Jeffrey G. Lawrence

Phylogenetic incongruence arising from fragmented speciation in enteric bacteria.

Evolutionary relationships among species are often assumed to be fundamentally unambiguous, where genes within a genome are thought to evolve in concert and phylogenetic incongruence between individual orthologs is attributed to idiosyncrasies in their evolution. We have identified substantial incongruence between the phylogenies of orthologous genes in Escherichia, Salmonella, and Citrobacter, or E. coli, E. fergusonii, and E. albertii. The source of incongruence was inferred to be recombination, because individual genes support conflicting topology more robustly than expected from stochastic sequence homoplasies. Clustering of phylogenetically informative sites on the genome indicated that the regions of recombination extended over several kilobases. Analysis of phylogenetically distant taxa resulted in consensus among individual gene phylogenies, suggesting that recombination is not ongoing; instead, conflicting relationships among genes in descendent taxa reflect recombination among their ancestors. Incongruence could have resulted from random assortment of ancestral polymorphisms if species were instantly created from the division of a recombining population. However, the estimated branch lengths in alternative phylogenies would require ancestral populations with far more diversity than is found in extant populations. Rather, these and previous data collectively suggest that genome-wide recombination rates decreased gradually, with variation in rate among loci, leading to pluralistic relationships among their descendent taxa.

Towards more robust methods of alien gene detection

Because the properties of horizontally-transferred genes will reflect the mutational proclivities of their donor genomes, they often show atypical compositional properties relative to native genes. Parametric methods use these discrepancies to identify bacterial genes recently acquired by horizontal transfer. However, compositional patterns of native genes vary stochastically, leaving no clear boundary between typical and atypical genes. As a result, while strongly atypical genes are readily identified as alien, genes of ambiguous character are poorly classified when a single threshold separates typical and atypical genes. This limitation affects all parametric methods that examine genes independently, and escaping it requires the use of additional genomic information. We propose that the performance of all parametric methods can be improved by using a multiple-threshold approach. First, strongly atypical alien genes and strongly typical native genes would be identified using conservative thresholds. Genes with ambiguous compositional features would then be classified by examining gene context, including the class (native or alien) of flanking genes. By including additional genomic information in a multiple-threshold framework, we observed a remarkable improvement in the performance of several popular, but algorithmically distinct, methods for alien gene detection.

Genomic Investigation Reveals Highly Conserved, Mosaic, Recombination Events Associated with Capsular Switching among Invasive Neisseria meningitidis Serogroup W Sequence Type (ST)-11 Strains

Neisseria meningitidis is an important cause of meningococcal disease globally. Sequence type (ST)-11 clonal complex (cc11) is a hypervirulent meningococcal lineage historically associated with serogroup C capsule and is believed to have acquired the W capsule through a C to W capsular switching event. We studied the sequence of capsule gene cluster (cps) and adjoining genomic regions of 524 invasive W cc11 strains isolated globally. We identified recombination breakpoints corresponding to two distinct recombination events within W cc11: A 8.4-kb recombinant region likely acquired from W cc22 including the sialic acid/glycosyl-transferase gene, csw resulted in a C→W change in capsular phenotype and a 13.7-kb recombinant segment likely acquired from Y cc23 lineage includes 4.5 kb of cps genes and 8.2 kb downstream of the cps cluster resulting in allelic changes in capsule translocation genes. A vast majority of W cc11 strains (497/524, 94.8%) retain both recombination events as evidenced by sharing identical or very closely related capsular allelic profiles. These data suggest that the W cc11 capsular switch involved two separate recombination events and that current global W cc11 meningococcal disease is caused by strains bearing this mosaic capsular switch.

Rapid Divergence of Two Classes of Haemophilus ducreyi

Haemophilus ducreyi, the etiologic agent of chancroid, expresses variants of several key virulence factors. While previous reports suggested that H. ducreyi strains formed two clonal populations, the differences between, and diversity within, these populations were unclear. To assess their variability, we examined sequence diversity at 11 H. ducreyi loci, including virulence and housekeeping genes, augmenting published data sets with PCR-amplified genes to acquire data for at least 10 strains at each locus. While sequences from all 11 loci place strains into two distinct groups, there was very little variation within each group. The difference between alleles of the two groups was variable and large at 3 loci encoding surface-exposed proteins (0.4 < K(S) < 1.3, where K(S) is divergence at synonymous sites) but consistently small at genes encoding cytoplasmic or periplasmic proteins (K(S) < 0.09). The data suggest that the two classes have recently diverged, that recombination has introduced variant alleles into at least 3 distinct loci, and that these alleles have been confined to one of the two classes. In addition, recombination is evident among alleles within, but not between, classes. Rather than clones of the same species, these properties indicate that the two classes may form distinct species.

Alternative pathways for siroheme synthesis in Klebsiella aerogenes.

Siroheme, the cofactor for sulfite and nitrite reductases, is formed by methylation, oxidation, and iron insertion into the tetrapyrrole uroporphyrinogen III (Uro-III). The CysG protein performs all three steps of siroheme biosynthesis in the enteric bacteria Escherichia coli and Salmonella enterica. In either taxon, cysG mutants cannot reduce sulfite to sulfide and require a source of sulfide or cysteine for growth. In addition, CysG-mediated methylation of Uro-III is required for de novo synthesis of cobalamin (coenzyme B(12)) in S. enterica. We have determined that cysG mutants of the related enteric bacterium Klebsiella aerogenes have no defect in the reduction of sulfite to sulfide. These data suggest that an alternative enzyme allows for siroheme biosynthesis in CysG-deficient strains of Klebsiella. However, Klebsiella cysG mutants fail to synthesize coenzyme B(12), suggesting that the alternative siroheme biosynthetic pathway proceeds by a different route. Gene cysF, encoding an alternative siroheme synthase homologous to CysG, has been identified by genetic analysis and lies within the cysFDNC operon; the cysF gene is absent from the E. coli and S. enterica genomes. While CysG is coregulated with the siroheme-dependent nitrite reductase, the cysF gene is regulated by sulfur starvation. Models for alternative regulation of the CysF and CysG siroheme synthases in Klebsiella and for the loss of the cysF gene from the ancestor of E. coli and S. enterica are presented.

Quantification of codon selection for comparative bacterial genomics

BackgroundStatistics measuring codon selection seek to compare genes by their sensitivity to selection for translational efficiency, but existing statistics lack a model for testing the significance of differences between genes. Here, we introduce a new statistic for measuring codon selection, the Adaptive Codon Enrichment (ACE).ResultsThis statistic represents codon usage bias in terms of a probabilistic distribution, quantifying the extent that preferred codons are over-represented in the gene of interest relative to the mean and variance that would result from stochastic sampling of codons. Expected codon frequencies are derived from the observed codon usage frequencies of a broad set of genes, such that they are likely to reflect nonselective, genome wide influences on codon usage (e.g. mutational biases). The relative adaptiveness of synonymous codons is deduced from the frequency of codon usage in a pre-selected set of genes relative to the expected frequency. The ACE can predict both transcript abundance during rapid growth and the rate of synonymous substitutions, with accuracy comparable to or greater than existing metrics. We further examine how the composition of reference gene sets affects the accuracy of the statistic, and suggest methods for selecting appropriate reference sets for any genome, including bacteriophages. Finally, we demonstrate that the ACE may naturally be extended to quantify the genome-wide influence of codon selection in a manner that is sensitive to a large fraction of codons in the genome. This reveals substantial variation among genomes, correlated with the tRNA gene number, even among groups of bacteria where previously proposed whole-genome measures show little variation.ConclusionsThe statistical framework of the ACE allows rigorous comparison of the level of codon selection acting on genes, both within a genome and between genomes.

Genetic Manipulation of Pathogenicity Loci in Non-Typhimurium Salmonella

The traditional genetic tools used in Salmonella enterica serovar Typhimurium rely heavily on a high-transducing mutant of bacteriophage P22. P22 recognizes its hosts by the structure of their O-antigens, which vary among serovars of Salmonella; therefore, it cannot be used in most non-Typhimurium Salmonella, including the majority of those causing food-borne illnesses in both humans and livestock. Bacteriophage P1 infects a variety of enteric bacteria, including galE mutants of serovar Typhimurium; however, the degree to which the presence of coimmune prophages, the lack of required attachment sites or the lack of host factors act as barriers to using phage P1 in natural isolates of Salmonella is unknown. Here, we show that recombineering can be used to make virtually any serovar of Salmonella susceptible to P1 infection; as a result, P1 can be utilized for facile genetic manipulation of non-Typhimurium Salmonella, including movement of very large pathogenicity islands. A toolkit for easy manipulation of non-Typhimurium serovars of Salmonella is described.

