Natural Strain Variation Reveals Diverse Biofilm Regulation in Squid-Colonizing Vibrio fischeri.

The mutualistic symbiont Vibrio fischeri builds a symbiotic biofilm during colonization of squid hosts. Regulation of the exopolysaccharide component, termed Syp, has been examined in strain ES114, where production is controlled by a phosphorelay that includes the inner membrane hybrid histidine kinase RscS. Most strains that lack RscS or encode divergent RscS proteins cannot colonize a squid host unless RscS from a squid symbiont is heterologously expressed. In this study, we examine V. fischeri isolates worldwide to understand the landscape of biofilm regulation during beneficial colonization. We provide a detailed study of three distinct evolutionary groups of V. fischeri and find that while the RscS-Syp biofilm pathway is required in one of the groups, two other groups of squid symbionts require Syp independent of RscS. Mediterranean squid symbionts, including V. fischeri SR5, colonize without an RscS homolog encoded by their genome. Additionally, group A V. fischeri strains, which form a tightly related clade of Hawaii isolates, have a frameshift in rscS and do not require the gene for squid colonization or competitive fitness. These same strains have a frameshift in sypE, and we provide evidence that this group A sypE allele leads to an upregulation in biofilm activity. Thus, this work describes the central importance of Syp biofilm in colonization of diverse isolates and demonstrates that significant evolutionary transitions correspond to regulatory changes in the syp pathway.IMPORTANCE Biofilms are surface-associated, matrix-encased bacterial aggregates that exhibit enhanced protection to antimicrobial agents. Previous work has established the importance of biofilm formation by a strain of luminous Vibrio fischeri bacteria as the bacteria colonize their host, the Hawaiian bobtail squid. In this study, expansion of this work to many natural isolates revealed that biofilm genes are universally required, yet there has been a shuffling of the regulators of those genes. This work provides evidence that even when bacterial behaviors are conserved, dynamic regulation of those behaviors can underlie evolution of the host colonization phenotype. Furthermore, this work emphasizes the importance of investigating natural diversity as we seek to understand molecular mechanisms in bacteria.

Generation and Validation of the iKp1289 Metabolic Model for Klebsiella pneumoniae KPPR1.

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.

Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology.

UNLABELLED: Many pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance. Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). The N. gonorrhoeae chromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transfer pilS sequences into the pilE locus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity. IMPORTANCE: Measuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation.

Neisseria gonorrhoeae MutS affects pilin antigenic variation through mismatch correction and not by pilE guanine quartet binding.

UNLABELLED: Many pathogens use homologous recombination to vary surface antigens to avoid immune surveillance. Neisseria gonorrhoeae achieves this in part by changing the properties of its surface pili in a process called pilin antigenic variation (AV). Pilin AV occurs by high-frequency gene conversion reactions that transfer silent pilS sequences into the expressed pilE locus and requires the formation of an upstream guanine quartet (G4) DNA structure to initiate this process. The MutS and MutL proteins of the mismatch correction (MMC) system act to correct mismatches after replication and prevent homeologous (i.e., partially homologous) recombination, but MutS orthologs can also bind to G4 structures. A previous study showed that mutation of MutS resulted in a 3-fold increase in pilin AV, which could be due to the loss of MutS antirecombination properties or loss of G4 binding. We tested two site-directed separation-of-function MutS mutants that are both predicted to bind to G4s but are not able to perform MMC. Pilus phase variation assays and DNA sequence analysis of pilE variants produced in these mutants showed that all three mutS mutants and a mutL mutant had similar increased frequencies of pilin AV. Moreover, the mutS mutants all showed similar increased levels of pilin AV-dependent synthetic lethality. These results show that antirecombination by MMC is the reason for the effect that MutS has on pilin AV and is not due to pilE G4 binding by MutS. IMPORTANCE: Neisseria gonorrhoeae continually changes its outer surface proteins to avoid recognition by the immune system. N. gonorrhoeae alters the antigenicity of the pilus by directed recombination between partially homologous pilin copies in a process that requires a guanine quartet (G4) structure. The MutS protein of the mismatch correction (MMC) system prevents recombination between partially homologous sequences and can also bind to G4s. We confirmed that loss of MMC increases the frequency of pilin antigenic variation and that two MutS mutants that are predicted to separate the two different functions of MutS inhibit pilin variation similarly to a complete-loss-of-function mutant, suggesting that interaction of MutS with the G4 structure is not a major factor in this process.

The genetics of Neisseria species.

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.

Replication fork inhibition in seqA mutants of Escherichia coli triggers replication fork breakage.

SeqA protein negatively regulates replication initiation in Escherichia 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 under-replication, indicating inhibition of replication forks. Pre-/post-labelling 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 the segregation fork running into the inhibited replication fork and snapping the latter at single-stranded DNA regions.

Neisseria gonorrhoeae RecQ helicase HRDC domains are essential for efficient binding and unwinding of the pilE guanine quartet structure required for pilin antigenic variation.

