ComE, a competence protein from Neisseria gonorrhoeae with DNA-binding activity

Neisseria gonorrhoeae is naturally able to take up exogenous DNA and undergo genetic transformation. This ability correlates with the presence of functional type IV pili, and uptake of DNA is dependent on the presence of a specific 10-bp sequence. Among the known competence factors in N. gonorrhoeae, none has been shown to interact with the incoming DNA. Here we describe ComE, a DNA-binding protein involved in neisserial competence. The gene comE was identified through similarity searches in the gonococcal genome sequence, using as the query ComEA, the DNA receptor in competent Bacillus subtilis. The gene comE is present in four identical copies in the genomes of both N. gonorrhoeae and Neisseria meningitidis, located downstream of each of the rRNA operons. Single-copy deletion of comE in N. gonorrhoeae did not have a measurable effect on competence, whereas serial deletions led to gradual decrease in transformation frequencies, reaching a 4 x 10(4)-fold reduction when all copies were deleted. Transformation deficiency correlated with impaired ability to take up exogenous DNA; however, the mutants presented normal piliation and twitching motility phenotype. The product of comE has 99 amino acids, with a predicted signal peptide; by immunodetection, a 8-kDa protein corresponding to processed ComE was observed in different strains of N. gonorrhoeae and N. meningitidis. Recombinant His-tagged ComE showed DNA binding activity, without any detectable sequence specificity. Thus, we identified a novel gonococcal DNA-binding competence factor which is necessary for DNA uptake and does not affect pilus biogenesis or function.     

Transformation of Streptococcus pneumoniae relies on DprA- and RecA-dependent protection of incoming DNA single strands

Seventy-five years after the discovery of transformation with Streptococcus pneumoniae, it is remarkable how little we know of the proteins that interact with incoming single strands in the early processing of transforming DNA. In this work, we used as donor DNA in transformation a radioactively labelled homologous fragment to examine the fate of the single-stranded (ssDNA) products of uptake in cells mutant for DprA or RecA, two proteins essential for transformation. Fifteen minutes after uptake, the labelling of specific chromosomal restriction fragments that demonstrated homologous integration in the wild type was not detected in dprA or recA cells, indicating that in the mutants incoming ssDNA could not be processed into recombinants. Investigation of the fate of donor label 1 min after uptake revealed that incoming ssDNA was immediately degraded in the absence of DprA or RecA. Our results demonstrate that incoming ssDNA requires active protection prior to the RecA-driven search for homology and that both DprA and RecA are needed for this protection.     

An inhibitor of DNA binding and uptake events dictates the proficiency of genetic transformation in Neisseria gonorrhoeae: mechanism of action and links to Type IV pilus expression

Although natural genetic transformation is a widely disseminated form of genetic exchange in prokaryotic species, the proficiencies with which DNA recognition, uptake and processing occur in nature vary greatly. However, the molecular factors and interactions underlying intra- and interspecies diversity in levels of competence for natural genetic transformation are poorly understood. In Neisseria gonorrhoeae, the Gram-negative aetiologic agent of gonorrhoea, DNA binding and uptake involve components required for Type IV pilus (Tfp) biogenesis as well as those which are structurally related to Tfp biogenesis components but dispensable for organelle expression. We demonstrate here that the gonococcal PilV protein, structurally related to Tfp pilin subunits, is an intrinsic inhibitor of natural genetic transformation which acts ultimately by reducing the levels of sequence-specific DNA uptake into the cell. Specifically, we show that DNA uptake is enhanced in strains bearing pilV mutations and reduced in strains overexpressing PilV. Furthermore, we show that PilV exerts its effect by acting as an antagonist of ComP, a positive effector of sequence-specific DNA binding. As it prevents the accumulation of ComP at a site where it can be purified by shear extraction of intact cells, the data are most consistent with PilV either obstructing ComP trafficking or altering ComP stability. In addition, we report that ComP and PilV play overlapping and partially redundant roles in Tfp biogenesis and document other genetic interactions between comP and pilV together with the pilE and pilT genes required for the expression of retractile Tfp. Together, the results reveal a novel mechanism by which the levels of competence are governed in prokaryotic species and suggest unique ways by which competence might be modulated.     

