Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Correspondence
Toshio Omori
aseigyo{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AB062506.
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INTRODUCTION |
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Since bacterial polysaccharides have various rheological properties, they are of industrial interest. Several kinds of bacterial polysaccharides such as acetan (Ojinnaka et al., 1996) and xanthan (Becker et al., 1998
), produced by Acetobacter xylinum and Xanthomonas campestris pv. campestris, respectively, have been used as food additives. In microbial processes, methane and methanol are attractive substrates because they can be supplied in large amounts at a low price. Therefore, EPS-producing methylotrophs also have been studied by several researchers. The component sugars and the optimal conditions for the production of EPSs by these methylotrophs have been well elucidated (Hou et al., 1978
; Wyss & Moreland, 1968
; Chida et al., 1983
). However, little is known about the molecular mechanism of the EPS synthesis by methylotrophs.
We have been studying the synthesis of EPS, designated as methanolan, by the obligate methylotroph Methylobacillus sp. strain 12S. Methanolan is a heteropolymer composed of glucosyl, galactosyl and mannosyl residues in the molar ratio 3 : 1 : 1 (Yoshida et al., 2000). In a previous study, we mutagenized strain 12S by Tn5 to produce mono- and/or oligosaccharide by single-step fermentation from methanol (Yoshida et al., 2000
). Eleven EPS-deficient (EPS-) mutants were obtained and three of them were found to accumulate significant amounts of glucose, erythrose, threose and a disaccharide-like compound in the medium. Among the mutants, strain Ma1 shows a stable deficiency in EPS production, although it cannot accumulate the mono- and/or oligosaccharide in the medium. Since strain Ma1 carries a single insertion of Tn5 in the genome, it is possible that Tn5 disrupts a primary gene for methanolan synthesis. Therefore, as the first step to reveal the molecular basis of methanolan synthesis by strain 12S, the flanking genes of the Tn5 insertion in the chromosome of strain Ma1 were isolated and characterized.
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METHODS |
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Measurement of enzymic activity of EpsQ, EpsS and EpsT.
For the amplification of epsQ, we used primers EpsQFr and EpsQRv (Table 2), which were identical to the sequence located 135115 bp upstream of the start codon, and complementary to the sequence located 323 bp downstream of the stop codon of epsQ, respectively. For the amplification of epsS, we used primers EpsSFr and EpsSRv, which were identical to the sequence located 3617 bp upstream of the start codon, and complementary to the sequence located 5978 bp downstream of the stop codon of epsS, respectively. For the amplification of epsT, we used primers EpsTFr and EpsTRv, which were identical to the sequence located 4627 bp upstream of the start codon, and complementary to the sequence located 2746 bp downstream of the stop codon of epsT, respectively. PCR cycling was done with the following conditions: 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 53 °C for 1 min and 72 °C for 1·5 min, followed by 10 min at 72 °C and holding at 4 °C. Each PCR product encoding epsQ, epsS and epsT was ligated into pT7Blue T-vector to give pTepsQ, pTepsS and pTepsT, respectively. Transformants of E. coli JM109 harbouring each plasmid were cultured for 10 h at 30 °C in 5 ml 2x YT medium supplemented with Ap and then transferred into 100 ml of the same fresh medium. After incubation for 3 h at 25 °C, 0·5 mM IPTG was added and further incubated for 3 h at 25 °C. The cells were harvested and washed twice with the same buffer as that used for each enzyme assay. For the enzyme assays of EpsQ, EpsS and EpsT, we used 50 mM Tris/HCl (pH 7·6), 10 mM Tris/HCl (pH 8·7) and 50 mM Tris/HCl (pH 8·0), respectively. The cell pellets were finally resuspended in 5 ml of the same buffer and lysed by sonication. After the cell debris was removed by centrifugation at 10 000 g for 10 min at 4 °C, the supernatant was used as a crude extract. Activities of GDP-mannose pyrophosphorylase (EC 2 . 7 . 7 . 13, GMPP) and UDP-galactose 4-epimerase (EC 5 . 1 . 3 . 2, UGE) in the crude extract were measured as described by Ohta et al. (2000)
and Moreno et al. (1981)
, respectively. Activity of UDP-glucose pyrophosphorylase (EC 2 . 7 . 7 . 9, UGP) was assayed by the method described by Koo et al. (2000)
with the following modifications. The assay was carried out in 500 µl reaction mixture containing 50 mM Tris/HCl (pH 8·0), 10 mM MgCl2, 2 mM UDP-glucose, 5 mM pyrophosphate (PPi), 20 µM glucose 1,6-bisphosphate, 0·3 mM NADP+, 10 mM NaF, 1 U phosphoglucomutase, 1 U glucose-6-phosphate dehydrogenase and 5 µl crude extract. The reaction was started by adding PPi and the absorbance was monitored at 340 nm, at 30 °C for 120 s. The protein concentration was determined using Protein Assay Kit II (Bio-Rad) with BSA as a standard.
Construction of a nonpolar Kmr cassette.
To disrupt the genes by the insertion of a drug-resistance cassette, which would allow the constitutive transcription of the downstream genes, a nonpolar Kmr cassette was constructed. pSUP5011 was used as a template DNA to amplify a DNA fragment encoding a Kmr gene with its promoter sequence but without the transcriptional terminal sequence by PCR. The KmFr primer (Table 2) was identical to the sequence located in the upstream region 141128 bp from the start codon of the Kmr gene. Primer KmRv (Table 2
) was complementary to the 14 bp sequence including the stop codon of the Kmr gene at the end. Both primers were designed to generate an EcoRV site at both ends of the PCR product. PCR cycling was done with the following conditions: 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 3 min, followed by 10 min at 72 °C and holding at 4 °C. The resultant 0·9 kb PCR product was ligated into pT7Blue T-vector to give pTKm, and the 0·9 kb EcoRV fragment was used as a Kmr cassette. To confirm that this cassette enhanced the transcription of downstream genes, we ligated the cassette into SmaI-digested pBBR1MCS3 to give pBBRMCS3K. The 1·4 kb XbaISacI fragment of pTepsQ, encoding epsQ, was further ligated downstream of the Kmr cassette of pBBRMCS3K in the same transcriptional direction, to give pBepsQ. A strain 12S derivative was transformed by the plasmid by electroporation according to the method described previously (Yoshida et al., 2001
). Transformants resistant to Km and tetracycline were selected. The GMPP activity of the crude extract obtained from the transformant was determined as described above.
Disruption of eps genes with the nonpolar Kmr cassette.
The plasmids used for gene disruption were constructed as described below, and its physical map is shown in Fig. 1 (b). For the disruption of epsA, pUMS12 was digested by EcoRV and ligated with the nonpolar Kmr cassette. At this step, it was confirmed that the transcriptional direction of both the cassette and each target eps gene were the same. The obtained plasmid was further digested by EcoRI and ligated with 1·9 kb EcoRISalI and 1·0 kb SalIEcoRI fragments of pUMS11 and pBMS21, respectively. The direction of the gene fragments was confirmed to be same as that of the native wild-type genome by restriction enzyme digestion. The resultant plasmid was designated as pMm
A and used for the disruption of epsA. For the disruption of epsB, pUMS11 was digested by EcoRV and ligated with the cassette. Then, the 2·7 kb EcoRIXbaI fragment of the resultant Kmr plasmid was ligated to a 1·4 kb KpnIEcoRI fragment of pUMS12 and KpnIXbaI digested pUC19 to give pMm
B. For the disruption of epsC, pBMS21 was digested by XhoI/KpnI, blunt-ended, and then self-ligated to delete the DraII site from the multi-cloning site of the vector. This plasmid was further digested by DraII, blunt-ended and then ligated with the cassette. The 1·9 kb SalIEcoRI fragment of the plasmid was ligated to the 3·7 kb EcoRISalI fragment of pUMS22 and SalI-digested pUC19 to give pMm
