Novel glycosyltransferase genes involved in the acetan biosynthesis of Acetobacter xylinum

Novel aceQ and aceR genes involved in the acetan biosynthesis of Acetobacter xylinum were newly isolated. The homology search with DNA Data Bank of Japan indicated that aceQ and aceR were glycosyltransferases. Their gene-disrupted mutants were obtained by homologous recombination using the tetracycline resistance gene and the electroporation method. By NMR and ESI-MS analyses, aceQ-disrupted mutant DQ was found to secrete a water-soluble polysaccharide harboring the -Man-GlcUA side chain and the aceR-disrupted mutant DR was found to secrete an acetan analog, lacking the terminal Rha residue. These results suggested that aceQ and aceR encode a glucosyltransferase and a rhamnosyltransferase, respectively. It was indicated that acetan analogs harboring various side chains can be generated easily by genetic engineering.     

Role of water-soluble polysaccharides in bacterial cellulose production

Acetobacter xylinum BPR2001 produces water-insoluble bacterial cellulose (BC) and a water-soluble polysaccharide called acetan in corn steep liquor-fructose medium. Acetobacter xylinum EP1, which is incapable of acetan production was derived by disrupting the aceA gene of BPR2001. The BC production by EP1 (2.88 g/L) was lower than that by BPR2001 (4.6 g/L) in baffled-flask culture. When purified acetan or agar was added to the medium from the start of cultivation, the BC production by EP1 was enhanced and the final BC yield of EP1 was almost the same as that of BPR2001. A similar improvement of BC production by EP1 by the addition of agar was also confirmed by cultivation in a 50-L airlift reactor. From these results, the role of acetan in BC production is associated with the increase in the viscosity of the culture medium which may hinder coagulation of BC and cells in the culture, thereby accelerating the growth of BPR2001 and BC production by BPR2001.     

Effects of acetan on production of bacterial cellulose by Acetobacter xylinum

Acetan is a water-soluble polysaccharide produced by a bacterial cellulose (BC) producer, Acetobacter xylinum. An acetan-nonproducing mutant, EP1, was generated from wild-type A. xylinum BPR2001 by the disruption of aceA, which may act to catalyze the first step of the acetan biosynthetic pathway in this bacterium. EP1 produced less BC than the wild-type strain. However, when EP1 was cultured in a medium containing acetan, BC production was stimulated and the final yield of BC was equivalent to that of BPR2001. The culture broth containing acetan was more viscous and the free cell number was higher than that of the broth without the polysaccharide, so acetan may hinder the coagulation of BC in the broth. The addition of 1.5 g/l agar also increased BC production; we concluded that acetan and BC syntheses were not directly related on the genetic level.     

Isolation and nucleotide sequence of the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase gene from Acetobacter xylinum.

A genetic locus from Acetobacter xylinum involved in acetan polysaccharide synthesis has been characterized. The chromosomal region was identified by screening a genomic library of A. xylinum in a Xanthomonas campestris mutant defective in xanthan polysaccharide synthesis. The A. xylinum cosmid clone can functionally complement a xanthan-negative mutant. The polymer produced by the recombinant strain was found to be indistinguishable from xanthan. Insertion mutagenesis and subcloning of the cosmid clone combined with complementation studies allowed the identification of a 2.3-kb fragment of A. xylinum chromosomal DNA. The nucleotide sequence of this fragment was analyzed and found to contain an open reading frame (aceA) of 1,182 bp encoding a protein of 43.2 kDa. Results from biochemical and genetic analyses strongly suggest that the aceA gene encodes the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase enzyme, which is responsible for the transfer of an alpha-mannosyl residue from GDP-Man to cellobiosyl-diphosphopolyprenol. A search for similarities with other known mannosyltransferases revealed that all bacterial alpha-mannosyltransferases have a short COOH-terminal amino acid sequence in common.     

Structure and conformation of a novel genetically engineered polysaccharide P2

A new exocellular polysaccharide (P2) has been produced by the manipulation of a glycosyl transferase gene (aceP) involved in the biosynthesis of the polysaccharide acetan by the bacterium Acetobacter xylinum strain CKE5. The P2 polysaccharide has been studied by methylation analysis, reductive cleavage, and 1H and 13C NMR spectroscopy. The data are consistent with the structure predicted when the aceP gene is deactivated: [Molecular structure: see text]. The effect of cooling on proton NMR line width indicates a coil-helix transition in P2 at about 70 degrees C.     

Synergistic interactions between the genetically modified bacterial polysaccharide P2 and carob or konjac mannan

Rheological studies have confirmed that the bacterial polysaccharide P2, a genetically modified variant of the Acetobacter xylinum polysaccharide acetan, undergoes synergistic gelation with either of the plant polysaccharides carob or konjac mannan. X-ray fibre diffraction data shows that P2 can form a 5-fold helical structure of pitch 4.7nm and an axial rise per disaccharide repeat of 0.92nm. Optical rotation data demonstrate that P2 undergoes a coil-helix transition in solution and that deacylation enhances the stability of the helical structure in solution. Studies made on mixtures prepared at different temperatures and ionic strengths suggest that denaturation of the P2 helix favours interaction and gelation. Deacetylation of P2 enhances gelation. X-ray diffraction data for oriented fibres prepared from deacetylated P2-konjac mannan mixed films reveal a 6-fold helical structure of pitch 5.54nm with an axial rise per disaccharide repeat also of 0.92nm. This mixed helix provides direct evidence for binding between the two polysaccharides. P2 contains two sites of acetylation: one on the backbone and one on the sidechain. The former site of acetylation inhibits helix formation for P2. It is suggested that this site of acetylation also inhibits formation of the mixed helix, explaining the enhanced gelation of mixtures on deacetylation.     

Identification, cloning and sequencing the aceA gene involved in acetan biosynthesis in Acetobacter xylinum

Abstract The aceA gene from Acetobacter xylinum was identified and cloned from a genomic DNA library. The complete DNA sequence was determined and computer analysis of the translated gene sequence revealed homology with the deduced amino acid sequence of gumD from Xanthomonas campestris . Therefore aceA is likely to encode the phosphate-prenyl glucose I -phosphate transferase catalyzing the first step in acetan biosynthesis in A. xylinum .     

Observation of the helical structure of the bacterial polysaccharide acetan by atomic force microscopy.

A method has been developed that has been found to give reproducible images of uncoated polysaccharides by Atomic Force Microscopy (AFM). Aqueous solutions of the polysaccharide are deposited as drops onto freshly cleaved mica surfaces, air dried, and then imaged under butanol. The method has been used to obtain images of the bacterial polysaccharide acetan. In regions within the deposited sample, where the molecules are aligned side-by-side, it has been possible to observe a periodic structure along the polysaccharide chain, attributable to the helical structure of acetan.     

Genetic analysis of acetan biosynthesis in Acetobacter xylinum: DNA sequence analysis of the aceM gene encoding an UDP-glucose dehydrogenase

Analysis of the nucleotide sequence of a 1592 bp region of Acetobacter xylinum genomic DNA involved in acetan biosynthesis revealed the presence of an open-reading frame (aceM) encoding a protein of 449 amino acids with a molecular weight of 48.5 kDa. The deduced amino acid sequence of aceM displayed high homology to the protein sequences of genes encoding UDP-glucose dehydrogenase (UGDH) activities from other organisms. AceM is likely to encode the UGDH involved in the biosynthesis of UDP-glucuronic acid required for acetanbiosynthesis. This revised version was published online in November 2006 with corrections to the Cover Date.     

Physicochemical characterization of an acetan variant secreted by Acetobacter xylinum strain CR1/4

Chemical mutagenesis has been used to produce mutants of Acetobacter xylinum NRRL B42 that are cellulose-negative and that produce variants of the acetan structure deficient in the side-chain sugar residues. The product of A. xylinum strain CR1/4 has been shown to possess a tetrasaccharide repeat unit with the side chain terminating in glucuronic acid. X-ray diffraction studies of oriented fibres suggest that the polysaccharide CR1/4 forms a fivefold helix with a pitch of 4.8 nm. Light-scattering studies on CR1/4 solutions suggest a molecular weight of 1.2 × 10⁶ with radii of gyration values of 86 nm (aqueous solution) and 67 nm (0.1 NaCl solution). The magnitude of the measured radii of gyration and the shape of the Holtzer plots suggest that CR1/4 can be described as a stiff coil. Preliminary differential scanning calorimetry data show melting behaviour consistent with order-disorder transitions of a charged helical structure. Rheological studies have revealed new synergistic interactions of CR1/4 with locus bean gum. Comparative studies of acetan and CR1/4 show that decreasing the length of the side chain enhances the solution viscosity.     

