Ó Springer 2006
World Journal of Microbiology & Biotechnology (2006) DOI 10.1007/s11274-005-9043-y
Proteomics of a plant growth-promoting rhizobacterium, Pseudomonas fluorescens MSP-393, subjected to salt shock Diby Paul, N. Dineshkumar and Sudha Nair* Microbiology Group, M. S. Swaminathan Research Foundation, Taramani Institutional Area, 600113, Chennai, India *Author for correspondence: tel.: +91-44-55299024, fax: 91-44-22541319, e-mail:
[email protected] Received 7 July 2005; accepted 12 August 2005
Keywords: Biocontrol, proteome analysis, Pseudomonas fluorescens, salt stress proteins
Summary Pseudomonas fluorescens MSP-393, a plant growth-promoting rhizobacterium is an efficient biocontrol agent in rice grown in saline soils of coastal ecosystems. To understand the mechanism of salt tolerance, proteome analysis of the bacterium was carried out employing two-dimensional gel electrophoresis and MALDI-TOF. This technique was used to investigate the regulation of gene product expression of P. fluorescens MSP-393 grown under high osmolarity and used peptide mass fingerprinting and in silico investigation to identify those proteins with altered expression. Among them 15 were assigned to proteins with known functions. Their roles in response to salt stress are discussed.
Introduction Bacteria are able to adapt to a certain range of changes in external osmolarity. One of its adaptations to counterbalance this osmotic difference is accumulation of low molecular weight hydrophilic molecules, which do not interfere with cell metabolism. Furthermore, bacteria take up suitable compounds from their surroundings (da Costa et al. 1998). Bacteria also initiate a program of gene expression in response to osmotic stress by high NaCl concentrations, which are manifested as a set of proteins produced in increased amounts in response to the stress (Volker et al. 1994). There are large numbers of specific proteins reported in various genera of bacteria that showed increase in their level of expression, upon adverse conditions such as heat, salt and nutrient limitations. In a post-genomic era, proteomics is one of the best strategies used to reveal the dynamic expressions of whole proteins in cells and their interactions. The term proteome is used here to describe the complex state of an organism under defined conditions rather than its complete protein repertoire. Due to its high resolution, twodimensional PAGE, combined with high throughput mass spectrometry and bioinformatics, is widely used for protein separation and identification, which is considered sufficiently discriminating to allow the unique identification of unknown proteins (Shen et al. 2002). Identification of differently displayed proteins could be
used to ascertain the genes responding to relative physiological actions, and clarify the functions of genes. Pseudomonas fluorescens MSP-393 is a plant growthpromoting rhizobacterium (PGPR), originally isolated from the saline soils of the coastal agricultural belt of southern coast of India. The strain has been identified as a potential biocontrol agent for bacterial blight disease caused by Xanthomonas oryzae pv. oryzae, in rice (Rangarajan et al. 2003). The bacterium maintained its root colonization and biocontrol potentials even in saline soils. This strain had been shortlisted from a collection of over a thousand isolates of rhizobacteria based on its salt tolerance (Rangarajan et al. 2003). Earlier studies have demonstrated the de novo synthesis of osmolytes by bacteria as a means of osmotolerance (data unpublished). The present study describes the proteins that are induced or repressed in P. fluorescens MSP-393 upon salt stress by using two-dimensional PAGE separation followed by matrix-assisted laser desorption ionization time-of-flight/mass spectrometry (MALDI-TOF/MS) to generate a distinctive peptide mass fingerprint. The whole experiment was done in duplicate and the results verified. Bioinformatics tools were employed to identify the proteins to understand the proteomics of salt stress in the bacterium. To our knowledge this is the first report on study of proteome analysis of a beneficial rhizobacterium, used in agricultural production.
D. Paul et al. Materials and methods Sample Preparation Fifty microlitres of mid-log-phase culture of P. fluorescens MSP-393 was inoculated to two flasks containing 100 ml of salt-minimal medium (MM). Upon midlog-phase growth at 28 °C, solid NaCl was added to one of the flasks to a final concentration of 500 mM in the medium. One hour after addition of NaCl, the cells were pelleted at 7000 rev/min for 10 min, washed three times with isotonic solution and pelleted again. The pellets were suspended in 1 ml of lysis buffer (0.01 M Tris–HCl, pH 7.4, 1 mM EDTA, 8 M urea, 10% glycerol, 0.5% Triton X-100, 6% Ampholytes, 0.05 M DTT and 2 mM PMSF). The cells were sonicated in ice 10 times, 30 s each with a gap of 1 min. The suspension was vortexed well and incubated at )20 °C for 1 h. The crude cell lysate was centrifuged at 13,000 rev/min for 10 min. The soluble proteins in the supernatant were precipitated with two volumes of a mixture of TCA:acetone (1:4) containing 20 nM DTT at )20 °C for 1 h. The pelleted protein was washed three times with 2 ml of acetone– DTT and air-dried. Analysis of cellular protein by two-dimensional PAGE Two-dimensional PAGE was carried out for protein samples obtained from normal and stressed cells in uniform conditions by the method of O’Farrell (1975). The first dimension was carried out using Immobilized Dry Strip Gels, pH 3–10/180 mm (Pharmacia) with the horizontal electrophoresis apparatus Multiphor II (Pharmacia). Rehydration-loading of the sample was performed with 2 mg of protein in 340 ll of rehydration buffer (8 M deionized urea, 2% CHAPS, 0.002% bromothymol blue (BTB), 20 mM DTT, 0.5% carrier ampholytes [Pharmalyte, pH 3–10; Pharmacia]) for 12 h. The isoelectric focusing (IEF) was carried out for 12 h with the following programme (power supply EPS 3500 XL, Pharmacia): Step 1: 500 V/500 V h)1; Step 2: 1000 V/1000 V h)1; Step 3: 3000 V/8000 V h)1; Step 4: 3000 V/32,000 V h)1. Focused IPG strips were equilibrated in equilibration buffer (8 M urea, 30% glycerol, 10% SDS, 0.002% BTB) containing 1% DTT for 15 min and subsequently for 15 min in the same buffer containing 2.5% iodoacetamide prior to loading on to the second dimension. The second dimension was carried out on running gels (12.5% polyacrylamide) in the presence of SDS as described by Laemmli (1970). The strips were embedded on the top of the SDS-gels by using 1% molten agarose in electrophoresis buffer (250 mM glycine, 25 mM Tris, 0.1% SDS). Electrophoresis was carried out at 30 mA per gel. Protein spots were visualized following silver staining and the well resolved and separated spots were numbered. The Mr of individual protein spots was determined by comparison with molecular weight markers. Gels were scanned at 30 dots/in and spot detection was carried out
using the software, Progenesis (Nonlinear Dynamics). The two-dimensional gels of normal and stressed cells were compared using this software and induced and repressed proteins were detected. In-gel proteolytic digestion of resolved proteins Individual proteins were excized from the gel, diced finely and treated with 100 volumes of 15 mM potassium ferricyanide–50 mM sodium thiosulphate for 20 min followed by washing with 100 volumes of double distilled water, four times. The gel pieces were washed with 100 ll of 50 mM ammonium bicarbonate–50% methanol for 20 min and dried in a vacuum centrifuge. The gel pieces were rehydrated in 150 ll reduction solution (10 mM DTT, 100 mM ammonium bicarbonate) for 30 min at 56°C. The reduction solution was discarded and 100 ll of alkylating solution (50 mM iodoacetamide, 100 mM ammonium bicarbonate) and incubated for 30 min in the dark at room temperature. The gel pieces were dehydrated with 100% acetonitrile for 5 min and dried in a vacuum centrifuge. The digestion was carried out by adding 25 ll of trypsin (40 ng/ ll) (Promega) prepared in 50 mM ammonium bicarbonate and incubating overnight at 37°C. The supernatant containing the peptides were taken out in a fresh microfuge tube. The remaining peptides from the gel pieces were extracted using 20 ll, 50% acetonitrile/1% trifluoroacetic acid in water. This was sonicated in a water bath for 25 min and the supernatant was taken out and added to the previously pooled peptide mixtures. MALDI-TOF MS Concentrated peptides were eluted in 3 ll of acetonitrile–0.1% trifluoroacetic acid (1:1) and analysed in MS mode using a Reflex III MALDI-TOF instrument (Bruker Daltonics, Bremen, Germany) to obtain the mass spectrum. Protein identification by peptide mass fingerprinting (PMF) The peptides mass spectrum of each protein was analysed and identified with database matching through http://www.matrixsciences.com. All searches were performed using a mass window between 1 and 100 kDa. The search parameters allowed for oxidation of methionine and carbamidomethylation of cysteine. The percentage similarity of aminoacids, Mr and pI were taken into consideration for identification of the proteins from bacteria of related species/genera.
