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.     

Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization.

The synthesis of an extracellular ribbon of cellulose in the bacterium Acetobacter xylinum takes place from linearly arranged, membrane-localized, cellulose-synthesizing and extrusion complexes that direct the coupled steps of polymerization and crystallization. To identify the different components involved in this process, we isolated an Acetobacter cellulose-synthesizing (acs) operon from this bacterium. Analysis of DNA sequence shows the presence of three genes in the acs operon, in which the first gene (acsAB) codes for a polypeptide with a molecular mass of 168 kDa, which was identified as the cellulose synthase. A single base change in the previously reported DNA sequence of this gene, resulting in a frameshift and synthesis of a larger protein, is described in the present paper, along with the sequences of the other two genes (acsC and acsD). The requirement of the acs operon genes for cellulose production was determined using site-determined TnphoA/Kanr GenBlock insertion mutants. Mutant analysis showed that while the acsAB and acsC genes were essential for cellulose production in vivo, the acsD mutant produced reduced amounts of two cellulose allomorphs (cellulose I and cellulose II), suggesting that the acsD gene is involved in cellulose crystallization. The role of the acs operon genes in determining the linear array of intramembranous particles, which are believed to be sites of cellulose synthesis, was investigated for the different mutants; however, this arrangement was observed only in cells that actively produced cellulose microfibrils, suggesting that it may be influenced by the crystallization of the nascent glucan chains.     

Physico-Mechanical Properties of Chemically Treated Bacterial (Acetobacter xylinum) Cellulose Membrane

Bacterial cellulose obtained through fermentation by the Acetobacter xylinum is of superior functional quality in comparison to plant cellulose. Various alkali treatment methods were used to process bio-chemically complex pellicle into a clean cellulose membrane/sheet. The effect of potassium hydroxide, sodium carbonate and potassium carbonate was found to be milder on the final cellulose product in contrast to the widely used sodium hydroxide treatment. These novel treatment methods also caused improvement in the tensile strength of the membranes in comparison to sodium hydroxide. The overall quality of the 0.1 M sodium carbonate- and potassium carbonate-treated cellulose was superior, as the membranes displayed maximum tensile strength and elongation next to the native membrane. The low tensile strength of sodium hydroxide-treated membrane is attributed to its higher swelling characteristics in alkali. Further, the low swelling property of sodium carbonate- and potassium carbonate-treated membranes resulted in their high oxygen transmission rates (low oxygen barrier). Hunter lab colour parameters were determined to assess the effect of different alkali treatments on the colour characteristics of the membranes. Further, based on the high mechanical strength and comparatively low oxygen transmission rates, the processed cellulose membranes may find application as a bio- packaging material for controlled atmosphere packaging, where hydrophilic membranes with high oxygen barrier and water vapour permeation are desirable.     

Alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum. Purification and regulatory properties.

The alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum was purified to homogeneity. It consists of three main polypeptide chains with a total molecular weight of about 2.4 X 10(6). It catalyzes the overall Mg2+ and thiamin pyrophosphate-dependent, NAD+- and CoA-linked oxidative decarboxylation of alpha-ketoglutarate, as well as the partial reactions characteristic of the three enzyme components described for the complex from other sources. Initial velocity studies revealed marked positive cooperativity for the substrate alpha-ketoglutarate (Hill coefficient (nH) = 2.0; concentration of ligand at half-maximum effect (S0.5) = 8 mM). The sigmoidal [alpha-ketoglutarate]-velocity relationship became hyperbolic upon addition of AMP or 3-acetylpyridine adenine dinucleotide (AcPyAD) or in the presence of high concentrations of NAD. S0.5 (alpha-ketoglutarate) decreased to 1 mM, but Vmax was unchanged. Saturation curves for NAD and AMP are sigmoidal (nH = 2) at low alpha-ketoglutarate concentrations and become hyperbolic at high alpha-ketoglutarate concentrations. As judged by S0.5, the relative efficiency of the allosteric effectors is AcPyAD greater than AMP greater than alpha-ketoglutarate- greater than NAD+. Half-maximal changes in nH, S0.5, and activation by AMP occur at a pH significantly different from that of half-maximal activity. A model for the allosteric behavior of the complex is proposed in which the first enzyme component of the complex (E1) is the site for the allosteric interactions and AMP is the primary positive modifier, whereas NAD and AcPyAD act as AMP analogues. The overall reaction is competitively inhibited by NADH with respect to NAD (K1 = 20 micronM) and by succinyl-CoA with respect of CoA (K1 = 3 micronM). The properties of the alpha-ketoglutarate dehydrogenase complex of A. xylinum appear to provide for appropriate partitioning of alpha-ketoglutarate carbon between competing pathways in response to the energy state of the cells.     

