The rates of pilin antigenic variation (Av) of two strains of Neisseria meningitidis were determined using an unbiased DNA sequencing assay. Strain MC58 underwent pilin Av at a rate similar to that of N. gonorrhoeae strain MS11 but lower than that of N. gonorrhoeae strain FA1090. Pilin Av was undetectable in strain FAM18.Neisseria meningitidis is a Gram-negative diplococcus that colonizes the nasopharynx of approximately 5 to 10% of the population and is usually nonpathogenic but can occasionally enter the bloodstream to cause septicemia and can eventually spread to the meninges, causing meningitis (15). Approximately 500,000 cases of meningococcal meningitis occur every year, with nearly 10% resulting in fatality (2).Type IV pili (TFP) are long filamentous structures protruding from the bacterial surface and are required for adherence of N. meningitidis to host cells (7). As with the TFP of the closely related pathogen Neisseria gonorrhoeae, the pili are able to undergo antigenic variation (Av). In N. gonorrhoeae, pilin Av occurs as a result of recombination between one of the multiple silent pilS copies and the expressed pilin gene (pilE). The pilS copies share significant regions of homology with pilE yet lack a promoter or ribosome-binding site and the initial 5Ј coding sequence. Pilin Av relies on RecA and the RecF-like recombination pathway to catalyze gene conversion, resulting in an altered pilE sequence, carrying part of the pilS donor, and the original unaltered pilS sequence (8, 9).While the frequency of pilin Av has been measured in N. gonorrhoeae (5,10,12), this process has never been quantified in N. meningitidis. Two sequenced strains were picked to measure pilin Av: serogroup B strain MC58 (sequence type complex), isolated from an invasive infection (14), and serogroup C strain FAM18 (ST-11 complex), which was isolated from a patient with septicemia (1). In both strains, the native recA gene was replaced with the very highly conserved N. gonorrhoeae recA6 construct, which allows regulation of expression with IPTG (isopropyl-␤-D-thiogalactopyranoside) (17). recA6 strains are RecA ϩ when grown with IPTG but are RecA Ϫ when grown without IPTG (17). These phenotypes were confirmed by measuring the UV sensitivities and DNA transformation competence levels of both strains with or without IPTG, and both strains were shown to be piliated by transmission electron microscopy (data not shown). Bacteria were grown at 37°C with 5% CO 2 on gonococcal medium base (GCB; Difco) plus Kellogg supplements I and II (11).The pilin Av sequencing assay was performed as described previously (5, 10, 12) with slight modifications. Briefly, FAM18 and MC58 were grown on solid GCB with 1 mM IPTG, allowing for the expression of RecA, for 22 h and 12.5 h, respectively, which was estimated to produce 20 generations. For FAM18, little or no pilin Av was expected since the G-quartetforming sequence required for pilin Av is degenerate in this strain (3). Therefore, two random progenitor colonies were picked from IPTG-enriched medi...

E. coli and Salmonella typhimurium are widely used bacterial hosts for genetic manipulation of DNA from prokaryotes and eukaryotes. Introduction of foreign DNA by electroporation or transduction into E. coli and Salmonella is limited by host restriction of incoming DNA by the recipient cells. Here, we describe a simple method that temporarily inactivates host restriction, allowing high-frequency DNA transfer. This technique might be readily applied to a wide range of bacteria to increase DNA transfer between strains and species.

Type IV pilus expression has been strongly implicated in the virulence of Neisseria gonorrhoeae, the causative agent of gonorrhea. In Neisseria, these pili undergo frequent antigenic variation (Av), which is presumed to allow reinfection of high-risk groups. Pilin Av is the result of RecA-mediated recombination events between the gene encoding the major pilin subunit (pilE) and multiple silent pilin locus (pilS) copies, utilizing a RecF-like recombination pathway. The role of RecBCD in pilin Av has been controversial. Previous studies measuring pilin Av in recB and recD mutants in two independent strains of N. gonorrhoeae (MS11 and FA1090) by indirect methods yielded conflicting results. In addition, these two laboratory strains have been suggested to express very different DNA repair capabilities. We show that the FA1090 and MS11 parental strains have similar abilities to repair DNA damage via UV-induced DNA damage, nalidixic acid-induced double-strand breaks, and methyl methanesulfonate-induced alkylation and that RecB and RecD are involved in the repair of these lesions. To test the role of the RecBCD pathway in pilin Av, the rate and frequency of pilin Av were directly measured by sequencing the pilE locus in randomly selected piliated progeny of both MS11 and FA1090 in recB and recD mutants. Our results definitively show that recB and recD mutants undergo pilin Av at rates similar to those of the parents in both strain backgrounds, demonstrating that efficient pilin Av is neither enhanced nor inhibited by the RecBCD complex.Homologous recombination is a widely conserved process used by all organisms to repair DNA damage and to mediate genetic transfer. Usually RecA or a RecA homologue is required for homologous recombination, but the other processing enzymes used to prepare DNA for recombination differ between organisms. Generally, double-stranded DNA (dsDNA) breaks are repaired by the RecBCD complex in gram-negative organisms and the AddAB complex in gram-positive organisms, although exceptions exist in which AddAB has been found in some proteobacteria (1,4,29,33,56). Single-stranded DNA (ssDNA) gaps are typically repaired by the RecF pathway, which is ubiquitous among the Bacteria and is part of a universal step in recombinational repair (31). While the RecBCD and RecF pathways have been extensively studied in Escherichia coli, they are less well understood in the humanspecific pathogen Neisseria gonorrhoeae. N. gonorrhoeae is a gram-negative bacterium that has been isolated only from infected humans and is believed to exist naturally only within the human population. Like E. coli, N. gonorrhoeae has a RecA homologue and possesses most of the genes that define the RecBCD and RecF pathways of E. coli (27).In E. coli, the RecBCD exonuclease begins to degrade DNA at the site of the break, and upon reaching a particular sequence, a chi site, the RecD component is inactivated, leaving RecBC to act as a helicase, processively unwinding the DNA, yielding ssDNA, which is the substrate for RecA (reviewed in refere...

