| 1. |
Ishida T,
Sugano Y,
Nakai T,
Shoda M,
( 2002 ) Effects of acetan on production of bacterial cellulose by Acetobacter xylinum. PMID : 12353627 : DOI : 10.1271/bbb.66.1677 Abstract >>
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.
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2. |
Kimura S,
Chen HP,
Saxena IM,
Brown RM,
Itoh T,
( 2001 ) Localization of c-di-GMP-binding protein with the linear terminal complexes of Acetobacter xylinum. PMID : 11544230 : DOI : 10.1128/JB.183.19.5668-5674.2001 PMC : PMC95459 Abstract >>
Specific labeling of a single row of cellulose-synthesizing complexes (terminal complexes, TC subunits, TCs, or TC arrays) in Acetobacter xylinum by antibodies raised against a 93-kDa protein (the cyclic dignanylic acid-binding protein) has been demonstrated by using the sodium dodecyl sulfate (SDS)-freeze-fracture labeling (FRL) technique. The antibodies to the 93-kDa protein specifically recognized the TC subunits on the protoplasmic fracture (PF) face of the outer membrane in A. xylinum; however, nonlabeled TCs were also observed. Two types of TC subunits (particles or pits) are observed on the PF face of the outer membrane: (i) immunogold-labeled TCs showing a line of depressions (pits) with an indistinct particle array and (ii) nonlabeled TC subunits with a distinct single row of particle arrays. The evidence indicates that the labeling patterns differ with respect to the presence or absence of certain TC subunits remaining attached to the replica after SDS treatment. This suggests the presence of at least two TC components, one in the outer membrane and the other in the cytoplasmic membrane. If the TC component in the outer membrane is preferentially fractured and remains attached to the ectoplasmic fracture face (or outer leaflet) of the outer membrane, subsequent replica formation reveals a pit or depression with positive antibody labeling on the PF face of the outer membrane. If the TC component in the outer membrane remains with the PF face (or inner leaflet) of the outer membrane, the innermost TC component is removed during SDS treatment and labeling does not occur. SDS-FRL of TCs in A. xylinum has enabled us to provide the first topological molecular analysis of component proteins in a cellulose-synthesizing TC structure in a prokaryotic organism.
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3. |
Amikam D,
Benziman M,
Gilles-Gonzalez MA,
Chang AL,
Tuckerman JR,
Gonzalez G,
Mayer R,
Weinhouse H,
Volman G,
( 2001 ) Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor. PMID : 11297407 : DOI : 10.1021/bi0100236 Abstract >>
The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis. This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG. Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme. Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity. O(2) regulation is due to deoxyheme being a better activator than oxyheme. AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain. The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors. The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold. The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher. Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger.
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4. |
Tajima K,
Tanio T,
Kobayashi Y,
Kohno H,
Fujiwara M,
Shiba T,
Erata T,
Munekata M,
Takai M,
( 2000 ) Cloning and sequencing of the levansucrase gene from Acetobacter xylinum NCI 1005. PMID : 10997873 : DOI : 10.1093/dnares/7.4.237 Abstract >>
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.
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5. |
Abdian PL,
Lellouch AC,
Gautier C,
Ielpi L,
Geremia RA,
( 2000 ) Identification of essential amino acids in the bacterial alpha -mannosyltransferase aceA. PMID : 11001941 : DOI : 10.1074/jbc.M007496200 Abstract >>
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.
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6. |
Gasson MJ,
Morris VJ,
Jay AJ,
Colquhoun IJ,
( 1999 ) Generation of a novel polysaccharide by inactivation of the aceP gene from the acetan biosynthetic pathway in Acetobacter xylinum. PMID : 10411277 : DOI : 10.1099/13500872-145-6-1499 Abstract >>
The acetan biosynthetic pathway in Acetobacter xylinum is an ideal model system for engineering novel bacterial polysaccharides. To genetically manipulate this pathway, an Acetobacter strain (CKE5), more susceptible to gene-transfer methodologies, was developed. A new gene, aceP, involved in acetan biosynthesis was identified, sequenced and shown to have homology at the amino acid level with beta-D-glucosyl transferases from a number of different organisms. Disruption of aceP in strain CKE5 confirmed the function assigned above and was used to engineer a novel polysaccharide with a pentasaccharide repeat unit.
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7. |
Roux M,
Ferreiro DU,
Dauphin-Dubois R,
Geremia RA,
( 1999 ) Expression and biochemical characterisation of recombinant AceA, a bacterial alpha-mannosyltransferase. PMID : 10485283 : DOI : 10.1007/s004380051040 Abstract >>
Biosynthesis of repeat-unit polysaccharides and N-linked glycans proceeds by sequential transfer of sugars from the appropriate sugar donor to an activated lipid carrier. The transfer of each sugar is catalysed by a specific glycosyltransferase. The molecular basis of the specificity of sugar addition is not yet well understood, mainly because of the difficulty of isolating these proteins. In this study, the aceA gene product expressed by Acetobacter xylinum, which is involved in the biosynthesis of the exopolysaccharide acetan, was overproduced in Escherichia coli and its function was characterised. The aceA ORF was subcloned into the expression vector pET29 in frame with the S.tag epitope. The recombinant protein was identified, and culture conditions were optimised for production of the soluble protein. The results of test reactions showed that AceA is able to transfer one alpha-mannose residue from GDP-mannose to cellobiose-P-P-lipid to produce alpha-mannose-cellobiose-P-P-lipid. AceA was not able to use free cellobiose as a substrate, indicating that the pyrophosphate-lipid moiety is needed for enzymatic activity.
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8. |
Onizuka T,
Ikeuchi M,
Inoue Y,
Akiyama H,
Hirano A,
Ishibashi M,
( 1999 ) Cloning of cellulose synthase genes from Acetobacter xylinum JCM 7664: implication of a novel set of cellulose synthase genes. PMID : 10382968 : DOI : 10.1093/dnares/6.2.109 Abstract >>
Three sets of cellulose synthase genes were cloned from a cellulose-producing bacterium Acetobacter xylinum JCM 7664. One set of genes (bcsAI/bcsBI/bcsCI/bcsDI) were highly conserved with the well-established type I genes in other strains of A. xylinum, while the other two (bcsABII-A, bcsABII-B) were homologous to the known type II (acsAII). Unexpectedly, they were immediately followed by a gene cluster of bcsX/bcsY/bcsCII/ORF569, likely forming an operon. Western blotting demonstrated that the BcsY protein accumulated in cells. Since BcsY showed striking similarities to a number of membrane-bound transacylases, it was hypothesized that the type II cellulose synthase produces acylated cellulose, which might be anchored on the cytoplasmic membrane. An insertion sequence of IS1380-type was found just upstream of the one type II gene (bcsABII-B), suggestive of nonfunctioning.
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9. |
Ryjenkov DA,
Simm R,
Römling U,
Gomelsky M,
( 2006 ) The PilZ domain is a receptor for the second messenger c-di-GMP: the PilZ domain protein YcgR controls motility in enterobacteria. PMID : 16920715 : DOI : 10.1074/jbc.C600179200 Abstract >>
The ubiquitous bacterial second messenger c-di-GMP controls exopolysaccharide synthesis, flagella- and pili-based motility, gene expression, and interactions of bacteria with eukaryotic hosts. With the exception of bacterial cellulose synthases, the identities of c-di-GMP receptors and end targets have remained unknown. Recently, Amikam and Galperin (Amikam, D., and Galperin, M. (2006) Bioinformatics 22, 3-6) hypothesized that the PilZ domains present in the BcsA subunits of bacterial cellulose synthases function in c-di-GMP binding. This hypothesis has been tested here using the Escherichia coli PilZ domain protein YcgR, its individual PilZ domain and the PilZ domain from Gluconacetobacter xylinus BcsA. YcgR was purified and found to bind c-di-GMP tightly and specifically, Kd 0.84 microm. Individual PilZ domains from YcgR and BcsA also bound c-di-GMP, albeit with lesser affinity, indicating that PilZ is sufficient for binding. The site-directed mutagenesis performed on YcgR implicated the most conserved residues in the PilZ domain directly in c-di-GMP binding. It is suggested that c-di-GMP binding to PilZ brings about conformational changes in the protein that stabilize the bound ligand and initiate the downstream signal transduction cascade. While the identity of the downstream partner(s) of YcgR remains unknown, it is shown that YcgR regulates flagellum-based motility in a c-di-GMP-dependent manner. The inactivation of ycgR improves swimming and swarming motility of the poorly motile yhjH mutants of Salmonella enterica serovar Typhimurium UMR1. Therefore, biochemical and genetic evidence presented here establishes PilZ as a long sought after c-di-GMP-binding domain and YcgR as a c-di-GMP receptor affecting motility in enterobacteria.
