1 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
2 Department of Biotechnology, Thailand Institute of Scientific and Technological Research, Khlong Luang, Pathumthani 12120, Thailand
Correspondence
Kazunobu Matsushita
kazunobu{at}yamaguchi-u.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 AB195173.
Supplementary tables showing the bacterial strains and PCR primers used in this study are available with the online version of this paper.
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INTRODUCTION |
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Acetic acid bacteria are obligately aerobic -Proteobacteria. They are oxidative bacteria that strongly oxidize ethanol to acetic acid and have thus been used for vinegar production. Such species maintain a high state of aeration by producing a pellicle which causes the cells to float on the medium surface in static culture. The pellicle is an assemblage of cells that are tightly associated with CPS on the cell surface. Many species of acetic acid bacteria produce CPS, which seems to be related to pellicle formation. Specifically, Acetobacter xylinum (now called Gluconacetobacter xylinus) is widely known to produce a pellicle consisting of bacterial cellulose and thus it is the most famous model organism for the study of cellulose biosynthesis (Brown et al., 1976
). In addition to ultrastructural and biochemical investigations, the operons involved in cellulose biosynthesis, such as the acs operon (Saxena et al., 1994
) and the bcs operon (Wong et al., 1990
), have already been studied. Thus, the pellicle produced by other Acetobacter or Gluconacetobacter species has been considered to consist of cellulose or cellulose-like material.
We previously reported that Acetobacter aceti IFO 3284 (reclassified as Acetobacter lovaniensis) produces two different types of colony on agar medium, a rough-surfaced colony (R strain) and a smooth-surfaced colony (S strain). The R strain can produce a pellicle polysaccharide which allows it to float on the medium surface in static culture, while the S strain cannot (Matsushita et al., 1992). The R and S strains are interconvertible by spontaneous mutation. The occurrence of such frequent mutation and the formation of two or more different types of colonies after serial transfer is well known in Acetobacter or Gluconacetobacter species, including G. xylinus (Shimwell & Carr, 1964
; Valla & Kjosbakken, 1982
). However, unlike G. xylinus, the R strain of A. lovaniensis produces a novel pellicle polysaccharide, which is a heteropolysaccharide composed of glucose and rhamnose (Moonmangmee et al., 2002a
). Furthermore, Acetobacter tropicalis SKU1100, a thermotolerant strain that can grow and form a thick pellicle in static conditions even at higher temperatures (3740 °C), produces a pellicle polysaccharide consisting of galactose, glucose and rhamnose (Moonmangmee et al., 2002b
).
In this study, we examined the polysaccharide production of A. tropicalis SKU1100 at the molecular level by searching for genes essential to pellicle formation. Since A. tropicalis also produces R and S strains, transposon mutagenesis was performed on an R strain isolated from its original culture. A mutant exhibiting a phenotype similar to the S strain was obtained and the genes involved in pellicle polysaccharide synthesis were isolated and characterized.
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METHODS |
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Isolation of R and S strains of A. tropicalis SKU1100.
A. tropicalis SKU1100 wild-type strain was cultured in potato medium with agitation. The culture was then diluted and spread onto potato agar, where almost all colonies were R-type, and an R-type colony was isolated as the R strain. To isolate the S-type colony, repeated shaking cultures were performed several times by transferring the culture to fresh potato medium every 24 h. The culture was then diluted and spread onto a plate from which an S-type colony was isolated as the S strain.
Comparison of growth behaviour.
The A. tropicalis SKU1100 R strain, the Pel mutant (see below) and the polB mutant (see below) were grown as seed cultures in potato medium for 24 h with agitation. For static culture, 25 µl of the seed culture was inoculated into 5 ml potato medium and cultivated statically at 30 °C. Cells were harvested from a single culture by centrifugation at 11 500 g in a microcentrifuge tube. Cell pellets were dried overnight at 120 °C and dry weight was measured. In the case of the shaking culture, 25 µl of the seed culture was inoculated into 5 ml potato medium and cultivated with agitation at 30 °C. Cell growth was measured as turbidity in Klett units.
DNA manipulation.
