylalanine, serine, acetamide, ethanol, glycerol, methanol, sorbitol, glycogen and peptone are not utilized. 
-Glucosidase and β-glucosidase are present. Weak reactions also detected for -galactosidase, β-galactosidase and β-glucuronidase. Alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, N-acetyl-β-glucosaminidase, -mannosidase and -fucosidase are not detected. Cells are resistant to ampicillin, cephalosporin C, chloramphenicol (25 µg ml–1), erythromycin, neomycin, penicillin G and tetracycline, but susceptible to anisomycin, aphidicolin, bacitracin, chloramphenicol (50 µg ml–1), novobiocin and rifamycin B. The ether lipids are diphytanyl derivatives. MK-8(VIII-H2) is the only respiratory lipoquinone present. The major polar lipids present are phosphatidylglycerol, methylated phosphatidylglycerophosphate, triglycosyl diether, sulfated diglycosyl diether and an unknown component. Phosphatidylglycerosulfate is absent. The DNA G+C content of the type strain is 61.7 mol%.
The type strain, SARL4BT (=DSM 18392T=JCM 14471T), was isolated from the brine–sediment interface of the Shaban Deep in the Red Sea.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Antunes, A. (2003). Microbial life at the anaerobic brine/seawater and sediment/brine interfaces. In Black Sea-Mediterranean-Red Sea, Cruise No. 52, January 2–March 27, 2002, pp. 50–52. Edited by J. Pätzold, G. Bohrmann & C. Hübscher. Hamburg: Universität Hamburg.
Antunes, A., Eder, W., Fareleira, P., Santos, H. & Huber, R. (2003). Salinisphaera shabanensis gen. nov., sp. nov., a novel, moderately halophilic bacterium from the brine-seawater interface of the Shaban Deep, Red Sea. Extremophiles 7, 29–34.[Medline]
Antunes, A., França, L., Rainey, F. A., Huber, R., Nobre, M. F., Edwards, K. J. & da Costa, M. S. (2007). Marinobacter salsuginis sp. nov., isolated from the brine-seawater interface of the Shaban Deep, Red Sea. Int J Syst Evol Microbiol 57, 1035–1040.
Balch, W. E., Fox, G. E., Magrum, L. J., Woese, C. R. & Wolfe, R. S. (1979). Methanogens: reevaluation of a unique biological group. Microbiol Rev 43, 260–296.
Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). A rapid method for the base ratio determination of bacterial DNA. Anal Biochem 81, 461–466.[CrossRef][Medline]
DasSarma, S. & Arora, P. (2001). Halophiles. In Encyclopedia of Life Sciences. Chichester: John Wiley. http://www.els.net
Degens, E. & Ross, D. A. (1969). Hot brines and recent heavy metal deposits in the Red Sea. New York: Springer.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Dussault, H. P. (1955). An improved technique for staining red halophilic bacteria. J Bacteriol 70, 484–485.
Dyall-Smith, M. (2001). The Halohandbook: Protocols for Halobacterial Genetics, edn 4.5. Melbourne: University of Melbourne.
Eder, W., Ludwig, W. & Huber, R. (1999). Novel 16S rRNA gene sequences retrieved from highly saline brine sediments of Kebrit Deep, Red Sea. Arch Microbiol 172, 213–218.[CrossRef][Medline]
Eder, W., Jahnke, L. L., Schmidt, M. & Huber, R. (2001). Microbial diversity of the brine-seawater interface of the Kebrit Deep, Red Sea, studied via 16S rRNA gene sequences and cultivation methods. Appl Environ Microbiol 67, 3077–3085.
Eder, W., Schmidt, M., Koch, M., Garbe-Schönberg, D. & Huber, R. (2002). Prokaryotic phylogenetic diversity and corresponding geochemical data of the brine-seawater interface of the Shaban Deep, Red Sea. Environ Microbiol 4, 758–763.[CrossRef][Medline]
Fiala, G., Woese, C. R., Langworthy, T. A. & Stetter, K. O. (1990). Flexistipes sinusarabici, a novel genus and species of eubacteria occurring in the Atlantis II Deep brines of the Red Sea. Arch Microbiol 154, 120–126.[CrossRef]
Huber, R., Burggraf, S., Mayer, T., Barns, S. M., Rossnagel, P. & Stetter, K. O. (1995). Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis. Nature 376, 57–58.[CrossRef][Medline]
Huber, R., Huber, H. & Stetter, K. O. (2000). Towards the ecology of hyperthermophiles: biotopes, new isolation strategies and novel metabolic properties. FEMS Microbiol Rev 24, 615–623.[CrossRef][Medline]
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Michaelis, W., Jenisch, A. & Richnow, H. H. (1990). Hydrothermal petroleum generation in Red Sea sediments from the Kebrit and Shaban Deeps. Appl Geochem 5, 103–114.[CrossRef]
Moissl, C., Rudolph, C., Rachel, R., Koch, M. & Huber, R. (2003). In situ growth of the novel SM1 euryarchaeon from a string-of-pearls-like microbial community in its cold biotope, its physical separation and insights into its structure and physiology. Arch Microbiol 180, 211–217.[CrossRef][Medline]
Nielsen, P., Fritze, D. & Priest, F. G. (1995). Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species. Microbiology 141, 1745–1761.
Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47, 233–238.
Ross, H. N. M. & Grant, W. D. (1985). Lipids in archaebacterial taxonomy. In Chemical Methods in Bacterial Systematics, pp. 289–300. Edited by M. Goodfellow & D. E. Minnikin. Orlando, FL: Academic Press.
Scientific Staff of Cruise Bannock 1984–12 (1985). Gypsum precipitation from cold brines in an anoxic basin in the eastern Mediterranean. Nature 314, 152–154.[CrossRef]
Smibert, R. M. & Krieg, N. R. (1981). General characterization. In Manual of Methods for General Bacteriology, pp. 409–443. Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American Society for Microbiology.
Tindall, B. J. (1989). Fully saturated menaquinones in the archaebacterium Pyrobaculum islandicum. FEMS Microbiol Lett 60, 251–254.[CrossRef]
Trüper, H. G. (1969). Bacterial sulfate reduction in the Red Sea hot brines. In Hot Brines and Recent Heavy Metal Deposits in the Red Sea, pp. 263–271. Edited by E. T. Degens & D. A. Ross. New York: Springer.
van der Wielen, P. W., Bolhuis, H., Borin, S., Daffonchio, D., Corselli, C., Giuliano, L., D'Auria, G., de Lange, G. J., Huebner, A. & other authors (2005). The enigma of prokaryotic life in deep hypersaline anoxic basins. Science 307, 121–123.
Vreeland, R. H., Straight, S., Krammes, J., Dougherty, K., Rosenzweig, W. D. & Kamekura, M. (2002). Halosimplex carlsbadense gen. nov., sp. nov., a unique halophilic archaeon, with three 16S rRNA genes, that grows only in defined medium with glycerol and acetate or pyruvate. Extremophiles 6, 445–452.[CrossRef][Medline]
Wainø, M., Tindall, B. J. & Ingvorsen, K. (2000). Halorhabdus utahensis gen. nov., sp. nov., an aerobic, extremely halophilic member of the Archaea from the Great Salt Lake, Utah. Int J Syst Evol Microbiol 50, 183–190.[Abstract]
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Wiesenburg, D. A., Brooks, J. M. & Bernard, B. B. (1985). Biogenic hydrocarbon gases and sulfate reduction in the Orca basin brine. Geochim Cosmochim Acta 49, 2069–2080.[CrossRef]
This article has been cited by other articles:
|
|
 |
|
  A. Oren, D. R. Arahal, and A. Ventosa Emended descriptions of genera of the family Halobacteriaceae Int J Syst Evol Microbiol, March 1, 2009; 59(3): 637 - 642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Antunes, F. A. Rainey, G. Wanner, M. Taborda, J. Patzold, M. F. Nobre, M. S. da Costa, and R. Huber A New Lineage of Halophilic, Wall-Less, Contractile Bacteria from a Brine-Filled Deep of the Red Sea J. Bacteriol., May 15, 2008; 190(10): 3580 - 3587. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
