产SEB金黄色葡萄球菌α-溶血毒素基因缺失菌株的构建

构建产肠毒素B(Staphylococcal enterotoxin B ,SEB)的金黄色葡萄球菌α-溶血毒素（α-hemolysin, α-HL)缺失菌株。首先构建用于α-HL基因敲除的同源重组质粒pMHL-α,经金黄色葡萄球菌RN4220修饰后再通过原生质体转入金黄色葡萄球菌SM-01。含重组质粒pMHL-α的金黄色葡萄球菌SM-01在42℃诱导条件下培养多代,最终筛选出α-溶血毒素基因缺失菌株。经序列分析和血平板溶血实验结果证明最终获得产SEB金黄色葡萄球菌α-HL缺失菌株。为野生型金黄色葡萄球菌的体内遗传操作及构建产超抗原药物金黄色葡萄球菌基因工程菌株提供了一定的理论基础和方法。     

Diversity and chemotaxis of soil bacteria with antifungal activity against Fusarium wilt of banana

The chemotactic response of bacteria to root exudates plays an important role in the colonization of bacteria in the rhizosphere. In this study, 420 strains of antifungal bacteria against Fusarium oxysporum f. sp. cubense (Foc) were screened for chemotaxis based on a cheA molecular diagnostic method. A total of 124 strains with antifungal efficiencies of 27.26-67.14?% generated a characteristic band of cheA. The chemotaxis of 97 bacterial strains producing a cheA band was confirmed using the drop assay and swarm plate assay using catechol, p-hydroxybenzoic acid, salicylic acid, and asparagine as the attractants. A phylogenetic analysis based on restriction fragment length polymorphisms (RFLPs) and 16S rDNA sequences indicated that the 124 chemotactic antagonists of Foc were affiliated with 18 species of Paenibacillaceae, Bacillaceae, Streptomycineae, Enterobacteriaceae, and Pseudomonadaceae. The chemical composition of banana root exudates were analyzed by GC-MS, and 62 compounds, including alkanes, alkenes, naphthalenes, benzenes, and alcohols, were evaluated. Five representative antagonists of Foc showed 1.76- to 7.75-fold higher chemotactic responses than the control to seven compounds in banana root exudates, as determination by capillary assays.     

Coagulase gene polymorphism of Staphylococcus aureus isolated from goat mastitis in Brazilian dairy herds

AIMS: To investigate the coagulase gene polymorphism of Staphylococcus aureus isolated from mastitic goat's milk. METHODS AND RESULTS: A typing procedure based on coagulase gene polymorphism was used to discriminate S. aureus isolated from goat mastitis. Thirty-six strains collected from goats belonging to herds from northern Ceara State and Serrana region of Rio de Janeiro State were analysed. Based on restriction fragment length polymorphism of 3' end coagulase gene, the goat strains were grouped into 11 types. In northern Ceara herds, the predominant type was found in 60% of the strains, while in the Serrana region herds the two most common accounting for 62.5% of the strains. CONCLUSIONS: The analysis of the coa gene proved to be useful for typing S. aureus from goat mastitis. Although the results showed that goats from the studied regions were infected by S. aureus strains harbouring more than one coagulase genotype, only one or two types predominated. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of the prevalent strains within a herd or region is a necessity. The important virulence factors could be identified in such strains and this information can then be used as a specific base to develop S. aureus mastitis control measures.     

