OBJECTIVETo construct an acid-sensitive mutant of Streptococcus mutans (S. mutans) by transposon mutagenesis and to find a new gene related to the acid tolerance of S. mutans.

METHODSThe transposon Tn917 was delivered into S. mutans UA159 by the temperature-sensitive plasmid pTV1-OK bearing Tn917 and transposition of Tn917 was induced after incubation at non-permissive temperature (42 degrees C). Transposants harboring Tn917 in the chromosome were screened for the selection of mutant that had diminished growth at low pH. Southern analysis was performed with EcoRI (no cut within Tn917) digests of S. mutans UA159 and the selected aid-sensitive mutant, with DIG-labeled probe of 4.3 kb KpnI fragment of pTV1-OK containing Tn917. Genetic backcross experiment was performed by transforming the genome of the mutant to another S. mutans strain MT8148 to determine the linkage of Tn917 insertion to the change of phenotype (acid-sensitivity). Comparison of the abilities to grow at low pH, the glycolytic pH drop and killing pH values were done between the acid-sensitive mutant and the parent strain. The asymmetric PCR method was used to obtain the fragment flanking Tn917 and the PCR products were cloned to pMD18-T vector for sequencing.

RESULTSOne mutant that showed no growth at pH 5.0 was isolated from 2 316 transposants and was named as b23. Southern analysis and genetic backcross experiment confirmed the linkage between single Tn917 insertion into the chromosome and the phenotypic change (acid sensitive). b23 was less acid tolerant than UA159 in that it showed poorer growth at low pH and higher glycolytic pH minimum and higher killing pH. BLAST results indicated that Tn917 inserted into the genome of S. mutans UA159 at the site of 996 123 bp.

CONCLUSIONAn acid-sensitive mutant of S. mutans was successfully constructed and a new gene that is responsible for the acid tolerance in S. mutans UA159 was revealed.

Genes, Bacterial ; Streptococcus mutans ; genetics ; Sucrose ; metabolism

Bacterial Typing Techniques ; Genes, Bacterial ; genetics ; Streptococcus pyogenes ; classification ; genetics

Bacteria ; genetics ; Bacterial Proteins ; secretion ; Bacterial Secretion Systems ; Databases, Factual ; Genes, Bacterial ; Genome, Bacterial

OBJECTIVEIn order to determine the function of PG0839 gene from Porphyromonas gingivalis (P. gingivalis) W83 strains, we intended to create a mutant in the PG0839 gene by homologous recombination.

METHODS1 584bp PG0839 gene fragment was amplified, digested by BamH I and EcoR I, purified and ligated to pUC19. The recombinant plasmid was designated as pPG0839-1. The erm cassette (2 101 bp) was inserted into the EcoR V restriction site of the PG0839 gene. The resultant recombinant plasmid, pPG0839-2, was used as a donor in the electroporation of P. gingivalis W83. After electroporated and selected on erythromycin brain heart infusion plates, a single colony was collected and designated as PG0839 gene-defective mutant.

RESULTSA mutant in PG0839 gene was created by insertional inactivation, and inactivation of PG0839 gene was confirmed by restriction endonuclease digestive, sequencing, polymerase chain reaction (PCR) and reverse transcription PCR.

CONCLUSIONA PG0839 gene-defective mutant was created successfully.

Base Sequence ; Genes, Bacterial ; Polymerase Chain Reaction ; Porphyromonas gingivalis

Bacillus subtilis is the focus of both academic and industrial research. Previous studies have reported a number of sequence variations in different B. subtilis strains. To uncover the genetic variation and evolutionary pressure in B. subtilis strains, we performed whole genome sequencing of two B. subtilis isolates, KM and CGMCC63528. Comparative genomic analyses of these two strains with other B. subtilis strains identified high sequence variations including large insertions, deletions and SNPs. Most SNPs in genes were synonymous and the average frequency of synonymous mutations was significantly higher than that of the non-synonymous mutations. Pan-genome analysis of B. subtilis strains showed that the core genome had lower dN/dS values than the accessory genome. Whole genome comparisons of these two isolates with other B. subtilis strains showed that strains in different subspecies have similar dN/dS values. Nucleotide diversity analysis showed that spizizenii subspecies have higher nucleotide diversity than subtilis subspecies. Our results indicate that genes in B. subtilis strains are under high purifying selection pressure. The evolutionary pressure in different subspecies of B. subtilis is complex.
Bacillus subtilis ; genetics ; Evolution, Molecular ; Genes, Bacterial ; Polymorphism, Single Nucleotide