C. For the disruption of epsK, epsD and epsE, pBMS16 was digested by NdeI, StuI and NruI, respectively, blunt-ended, and then ligated with the cassette to give pMm
K, pMm
D and pMm
E, respectively. For the disruption of epsF, pBMS622SalSac was digested by PmaCI and ligated with the cassette to give pMm
F. For the disruption of epsG and epsI, pBMS622 was digested by BglII/NdeI, blunt-ended, and then ligated with the cassette to give pMm
G and pMm
I, respectively. For the disruption of epsH, pBMS622PH was digested by EcoRI, blunt-ended and ligated with the cassette to give pMm
H. For the disruption of epsJ, pUMS62PSm was digested by EcoRV and ligated with the cassette to give pMm
J. For the disruption of epsN, pBMS64BEV was digested by SmaI and ligated with the cassette to give pMm
N. For the disruption of epsO, pUMS63 was digested by EcoRV and ligated with the cassette to give pMm
O. For the disruption of epsP, pUMS63 was digested by PmaCI and ligated with the cassette to give pMm
P. For the disruption of epsR, pBMS7Sal was digested by NdeI and ligated with the cassette to give pMm
R.
Strain 12S was transformed by these plasmids by electroporation, and then the Kmr transformants were selected. The Kmr and Ap-sensitive (Aps) transformants were further selected to obtain mutants in which the intact eps gene was replaced with the corresponding gene disrupted by the nonpolar Kmr cassette as a result of a double crossover. The disruption of the target gene was confirmed by Southern hybridization using the Kmr gene, vector gene and the target eps genes as probes.
Construction of transformants in which epsA is expressed constitutively.
pUMS1 was used as a template to amplify a gene fragment encoding epsA and its native ribosome-binding site by PCR. We used the primers EpsAFr and EpsARv, which were identical to the sequence located 6443 bp upstream of the start codon, and complementary to the sequence located 422 bp downstream from the stop codon of epsA, respectively. PCR cycling was done with the following conditions: 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 50 °C for 30 s and 70 °C for 60 s, followed by 10 min at 72 °C and holding at 4 °C. The resultant 0·9 kb PCR product was ligated into pT7Blue T-vector to give pTepsA. Then, the 0·9 kb HincIISmaI fragment of the plasmid was inserted into the DraI site of pBBR122 to place epsA downstream of the constitutive promoter of the chloramphenicol-resistance gene in the same transcriptional direction. Then the resultant plasmid, designated pBepsA, was introduced into strain 12S by electroporation.
Measurement of EPS production by strain 12S and its derivatives.
Strain 12S, specific eps gene-disrupted mutants and the transformant were grown in 10 ml MMe medium at 30 °C as described above. After 7 days cultivation, the cells were removed by centrifugation at 8000 g for 20 min at 4 °C. The total amount of hexose in the supernatant solution was determined by the phenol/sulfuric acid method as described by Dubois et al. (1956) using glucose as a standard.
Determination of glycosyltransferase activity.
