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

Conjugation of plasmids from is essential for the genetic manipulation of spp. To facilitate intergeneric conjugation from to 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.

Production of recombineering substrates with standard-size PCR primers.

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 (c.

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.

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.

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.

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.

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.

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.

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.

Interaction between leukotoxin and Cu,Zn superoxide dismutase in Aggregatibacter actinomycetemcomitans.

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.

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.

The tad locus: postcards from the widespread colonization island.

The Tad (tight adherence) macromolecular transport system, which is present in many bacterial and archaeal species, represents an ancient and major new subtype of type II secretion. The tad genes are present on a genomic island named the widespread colonization island (WCI), and encode the machinery that is required for the assembly of adhesive Flp (fimbrial low-molecular-weight protein) pili. The tad genes are essential for biofilm formation, colonization and pathogenesis in the genera Aggregatibacter (Actinobacillus), Haemophilus, Pasteurella, Pseudomonas, Yersinia, Caulobacter and perhaps others.

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.

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.

ORFcurator: molecular curation of genes and gene clusters in prokaryotic organisms.

Unlabelled: The ability to detect clusters of functionally related genes in multiple microbial genomes has enormous potential for enhancing studies on gene function and microbial evolution. The staggering amount of new genome sequence data presents a largely untapped resource for gene cluster discovery. To date, gene cluster analysis has not been fully automated, and one must rely on manual, tedious and time-consuming manipulation of sequences.

VEX-capture: a new technique that allows in vivo excision, cloning, and broad-host-range transfer of large bacterial genomic DNA segments.

We have developed a novel and easily performed procedure for the targeted excision, cloning, and broad-host-range transfer of large bacterial genomic DNA segments. This procedure, called Vector-mediated excision and Capture (VEX-Capture), represents a new molecular tool for the convenient manipulation and exchange of large (20-40+ kb) bacterial genomic fragments. VEX-Capture utilizes lox/Cre-mediated site-specific recombination for excision of the targeted genomic segment and homologous recombination for cloning of the excised DNA section onto a self-transmissible, broad-host-range IncP plasmid.

Tight-adherence genes of Actinobacillus actinomycetemcomitans are required for virulence in a rat model.

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that has been associated with localized aggressive periodontitis and infections of the heart, brain, and urinary tract. Wild-type clinical isolates have the remarkable ability to adhere tenaciously and nonspecifically to solid surfaces such as glass, plastic, and hydroxyapatite. Adherence by A.

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.

An automated phylogenetic key for classifying homeoboxes.

When novel gene sequences are discovered, they are usually identified, classified, and annotated based on aggregate measures of sequence similarity. This method is prone to errors, however. Phylogenetic analysis is a more accurate basis for gene classification and ortholog identification, but it is relatively labor-intensive and computationally demanding.

Purification of secreted leukotoxin (LtxA) from Actinobacillus actinomycetemcomitans.

The RTX (repeats in toxin) family of toxins is important in the pathogenesis of many Gram-negative bacteria. The oral and systemic human pathogen Actinobacillus actinomycetemcomitans produces a member of this family known as leukotoxin (LtxA). Previously, we found that LtxA is secreted into culture supernatants of A.

Host-specific incompatibility by 9-bp direct repeats indicates a role in the maintenance of broad-host-range plasmid RK2.

Broad-host-range incompatibility group P (IncP) plasmids RK2 and R751 have 9-bp direct repeats (DR) of unknown function located between their kilC and kilE loci. The nucleotide sequences of the 9-bp repeats are different for RK2 (an IncPalpha group plasmid) and R751 (IncPbeta group), but both DR regions are organized similarly, including an 11-bp spacer with identical 5'-CGCCA-3' cores and an adjacent binding site for KorB, a known partition protein and transcriptional repressor. The occurrence of similarly arranged DR elements with different repeat sequences is suggestive of an important plasmid-specific function for the DR regions.

The active partition gene incC of IncP plasmids is required for stable maintenance in a broad range of hosts.

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 O(B), the DNA target for KorB, which occurs at multiple locations on IncP plasmids.

Phenotypic variation in Actinobacillus actinomycetemcomitans during laboratory growth: implications for virulence.

This study examined alteration of specific virulence traits associated with phenotypic changes seen when a low-passage disease-associated and well maintained parent strain of Actinobacillus actinomycetemcomitans was compared to a laboratory-grown spontaneous variant/mutant. Clinical isolates of A. actinomycetemcomitans recovered from periodontitis patients typically grow as rough, adherent colonies on primary culture but undergo transformation to smooth, non-adherent colonies following repeated passage in vitro.