Induction of orientation of bacterial cellulose microfibrils by a novel terpenoid from Acetobacter xylinum

1. The bacterium Acetobacter xylinum produces extracellular cellulose microfibrils that form a pellicle in the medium enmeshing the bacterial cells. These microfibrils may show some localized alignment, which can be seen as birefringence when the culture is viewed between crossed Polaroid sheets. 2. An increase in birefringence can be induced by the addition of small amounts of certain classes of lipids, particularly sterols, to the cultures. 3. A crude lipid extract from Acetobacter cells induced greatly increased birefringence when added to fresh cultures of this organism. 4. When the bacterial lipids were fractionated, most of the activity was recovered in a complex, polar lipid. The lipid is secreted into the medium during growth and is unstable. The non-saponifiable portion of this lipid is shown to be a 1:1 mixture of a saturated and a monounsaturated C₃₅ tetrahydroxy terpene with a hopane ring system in the accompanying paper by Förster et al. (1973). The saturated molecule is referred to as tetrahydroxybacteriohopane. 5. Tetrahydroxybacteriohopane is itself capable of inducing birefringence in cultures as is 22-hydroxyhopane, which was also isolated from the non-saponifiable fraction of the total lipids. 6. The mechanism of induction of birefringence (orientation of microfibrils) is not known. This is unlikely to be a specific effect, since all the above compounds are active (intact lipid, tetrahydroxybacteriohopane, 22-hydroxyhopane), as are other classes of lipid. It is suggested, however, that a common mechanism may be involved and that similar compounds may be concerned with control of microfibril alignment in the cells of higher plants.     

Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O(2) tension maximizes bacterial cellulose pellicle production

Vitreoscilla hemoglobin (VHb) was constitutively expressed in Acetobacter xylinum to enhance bacterial cellulose (BC) production. A pronounced enhancement of BC production in static culture was observed. Reducing O(2) tension in gaseous phase of the culture by tightly sealing the culture tube could also enhance BC production by 70%. O(2) tension in gaseous phase reduced from 21 to 15% in the sealed and static culture of VHb-expressing A. xylinum after 7 days cultivation, while 7.36g/l of BC with yield of 0.44 were obtained. BC pellicle production by VHb-expressing A. xylinum was successfully scaled-up in a sealed 4l disposable zip lock plastic bag with BC yield of 0.38 and concentration of 6.73g/l.     

Additional properties of a soluble polymer of glucose from cultures of Acetobacter xylinum

The results of differential, thermal analysis of a soluble, beta (1 leads to 2)-branched, beta (1 leads to 4)-D-glucan isolated from cultures of Acetobacter xylinum are consistent with previous conclusions about its structure. The O-acetyl content of the polymer is 8.3% which corresponds to a maximum substitution of one acetyl group per three glucose residues. Proton nuclear magnetic resonance spectra confirm that all the glycosidic bonds are beta linkages. Some preparations of the polymer are contaminated by another polymer containing mannose and rhamnose. No evidence was obtained to support a previous suggestion that the branched D-glucan is a precursor of bacterial cellulose and this suggestion is now withdrawn.     

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.     

Characterization of the Biosynthetic Pathway of Cellulose from Glucose and Fructose in Acetobacter xylinum

For characterization of the biosynthetic pathway of cellulose in a cellulose-producing Acetobacter xylinum strain BPR2001, the activities of several enzymes were measured. The activity of phosphoglucose isomerase catalyzing the conversion of fructose-6- phosphate into glucose-6-phosphate was greatly increased by fructose in the medium. The UDP-glucose pyrophosphorylase activity catalyzing the synthesis of UDP-glucose was very high in strain BPR2001, consistent with the idea that this is the key enzyme in cellulose biosynthesis. Strain BPR2001 was found to have a fructose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS).     

The effect of pH, sucrose and ammonium sulphate concentrations on the production of bacterial cellulose (Nata-de-coco) by Acetobacter xylinum

The effect of pH, sucrose and ammonium sulphate concentrations on the production of nata-de-coco, a form of bacterial cellulose, by Acetobacter xylinum was studied. Comparisons for physical properties like thickness, wet weight, water-holding capacity (WHC), moisture content and hardness, a textural parameter were done on nata-de-coco grown in tender coconut water medium supplemented with varying concentrations of sucrose and ammonium sulphate at different pH values. The results were analysed by fitting a second-order polynomial regression equation. Response surface methodology was used to study the effect of the three variables. The study showed that A. xylinum could effectively use sucrose as the sole carbon source in coconut water medium and that cellulose production was more dependent on pH than either sucrose or ammonium sulphate concentrations. Maximum thickness of nata was obtained at pH 4.0 with 10% sucrose and 0.5% ammonium sulphate concentrations. These conditions also produced good quality nata-de-coco with a smooth surface and soft chewy texture. The study will enable efficient utilization of coconut water, a hitherto wasted byproduct of coconut industry and will also provide a new product dimension to the aggrieved coconut farmers who are not getting the right price for their product.     

Utilization of the buffering capacity of corn steep liquor in bacterial cellulose production by Acetobacter xylinum

Acetobacter xylinum BPR2001 produces water-insoluble bacterial cellulose (BC). Using a pH sensor for the accurate control of pH, which is one of the most critical factors for efficient BC production, is difficult especially in a baffled shake-flask and an airlift reactor. The buffering capacity of corn steep liquor (CSL) was estimated by measuring (buffering capacity) values in advance and was used to maintain the pH within the optimal range during the production of BC. When CSL was added to either a shake-flask, a stirred-tank reactor or an airlift reactor, BC production was almost the same as that in cultivations where pH was controlled manually or by a pH sensor.     

Production of bacterial cellulose by Acetobacter xylinum BPR2001 using molasses medium in a jar fermentor

Bacterial cellulose (BC) production by Acetobacter xylinum subsp. sucrofermentans BPR2001 using molasses medium was carried out in a jar fermentor. When molasses was subjected to H₂SO₄-heat treatment, the maximum BC concentration increased to 76% more than that achieved using untreated molasses, and the specific growth rate increased 2-fold. When the initial sugar concentrations in the H₂SO₄-heat treated molasses were varied from 23 g/l to 72 g/l, BC concentration, production rate, and yield were maximum at sugar concentrations of 23 g/l and 37 g/l, and production of by-products, such as polysaccharides and CO₂, was lower than at sugar concentrations of 48 g/l and 72 g/l, indicating that maintaining a lower molasses concentration is essential for efficient BC production in jar fermentors, this being due mainly to the complex nature of molasses. Molasses has a clear advantage over pure sugars as a carbon source from an economic viewpoint.     

木醋杆菌纤维素合成操纵子的克隆及棉花转化

革兰氏阴性菌木醋杆菌 (Acetobacterxylinum (Brown)Yamada)合成一种由纤维素微纤丝组成的胞外带状物。与高等植物纤维素相比 ,它具有独特的结构和机械性能。根据从木醋杆菌ATCC 5 35 82克隆的acs纤维素合成操纵子序列设计引物 ,用PCR的方法从木醋杆菌Ay2 0 1中克隆了ayacs纤维素合成操纵子的全部 4个基因。序列比较发现 ,两者高度同源。将连上CaMV 35S启动子的acsA、acsB克隆到植物表达载体pCAMBIA 130 1上 ,acsC、acsD克隆到pCOB30 2_3中。然后通过花粉管通道法转化棉花 (Gossypiumhirsutum)胚珠 ,收获的种子在含有卡那霉素和除草剂的双抗培养基上进行筛选。PCR检测发现 934粒种子中有 5棵植株含有全部 4个基因。这是首次将编码 4个功能蛋白的细菌操纵子成功地转入棉花     

