Decoupling of divergent gene regulation by sequence-specific DNA binding factors.

Divergent gene pairs (DGPs) are abundant in eukaryotic genomes. Since two genes in a DGP potentially share the same regulatory sequence, one might expect that they should be co-regulated. However, an inspection of yeast DGPs containing cell-cycle or stress response genes revealed that most DGPs are differentially-regulated. The mechanism underlying DGP differential regulation is not understood. Here, we showed that co- versus differential regulation cannot be explained by genetic features including promoter length, binding site orientation, TATA elements, nucleosome distribution, or presence of non-coding RNAs. Using time-lapse fluorescence microscopy, we carried out an in-depth study of a differentially regulated DGP, PFK26-MOB1. We found that their differential regulation is mainly achieved through two DNA-binding factors, Tbf1 and Mcm1. Similar to 'enhancer-blocking insulators' in higher eukaryotes, these factors shield the proximal promoter from the action of more distant transcription regulators. We confirmed the blockage function of Tbf1 using synthetic promoters. We further presented evidence that the blockage mechanism is widely used among genome-wide DGPs. Besides elucidating the DGP regulatory mechanism, our work revealed a novel class of insulators in yeast.

Stochastic expression and epigenetic memory at the yeast HO promoter.

Eukaryotic gene regulation usually involves sequence-specific transcription factors and sequence-nonspecific cofactors. A large effort has been made to understand how these factors affect the average gene expression level among a population. However, little is known about how they regulate gene expression in individual cells. In this work, we address this question by mutating multiple factors in the regulatory pathway of the yeast HO promoter (HOpr) and probing the corresponding promoter activity in single cells using time-lapse fluorescence microscopy. We show that the HOpr fires in an "on/off" fashion in WT cells as well as in different genetic backgrounds. Many chromatin-related cofactors that affect the average level of HO expression do not actually affect the firing amplitude of the HOpr; instead, they affect the firing frequency among individual cell cycles. With certain mutations, the bimodal expression exhibits short-term epigenetic memory across the mitotic boundary. This memory is propagated in "cis" and reflects enhanced activator binding after a previous "on" cycle. We present evidence that the memory results from slow turnover of the histone acetylation marks.

Molecular Types of Methicillin-Resistant Staphylococcus aureus and Methicillin-Sensitive S. aureus Strains Causing Skin and Soft Tissue Infections and Nasal Colonization, Identified in Community Health Centers in New York City.

In November 2011, The Rockefeller University Center for Clinical and Translational Science (CCTS), the Laboratory of Microbiology and Infectious Diseases, and Clinical Directors Network (CDN) launched a research and learning collaborative project with six community health centers in the New York City metropolitan area to determine the nature (clonal type) of community-acquired Staphylococcus aureus strains causing skin and soft tissue infections (SSTIs). Between November 2011 and March 2013, wound and nasal samples from 129 patients with active SSTIs suspicious for S. aureus were collected and characterized by molecular typing techniques. In 63 of 129 patients, the skin wounds were infected by S. aureus: methicillin-resistant S. aureus (MRSA) was recovered from 39 wounds and methicillin-sensitive S. aureus (MSSA) was recovered from 24. Most-46 of the 63-wound isolates belonged to the CC8/Panton-Valentine leukocidin-positive (PVL(+)) group of S. aureus clone USA300: 34 of these strains were MRSA and 12 were MSSA. Of the 63 patients with S. aureus infections, 30 were also colonized by S. aureus in the nares: 16 of the colonizing isolates were MRSA, and 14 were MSSA, and the majority of the colonizing isolates belonged to the USA300 clonal group. In most cases (70%), the colonizing isolate belonged to the same clonal type as the strain involved with the infection. In three of the patients, the identity of invasive and colonizing MRSA isolates was further documented by whole-genome sequencing.

Genetic pathway in acquisition and loss of vancomycin resistance in a methicillin resistant Staphylococcus aureus (MRSA) strain of clonal type USA300.

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible "parental" strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a "stealth" strategy to evade detection by the host immune system.

The evolution of antibiotic resistance in an incurable and ultimately fatal infection: A retrospective case study.