Strain 12S and epsB-disrupted mutant MmB were grown in 5 ml MMe medium overnight and then diluted 100-fold in 100 ml fresh MMe medium. After incubation until mid-exponential phase (OD600 0·61·0), the cells were harvested by centrifugation at 8000 g for 15 min at 4 °C, resuspended in 10 ml buffer A [1·2 M sucrose, 50 mM Tris/HCl (pH 8·0), 1 mM EDTA, 20 mg lysozyme] and incubated for 5 min at 25 °C. Spheroplasts were collected by centrifugation at 8000 g for 15 min at 4 °C, resuspended in 20 ml buffer B [20 mM Tris/HCl (pH 8·0), 1 mM EDTA, 200 µM PMSF] and sonicated for 2 min. Debris was removed by centrifugation at 8000 g for 20 min at 4 °C and then membranes in the collected supernatant solution were obtained by recentrifugation at 30 000 g for 1 h at 0 °C. Membranes were washed once in 20 ml buffer C [50 mM Tris/acetate (pH 8·3), 1 mM EDTA, 200 µM PMSF], recentrifuged and finally resuspended in 0·5 ml buffer C. The glycosyltransferase activities of the membrane fractions were measured as described by Kolkman et al. (1996)
with some modifications. The activity was measured by adding 10 mM MgCl2 and 1·5 µM UDP-D-[U-14C]glucose (1110 MBq mmol-1; NEN Life Science), UDP-D-[U-14C]galactose (1110 MBq mmol-1; Amersham Pharmacia Biotech) or GDP-D-[U-14C]mannose (1110 MBq mmol-1; Amersham Pharmacia Biotech) to membrane samples in a final volume of 50 µl. The concentration of protein was approximately 1·4 mg ml-1. After 1 h incubation at 12 °C, the reaction was stopped by addition of 1 ml chloroform/methanol (2 : 1). Then the solution was extracted three times with 0·2 ml PSUP (1·5 ml chloroform, 25 ml methanol, 23·5 ml water and 0·183 g KCl) to remove the unreacted substrates. The incorporation of 14C-labelled glycolipid into the organic phase was measured with a scintillation counter (Aloka). The results are presented as the means of three independent assays ±SD.
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RESULTS |
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ORF9 and ORF10 contain putative transmembrane regions and a cytoplasmic ATP-binding domain, respectively, and display homology to a large group of proteins that regulate the chain length of EPS, CPS and LPS O antigen, such as ExoP from Sinorhizobium meliloti and Wzc from E. coli (Gonzalez et al., 1998; Vincent et al., 1999
). The homologues of Gram-negative bacteria are mostly a single peptide, though those of Gram-positive bacteria are separated into two polypeptides (Morona et al., 2000
) as in the case of ORF9 and ORF10 of strain 12S. Since mutation of exoP results in the synthesis of low but not high molecular mass succinoglycan, as well as an increase in free repeating units in the medium (Becker et al., 1995
), the homologues are proposed to play a role in high-level polymerization of bacterial polysaccharide.
The functions of ORFs L, 11, 12 and 14 could not be predicted on the basis of the homologies. However, ORF11 and ORF14 contained more than eight transmembrane regions, suggesting that they are probably located at the cytoplasmic membrane, possibly as a complex. Wzx and Wzy also contain more than nine transmembrane regions, and they participate in the Wzy-dependent system as a flippase to transfer the assembled lipid-linked repeating units across the cytoplasmic membrane and polymerase to ligate the repeating units to the nascent polysaccharide in the periplasm. Furthermore, Wzx and Wzy homologues isolated from many CPS- and LPS-producing bacteria have little primary sequence similarity, but have similar hydropathy profiles (Drummelsmith & Whitfield, 1999; Morona et al., 1994
). Unambiguous differentiation of Wzy and Wzx homologues is not feasible based on the sequence similarities, however comparison of the hydropathy profiles leads us to predict that ORF11 and ORF14 participate in methanolan biosynthesis as either the flippase or polymerase.
The location of ORFL and ORF12 in the periplasmic space or outer membrane was also predicted on the basis of the existence of a putative cleavable signal sequence in the N terminus, but no other hydrophobic region. This location indicated their possible role as export proteins in methanolan synthesis, similar to ORF8.
NDP-hexose pyrophosphorylase- and NDP-hexose epimerase-genes/ORFs 18, 20 and 21
The entire putative amino acid sequence of ORFs 18, 20 and 21 displayed high homology along their entire length with known GMPP, UGE and UGP, respectively (Table 3). Since methanolan is composed of mannose, galactose and glucose, these proteins probably participate in the activation of these sugars.