Gene Expression Regulation, Bacterial ; Genes, Regulator ; Humans ; Streptococcus mutans ; genetics

Temperature is an important environmental factor that can greatly influence the cultivation of Auricularia cornea. In this study, lignin peroxidase, laccase, manganese peroxidase, and cellulose in A. cornea fruiting bodies were tested under five different temperatures (20 °C, 25 °C, 30 °C, 35 °C, and 40 °C) in three different culture periods (10 days, 20 days and 30 days). In addition, the V4 region of bacterial 16S rRNA genes in the substrate of A. cornea cultivated for 30 days at different temperatures were sequenced using next-generation sequencing technology to explore the structure and diversity of bacterial communities in the substrate. Temperature and culture days had a significant effect on the activities of the four enzymes, and changes in activity were not synchronized with changes in temperature and culture days. Overall, we obtained 487,694 sequences from 15 samples and assigned them to 16 bacterial phyla. Bacterial community composition and structure in the substrate changed when the temperature was above 35 °C. The relative abundances of some bacteria were significantly affected by temperature. A total of 35 genera at five temperatures in the substrate were correlated, and 41 functional pathways were predicted in the study. Bacterial genes associated with the membrane transport pathway had the highest average abundance (16.16%), and this increased at 35 °C and 40 °C. Generally, different temperatures had impacts on the physiological activity of A. cornea and the bacterial community in the substrate; therefore, the data presented herein should facilitate cultivation of A. cornea.
Bacteria ; Cellulose ; Cornea* ; Fruit ; Genes, Bacterial ; Genes, rRNA ; Laccase ; Lignin ; Manganese ; Membranes ; Peroxidase

The global regulatory gene, nsdA, negatively regulates antibiotics production in Streptomyces coelicolor. Southern blot experiment, using an nsdA fragment of S. coelicolor as probe, indicated that nsdA gene existed in many Streptomyces. Primers were designed based on the published sequences of S. coelicolor and S. avermitilis. PCR amplification and sequencing showed that nsdA in Streptomyces was conservative and that of S. lividans ZX64 has a 100% identity in the nucleotide sequence comparing with that of S. coelicolor A3 (2). The nsdA disrupted mutant of S. lividans was constructed named as WQ2. WQ2 was able to produce actinorhodin but the wild-type strain ZX64 did not, which has a silent gene cluster contributing to the biosynthesis of actinorhodin. However, the ability was lost when another copy of the wild nsdA gene was introduced into WQ2. All the results above indicate that nsdA homologous gene is wildly existent and conserved in Streptomyces. And it plays a role in negatively regulating the actinorhodin synthesis in S. lividans and disruption of it can activate the silent gene cluster.
Anti-Bacterial Agents ; biosynthesis ; Blotting, Southern ; Genes, Bacterial ; physiology ; Genes, Regulator ; physiology ; Multigene Family ; Streptomyces lividans ; genetics

OBJECTIVETo study the changes in expression of quorum sensing gene aba I in Acinetobacter baumannii (AB) strains isolated from burn patients during biofilm formation process, and its influences on the extracellular matrix of biofilm and drug resistance of AB.

METHODSSix drug-resistant and five drug-sensitive AB strains isolated from wound excretion, blood and venous catheter were collected from burn patients hospitalized in Ruijin hospital of Shanghai Jiao Tong University School of Medicine from January to October 2011. The AB standard strain ATCC 19606 was used as control. (1) Clinical strains and standard strain were normally cultured 10, 24, and 48 h respectively in vitro. The bacteria samples were stained with propidium iodide to measure biofilm thickness with confocal laser scanning microscope. (2) Clinical strains and standard strain were cultured in tubes 10, 24, and 48 h respectively in vitro under shaking condition. The bacteria floating in the medium were regarded as free bacteria, while those adhered to the tube wall as the bacteria within biofilm (biofilm bacteria). Relative expression value of genes aba I and pgaB was detected by real-time fluorescent quantitative PCR with the expression value of the standard strain set at 1. Data were processed with analysis of variance.