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10. |
Mayer R,
Ross P,
Weinhouse H,
Amikam D,
Volman G,
Ohana P,
Calhoon RD,
Wong HC,
Emerick AW,
Benziman M,
( 1991 ) Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants. PMID : 1647035 : DOI : 10.1073/pnas.88.12.5472 PMC : PMC51895 Abstract >>
To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either [32P]c-di-GMP or [alpha-32P]UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene. We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.
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11. |
Trcek J,
( 2005 ) Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene. PMID : 16261863 : DOI : 10.1016/j.syapm.2005.05.001 Abstract >>
Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S-23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNAIle and tRNAAla, the sequences of 16S-23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. acetispecific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for revealing the misclassification of strain IFO 3283 into the species A. aceti.
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12. |
Bae SO,
Sugano Y,
Ohi K,
Shoda M,
( 2004 ) Features of bacterial cellulose synthesis in a mutant generated by disruption of the diguanylate cyclase 1 gene of Acetobacter xylinum BPR 2001. PMID : 15042328 : DOI : 10.1007/s00253-004-1593-7 Abstract >>
The diguanylate cyclase 1 (DGC1) (dgc1) gene in Acetobacter xylinum BPR 2001--a bacterial cellulose (BC) producer--was cloned and sequenced, and a DGC1 gene-disrupted mutant, strain DD, was constructed. The production and structural characteristics of the BC formed by DD were compared with those of the parental strain BPR 2001. BC production by DD was almost the same as that by BPR 2001 in static cultivation and in shake flask cultivation. However, in a jar fermentor DD produced about 36% more BC than the parental strain. DD produced suspended particle materials that cannot aggregate owing to their random structural characteristics in static cultivation; more uniformly dispersed BC pellicles and smaller BC pellets are produced on average in a jar fermentor, as reflected by the higher BC production by DD than by the parental strain in a jar fermentor. Micrographs of BC produced by DD revealed that the width of cellulose ribbons assemblies decreased as a result of differences in the ultrastructure and mechanism of formation of BC between the two strains. These results reveal that disruption of the dgc1 gene, which catalyzes synthesis of c-di-GMP (an effector of BC synthase), is not fatal for BC synthesis, although it affects BC structure.
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13. |
Saxena IM,
Lin FC,
Brown RM,
( 1990 ) Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum. PMID : 2151718 : DOI : 10.1007/bf00016118 Abstract >>
The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb Hind III fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the Hind III fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.
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14. |
Lin FC,
Brown RM,
Drake RR,
Haley BE,
( 1990 ) Identification of the uridine 5'-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc. PMID : 2138620 : Abstract >>
Photoaffinity labeling of purified cellulose synthase with [beta-32P]5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of [beta-32P]5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.
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15. |
Wong HC,
Fear AL,
Calhoon RD,
Eichinger GH,
Mayer R,
Amikam D,
Benziman M,
Gelfand DH,
Meade JH,
Emerick AW,
( 1990 ) Genetic organization of the cellulose synthase operon in Acetobacter xylinum. PMID : 2146681 : DOI : 10.1073/pnas.87.20.8130 PMC : PMC54906 Abstract >>
An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum.
