Comparative Transcriptional Profiling of Bacillus cereus Sensu Lato Strains during Growth in CO2-Bicarbonate and Aerobic Atmospheres

Bacillus species are spore-forming bacteria that are ubiquitous in the environment and display a range of virulent and avirulent phenotypes. This range is particularly evident in the Bacillus cereus sensu lato group; where closely related strains cause anthrax, food-borne illnesses, and pneumonia, but can also be non-pathogenic. Although much of this phenotypic range can be attributed to the presence or absence of a few key virulence factors, there are other virulence-associated loci that are conserved throughout the B. cereus group, and we hypothesized that these genes may be regulated differently in pathogenic and non-pathogenic strains.Here we report transcriptional profiles of three closely related but phenotypically unique members of the Bacillus cereus group--a pneumonia-causing B. cereus strain (G9241), an attenuated strain of B. anthracis (Sterne 34F(2)), and an avirulent B. cereus strain (10987)--during exponential growth in two distinct atmospheric environments: 14% CO(2)/bicarbonate and ambient air. We show that the disease-causing Bacillus strains undergo more distinctive transcriptional changes between the two environments, and that the expression of plasmid-encoded virulence genes was increased exclusively in the CO(2) environment. We observed a core of conserved metabolic genes that were differentially expressed in all three strains in both conditions. Additionally, the expression profiles of putative virulence genes in G9241 suggest that this strain, unlike Bacillus anthracis, may regulate gene expression with both PlcR and AtxA transcriptional regulators, each acting in a different environment.We have shown that homologous and even identical genes within the genomes of three closely related members of the B. cereus sensu lato group are in some instances regulated very differently, and that these differences can have important implications for virulence. This study provides insights into the evolution of the B. cereus group, and highlights the importance of looking beyond differences in gene content in comparative genomics studies

Estimating the burden of disease attributable to four selected environmental risk factors in South Africa

The first South African National Burden of Disease study quantified the underlying causes of premature mortality and morbidity experienced in South Africa in the year 2000. This was followed by a Comparative Risk Assessment to estimate the contributions of 17 selected risk factors to burden of disease in South Africa. This paper describes the health impact of exposure to four selected environmental risk factors: unsafe water, sanitation and hygiene; indoor air pollution from household use of solid fuels; urban outdoor air pollution and lead exposure.The study followed World Health Organization comparative risk assessment methodology. Population-attributable fractions were calculated and applied to revised burden of disease estimates (deaths and disability adjusted life years, [DALYs]) from the South African Burden of Disease study to obtain the attributable burden for each selected risk factor. The burden attributable to the joint effect of the four environmental risk factors was also estimated taking into account competing risks and common pathways. Monte Carlo simulation-modeling techniques were used to quantify sampling, uncertainty.Almost 24 000 deaths were attributable to the joint effect of these four environmental risk factors, accounting for 4.6% (95% uncertainty interval 3.8-5.3%) of all deaths in South Africa in 2000. Overall the burden due to these environmental risks was equivalent to 3.7% (95% uncertainty interval 3.4-4.0%) of the total disease burden for South Africa, with unsafe water sanitation and hygiene the main contributor to joint burden. The joint attributable burden was especially high in children under 5 years of age, accounting for 10.8% of total deaths in this age group and 9.7% of burden of disease.This study highlights the public health impact of exposure to environmental risks and the significant burden of preventable disease attributable to exposure to these four major environmental risk factors in South Africa. Evidence-based policies and programs must be developed and implemented to address these risk factors at individual, household, and community levels

Theoretical Analysis of the Stress Induced B-Z Transition in Superhelical DNA

We present a method to calculate the propensities of regions within a DNA molecule to transition from B-form to Z-form under negative superhelical stresses. We use statistical mechanics to analyze the competition that occurs among all susceptible Z-forming regions at thermodynamic equilibrium in a superhelically stressed DNA of specified sequence. This method, which we call SIBZ, is similar to the SIDD algorithm that was previously developed to analyze superhelical duplex destabilization. A state of the system is determined by assigning to each base pair either the B- or the Z-conformation, accounting for the dinucleotide repeat unit of Z-DNA. The free energy of a state is comprised of the nucleation energy, the sequence-dependent B-Z transition energy, and the energy associated with the residual superhelicity remaining after the change of twist due to transition. Using this information, SIBZ calculates the equilibrium B-Z transition probability of each base pair in the sequence. This can be done at any physiologically reasonable level of negative superhelicity. We use SIBZ to analyze a variety of representative genomic DNA sequences. We show that the dominant Z-DNA forming regions in a sequence can compete in highly complex ways as the superhelicity level changes. Despite having no tunable parameters, the predictions of SIBZ agree precisely with experimental results, both for the onset of transition in plasmids containing introduced Z-forming sequences and for the locations of Z-forming regions in genomic sequences. We calculate the transition profiles of 5 kb regions taken from each of 12,841 mouse genes and centered on the transcription start site (TSS). We find a substantial increase in the frequency of Z-forming regions immediately upstream from the TSS. The approach developed here has the potential to illuminate the occurrence of Z-form regions in vivo, and the possible roles this transition may play in biological processes

