Sequence and origin of the Streptomyces intergenetic-conjugation helper plasmid pUZ8002.

Conjugation of plasmids from Escherichia coli is essential for the genetic manipulation of Streptomyces spp. To facilitate intergeneric conjugation from E. coli to Streptomyces the conjugative machinery required for genetic transfer is usually provided by the non-transferable helper plasmid, pUZ8002. Here we present the complete nucleotide sequence of pUZ8002, describe the previously undocumented creation process, and provide details of the sequence relative to the parental pUZ8 plasmid and another previously published pUZ8002 sequence.

The product of tadZ, a new member of the parA/minD superfamily, localizes to a pole in Aggregatibacter actinomycetemcomitans.

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.

Structure of the pilus assembly protein TadZ from Eubacterium rectale: implications for polar localization.

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 Eubacterium rectale (ErTadZ), in complex with ATP and Mg(2+) , 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 modelling 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 an early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.

The Widespread Colonization Island of Actinobacillus actinomycetemcomitans.

Genomic islands, such as pathogenicity islands, contribute to the evolution and diversification of microbial life. Here we report on the Widespread Colonization Island, which encompasses the tad (tight adherence) locus for colonization of surfaces and biofilm formation by the human pathogen Actinobacillus actinomycetemcomitans. At least 12 of the 14 genes at the tad locus are required for tenacious biofilm formation and synthesis of bundled Flp pili (fibrils) that mediate adherence. The pilin subunit, Flp1, remains inside the cell in tad-locus mutants, indicating that these genes encode a secretion system for export and assembly of fibrils. We found tad-related regions in a wide variety of Bacterial and Archaeal species, and their sequence characteristics indicate possible horizontal transfer. To test the hypothesis of horizontal transfer, we compared the phylogeny of the tad locus to a robust organismal phylogeny using statistical tests of congruence and tree reconciliation techniques. Our analysis strongly supports a complex history of gene shuffling by recombination and multiple horizontal transfers, duplications and losses. We present evidence for a specific horizontal transfer event leading to the establishment of this region as a determinant of disease.

Different phenotypes of Walker-like A box mutants of ParA homolog IncC of broad-host-range IncP plasmids.

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 (O(B)) 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 Mg²âº 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.

Complete genome sequence of Aggregatibacter (Haemophilus) aphrophilus NJ8700.

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.

Transcriptional regulation of the tad locus in Aggregatibacter actinomycetemcomitans: a termination cascade.

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.

Outer membrane components of the Tad (tight adherence) secreton of Aggregatibacter actinomycetemcomitans.

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.

Bacterial conjugation in the cytoplasm of mouse cells.

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.

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans.

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.