Identification and characterization of a monofunctional glycosyltransferase from Staphylococcus aureus

A gene (mgt) encoding a monofunctional glycosyltransferase (MGT) from Staphylococcus aureus has been identified. This first reported gram-positive MGT shared significant homology with several MGTs from gram-negative bacteria and the N-terminal glycosyltransferase domain of class A high-molecular-mass penicillin-binding proteins from different species. S. aureus MGT contained an N-terminal hydrophobic domain perhaps involved with membrane association. It was expressed in Escherichia coli cells as a truncated protein lacking the hydrophobic domain and purified to homogeneity. Analysis by circular dichroism revealed that secondary structural elements of purified truncated S. aureus MGT were consistent with predicted structural elements, indicating that the protein might exhibit the expected folding. In addition, purified S. aureus MGT catalyzed incorporation of UDP-N-acetylglucosamine into peptidoglycan, proving that it was enzymatically active. MGT activity was inhibited by moenomycin A, and the reaction product was sensitive to lysozyme treatment. Moreover, a protein matching the calculated molecular weight of S. aureus MGT was identified from an S. aureus cell lysate using antibodies developed against purified MGT. Taken together, our results suggest that this enzyme is natively present in S. aureus cells and that it may play a role in bacterial cell wall biosynthesis.     

Molecular analysis of microbial population transition associated with the start of denitrification in a wastewater treatment process

AIMS: The objective of this study is to determine the bacteria playing an important role in denitrification by monitoring the molecular dynamics accompanying the start of denitrification. METHODS AND RESULTS: cDNA reverse-transcribed from 16S rRNA was amplified with fluorescent labelled primer for terminal restriction fragment length polymorphism (T-RFLP) analysis and an unlabelled primer for cloning analysis. The terminal restriction fragments (T-RFs) that increased in association with the start of denitrification were determined. These T-RFs were identified by in silico analysis of 16S rRNA sequences obtained from cloning. As a result, it was clearly observed that the bacteria belonging to the genera Hydrogenophaga and Acidovorax increased in number after the start of denitrification. CONCLUSIONS: It was demonstrated that T-RFLP analysis targeting 16S rRNA is appropriate for the daily monitoring of a bacterial community to control wastewater treatment processes. Combination of the results of T-RFLP analysis and 16S rRNA clone library indicated that the bacteria belonging to the genera Hydrogenophaga and Acidovorax play an important role in denitrification. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide new insight to the 16S rRNA level of active denitrifying bacteria in wastewater treatment processes.     

金黄色葡萄球菌hmp基因缺失突变株的构建及抗一氧化氮能力分析

摘要：【目的】:构建金黄色葡萄球菌RN6390黄素血红蛋白(flavohaemoglobin, HMP)基因缺失突变株,研究其抗一氧化氮(Nitric Oxide, NO) 能力及其在细菌生物被膜形成中的作用。【方法】：根据同源重组技术的原理,利用PCR扩增RN6390的hmp基因上下游同源臂,经过抗生素和温度交替培养筛选hmp基因缺失突变株,利用基因组PCR、定量PCR对突变菌株进行鉴定。以硝普钠（SNP）为NO供体,检测了hmp基因缺失菌株的抗NO能力,并初步研究了hmp基因在生物被膜形成中的作用。【结果】：成功构建了RN6390的hmp基因缺失突变株,外源NO能够诱导菌株hmp基因的表达,hmp基因缺失菌株抗NO能力明显下降,但其生物被膜形成能力有明显提高。【结论】：获得了RN6390的hmp基因缺失突变株,该突变株的获得为进一步研究hmp基因的生物功能,以及细菌内源性NO的作用奠定了良好的技术平台。     

Interactions between cationic liposomes and bacteria: the physical-chemistry of the bactericidal action.

The bactericidal effect of dioctadecyldimethylammonium bromide (DODAB), a liposome forming synthetic amphiphile, is further evaluated for Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in order to establish susceptibilities of different bacteria species towards DODAB at a fixed viable bacteria concentration (2.5 x 10(7) viable bacteria/mL). For the four species, susceptibility towards DODAB increases from E. coli to S. aureus in the order above. Typically, cell viability decreases to 5% over 1 h of interaction time at DODAB concentrations equal to 50 and 5 microm for E. coli and S. aureus, respectively. At charge neutralization of the bacterial cell, bacteria flocculation by DODAB vesicles is shown to be a diffusion-controlled process. Bacteria flocculation does not yield underestimated counts of colony forming units possibly because dilution procedures done before plating cause deflocculation. The effect of vesicle size on cell viability demonstrates that large vesicles, due to their higher affinity constant for the bacteria (45.20 m(-)) relative to the small vesicles (0.14 m(-)), kill E. coli at smaller DODAB concentrations. For E. coli and S. aureus, simultaneous determination of cell viability and electrophoretic mobility as a function of DODAB concentration yields a very good correlation between cell surface charge and cell viability. Negatively charged cells are 100% viable whereas positively charged cells do not survive. The results show a clear correlation between simple adsorption of entire vesicles generating a positive charge on the cell surfaces and cell death.     

Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9

A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.     

Rapid genetic screening for hemochromatosis using automated SSCP-based capillary electrophoresis (SSCP-CE).

Hereditary hemochromatosis (HHC) represents an autosomal recessive disease in which increased iron absorption causes iron overload and irreversible tissue damage. The recently detected association between two point mutations in the HFE gene on chromosome 6p and HHC has made it possible to screen for the disease before the onset of irreversible tissue damage. Conventional genetic testing is based on restriction fragment-length polymorphisms (RFLP) using two endonuclease recognition sites in codon 63 or 282, respectively. In this study, we have adapted single-strand conformation polymorphism analysis for capillary electrophoresis (SSCP-CE) to detect homozygote or heterozygote point mutations. Two HFE gene fragments spanning codons 63 and 282 were amplified by a duplex PCR using genomic DNA from peripheral blood or from tissue sections of paraffin-embedded liver biopsies as template. Thereby, rapid genotyping of both HFE mutations was achieved with a single PCR, omitting the need of further analysis by restriction digest. Eighty-five patients with liver disease and/or suspected iron overload were genotyped using SSCP-CE, and all results were verified by conventional RFLP analysis. In summary, SSCP-CE proved to be a reliable, cost-effective, sensitive and rapid method for genotyping HFE mutations. This method will further facilitate high-throughput genetic screening using capillary array electrophoretic devices.     

金黄色葡萄球菌甲氧西林耐药辅助基因femB的克隆和原核表达

金黄色葡萄球菌femB基因与甲氧西林高水平耐药密切相关,可能成为开发抗MRSA药物的新靶位.以金葡菌基因组DNA为模板,PCR扩增femB全长基因,所得片段与pGM-T载体连接并转化感受态大肠杆菌DH5α,阳性克隆以PCR、双酶切及测序鉴定.将鉴定正确的目的片段定向克隆到pGEX-4T-1表达载体中,转化至大肠杆菌BL21后经IPTG诱导表达GST/FemB融合蛋白;采用SDS-PAGE及Western blot对融合蛋白进行鉴定.结果显示,重组质粒在宿主菌中获得了高效表达,融合蛋白相对分子质量为75 kD,该融合蛋白可与抗GST-tag抗体特异结合;表明femB基因的原核表达系统构建成功,为进一步研究其生物学功能奠定了基础.     

Structural evidence of α-aminoacylated lipoproteins of Staphylococcus aureus

Bacterial lipoproteins are known to be diacylated or triacylated and activate mammalian immune cells via Toll-like receptor 2/6 or 2/1 heterodimer. Because the genomes of low G+C content gram-positive bacteria, such as Staphylococcus aureus, do not contain Escherichia coli-type apolipoprotein N-acyltransferase, an enzyme converting diacylated lipoproteins into triacylated forms, it has been widely believed that native lipoproteins of S. aureus are diacylated. However, we recently demonstrated that one lipoprotein SitC purified from S. aureus RN4220 strain was triacylated. Almost simultaneously, another group reported that another lipoprotein SA2202 purified from S. aureus SA113 strain was diacylated. The determination of exact lipidated structures of S. aureus lipoproteins is thus crucial for elucidating the molecular basis of host-microorganism interactions. Toward this purpose, we intensively used MS-based analyses. Here, we demonstrate that SitC lipoprotein of S. aureus RN4220 strain has two lipoprotein lipase-labile O-esterified fatty acids and one lipoprotein lipase-resistant fatty acid. Further MS/MS analysis of the lipoprotein lipase digest revealed that the lipoprotein lipase-resistant fatty acid was acylated to α-amino group of the N-terminal cysteine residue of SitC. Triacylated forms of SitC with various length fatty acids were also confirmed in cell lysate of the RN4220 and Triton X-114 phase in three other S. aureus strains, including SA113 strain and one Staphylococcus epidermidis strain. Moreover, four other major lipoproteins including SA2202 in S. aureus strains were identified as N-acylated. These results strongly suggest that lipoproteins of S. aureus are mainly in the N-acylated triacyl form.     

Characterization of Staphylococcus aureus strains isolated from raw milk of bovine subclinical mastitis in Tehran and Mashhad

Staphylococcus aureus is considered one of the most important food borne pathogens. A total of 111 isolates of S. aureus were cultured from raw milk samples during January 2009 to June 2009 from Tehran and Mashhad. The coagulase gene polymorphism and the prevalence of classical enterotoxin genes of S. aureus strains were determined by PCR-RFLP (restriction fragment length polymorphism) and Multiplex-PCR. Disk diffusion method was used to determine the susceptibility of isolates to antimicrobial agents as instructed by Clinical and Laboratory Standards Institute. Sixty-seven % of the isolates harboured one or more enterotoxin genes. The most prevalent gene was sec, found in 59 % of the isolates. Approximately 8% of the isolates were positive for sea, seb and sed genes. Only one isolate had see gene. The rate of coexistence of enterotoxin genes was 14%. All S. aureus isolates were susceptible to ciprofloxacin, gentamicin, imipenem, minocycline, oxacillin and vancomycin. They were resistant to ampicillin (64%), penicillin (56%), clindamycin (22%), tetracycline (22%), doxycycline (19%), teicoplanin (13%), rifampin (2%) and trimethoprim-sulfamethoxazole (2%). On the basis of coagulase gene analysis of 111 S. aureus isolates, the PCR products of 56 isolates were digested with Alu I that produced three distinct patterns. These data indicate the high prevalence of enterotoxigenic S. aureus in raw bovine milk in Tehran and Mashhad, and highlight the importance of proper quality control of dairy products for public health.     

A Staphylococcus aureus plasmid that specifies constitutive macrolide-lincosamide-streptogramin B resistance contains a novel deletion in the ermC attenuator

A 2.5 kb plasmid, pA22, isolated from a naturally occurring S. aureus strain confers constitutive MLS-resistance. By restriction enzyme analysis, pA22 is indistinguishable from the S. aureus inducible MLS-resistance conferring plasmid, pT48, apart froma small deletion. DNA sequencing showed that the deletion, is in the leader/attenuator region of the ermC (MLS-resistance) gene and removes some of the complementary repeat regions required by the translational attenuation model in pT48 for inducible ermC expression. The deletion in plasmid pA22 is different from that found in similar 2.5kb constitutive MLS-resistance plasmids in other Gram-positive bacteria. It is suggested that plasmids conferring the constitutive phenotype have evolved from an inducible ancestor on several independent occasions.     

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.     

