Genome Sequence of a Cellulose-Producing Bacterium,Gluconacetobacter hansenii ATCC 23769

The Gram-negative bacterium Gluconacetobacter hansenii is considered a model organism for studying cellulose synthesis. We have determined the genome sequence of strain ATCC 23769.Plants produce cellulose, an unbranched chain of β-1,4-linked glucose units, as a structural polysaccharide. It is the most abundant polymer on earth, recently receiving much interest due to its potential use as a feedstock for bioethanol. Bacteria also produce cellulose. Among these, Gluconacetobacter hansenii (previously named Acetobacter xylinus) (4) has been extensively characterized and is a model system for cellulose biosynthesis (1, 2, 7). G. hansenii produces extracellular cellulose that is devoid of lignin or hemicellulose, making it an excellent source for pure cellulose. A lack of a completely sequenced genome for this organism has been a limiting factor in identifying other key proteins involved in cellulose synthesis.The whole-genome sequencing of G. hansenii ATCC 23769 was performed using the 454 FLX-Titanium pyrosequencing technology (5). A combinatorial sequencing approach using 489,201 reads obtained from the shotgun library and 195,088 reads from an 8-kb pair end library (3) produced a total of 221,294,116 bp. These reads were assembled using the Newbler assembler, producing 88 large contigs (>500 bp) and a chromosome-sized scaffold of 3,646,142 bp with an average coverage of ×50.5. This scaffold contained exclusively chromosomal DNA and no plasmid sequences. The gaps in the large scaffold were filled by primer walking and subsequent sequencing of the PCR products. The resulting high-quality draft assembly, consisting of a large scaffold with 71 contigs, was annotated using the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) service of the National Institute of Biotechnology Information (NCBI).The chromosomal sequence of G. hansenii 23769 contains 3,547,122 bp, with a G+C content of 59%. The genome contains 3,351 genes, of which 3,308 are protein-encoding genes, accounting for 84% of the genome. There are 43 genes for tRNAs and 2 rRNA loci. The genes encoding proteins involved in cellulose synthesis are in an operon consisting of acsAB (GXY_04277), acsC (GXY_04282), and acsD (GXY_04292), as previously shown by Saxena et al. (7). Interestingly, there are two additional copies of acsAB, GXY_08864 and GXY_14452, which share 69% and 72% sequence identity, respectively, with the acsAB genes in the operon; the deduced amino acid sequences are 40% and 46% identical, respectively, with that deduced from acsAB in the operon. There are also two additional copies of acsC, GXY_08869 and GXY_014472, which share 72% and 65% DNA sequence identity, respectively, with the acsC gene in the operon; the deduced amino acid sequences share 28% and 30% amino acid identity, respectively, with that deduced from acsC. acsAB (GXY_08864) and acsC (GXY_08869) are only 17 bp apart, less than the distance (66 bp) between the acsAB and acsC genes in the operon. acsAB (GXY_14452) and acsC (GXY_14472) are separated by 3,299 bp, with three genes in between. However, acsD is present only in the operon, not duplicated elsewhere in the genome. The genome also contains three genes encoding diguanylate cyclase, as previously reported by Tal et al. (8). Diguanylate cyclase catalyzes the formation of cyclic di-GMP, a second messenger in bacteria that functions as an allosteric activator of cellulase synthase AcsAB (6).     

Isolation and Characterization of the Strains Producing Bacterial Cellulose

Microbiology - Two new strains producing bacterial cellulose were isolated from the Kombucha and Tibicos symbiotic communities. Based on the 16S rRNA gene analysis and the morphological,...     

Characterization of Cellulose Production by a Gluconacetobacter xylinus Strain from Kombucha

The aims of this work were to characterize and improve cellulose production by a Gluconoacetobacter xylinus strain isolated from Kombucha and determine the purity and some structural features of the cellulose from this strain. Cellulose yield in tea medium with both black tea and green tea and in Hestrin and Schramm (HS) medium under both static and agitated cultures was compared. In the tea medium, the highest cellulose yield was obtained with green tea (~0.20 g/L) rather than black tea (~0.14 g/L). Yield in HS was higher (~0.28 g/L) but did not differ between static and agitated incubation. ¹H-NMR and ¹³C-NMR spectroscopy indicated that the cellulose is pure (free of acetan) and has high crystallinity, respectively. Cellulose yield was improved by changing the type and level of carbon and nitrogen source in the HS medium. A high yield of ~2.64 g/L was obtained with mannitol at 20 g/L and corn steep liquor at 40 g/L in combination. In the tea medium, tea at a level of 3 g/L gave the highest cellulose yield and the addition of 3 g/L of tea to the HS medium increased cellulose yield to 3.34 g/L. In conclusion, the G. xylinus strain from Kombucha had different cellulose-producing characteristics than previous strains isolated from fruit. Cellulose was produced in a pure form and showed high potential applicability. Our studies extensively characterized cellulose production from a G. xylinus strain from Kombucha for the first time, indicating both similarities and differences to strains from different sources.     

Isolation and Characterization of Two Groups of Novel Marine Bacteria Producing Violacein

Thirteen strains of novel marine bacteria producing a purple pigment were isolated from the Pacific coast of Japan. They were divided into two groups based on their 16S ribosomal RNA gene sequences, and both groups of bacteria belonged to the genus Pseudoalteromonas. The UV-visible spectrum of the pigment was identical to those of violacein, a pigment produced by several species of bacteria including Chromobacterium violaceum, an opportunistic pathogen. Further analysis of the chemical structure of the pigment by mass spectroscopy and nuclear magnetic resonance spectroscopy showed that the pigment was violacein. The high purity of violacein in the crude extract enabled us to employ simple and nonpolluting procedures to purify the pigment. Isolated bacteria may be useful as a C. violaceum substitute for the safe production of violacein.     

Some Properties of Two Autolytic-Defective Mutants of Streptococcus faecalis ATCC 9790

The isolation and some properties of two mutants of Streptococcus faecalis ATCC 9790 (S. faecium) which autolyze at a much slower rate than the wild type are described. Compared with the wild type, mutant E71 autolyzed more slowly, contained less active but more latent autolysin in the isolated wall fraction, and possessed a wall of very similar chemical composition and degree of cross-bridging. Ultrastructural studies of exponential phase cells showed that cells of E71 were on the average slightly longer and had slightly thickened walls compared to the wild type. Mutant E81 autolyzed much more slowly, grew exponentially in long chains (8 to 40 cells compared with mainly diplococci), contained much less active and latent autolysin in the wall, and possessed a wall of very similar chemical composition but with about twice the content of N-terminal groups. Mutant E81 walls were more susceptible to isolated autolysin but possessed an autolysin of the same specificity as the wild type. Ultrastructurally E81 cells were, on the average, significantly longer and had thicker walls than the wild type. Mutant E71 may be partially blocked at either transport of autolysin to the wall or in conversion of latent to active autolysin. The pleitropic effects noted in mutant E81 have been taken to suggest a possible membrane defect and to support the role of the autolysin in cell separation.     

Isolation and Characterization of Aminopeptidase-Deficient Lactobacillus bulgaricus Mutants

Lactobacillus bulgaricus CNRZ 397 is able to hydrolyze many amino-acyl- and dipeptidyl-β-naphthylamides. Analysis of heat inactivation kinetics, protease inhibitor effects, and the subcellular location of aminopeptidase (AP) activities from the parental strain and mutant derivatives dificient in alanyl- or leucyl-β-naphthylamide hydrolysis pointed out the existence of four APs. All mutants isolated were totally deficient in AP II, a cell wall metallo-enzyme with a broad substrate specificity but that is specifically responsible for lysyl-AP activity and is characterized by a molecular mass of 95,000 daltons. AP I and AP III are cytoplasmic enzymes that exhibit arginyl-AP activity; both enzymes are inducible during growth in rich peptide MRS medium (Difco Laboratories, Detroit, Mich.). The existence of a fourth AP (AP IV) that is involved in leucyl-AP activity was suggested. Moreover, we showed that X-prolyl-dipeptidyl-AP activity, which was not catalyzed by an AP, involved an enzyme(s) that is controlled by a regulatory mechanism that is common to that of AP II.     

Isolation and Characterization of Sendai Virus Temperature-Sensitive Mutants

Ten temperature-sensitive mutants of Sendai virus, a paramyxovirus, were isolated and partially characterized. The mutants replicated in chicken embryo lung cells at 30 C, but not at 38 C; wild-type virus grew equally well at both temperatures. Complementation tests divided the mutants into seven groups. Six groups synthesized neither infectious virus nor RNA when incubated at 38 C from the beginning of infection. Temperature shift-up experiments demonstrated that three of these complementation groups were blocked in early steps required for RNA synthesis, but these gene functions were not needed throughout the replicative cycle. In contrast, the other three RNA-negative complementation groups were defective throughout the replicative cycle in functions required for virus-specific RNA synthesis. Only one mutant, which complemented all of the above, synthesized RNA but not infectious virus when placed at 38 C; the hemagglutinin of this mutant functioned only at the permissive temperature.     