A likelihood approach to classifying fluorescent events collected by multicolor flow cytometry

Flow cytometry is an effective tool for enumerating fluorescently-labeled microbes recovered from natural environments. However, low signal strength and the presence of fluorescent, non-cellular particles complicate the separation of cellular events from noise. Existing classification methods rely on the arbitrary placement of noise thresholds, resulting in potentially high rates of misclassification of fluorescent cells, thus precluding the robust estimation of the proportions of classes of fluorescent cells. Here we present a method for objectively separating signal from noise. Rather than setting an arbitrary noise threshold, the Z-scoring approach uses the Gaussian distribution of signal strength (a) to locate noise threshold for individual fluorophores, (b) to predict the likelihood of different fluorescent genotypes in producing the signal observed, and (c) to normalize the fraction of cellular events count for each fluorescent cell class. The likelihood framework allows rejection of alternative genotypes, leading to robust and reliable classification of fluorescent cells. Use of Z-scoring in classification of cells expressing multiple fluorophores, use of spillover in actively scoring events, and the successful classification of multiple fluorophores using a single detector within a flow cytometer are discussed. A software package that performs Z-scoring for cells labeled with one or more fluorophores is described.

Chromosome architecture constrains horizontal gene transfer in bacteria

Despite significant frequencies of lateral gene transfer between species, higher taxonomic groups of bacteria show ecological and phenotypic cohesion. This suggests that barriers prevent panmictic dissemination of genes via lateral gene transfer. We have proposed that most bacterial genomes have a functional architecture imposed by Architecture IMparting Sequences (AIMS). AIMS are defined as 8 base pair sequences preferentially abundant on leading strands, whose abundance and strand-bias are positively correlated with proximity to the replication terminus. We determined that inversions whose endpoints lie within a single chromosome arm, which would reverse the polarity of AIMS in the inverted region, are both shorter and less frequent near the replication terminus. This distribution is consistent with the increased selection on AIMS function in this region, thus constraining DNA rearrangement. To test the hypothesis that AIMS also constrain DNA transfer between genomes, AIMS were identified in genomes while ignoring atypical, potentially laterally-transferred genes. The strand-bias of AIMS within recently acquired genes was negatively correlated with the distance of those genes from their genome’s replication terminus. This suggests that selection for AIMS function prevents the acquisition of genes whose AIMS are not found predominantly in the permissive orientation. This constraint has led to the loss of at least 18% of genes acquired by transfer in the terminus-proximal region. We used completely sequenced genomes to produce a predictive road map of paths of expected horizontal gene transfer between species based on AIMS compatibility between donor and recipient genomes. These results support a model whereby organisms retain introgressed genes only if the benefits conferred by their encoded functions outweigh the detriments incurred by the presence of foreign DNA lacking genome-wide architectural information.

Selection, periodicity and potential function for Highly Iterative Palindrome-1 (HIP1) in cyanobacterial genomes

Abstract Highly Iterated Palindrome 1 (HIP1, GCGATCGC) is hyper-abundant in most cyanobacterial genomes. In some cyanobacteria, average HIP1 abundance exceeds one motif per gene. Such high abundance suggests a significant role in cyanobacterial biology. However, 20 years of study have not revealed whether HIP1 has a function, much less what that function might be. We show that HIP1 is 15- to 300-fold over-represented in genomes analyzed. More importantly, HIP1 sites are conserved both within and between open reading frames, suggesting that their overabundance is maintained by selection rather than by continual replenishment by neutral processes, such as biased DNA repair. This evidence for selection suggests a functional role for HIP1. No evidence was found to support a functional role as a peptide or RNA motif or a role in the regulation of gene expression. Rather, we demonstrate that the distribution of HIP1 along cyanobacterial chromosomes is significantly periodic, with periods ranging from 10 to 90 kb, consistent in scale with periodicities reported for co-regulated, co-expressed and evolutionarily correlated genes. The periodicity we observe is also comparable in scale to chromosomal interaction domains previously described in other bacteria. In this context, our findings imply HIP1 functions associated with chromosome and nucleoid structure.