Neisseria gonorrhoeae and Neisseria meningitidis are closely related organisms that cause the sexually transmitted infection gonorrhea and serious bacterial meningitis and septicemia, respectively. Both species possess multiple mechanisms to alter the expression of surface-exposed proteins through the processes of phase and antigenic variation. This potential for wide variability in surface-exposed structures allows the organisms to always have subpopulations of divergent antigenic types to avoid immune surveillance and to contribute to functional variation. Additionally, the Neisseria are naturally competent for DNA transformation, which is their main means of genetic exchange. Although bacteriophages and plasmids are present in this genus, they are not as effective as DNA transformation for horizontal genetic exchange. There are barriers to genetic transfer, such as restriction-modification systems and CRISPR loci, that limit particular types of exchange. These host-restricted pathogens illustrate the rich complexity of genetics that can help define the similarities and differences of closely related organisms.

RecA-and RecBC-catalyzed repair in eubacteria assembles chromosomes fragmented by double-strand breaks. We propose that recA mutants, being unable to repair fragmented chromosomes, depend on various strategies designed to avoid chromosomal fragmentation. To identify chromosomal fragmentation-avoidance strategies, we screened for Escherichia coli mutants synthetically inhibited in combination with recA inactivation by identifying clones unable to lose a plasmid carrying the recA ؉ gene. Using this screen, we have isolated several RecA-dependent mutants and assigned them to three distinct areas of metabolism. The tdk and rdgB mutants affect synthesis of DNA precursors. The fur, ubiE, and ubiH mutants are likely to have increased levels of reactive oxygen species. The seqA, topA mutants and an insertion in smtA perturbing the downstream mukFEB genes affect nucleoid administration. All isolated mutants show varying degree of SOS induction, indicating elevated levels of chromosomal lesions. As predicted, mutants in rdgB, seqA, smtA, topA, and fur show increased levels of chromosomal fragmentation in recBC mutant conditions. Future characterization of these RecA-dependent mutants will define mechanisms of chromosomal fragmentation avoidance. C hromosomal fragmentation due to double-strand DNA breaks and disintegrated replication forks is a major contributor to genome instability in all organisms (1, 2). The two major pathways used by eukaryotic cells to repair fragmented chromosomes are nonhomologous end joining and homologous recombination (3). Nonhomologous end joining is an imprecise repair, frequently involving loss of genetic information, but it is nevertheless an efficient way to repair double-strand breaks in cells of higher eukaryotes (4). However, disintegrated replication forks, having a single double-strand end, cannot be reassembled by nonhomologous end joining and require error-free recombinational repair (5, 6). The importance of recombinational repair in higher eukaryotes is illustrated by the inviability of recombinational repair mutants in mice (7) and by the rapid death of recombinational repair-defective vertebrate cells due to chromosomal fragmentation (8). In the model eubacterium Escherichia coli, recombinational repair of fragmented chromosomes is catalyzed by the RecA and RecBCD enzymes (9). RecBCD is an exonuclease͞helicase that prepares the doublestrand DNA ends of the break for RecA polymerization (10). RecA filament catalyzes homologous strand exchange of the broken DNA duplex with an intact sister duplex, thus creating an opportunity for double-strand break repair (11).In recA or recBC mutants of E. coli, double-strand DNA breaks are not repaired (12) and cause chromosomal loss and cell death (13,14). However, recA mutant E. coli strains are still 50% viable (15, 16), which indicates that the chromosomal fragmentation is not a frequent event in E. coli and suggests the existence of strategies designed to avoid chromosomal fragmentation. Inactivation of one of these hypothetical avoidance a...

We investigated the characteristics of a lambdoid prophage, nicknamed Lula, contaminating E. coli strains from several sources, that allowed it to spread horizontally in the laboratory environment. We found that new Lula infections are inconspicuous; at the same time, Lula lysogens carry unusually high titers of the phage in their cultures, making them extremely infectious. In addition, Lula prophage interferes with P1 phage development and induces its own lytic development in response to P1 infection, turning P1 transduction into an efficient vehicle of Lula spread. Thus, using Lula prophage as a model, we reveal the following principles of survival and reproduction in the laboratory environment: 1) stealth (via laboratory material commensality), 2) stability (via resistance to specific protocols), 3) infectivity (via covert yet aggressive productivity and laboratory protocol hitchhiking). Lula, which turned out to be identical to bacteriophage phi80, also provides an insight into a surprising persistence of T1-like contamination in BAC libraries.

Klebsiella pneumoniae has a reputation for causing a wide range of infectious conditions, with numerous highly virulent and antibiotic-resistant strains. Metabolic models have the potential to provide insights into the growth behavior, nutrient requirements, essential genes, and candidate drug targets in these strains. Here we develop a metabolic model for KPPR1, a highly virulent strain of K. pneumoniae. We apply a combination of Biolog phenotype data and fitness data to validate and refine our KPPR1 model. The final model displays a predictive accuracy of 75% in identifying potential carbon and nitrogen sources for K. pneumoniae and of 99% in predicting nonessential genes in rich media. We demonstrate how this model is useful in studying the differences in the metabolic capabilities of the low-virulence MGH 78578 strain and the highly virulent KPPR1 strain. For example, we demonstrate that these strains differ in carbohydrate metabolism, including the ability to metabolize dulcitol as a primary carbon source. Our model makes numerous other predictions for follow-up verification and analysis.

SeqA protein negatively regulates replication initiation in E. coli and is also proposed to organize maturation and segregation of the newly-replicated DNA. The seqA mutants suffer from chromosomal fragmentation; since this fragmentation is attributed to defective segregation or nucleoid compaction, two-ended breaks are expected. Instead, we show that, in SeqA’s absence, chromosomes mostly suffer one-ended DNA breaks, indicating disintegration of replication forks. We further show that replication forks are unexpectedly slow in seqA mutants. Quantitative kinetics of origin and terminus replication from aligned chromosomes not only confirm origin overinitiation in seqA mutants, but also reveal terminus underreplication, indicating inhibition of replication forks. Pre/post-labeling studies of the chromosomal fragmentation in seqA mutants suggest events involving single forks, rather than pairs of forks from consecutive rounds rear-ending into each other. We suggest that, in the absence of SeqA, the sister-chromatid cohesion “safety spacer” is destabilized and completely disappears if the replication fork is inhibited, leading to segregation fork running into the inhibited replication fork and snapping it at single-stranded DNA regions.