Genetic analysis of the requirement for flp-2, tadV, and rcpB in Actinobacillus actinomycetemcomitans biofilm formation.

The tad locus of Actinobacillus actinomycetemcomitans encodes a molecular transport system required for tenacious, nonspecific adherence to surfaces and formation of extremely strong biofilms. This locus is dedicated to the biogenesis of Flp pili, which are required for colonization and virulence. We have previously shown that 11 of the 14 tad locus genes are required for adherence and Flp pilus production. Here, we present genetic and phylogenetic analyses of flp-2, tadV, and rcpB genes in biofilm formation. We show that tadV, predicted to encode prepilin peptidase, is required for adherence. In contrast, targeted insertional inactivation of flp-2, a gene closely related to the prepillin gene flp-1, did not abrogate biofilm formation. Expression studies did not detect Flp2-T7 protein under standard laboratory conditions. We present phylogenetic data showing that there is no significant evidence for natural selection in the available flp-2 sequences from A. actinomycetemcomitans, suggesting that flp-2 does not play a significant role in the biology of this organism. Mutants with insertions at the 3' end of rcpB formed biofilms equivalent to wild-type A. actinomycetemcomitans. Surprisingly, 5' end chromosomal insertion mutants in rcpB were obtained only when a wild-type copy of the rcpB gene was provided in trans or when the Tad secretion system was inactivated. Together, our results strongly suggest that A. actinomycetemcomitans rcpB is essential in the context of a functional tad locus. These data show three different phenotypes for the three genes.

Characteristic attributes in cancer microarrays.

Rapid advances in genome sequencing and gene expression microarray technologies are providing unprecedented opportunities to identify specific genes involved in complex biological processes, such as development, signal transduction, and disease. The vast amount of data generated by these technologies has presented new challenges in bioinformatics. To help organize and interpret microarray data, new and efficient computational methods are needed to: (1) distinguish accurately between different biological or clinical categories (e.g., malignant vs. benign), and (2) identify specific genes that play a role in determining those categories. Here we present a novel and simple method that exhaustively scans microarray data for unambiguous gene expression patterns. Such patterns of data can be used as the basis for classification into biological or clinical categories. The method, termed the Characteristic Attribute Organization System (CAOS), is derived from fundamental precepts in systematic biology. In CAOS we define two types of characteristic attributes ('pure' and 'private') that may exist in gene expression microarray data. We also consider additional attributes ('compound') that are composed of expression states of more than one gene that are not characteristic on their own. CAOS was tested on three well-known cancer DNA microarray data sets for its ability to classify new microarray samples. We found CAOS to be a highly accurate and robust class prediction technique. In addition, CAOS identified specific genes, not emphasized in other analyses, that may be crucial to the biology of certain types of cancer. The success of CAOS in this study has significant implications for basic research and the future development of reliable methods for clinical diagnostic tools.

Genes for tight adherence of Actinobacillus actinomycetemcomitans: from plaque to plague to pond scum.

The Gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans forms an extremely tenacious biofilm on solid surfaces such as glass, plastic and hydroxyapatite. This characteristic is likely to be important for colonization of the oral cavity and initiation of a potentially devastating form of periodontal disease. Genetic analysis has revealed a cluster of tad genes responsible for tight adherence to surfaces. Evidence indicates that the tad genes are part of a locus encoding a novel secretion system for the assembly and release of long, bundled Flp pili. Remarkably similar tad loci appear in the genomes of a wide variety of Gram-negative and Gram-positive bacteria, including many significant pathogens, and in Archaea. We propose that the tad loci are important for microbial colonization in a variety of environmental niches.

Nonspecific adherence and fibril biogenesis by Actinobacillus actinomycetemcomitans: TadA protein is an ATPase.

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.

A small protein-protein interaction domain common to KlcB and global regulators KorA and TrbA of promiscuous IncP plasmids.