美洲大蠊成虫肠道抗细菌共生放线菌的筛选及鉴定

【目的】鉴于野外美洲大蠊Periplaneta americana对恶劣环境适应性强,本研究旨在从云南省大理州的野外美洲大蠊成虫肠道中分离、筛选出抗细菌活性放线菌,为抗生素开发提供菌种资源。【方法】采用涂布平板法和平板划线法对美洲大蠊成虫肠道放线菌进行分离;以金黄色葡萄球菌Staphylococcus aureus、耐甲氧西林金黄色葡萄球菌、铜绿假单胞菌Pseudomonas aeruginosa、粪肠球菌Enterococcus faecalis、大肠杆菌Escherichia coli和鼠伤寒沙门氏菌Salmonella typhimurium共6种人体病原细菌为指示菌株,采用牛津杯法对分离自这些放线菌的次生代谢产物进行抗菌活性测定;通过形态学特征和16S rRNA基因序列分析,对具有广谱和明显抗菌活性的放线菌进行鉴定,并经16SrRNA基因序列的BlAST同源性比对及系统发育分析确定它们的分类地位。【结果】从美洲大蠊成虫肠道共分离获得41株放线菌。抗菌活性测定结果表明,34株(82.9%)放线菌对至少1种指示病原细菌具有抑制作用,其中有7株对3种以上病原细菌具有抑制作用,9株表现...     

Enhanced conjugative transfer of plasmid DNA from Escherichia coli to Staphylococcus aureus and Listeria monocytogenes

Abstract Transfer of mobilizable shuttle cloning vectors by conjugation from Escherichia coli to Staphylococcus aureus occurred at a very low frequency (10⁻⁹ transconjugants per donor colony-forming unit after the mating period). It was observed that subinhibitory concentrations of penicillins (oxacillin or penicillin G) in the mating medium resulted in increased transfer frequency by conjugation of the shuttle vector pAT18 from E. coli SM10 to S. aureus 80CR5 Str (54-fold) and to Listeria monocytogenes LO17RF (45-fold). These results were interpreted as indicating that the cell wall of Gram-positive bacteria constitutes an important barrier for conjugative transfer of genetic information demonstrated that presence of a restriction system(s) in S. aureus recipients represented a major barrier to introduction of foreign DNA.     

Direct and fast capillary zone electrophoretic method for the determination of Gleevec and its main metabolite in human urine

A capillary zone electrophoretic (CZE) method was investigated for the determination of Gleevec and its main metabolite (N-demethylated piperazine derivative) in human urine using a fused-silica capillary (75 microm I.D.x60 cm total length, 10 cm effective length). The separation was performed with an hydrodynamic injection time of 10 s (0.5 p.s.i.) a voltage of -25 kV, a capillary temperature of 25 degrees C and a 100 mM phosphoric acid adjusted to pH 2 with the addition of triethanolamine. Under these conditions, the analysis takes about 5 min. A linear response over the 0.4-30.0 mg l(-1) concentration range was investigated for two compounds. A dilution of the sample was the only step necessary before the electrophoresis analysis. Detection limits of 0.1 mg l(-1) for Gleevec and its metabolite (S/N=3) were obtained. The developed method is easy, rapid and sensitive and has been applied to determine Gleevec and its main metabolite in clinical urine samples.     

ADSRRS-fingerprinting and PCR MP techniques for studies of intraspecies genetic relatedness in Staphylococcus aureus

The present study was designed to evaluate the usefulness of two novel molecular typing methods, amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and the PCR melting profile (PCR MP), for Staphylococcus aureus strain differentiation. Thirty-seven S. aureus strains isolated from patients with a history of furunculosis were studied. The strains were identified by determining several phenotypic properties and were genotyped using three differentiation methods: macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE), ADSRRS-fingerprinting, and PCR MP technique. In some cases the results obtained showed that the S. aureus isolated from the nose was identical to the one from the furuncle of the same patient. The same genotype was also identified for S. aureus strains isolated from two different members of a family with a history of recurrent furunculosis, although the active lesions were present in only one of them when the investigation was done. Results from strain genotyping illustrated that the recently developed ADSRRS-fingerprinting and PCR MP techniques are useful for studies of intraspecies genetic relatedness of S. aureus strains. They are as effective in discriminating closely related strains as the PFGE method, which is currently considered to be "the gold standard" for epidemiological studies.