Results and discussion Current practices for the identification of proteins from two-dimensional gels frequently involve identification of
Spot No.
10i
26i
39i
41i
42i
71i
72i
73i
76r
SI No.
1
2
3
4
5
6
7
8
9
YP_071781.1
S26423
Q89LB1
CAG37678
E87437
G83193
AF3273
NP_249476
Q6FFE7
NCBI Acc No.
Putative ABC iron siderophore transporter, fused permease and ATPase domains – Yersinia pseu dotuberculosis IP 32953
Heat shock protein 60 – Yersinia enterocolitica
60 kDa chaperonin (Protein Cpn60) (groEL protein) – Brady rhizobium japonicum
ABC transporter, ATP – binding protein CC1518 [imported] – Caulobacter crescentus Probable tryptophan synthase, beta subunit – Desulfotalea psy chrophila
Survival protein SurE PA3625 [imported] – Pseudomonas aerugin osa (strain PAO1)
Probable acyl carrier protein phosphodiesterase [Pseudomonas aeruginosaPAO1] Ribosomal protein L11 methyl transferase (EC 2.1.1) [imported] – Brucella melitensis (strain 16M)
Regulatory protein, for nitrogen assimilation P-II 2, for glutamine synthetase – Acinetobacter sp. (strain ADP1).
Functional category
Coverage (%)
13
14
9
10
21
23
24
15
55
a
6.37
4.87
5.12
5.54
6.45
5.25
5
5.8
5.43
pI
65.77
57.75
57.21
43.44
25.98
26.47
23.93
23.03
12.18
Mr
5
7
5
4
3
4
4
3
5
Peptides matched
b
Table 1. P. fluorescens MSP-393 whole cell proteins up- or down-regulated upon osmotic (0.5 M) shock at late logarithmic phase of growth
1–12 3–17 18–38 48–58 91–103 20–37 21–30 4–20 70–82 82–99 161–171 190–202 39–55 128–140 141–154 149–168 46–64 119–139 143–154 38–52 38–52 132–145 251–262 118–139 119–132 123–139 198–210 346–362 2–15 226–242 365–380 444–454 529–550 529–550 532–550 76–93 96–122 103–110 195–213 420–432
Start–end positions
MKLVTAIVKPFK LVTAIVKPFKLDDVR EALSEIGVQGITVTEVKGFGR GAEYVVDFLPK IFVTNLEQVIRIR LAEVFLAAYR ILAVHASPRGERSQSRR RLAEVFLAAYREAHPQAR YILDHPECVAGKR RVLDFASGSGLVAIAAMK LIPWFTKLAER LQQLAIYTVPVTR SGASSSLTLDRPLHPQR LTDNLPTAMHFAR LLVSAHERLAVPPR LAVPPRTVLNVNIPNLPLDR STTLQAILGFVRPSAGRVR AFGTVGLAEEAWDRRVSGFSK QKTAIALAVLRR TDPEFRAELDYYMKK TDPEFRAELDYYMKK CTVYMGKVDVERQK DVQLIGVEPAGR RGLDRGTQAAIAALRAMATPVK GLDRGTQAAIAALR GTQAAIAALRAMATPVK GFLSPYFITDADR LGQIRVEIEKTTSDYDR AAKDVKFGNDARIK KISNIREMLPVLEAVAK LQERVAKLAGGVAVIK VALRAMESPLR DDKGADMGAGGMGGMGGMGGMM DDKGADMGAGGMGGMGGMGGMM GADMGAGGMGGMGGMGGMM VLYFHVAIIGFNAGAKIR LGEHLRRLPMGFFYRSDMSSVNNTLLK LPMGFFYR LNDTLMEYIDGLKELKAYR SYVLNETIANNLR
Peptide sequence of matched fragment
Salt Shock Proteins in Pseudomonas fluorescens
81i
82i
83i
84r
87i
88i
10
11
12
13
14
15
Q9F5M0
AAO58495
G83402
CAE10686
ZP 00196213
NP_773639.1
NCBI Acc No.
Flagellar M-Ring protein – Zymomonas mobilis
Probable Acyl-Coa Dehydrogenase Oxidoreductase Protein [Ralstonia solanacearum GMI1000]
Ribose transport protein RbsA PA1947 [imported] – Pseudomonas aeruginosa (strain PAO1)
Superfamily I DNA and RNA he licases [Geobacter metallireducens GS-15]
Methionyl-tRNA synthetase [Mesorhizobium sp. BNC1].