The kor regulon of broad host-range, incompatibility group P (IncP) plasmids uses the KorA, KorB, and KorC repressors to regulate expression of genes for replication, conjugation, segregation, and host range. One operon, kilC, encodes the KorC repressor and two genes of unknown function (klcA and klcB). The predicted sequences of the 51.1 kDa KlcB protein, the 11.3 kDa KorA repressor, and another small (13.5 kDa) regulatory protein, TrbA, show a highly related 35 amino acid residue segment (V-L-P domain). We found that induction of the klcB gene is toxic to Escherichia coli host cells harboring an IncP plasmid. We confirmed a model in which the V-L-P domain of KlcB interacts directly with the V-L-P domain of KorA to derepress KorA-regulated operons, thereby allowing expression of toxic genes. First, a lacZ reporter fused to the kleA promoter, which is regulated by KorA and KorC, revealed that klcB induction specifically releases KorA-repression but has no effect on KorC repression. Second, induced expression of the V-L-P domains from KorA or KlcB is sufficient to release KorA-repression at the kleA promoter. Third, purified GST-KlcB fusion protein interacts specifically with His-tagged KorA. Fourth, fusion of the V-L-P domains of KorA and TrbA and full-length KlcB polypeptide to the DNA-binding domain of bacteriophage lambda repressor leads to the formation of functional, dimeric repressors, and mutations that alter conserved residues of the V-L-P domain adversely affect dimerization. Fifth, crosslinking experiments demonstrated that the V-L-P domain of KorA is able to dimerize in solution and form heterodimers in mixtures with full-length KorA polypeptide. These findings show that the V-L-P domain is a protein-protein interaction module that is likely to be responsible for dimerization of KorA and TrbA, and important for KlcB dimerization. We speculate on the possible significance of KlcB-KorA heterodimers in IncP plasmid maintenance.

flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a Gram-negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non-specific, tenacious adherence to surfaces. Previous investigations have implicated an abundant, small ( approximately 6.5 kDa), fibril-associated protein (Flp/Fap) as the primary fibril subunit. Here, we report studies on fibril structure and on the function and evolution of Flp. High-resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5- to 7-nm-diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria. Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles. A modified version of the CU1000N Flp1 polypeptide with the T7-TAG epitope fused to its C-terminus was expressed in the wild-type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7-TAG. To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903phikan to isolate insertion mutations in the flp-1 gene (formerly designated flp). Mutants with insertions early in flp-1 fail to produce fibrils and do not adhere to surfaces. Both fibril production and adherence were restored by cloned flp-1 in trans, thus providing the first evidence that flp-1 is required for fibril formation and tight, non-specific adherence. One mutant was found to have an insertion near the 3' end of flp-1 that results in the expression of a truncated and altered C-terminus of Flp1. This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild-type bacteria. These findings and related observations with the Flp1-T7-TAG protein indicate that the C-terminus of Flp1 is important for the bundling and adherence properties of pili. Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp-1 to be a member of a novel and widespread subfamily of type IV prepilin genes. Thus, Flp pili are likely to be expressed by diverse bacterial species. Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp-like genes, including the open reading frame (flp-2, previously designated orfA) immediately downstream of flp-1 in A. actinomycetemcomitans. The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.

Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification.

Macromolecular transport systems in bacteria currently are classified by function and sequence comparisons into five basic types. In this classification system, type II and type IV secretion systems both possess members of a superfamily of genes for putative NTP hydrolase (NTPase) proteins that are strikingly similar in structure, function, and sequence. These include VirB11, TrbB, TraG, GspE, PilB, PilT, and ComG1. The predicted protein product of tadA, a recently discovered gene required for tenacious adherence of Actinobacillus actinomycetemcomitans, also has significant sequence similarity to members of this superfamily and to several unclassified and uncharacterized gene products of both Archaea and Bacteria. To understand the relationship of tadA and tadA-like genes to those encoding the putative NTPases of type II/IV secretion, we used a phylogenetic approach to obtain a genealogy of 148 NTPase genes and reconstruct a scenario of gene superfamily evolution. In this phylogeny, clear distinctions can be made between type II and type IV families and their constituent subfamilies. In addition, the subgroup containing tadA constitutes a novel and extremely widespread subfamily of the family encompassing all putative NTPases of type IV secretion systems. We report diagnostic amino acid residue positions for each major monophyletic family and subfamily in the phylogenetic tree, and we propose an easy method for precisely classifying and naming putative NTPase genes based on phylogeny. This molecular key-based method can be applied to other gene superfamilies and represents a valuable tool for genome analysis.