Transcriptional regulator-like genes/ORF0 and ORFK
ORF0 displays a characteristic feature of bacterial transcriptional regulators belonging to the LuxR superfamily (Wehland & Bernhard, 2000). Although the predicted sequence of the N-terminal region showed no relation to reported proteins, significant homology was found in the C-terminal region corresponding to the DNA-binding domain (helixturnhelix motif) conserved among the LuxR superfamily (Velasco et al., 1998
) (Fig. 3
). By contrast, ORFK did not contain the DNA-binding motif, though the N-terminal region contained the cAMP-binding domain specific for the regulatory proteins involved in the transcription of catabolite genes, such as catabolite repression protein (CRP) of Klebsiella pneumoniae (Meyer et al., 2001
).
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Others/ORFs 3, 4 and 23
ORF3 and ORF4 were found downstream of region I in the opposite direction of transcription from epsA to epsC. Although ORF3 did not show homology with any reported proteins, over the entire length of the predicted protein sequence ORF4 showed strong similarity with proteins belonging to the inositol monophosphatase (IMPase) family. Metal- and phosphate-binding motifs conserved among diverse prokaryotic and eukaryotic IMPases were also found in ORF4 (data not shown). Since the IMPase that catalyses the hydrolysis of the ester bound in myo-inositol 1- (or 4)-phosphate has never been reported to participate in the synthesis of polysaccharide, ORF4 is probably not involved in EPS synthesis. The putative sequence of ORF23 displayed high homology with the DNA mismatch repair protein MutS, indicating that this ORF is not involved in EPS synthesis.
Based on the features described above, ORFs 0, 1, 2, K, L, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 were designated epsA, epsB, epsC, epsK, epsL, epsD, epsE, epsF, epsG, epsH, epsI, epsJ, epsM, epsN, epsO, epsP, epsQ, epsR, epsS, epsT and epsU, respectively (Fig. 1a).
Enzymic activities of EpsQ, EpsS and EpsT expressed in E. coli
To identify the enzymic activity of EpsQ, EpsS and EpsT, each gene cloned in an expression vector was introduced into E. coli JM109, and the activities of GMPP, UGE and UGP were compared to that of E. coli harbouring the pT7Blue T-vector control. We observed 5·3, 5·0 and 3·6 times higher activity of GMPP, UGE and UGP in the transformants harbouring pTepsQ [2·3x102±9·7 nmol min-1 (ng protein)-1], pTepsS [2·0x102±27 nmol min-1 (ng protein)-1] and pTepsT [2·2x102±9·4 nmol min-1 (ng protein)-1], respectively (data given are the means±SD of two independent determinations), revealing that epsQ, epsS and epsT encode GMPP, UGE and UGP, respectively. Since methanolan is composed of glucose, galactose and mannose (Yoshida et al., 2000), EpsQ, EpsS and EpsT were probably responsible for the synthesis of GDP-mannose, UDP-galactose and UDP-glucose, respectively.
Gene disruption analysis using the nonpolar Kmr cassette
To confirm that the nonpolar Kmr cassette constructed in this study had no polar effects on the downstream genes, pBepsQ, which encodes epsQ downstream of the cassette, was made. The GMPP activity of the crude extract prepared from the transformant harbouring pBepsQ was 13·3 times higher [2·0x102±0·9 nmol min-1 (ng protein)-1] than that of the wild-type strain harbouring pBBRMCS3K [1·5x10±5·0 nmol min-1 (ng protein)-1] (data given are the means±SD of two independent determinations), revealing that the cassette enhances the transcription of downstream gene. Therefore, single genes were disrupted by inserting the nonpolar Kmr cassette into the epsABCKDEFGHIJNOP and epsR genes, and methanolan synthesis was assayed. epsM and epsL disruptants could not be obtained, even though the experiments were repeated several times. Fig. 4 shows the amount of EPS produced by the wild-type strain and the specific mutants. Mm
B, Mm
C, Mm
E, Mm
F, Mm
G, Mm
J, Mm
N, Mm
O, Mm
P and Mm
R not only showed an altered non-mucoid morphology, but also decreased EPS production to less than 19 % of the wild-type strain. The amount of EPS produced by these mutants was as small as that by most EPS- mutants constructed by Tn5 insertion (Yoshida et al., 2000
), indicating that the sugars detected in spent medium might come from cell debris. Therefore, it was confirmed that epsBCEFGJNOP and epsR are essential for the synthesis of methanolan. By contrast, the disruption of epsK, epsD and epsH did not significantly alter the ability to produce EPS, suggesting that these genes are dispensable for methanolan synthesis. On the other hand, Mm
A and Mm
I showed a moderate phenotype among the mutants and still produced half the amount of EPS in comparison to the wild-type strain, suggesting that EpsA and EpsI participate in methanolan synthesis, but are not indispensable.