Background and objectives: The processes by which pathogens evolve within a host dictate the efficacy of treatment strategies designed to slow antibiotic resistance evolution and influence population-wide resistance levels. The aim of this study is to describe the underlying genetic and phenotypic changes leading to antibiotic resistance within a patient who died as resistance evolved to available antibiotics. We assess whether robust patterns of collateral sensitivity and response to combinations existed that might have been leveraged to improve therapy. Methodology: We used whole-genome sequencing of nine isolates taken from this patient over 279 days of a chronic infection with Enterobacter hormaechei, and systematically measured changes in resistance against five of the most relevant drugs considered for treatment. Results: The entirety of the genetic change is consistent with de novo mutations and plasmid loss events, without acquisition of foreign genetic material via horizontal gene transfer. The nine isolates fall into three genetically distinct lineages, with early evolutionary trajectories being supplanted by previously unobserved multi-step evolutionary trajectories. Importantly, although the population evolved resistance to all the antibiotics used to treat the infection, no single isolate was resistant to all antibiotics. Evidence of collateral sensitivity and response to combinations therapy revealed inconsistent patterns across this diversifying population. Conclusions: Translating antibiotic resistance management strategies from theoretical and laboratory data to clinical situations, such as this, will require managing diverse population with unpredictable resistance trajectories.

Intra-Urban Variation of Intimate Partner Violence Against Women and Men in Kenya: Evidence from the 2014 Kenya Demographic and Health Survey.

Although urban areas are diverse and urban inequities are well documented, surveys commonly differentiate intimate partner violence (IPV) rates only by urban versus rural residence. This study compared rates of current IPV victimization among women and men by urban residence (informal and formal settlements). Data from the 2014 Kenya Demographic and Health Survey, consisting of an ever-married sample of 1,613 women (age 15-49 years) and 1,321 men (age 15-54 years), were analyzed. Multilevel logistic regression was applied to female and male data separately to quantify the associations between residence and any current IPV while controlling for regional variation and other factors. Results show gendered patterns of intra-urban variation in IPV occurrence, with the greatest burden of IPV identified among women in informal settlements (across all types of violence). Unadjusted analyses suggest residing in informal settlements is associated with any current IPV against women, but not men, compared with their counterparts in formal urban settlements. This correlation is not statistically significant when adjusting for women's education level in multivariate analysis. In addition, reporting father beat mother, use of current physical violence against partner, partner's alcohol use, and marital status are associated with any current IPV against women and men. IPV gets marginal attention in urban violence and urban health research, and our results highlight the importance of spatially disaggregate IPV data-beyond the rural-urban divide-to inform policy and programming. Future research may utilize intersectional and syndemic approaches to investigate the complexity of IPV and clustering with other forms of violence and other health issues in different urban settings, especially among marginalized residents in informal urban settings.

Analysis of a Cell Wall Mutant Highlights Rho-Dependent Genome Amplification Events in Staphylococcus aureus.

In a study of antibiotic resistance in Staphylococcus aureus, specific cell wall mutants were previously generated for the peptidoglycan biosynthesis gene murF, by the insertion of an integrative plasmid. A collection of 30 independent mutants was obtained, and all harbored a variable number of copies of the inserted plasmid, arranged in tandem in the chromosome. Of the 30 mutants, only 3, F9, F20 and F26, with a lower number of plasmid copies, showed an altered peptidoglycan structure, lower resistance to ß-lactams and a different loss-of-function mutation in rho gene, that encodes a transcription termination factor. The rho mutations were found to correlate with the level of oxacillin resistance, since genetic complementation with rho gene reestablished the resistance and cell wall parental profile in F9, F20 and F26 strains. Furthermore, complementation with rho resulted in the amplification of the number of plasmid tandem repeats, suggesting that Rho enabled events of recombination that favored a rearrangement in the chromosome in the region of the impaired murF gene. Although the full mechanism of reversion of the cell wall damage was not fully elucidated, we showed that Rho is involved in the recombination process that mediates the tandem amplification of exogeneous DNA fragments inserted into the chromosome. IMPORTANCE The cell wall of bacteria, namely, peptidoglycan, is the target of several antibiotic classes such as ß-lactams. Staphylococcus aureus is well known for its capacity to adapt to antibiotic stress and develop resistance strategies, namely, to ß-lactams. In this context, the construction of cell wall mutants provides useful models to study the development of such resistance mechanisms. Here, we characterized a collection of independent mutants, impaired in the same peptidoglycan biosynthetic step, obtained through the insertion of a plasmid in the coding region of murF gene. S. aureus demonstrated the capacity to overcome the cell wall damage by amplifying the copy number of the inserted plasmid, through an undescribed mechanism that involves the Rho transcription termination factor.

Genome Sequence of Elizabethkingia anophelis Strain EaAs1, Isolated from the Asian Malaria Mosquito Anopheles stephensi.

We sequenced the genome of a strain of the Gram-negative bacterial species Elizabethkingia anophelis, which is an important component of the Anopheles mosquito microbiome. This genome sequence will add to the list of resources used to examine host-microbe interactions in mosquitoes.

Genome Sequences of Staphylococcus hominis Strains ShAs1, ShAs2, and ShAs3, Isolated from the Asian Malaria Mosquito Anopheles stephensi.

Staphylococcus hominis is a culturable component of the bacterial microbiome of Anopheles stephensi. Here, we present the annotated draft genome sequences of three S. hominis isolates from A. stephensi. These genomic resources will facilitate experiments to further our understanding of the role of bacteria in mosquito biology.

Genome Sequence of Stenotrophomonas maltophilia Strain SmAs1, Isolated From the Asian Malaria Mosquito Anopheles stephensi.

An isolate of Stenotrophomonas maltophilia was cultured from the Asian malaria vector Anopheles stephensi. Here, we present the annotated draft genome sequence of this S. maltophilia strain. This genomic resource will facilitate further characterization of bacteria associated with mosquitoes.

Comparison of O-Antigen Gene Clusters of All O-Serogroups of Escherichia coli and Proposal for Adopting a New Nomenclature for O-Typing.

Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interactions. Because they are highly immunogenic and display antigenic specificity unique for each strain, O-antigens are the biomarkers for designating O-types. Immunologically, 185 O-serogroups and 11 OX-groups exist for classification. Conventional serotyping for O-typing entails agglutination reactions between the O-antigen and antisera generated against each O-group. The procedure is labor intensive, not always accurate, and exhibits equivocal results. In this report, we present the sequences of 71 O-antigen gene clusters (O-AGC) and a comparison of all 196 O- and OX-groups. Many of the designated O-types, applied for classification over several decades, exhibited similar nucleotide sequences of the O-AGCs and cross-reacted serologically. Some O-AGCs carried insertion sequences and others had only a few nucleotide differences between them. Thus, based on these findings, it is proposed that several of the E. coli O-groups may be merged. Knowledge of the O-AGC sequences facilitates the development of molecular diagnostic platforms that are rapid, accurate, and reliable that can replace conventional serotyping. Additionally, with the scientific knowledge presented, new frontiers in the discovery of biomarkers, understanding the roles of O-antigens in the innate and adaptive immune system and pathogenesis, the development of glycoconjugate vaccines, and other investigations, can be explored.

Correction: Comparison of O-Antigen Gene Clusters of All O-Serogroups of Escherichia coli and Proposal for Adopting a New Nomenclature for O-Typing.

[This corrects the article DOI: 10.1371/journal.pone.0147434.].

Complete Genome Sequence of SS52, a Strain of Escherichia coli O157:H7 Recovered from Supershedder Cattle.

Shiga toxin-producing Escherichia coli O157:H7 causes foodborne infections, and cattle are the primary reservoir. Some animals, known as supershedders, excrete orders of magnitude more E. coli O157:H7 in the feces than normal. Here, we report the complete genome sequence of the SS52 supershedder strain of E. coli O157:H7.

Comparative analysis of super-shedder strains of Escherichia coli O157:H7 reveals distinctive genomic features and a strongly aggregative adherent phenotype on bovine rectoanal junction squamous epithelial cells.

Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as "super-shedders" (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

Genome wide identification of regulatory motifs in Bacillus subtilis.

BACKGROUND: To explain the vastly different phenotypes exhibited by the same organism under different conditions, it is essential that we understand how the organism's genes are coordinately regulated. While there are many excellent tools for predicting sequences encoding proteins or RNA genes, few algorithms exist to predict regulatory sequences on a genome wide scale with no prior information. RESULTS: To identify motifs involved in the control of transcription, an algorithm was developed that searches upstream of operons for improbably frequent dimers. The algorithm was applied to the B. subtilis genome, which is predicted to encode for approximately 200 DNA binding proteins. The dimers found to be over-represented could be clustered into 317 distinct groups, each thought to represent a class of motifs uniquely recognized by some transcription factor. For each cluster of dimers, a representative weight matrix was derived and scored over the regions upstream of the operons to predict the sites recognized by the cluster's factor, and a putative regulon of the operons immediately downstream of the sites was inferred. The distribution in number of operons per predicted regulon is comparable to that for well characterized transcription factors. The most highly over-represented dimers matched sigmaA, the T-box, and sigmaW sites. We have evidence to suggest that at least 52 of our clusters of dimers represent actual regulatory motifs, based on the groups' weight matrix matches to experimentally characterized sites, the functional similarity of the component operons of the groups' regulons, and the positional biases of the weight matrix matches. All predictions are assigned a significance value, and thresholds are set to avoid false positives. Where possible, we examine our false negatives, drawing examples from known regulatory motifs and regulons inferred from RNA expression data. CONCLUSIONS: We have demonstrated that in the case of B. subtilis our algorithm allows for the genome wide identification of regulatory sites. As well as recovering known sites, we predict new sites of yet uncharacterized factors. Results can be viewed at http://www.physics.rockefeller.edu/~mwangi/.

Complete Genome Sequence of Mycobacterium avium subsp. paratuberculosis, Isolated from Human Breast Milk.

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease in ruminants and has also been associated with human Crohn's disease. We report the complete genome sequence of M. avium subsp. paratuberculosis, isolated from the breast milk of a Crohn's disease patient. This sequence has high identity with characterized strains recovered from cattle.

The mechanism of heterogeneous beta-lactam resistance in MRSA: key role of the stringent stress response.

All methicillin resistant S. aureus (MRSA) strains carry an acquired genetic determinant--mecA or mecC--which encode for a low affinity penicillin binding protein -PBP2A or PBP2A'--that can continue the catalysis of peptidoglycan transpeptidation in the presence of high concentrations of beta-lactam antibiotics which would inhibit the native PBPs normally involved with the synthesis of staphylococcal cell wall peptidoglycan. In contrast to this common genetic and biochemical mechanism carried by all MRSA strains, the level of beta-lactam antibiotic resistance shows a very wide strain to strain variation, the mechanism of which has remained poorly understood. The overwhelming majority of MRSA strains produce a unique--heterogeneous--phenotype in which the great majority of the bacteria exhibit very poor resistance often close to the MIC value of susceptible S. aureus strains. However, cultures of such heterogeneously resistant MRSA strains also contain subpopulations of bacteria with extremely high beta-lactam MIC values and the resistance level and frequency of the highly resistant cells in such strain is a characteristic of the particular MRSA clone. In the study described in this communication, we used a variety of experimental models to understand the mechanism of heterogeneous beta-lactam resistance. Methicillin-susceptible S. aureus (MSSA) that received the mecA determinant in the laboratory either on a plasmid or in the form of a chromosomal SCCmec cassette, generated heterogeneously resistant cultures and the highly resistant subpopulations that emerged in these models had increased levels of PBP2A and were composed of bacteria in which the stringent stress response was induced. Each of the major heterogeneously resistant clones of MRSA clinical isolates could be converted to express high level and homogeneous resistance if the growth medium contained an inducer of the stringent stress response.

Whole-genome sequencing reveals a link between ß-lactam resistance and synthetases of the alarmone (p)ppGpp in Staphylococcus aureus.

The overwhelming majority of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates exhibit a peculiar heterogeneous resistance to ß-lactam antibiotics: in cultures of such strains, the majority of cells display only a low level of methicillin resistance--often close to the MIC breakpoint of susceptible strains. Yet, in the same cultures, subpopulations of bacteria exhibiting very high levels of resistance are also present with variable frequencies, which are characteristic of the particular MRSA lineage. The mechanism of heterogeneous resistance is not understood. We describe here an experimental system for exploring the mechanism of heterogeneous resistance. Copies of the resistance gene mecA cloned into a temperature-sensitive plasmid were introduced into the fully sequenced methicillin-susceptible clinical isolate S. aureus strain 476. Transductants of strain 476 expressed methicillin resistance in a heterogeneous fashion: the great majority of cells showed only low MIC (0.75 µg/ml) for the antibiotic, but a minority population of highly resistant bacteria (MIC >300 µg/ml) was also present with a frequency of ∼10(-4). The genetic backgrounds of the majority and minority cells were compared by whole-genome sequencing: the only differences detectable were two point mutations in relA of the highly resistant minority population of bacteria. The relA gene codes for the synthesis of (p)ppGpp, an effector of the stringent stress response. Titration of (p)ppGpp showed increased amounts of this effector in the highly resistant cells. Involvement of (p)ppGpp synthesis genes may explain some of the perplexing aspects of ß-lactam resistance in MRSA, since many environmental and genetic changes can modulate cellular levels of (p)ppGpp.

Transcriptome-wide sequencing reveals numerous APOBEC1 mRNA-editing targets in transcript 3' UTRs.

Apolipoprotein B-editing enzyme, catalytic polypeptide-1 (APOBEC1) is a cytidine deaminase initially identified by its activity in converting a specific cytidine (C) to uridine (U) in apolipoprotein B (apoB) mRNA transcripts in the small intestine. Editing results in the translation of a truncated apoB isoform with distinct functions in lipid transport. To address the possibility that APOBEC1 edits additional mRNAs, we developed a transcriptome-wide comparative RNA sequencing (RNA-Seq) screen. We identified and validated 32 previously undescribed mRNA targets of APOBEC1 editing, all of which are located in AU-rich segments of transcript 3' untranslated regions (3' UTRs). Further analysis established several characteristic sequence features of editing targets, which were predictive for the identification of additional APOBEC1 substrates. The transcriptomics approach to RNA editing presented here dramatically expands the list of APOBEC1 mRNA editing targets and reveals a novel cellular mechanism for the modification of transcript 3' UTRs.

Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing.

The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.