Incompatibility protein IncC and global regulator KorB interact in active partition of promiscuous plasmid RK2.

Replication of the broad-host-range, IncPalpha 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 (O(B)). 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.

Nonspecific adherence by Actinobacillus actinomycetemcomitans requires genes widespread in bacteria and archaea.

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 IS903phikan 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.

Secretion of RTX leukotoxin by Actinobacillus actinomycetemcomitans.

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.

Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans.

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, IS903phikan, 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. IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903phikan 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 IS903phikan 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 IS903phikan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.

Tenacious adhesion of Actinobacillus actinomycetemcomitans strain CU1000 to salivary-coated hydroxyapatite.

Adherence of Actinobacillus actinomycetemcomitans to hard-tissue surfaces was evaluated by comparing a phenotypically stable, well-maintained clinical isolate (strain CU1000) to Streptococcus gordonii G9B, an extensively studied oral-colonizing bacterium. Standard innocula of radiolabelled bacteria were added to saliva-coated hydroxyapatite (SHA) and the ratio of bound to unbound cells counted. Several other clinical isolates as well as laboratory strain Y4 were studied. In other experiments, cell detachment from SHA was compared in static and shaking vessels to calculate controlled desorption of cells over time. A sonic-displacement assay was used to measure avidity of binding to HA and SHA. To better define the attachment properties of CU1000, bacteria were treated with a variety of agents including detergents, salts and enzymes before or after incubation with SHA. Results indicated that CU1000 bound better than G9B (a minimum of 10-fold greater; p < or = 0.05) and did not desorb from SHA, while G9B desorbed to equilibrium in 4 h. Furthermore, Langmuir isotherm calculations indicated that, unlike G9B, CU1000 did not follow second-order adsorption kinetics and thus did not achieve saturation. In addition, of the agents tested only periodate reduced attachment and resulted in detachment of CU1000 from surfaces. These experiments suggest that clinical isolates of A. actinomycetemcomitans possess unique binding properties that promote adsorption to and impede desorption from SHA. The characteristics described for the actinobacillus in this study have been previously underestimated, appear to be mediated by glycoconjugates, and may resemble attachment described for several biofilm-forming, non-oral pathogens.

The kilE locus of promiscuous IncP alpha plasmid RK2 is required for stable maintenance in Pseudomonas aeruginosa.

Eight coordinately regulated operons constitute the kor regulon of the IncP alpha plasmid RK2. Three operons specify functions required for replication initiation, conjugative transfer, and control of gene expression. The functions of the other operons, including those of the four coregulated operons that compose the kilA, kilC, and kilE loci, have not been determined. Here, we present the first evidence that a kil determinant is involved in IncP plasmid maintenance. Elevation of KorC levels specifically to reduce the expression of the KorC-regulated kilC and kilE operons severely affected the maintenance of both the IncP alpha plasmid RK2lac and the IncP beta plasmid R751 in Pseudomonas aeruginosa but had little effect on plasmid maintenance in Escherichia coli. Precise deletion of the two kilE operons from RK2lac was achieved with the VEX mutagenesis system for large genomes. The resulting plasmid showed significant loss of stability in P. aeruginosa only. The defect could be complemented by reintroduction of kilE at a different position on the plasmid. The instability of the RK2lac delta kilE mutant did not result from a reduction in average plasmid copy number, reduced expression of kilC, decreased conjugative transfer, or loss of the korE regulator. We found that both the par and kilE loci are required for full stability of RK2lac in P. aeruginosa and that the par and kilE functions act independently. These results demonstrate a critical role for the kilE locus in the stable inheritance of RK2 in P. aeruginosa.

A potential new gene (tccA) on IncP plasmid RK2 and transposon Tn1721: relationship of its product to the TrwC relaxase/helicase of IncW plasmid R388.

Computational analysis of the fully sequenced 60-kb genome of broad-host-range IncP alpha plasmid RK2 revealed a previously unreported potential protein-coding sequence, an 80-codon open reading frame (tccA), located in the region between the vegetative origin of replication (oriV) and the tetR gene of the tetracycline resistance determinant. The coding region is also present in the transposon Tn1721 tet region, which is nearly identical to the tet region of RK2. Remarkably, the predicted polypeptide product of the coding region displays 56% identity and 72% similarity with the C-terminal domain of the TrwC relaxase/helicase protein of IncW plasmid R388.

Mechanism of retrotransfer in conjugation: prior transfer of the conjugative plasmid is required.

Bacterial conjugation normally involves the unidirectional transfer of DNA from donor to recipient. Occasionally, conjugation results in the transfer of DNA from recipient to donor, a phenomenon known as retrotransfer. Two distinct models have been generally considered for the mechanism of retrotransfer. In the two-way conduction model, no transfer of the conjugative plasmid is required. The establishment of a single conjugation bridge between donor and recipient is sufficient for the transfer of DNA in both directions. In the one-way conduction model, transfer of the conjugative plasmid to the recipient is required to allow the synthesis of a new conjugation bridge for the transfer of DNA from recipient to donor. We have tested these models by the construction of a mutant of the self-transmissible, IncP plasmid RK2lac that allows the establishement of the conjugation bridge but is incapable of self-transfer. Four nucleotides of the nic region of the origin of transfer (oriT) were changed directly in the 67-kb plasmid RK2lac by a simple adaptation of the vector-mediated excision (VEX) strategy for precision mutagenesis of large plasmids (E. K.Ayres, V. J. Thomson, G. Merino, D. Balderes, and D. H. Figurski, J. Mol. Biol. 230:174-185, 1993). The resulting RK2lac oriT1 mutant plasmid mobilizes IncQ or IncP oriT+ plasmids efficiently but transfers itself at a frequency which is 10(4)-fold less than that of the wild type. Whereas the wild-type RK2lac oriT+ plasmid promotes the retrotransfer of an IncQ plasmid from Escherichia coli or Pseudomonas aeruginosa recipients, the RK2lac oriT1 mutant is severely defective in retrotransfer. Therefore, retrotransfer requires prior transfer of the conjugative plasmid to the recipient. The results prove that retrotransfer occurs by two sequential DNA transfer events.

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2.

The par region of the broad-host-range, IncP alpha plasmid RK2 has been implicated as a stability determinant by its ability to enhance the maintenance of mini-RK2 plasmids or heterologous replicons in a growing population of host cells. The region consists of two operons: parCBA, which encodes a multimer resolution system, and parDE, which specifies a postsegregational response mechanism that is toxic to plasmidless segregants. To assess the importance of this region to the stable maintenance of the complete RK2 plasmid in different hosts, we used the vector-mediated excision (VEX) deletion system to specifically remove the entire par region or each operon separately from an otherwise intact RK2 plasmid carrying a lacZ marker. The par region was found to be important to stable maintenance of RK2lac (pRK2526) in Escherichia coli and five other gram-negative hosts (Agrobacterium tumefaciens, Azotobacter vinelandii, Acinetobacter calcoaceticus, Caulobacter crescentus, and Pseudomonas aeruginosa). However, the relative importance of the parCBA and parDE operons varied from host to host. Deletion of parDE had no effect on the maintenance of pRK2526 in A. calcoaceticus, but it severely reduced pRK2526 maintenance in A. vinelandii and resulted in significant instability in the other hosts. Deletion of parCBA did not alter pRK2526 stability in E. coli, A. tumefaciens, or A. vinelandii but severely reduced plasmid maintenance in A. calcoaceticus and P. aeruginosa. In the latter two hosts and C. crescentus, the delta parCBA mutant caused a notable reduction in growth rate in the absence of selection for the plasmid, indicating that instability resulting from the absence of parCBA may trigger the postsegregational response mediated by parDE. We also examined the effect of the conjugal transfer system on RK2 maintenance in E. coli. Transfer-defective traJ and traG mutants of pRK2526 were stably maintained in rapidly growing broth cultures. On solid medium, which should be optimal for IncP-mediated conjugation, colonies from cells containing the pRK2526 tra mutants displayed significant numbers of white (Lac-) sectors on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates, whereas sectors appeared rarely in colonies from tra+ plasmid-containing cells. Both the traJ and traG mutations further reduced the maintenance of the already unstable deltapar derivative. Thus, these experiments with defined mutations in an intact RK2 plasmid have revealed (i) that the par region allows RK2 to adapt to the different requirements for stable maintenance in various hosts and (ii) that conjugal transfer can contribute to the maintenance of RK2 in a growing population, particularly under conditions that are favorable to RK2 transfer.

Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids.

The kil-kor regulon was first identified on the broad-host-range IncP alpha plasmid RK2 by the presence of multiple kil loci (kilA, kilB, kilC, and recently kilE) that are lethal to Escherichia coli host cells in the absence of regulation by kor functions in various combinations. Whereas the kilB operon is required for mating-pair formation during conjugation, the functions encoded by the other kil loci are not known. They are not essential for replication or conjugal transfer, but their coregulation with replication and transfer genes indicates that they are likely to be important for RK2. In this report, we describe molecular and genetic studies on kilC. We determined the nucleotide sequence of the kilC region, which is located between the origin of vegetative replication (oriV) and transposon Tn1 on RK2. Primer extension analysis identified the transcriptional start site and showed that a sequence corresponding to a strong sigma 70 promoter is functional. The abundance of RNA initiated from the kilC promoter is reduced in the presence of korA and korC, as predicted from genetic analysis of kilC regulation. The first gene of the kilC operon (klcA) is sufficient to express the host-lethal phenotype of the kilC determinant in the absence of korA and korC. By comparing RK2 to the related IncP alpha plasmids pUZ8 and R995, we determined that the Tn1 transposon in RK2 interrupts a gene (klcB) immediately downstream of klcA. Thus, the kilC determinant is normally part of an autoregulated operon of three genes: klcA, klcB, and korC. klcA is predicted to encode a 15,856-Da polypeptide that is related to the ArdB antirestriction protein of the IncN plasmid pKM101, suggesting a role for klcA in the broad host ranges of IncP alpha plasmids. The predicted product of the uninterrupted klcB gene is a polypeptide of 51,133 Da that contains a segment with significant similarity to the RK2 regulatory proteins KorA and TrbA. Located 145 bp upstream of the kilC promoter is a 10th copy of the 17-bp oriV iteron sequence in inverted orientation relative to that of the other nine iterons of oriV. Iteron 10 is identical to the "orphan" iteron 1, and both have identical 6-bp flanking sequences that make them likely to be strong binding sites for the TrfA replication initiator protein. The locations and relative orientation of orphan iterons 10 and 1 raise the possibility that these iterons promote the formation of a DNA loop via protein-protein interactions by bound TrfA and lead us to propose that they demarcate the functional origin of replication. This analysis of the kilC region and our previous studies on the other kil loci of RK2 have revealed that the region between oriV and the korABF operon in wild-type IncP alpha plasmids is saturated by the kilC, kilE, and kilA loci arranged in four kor-regulated operons encoding a total of 12 genes.

Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis.

The IncP alpha promiscuous plasmid (R18, R68, RK2, RP1 and RP4) comprises 60,099 bp of nucleotide sequence, encoding at least 74 genes. About 40 kb of the genome, designated the IncP core and including all essential replication and transfer functions, can be aligned with equivalent sequences in the IncP beta plasmid R751. The compiled IncP alpha sequence revealed several previously unidentified reading frames that are potential genes. IncP alpha plasmids carry genetic information very efficiently: the coding sequences of the genes are closely packed but rarely overlap, and occupy almost 86% of the genome's nucleotide sequence. All of the 74 genes should be expressed, although there is as yet experimental evidence for expression of only 60 of them. Six examples of tandem-in-frame initiation sites specifying two gene products each are known. Two overlapping gene arrangements occupy different reading frames of the same region. Intergenic regions include most of the 25 promoters; transcripts are usually polycistronic. Translation of most of the open reading frames seems to be initiated independently, each from its own ribosomal binding and initiation site, although, a few cases of coupled translation have been reported. The most frequently used initiation codon is AUG but translation for a few open reading frames begins at GUG or UUG. The most common stop-codon is UGA followed by UAA and then UAG. Regulatory circuits are complex and largely dependent on two components of the central control operon. KorA and KorB are transcriptional repressors controlling at least seven operons. KorA and KorB act synergistically in several cases by recognizing and binding to conserved nucleotide sequences. Twelve KorB binding sites were found around the IncP alpha sequence and these are conserved in R751 (IncP beta) with respect to both sequence and location. Replication of IncP alpha plasmids requires oriV and the plasmid-encoded initiator protein TrfA in combination with the host-encoded replication machinery. Conjugative plasmid transfer depends on two separate regions occupying about half of the genome. The primary segregational stability system designated Par/Mrs consists of a putative site-specific recombinase, a possible partitioning apparatus and a post-segregational lethality mechanism, all encoded in two divergent operons. Proteins related to the products of F sop and P1 par partitioning genes are separately encoded in the central control operon.

Prevalence and distribution of bacteriophage phi Aa DNA in strains of Actinobacillus actinomycetemcomitans.

phi Aa is a bacteriophage that was originally isolated by induction of a lysogenic strain of the oral bacterium Actinobacillus actinomycetemcomitans. Since the discovery of phage phi Aa, additional phages infecting several other strains of A. actinomycetemcomitans have been identified. To determine the prevalence of phi Aa or phi Aa-related temperate phages in this species, a phi Aa-specific DNA probe was prepared to screen for homologous sequences among 42 strains of A. actinomycetemcomitans. Fourteen (33%) of the 42 strains examined contained DNA sequences that hybridized with the phage phi Aa probe. A bacteriophage designated phi Aa33384 was isolated by induction from one of the strains (ATCC 33384) that contained a sequence that hybridized with the phi Aa probe. The phi Aa probe hybridized with the DNA extracted from bacteriophage phi Aa33384. The distribution of the phage phi Aa sequence among A. actinomycetemcomitans serotypes was 5/13 (38%) of the serotype a strains, 0/16 (0%) of the serotype b strains, and 9/13 (69%) of the serotype c strains. The results of this investigation suggest that the target sequence prepared from the phage phi Aa genome is fairly common in the A. actinomycetemcomitans chromosome, and that the sequence is distributed among the A. actinomycetemcomitans serotypes in a seemingly nonrandom manner.

Host-inhibitory functions encoded by promiscuous plasmids. Transient arrest of Escherichia coli segregants that fail to inherit plasmid RK2.

An effective strategy for a plasmid to be maintained in a growing population of bacteria is to prevent the proliferation of newly formed daughter cells that lack the plasmid. In this study, we determined that the promiscuous, incompatibility group P plasmid RK2 encodes such a system. To induce large numbers of RK2-free segregants of an RK2-containing host, we designed a genetic system that made RK2 replication temperature-sensitive. A double amber mutant of the trfA replication initiator gene was constructed to complement a trfA deletion mutant of an otherwise wild-type RK2 in an Escherichia coli supD(Ts) strain. At the permissive temperature (30 degrees C), RK2 was maintained stably in the strain. Shifting the cells to the non-permissive temperature (42 degrees C) resulted in the nearly synchronous appearance of cells lacking RK2. We found that the number of viable cells rose only slowly over a period of six hours, during which time the cells formed long filaments reaching 20 to 40 times the length of a normal E. coli cell. After six hours, the arrested cells regained the ability to divide and multiply exponentially, and the filaments were eventually reduced to normal-sized cells. Neither arrest nor filamentation required the host recA function. Inhibiting the replication of an RK2 mutant deleted for the par stability locus also induced the arrest of segregants lacking the plasmid, but the arrested cells were not filamented. Our results demonstrate that RK2 encodes at least two functions that are activated in a plasmidless segregant: (1) a filamentation-inducing function specified by the par stability locus, and (2) a post-segregational arrest function (psa) that inhibits the proliferation of plasmidless segregants independent of par. We discuss the possible roles of these functions in the stable maintenance of RK2.