Characterization of Tn5-Induced Mutants of Xenorhabdus nematophilus ATCC 19061

A negative-selection vector, pHX1, was constructed for use in transposon mutagenesis of Xenorhabdus nematophilus ATCC 19061. pHX1 contains the Bacillus subtilis levansucrase gene which confers sucrose sensitivity. In addition, various Tn5-containing plasmids with different replication origins were transferred by conjugation from Escherichia coli into X. nematophilus ATCC 19061, and one of these plasmids, pGS9, yields Tn5 insertion mutants of X. nematophilus ATCC 19061. By using these two delivery vehicles, more than 250 putative Tn5 insertion mutants of X. nematophilus ATCC 19061 were isolated and were then characterized. Mutants that were altered in bromothymol blue adsorption, ability to lyse sheep erythrocytes, production of antibiotics on a variety of media, and virulence for Galleria mellonella were found.     

乳酸抗性酵母的筛选及其生长特性的研究

以酿酒酵母 (saccharomycesceevisiae)单倍体YNN -2 7(αtep ura )为亲株 ,在含有 4 %乳酸的梯度平板上直接进行紫外线诱变处理 ,筛选到突变株YNN -2 7-2 4。通过对该突变株乳酸抗性产生原因分析、在含有不同浓度的乳酸和潮霉素B(hygromycinB)的YPDL和YPDLH培养基中的重复特性的研究发现 ,该突变株对乳酸和潮霉素B产生的抗性 ,不是因对环境条件的适应而产生 ,而是由基因突变所引起。与突变株YNN2 7-2 4相比 ,乳酸对亲株生长的影响在于延长了其生长的延迟期 ,而其生长速率没有发生改变。用Mini-photo 51 8测定供试菌株在生长过程中的吸光度 ( 660nm)以研究酵母菌的生长特性 ,是一种行之有效的方法 ,具有较高的灵敏度和较好的再现性。     

Cloning and sequencing of the beta-glucosidase gene from Acetobacter xylinum ATCC 23769.

The beta-glucosidase gene (bglxA) was cloned from the genomic DNA of Acetobacter xylinum ATCC 23769 and its nucleotide sequence (2200 bp) was determined. This bglxA gene was present downstream of the cellulose synthase operon and coded for a polypeptide of molecular mass 79 kDa. The overexpression of the beta-glucosidase in A. xylinum caused a tenfold increase in activity compared to the wild-type strain. In addition, the action pattern of the enzyme was identified as G3ase activity. The deduced amino acid sequence of the bglxA gene showed 72.3%, 49.6%, and 45.1% identity with the beta-glucosidases from A. xylinum subsp. sucrofermentans, Cellvibrio gilvus, and Mycobacterium tuberculosis, respectively. Based on amino acid sequence similarities, the beta-glucosidase (BglxA) was assigned to family 3 of the glycosyl hydrolases.     

Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii

Environmental protection through biological mechanisms that aid in the reductive immobilization of toxic metals (e.g., chromate and uranyl) has been identified to involve specific NADH-dependent flavoproteins that promote cell viability. To understand the enzyme mechanisms responsible for metal reduction, the enzyme kinetics of a putative chromate reductase from Gluconacetobacter hansenii (Gh-ChrR) was measured and the crystal structure of the protein determined at 2.25 Å resolution. Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions. Kinetic measurements indicate that NADH acts as a substrate inhibitor; catalysis requires chromate binding prior to NADH association. The crystal structure of Gh-ChrR shows the protein is a homotetramer with one bound flavin mononucleotide (FMN) per subunit. A bound anion is visualized proximal to the FMN at the interface between adjacent subunits within a cationic pocket, which is positioned at an optimal distance for hydride transfer. Site-directed substitutions of residues proposed to involve in both NADH and metal anion binding (N85A or R101A) result in 90–95% reductions in enzyme efficiencies for NADH-dependent chromate reduction. In comparison site-directed substitution of a residue (S118A) participating in the coordination of FMN in the active site results in only modest (50%) reductions in catalytic efficiencies, consistent with the presence of a multitude of side chains that position the FMN in the active site. The proposed proximity relationships between metal anion binding site and enzyme cofactors is discussed in terms of rational design principles for the use of enzymes in chromate and uranyl bioremediation.     

