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1.  Production of Recombineering Substrates With Standard-size PCR Primers 
FEMS microbiology letters  2012;337(2):97-103.
Recombineering is a powerful method for DNA manipulation. It has advantages over restriction endonuclease-based methods and is usually rapid. Typically recombineering uses long PCR primers (~65 bases), each of which contains a small region of target homology (~45 bases). We have developed a simple, albeit somewhat less rapid, strategy to create recombineering substrates that can use primers of ≤35 bases for all steps. The regions of homology can be several hundred base pairs in length to (1) increase the chance of obtaining the desired clone and/or (2) allow coliphage-based recombineering in some non-Escherichia coli bacteria. The method uses cloning techniques to construct a template for the generation of the recombineering substrate. Because the template is made from cloned DNA segments, the segments (including those for the homology regions) can be readily changed. During construction of the template plasmid, potential background transformants arising from the vector without insert are significantly reduced by cloning each segment with two restriction endonucleases that produce non-compatible ends. We have used this method to change the bla gene of pACYC177 to aadA, to add the MCS-lacZα region from pBBR1MCS to IncQ plasmid vectors, and to make an oriTIncP-aacC1 cassette and add it to a plasmid.
doi:10.1111/1574-6968.12016
PMCID: PMC3510332  PMID: 23003673
λ red; recET; genetic engineering; DNA cloning; homologous recombination
2.  Different Phenotypes of Walker-Like A Box Mutants of ParA Homolog IncC of Broad-Host-Range IncP Plasmids 
Plasmid  2012;68(2):93-104.
The promiscuous IncPα plasmids RK2 and R995 encode a broad-host-range partition system, whose essential components include the incC and korB genes and a DNA site (OB) to which the korB product binds. IncC2, the smaller of the two incC products, is sufficient for stabilization of R995ΔincC. It is a member of the type Ia ParA family of partition ATPases. To better understand the role of ATP in partition, we constructed three alanine-substitution mutants of IncC2. Each mutation changed a different residue of the Walker-like ATP-binding and hydrolysis motif, including a lysine (K10) conserved solely among members of the ParA and MinD families. All three IncC2 mutants were defective in plasmid partition, but they differed from one another in other respects. The IncC2 T16A mutant, predicted to be defective in Mg2+ coordination, was severely impaired in all activities tested. IncC2 K10A, predicted to be defective in ATP hydrolysis, mediated enhanced incompatibility with R995 derivatives. IncC2 K15A, predicted to be defective in ATP binding, exhibited two distinct incompatibility properties depending on the genotype of the target plasmid. When in trans to plasmids carrying a complementable incC deletion, IncC2 K15A caused dramatic plasmid loss, even at low levels of expression. In trans to wild-type R995 or to R995ΔincC carrying a functional P1 partition system, IncC2 K15A-mediated incompatibility was significantly less than that caused by wild-type IncC2. All three Walker-like A box mutants were also defective for the host toxicity that normally results from co-overexpression of incC and korB. The phenotypes of the mutants support a model in which nucleotide hydrolysis is required for separation of paired plasmid complexes and possible interaction with a host factor.
doi:10.1016/j.plasmid.2012.04.003
PMCID: PMC3418066  PMID: 22579980
KorB; partition; segregation; RK2; R995; ATPase
3.  Structure of the pilus assembly protein TadZ from Eubacterium rectale: Implications for polar localization 
Molecular Microbiology  2012;83(4):712-727.
Summary
The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from E. rectale (ErTadZ), in complex with ATP and Mg2+, was determined to 2.1 Å resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker-A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N-terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped-down “active site”. Homology modeling of the N-terminal atypical receiver domain of CpaE indicates that it has a conserved protein-protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.
doi:10.1111/j.1365-2958.2011.07954.x
PMCID: PMC3272108  PMID: 22211578
Type IV pili assembly; TadZ; atypical receiver domain; atypical ATPase; localization factor
4.  The product of tadZ, a new member of the parA/minD superfamily, localizes to a pole in Aggregatibacter actinomycetemcomitans 
Molecular Microbiology  2012;83(4):694-711.
