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ABSTRACT: Escherichia coli is a Gram-negative bacterium, commonly used in both teaching and research laboratories. This unit includes protocols for the growth and maintenance of E. coli in any teaching- or research-associated laboratory. Curr. Protoc. Microbiol. 27:5A.4.1-5A.4.9. © 2012 by John Wiley & Sons, Inc.
Current protocols in microbiology 11/2012; Chapter 5:Unit5A.4.
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ABSTRACT: The vibriocidal titer assay can be used to detect antibodies against Vibrio cholerae in serum samples, serving as an indicator of prior infection and potential protection against cholera. The assay can be utilized in research and clinical settings to test the effectiveness of vaccines, and also in epidemiological studies relevant to cholera transmission and surveillance. This unit outlines the steps involved in conducting an easily interpreted colorimetric vibriocidal titer assay with a relatively short turnaround time for results of around 8 hr, with final result observations in 24 hr. The assay can also be easily scaled up or down to accommodate as many or as few serum samples available and is not V. cholerae strain specific.
Current protocols in microbiology 11/2011; Chapter 6:Unit6A.3.
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ABSTRACT: Vibrio cholerae serogroup O1, the causative agent of the diarrheal disease cholera, is divided into two biotypes: classical and El Tor. Both biotypes produce the major virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). Although possessing genotypic and phenotypic differences, El Tor biotype strains displaying classical biotype traits have been reported and subsequently were dubbed El Tor variants. Of particular interest are reports of El Tor variants that produce various levels of CT, including levels typical of classical biotype strains. Here, we report the characterization of 10 clinical isolates from the International Centre for Diarrhoeal Disease Research, Bangladesh, and a representative strain from the 2010 Haiti cholera outbreak. We observed that all 11 strains produced increased CT (2- to 10-fold) compared to that of wild-type El Tor strains under in vitro inducing conditions, but they possessed various TcpA and ToxT expression profiles. Particularly, El Tor variant MQ1795, which produced the highest level of CT and very high levels of TcpA and ToxT, demonstrated hypervirulence compared to the virulence of El Tor wild-type strains in the infant mouse cholera model. Additional genotypic and phenotypic tests were conducted to characterize the variants, including an assessment of biotype-distinguishing characteristics. Notably, the sequencing of ctxB in some El Tor variants revealed two copies of classical ctxB, one per chromosome, contrary to previous reports that located ctxAB only on the large chromosome of El Tor biotype strains.
Journal of clinical microbiology 08/2011; 49(11):3739-49. · 4.16 Impact Factor
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ABSTRACT: Whole-genome sequencing, also known as deep sequencing, is becoming a more affordable and efficient way to identify SNP mutations, deletions, and insertions in DNA sequences across several different strains. Two major obstacles preventing the widespread use of deep sequencers are the costs involved in services used to prepare DNA libraries for sequencing and the overall accuracy of the sequencing data. This unit describes the preparation of DNA libraries for multiplexed paired-end sequencing using the Illumina GA series sequencer. Self-preparation of DNA libraries can help reduce overall expenses, especially if optimization is required for the different samples, and use of the Illumina GA Sequencer can improve the quality of the data.
Current protocols in microbiology 02/2011; Chapter 1:Unit 1E.4.
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ABSTRACT: Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the alpha-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Tel(r)) and trimethoprim (Tp(r)) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Tel(r)-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tp(r)), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Tel(r)) and pFLP-AB5 (Tp(r)). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the Delta betBA::Tel(r)-FRT and Delta betBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.
Applied and environmental microbiology 08/2008; 74(14):4498-508. · 3.69 Impact Factor
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ABSTRACT: Beta-oxidative enzymes for fatty acid degradation (Fad) of long-chain fatty acids (LCFAs) are induced in vivo during lung infection in cystic fibrosis patients, and this may contribute to nutrient acquisition and pathogenesis of Pseudomonas aeruginosa. The promoter region of one P. aeruginosa beta-oxidation operon, fadBA5 (PA3014 and PA3013), was mapped. Focusing on the transposon mutagenesis of strain PAO1 carrying the P(fadBA5)-lacZ fusion, a regulator for the fadBA5 operon was identified to be PsrA (PA3006). Transcriptome analysis of the DeltapsrA mutant indicated its importance in regulating beta-oxidative enzymes. These microarray data were confirmed by real-time RT-PCR analyses of the fadB5 and lipA (encoding a lipase) genes. Induction of the fadBA5 operon was demonstrated to respond to novel LCFA signals, and this induction required the presence of PsrA, suggesting that LCFAs bind to PsrA to derepress fadBA5. Electrophoretic mobility shift assays indicate specific binding of PsrA to the fadBA5 promoter region. This binding is disrupted by specific LCFAs (C(18:1)(Delta9), C(16:0), C(14:0) and, to a lesser extent, C(12:0)), but not by other medium- or short-chain fatty acids or the first intermediate of beta-oxidation, acyl-CoA. It is shown here that PsrA is a fadBA5 regulator that binds and responds to LCFA signals in P. aeruginosa.
Microbiology 06/2008; 154(Pt 6):1584-98. · 3.06 Impact Factor
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ABSTRACT: Without prior knowledge of the promoters of various genes in bacteria, it can be difficult to study gene regulation using reporter-gene fusions. Regulation studies of promoters are ideal at their native locus, which do not require prior knowledge of promoter regions. Based on a previous study with FRT-lacZ-KmR constructs, we constructed two novel FRT-lacZ-GmR plasmids. This allows easy engineering of Pseudomonas aeruginosa reporter-gene fusions, post-mutant construction, with the Flp-FRT system. We demonstrate the usefulness of one of these FRT-lacZ-GmR plasmids to study the regulation of the fadAB1 operon in P. aeruginosa at its native locus. The fadAB1 operon, involved in fatty acid (FA) degradation, was significantly induced in the presence of several medium chain-length fatty acids (MCFA) and, to a lesser degree, long chain-length fatty acids (LCFA). In addition to the previous work on the FRT-lacZ-KmR tools, these new constructs increase the repertoire of tools that can be applied to P. aeruginosa or other species and strains of bacteria where kanamycin resistance may not be appropriate.
Plasmid 04/2008; 59(2):111-8. · 1.52 Impact Factor
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ABSTRACT: One of the hallmarks of Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients is very-high-cell-density (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by the quorum-sensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection are largely unknown. Here, we performed microarray studies of P. aeruginosa directly isolated from the lungs of CF patients to demonstrate its metabolic capability and virulence in vivo. In vivo microarray data, confirmed by real-time reverse transcription-PCR, indicated that the P. aeruginosa population expressed several genes for virulence, drug resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The most abundant lung surfactant lipid molecule, phosphatidylcholine (PC), induces key genes of P. aeruginosa pertinent to PC degradation in vitro as well as in vivo within the lungs of CF patients. The results support recent research indicating that P. aeruginosa exists in the lungs of CF patients as a diverse population with full virulence potential. The data also indicate that there is deregulation of several pathways, suggesting that there is in vivo evolution by deregulation of a large portion of the transcriptome during chronic infection in CF patients. To our knowledge, this is the first in vivo transcriptome analysis of P. aeruginosa in a natural infection in CF patients, and the results indicate several important aspects of P. aeruginosa pathogenesis, drug resistance, nutrient utilization, and general metabolism within the lungs of CF patients.
Infection and Immunity 12/2007; 75(11):5313-24. · 4.16 Impact Factor
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ABSTRACT: The T7-expression system has been very useful for protein expression in Escherichia coli. However, it is often desirable to over-express proteins in species other than E. coli. Here, we constructed an inducible broad-host-range T7-expression transposon, which allows simple one-step construction of T7-expression strains in various species, providing the option to over-express proteins of interest in a broader host-range. This transposon contains the T7 RNA polymerase driven by the lacUV5 promoter, which is repressed by the lac-repressor. Leaky expression is prevented by the presence of T7-lysozyme on this construct. The complete T7-expression system is flanked by mariner transposon repeats of the suicidal R6Kgammaori plasmid, pBT20-Deltabla. Stable integration of the whole system is possible by a one-step selection for a Flp-excisable Gm(R)-marker. We showed the engineering of E. coli, Pseudomonas aeruginosa, Erwinia carotovora, Salmonella choleraesuis, Agrobacterium tumefaciens, and Chromobacterium violaceum strains with this construct and demonstrated the expression of the Burkholderia pseudomallei Asd protein in these hosts, by induction with isopropyl-beta-d-thiogalactopyranoside (IPTG).
Protein Expression and Purification 11/2007; 55(2):325-33. · 1.59 Impact Factor
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ABSTRACT: The small multidrug resistance protein family has two subclasses. In this study we used a mutation approach to see what is necessary to convert a SUG subgroup member into a quaternary ammonium compound (QAC) transporter. We chose four key residues (H24, M39, I43, and A44) conserved within SUGs but conserved differently within the QAC transporters. Altogether, seven mutants were generated in Citrobacter freundii SugE. Surprisingly, the mutated SugE demonstrated an increased sensitivity to representative QACs. Additionally, ethidium uptake is found to be more prominent in the hypersensitive mutants. We conducted orientation studies using topology reporter gene fusions which indicated that SugE and the QAC transporter EmrE both have their N- and C-termini in the cytoplasm as predicted. The results imply that SugE can be converted to a QAC transporter with only a single mutation. However, because hypersensitivity was observed, the SugE mutant proteins are behaving as importers rather than as exporters.
Biochemical and Biophysical Research Communications 01/2004; 312(4):914-21. · 2.48 Impact Factor
