An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

BACKGROUND: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process. RESULTS: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family. CONCLUSION: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria

Evidence of MexT-Independent Overexpression of MexEF-OprN Multidrug Efflux Pump of Pseudomonas aeruginosa in Presence of Metabolic Stress

The Pseudomonas aeruginosa MexEF-OprN efflux pump confers resistance to clinically significant antibiotics. Regulation of mexEF-oprN operon expression is multifaceted with the MexT activator being one of the most prominent regulatory proteins.We have exploited the impaired metabolic fitness of a P. aeruginosa mutant strain lacking several efflux pump of the resistance nodulation cell division superfamily and the TolC homolog OpmH, and isolated derivatives (large colony variants) that regained fitness by incubation on nutrient-rich medium in the absence of antibiotics. Although the mexEF-oprN operon is uninducible in this mutant due to a 8-bp mexT insertion present in some P. aeruginosa PAO1 strains, the large colony variants expressed high levels of MexEF-OprN. Unlike large colony variants obtained after plating on antibiotic containing medium which expressed mexEF-oprN in a MexT-dependent fashion as evidenced by clean excision of the 8-bp insertion from mexT, mexEF-oprN expression was MexT-independent in the large colony variants obtained by plating on LB alone since the mexT gene remained inactivated. A search for possible regulators of mexEF-oprN expression using transposon mutagenesis and genomic library expression approaches yielded several candidates but proved inconclusive.Our results show that antibiotic and metabolic stress lead to up-regulation of MexEF-OprN expression via different mechanisms and that MexEF-OprN does not only extrude antimicrobials but rather serves other important metabolic functions

Identification and DNA sequence of tdcR , a positive regulatory gene of the tdc operon of Escherichia coli

Efficient in vivo expression of the biodegradative threonine dehydratase ( tdc ) operon of Escherichia coli is dependent on a regulatory gene, tdcR . The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of M r 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans -acting positive activator for the tdc operon.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47570/1/438_2004_Article_BF00332418.pd

A novel phosphate-regulated expression vector in Escherichia coli

The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and [beta]-galactosidase ([beta]Gal), respectively. Enzyme activities were virtually completely repressed in the presencee of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (pugp -directed [beta]Gal synthesis was approx. 80% of that directed by directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28559/1/0000361.pd

A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids

<p>Abstract</p> <p>Background</p> <p>The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K plasmid and derivatives harboring its γ origin of replication (<it>ori</it>_R6Kγ) is dependent on the <it>pir </it>gene-encoded π protein. Originally encoded by R6K, this protein is usually provided <it>in trans </it>in hosts engineered to support replication of plasmids harboring <it>ori</it>_R6Kγ. In <it>Escherichia coli </it>this is commonly achieved by chromosomal integration of <it>pir </it>either via lysogenization with a λ<it>pir </it>phage or homologous recombination at a pre-determined locus.</p> <p>Findings</p> <p>Current methods for construction of host strains for <it>ori</it>_R6Kγ-containing plasmids involve procedures that do not allow selection for presence of the <it>pir </it>gene and require cumbersome and time-consuming screening steps. In this study, we established a mini-Tn<it>7</it>-based method for rapid and reliable construction of <it>pir</it>⁺host strains. Using a curable mini-Tn<it>7 </it>delivery plasmid, <it>pir </it>expressing derivatives of several commonly used <it>E. coli </it>cloning and mobilizer strains were isolated using both the wild-type <it>pir⁺</it>gene as well as the copy-up <it>pir-116 </it>allele. In addition, we isolated <it>pir</it>⁺and <it>pir-116 </it>expressing derivatives of a clinical isolate of <it>Salmonella enterica </it>serovar Typhimurium. In both <it>E. coli </it>and <it>S. enterica </it>serovar Typhimurium, the presence of the <it>pir⁺</it>wild-type or <it>pir-116 </it>alleles allowed the replication of <it>ori</it>_R6Kγ-containing plasmids.</p> <p>Conclusions</p> <p>A mini-Tn<it>7 </it>system was employed for rapid and reliable engineering of <it>E. coli </it>and <it>S. enterica </it>serovar Typhimurium host strains for plasmids containing <it>ori</it>_R6Kγ. Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with <it>ori</it>_R6Kγ.</p

Does microbicide use in consumer products promote antimicrobial resistance? A critical review and recommendations for a cohesive approach to risk assessment

The increasing use of microbicides in consumer products is raising concerns related to enhanced microbicide resistance in bacteria and potential cross resistance to antibiotics. The recently published documents on this topic from the European Commission have spawned much interest to better understand the true extent of the putative links for the benefit of the manufacturers, regulators, and consumers alike. This white paper is based on a 2-day workshop (SEAC-Unilever, Bedford, United Kingdom; June 2012) in the fields of microbicide usage and resistance. It identifies gaps in our knowledge and also makes specific recommendations for harmonization of key terms and refinement/standardization of methods for testing microbicide resistance to better assess the impact and possible links with cross resistance to antibiotics. It also calls for a better cohesion in research in this field. Such information is crucial to developing any risk assessment framework on microbicide use notably in consumer products. The article also identifies key research questions where there are inadequate data, which, if addressed, could promote improved knowledge and understanding to assess any related risks for consumer and environmental safety

Molecular Investigations of PenA-mediated β-lactam Resistance in Burkholderia pseudomallei

Burkholderia pseudomallei is the etiological agent of melioidosis. Because of the bacterium’s intrinsic resistance and propensity to establish latent infections, melioidosis therapy is complicated and prolonged. Newer generation β-lactams, specifically ceftazidime, are used for acute phase therapy, but resistance to this cephalosporin has been observed. The chromosomally encoded penA gene encodes a putative twin arginine translocase (TAT)-secreted β-lactamase, and penA mutations have been implicated in ceftazidime resistance in clinical isolates. However, the role of PenA in resistance has not yet been systematically studied in isogenetic B. pseudomallei mutant backgrounds. We investigated the effects of penA deletion, point mutations, and up-regulation, as well as tat operon deletion and PenA TAT-signal sequence mutations. These experiments were made possible by employing a B. pseudomallei strain that is excluded from Select Agent regulations. Deletion of penA significantly (>4-fold) reduced the susceptibility to six of the nine β-lactams tested and ≥16-fold for ampicillin, amoxicillin, and carbenicillin. Overexpression of penA by single-copy, chromosomal expression of the gene under control of the inducible Ptac promoter, increased resistance levels for all β-lactams tested 2- to 10-fold. Recreation of the C69Y and P167S PenA amino acid substitutions previously observed in resistant clinical isolates increased resistance to ceftazidime by ≥85- and 5- to 8-fold, respectively. Similarly, a S72F substitution resulted in a 4-fold increase in resistance to amoxicillin and clavulanic acid. Susceptibility assays with PenA TAT-signal sequence and ΔtatABC mutants, as well as Western blot analysis, confirmed that PenA is a TAT secreted enzyme and not periplasmic but associated with the spheroplastic cell fraction. Lastly, we determined that two LysR-family regulators encoded by genes adjacent to penA do not play a role in transcriptional regulation of penA expression

In vivo Bioluminescence Imaging of Burkholderia mallei Respiratory Infection and Treatment in the Mouse Model

Bioluminescent imaging (BLI) technology is a powerful tool for monitoring infectious disease progression and treatment approaches. BLI is particularly useful for tracking fastidious intracellular pathogens that might be difficult to recover from certain organs. Burkholderia mallei, the causative agent of glanders, is a facultative intracellular pathogen and has been classified by the CDC as a Category B select agent due to its highly infectious nature and potential use as a biological weapon. Very little is known regarding pathogenesis or treatment of glanders. We investigated the use of bioluminescent reporter constructs to monitor the dynamics of infection as well as the efficacy of therapeutics for B. mallei in real-time. A stable luminescent reporter B. mallei strain was created using the pUTmini-Tn5::luxKm2 plasmid and used to monitor glanders in the BALB/c murine model. Mice were infected via the intranasal route with 5 × 103 bacteria and monitored by BLI at 24, 48, and 72 h. We verified that our reporter construct maintained similar virulence and growth kinetics compared to wild-type B. mallei and confirmed that it maintains luminescent stability in the presence or absence of antibiotic selection. The luminescent signal was initially seen in the lungs, and progressed to the liver and spleen over the course of infection. We demonstrated that antibiotic treatment 24 h post-infection resulted in reduction of bioluminescence that can be attributed to decreased bacterial burden in target organs. These findings suggest that BLI can be used to monitor disease progression and efficacy of therapeutics during glanders infections. Finally, we report an alternative method to mini-Tn5::luxKm2 transposon using mini-Tn7-lux elements that insert site-specifically at known genomic attachment sites and that can also be used to tag bacteria

Molecular Basis of Rare Aminoglycoside Susceptibility and Pathogenesis of Burkholderia pseudomallei Clinical Isolates from Thailand

Burkholderia pseudomallei is the etiologic agent of melioidosis, an emerging tropical disease. Because of low infectious dose, broad-host-range infectivity, intrinsic antibiotic resistance and historic precedent as a bioweapon, B. pseudomallei was listed in the United States as a Select Agent and Priority Pathogen of biodefense concern by the US Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases. The mechanisms governing antibiotic resistance and/or susceptibility and virulence in this bacterium are not well understood. Most clinical and environmental B. pseudomallei isolates are highly resistant to aminoglycosides, but susceptible variants do exist. The results of our studies with three such variants from Thailand reveal that lack of expression or deletion of an efflux pump is responsible for this susceptibility. The large deletion present in one strain not only removes an efflux pump but also several putative virulence genes, including an entire siderophore gene cluster. Despite this deletion, the strain is fully virulent in an acute mouse melioidosis model. In summary, our findings shed light on mechanisms of antibiotic resistance and pathogenesis. They also validate the previously advocated use of laboratory-constructed, aminoglycoside susceptible efflux pump mutants in genetic manipulation experiments