Summary
Aggregatibacter actinomycetemcomitans establishes a tenacious biofilm that is important for periodontal disease. The tad locus encodes the components for the secretion and biogenesis of Flp pili, which are necessary for the biofilm to form. TadZ is required, but its function has been elusive. We show that tadZ genes belong to the parA/minD superfamily of genes and that TadZ from A. actinomycetemcomitans (AaTadZ) forms a polar focus in the cell independent of any other tad locus protein. Mutations indicate that regions in AaTadZ are required for polar localization and biofilm formation. We show that AaTadZ dimerizes and that all TadZ proteins are predicted to have a Walker-like A box. However, they all lack the conserved lysine at position 6 (K6) present in the canonical Walker-like A box. When the alanine residue (A6) in the atypical Walker-like A box of AaTadZ was converted to lysine, the mutant protein remained able to dimerize and localize, but it was unable to allow the formation of a biofilm. Another essential biofilm protein, the ATPase (AaTadA), also localizes to a pole. However, its correct localization depends on the presence of AaTadZ. We suggest that the TadZ proteins mediate polar localization of the Tad secretion apparatus.
doi:10.1111/j.1365-2958.2011.07955.x
PMCID: PMC3305808  PMID: 22239271
type IV pili; adherence; biofilm; Actinobacillus; tad; Walker-like A box
5.  Complete Genome Sequence of Aggregatibacter (Haemophilus) aphrophilus NJ8700▿  
Journal of Bacteriology  2009;191(14):4693-4694.
We report the finished and annotated genome sequence of Aggregatibacter aphrophilus strain NJ8700, a strain isolated from the oral flora of a healthy individual, and discuss characteristics that may affect its dual roles in human health and disease. This strain has a rough appearance, and its genome contains genes encoding a type VI secretion system and several factors that may participate in host colonization.
doi:10.1128/JB.00447-09
PMCID: PMC2704734  PMID: 19447908
6.  Bacterial Conjugation in the Cytoplasm of Mouse Cells▿  
Infection and Immunity  2008;76(11):5110-5119.
Intracellular pathogenic organisms such as salmonellae and shigellae are able to evade the effects of many antibiotics because the drugs are not able to penetrate the plasma membrane. In addition, these bacteria may be able to transfer genes within cells while protected from the action of drugs. The primary mode by which virulence and antibiotic resistance genes are spread is bacterial conjugation. Salmonellae have been shown to be competent for conjugation in the vacuoles of cultured mammalian cells. We now show that the conjugation machinery is also functional in the mammalian cytosol. Specially constructed Escherichia coli strains expressing Shigella flexneri plasmid and chromosomal virulence factors for escape from vacuoles and synthesizing the invasin protein from Yersinia pseudotuberculosis to enhance cellular entry were able to enter 3T3 cells and escape from the phagocytic vacuole. One bacterial strain (the donor) of each pair to be introduced sequentially into mammalian cells had a conjugative plasmid. We found that this plasmid could be transferred at high frequency. Conjugation in the cytoplasm of cells may well be a general phenomenon.
doi:10.1128/IAI.00445-08
PMCID: PMC2573342  PMID: 18765719
7.  Transcriptional Regulation of the tad Locus in Aggregatibacter actinomycetemcomitans: a Termination Cascade▿  
Journal of Bacteriology  2008;190(11):3859-3868.
The tad (tight adherence) locus of Aggregatibacter actinomycetemcomitans includes genes for the biogenesis of Flp pili, which are necessary for bacterial adhesion to surfaces, biofilm formation, and pathogenesis. Although studies have elucidated the functions of some of the Tad proteins, little is known about the regulation of the tad locus in A. actinomycetemcomitans. A promoter upstream of the tad locus was previously identified and shown to function in Escherichia coli. Using a specially constructed reporter plasmid, we show here that this promoter (tadp) functions in A. actinomycetemcomitans. To study expression of the pilin gene (flp-1) relative to that of tad secretion complex genes, we used Northern hybridization analysis and a lacZ reporter assay. We identified three terminators, two of which (T1 and T2) can explain flp-1 mRNA abundance, while the third (T3) is at the end of the locus. T1 and T3 have the appearance and behavior of intrinsic terminators, while T2 has a different structure and is inhibited by bicyclomycin, indicating that T2 is probably Rho dependent. To help achieve the appropriate stoichiometry of the Tad proteins, we show that a transcriptional-termination cascade is important to the proper expression of the tad genes. These data indicate a previously unreported mechanism of regulation in A. actinomycetemcomitans and lead to a more complete understanding of its Flp pilus biogenesis.