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER with Special Attention to Eggshell Mutants

To study genes that function mainly or exclusively during oogenesis, we have isolated and analyzed female-sterile mutations, with special emphasis on those that affect eggshell formation. Following treatment that induced 61 to 66% lethals, 8.1% of the 1071 X chromosomes tested carried recessive female sterility mutations (87 isolates), and 8.0% carried partial female-sterile mutations (86 isolates), respectively. In addition, three dominant female steriles were recovered. Some of the mutants had very low fecundity, and others laid morphologically normal eggs that failed to develop. A third category included 29 mutants that laid eggs with morphological abnormalities: 26 were female steriles, two were partial female steriles and one was fertile. Mutants of this third category were characterized in some detail and compared with 40 previously isolated mutants that laid similarly abnormal eggs. Approximately 28–31 complementation groups with morphological abnormalities were detected, some of which were large allelic series (11, 9, 7, 6 and 5 alleles). Twenty-four groups were mapped genetically or cytogenetically, and 21 were partially characterized by ultrastructural and biochemical procedures. Of the latter, one group showed clear deficiency of yolk proteins, and nine showed prominent ultrastructural defects in the chorion (at least eight accompanied by deficiencies in characterized chorion proteins). At least six groups with clear-cut effects were found at loci not previously identified with known chorion structural genes.     

Isolation and Characterization of NaCl-Sensitive Mutants of Caulobacter crescentus

An attempt to characterize Caulobacter crescentus genes important for the response to high concentrations of NaCl was initiated by the isolation of mutants defective in survival in the presence of 85 mM NaCl. A transposon Tn5 library was screened, and five strains which contained different genes disrupted by the transposon were isolated. Three of the mutants had the Tn5 in genes involved in lipopolysaccharide biosynthesis, one had the Tn5 in the nhaA gene, which encodes a Na⁺/H⁺ antiporter, and one had the Tn5 in the ppiD gene, which encodes a peptidyl-prolyl cis-trans isomerase. All the mutant strains showed severe growth arrest in the presence of 85 mM NaCl, but only the nhaA mutant showed decreased viability under these conditions. All the mutants except the nhaA mutant showed a slightly reduced viability in the presence of 40 mM KCl, but all the strains showed a more severe reduction in viability in the presence of 150 mM sucrose, suggesting that they are defective in responding to osmotic shock. The promoter regions of each disrupted gene were cloned in lacZ reporter vectors, and the pattern of expression in response to NaCl and sucrose was determined; this showed that both agents induced ppiD and nhaA gene expression but did not induce the other genes. Furthermore, the ppiD gene was not induced by heat shock, indicating that it does not belong to the σ³² regulon, as opposed to what was observed for its Escherichia coli homolog.     

Isolation and Characterization of Low-Kynureninase Mutants of Neurospora crassa

Two kynureninase activities are known in Neurospora crassa, one of which (kynureninase I) is inducible, the other (kynureninase II) being constitutive. A method is described for the isolation of low-kynureninase mutants of N. crassa. When grown on an inducer, the mutants show significantly less kynureninase I activity compared with wild type, whereas constitutive kynureninase II activity is unaffected. Since a low level of kynureninase I activity remains in the mutants examined, the mutations may be in a regulatory gene or genes. Other experiments are described concerning the molecular weights of the two enzymes and the intracellular localization and specificity of kynureninase II.     

Isolation and Characterization of Chemotaxis Mutants of Chlamydomonas reinhardtii

Zoospores of Chlamydomonas reinhardtii exhibit chemotaxis towards maltose, sucrose, xylose, mannitol, and ammonium. Ten independent mutants defective in chemotaxis towards sugars have been isolated. These mutants form five phenotypic classes. Genetic analysis of two mutant strains defective in chemotaxis to maltose (CHE1, CHE3) and two mutant strains defective in chemotaxis to sucrose (CHE2, CHE4) indicated that the defect in them depended on single nuclear recessive mutant alleles. Mutations mal1, mal2, suc1, and suc2 represent four chemotactic loci that are unlinked to the marker mt located on the linkage group VI. Four loci are unlinked to each other. These observations suggest that the mal and the suc loci do not constitute a spatially single functional group.     

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad52, rad55 and rad57 were found amoung these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups.