Construction and characterization of a recA mutant of Thiobacillus ferrooxidans by marker exchange mutagenesis

To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T. ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out the T. ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020 recA gene disrupted by a kanamycin resistance gene was transferred from E. coli to T. ferrooxidans ATCC 33020 by conjugation under the best conditions determined. The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the recA gene, indicating that the recA::Omega-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and gamma irradiation compared to the wild-type strain. However, the T. ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in T. ferrooxidans.     

Production of unmarked mutations in mycobacteria using site-specific recombination

Gene disruption experiments play an important role in the functional characterization of genes in mycobacteria and rely mostly on the use of one or two antibiotic resistance markers. We have developed a system for mycobacteria which features both the advantages of the use of antibiotic resistance markers for gene disruption experiments and the ability to efficiently rescue the marker leaving an unmarked mutation on the chromosome. This new genetic tool relies on the transposon gammadelta site-specific recombination system. A res-OmegaKm-res cassette was used to generate an insertional mutation by allelic exchange both in Mycobacterium smegmatis and Mycobacterium bovis BCG. Upon expression in the mutated strains of tnpR, the transposon gammadelta resolvase gene, res-OmegaKm-res, was excised efficiently leaving behind a single res sequence at the mutated locus. A plasmid was engineered allowing expression of tnpR from an easily curable mycobacterial vector. This system will be useful for simple construction of unmarked mutations or repeated use of the same antibiotic marker to generate multiple mutants.     

Genetic and physiological characterization of 23S rRNA and ftsJ mutants of Borrelia burgdorferi isolated by mariner transposition

Borrelia burgdorferi contains one 16S rRNA gene and two tandem sets of 23S and 5S rRNA genes located in a single chromosomal region. This unusual rRNA gene organization has been speculated to be involved in the slow growth of this organism. Because we were repeatedly unable to isolate a 23S ribosomal mutant in B. burgdorferi by allelic exchange, we developed a transposition mutagenesis system for this bacterium. To this end, Himar1 transposase is expressed in B. burgdorferi from a resident plasmid containing an erythromycin resistance marker, and this strain is then electroporated with suicide plasmids containing mariner transposons and kanamycin resistance genes expressible in B. burgdorferi. This system permitted us to generate hundreds of erythromycin/kanamycin-resistant B. burgdorferi clones with each of three suicide plasmids. DNA sequencing of several kanamycin-resistant clones generated with one of the suicide plasmids showed stable and random insertion of the transposon into the B. burgdorferi chromosomal and plasmid genome. One mutant was inactivated in rrlA (23S rRNA), another in ftsJ (rrmJ). rrlA disruption had no effect on growth rate under a wide range of culture conditions, but disruption of ftsJ interfered significantly with growth rate and bacterial morphology. These data show it is possible to isolate random and stable B. burgdorferi transposition mutants for physiological analysis of this pathogenic spirochete.     

Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria

Complete genome sequences are now available for many bacterial species that lack sophisticated genetic tools. We describe the development of a broad-host-range cre-lox system that allows antibiotic marker recycling in a variety of gram-negative bacteria. This system consists of an allelic exchange vector bearing a kanamycin cassette flanked by loxP sites and a tetracycline-resistant IncP plasmid that provides expression of the Cre recombinase. We demonstrate this system by generating unmarked deletions of genes in two different bacteria, Methylobacterium extorquens AM1 and Burkholderia fungorum LB400. This new antibiotic marker recycling system offers the possibility of creating unmarked mutants in a wide variety of gram-negative bacteria. Furthermore, marker recycling allows the generation of strains bearing multiple genetic manipulations in organisms for which few antibiotic markers are currently available.     

Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in pseudomonas aeruginosa

The complete genome of the bacterial pathogen Pseudomonas aeruginosa has now been sequenced, allowing gene deletion, one of the most frequently used methods in gene function study, to be fully exploited. In this study, we combine the sacB-based negative selection system with a cre-lox antibiotic marker recycling method. This methodology allows allelic exchange between a target gene and a gentamicin cassette flanked by the two lox sequences. A tetracycline plasmid expressing the cre recombinase is then introduced in the mutant strain to catalyze the excision of the lox-flanked resistance marker. We demonstrate here the efficiency of the combination of these two methods in P. aeruginosa by successively deleting ExoS and ExoT, which are two genetically independent toxins of the type-three secretion system (TTSS). This functional cre-lox recycling antibiotic marker system can create P. aeruginosa strains with multiple mutations without modifying the antibiotic resistance profile when compared to the parental strain.     

Consecutive gene deletions in Mycobacterium smegmatis using the yeast FLP recombinase

Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants, because there are only two efficient resistance markers available for allelic exchange experiments. We have established a system based on the Flp recombinase of the yeast Saccharomyces cerevisiae for use in the nonpathogenic model organism Mycobacterium smegmatis. This system consists of a hygromycin resistance cassette flanked by two Flp recognition targets (FRT) in direct orientation and a curable plasmid for expression of the flp gene. The FRT-hyg-FRT cassette was used on a suicide plasmid and on a conditionally replicating plasmid to delete two of the four known porin genes of M. smegmatis, mspA and mspC, respectively, by homologous recombination. The hyg gene was specifically removed from the chromosome of both mutants upon expression of the flp gene. Based on the marker-less mspC mutant strain, a double knock-out mutant lacking also mspA was obtained using the same strategy. Thus, by a fast and efficient two-step procedure, each of the porin genes was replaced by a single FRT site, which can be further used for site-specific integration. These results show that the Flp/FRT system is a suitable genetic tool for constructing unmarked mutations and for the analysis of redundant genes by consecutive gene deletions in M. smegmatis.     

Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon

To facilitate efficient allelic exchange of genetic information into a wild-type strain background, we improved upon and merged approaches using a temperature-sensitive plasmid and a counter-selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild-type chromosomal sequences, at either the fimB-A or lacZ-A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn5, but, unlike another similar cassette, it lacks IS1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature-sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non-selective conditions at 42 degrees C, and then in the presence of sucrose at 30 degrees C, allowing positive selection for both plasmid excision and curing. Despite marked locus-specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non-essential parts of the E. coli chromosome.     

Improved antibiotic resistance gene cassette for marker exchange mutagenesis in Ralstonia solanacearum and Burkholderia species

Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.     

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non-motile mutants deficient in endoflagella

Leptospira spp. offer many advantages as model bacteria for the study of spirochaetes. However, homologous recombination between introduced DNA and the corresponding chromosomal loci has never been demonstrated. A unique feature of spirochaetes is the presence of endoflagella between the outer membrane sheath and the cell cylinder. We chose the flaB flagellin gene, constituting the flagellar core, as a target for gene inactivation in the saprophyte Leptospira biflexa. The amino acid sequence of the FlaB protein of L. biflexa was most similar to those of spirochaetes Brachyspira hyodysenteriae (agent of swine dysentery), Leptospira interrogans (agent of leptospirosis) and Treponema pallidum (agent of syphilis). A suicide vector containing the L. biflexa flaB gene disrupted by a kanamycin marker was UV irradiated or alkali denatured before electroporation. This methodology allowed the selection of many kanamycin-resistant colonies resulting from single and double cross-over events at the flaB locus. The double recombinant mutants are non-motile, as visualized in both liquid and semi-solid media. In addition, a flaB mutant selected for further analysis was shown to be deficient in endoflagella by electron microscopy. However, most of the transformants had resulted from a single homologous recombination event, giving rise to the integration of the suicide vector. We evaluated the effect of the sacB and rpsL genes in L. biflexa as potential counterselectable markers for allelic exchange, and then used the rpsL system for the positive selection of flaB double recombinants in a streptomycin-resistant strain. Like the flaB mutant studied above, the Strr double cross-over mutant was non-motile and deficient in endoflagella. Our results demonstrate that FlaB is involved in flagella assembly and motility. They also show the feasibility of performing allelic replacement in Leptospira spp. by homologous recombination.     

Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation

The use of antibiotic-resistance genes as selectable markers in transgenic organisms is coming under increased scrutiny, for fear that they may spread to human pathogens, thereby reducing the effectiveness of antibiotic therapy. A current Pseudomonas fluorescens protein expression system uses a tetracycline resistance gene (tetR/tetA) to maintain an expression plasmid under control of a repressible promoter and a kanamycin resistance gene (kanR) to maintain a plasmid carrying a repressor gene. We investigated using auxotrophic markers to replace these two antibiotic resistance genes: pyrF (encoding orotidine-5'-phosphate decarboxylase) in place of tetR/tetA and proC (encoding pyrroline-5-carboxylate reductase) in place of kanR, complementing their respective precise chromosomal deletions created by allele exchange using a suicide vector carrying pyrF as a counterselectable marker. The resulting strains, devoid of antibiotic-resistance genes, were shown to achieve high productivity of nitrilase and thermostable alpha-amylase equal to that of the former antibiotic-resistant production host. The production plasmids were stable. The pyrF (uracil-dependent) background of the production host strain also allows us to sequentially alter the genome to incorporate other desired genomic changes, deletions, or insertions using 5'-fluoroorotic acid counterselection, restoring the selectable marker after each step.     

产生无标记农杆菌突变体方法的建立及优化

农杆菌已经用作许多生物过程研究的模型细菌,为了解析这些生物过程的分子机理,对农杆菌的某些基因进行突变就显得非常重要．以自杀性基因sacB作为反向可选择性标记基因,利用同源重组的原理,建立了一种可对农杆菌基因进行准确插入、删除和位点置换的突变方法,所获突变体不带任何不需要的外源DNA序列．通过详细研究同源序列的长度对农杆菌同源重组效率和突变体产生概率的影响,以及对农杆菌中的同源重组机理的分析,提出了优化该突变体产生方法的方案,即通过设计不等长的上下游同源序列和选择其中一种类型的单交换重组体来筛选二次交换重组体的方法,可以显著地提高理想突变体的产生概率．研究结果对如何提高突变体的产生概率和减少突变体筛选的工作量具重要的参考价值．利用该方法成功地获得了两个基因被同时删除而且不含抗性标记的农杆菌突变株．     

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

Taurine: new implications for an old amino acid

We describe here a technique for allelic exchange in Francisella tularensis subsp. novicida utilizing polymerase chain reaction (PCR) products. Linear PCR fragments containing gene deletions with an erythromycin resistance cassette insertion were transformed into F. tularensis. The subsequent ErmR progeny were found to have undergone allelic exchange at the correct location in the genome; the minimum flanking homology necessary was 500 bp. This technique was used to create mglA, iglC, bla, and tul4 mutants in F. tularensis subsp. novicida strains. The mglA and iglC mutants were defective for intramacrophage growth, and the tul4 mutant lacked detectable Tul4 by Western immunoblot, as expected. Interestingly, the bla mutant maintained resistance to ampicillin, indicating the presence of multiple ampicillin resistance genes in F. tularensis.     

Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic resistance markers using a two gene cassette with positive and negative selection.

The pathogenesis of infections caused by Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can be studied using experimental infection of human male volunteers. The desire to avoid introducing new antibiotic resistance markers into strains to be used in human experimental infection has complicated the construction of genetically defined mutants in which expression of potential virulence factors is inactivated. To facilitate construction of such mutants, we have used a two-step mutagenesis strategy that allows for gene replacements without introducing new selectable markers into the final strain. The method uses a two-gene cassette containing both a selectable marker (ermC') and a counterselectable marker (rpsL). The cassette is cloned into the gene of interest and used to replace the wild-type gene on the chromosome by allelic exchange. A second transformation replaces the cassette-containing version of the gene with an engineered version with an unmarked deletion or other mutation. The rpsL gene of Escherichia coli functioned for the counterselection in the gonococcus, albeit with low efficiency. To improve the efficiency of the counterselection, we cloned the gonococcal rpsL gene and incorporated it into the cassette. This technique has been successful in creating defined mutants for human challenge, and also circumvents the limitation in the number of different selectable markers that are useful in Neisseria species.     

Efficient targeted mutagenesis in Borrelia burgdorferi

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A(1). The utility of the coumermycin-resistant gyrB(r) gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.     

Development of a system for genetic manipulation of Bartonella bacilliformis.

Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all Bartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes ClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan(r) clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.     

鲍曼不动杆菌无痕基因敲除及hcp基因相关功能

[背景]鲍曼不动杆菌耐药严重,基因敲除是研究细菌毒力与耐药的重要方式.但现有的大部分细菌基因敲除方法基于抗生素抗性筛选,导致不适用于多重耐药菌株的基因敲除.[目的]旨在建立一种非依赖于抗生素抗性筛选的方法,用于敲除多重耐药鲍曼不动杆菌基因.[方法]运用同源 重组和自杀载体pMo 130-TelR对亚碲酸钾的抗性,使用两...     

Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene

We established a novel and convenient method to construct a ura3 strain (ura3/ura3) of the asporogenous and diploid yeast, Candida tropicalis, that produces dicarboxylic acid. One copy of the URA3 gene was disrupted using a mutated hygromycin B resistance gene (HYG#). The obtained hygromycin-resistant strain was further transformed with a URA3 disruption cassette and selected on a plate containing 5-fluoroorotic acid. The obtained strains were analyzed and the disruption of the gene was confirmed by PCR and Southern blot analysis. The results showed that the strains were obtained in which allelic URA3 genes were simultaneously disrupted. Furthermore, we established a cotransformation method for this gene disruption, using HYG# in C. tropicalis. In order to disrupt the allelic POX4 genes (encoding acyl-CoA oxidase) of dicarboxylic acid-producing strains, the ARS plasmid (which contained HYG#) and a POX4 disruption cassette (which carried the LAC4 gene encoding beta-galactosidase of Kluyveromyces lactis) were simultaneously introduced by transformation. As a result, the allelic POX4 gene was successfully disrupted.