doi:10.1128/JB.00128-08
PMCID: PMC2395055  PMID: 18375561
8.  tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans 
Gene  2007;399(1):53-64.
tfoX (sxy) is a regulatory gene needed to turn on competence genes. Aggregatibacter (Actinobacillus) actinomycetemcomitans has a tfoX gene that is important for transformation. We cloned this gene on an IncQ plasmid downstream of the inducible tac promoter. When this plasmid was resident in cells of A. actinomycetemcomitans and tfoX was induced, the cells became competent for transformation. Several strains of A. actinomycetemcomitans, including different serotypes, as well as rough (adherent) and isogenic smooth (nonadherent) forms were tested. Only our two serotype f strains failed to be transformed. With the other strains, we could easily get transformants with extrachromosomal plasmid DNA when closed circular, replicative plasmid carrying an uptake signal sequence (USS) was used. When a replicative plasmid carrying a USS and cloned DNA from the chromosome of A. actinomycetemcomitans was linearized by digestion with a restriction endonuclease or when genomic DNA was used directly, the outcome was allelic exchange. To facilitate allelic exchange, we constructed a suicide plasmid (pMB78) that does not replicate in A. actinomycetemcomitans and carries a region with two inverted copies of a USS. This vector gave allelic exchange in the presence of cloned and induced tfoX easily and without digestion. Using transposon insertions in cloned katA DNA, we found that as little as 78 bp of homology at one of the ends was sufficient for that end to participate in allelic exchange. The cloning and induction of tfoX makes it possible to transform nearly any strain of A. actinomycetemcomitans, and allelic exchange has proven to be important for site-directed mutagenesis.
doi:10.1016/j.gene.2007.04.026
PMCID: PMC2080652  PMID: 17561357
periodontitis; pathogen; USS; allelic exchange; site-directed mutagenesis; competence
9.  Outer Membrane Components of the Tad (Tight Adherence) Secreton of Aggregatibacter actinomycetemcomitans▿ †  
Journal of Bacteriology  2007;190(3):980-990.
Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins.
doi:10.1128/JB.01347-07
PMCID: PMC2223556  PMID: 18055598
10.  Interaction between Leukotoxin and Cu,Zn Superoxide Dismutase in Aggregatibacter actinomycetemcomitans▿  
Infection and Immunity  2007;75(9):4490-4497.
Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative oral pathogen that is the etiologic agent of localized aggressive periodontitis and systemic infections. A. actinomycetemcomitans produces leukotoxin (LtxA), which is a member of the RTX (repeats in toxin) family of secreted bacterial toxins and is known to target human leukocytes and erythrocytes. To better understand how LtxA functions as a virulence factor, we sought to detect and study potential A. actinomycetemcomitans proteins that interact with LtxA. We found that Cu,Zn superoxide dismutase (SOD) interacts specifically with LtxA. Cu,Zn SOD was purified from A. actinomycetemcomitans to homogeneity and remained enzymatically active. Purified Cu,Zn SOD allowed us to isolate highly specific anti-Cu,Zn SOD antibody and this antibody was used to further confirm protein interaction. Cu,Zn SOD-deficient mutants displayed decreased survival in the presence of reactive oxygen and nitrogen species and could be complemented with wild-type Cu,Zn SOD in trans. We suggest that A. actinomycetemcomitans Cu,Zn SOD may protect both bacteria and LtxA from reactive species produced by host inflammatory cells during disease. This is the first example of a protein-protein interaction involving a bacterial Cu,Zn SOD.
doi:10.1128/IAI.00288-07
PMCID: PMC1951164  PMID: 17635874
11.  The TadV Protein of Actinobacillus actinomycetemcomitans Is a Novel Aspartic Acid Prepilin Peptidase Required for Maturation of the Flp1 Pilin and TadE and TadF Pseudopilins†  
Journal of Bacteriology  2006;188(19):6899-6914.
The tad locus of Actinobacillus actinomycetemcomitans encodes genes for the biogenesis of Flp pili, which allow the bacterium to adhere tenaciously to surfaces and form strong biofilms. Although tad (tight adherence) loci are widespread among bacterial and archaeal species, very little is known about the functions of the individual components of the Tad secretion apparatus. Here we characterize the mechanism by which the pre-Flp1 prepilin is processed to the mature pilus subunit. We demonstrate that the tadV gene encodes a prepilin peptidase that is both necessary and sufficient for proteolytic maturation of Flp1. TadV was also found to be required for maturation of the TadE and TadF pilin-like proteins, which we term pseudopilins. Using site-directed mutagenesis, we show that processing of pre-Flp1, pre-TadE, and pre-TadF is required for biofilm formation. Mutation of a highly conserved glutamic acid residue at position +5 of Flp1, relative to the cleavage site, resulted in a processed pilin that was blocked in assembly. In contrast, identical mutations in TadE or TadF had no effect on biofilm formation, indicating that the mechanisms by which Flp1 pilin and the pseudopilins function are distinct. We also determined that two conserved aspartic acid residues in TadV are critical for function of the prepilin peptidase. Together, our results indicate that the A. actinomycetemcomitans TadV protein is a member of a novel subclass of nonmethylating aspartic acid prepilin peptidases.
doi:10.1128/JB.00690-06
PMCID: PMC1595517  PMID: 16980493
12.  The Active Partition Gene incC of IncP Plasmids Is Required for Stable Maintenance in a Broad Range of Hosts 
Journal of Bacteriology  2002;184(6):1788-1793.
Plasmids of incompatibility group P (IncP) are capable of replication and stable inheritance in a wide variety of gram-negative bacteria. Three determinants of IncP plasmids are components of an active partition locus that is predicted to function in the segregation of plasmid copies to daughter cells. These determinants are incC, which codes for a member of the ParA family of partition ATPases; korB, which specifies a DNA-binding protein that also functions as a global transcriptional repressor; and OB, the DNA target for KorB, which occurs at multiple locations on IncP plasmids. To determine the importance and host range of the IncC/KorB partition system in the maintenance of IncP plasmids, we constructed an in-frame deletion of incC in the otherwise intact 60-kb IncPα plasmid R995. R995ΔincC was found to be highly unstable in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Agrobacterium tumefaciens, and Acinetobacter calcoaceticus, whereas wild-type R995 is stable in all these hosts. In addition, R995ΔincC could not be established in Actinobacillus actinomycetemcomitans. trans-Complementation analysis showed that the coding region for IncC2 polypeptide, which is expressed from an internal translational start within the incC gene, was sufficient to restore stable maintenance to wild-type levels. The results show that the IncC/KorB active partition system of IncP plasmids is remarkably proficient for stable maintenance in diverse bacteria.
doi:10.1128/JB.184.6.1788-1793.2002
PMCID: PMC134889  PMID: 11872733
13.  Nonspecific Adherence and Fibril Biogenesis by Actinobacillus actinomycetemcomitans: TadA Protein Is an ATPase 
Journal of Bacteriology  2001;183(20):5927-5936.
Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.
doi:10.1128/JB.183.20.5927-5936.2001
PMCID: PMC99671  PMID: 11566992
15.  Secretion of RTX Leukotoxin by Actinobacillus actinomycetemcomitans 
Infection and Immunity  2000;68(11):6094-6100.
Actinobacillus actinomycetemcomitans, the etiologic agent for localized juvenile periodontitis and certain other human infections, such as endocarditis, expresses a leukotoxin that acts on polymorphonuclear leukocytes and macrophages. Leukotoxin is a member of the highly conserved repeat toxin (RTX) family of bacterial toxins expressed by a variety of pathogenic bacteria. While the RTX toxins of other bacterial species are secreted, the leukotoxin of A. actinomycetemcomitans is thought to remain associated with the bacterial cell. We have examined leukotoxin production and localization in rough (adherent) and smooth (nonadherent) strains of A. actinomycetemcomitans. We found that leukotoxin expressed by the rough, adherent, clinical isolate CU1000N is indeed cell associated, as expected. However, we were surprised to find that smooth, nonadherent strains of A. actinomycetemcomitans, including Y4, JP2 (a strain expressing a high level of toxin), and CU1060N (an isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin into the culture supernatants during early stages of growth. After longer times of incubation, leukotoxin disappears from the supernatants, and its loss is accompanied by the appearance of a number of low-molecular-weight polypeptides. The secreted leukotoxin is active, as evidenced by its ability to kill HL-60 cells in vitro. We found that the growth phase and initial pH of the growth medium significantly affect the abundance of secreted leukotoxin, and we have developed a rapid (<2 h) method to partially purify large amounts of leukotoxin. Remarkably, mutations in the tad genes, which are required for tight nonspecific adherence of A. actinomycetemcomitans to surfaces, cause leukotoxin to be released from the bacterial cell. These studies show that A. actinomycetemcomitans has the potential to secrete abundant leukotoxin. It is therefore appropriate to consider a possible role for leukotoxin secretion in the pathogenesis of A. actinomycetemcomitans.
PMCID: PMC97685  PMID: 11035711
16.  Nonspecific Adherence by Actinobacillus actinomycetemcomitans Requires Genes Widespread in Bacteria and Archaea 
Journal of Bacteriology  2000;182(21):6169-6176.
The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903φkan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad−). Unlike wild-type cells, Tad− mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad-like genes have a significant role in microbial colonization.
PMCID: PMC94753  PMID: 11029439
17.  Incompatibility Protein IncC and Global Regulator KorB Interact in Active Partition of Promiscuous Plasmid RK2 
Journal of Bacteriology  2000;182(21):6014-6026.
Replication of the broad-host-range, IncPα plasmid RK2 requires two plasmid loci: trfA, the replication initiator gene, and oriV, the origin of replication. While these determinants are sufficient for replication in a wide variety of bacteria, they do not confer the stable maintenance of parental RK2 observed in its hosts. The product of the incC gene has been proposed to function in the stable maintenance of RK2 because of its relatedness to the ParA family of ATPases, some of which are known to be involved in the active partition of plasmid and chromosomal DNA. Here we show that IncC has the properties expected of a component of an active partition system. The smaller polypeptide product of incC (IncC2) exhibits a strong, replicon-independent incompatibility phenotype with RK2. This incompatibility phenotype requires the global transcriptional repressor, KorB, and the target for incC-mediated incompatibility is a KorB-binding site (OB). We found that KorB and IncC interact in vivo by using the yeast two-hybrid system and in vitro by using partially purified proteins. Elevated expression of the incC and korB genes individually has no obvious effect on Escherichia coli cell growth, but their simultaneous overexpression is toxic, indicating a possible interaction of IncC-KorB complexes with a vital host target. A region of RK2 bearing incC, korB, and multiple KorB-binding sites is able to stabilize an unstable, heterologous plasmid in an incC-dependent manner. Finally, elevated levels of IncC2 cause RK2 to aggregate, indicating a possible role for IncC in plasmid pairing. These findings demonstrate that IncC, KorB, and at least one KorB-binding site are components of an active partition system for the promiscuous plasmid RK2.
PMCID: PMC94734  PMID: 11029420
18.  Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans 
Journal of Bacteriology  1999;181(23):7298-7307.
Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903φkan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903φkan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903φkan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903φkan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903φkan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903φkan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903φkan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.
PMCID: PMC103693  PMID: 10572134

Results 1-18 (18)