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

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.

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

Streptomycetes are Gram-positive bacteria of Actinomycetales. These organisms can produce many economically important secondary metabolites. With the development of molecular biology, gene sequencing technology and synthetic biology, people gained a better understanding of the Streptomyces family. The means to transform genome on the molecular level is also increasing. By simplifying the Streptomyces genome rationally and efficiently, it will improve the yield and quality of the metabolites as well as reduce the consumption of the substrates. Markerless knockout is an important way to carry out genetic modification. Here we describe novel genome modification techniques developed for Streptomyces in recent years with focus on the markerless knockout technologies.
Chromosomes, Bacterial ; genetics ; Gene Knockout Techniques ; methods ; Genes, Bacterial ; genetics ; Streptomyces ; genetics

Antimicrobial resistance has become a major public health issue of global concern. Conjugation is an important way for fast spreading drug-resistant plasmids, during which the type Ⅳ pili plays an important role. Type Ⅳ pili can adhere on the surfaces of host cell and other medium, facilitating formation of bacterial biofilms, bacterial aggregations and microcolonies, and is also a critical factor in liquid conjugation. PilV is an adhesin-type protein found on the tip of type Ⅳ pili encoded by plasmid R64, and can recognize the lipopolysaccharid (LPS) molecules that locate on bacterial membrane. The shufflon is a clustered inversion region that diversifies the PilV protein, which consequently affects the recipient recognition and conjugation frequency in liquid mating. The shufflon was firstly discovered on an IncI1 plasmid R64 and has been identified subsequently in plasmids IncI2, IncK and IncZ, as well as the pathogenicity island of Salmonella typhi. The shufflon consists of four segments including A, B, C, and D, and a specific recombination site named sfx. The shufflon is regulated by its downstream-located recombinase-encoding gene rci, and different rearrangements of the shufflon region in different plasmids were observed. Mobile colistin resistance gene mcr-1, which has attracted substantial attentions recently, is mainly located in IncI2 plasmid. The shufflon may be one of the contributors to fast spread of mcr-1. Herein, we reviewed the discovery, structure, function and prevalence of plasmid mediated shufflon, aiming to provide a theoretical basis on transmission mechanism and control strategy of drug-resistant plasmids.
Plasmids/genetics* ; Proteins/genetics* ; Bacteria/genetics* ; Recombinases ; Genes, Bacterial ; Anti-Bacterial Agents

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

The minimum life is one of the most important research topics in synthetic biology. Minimizing a genome while at the same time maintaining an optimal growth of the cells is one of the important research objectives in metabolic engineering. Here we propose a genome minimization method based on genome scale metabolic network analysis. The metabolic network is minimized by first deleting the zero flux reactions from flux variability analysis, and then by repeatedly calculating the optimal growth rates after combinatorial deletion of the non-essential genes in the reduced network. We applied this method to the classic E. coli metabolic network model ---iAF1260 and successfully reduced the number of genes in the model from 1 260 to 312 while maintaining the optimal growth rate unaffected. We also analyzed the metabolic pathways in the network with the minimized number of genes. The results provide some guidance for the design of wet experiments to obtain an E. coli minimal genome.
Escherichia coli ; genetics ; metabolism ; Genes, Bacterial ; Genome, Bacterial ; genetics ; Metabolic Engineering ; Metabolic Networks and Pathways

OBJECTIVETo identify the differential genes in Porphyromonas gingivalis (P.gingivalis) highly toxic strain W83 and minimally toxic strain ATCC 33277.

METHODSUsing suppression subtractive hybridization (SSH) to compare P.gingivalis highly toxic strain W83 (tester) and minimally toxic strain ATCC 33277 (driver). The chromosomal DNAs were purified from P.gingivalis W83 and P.gingivalis ATCC 33277, and digested by restriction enzyme RsaI. The tester DNA samples were separated and ligated with adaptor 1 and adaptor 2R. Two subtractive hybridization and PCR profile were performed. Tester-specific DNAs also were selectively amplified. The mixture of subtracted DNA fragments were ligated with pMD-18T vector and transformed to competent cells E.coli JM109. The differential subtraction library was established. The positive clones were identified by PCR and then sequenced, and searched homologically.

RESULTSSubtractive library which had high subtractive efficiency was successfully set up and 36 positive clones were screened by SSH. The fragments from 88 bp to 372 bp were enriched in P.gingivalis highly toxic strain W83 sequences which were absent from P.gingivalis ATCC 33277. Through dot blot analysis confirmed that all these fragments were present in P.gingivalis W83 but absent from ATCC 33277. The GenBank homology search indicated that among them, several genes were associated with two paralogous regions of the chromosome; Some genes are associated with evasion of P.gingivalis W83; Another gene was related to antibiotic resistance and the products of some genes were virulence and acquisition of peptides.

CONCLUSIONSComparative whole-genome analysis of highly toxic and minimally toxic strains of P.gingivalis has identified the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277. These genes may provide an important clue for studying the mechanism of occurrence and development of periodontal disease.

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