Deciphering the Code for Retroviral Integration Target Site Selection

Upon cell invasion, retroviruses generate a DNA copy of their RNA genome and integrate retroviral cDNA within host chromosomal DNA. Integration occurs throughout the host cell genome, but target site selection is not random. Each subgroup of retrovirus is distinguished from the others by attraction to particular features on chromosomes. Despite extensive efforts to identify host factors that interact with retrovirion components or chromosome features predictive of integration, little is known about how integration sites are selected. We attempted to identify markers predictive of retroviral integration by exploiting Precision-Recall methods for extracting information from highly skewed datasets to derive robust and discriminating measures of association. ChIPSeq datasets for more than 60 factors were compared with 14 retroviral integration datasets. When compared with MLV, PERV or XMRV integration sites, strong association was observed with STAT1, acetylation of H3 and H4 at several positions, and methylation of H2AZ, H3K4, and K9. By combining peaks from ChIPSeq datasets, a supermarker was identified that localized within 2 kB of 75% of MLV proviruses and detected differences in integration preferences among different cell types. The supermarker predicted the likelihood of integration within specific chromosomal regions in a cell-type specific manner, yielding probabilities for integration into proto-oncogene LMO2 identical to experimentally determined values. The supermarker thus identifies chromosomal features highly favored for retroviral integration, provides clues to the mechanism by which retrovirus integration sites are selected, and offers a tool for predicting cell-type specific proto-oncogene activation by retroviruses

Drilling Predation on Serpulid Polychaetes (Ditrupa arietina) from the Pliocene of the Cope Basin, Murcia Region, Southeastern Spain

We report quantitative analyses of drilling predation on the free-living, tube-dwelling serpulid polychaete Ditrupa arietina from the Cope Cabo marine succession (Pliocene, Spain). Tubes of D. arietina are abundant in the sampled units: 9 bulk samples from 5 horizons yielded ∼5925 specimens of D. arietina. Except for fragmentation, tubes were well preserved. Complete specimens ranged from 3.1 to 13.4 mm in length and displayed allometric growth patterns, with larger specimens being relatively slimmer. Drilled Ditrupa tubes were observed in all samples. Drillholes, identified as Oichnus paraboloides, were characterized by circular to elliptical outline (drillhole eccentricity increased with its diameter), parabolic vertical profile, outer diameter larger than inner diameter, penetration of one tube wall only, narrow range of drill-hole sizes, and non-random (anterior) distribution of drillholes. A total of 233 drilled specimens were identified, with drilling frequencies varying across horizons from 2.7% to 21% (3.9% for pooled data). Many tube fragments were broken across a drillhole suggesting that the reported frequencies are conservative and that biologically-facilitated (drill-hole induced) fragmentation hampers fossil preservation of complete serpulid tubes. No failed or repaired holes were observed. Multiple complete drillholes were present (3.9%). Drilled specimens were significantly smaller than undrilled specimens and tube length and drill-hole diameter were weakly correlated. The results suggest that drillholes were produced by a size-selective, site-stereotypic predatory organism of unknown affinity. The qualitative and quantitative patterns reported here are mostly consistent with previous reports on recent and fossil Ditrupa and reveal parallels with drilling patterns documented for scaphopod mollusks, a group that is ecologically and morphologically similar to Ditrupa. Consistent with previous studies, the results suggest that free-dwelling serpulid polychaetes are preyed upon by drilling predators and may provide a viable source of data on biotic interactions in the fossil record

Comparative (Meta)genomic Analysis and Ecological Profiling of Human Gut-Specific Bacteriophage φB124-14

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome

HIV Integration Targeting: A Pathway Involving Transportin-3 and the Nuclear Pore Protein RanBP2

Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration