Integrating balanced mevalonate pathway into chromosome for improving lycopene production in Escherichia coli / 生物工程学报

Isoprenoids are all derived from two five-carbon building blocks called isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized either by the mevalonate (MVA) pathway or 2-C-methyld-D-erythritol-4-phosphate (MEP) pathway. In this study, the MVA pathway genes were integrated into the chromosome of LYC101, in which the expression of key genes in the MEP synthesis pathway and lycopene synthesis pathway were optimized by artificial regulatory parts, to further improve the production of isoprenoids in Escherichia coli. The plasmids pALV23 and pALV145 were screened from a plasmid library that constructed by using the RBS library to link the genes of the MVA pathway, which greatly increased the production of β-carotene. The effects of plasmids pALV23 and pALV145 on the lycopene production in low and high lycopene production strain, LYC001 and LYC101, were compared, respectively. The production of lycopene was increased by plasmids pALV23 and pALV145 in both strains. In high lycopene production strain LYC101, pALV23 produced more lycopene than pALV145. Then, the MVA gene together of promoter of pALV23 was integrated into the chromosome of LYC101 at poxB site using method of homologous recombination helped by CRISPR-Cas9 system, resulted in genetically stable strain, LYC102. The yield of lycopene of LYC102 was 40.9 mg/g DCW, 1.19-folds higher than that of LYC101, and 20% more than that of LYC101 with pALV23. Simultaneous expression of MVA pathway and MEP pathway in recombinant E. coli can effectively increase the yield of terpenoids. In this study, a plasmid-free, genetically stable, high-yielding lycopene strain was constructed, which could be used for industrialization. Also, the platform strain can be used for the synthesis of other terpenoids.

Bases moleculares de la resistencia a meticilina en Staphylococcus aureus / Molecular basis of methicillin-resistance in Staphylococcus aureus

Resumen Desde el inicio de la era antimicrobiana se han ido seleccionando gradualmente cepas de Staphylococcus aureus resistentes a antimicrobianos de amplio uso clínico. Es así como en 1960 se describen en Inglaterra las primeras cepas resistentes a meticilina, y algunos años después son informadas en hospitales de Chile. Actualmente, S. aureus resistente a penicilinas antiestafilocóccicas es endémico en los hospitales de nuestro país y del mundo, siendo responsable de una alta morbimortalidad. La resistencia es mediada habitualmente por la síntesis de una nueva transpeptidasa, denominada PBP2a o PBP2' que posee menos afinidad por el β-lactámico, y es la que mantiene la síntesis de peptidoglicano en presencia del antimicrobiano. Esta nueva enzima se encuentra codificada en el gen mecA, a su vez inserto en un cassette cromosomal con estructura de isla genómica, de los cuales existen varios tipos y subtipos. La resistencia a meticilina se encuentra regulada, principalmente, por un mecanismo de inducción de la expresión del gen en presencia del β-lactámico, a través de un receptor de membrana y un represor de la expresión. Si bien se han descrito mecanismos generadores de resistencia a meticilina mec independientes, son categóricamente menos frecuentes.

Staphylococcus aureus isolates resistant to several antimicrobials have been gradually emerged since the beginning of the antibiotic era. Consequently, the first isolation of methicillin-resistant S. aureus occurred in 1960, which was described a few years later in Chile. Currently, S. aureus resistant to antistaphylococcal penicillins is endemic in Chilean hospitals and worldwide, being responsible for a high burden of morbidity and mortality. This resistance is mediated by the expression of a new transpeptidase, named PBP2a or PBP2', which possesses lower affinity for the β-lactam antibiotics, allowing the synthesis of peptidoglycan even in presence of these antimicrobial agents. This new enzyme is encoded by the mecA gene, itself embedded in a chromosomal cassette displaying a genomic island structure, of which there are several types and subtypes. Methicillin resistance is mainly regulated by an induction mechanism activated in the presence of β-lactams, through a membrane receptor and a repressor of the gene expression. Although mec-independent methicillin resistance mechanisms have been described, they are clearly infrequent.