RESULTS(1) At post culture hour (PCH) 10, 24, 48, biofilm thickness of clinical strains was thicker than that of standard strain; biofilm thickness of drug-resistant strains [(28.8 ± 0.6), (31.7 ± 1.1), and (38.1 ± 3.1) µm] was respectively thicker than that of drug-sensitive strains [(17.1 ± 0.4), (20.1 ± 1.6), and (25.8 ± 1.7) µm, with F value respectively 1274.38, 206.60, and 61.73, P values all below 0.05]. (2) Biofilm bacteria: at PCH 10, 24, 48, expression values of aba I in drug-resistant strains (6.6 ± 1.7, 25.7 ± 3.5, 9.8 ± 3.6) were much higher than those of drug-sensitive strains (2.7 ± 1.0, 15.0 ± 3.5, 4.7 ± 3.2, with F value respectively 21.82, 25.24, and 6.22, P values all below 0.05); expression values of pgaB in drug-resistant strains (37.4 ± 1.1, 44.5 ± 3.6, 33.1 ± 11.5) were obviously higher than those of drug-sensitive strains (14.6 ± 0.8, 20.0 ± 6.9, 18.7 ± 6.8, with F value respectively 1488.44, 57.26, and 6.01, P values all below 0.05). (3) Free bacteria: at PCH 10, 24, 48, there was no significant statistical difference between drug-resistant strains and drug-sensitive strains in expression value of aba I (with F value respectively 0.24, 2.33, and 0.11, P values all above 0.05); expression values of pgaB in drug-resistant strains (13.8 ± 3.8, 12.5 ± 2.9, 23.7 ± 2.1) were obviously higher than those of drug-sensitive strains (7.0 ± 5.9, 5.0 ± 1.3, 15.6 ± 6.7, with F value respectively 5.44, 28.42, and 7.76, P values all below 0.05). (4) Comparison between biofilm bacteria and free bacteria in resistant strains: expression value of aba I in biofilm bacteria at each time point was respectively higher than that of free bacteria (with F value respectively 43.69, 286.61, and 9.98, P values all below 0.05); expression values of pgaB in biofilm bacteria at PCH 10, 24 were higher than those in free bacteria (with F value respectively 214.26 and 283.20, P values below 0.05). (5) Comparison between biofilm bacteria and free bacteria in sensitive strains: expression value of aba I in BF bacteria at PCH 24 was higher than that of free bacteria (F = 70.28, P < 0.05); expression values of pgaB in biofilm bacteria at PCH 10, 24 were higher than those of free bacteria (with F value respectively 8.03 and 22.62, P values below 0.05).

CONCLUSIONSDuring biofilm formation process, the increasing expression of quorum sensing gene aba I in drug-resistant AB strains isolated from burn patients may up-regulate the expression of gene pgaB, which leads to high production of extracellular matrix and biofilm formation, and enhances drug resistance of AB.

Acinetobacter Infections ; Acinetobacter baumannii ; genetics ; isolation & purification ; Biofilms ; Burns ; microbiology ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans

Xanthomonas campestris pv. campestris (Xcc), the pathogenic agent of black rot disease in cruciferous plants, produces large amount of extracellular polysaccharide (EPS), which has found wide applications in industry. For the great commercial value of the xanthan gum, many of the genes involved in EPS biosynthesis have been cloned and the mechanism of EPS biosynthesis also has been studied. In order to clone genes involved in EPS biosynthesis, Xcc wild-type strain 8004 was mutagenized with transposon Tn5 gusA5, and a number of EPS-defective mutants were isolated in our previous work. The Tn5 gusA5 inserted sites of these mutants were located by using thermal asymmetric interlaced PCR, and results showed that two EPS-defective mutants were insertion mutants of the gene wxcA which involved in lipopolysaccharide (LPS) biosynthesis. The gene wxcA involved in lipopolysaccharide biosynthesis but dose not extracellular polysaccharide in others' report. wxcA::Tn5 gusA5 mutant 021C12, the polar mutant, was complemented with recombinant plasmid pLATC8570 harboring an intact wxcA gene in this work, but the yield of EPS of the wxcA::Tn5 gusA5 mutant was not restored. In order to identify the function of wxcA gene of Xcc 8004 strain, the gene wxcA was deleted by gene replacement strategy, and the no-polar mutant of wxcA was obtained. DeltawxcA mutant strain, named Xcc 8570, was confirmed by using both PCR and southern analysis. Beside the LPS biosynthesis of deltawxcA mutant was affected, The EPS yield of deltawxcA mutant strain reduced by 50% as compared with the wild-type strain 8004. DeltawxcA mutant could be complemented in trans with the intact wxcA gene, and the EPS yield of the mutant was restored. The combined data showed that wxcA gene not only involved in LPS biosynthesis but also EPS yield in Xcc 8004 strain.
Cell Proliferation ; Genes, Bacterial ; physiology ; Lipopolysaccharides ; biosynthesis ; Mutation ; Polysaccharides, Bacterial ; biosynthesis ; Xanthomonas campestris ; genetics