Variations in genome size and gene order were observed in archival Salmonella enterica serovar Typhimurium cultures stored for over 40 years. In one strain, microarray analysis revealed a large, stable amplification. PCR analysis of the same strain revealed a genomic duplication that underwent a translocation. Other strains had smaller duplications and deletions. These results demonstrate that storage in stabs over time at room temperature not only allows for further bacterial growth but also may produce an environment that selects for a variety of mutations, including genomic rearrangements.Several investigators (3, 4, 8-10, 15, 16, 26) have examined genetic changes among survivors within populations of cells present in a harsh environmental habitat. In addition to the jettisoning of "excess baggage" genes, as postulated by Koch (17), it might be anticipated that certain genetic rearrangements, including amplifications, could increase the fitness of cells in the population. We have a unique set of several thousand bacterial samples that have experienced unfavorable growth conditions during 4 decades of storage in the presence of limiting nutrients and increasing metabolic by-products.This extensive collection of Salmonella enterica serovar Typhimurium LT2 auxotrophic mutants accumulated by Miloslav Demerec and associates decades ago was the basis of inter-and intragenic chromosomal mapping (5-7). Upon inoculation over 4 decades ago, low reversion rates of less than 1 in 10 8 cells were recorded, indicating the stability of the auxotrophic phenotype upon inoculation into stabs. To date, 421 of these stabbed cultures that had been stored at room temperature have been opened, and each yielded colony formers, suggesting survival strategies that are currently not well understood.Tests performed on 14 serovar Typhimurium LT2 strains included DNA-DNA hybridization analysis to compare the archived strains with wild-type serovar Typhimurium LT2 and PCR analysis to observe any change in genomic rearrangements at rRNA (rrn) operons. Five of these are listed in Table 1.While nine of the strains had no detectable genomic anomalies, a few strains had relatively small chromosomal amplifications and deletions. Interestingly, one strain had a largescale genomic rearrangement and amplification. Note that these assays were performed on LT2 strains that do not have mutator phenotypes (data not shown). Archival serovar Typhimurium LT7 strains, which have a mutator phenotype (19), have previously been shown to have undergone genomic rearrangements (8).Previous work has shown that chromosomal duplications can occur at relatively high frequencies in the presence of limited carbon sources but that these duplications typically are not maintained for long periods of time once the strains are grown in an abundance of the previously limited nutrient (28). Additionally, other investigators have shown that stable genomic rearrangements due to recombination at rRNA (rrn) operons are frequently found in natural isolates of Salmonella enteric...

Type IV pili are required for virulence in Neisseria gonorrhoeae, as they are involved in adherence to host epithelium, twitching motility, and DNA transformation. The outer membrane secretin PilQ forms a homododecameric ring through which the pilus is proposed to be secreted. pilQ null mutants are nonpiliated, and thus, all pilus-dependent functions are eliminated. Mutagenesis was performed on the middle one-third of pilQ, and mutants with colony morphologies consistent with the colony morphology of nonpiliated or underpiliated bacteria were selected. Nineteen mutants, each with a single amino acid substitution, were isolated and displayed diverse phenotypes in terms of PilQ multimer stability, pilus expression, transformation efficiency, and host cell adherence. The 19 mutants were grouped into five phenotypic classes based on functionality. Four of the five mutant classes fit the current model of pilus functionality, which proposes that a functional pilus assembly apparatus, not necessarily full-length pili, is required for transformation, while high levels of displayed pili are required for adherence. One class, despite having an underpiliated colony morphology, expressed high levels of pili yet adhered poorly, demonstrating that pilus expression is necessary but not sufficient for adherence and indicating that PilQ may be directly involved in host cell adherence. The collection of phenotypes expressed by these mutants suggests that PilQ has an active role in pilus expression and function.