Flagellar M-ring protein – Bradyrhizobium japonicum
Functional category
8
18
21
13
12
18
Coverage (%)
a
b
Coverage: the percentage aminoacid coverage (peptides observed/theoretical from sequence data). Peptides matched: number of peptides observed in mass spectra contributing to the score.
a
Spot No.
SI No.
Table 1. Continued
6.41
5.8
6.13
5.9
5.3
5.82
pI
66.56
67.90
56.03
77.83
58.12
60.02
Mr
6
6
6
7
4
7
b Peptides matched
107–127 143–158 159–172 177–197 284–295 296–306 369–377 251–263 116–130 101–115 193–210 572–591 602–612 603–613 625–636 614–625 356–368 63–77 482–497 257–277 165–178 214–230 322–342 343–367 450–477 462–484 530–541 269–286 463–481 9–26 519–41 379–399 88–110 603–625 37–40 43–51 119–134 125–134 158–169 599–612
Start–end positions MLLAEKGLPTSANSGYELFDK LRALEGEIARTVQLMK GVKAARVHIVMPVR ATQQPPSASVVLRTDGAIEAR QTNETVYDPEGR AERSVRNVREK LFIAVLVNR WRYWPADVHIIGK GDIYLGGYSGWYSVR HRTASQAIWKAMADR NEIVSFVRSGLRDLSISR IANGLTEDNSNSSAAQAEKK RLCYVGITRAR LCYVGITRARR KYGKLLERQPSR HLTITRCLHRKK TNSQSRAFEEQLR AAGEMKERVRELVGR VKGFYDRIGLEEELYR GADVGNILDFERDYPGCRVVK LLILDEPTAMLTAR IAVLRDGRLVCVEPIER LSLRSPADSVRQGVALITEDR KGEGLLLDQSISANLALGNLPALAR GKALVVVSSDLRELMLICDRIGVLSAGR ELMLICDRIGVLSAGRMVDTFER VTQQLWAAGDPR GTTNCLLNFGEGTQFRPK LNPIHEGTHGIQGLDLLGR RDLSFVLYEWLDVEALT R ALGMAAQSLAKVTQQLWAAGDPR AYVEGALGLNLYCAKLVDEER AGLMAAGQDFERGGMQLPTVVEK TGPQLALLASLDTTTLDMQDAWF VYER DFVQQPAIK EFYKARDLLAQQGLPK DLLAQQGLPK RLRAAREVVLAR AALVVHDMMRSDLK
Peptide sequence of matched fragment
D. Paul et al.
Salt Shock Proteins in Pseudomonas fluorescens proteins on the basis of their homology with those derived from the same or another closely related species or genus for which genomic sequence data are available (Joanna et al. 2001). The present study showed significant changes in the protein patterns in the twodimensional PAGE profiles of salt-stressed and normal cells. Although the entire proteome of MSP-393 was not represented in our two-dimensional maps, 22 distinctly different proteins were found to be regulated (up or down) in P. fluorescens MSP-393, upon salt shock (Table 1). With peptide mass fingerprinting and using bioinformatics tools, function was assigned to 13 induced proteins and two repressed proteins. All of the proteins identified had molecular weights and isoelectric points between 12 and 77 kDa and between 4 and 7 pI, respectively. The functions of seven proteins were not determined as there were no significant matches in the protein database. The majority of proteins identified were homologous to stress proteins in prokaryotes. The protein 10i showed high homology to the enzyme, glutamine synthetase which is required for osmolyte distribution and plays a key role in the synthesis of glutamate, a prominent osmolyte in bacteria (Robinson 1999; Robinson et al. 2001). Our previous studies have shown that this strain synthesized 5 times more glutamic acid upon exposure to 300 mM of NaCl (data unpublished). One of the induced proteins (26i) was found to be acyl carrier protein (ACP), which is reported to be an essential component of enzyme systems for the biosynthesis of membrane components (Anderson & Raetz 1987) while another induced protein, ABC transporter (42i) is reported to form a super family of diverse membrane proteins (Glaasker et al. 1998). ACP has not been reported as a salt-shock protein, but it could be speculated that it is important for osmotolerance, as membrane-components are in contact with the high salt concentrations in the medium. It could also be corroborated with the high root-colonization potential of the strain even in saline soils. Protein 41i is a survival protein (SurE). It is encoded by surE and is essential for stationary-phase survival and for survival of osmotic stress (2.5 M NaCl) in bacteria (Jonathan et al. 1998). Also there was an upregulation of synthesis of ribosomal proteins (39i) and tryptophan synthase (71i) in MSP-393 upon salt shock, the roles of which as salt-shock proteins have been reported (Yale & Bohnert 2001, Schmalisch et al. 2002). A 60 kDa chaperonin was identified that was up-regulated in MSP-393 in response to salt shock. Chaperonins are pivotal in the refolding of denatured proteins (Hartl et al. 1994) and are considered to be important stress proteins. Heat-shock proteins are highly conserved families of proteins, which play essential roles in the folding, unfolding, and transport of proteins within both prokaryotic and eukaryotic cells (Fenton & Horwich 1997). ABC iron siderophores transporter (Protein 76r) has been reported to be a stress-regulated protein (Crosa 1997) and with MSP393, it was repressed, perhaps due to reduced cell