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16. |
Brede G,
Fjaervik E,
Valla S,
( 1991 ) Nucleotide sequence and expression analysis of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene. PMID : 1938907 : DOI : 10.1128/jb.173.21.7042-7045.1991 PMC : PMC209064 Abstract >>
The nucleotide sequence of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene was determined; this is the first procaryotic uridine diphosphoglucose pyrophosphorylase gene sequence reported. The sequence data indicated that the gene product consists of 284 amino acids. This finding was consistent with the results obtained by expression analysis in vivo and in vitro in Escherichia coli.
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17. |
Saxena IM,
Lin FC,
Brown RM,
( 1991 ) Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum. PMID : 1830823 : DOI : 10.1007/bf00016067 Abstract >>
DNA sequencing of the region downstream of the cellulose synthase catalytic subunit gene of Acetobacter xylinum led to the identification of an open reading frame coding for a polypeptide of 86 kDa. The deduced amino acid sequence of this polypeptide matches from position 27 to 40 with the N-terminal amino acid sequence determined for a 93 kDa polypeptide that copurifies with the cellulose synthase catalytic subunit during purification of cellulose synthase. The cellulose synthase catalytic subunit gene and the gene encoding the 93 kDa polypeptide, along with other genes probably, are organized as an operon for cellulose biosynthesis in which the first gene is the catalytic subunit gene and the second gene codes for the 93 kDa polypeptide. The function of the 93 kDa polypeptide is not clear at present, however it appears to be tightly associated with the cellulose synthase catalytic subunit. Sequence analysis of the polypeptide shows that it is a membrane protein with a signal sequence at the N-terminal end and a transmembrane helix in the C-terminal region for anchoring it into the membrane.
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18. |
Valera MJ,
Torija MJ,
Mas A,
Mateo E,
( 2015 ) Cellulose production and cellulose synthase gene detection in acetic acid bacteria. PMID : 25381910 : DOI : 10.1007/s00253-014-6198-1 Abstract >>
The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter, Komagataeibacter, Gluconacetobacter, and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.
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19. |
Yadav V,
Panilaitis B,
Shi H,
Numuta K,
Lee K,
Kaplan DL,
( 2011 ) N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus. PMID : 21655093 : DOI : 10.1371/journal.pone.0018099 PMC : PMC3107205 Abstract >>
Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as �GnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.
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20. |
( 1997 ) Cloning of the aceF gene encoding the phosphomannose isomerase and GDP-mannose pyrophosphorylase activities involved in acetan biosynthesis in Acetobacter xylinum. PMID : 9311139 : DOI : 10.1111/j.1574-6968.1997.tb12672.x Abstract >>
The aceF 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 xanB from Xanthomonas campestris. Therefore aceF is likely to encode a bifunctional enzyme with mannose-6-phosphate isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) activities. PMI and GMP activities were detected in strains of Escherichia coli expressing the cloned aceF gene.
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21. |
( 1996 ) Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum: nucleotide sequence analysis of the aceB, aceC, aceD and aceE genes. PMID : 8988363 : Abstract >>
Sequence analysis of a 5.323 kb chromosomal DNA fragment from Acetobacter xylinum involved in the biosynthesis of the exopolysaccharide acetan, revealed the presence of four ace genes designated aceB, aceC, aceD and aceE. Comparison of translated gene sequences to the databanks was used to assign putative gene functions. AceB displayed strong homology to a glucose-diphosphoprenyl beta, D-glucose transferase from Xanthomonas campestris, while AceC was homologous to a cellobiosyl-diphosphoprenyl alpha, D-mannose transferase from the same organism. Thus these genes encode enzymes catalyzing the second and third steps of the acetan biosynthetic pathway. AceD and AceE were homologous to ExoP and ExoT respectively from Rhizobium meliloti and are likely to be involved in acetan polymerization and export.