Characterization of biofilm formation, antimicrobial resistance, and staphylococcal cassette chromosome mec analysis of methicillin resistant Staphylococcus hominis from blood cultures of children

Abstract INTRODUCTION: Methicillin resistant Staphylococcus hominis (MRSHo) has been recognized as an important human pathogen, particularly in immunocompromised patients. METHODS: A total of 19 S. hominis isolates were collected from children at the Children's Medical Centre, Tehran, Iran, from March 2012 to February 2013. MRSHo susceptibility against 13 antimicrobial and 3 antiseptic agents was determined using disk diffusion (DAD) and minimum inhibitory concentration (MIC), respectively. All isolates were subjected to polymerase chain reaction (PCR) assay for 15 distinct resistance genes, staphylococcal cassette chromosome mec (SCCmec), and arginine catabolic mobile elements (ACMEs). Biofilm production of the isolates was determined using a colorimetric microtiter plate assay. RESULTS: Of the 19 isolates, 16 were resistant to oxacillin and harbored mecA. High resistance was also observed against trimethoprim/sulfamethoxazole (81.2%). All MRSHo isolates were susceptible to the three disinfectants tested (Septicidine-PC, Septi turbo, and Sayacept-HP). In total, 15 (78.9%) isolates produced biofilms. Three isolates had SCCmec types (V and VIII), 13 were untypable (UT), and 5 had ACME type II. CONCLUSIONS: The results indicate that MRSHo with high antibiotic resistance and unknown SCCmec might become a serious problem in the future for the treatment of patients such as children.

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity

Abstract: INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

Markerless DNA deletion based on Red recombination and in vivo I-Sec I endonuclease cleavage in Escherichia coli chromosome / 生物工程学报

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.

Tetracycline and trimethoprim/sulfamethoxazole at clinical laboratory: Can they help to characterize Staphylococcus aureus carrying different SCCmec types?

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) can be difficult to detect at the clinical practice. METHODS: We analyzed 140 MRSA isolates from inpatients to correlate the antimicrobial susceptibility with the SCCmec types. RESULTS: Type III (n = 63) isolates were more resistant to ciprofloxacin, clindamycin, cloramphenicol, erythromycin, gentamicin, and rifampin than type IV (n = 65) ones (p < 0.05). Moreover, type IV isolates were susceptible to tetracycline (100%) and trimethoprim/sulfamethoxazole (98%), while type III isolates presented resistance to them. CONCLUSIONS: In regions where these SCCmec types are prevalent, the detection of specific resistant phenotypes could help to predict them, mainly when there are no technical conditions to SCCmec typing.

Progress in markerless knockout of chromosome genes in Streptomycetes / 生物工程学报

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.

The molecular physiological and genetic mechanisms underlying the superb efficacy of quinolones / 药学学报

The fluoroquinolones are the most widely used broad-spectrum antibiotics, accounting for 18% of global antibacterial market share. They can kill bacteria rapidly with variety of derivatives available. Different quinolones vary significantly in rate and spectrum of killing, oxygen requirement for metabolism and reliance upon protein synthesis. Further understanding the sophisticated mechanisms of action of this important antibiotic family based on the molecular genetic response of bacteria can facilitate the discovery of better quinolone derivatives. Factors such as SOS response, bacterial toxin-antitoxin system, programmed death, chromosome fragmentation and reactive oxygen have been implicated in the action to some extent. "Two steps characteristic" of quinolones killing is also emphasized, which might inspire future better quinolones modification.

A new multilocus sequence analysis scheme for Mycobacterium tuberculosis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis (M. tuberculosis) multilocus sequence analysis (MLSA) was established for the phylogenetic and epidemiology analysis.</p><p><b>METHODS</b>To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods.</p><p><b>RESULTS</b>After comparison of the genome, seven high discrimination gene loci (recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR) were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types (STs) were identified. Based on the Hunter-Gaston Index (HGI), MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types (ST) came into a main cluster, showing the major clonal complexes in those 44 strains.</p><p><b>CONCLUSION</b>MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.</p>

Long-range correlations in prokaryotic chromosomes.

One of the fascinating properties of the DNA sequences of prokaryotic and eukaryotic chromosomes is that they possess long-range order. Computational methods like spectral analysis, mutual information and DNA random walks have been used to probe long-range order via-long range correlations. This work attempts to show the advantage of using the Information Theoretic measure of mutual information for this purpose. A number Mu is found which indicates the existence of long-range order. Mu is the ratio between the value of mutual information function between two nucleotides of a DNA sequence separated by a large distance of 100 kilobases to the value expected from a randomized sequence of the same DNA. It is found that in spite of the constant shuffling of nucleotides due to insertion, deletion, inversion and recombination that occur during evolution, the chromosomal structure of prokaryotes is not always mosaic. While all archaeal chromosomes show mosaic structure and lack long-range order, a sizable fraction of the bacterial chromosomes do possess long-range order. A statistical multivariate analysis has been done to find which of the physical variables like genome size or GC% affects the organization of the chromosome or correlates with the long-range order. The existence of long-range order in bacterial chromosomes could be directly correlated to the degree of gene strand bias shown by it. Firmicutes which have low GC content also have pronounced strand bias and show long-range correlations. It is observed that the occurrence of long-range order in bacteria is independent of genome size, but depends on its GC content and gene strand bias.

Determination of staphylococcal exotoxins, SCCmec types, and genetic relatedness of Staphylococcus intermedius group isolates from veterinary staff, companion animals, and hospital environments in Korea

The Staphylococcus (S.) intermedius group (SIG) has been a main research subject in recent years. S. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases. To prevent SIG-associated infection and disease outbreaks, identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec (SCCmec) types among SIG isolates may be helpful. In this study, it was found that a single isolate (one out of 178 SIG isolates examined) harbored the canine enterotoxin SEC gene. However, the S. intermedius exfoliative toxin gene was found in 166 SIG isolates although the S. aureus-derived exfoliative toxin genes, such as eta, etb and etd, were not detected. SCCmec typing resulted in classifying one isolate as SCCmec type IV, 41 isolates as type V (including three S. intermedius isolates), and 10 isolates as non-classifiable. Genetic relatedness of all S. pseudintermedius isolates recovered from veterinary staff, companion animals, and hospital environments was determined by pulsed-field gel electrophoresis. Strains having the same band patterns were detected in S. pseudintermedius isolates collected at 13 and 18 months, suggesting possible colonization and/or expansion of a specific S. pseudintermedius strain in a veterinary hospital.

Structural variation of the superintegron in the toxigenic Vibrio cholerae O1 El Tor / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To understand the genetic structures and variations of the superintegron (SI) in Vibrio cholerae isolated in the seventh cholera pandemic.</p><p><b>METHODS</b>Polymerase chain reaction scanning and fragment sequencing were used. Sixty toxigenic V. cholerae O1 El Tor strains isolated between 1961 and 2008 were analyzed.</p><p><b>RESULTS</b>Some variations were found, including insertions, replacements, and deletions. Most of the deletions were probably the result of recombination between V. cholerae repeat sequences. The majority of the variations clustered together. The SIs of the strains isolated in the 1960s and 1970s showed more diversity, whereas SI cassette variations in strains isolated in the 1990s and after were lower, with ∼24 kb signature sequence deletion. This indicates the predominant SI in the host during the epidemic in the 1990s and after. The insertion cassettes suggested the mobilization from the SIs of other V. cholerae serogroups and Vibrio mimicus.</p><p><b>CONCLUSION</b>The study revealed that structural variations of SIs were obvious in the strains isolated in epidemics in different decades, whereas the divergence was based on syntenic structure of SIs in these El Tor strains. Also, the continuing cassette flows in the SIs of the host strains during the seventh cholera pandemics were displayed.</p>

Investigation of the prevalence of patients co-colonized or infected with methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci in China: a hospital-based study / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Nosocomial infection caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) could lead to increased morbidity and mortality. In 2006, VRE nosocomial spread became a reality in our hospital since the first VRE nosocomial infection in 2003. Little is known about the prevalence of coexistence with VRE and MRSA in the patients. The primary objective of the study was to identify the molecular characteristics of epidemic MRSA clones in our hospital and the prevalence of the coexistence with MRSA and VRE in same patients during the 2-year period, 2006 - 2007.</p><p><b>METHODS</b>The clinical features, laboratory test results, and therapeutic outcomes of 129 cases who isolated MRSA collected from January 2006 to December 2007 were retrospectively analyzed. Polymerase chain reaction (PCR) was used to determine mecA-femB type and staphylococcal cassette chromosome mec (SCCmec) type. All the participants were screened for clinical and microbiological data to identify the coexistence of VRE strains with MRSA.</p><p><b>RESULTS</b>One hundred and twenty-nine MRSA isolates were included in the study: 71 (55%) from the intensive care unit, 35 (27.2%) from the surgical wards and 23 (17.8%) from the medical wards. The most frequent source of isolation of MRSA was sputum (76.7%). From seven patients we isolated MRSA and VRE (E. faecium) simultaneously during their inpatient stay. One hundred and twenty-seven (127/129, 98.4%) MRSA isolates harboured SCCmec type III, only 2 MRSA strains contained SCCmec type II. All of the 129 MRSA isolates remained sensitive to vancomycin, teicoplanin and linezolid. Higher sensitivity rates were noted for chloramphenicol 99.2% (128/129). Only 20.2% (26/129) of the MRSA isolates were sensitive to rifampin. All isolates presented resistance to multiple antimicrobial agents with high minimum inhibitory concentrations (MICs), including: beta-lactams (penicillin, oxacillin, cefoxitin, and cefazolin), tetracycline, erythromycin, gentamicin, and quinolones (ciprofloxacin, levofloxacin, and moxifloxacin).</p><p><b>CONCLUSIONS</b>The predominant MRSA clone at Beijing Chaoyang Hospital from 2006 to 2007 had the type III SCCmec element. All of the MRSA isolates were multiresistant to antimicrobial agents. Emergence of coexistence of MRSA and VRE in the same patient was not rare. Physicians should pay more attention to infections resulting from MRSA and VRE. Aggressive infection control measures should be taken to prevent the transmission of the multidrug resistance organism.</p>

Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3) located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00° values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

Heterologous genes expression on Escherichia coli chromosome lac operon using Red recombination / 生物工程学报

To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.

The involvement of tetA and tetE tetracycline resistance genes in plasmid and chromosomal resistance of Aeromonas in Brazilian strains

This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5 percent of the samples were positive for tetA, and also 37.5 percent were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.

In silico comparison of bacterial strains using mutual information.

Fast-sequencing throughput methods have increased the number of completely sequenced bacterial genomes to about 400 by December 2006, with the number increasing rapidly. These include several strains. In silico methods of comparative genomics are of use in categorizing and phylogenetically sorting these bacteria. Various word-based tools have been used for quantifying the similarities and differences between entire genomes. The simple di-nucleotide frequency comparison, codon specificity and k-mer repeat detection are among some of the well-known methods. In this paper, we show that the Mutual Information function, which is a measure of correlations and a concept from Information Theory, is very effective in determining the similarities and differences among genome sequences of various strains of bacteria such as the plant pathogen Xylella fastidiosa, marine Cyanobacteria Prochlorococcus marinus or animal and human pathogens such as species of Ehrlichia and Legionella. The short-range three-base periodicity, small sequence repeats and long-range correlations taken together constitute a genome signature that can be used as a technique for identifying new bacterial strains with the help of strains already catalogued in the database. There have been several applications of using the Mutual Information function as a measure of correlations in genomics but this is the first whole genome analysis done to detect strain similarities and differences.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Isolates with Toxic Shock Syndrome Toxin and Staphylococcal Enterotoxin C genes / 대한진단검사의학회지

BACKGROUND: Many methicillin-resistant Staphylococcus aureus (MRSA) isolates in Korea possess a specific profile of staphylococcal enterotoxins in that the toxic shock syndrome toxin gene (tst) coexists with the staphylococcal enterotoxin C gene (sec). Because the analysis of staphylococcal cassette chromosome mec (SCCmec), a mobile genetic element mecA gene encoding methicillin resistance, showed that majority of these are SCCmec type II, these MRSA isolates with tst and sec may be genetically related with each other. This study was performed to investigate the genetic relatedness of tstand sec-harboring MRSA strains isolated in Korea by using pulsed-field gel electrophoresis (PFGE). METHODS: A total of 59 strains of MRSA isolates of SCCmec type II possessing tst and sec were selected for PFGE and phylogenetic analyses. These isolates were collected from 13 health care facilities during nationwide surveillance of antimicrobial resistance in 2002. RESULTS: The 59 MRSA isolates were clustered into 11 PFGE types, including one major group of 26 strains (44.1%) isolated from 7 healthcare facilities. Seven PFGE types contained 2 or more isolates each, comprising 55 isolates in total. CONCLUSIONS: Most of SCCmec type II MRSA isolates containing tst and sec showed closely related PFGE patterns. Moreover, MRSA isolates collected from different healthcare facilities showed identical PFGE patterns. These findings suggested a clonal spread of MRSA strains possessing tst and sec in Korean hospitals.

SCCmec typing and detection of VISA-related genes in methicillin-resistant Staphylococcus aureus clinical strains from Kobe University Hospital, Japan.

A total of 50 clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) were collected from Kobe University Hospital in 2003. Molecular typing of SCCmec was performed by multiplex polymerase chain reaction (PCR) and the presence of six genes (vraR, vraG, vraA, vraF, fruA, and fruB) associated with vancomycin (VCM) resistance was examined by simple PCR analysis. Out of 50 MRSA strains isolated 47 strains contained Type II SCCmec and the remaining contained Type IV SCCmec. Thirty seven strains contained pUB110 plasmid. VraA was present in 69% of the strains, vraF in 10%, vraG in 53%, and vraR in 16%. Noteworthy, strains without pUB110 contained vraR in relatively higher frequency (31%) compared with strains with pUB110 (11%).

Allele-specific suppression of the temperature sensitivity of fitA/fitB mutants of Escherichia coli by a new mutation (fitC4): isolation, characterization and its implications in transcription control.

The temperature sensitive transcription defective mutant of Escherichia coli originally called fitA76 has been shown to harbour two missense mutations namely pheS5 and fit95. In order to obtain a suppressor of fitA76, possibly mapping in rpoD locus, a Ts+ derivative (JV4) was isolated from a fitA76 mutant. It was found that JV4 neither harbours the lesions present in the original fitA76 nor a suppressor that maps in or near rpoD. We show that JV4 harbours a modified form of fitA76 (designated fitA76*) together with its suppressor. The results presented here indicate that the fit95 lesion is intact in the fitA76* mutant and the modification should be at the position of pheS5. Based on the cotransduction of the suppressor mutation and/or its wild type allele with pps, aroD and zdj-3124::Tn10 kan we have mapped its location to 39.01 min on the E. coli chromosome. We tentatively designate the locus defined by this new extragenic suppressor as fitC and the suppressor allele as fitC4. While fitC4 could suppress the Ts phenotype of fitA76* present in JV4, it fails to suppress the Ts phenotype of the original fitA76 mutant (harbouring pheS5 and fit95). Also fitC4 could suppress the Ts phenotype of a strain harbouring only pheS5. Interestingly, the fitC4 Ts phenotype could also be suppressed by fit95. The pattern of decay of pulse labelled RNA in the strains harbouring fitC4 and the fitA76* resembles that of the original fitA76 mutant implying a transcription defect similar to that of fitA76 in both these mutants. The implications of these findings with special reference to transcription control by Fit factors in vivo are discussed.