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Glycosyltransferase activity of EpsB
Since the putative location at the inner membrane and the sequence homology strongly indicated that EpsB might participate in the first transfer of a sugar residue in methanolan synthesis, we compared membrane proteins of strain 12S and epsB-disrupted mutant MmB for their ability to incorporate UDP-[14C]glucose, GDP-[14C]mannose and UDP-[14C]galactose into the lipid carrier. A significant amount of glucose [1400±8 c.p.m. (mg membrane protein)-1] and a relatively low amount of galactose [77±8 c.p.m. (mg membrane protein)-1], were incorporated into the membranes prepared from the wild-type strain. No incorporation of mannose was detected. The reason why galactose was incorporated into the lipid carrier may be that UDP-[14C]galactose could be transformed to UDP-[14C]glucose by UGE present in strain 12S. Therefore, we conclude that the initial sugar transferred onto the phosphoryl lipid carrier is most likely to be glucose. Membranes prepared from the epsB mutant Mm
B incorporated a negligible amount of UDP-glucose [2±2 c.p.m. (mg membrane protein)-1] and no incorporation of GDP-galactose and GDP-mannose was observed. These results strongly suggest that EpsB is an initial glucosyltransferase.
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DISCUSSION |
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In the Wzy-dependent system, the repeating unit constructed onto the lipid carrier in the cytoplasm is probably transported to the periplasm by flippase, Wzx, and then added to the nascent polysaccharide by polymerase, Wzy (Whitfield, 1995). EpsM and EpsH are proposed to play similar roles to Wzy and Wzx, since their hydropathy profiles are similar to these proteins (Drummelsmith & Whitfield, 1999
). Though the transport of polysaccharide across the outer membrane is reportedly catalysed by Wza in this system, how a polysaccharide is exported through the periplasm is not well defined (Drummelsmith & Whitfield, 2000
). EpsI, which is probably located in the periplasm, is proposed to participate in this export process, although disruption of epsI did not cause a complete loss of EPS production. EpsL is also proposed to be a transporter located at the periplasm or outer membrane. However, its participation in methanolan synthesis is not clear because a specific mutant could not be obtained. To elucidate whether and how EpsEI and EpsL export the nascent methanolan outside of the cells, the location of these proteins should be determined in a further study.
EpsQS and EpsT are possibly enzymes involved in the synthesis of activated sugars. EpsQ and EpsT appear to synthesize GDP-mannose and UDP-glucose via a condensation of corresponding hexose 1-phosphate and NTP, respectively. EpsS is predicted to epimerize UDP-glucose to UDP-galactose.
EpsC appears to be required for methanolan synthesis on the basis of gene disruption. However, epsC homologues are rarely found in the CPS synthesis gene cluster (Llull et al., 1998) and the function of the product has not been determined. Therefore a function of EpsC could not be proposed based on its sequence. Further research is needed to clarify the role of EpsC in methanolan synthesis.