The extraction of plasmid and genomic DNA from A. tropicalis strains was performed according to a standard protocol (Sambrook et al., 1989). The extraction of plasmid DNA from E. coli and A. tropicalis was performed with a QIAprep Spin Miniprep Kit (Qiagen), where an incubation step at 37 °C for 30 min with 20 µg lysozyme ml1 was included in the case of A. tropicalis. PCR was carried out in a 25 µl reaction volume (puReTaq Ready-To-Go PCR beads; Amersham Biosciences) using a GeneAmp PCR System 2400 (PerkinElmer). Agarose gel electrophoresis was performed in a 0·8 % agarose gel in TBE buffer (89 mM Tris/HCl,pH 8·0, 89 mM boric acid, 2 mM EDTA) and DNA excised from the gel was purified using a QIAquick Gel Extraction Kit (Qiagen). The PCR primers used in this study are listed in supplementary Table B (available with the online version of this paper).
Transposon mutagenesis.
Random insertion of transposon Tn10 into the DNA of E. coli S17-1 harbouring pSUP2021 was carried out after infection with the transposon vehicle phage NK1323 (Reddy & Gowrishankar, 2000
) as described by Sambrook et al. (1989)
. Tcr colonies were then screened and their plasmids were extracted from Tcr colonies. Mixtures of the plasmids were transformed into E. coli DH5
. Then, a Tcr clone was selected and pSUP2021 containing Tn10, pSUP2021Tn10, was isolated. pSUP2021Tn10 was transformed into an E. coli S17-1 donor strain for conjugal mating and transferred to the A. tropicalis SKU1100 R strain via conjugation. The recipient A. tropicalis strain and E. coli S17-1 harbouring pSUP2021Tn10 were grown to exponential phase (
108 c.f.u. ml1) in potato medium and LB broth, respectively, and then mixed at a ratio of 3 : 2. The mixture was centrifuged for 5 min at 2460 g, and the pellets were resuspended in 100 µl potato medium and put onto a potato agar plate as a single spot. After incubation at 30 °C for 10 h, the cells were suspended in 1 ml distilled water and spread onto YPG agar containing 0·2 % acetic acid and Tc. Tcr colonies appeared after 3 days' incubation at 30 °C. A smooth-surfaced colony was obtained and subsequently used for further experiments.
Southern hybridization.
Chromosomal DNA extracted from the A. tropicalis R strain and its transposon mutants was completely digested with SphI, which does not cut the sequence of Tn10, and separated by agarose gel electrophoresis. Afterwards, the DNA was transferred to a nylon membrane by capillary blotting (Sambrook et al., 1989) and fixed to the membrane by UV irradiation. The 1·1 kb PCR product for Tn10 that was amplified from pSUP2021Tn10 using primers TnL and TnR was used as a hybridization probe. Hybridization was carried out with ECL Direct Labelling and Detection Systems (Amersham Biosciences) according to the manufacturer's manual.
Cloning of the flanking sequence of the Tn10 insertion site.
Transposon flanking regions were cloned by the in vitro cloning method, where PCR is carried out between one primer that anneals to the known sequence (transposon DNA) and a second primer that anneals to the cassette DNA sequence (DNA cassette, LA PCR in vitro Cloning Kit; Takara) ligated to both sides of the DNA fragment and the intervening, unknown sequences are amplified. The chromosomal DNA of the Pel mutant (see below) was digested with BamHI and ligated with a Sau3AI DNA cassette. Approximately 100 ng of the ligated DNA was used as template in each 25 µl PCR reaction with 10 pmol each PCR primer, C1/T1 or C1/T3. C1 and C2 were the primers for the cassette, whereas T1 and T2, and T3 and T4 were designed based on the transposon sequence at the 5' and 3' ends, respectively. The reactions (30 cycles) were carried out using PCR conditions of 94 °C for 30 s, 65 °C for 1 min and 72 °C for 3 min. After completion of the first PCR, each reaction mixture was diluted 100-fold and 1 µl of the diluted solution was then used as template for the second PCR, where primers C2/T2 or C2/T4 were used instead of C1/T1 and C1/T3 used in the first PCR, respectively. Furthermore, to determine the upstream sequence of the BamHI site, a DNA fragment ligated to a SalI DNA cassette was amplified with primers U1/U2 instead of T1/T2, respectively. Also, to determine the sequence downstream from the BamHI site, a DNA fragment ligated to an SphI DNA cassette was amplified with primers D1/D2 instead of T3/T4, respectively. D1 and D2 primers were designed based on the 5'-terminal sequence of the pTL1 insertion, whereas U1 and U2 were designed based on the 3'-terminal sequences of the pTR1 insertion. The PCR products were separated in 0·8 % agarose gel, purified and ligated into pGEM-T Easy Vector (Promega) to create pTR2, pTR1, pTL1 and pTL2 with the primer sets of C2/U2, C2/T2, C2/T4, and C2/D2, respectively (see Fig. 4), and then subjected to sequence analysis.
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Complementation analysis.
The DNA fragment bearing the polE gene (see Fig. 4) was amplified from the A. tropicalis SKU1100 R strain with primers WFr and WRv using PCR conditions (30 cycles) of 94 °C for 30 s, 64 °C for 1 min and 72 °C for 2 min and were then ligated into the pGEM-T vector. A 1·5 kb EcoRI fragment was then cloned into the EcoRI site of a broad-host-range plasmid, pCM62 (Tcr), resulting in pCMPolE. The plasmid was digested with BamHI, blunt-ended and ligated with the 0·9 kb EcoRV fragment of the non-polar Kmr cassette from pTKm (Yoshida et al., 2003
) in the same transcriptional direction as polE to give pCMPolEKm. This plasmid was first transformed into E. coli S17-1 and then incorporated into the Pel mutant via conjugative transfer as described above. The transconjugants were selected on YPG agar containing 0·2 % acetic acid and 75 µg Km ml1. pCMpolE was also transformed into the S strain by conjugation. The transconjugants were then directly selected on YPG agar containing 0·2 % acetic acid and Tc.
Disruption and complementation of polB gene.
To disrupt polB (see Fig. 4), the polB gene region was amplified by PCR from SKU1100 R strain chromosomal DNA using primers PolBFr and PolBRv. PCR cycling was done with the following conditions: 94 °C for 5 min, then 25 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 3 min, followed by 7 min at 72 °C. The resultant 2·5 kb PCR product was ligated into pGEM-T vector to give pTPolB. The plasmid was digested with BamHI, blunt-ended and ligated with the 0·9 kb EcoRV fragment of the non-polar Kmr cassette, as described above. At this step, it was confirmed that the transcriptional direction of both the cassette and the target polB gene was the same. The resultant plasmid, pT
PolB, was digested with EcoRI and the 3·4 kb fragment was used for the disruption of polB. The SKU1100 R strain was electroporated with approximately 0·2 µg DNA fragment and Kmr transformants were selected on YPG medium containing Km. The disruption of the target gene was confirmed by PCR. The disruption mutant obtained was designated
polB. For the complementation of
polB, a 2·5 kb EcoRI fragment of pTPolB was inserted into the EcoRI site of pCM62, to place polB downstream of the lac promoter in the same transcriptional direction to give pCMPolB. The plasmid was then electroporated into the
polB mutant and the transformants were selected on YPG medium containing Km and Tc. The complemented strain was named
polB(pCMpolB) and the incorporation of plasmid was confirmed by agarose gel electrophoresis (data not shown).
Determination of sugar composition.
A. tropicalis strains were cultured in potato medium by shaking (200 r.p.m.) with a rotary shaker at 30 °C for 24 h. The cells were then collected by centrifugation and washed twice with distilled water, whereas the culture supernatants were mixed with 2 vols 2-propanol to precipitate any polysaccharides. Approximately 50 mg of the cells and 20 mg of the crude precipitated polysaccharide were hydrolysed in an aqueous solution of 2 M trifluoroacetic acid (TFA) for 2 h at 120 °C in a glass screw-capped vial. The resultant solutions were subsequently taken to dryness at 40 °C in a vacuum-centrifuged evaporator. The dried products were then dissolved in 1 ml distilled water and evaporated to dryness again. Next the pellets were dissolved in 100 µl distilled water and 2 µl of the suspensions were applied onto a silica gel plate (silica gel60; Merck). After being developed with a solvent system of 1-propanol/distilled water (85 : 15, v/v), the plate was sprayed with 5 % sulfuric acid in ethanol and baked for 10 min at 100 °C to visualize sugar spots.
Other analytical methods.
Scanning electron microscopy (SEM) was performed by using a JEOL JSM6100 scanning electron microscope with the cells prepared as described previously (Moonmangmee et al., 2002a). Sugar content was measured by the phenol/sulfuric acid method (Dubois et al., 1956
) using glucose as standard. Before analysis, A. tropicalis cells were washed twice with distilled water while the culture medium was dialysed against distilled water. Protein content was measured by the modified Lowry method (Dulley & Grieve, 1975
) using bovine serum albumin as a standard.
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RESULTS |
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Furthermore, to ascertain whether the Pel mutation affects both LPS synthesis and pellicle formation, we compared the LPS profiles between the R strain and the Pel mutant by 15 % SDS-PAGE. No difference in the mobility of LPS from both strains was observed (data not shown).
Identification of the transposon-inserted gene and its flanking genes in the Pel mutant
To determine the gene in which the transposon insertion occurred in the chromosome of the Pel mutant, in vitro cloning was performed, as described in Methods. Conceptual translation of the nucleotide sequence revealed the presence of an ORF, designated polE, which encodes a protein consisting of 321 aa. Tn10 was inserted at a position 557 bp from the start codon. Upstream of polE, a promoter-like sequence (35 TTTTCT and 10 TAGAAA) and four other ORFs, designated polABCD, were found; downstream an inverted repeat sequence (5'-AGAAGGAGGAGGCCTCCTTCT-3') which might work as a rho-independent terminator was found. Thus, polABCDE is expected to form an operon (Fig. 4). The deduced amino acid sequences of these genes showed a high level of homology to those of enzymes involved in polysaccharide synthesis, present in various micro-organisms (Table 2
). polABCD showed significant homology to the rfbBACD genes involved in the dTDP-rhamnose synthesis pathway in Gram-negative bacteria (Boels et al., 2004
; Tsukioka et al., 1997
; Mitchison et al., 1997
; Marolda & Valvano, 1995
), which encode the enzymes dTDP-glucose-4,6-dehydratase, glucose-1-phosphate thymidyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and dTDP-4-dehydrorhamnose reductase, respectively. Conversely, polE showed relatively low homology to glycosyltransferases.
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Since the Pel mutant exhibited a phenotype similar to the S strain (the formation of smooth and slimy colonies on agar plates, the absence of pellicle formation in static culture and the secretion of polysaccharides into the culture medium), the S strain may similarly have a mutation in the polE gene. To confirm this, the S strain was conjugated with pCMPolE and all the colonies that appeared on the selective plate displayed a rough surface and grew in static culture by producing a pellicle (data not shown).
polB mutant lacking both CPS and EPS productions
To identify the role of genes upstream of polE, the polB gene, which was expected to encode a glucose-1-phosphate thymidyltransferase, was disrupted with a non-polar Km cassette; this cassette has been reported to create non-polar insertions during gene disruption (Yoshida et al., 2003). Interestingly, the
polB mutant forms colonies which are flat and shiny, but not slimy and smooth on agar medium [Fig. 1A
(e)]. It could not form pellicle under static culture [Fig. 1B
(e)] and there was no amorphous material on the cell surface (Fig. 2c
), indicating that this strain had lost CPS production. Unlike the S or Pel strains, however, the
polB strain exhibited low sugar content in both cells and culture medium (Table 1
), and no EPS, which can be precipitated by alcohol, was detected in the culture medium. In TLC analysis of the acid-hydrolysed sample from
polB cells, no rhamnose spot was detected, while only weak galactose and glucose spots were seen (Fig. 3
). All the phenotypes resulting from polB gene disruption could be recovered with plasmid pCMPolB which contained polB, indicating that polB is involved in CPS production.
Physiological effect of Pel and polB mutation
Since Pel and polB mutants were defective in pellicle formation and CPS production, we compared the growth of these mutants with the R strain in static and shaking cultures (Fig. 5
). The results showed that Pel and
polB mutants, unlike the R strain, could not grow well in static culture, while all strains grew well in shaking culture. The S strain is also unable to grow in static culture, but can grow in shaking culture. Since a pellicle is expected to have the ability to resist some stress, we also compared growth in the presence of acetic acid, a typical stress agent for acetic acid bacteria. As shown in Fig. 5(c)
, both Pel and
polB mutants, as well as the S strain, were more sensitive to acetic acid than the R strain.
polB could not grow well with 0·5 % acetic acid and failed to grow with 1 % acetic acid, while the Pel mutant, similar to the S strain, could grow slightly in the presence of 0·5 % acetic acid but only marginally with 1 % acetic acid.
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DISCUSSION |
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In this study, we investigated genes related to polysaccharide synthesis and pellicle formation in A. tropicalis SKU1100, and also the mechanism by which the R strain loses the ability to produce polysaccharide and is easily converted into the S strain. In A. tropicalis SKU1100, the S strain isolated from the original R strain did not contain CPS attached to the cell, but secreted polysaccharide which had an identical sugar composition as the CPS into the culture medium. This contrasts with the A. lovaniensis IFO3284 S strain (Moonmangmee et al., 2002a) and
polB mutant of A. tropicalis which do not produce any CPS or EPS. Although no growth differences were seen among R strain, S strain, Pel mutant and
polB mutant cells under shaking conditions, the S strain, Pel mutant and
polB mutant demonstrated virtually no growth in static culture, suggesting that CPS production is essential not only for pellicle formation but also for growth of A. tropicalis SKU1100 in static culture. More interestingly, the S strain, Pel mutant and
polB mutant were more sensitive to acetic acid than the R strain, which is more critical for
polB as it produces neither CPS nor EPS. Thus CPS may serve as a barrier, protecting R strain cells from acetic acid stress, reminiscent of a biofilm in relation to antibiotic resistance.
To identify the genes involved in pellicle formation or pellicle polysaccharide synthesis in A. tropicalis, transposon mutagenesis was performed on the R strain. A transposon mutant (Pel) exhibited the same smooth-surface colony morphology and phenotype as the S strain. An ORF, polE, was identified at the transposon insertion site of the Pel mutant. Four other ORFs, polABCD, were found upstream of polE, and the ORFs were tightly linked in the same orientation, implying that polABCDE has an operon structure. polABCD showed significant homology to the rfbBACD genes involved in the dTDP-rhamnose synthesis pathway, which is expected to consist of the following four sequential reactions; glucose 1-phosphate+dTTP is converted to dTDP-glucose (RfbA), then to dTDP-4-keto-6-deoxy-D-glucose (RfbB), then to dTDP-6-deoxy-L-mannose (rfbc) and finally to dtdp-L-rhamnose (RfbD). We found that mutation of polB of A. tropicalis SKU1100 led to completely defective CPS and EPS synthesis. Since the structure of A. tropicalis CPS is expected to have a main chain consisting of rhamnose and many branches with glucose and galactose residues (S. Moonmangmee and others, unpublished), it is reasonable that a defect in dTDP-rhamnose synthesis would eliminate the production of CPS.
On the other hand, PolE exhibited a relatively low level of homology to other glycosyltransferase homologues, compared to PolABCD. However, there are PolE homologues (2546 % identity) among putative glycosytransferases of the -Proteobacteria, close to the genus Acetobacter, and also among putative rhamnosyltransferases from lactic acid bacteria (Table 2
). Furthermore, similar to polE which is located directly downstream of polABCD, these homologous genes, except for the Nitrosomonas enzyme, are also located within the rfbBACD gene cluster. Thus, it is expected that polE encodes a rhamnosyltransferase, which transfers a rhamnosyl residue onto an the oligosaccharide unit being synthesized, working together with the rfbBACD gene products. However, this may not be the case, because the Pel mutant (
polE) still produces EPS with the same sugar composition as the wild-type CPS, although the purified EPS was shown to have a different molecular mass (unpublished data). Despite a relatively low homology, these PolE homologues have four clear consensus motifs: DDGSxD and DQDDxW near the N-terminal region followed by HDWxx and xYRQH present downstream, as shown in Fig. 7
. Although the former two motifs are similar to, but not the same as, domain A (putative catalytic sites) of ExoU and HasA families of
-glycosyltransferases, the latter two consensus motifs downstream of domain A are rather different from domain B (putative substrate-binding sites) of the same families (Keenleyside & Whitfield, 1996
). Thus, it cannot be concluded that PolE homologues are a kind of glycosyltransferase. In addition, PolE has very low homology (14 % identity/49 % similarity) to a functionally identified rhamnosyltransferase (WbbL) of Serratia marcescens (Rubires et al., 1997
), which does not have any PolE consensus motifs. Thus, PolE seems not to work as a rhamnosyltransferase, but rather has some other function. Based on the results presented in this work, it is conceivable that PolE may be involved in the association of CPS to the cell surface in the R strain. In E. coli Group 1 CPS, Wzi is an outer-membrane protein that plays a role (direct or indirect) in surface assembly of the CPS (Whitfield & Paiment, 2003
). The exact role of Wzi is still unknown, but Wzi mutants retain a mucoid phenotype showing a significant reduction in surface-associated CPS and a corresponding increase in medium EPS, which is similar to the case of polE disruption in A. tropicalis SKU1100. However, the amino acid sequence of PolE displays a very low homology to that of Wzi, and the secondary structure of PolE predicted using PSORT (http://psort.nibb.ac.jp) suggests that it has a transmembrane region but is an inner membrane protein. Thus, PolE could have some specific role in CPS formation, but working in a different way to Wzi.
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ACKNOWLEDGEMENTS |
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Received 18 July 2005;
revised 19 September 2005;
accepted 20 September 2005.
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