metabolism. Two of the protein spots had homology to the same protein: the M-ring of flagellar assembly has been reported to be a general flagellar chaperone (Tohru et al. 2000), which explains the up-regulation of the gene in MSP-393 upon high osmolarity. The induced protein 82i in P. fluorescens MSP-393 is a methionyl-tRNA synthetase (MetRS), which is reported to be associated with oxidative stress or timing of cell division (Kisseleva et al. 1998). There has been no report of this enzyme activation in prokaryotes upon salt stress. There was an up-regulation of DNA and RNA helicases upon salt shock in MSP-393. RNA helicases have been implicated in enabling bacteria to survive cold-shock and grow at low temperature (Julianne et al. 2000). DNA helicase as salt-shock protein has been reported in plants, though not in prokaryotes (Narendra 2005). Protein 84r which was repressed upon salt shock in P. fluorescens MSP-393, is a ribose transport protein (RbsA). RbsA is involved in active transport of ribose across the cytoplasmic membrane in Escherichia coli (Buckel et al. 1986). The repression of this in MSP-393 indicates the reduced transport of sugars across the membrane upon salt stress. Protein 87i is an acyl-CoA dehydrogenase oxidoreductase protein, which is not only one of the key enzymes of glycometabolism, but is also important for carnitine degradation to c-butyrobetaine. It is known that carnitine and c-butyrobetaine have a structure similar to glycine betaine (Walt & Kahn 2002) and thus are considered important osmoprotectants. The results clearly demonstrate that the expression of proteins from a number of functional categories is modulated in P. fluorescens MSP-393 at high osmolarity. These stress-alleviation proteins must be playing a major role in helping the bacterium to maintain its metabolism unaltered considerably, thus delivering the plant growthpromoting and biocontrol properties in saline soils. Plant growth promotion by rhizobacteria has been reported to be by the production of plant growth regulators, suppression of deleterious organisms and promotion of the availability and uptake of mineral nutrients (Kloepper et al. 1980) and biological control results from production of metabolites, which directly inhibit the pathogen, such as antibiotics, hydrogen cyanide, iron-chelating siderophores, and cell wall-degrading enzymes and also by induction of systemic resistance to the plant (Defago et al. 1990). All this varied complex metabolic machinery in the bacterium needs to be maintained even at high osmolarity in order to maintain these beneficial attributes. It could be ascertained that the osmotolerance mechanisms of MSP-393 viz. de novo synthesis of osmolytes and salt stress proteins effectively nullified the detrimental effects of high osmolarity.
Conclusions Salt tolerance is an inevitable property for a biocontrol bacterium aimed for use in saline agricultural soils.
D. Paul et al. P. fluorescens MSP-393, a potential biocontrol agent against bacterial blight disease of rice is salt tolerant. The study revealed the roles of different salt stress proteins in the bacterium. Research focused on elucidating the molecular mechanisms of salt-tolerance of rhizobacteria and screening salt-tolerant strains is of great importance for development and improvement of agricultural production as the saline areas under agriculture are increasing every year, across the globe.
Acknowledgements The financial aid from the Department of Biotechnology, Government of India and MALDI-TOF facility from Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India are gratefully acknowledged.
References Anderson, M.S. & Raetz, C.R.H. 1987 Biosynthesis of lipid A precursors in Escherichia coli, a cytoplasmic acyltransferase that converts UDP-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. Journal of Biological Chemistry 262, 5159–5169. Buckel, S.D., Bell, A.W., Mohana Rao, J.K. & Hermodson, M.A. 1986 An analysis of the structure of the product of the rbsA gene of Escherichia coli K12. Journal of Biological Chemistry 261, 7659– 7662. Crosa, J.H. 1997 Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria. Microbiology and Molecular Biology Reviews 61, 319–336. da Costa, M.S., Santos, H. & Galinski, E.A. 1998 An overview of the role and diversity of compatible solutes in Bacteria and Archaea. Advances in Biochemical Engineering/Biotechnology 61, 117–53. Defago, G., Berling, C.H., Burger, U., Haas, D., Kahr, G., Keel, C., Voisard, C., Wirthner, P. & Wurthrich, B. 1990 Suppression of black root rot of tobacco and other root diseases by strains of Pseudomonas fluorescens: potential applications and mechanisms. In Biological Control of Soil-Borne Pathogens, ed. Hornby D. (ed.) pp. 93–108, Wallingford, UK: CAB International. 0851986374. Fenton, W.A. & Horwich, A.L. 1997 GroEL-mediated protein folding. Protein Science 6, 743–760. Glaasker, E., Heuberger, E.H.M.L., Konings, W.N. & Poolman, B. 1998 Mechanism of osmotic activation of the quaternary ammonium compound transporter (Qac T) of L. plantarum. Journal of Bacteriology 180, 5540–5546. Hartl, F.U., Hlodan, R. & Langer, T. 1994 Molecular chaperones in protein folding: the art of avoiding sticky situations. Trends in Biochemical Sciences 19, 20–25.
Joanna, C.W., Karen, A., Homer, & Beighton, D. 2001 Altered protein expression of Streptococcus oralis cultured at low pH revealed by two-dimensional gel electrophoresis. Applied and Environmental Microbiology 67, 3396–3405. Jonathan, E.V., Jeffrey, K.I. & Clarke, S. 1998 Mutations in the Escherichia coli surE gene increase isoaspartyl accumulation in a strain lacking the pcm repair methyltransferase but suppress stress-survival phenotypes. FEMS Microbiology Letters 167, 19–25. Julianne, L., Thomas, T. & Cavicchioli, R. 2000 Low temperature regulated DEAD-box RNA helicase from the antarctic archaeon, Methanococcoides burtonii. Journal of Molecular Biology 297, 553– 567. Kisseleva, L.L., Justesenc, J., Alexey, D., Wolfson, Z. & Yu Frolovaa, L. 1998 Diadenosine oligophosphates (ApnA), a novel class of signalling molecules? FEBS Letters 427, 157–163. Kloepper, J.W., Schroth, M.N. & Miller, T.D. 1980 Effect of rhizosphere colonization by plant growth promoting rhizobacteria on potato development and yield. Phytopathology 70, 1078–1082. Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Narendra, T. 2005 Unwinding after high salinity stress: development of salinity tolerant plant without affecting yield. ISB News Report, March 2005. O’Farrell, P.H. 1975 High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250, 4007–4021. Rangarajan, S., Saleena, L.M., Vasudevan, P. & Nair, S. 2003 Biological suppression of rice diseases by Pseudomonas spp. under saline soil conditions. Plant and Soil 251, 73–82. Robinson, P.M. 1999 The enzymology of osmoregulation in archaea: glutamine synthetase and b-glutamine production. PhD Thesis Boston College DAI-B 60/06 p. 2677. Robinson, P., Neelon, K., Schreier, H.J. & Roberts, M.F. 2001 bGlutamate as a substrate for glutamine synthetase. Applied and Environmental Microbiology 67, 4458–4463. Schmalisch, M., Langbein, I. & Stulke, J. 2002 The general stress protein Ctc of Bacillus subtilis is a ribosomal protein. Journal of Molecular Microbiology and Biotechnology 4, 495–501. Shen, S.H., Matsubae, M. & Takao, T. 2002 A proteomic analysis of leaf sheaths from rice. Journal of Biochemistry 132, 613–620. Tohru, M., Chu, R., Yamaguchi, S. & Macnab, R.M. 2000 Role of FliJ in flagellar protein export in Salmonella. Journal of Bacteriology 182, 4207–4215. Volker, U., Engelmann, S., Maul, B., Riethdorf, S., Volker, A., Schmid, R., Mach, H. & Hecker, M. 1994 Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology 140, 741–752. Walt, A. & Kahn, M.L. 2002 The fixA and fixB gene are necessary for anaerobic carnitine reduction in Escherichia coli. Journal of Bacteriology 184, 4044–4047. Yale, J. & Bohnert, H.J. 2001 Transcript expression in Saccharomyces cerevisiae at high salinity. Journal of Biological Chemistry 276, 15996–16007.