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22. |
( 1996 ) Towards a classification of glycosyltransferases based on amino acid sequence similarities: prokaryotic alpha-mannosyltransferases. PMID : 8761462 : DOI : 10.1042/bj3180133 PMC : PMC1217598 Abstract >>
A number of genes encoding bacterial glycosyltransferases have been sequenced during the last few years, but their low sequence similarity has prevented a straightforward grouping of these enzymes into families. The sequences of several bacterial alpha-mannosyltransferases have been compared using current alignment algorithms as well as hydrophobic cluster analysis (HCA). These sequences show a similarity which is significant but too low to be reliably aligned using automatic alignment methods. However, a region spanning approx. 270 residues in these proteins could be aligned by HCA, and several invariant amino acid residues were identified. These features were also found in several other glycosyltransferases, as well as in proteins of unknown function present in sequence databases. This similarity most probably reflects the existence of a family of proteins with conserved structural and mechanistic features. It is argued that the present IUBMB classification of glycosyltransferases could be complemented by a classification of these enzymes based on sequence similarities analogous to that which we proposed for glycosyl hydrolases [Henrissat, B. (1991) Biochem. J. 280, 309-316].
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23. |
( 1996 ) Identification, cloning and sequencing the aceA gene involved in acetan biosynthesis in Acetobacter xylinum. PMID : 8935665 : DOI : 10.1111/j.1574-6968.1996.tb08092.x 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 l-phosphate transferase catalyzing the first step in acetan biosynthesis in A. xylinum.
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24. |
( 1996 ) Isolation and nucleotide sequence of the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase gene from Acetobacter xylinum. PMID : 8759843 : DOI : 10.1128/jb.178.16.4814-4821.1996 PMC : PMC178262 Abstract >>
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.
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25. |
( 1994 ) Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization. PMID : 8083166 : DOI : 10.1128/jb.176.18.5735-5752.1994 PMC : PMC196778 Abstract >>
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.
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26. |
( 1994 ) IS1032 from Acetobacter xylinum, a new mobile insertion sequence. PMID : 7991672 : Abstract >>
IS1031 elements constitute a family of related insertion sequences (IS) in Acetobacter xylinum strains. A new IS1031-related element, IS1032, was isolated from A. xylinum ATCC 23770. Southern hybridization analysis showed that one or more sequences similar to IS1032 are present in most of the A. xylinum strains examined. In addition, one copy was detected in Acetobacter aceti ATCC 15973. The transposition of IS1032 was evident from the appearance of an extra insertion in a spontaneous exopolysaccharide-negative mutant of A. xylinum ATCC 23770. IS1032 consists of 916 bp and has imperfect terminal inverted repeats of 14 bp (IR-Left) and 16 bp (IR-Right). A 3-bp target sequence is duplicated upon insertion. IS1032 displays a single open reading frame, encoding a putative 276-amino-acid protein sharing 58% identity with the corresponding protein encoded by IS1031. Thus, IS1032 is a member of the IS1031 family in A. xylinum. A striking degree of nucleotide sequence similarity between IS1032 and ISRm4 from Rhizobium meliloti was found. Furthermore, the IS1031-family transposases also display stretches of amino acid sequence similarities with putative transposases encoded by IS elements from other species.
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27. |
Saxena IM,
Brown RM,
Fevre M,
Geremia RA,
Henrissat B,
( 1995 ) Multidomain architecture of beta-glycosyl transferases: implications for mechanism of action. PMID : 7883697 : DOI : 10.1128/jb.177.6.1419-1424.1995 PMC : PMC176755 Abstract >>
N/A
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28. |
( 1994 ) Nucleotide sequence and expression analysis of the Acetobacter xylinum phosphoglucomutase gene. PMID : 8025683 : DOI : 10.1099/13500872-140-5-1183 Abstract >>
The Acetobacter xylinum gene (celB) encoding phosphoglucomutase (EC 5.4.2.2) has previously been cloned by complementation of cellulose-negative mutants. In the present report the nucleotide sequence of a 2.0 kb DNA fragment containing celB is described. Expression analysis using the bacteriophage T7 RNA polymerase promoter phi 10 resulted in identification of a probable translational start codon of celB, and this conclusion was confirmed by N-terminal amino acid sequencing of the recombinant protein. From the nucleotide sequence data it was deduced that celB encodes a protein with a calculated molecular mass of 59.6 kDa. A protein of similar size was visualized after in vitro transcription and translation, using the cloned 2.0 kb fragment as template. The results of an amino acid sequence comparison and a biochemical analysis indicated that the CelB protein is structurally and functionally related to the previously characterized human and rabbit phosphoglucomutases.
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29. |
Okamoto T,
Yamano S,
Ikeaga H,
Nakamura K,
( 1994 ) Cloning of the Acetobacter xylinum cellulase gene and its expression in Escherichia coli and Zymomonas mobilis. PMID : 7765731 : Abstract >>
A DNA fragment corresponding to carboxymethylcellulase activity of Acetobacter xylinum IFO 3288 was isolated and cloned in Escherichia coli, and the DNA sequence was determined. The DNA fragment sequenced had an open-reading frame of 654 base pairs that encoded a protein of 218 amino acid residues with a deduced molecular mass of 23,996 Da. The protein encoded in the DNA fragment expressed in E. coli hydrolyzed a carboxymethylcellulose. This gene was subcloned into the shuttle vector [pZA22; Misawa et al. (1986) Agric Biol Chem 50:3201-3203] between Zymomonas mobilis and E. coli. The recombinant plasmid pZAAC21 was introduced into Z. mobilis IFO 13756 by electroporation. The carboxymethylcellulase gene was efficiently expressed in both bacteria, although the level of expression in Z. mobilis was ten times greater than that in E. coli. Approximately 75% of the total carboxymethylcellulase activity detected in Z. mobilis was located in the periplasmic space (outside of the cytoplasmic space). Enzyme activity was not detected in the periplasmic space, but in the cytoplasm of E. coli.
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30. |
( 1998 ) Control of expression by the cellulose synthase (bcsA) promoter region from Acetobacter xylinum BPR 2001. PMID : 9630539 : DOI : 10.1016/s0378-1119(98)00191-7 Abstract >>
The 5' upstream region (about 3.1kb) of the cellulose synthase operon (bcs operon) has been isolated by cloning from Acetobacter xylinum strain BPR 2001. The expression level of the upstream region was determined using sucrose synthase cDNA as a reporter gene in the shuttle vector pSA19. The expression occurred with the 1.1-kb upstream sequence from the ATG start codon of the bcs operon but not with the 241-bp upstream sequence in A. xylinum, although neither the 1.1-kb nor the 241-bp upstream sequence caused any expression as a promoter in Escherichia coli. The level of expression with the 1. 1-kb upstream sequence in A. aceti was 75% of that in A. xylinum. These results suggest that the upstream region functions as a specific promoter for the Acetobacter genus. The expression was reduced by the introduction of the 241-bp upstream region between the lac promoter and the reporter gene in E. coli and was not detected in A. xylinum. This suggests that the short upstream region composed of 241bp contains the site(s) which causes a negative regulation on the transcription for bcs operon. The production of recombinant protein with the ribosome-binding site (RBS) of A. xylinum obtained from the bcs operon, was reduced to about half in E. coli, and that with the site of the lac promoter was also reduced to about half in A. xylinum. This shows that a species-specific predominance occurs during interaction between mRNA and 16S rRNA in the RBS between A. xylinum and E. coli.
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31. |
( 1998 ) Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes. PMID : 9721278 : PMC : PMC107450 Abstract >>
Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of beta-1,4-glucan (cellulose) synthase in Acetobacter xylinum. The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate, cdg1, cdg2, and cdg3. Within each cdg operon, a pdeA gene lies upstream of a dgc gene. cdg1 contains two additional flanking genes, cdg1a and cdg1d. cdg1a encodes a putative transcriptional activator, similar to AadR of Rhodopseudomonas palustris and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the cdg operons, such that cdg1 contributes 80% of cellular DGC and PDEA activities and cdg2 and cdg3 contribute 15 and 5%, respectively. Disruption of dgc genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process.
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