The strict human pathogen Neisseria gonorrhoeae utilizes homologous recombination to antigenically vary the pilus, thus evading the host immune response. High-frequency gene conversion reactions between many silent pilin loci and the expressed pilin locus (pilE) allow for numerous pilus variants per strain to be produced from a single strain. For pilin antigenic variation (Av) to occur, a guanine quartet (G4) structure must form upstream of pilE. The RecQ helicase is one of several recombination or repair enzymes required for efficient levels of pilin Av, and RecQ family members have been shown to bind to and unwind G4 structures. Additionally, the vast majority of RecQ helicase family members encode one "helicase and RNase D C-terminal" (HRDC) domain, whereas the N. gonorrhoeae RecQ helicase gene encodes three HRDC domains, which are critical for pilin Av. Here, we confirm that deletion of RecQ HRDC domains 2 and 3 causes a decrease in the frequency of pilin Av comparable to that obtained with a functional knockout. We demonstrate that the N. gonorrhoeae RecQ helicase can bind and unwind the pilE G4 structure. Deletion of the RecQ HRDC domains 2 and 3 resulted in a decrease in G4 structure binding and unwinding. These data suggest that the decrease in pilin Av observed in the RecQ HRDC domain 2 and 3 deletion mutant is a result of the enzyme's inability to efficiently bind and unwind the pilE G4 structure.

Genome of Enterobacteriophage Lula/phi80 and insights into its ability to spread in the laboratory environment.

The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80's genome layout copies that of lambda, yet homology with other lambdoid phages is mostly limited to the capsid genes. Lula/phi80's DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage's damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80 lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80 SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi80, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion.

Unauthorized horizontal spread in the laboratory environment: the tactics of Lula, a temperate lambdoid bacteriophage of Escherichia coli.

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.

Reduced lipopolysaccharide phosphorylation in Escherichia coli lowers the elevated ori/ter ratio in seqA mutants.

The seqA defect in Escherichia coli increases the ori/ter ratio and causes chromosomal fragmentation, making seqA mutants dependent on recombinational repair (the seqA recA colethality). To understand the nature of this chromosomal fragmentation, we characterized DeltaseqA mutants and isolated suppressors of the DeltaseqA recA lethality. We demonstrate that our DeltaseqA alleles have normal function of the downstream pgm gene and normal ratios of the major phospholipids in the membranes, but increased surface lipopolysaccharide (LPS) phosphorylation. The predominant class of DeltaseqA recA suppressors disrupts the rfaQGP genes, reducing phosphorylation of the inner core region of LPS. The rfaQGP suppressors also reduce the elevated ori/ter ratio of the DeltaseqA mutants but, unexpectedly, the suppressed mutants still exhibit the high levels of chromosomal fragmentation and SOS induction, characteristic of the DeltaseqA mutants. We also found that colethality of rfaP with defects in the production of acidic phospholipids is suppressed by alternative initiation of chromosomal replication, suggesting that LPS phosphorylation stimulates replication initiation. The rfaQGP suppression of the seqA recA lethality provides genetic support for the surprising physical evidence that the oriC DNA forms complexes with the outer membrane.

The mutT defect does not elevate chromosomal fragmentation in Escherichia coli because of the surprisingly low levels of MutM/MutY-recognized DNA modifications.

Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT-->CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we found mutT recBC double mutants viable with no signs of chromosomal fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated with MutM in vitro, shows no increased relaxation, indicating no additional 8-oxo-G modifications. Our DeltamutT allele elevates the AT-->CG transversion rate 27,000-fold, consistent with published reports. However, the rate of AT-->CG transversions in our mutT(+) progenitor strain is some two orders of magnitude lower than in previous studies, which lowers the absolute rate of mutagenesis in DeltamutT derivatives, translating into less than four 8-oxo-G modifications per genome equivalent, which is too low to cause the expected effects. Introduction of various additional mutations in the DeltamutT strain or treatment with oxidative agents failed to increase the mutagenesis even twofold. We conclude that, in contrast to the previous studies, there is not enough 8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would trigger chromosomal fragmentation.

Translational control of tetracycline resistance and conjugation in the Bacteroides conjugative transposon CTnDOT.

The tetQ-rteA-rteB operon of the Bacteroides conjugative transposon CTnDOT is responsible for tetracycline control of the excision and transfer of CTnDOT. Previous studies revealed that tetracycline control of this operon occurred at the translational level and involved a hairpin structure located within the 130-base leader sequence that lies between the promoter of tetQ and the start codon of the gene. This hairpin structure is formed by two sequences, designated Hp1 and Hp8. Hp8 contains the ribosome binding site for tetQ. Examination of the leader region sequence revealed three sequences that might encode a leader peptide. One was only 3 amino acids long. The other two were 16 amino acids long. By introducing stop codons into the peptide coding regions, we have now shown that the 3-amino-acid peptide is the one that is essential for tetracycline control. Between Hp1 and Hp8 lies an 85-bp region that contains other possible RNA hairpin structures. Deletion analysis of this intervening DNA segment has now identified a sequence, designated Hp2, which is essential for tetracycline regulation. This sequence could form a short hairpin structure with Hp1. Mutations that made the Hp1-Hp2 structure more stable caused nearly constitutively high expression of the operon. Thus, stalling of ribosomes on the 3-amino-acid leader peptide could favor formation of the Hp1-Hp2 structure and thus preclude formation of the Hp1-Hp8 structure, releasing the ribosome binding site of tetQ. Finally, comparison of the CTnDOT tetQ leader regions with upstream regions of five tetQ genes found in other elements reveals that the sequences are virtually identical, suggesting that translational attenuation is responsible for control of tetracycline resistance in these other cases as well.

RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation.

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.