Tracing the Evolution of Competence in Haemophilus influenzae

Natural competence is the genetically encoded ability of some bacteria to take up DNA from the environment. Although most of the incoming DNA is degraded, occasionally intact homologous fragments can recombine with the chromosome, displacing one resident strand. This potential to use DNA as a source of both nutrients and genetic novelty has important implications for the ecology and evolution of competent bacteria. However, it is not known how frequently competence changes during evolution, or whether non-competent strains can persist for long periods of time. We have previously studied competence in H. influenzae and found that both the amount of DNA taken up and the amount recombined varies extensively between different strains. In addition, several strains are unable to become competent, suggesting that competence has been lost at least once. To investigate how many times competence has increased or decreased during the divergence of these strains, we inferred the evolutionary relationships of strains using the largest datasets currently available. However, despite the use of three datasets and multiple inference methods, few nodes were resolved with high support, perhaps due to extensive mixing by recombination. Tracing the evolution of competence in those clades that were well supported identified changes in DNA uptake and/or transformation in most strains. The recency of these events suggests that competence has changed frequently during evolution but the poor support of basal relationships precludes the determination of whether non-competent strains can persist for long periods of time. In some strains, changes in transformation have occurred that cannot be due to changes in DNA uptake, suggesting that selection can act on transformation independent of DNA uptake.     

Natural DNA uptake by Escherichia coli

Escherichia coli has homologues of the competence genes other species use for DNA uptake and processing, but natural competence and transformation have never been detected. Although we previously showed that these genes are induced by the competence regulator Sxy as in other gamma-proteobacteria, no conditions are known that naturally induce sxy expression. We have now tested whether the competence gene homologues encode a functional DNA uptake machinery and whether DNA uptake leads to recombination, by investigating the effects of plasmid-borne sxy expression on natural competence in a wide variety of E. coli strains. High- and low-level sxy expression alone did not induce transformation in any of the strains tested, despite varying the transforming DNA, its concentration, and the incubation conditions used. Direct measurements of uptake of radiolabelled DNA were below the limit of detection, however transformants were readily detected when recombination functions were provided by the lambda Red recombinase. This is the first demonstration that E. coli sxy expression can induce natural DNA uptake and that E. coli's competence genes do encode a functional uptake machinery. However, the amount of transformation cells undergo is limited both by low levels of DNA uptake and by inefficient DNA processing/recombination.     

The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation

The expression of type IV pili (Tfp) by Neisseria gonorrhoeae has been shown to be essential for natural genetic transformation at the level of sequence-specific uptake of DNA. All previously characterized mutants defective in this step of transformation either lack Tfp or are altered in the expression of Tfp-associated properties, such as twitching motility, autoagglutination and the ability to bind to human epithelial cells. To examine the basis for this relationship, we identified potential genes encoding polypeptides sharing structural similarities to PilE, the Tfp subunit, within the N. gonorrhoeae genome sequence database. We found that disruption of one such gene, designated comP (for competence-associated prepilin), leads to a severe defect in the capacity to take up DNA in a sequence-specific manner, but does not alter Tfp biogenesis or expression of the Tfp-associated properties of auto-agglutination, twitching motility and human epithelial cell adherence. Indirect evidence based on immunodetection suggests that ComP is expressed at very low levels relative to that of PilE. The process of DNA uptake in gonococci, therefore, is now known to require the expression of at least three distinct components: Tfp, the recently identified PilT protein and ComP.     

Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance

Sparling, Philip F. (Communicable Disease Center, Atlanta, Ga.). Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J. Bacteriol. 92:1364-1371. 1966.-Eight strains of Neisseria gonorrhoeae were transformed to streptomycin resistance by deoxyribonucleic acid (DNA) extracted from a streptomycin-resistant strain of N. gonorrhoeae. In all strains, competence was greatest in the naturally occurring, virulent clonal types 1 and 2, which gave transformation frequencies up to 1%. Clonal types 3 and 4, which arise on laboratory transfer and are avirulent, gave maximal transformation frequencies of 0.00005%. Competence was maximal in lag and early log phases of growth, but was maintained throughout the growth cycle. A complex broth was required for the physiological expression of competence. The kinetics of DNA uptake, dose-response curve of DNA versus transformants, time required for phenotypic expression, and other features were similar to those in other bacterial transformation systems.     

A competence regulon in Streptococcus pneumoniae revealed by genomic analysis

Transformation in bacteria is the uptake and incorporation of exogenous DNA into a cell's genome. Several species transform naturally during a regulated state defined as competence. Genetic elements in Streptococcus pneumoniae induced during transformation were identified by combining a genetic screen with genomic analysis. Six loci were discovered that composed a competence-induced regulon. These loci shared a consensus promoter sequence and encoded proteins, some of which were similar to proteins involved in DNA processing during transformation in other bacteria. Each locus was induced during competence and essential for genetic transformation.     

The dprA gene is required for natural transformation of Helicobacter pylori

Genetic recombination in Helicobacter pylori is believed to be involved in host adaptation of this gastric pathogen and uptake of DNA by natural transformation can result in changes in virulence factors as well as antigenic variation. To elucidate the mechanisms involved in natural transformation we tested two genes with homology to known competence genes (dprA and traG) for their role in this process. Insertion mutants in these genes were constructed in two different H. pylori strains and their competence by natural transformation was compared to the wild-type. Mutation of the traG homolog did not reduce competence. Mutation of the dprA gene, however, severely impaired natural transformation both with plasmid and chromosomal DNA. Our data indicate that dprA and comB3 are essential parts of a common pathway for chromosomal and plasmid transformation.     

Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation

We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.     

Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili

We have recently described the expression of two pili of different lengths on the surface of Legionella pneumophila (B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768-1775, 1998). Production of long pili requires a functional pilEL locus, encoding a type IV pilin protein. Since type IV pili in Neisseria gonorrhoeae are associated with competence for DNA transformation, we examined the competence of L. pneumophila for DNA transformation under conditions that allowed the expression of type IV pili. We show that L. pneumophila is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing L. pneumophila DNA. Many different L. pneumophila loci are able to transform L. pneumophila after addition of plasmid DNA, including gspA, ppa, asd, and pilEL. The transformation frequency is reduced when competing DNA containing either L. pneumophila DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and a pilEL mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of pilEL alone. We conclude that competence for DNA transformation in L. pneumophila is associated with expression of the type IV pilus and results in recombination of L. pneumophila DNA into the chromosome. Since expression of type IV pili also facilitates attachment of L. pneumophila to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili).     

细菌自然转化的分子机制研究进展

自然环境中,具备自然转化能力的细菌可以自发地从外界获取DNA,从而获得新的遗传性状。为能够自然地被转化,细菌需首先建立一个被称作感受态的生理状态并在此状态下表达DNA摄取和加工相关的基因。DNA摄取基因的表达产物可组装一个能将外源DNA摄入细胞质的蛋白复合物。在细胞质中,进入的DNA可同基因组DNA发生同源重组或建立成一个独立的质粒。一般DNA摄入细胞的过程可分为两个阶段,即从外部基质到细胞周质和跨细胞内膜的转运。近年来,包括作者在内的研究人员发现大肠杆菌中存在新的自然质粒转化模式。本文将首先综述近年来细菌自然转化的分子机制,随后简要介绍大肠杆菌中独特的自然质粒转化模式。     

Quorum sensing contributes to natural transformation of Vibrio cholerae in a species-specific manner

Although it is a human pathogen, Vibrio cholerae is a regular member of aquatic habitats, such as coastal regions and estuaries. Within these environments, V. cholerae often takes advantage of the abundance of zooplankton and their chitinous molts as a nutritious surface on which the bacteria can form biofilms. Chitin also induces the developmental program of natural competence for transformation in several species of the genus Vibrio. In this study, we show that V. cholerae does not distinguish between species-specific and non-species-specific DNA at the level of DNA uptake. This is in contrast to what has been shown for other Gram-negative bacteria, such as Neisseria gonorrhoeae and Haemophilus influenzae. However, species specificity with respect to natural transformation still occurs in V. cholerae. This is based on a positive correlation between quorum sensing and natural transformation. Using mutant-strain analysis, cross-feeding experiments, and synthetic cholera autoinducer-1 (CAI-1), we provide strong evidence that the species-specific signaling molecule CAI-1 plays a major role in natural competence for transformation. We suggest that CAI-1 can be considered a competence pheromone.     

Transformation of Xanthomonas campestris pathovar campestris with plasmid DNA

Procedures for the introduction of plasmid DNA into Gram-negative bacteria have been adapted and optimized to permit transformation of the plant pathogen Xanthomonas campestris pathovar campestris with the cloning vector pKT230 and other broad-host-range plasmids. The technique involves CaCl2-induced competence and heat shock and is similar to that routinely used for Escherichia coli. Wild-type X. c. campestris strains appear to restrict incoming unmodified DNA, so that plasmid DNA for transformation must be prepared from X. c. campestris (into which it has previously been introduced by conjugation). To overcome this disadvantage a restriction-deficient mutant has been isolated.     

Transformation in Escherichia coli: stages in the process.

Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported. E. coli can be rendered competent for DNA uptake by a temperature shock (0 degrees C leads to 42 degrees C leads to 0 degrees C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions. Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2+ is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0 degrees C. When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred. This suggests that competence induction and uptake may be regarded as separate stages. Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence. After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected. It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2+ and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency. This incubation probably causes recovery of physiologically labile cells.     

Analysis of cell variants showing differential susceptibilities to radiation- or chemical-induced neoplastic transformation: differences in their responses to growth factors

The induction of DNA synthesis in quiescent, density-arrested Balb/c 3T3 cells is known to be controlled by the sequential action of at least two functionally distinct sets of growth factors, so-called "competence factors" and "progression factors." Here we examined this induction pathway in Balb/c 3T3 A31-I variants, which showed differential susceptibilities to radiation- and chemical-induced neoplastic transformation despite their similar susceptibilities to radiation- or chemical-induced cell killing and mutagenesis. DNA synthesis was acquired only with the exposure to progression factors in a highly susceptible cell variant (A31-1-13) whereas both competence factors and progression factors were required for a less susceptible cell variant (A31-I-1). The competent state constitutively produced by an autologous mechanism in the highly transformation-susceptible A31-I-13 cells suggests the existence of an endogenous promoter that acts for the expression of the transformed phenotype in an autocrine fashion when the cells have been initiated by radiation or chemical carcinogens. The growth factor requirements acting as a determining factor for susceptibilities to transformation are discussed.     

A genetic screen identifies genes and sites involved in pilin antigenic variation in Neisseria gonorrhoeae

It has previously been shown that the frequency of pilin antigenic variation in Neisseria gonorrhoeae (the gonococcus, Gc) is regulated by iron availability. To identify factors involved in pilin variation in an iron-dependent or an iron-independent manner, we conducted a genetic screen of transposon-mutated gonococci using a pilus-dependent colony morphology phenotype to detect antigenic variation deficient mutants. Forty-six total mutants representing insertions in 30 different genes were shown to have reduced colony morphology changes resulting from impaired pilin variation. Five mutants exhibited an iron-dependent decrease in pilin variation, while the remaining 41 displayed an iron-independent decrease in pilin variation. Based on the levels of antigenic variation impairment, we defined the genes as being essential for, important for, or involved in antigenic variation. DNA repair and DNA transformation frequencies of each mutant were measured to determine whether other recombination-based processes were also affected in the mutants. Each mutant was placed into one of six classes based on their pilin variation, DNA repair and DNA transformation phenotypes. Among the many genes identified, recR is shown to be an additional member of the gonococcal RecF-like recombination pathway. In addition, recG and ruvA represent the first evidence that the processing of Holliday junctions is required for pilin antigenic variation. Moreover, two independent insertions in a non-coding region upstream of the pilE gene suggest that cis-acting sequences important for pilin variation are found in that region. Finally, insertions that effect expression of the thrB and thrC genes suggest that molecules in the threonine biosynthetic pathway are important for pilin variation. Many of the other genes identified in this genetic screen do not have an obvious role in pilin variation, DNA repair, or DNA transformation.