EpsU showed homology with proteins involved in the degradation of cell walls, indicating that it could degrade methanolan. A polysaccharide-degrading enzyme is often encoded in EPS biosynthesis gene clusters, as in the case of Pseudomonas aeruginosa (Monday & Schiller, 1996). However, in preliminary experiments, we could not detect activity of EpsU expressed in E. coli to degrade methanolan (data not shown). Further research is needed to clarify the function of EpsU.
It was unexpected to find epsD, which contains a motif specific for various PPIs, in the methanolan biosynthesis gene cluster. PPI catalyses the folding of proteins which are located in or exported through the periplasm, via the rotation around the prolyl peptidyl bond. One of the best-studied PPIs, SurA, reportedly suppresses a htrM (rfaD)-null mutant that is deficient in LPS production (Missiakas et al., 1996). The restoration of LPS production by overexpression of SurA appears to be associated with its ability to catalyse the folding of outer-membrane proteins immediately after translocation across the cytoplasmic membrane. Since many proteins located outside of the cytoplasmic membrane are involved in EPS synthesis as transporters or polymerases, it is not surprising that a folding catalyst is encoded in the same cluster.
EpsA contained the consensus sequence of the LuxR superfamily, and showed homology with RcsA homologues, which are frequently observed among the EPS- and CPS-producing Gram-negative bacteria such as K. pneumoniae and E. amylovora (Allen et al., 1987; Bugert & Geider, 1995
). The rcs system including RcsA as the positive transcriptional regulator has been elucidated as the regulatory system for the colanic acid synthesis in E. coli K-12 (Gottesman & Stout, 1991
). This system has two positive regulators (RcsA and RcsB) and two negative regulators (RcsC and Lon). RcsB and RcsC are proposed to be the effector and sensor, respectively, of a two-component regulatory system. RcsA is an auxiliary factor that may interact with activated RcsB to form a heterodimer required for increased transcription of the cps genes. In addition, the homodimer of RcsB also can promote the transcription of the cps genes at a low level. Since the disruption of epsA did not result in the complete loss of the ability to produce EPS, it is likely that unknown proteins also regulate methanolan synthesis independently of EpsA, like RcsB in the rcs system. Since the constitutive expression of epsA in strain 12S also increased methanolan production, it is strongly suggested that EpsA acts as a positive regulator for the transcription of eps genes in the same manner as the rcs system.
EpsK is probably involved in the regulation of EPS synthesis, because a predicted cAMP-binding domain, which is conserved among various regulatory proteins of prokaryotes and eukaryotes, was found at the N terminus. The cAMP-receptor proteins found in prokaryotes are known to regulate the transcription of specific catabolite genes (Meyer et al., 2001). These proteins conserve not only the cAMP-binding domain, but also a helixturnhelix motif to bind to DNA. Since EpsK contains only the cAMP-binding domain, it is unlikely that EpsK has the same function as these homologues. However, because cAMP is a well-known effector in maintaining the catabolite balance in prokaryotes, it is possible that the carbon flux for the synthesis of methanolan is also regulated by the cAMP-dependent network, in which EpsK participates as one of the components of the regulatory unit.
In methylotrophs, C1 compounds are oxidized to formaldehyde, which is further oxidized to carbon dioxide or assimilated (Attwood & Quayle, 1984). Furthermore, it is probable that C1 compounds penetrate the cell membrane of bacteria because of their small size. Therefore, when methylotrophs grow in the presence of excess amounts of C1 compounds, it may be necessary to prevent formaldehyde accumulation by controlling the balance between the production and utilization of this toxic metabolite (Attwood & Quayle, 1984
). One reason why a large number of methylotrophs are able to produce EPS might be that EPS synthesis is an efficient way of converting formaldehyde into non-toxic compounds (Linton et al., 1986
; Southgate & Goodwin, 1989
). This study indicates that methanolan synthesis by strain 12S might be regulated through an rcs-like system. Further investigation of the regulation system of EPS synthesis in strain 12S is needed to clarify the relation between C1 compound metabolism and EPS synthesis.
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Received 29 July 2002;
revised 23 September 2002;
accepted 22 October 2002.
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