木醋杆菌纤维素合成操纵子的克隆及棉花转化

革兰氏阴性菌木醋杆菌(Acetobacter xylinum (Brown) Yamada)合成一种由纤维素微纤丝组成的胞外带状物.与高等植物纤维素相比,它具有独特的结构和机械性能.根据从木醋杆菌ATCC 53582克隆的acs纤维素合成操纵子序列设计引物, 用PCR的方法从木醋杆菌Ay201中克隆了ayacs纤维素合成操纵子的全部4个基因.序列比较发现,两者高度同源.将连上CaMV 35S启动子的acsA、acsB克隆到植物表达载体pCAMBIA 1301上,acsC、acsD克隆到pCOB302-3中.然后通过花粉管通道法转化棉花(Gossypium hirsutum)胚珠,收获的种子在含有卡那霉素和除草剂的双抗培养基上进行筛选.PCR检测发现934粒种子中有5棵植株含有全部4个基因.这是首次将编码4个功能蛋白的细菌操纵子成功地转入棉花.     

Visualization of pores (export sites) correlated with cellulose production in the envelope of the gram-negative bacterium Acetobacter xylinum

The Gram-negative bacterium Acetobacter xylinum assembles a cellulse ribbon composed of a number of microfibrils in the longitudinal axis of its envelope. The zone of ribbon assembly was investigated by freeze-etch electron microscopy. Freeze-etching revealed, beneath the cellulose ribbons, a linear array of pores on the lipopolysaccharide membrane. These pores have a rim diameter of 120--150 A and a central hole or deepening of approximately 35 A. The axes of pore arrays closely coincide with linear arrays of 100 A particles on the E- and P-faces of the fractured lipopolysaccharide membranes. Pores and particles in the lipopolysaccharide membrane are probably congruent. The pores are hypothesized to be the export sites (penetration sites) for cellulose.     

Influence of 1‐methylcyclopropene (1‐MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture

Aims: Bacterial cellulose is an extracellular polysaccharide secreted by Acetobacter xylinum, which has become a novel material increasingly used in food and medical industries. However, its broad application is limited by its low yield and high cost. 1‐Methylcyclopropene (1‐MCP) is a potent inhibitor to either exogenous or endogenous ethylene during the biological senescence of plants, which has been broadly applied in commercial preservation of fruits and vegetables. The purpose of this study was to investigate the effects of 1‐MCP on both the growth of Acet.  xylinum and its cellulose production to demonstrate the potential enhancement of bacterial cellulose yield. Methods and Results: Three groups of samples were fermented under agitated culture with 125 rev min^?1 rotational speed. To the culture media, 0·14 mg of 1‐MCP contained in 100 mg dextrose powder was added on assigned days or on the first culture day only. Results from the measurement of bacterial cell concentration and bacterial cellulose yield at the end of a 12‐day culture demonstrated that cultures excluding 1‐MCP displayed a higher cell concentration and a lower cellulose production, while cultures containing 1‐MCP produced 15·6% more cellulose (1‐MCP added on day 1) and 25·4% (1‐MCP added on each assigned day) with less biomass. Conclusions: 1‐MCP was able to affect the growth of Acet. xylinum cells and resulted in increasing bacterial cellulose yield up to 25·4% over controls, which did not contain 1‐MCP. Significance and Impact of the Study: This was the first study to use the growth inhibitor of plants to investigate its effects on bacterial growth and production. It also demonstrated a significant enhancement of bacterial cellulose yield by the addition of 1‐MCP during the common agitated culture of Acet. xylinum.     

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.