Cloning and sequencing of the levansucrase gene from Acetobacter xylinum NCI 1005.

The levansucrase gene (lsxA) was cloned from the genomic DNA of Acetobacter xylinum NCI 1005, and the nucleotide sequence of the lsxA gene (1,293 bp) was determined. The deduced amino acid sequence of the lsxA gene showed 57.4% and 46.2% identity with the levansucrases from Zymomonas mobilis and Erwinia amylovora, respectively, while only 35.2% identity with that from Acetobacter diazotrophicus. The gene product of lsxA (LsxA) that was overproduced in E. coli coded for a polypeptide of molecular mass 47 kDa. The LsxA released glucose and produced polysaccharide from sucrose, the structure of which was analyzed by nuclear magnetic resonance spectroscopy and determined to be a beta-(2,6)-linked polyfructan.     

Microbial cellulose as a specialty chemical

Microbial polysaccharides are extensively used commercially as gelling or suspending agents, as protective colloids or as thickening agents. Until recently, microbial cellulose producing systems such as Acetobacter xylinum, had been used largely as model systems for the study of cellulose biosynthesis. Current advances in molecular biology and biochemical engineering promise to usher microbial cellulose into the specialty chemical market. This review will highlight some of the recent progress made in our understanding of microbial cellulose biochemistry and biosynthesis, describe some of its inherent virtues and identify current unique applications of this versatile biopolymer.     

Identification of a second cellulose synthase gene (acsAII) in Acetobacter xylinum.

A second cellulose synthase gene (acsAII) coding for a 175-kDa polypeptide that is similar in size and sequence to the acsAB gene product has been identified in Acetobacter xylinum AY201. Evidence for the presence of this gene was obtained during analysis of A. xylinum mutants in which the acsAB gene was disrupted (I.M. Saxena, K. Kudlicka, K. Okuda, and R.M. Brown, Jr., J. Bacteriol. 176:5735-5752, 1994). Although these mutants produced no detectable cellulose, they exhibited significant cellulose synthase activity in vitro. The acsAII gene was isolated by using an acsAB gene fragment as a probe. The acsAII gene coded for cellulose synthase activity as determined from sequence analysis and study of mutants in which this gene was disrupted. A mutant in which only the acsAII gene was disrupted showed no significant differences in either the in vivo cellulose production or the in vitro cellulose synthase activity compared with wild-type cells. Mutants in which both the acsAII and acsAB genes were disrupted produced no cellulose in vivo and exhibited negligible cellulose synthase activity in vitro, thus confirming that the cellulose synthase activity observed in the acsAB mutants was coded by the acsAII gene. These results establish the presence of an additional gene for cellulose synthase expressed in cells of A. xylinum, yet this gene is not required for cellulose production when cells are grown under laboratory conditions.     

Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.     

Effect of addition of water-soluble polysaccharides on bacterial cellulose production in a 50-L airlift reactor

Bacterial cellulose (BC) production was carried out in a batch cultivation of Acetobacter xylinum in a 50-L internal loop airlift reactor by addition of water-soluble polysaccharides into the medium. When 0.1% (w/w) agar was added, BC production reached 8.7 g/L compared with 6.3 g/L in the control, and duration of the cultivation period to reach the maximum concentration of BC was almost half of that without addition of polysaccharides. During cultivation, BC was formed into pellets whose size was smaller when the productivity of BC was higher, indicating that increase in the relative viscosity by addition of polysaccharides hindered formation of large clumps of BC and increase in the volumetric oxygen transfer coefficient at high flow rate led to increase in BC productivity.     

细菌纤维素的研究进展

细菌纤维素是由醋酸杆菌属、根瘤菌属、土壤杆菌属、八叠球菌属等的某些细菌在一定条件下产生的,其中最有代表性的细菌是木醋杆菌。与传统植物纤维素相比,细菌纤维素具有很高的化学纯度。主要介绍细菌纤维素性质、生物合成的方法及其在食品工业、造纸工业和作为一种生物材料在医学工程等方面的应用。     

An Acetobacter xylinum insertion sequence element associated with inactivation of cellulose production.

An insertion sequence (IS) element, IS1031, caused insertions associated with spontaneous cellulose deficient (Cel-) mutants of Acetobacter xylinum ATCC 23769. The element was discovered during hybridization analysis of DNAs from Cel- mutants of A. xylinum ATCC 23769 with pAXC145, an indigenous plasmid from a Cel- mutant of A. xylinum NRCC 17005. An IS element, IS1031B, apparently identical to IS1031, was identified on pAXC145. IS1031 is about 950 bp. DNA sequencing showed that the two elements had identical termini with inverted repeats of 24 bp containing two mismatches and that they generated 3-bp target sequence duplications. The A. xylinum ATCC 23769 wild type carries seven copies of IS1031. Southern hybridization showed that 8 of 17 independently isolated spontaneous Cel- mutants of ATCC 23769 contained insertions of an element homologous to IS1031. Most insertions were in unique sites, indicating low insertion specificity. Significantly, two insertions were 0.5 kb upstream of a recently identified cellulose synthase gene. Attempts to isolate spontaneous cellulose-producing revertants of these two Cel- insertion mutants by selection in static cultures were unsuccessful. Instead, pseudorevertants that made waxlike films in the liquid-air interface were obtained. The two pseudorevertants carried new insertions of an IS1031-like element in nonidentical sites of the genome without excision of the previous insertions. Taken together, these results suggest that indigenous IS elements contribute to genetic instability in A. xylinum. The elements might also be useful as genetic tools in this organism and related species.     

Phytanyl-pyrophosphate-linked substrate for a bacterial alpha-mannosyltransferase

The biochemical characterization of bacterial glycosyltransferases involved in the assembly of cell-wall-associated polysaccharides is often hindered by the lack of the appropriate undecaprenyl-pyrophosphate-linked acceptor substrate. In order to find a suitable synthetic substrate for the alpha1,3-mannosyltransferase AceA from Acetobacter xylinum, phytanyl-pyrophosphate-linked cellobiose was prepared. In the presence of GDP-[14C]mannose and recombinant AceA, the phytanyl-pyrophosphate-linked cellobiose afforded a 14C-labeled trisaccharide that was sensitive to alpha-mannosidase degradation in a fashion analogous to the natural undecaprenyl-pyrophosphate-linked cellobiose substrate. These results suggest that phytanyl-pyrophosphate-linked oligosaccharides may be useful substrates for other important bacterial glycosyltransferases.     

Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation

M ichiels , K., V erreth , C. & V anderleyden , J. 1990. Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation. Journal of Applied Bacteriology 69 , 705–711.
Surface polysaccharide production by Azospirillum is demonstrated by fluorescence of colonies grown on media containing the fluorescent dye Calcofluor, which binds to β-linked polysaccharides. Mutants showing decreased and increased levels of fluorescence are obtained from Azospirillum lipoferum strain Sp59b by chemical mutagenesis, and from A. brasilense strain 7030 by Tn5 mutagenesis.
The A. brasilense 7030 fluorescence mutants produce wild-type levels of exo-polysaccharide in their culture supernatant fluids, but are affected in flocculation in liquid culture. On the basis of these observations, we postulate that an A. brasilense surface polysaccharide, different from the exopolysaccharide, is involved in both Calcofluor staining and flocculation.
It is shown by DNA hybridization that the genetic loci affected in the A. brasilense 7030 fluorescence mutants are different from the A. brasilense exoB and exoC loci, which are involved in exopolysaccharide production.     

Identification of essential amino acids in the bacterial alpha -mannosyltransferase aceA

The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue.