Bronwyn G. Butcher

The Chromosomal Arsenic Resistance Genes of Thiobacillus ferrooxidans Have an Unusual Arrangement and Confer Increased Arsenic and Antimony Resistance to Escherichia coli

ABSTRACT The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC,arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) andarsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidansis gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functionaltrxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR andarsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.

Identification of Bacillus subtilis σW-dependent genes that provide intrinsic resistance to antimicrobial compounds produced by Bacilli

Bacillus subtilis produces many antibiotics of varying structures and specificity. Here we identify a prominent role for σW, an extracytoplasmic function (ECF) sigma factor, in providing intrinsic resistance to antimicrobial compounds produced by other Bacilli. By using a panel of B. subtilis mutants disrupted for each of the 30 known σW‐dependent operons we identified resistance genes for at least three different antimicrobial compounds. The ydbST and fosB genes contribute to resistance to antimicrobial compound(s) produced by B. amyloliquefaciens FZB42, the yqeZyqfAB operon provides resistance to the SPβ prophage‐encoded bacteriocin sublancin, and the yknWXYZ operon and yfhL provide resistance to the antimicrobial peptide SdpC. YfhL encodes a paralogue of SdpI, a membrane protein that provides immunity to SdpC. In competition experiments, we identify σW as a key factor in allowing B. subtilis to resist antibiotic killing and encroachment by competing strains. Together with the previous observation that σW provides inducible resistance against the Streptomyces antibiotic fosfomycin, these studies support the notion that σW controls an antibiosis regulon important in the microbial ecology of soil bacteria.

Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity

To fully understand how bacteria respond to their environment, it is essential to assess genome-wide transcriptional activity. New high-throughput sequencing technologies make it possible to query the transcriptome of an organism in an efficient unbiased manner. We applied a strand-specific method to sequence bacterial transcripts using Illuminas high-throughput sequencing technology. The resulting sequences were used to construct genome-wide transcriptional profiles. Novel bioinformatics analyses were developed and used in combination with proteomics data for the qualitative classification of transcriptional activity in defined regions. As expected, most transcriptional activity was consistent with predictions from the genome annotation. Importantly, we identified and confirmed transcriptional activity in areas of the genome inconsistent with the annotation and in unannotated regions. Further analyses revealed potential RpoN-dependent promoter sequences upstream of several noncoding RNAs (ncRNAs), suggesting a role for these ncRNAs in RpoN-dependent phenotypes. We were also able to validate a number of transcriptional start sites, many of which were consistent with predicted promoter motifs. Overall, our approach provides an efficient way to survey global transcriptional activity in bacteria and enables rapid discovery of specific areas in the genome that merit further investigation.

Plumage Genes and Little Else Distinguish the Genomes of Hybridizing Warblers

When related taxa hybridize extensively, their genomes may become increasingly homogenized over time. This mixing via hybridization creates conservation challenges when it reduces genetic or phenotypic diversity and when it endangers previously distinct species via genetic swamping [1]. However, hybridization also facilitates admixture mapping of traits that distinguish each species and the associated genes that maintain distinctiveness despite ongoing gene flow [2]. We address these dual aspects of hybridization in the golden-winged/blue-winged warbler complex, two phenotypically divergent warblers that are indistinguishable using traditional molecular markers and that draw substantial conservation attention [3-5]. Whole-genome comparisons show that differentiation is extremely low: only six small genomic regions exhibit strong differences. Four of these divergence peaks occur in proximity to genes known to be involved in feather development or pigmentation: agouti signaling protein (ASIP), follistatin (FST), ecodysplasin (EDA), wingless-related integration site (Wnt), and beta-carotene oxygenase 2 (BCO2). Throat coloration-the most striking plumage difference between these warblers-is perfectly associated with the promoter region of agouti, and genotypes at this locus obey simple Mendelian recessive inheritance of the black-throated phenotype characteristic of golden-winged warblers. The more general pattern of genomic similarity between these warblers likely results from a protracted period of hybridization, contradicting the broadly accepted hypothesis that admixture results from solely anthropogenic habitat change in the past two centuries [4]. Considered in concert, these results are relevant to both the genetic architecture of avian feather pigmentation and the evolutionary history and conservation challenges associated with these declining songbirds.

Characterization of the Fur Regulon in Pseudomonas syringae pv. tomato DC3000

The plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) is found in a wide variety of environments and must monitor and respond to various environmental signals such as the availability of iron, an essential element for bacterial growth. An important regulator of iron homeostasis is Fur (ferric uptake regulator), and here we present the first study of the Fur regulon in DC3000. Using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq), 312 chromosomal regions were highly enriched by coimmunoprecipitation with a C-terminally tagged Fur protein. Integration of these data with previous microarray and global transcriptome analyses allowed us to expand the putative DC3000 Fur regulon to include genes both repressed and activated in the presence of bioavailable iron. Using nonradioactive DNase I footprinting, we confirmed Fur binding in 41 regions, including upstream of 11 iron-repressed genes and the iron-activated genes encoding two bacterioferritins (PSPTO_0653 and PSPTO_4160), a ParA protein (PSPTO_0855), and a two-component system (TCS) (PSPTO_3382 to PSPTO_3380).

Genome-Wide Identification of Transcriptional Start Sites in the Plant Pathogen Pseudomonas syringae pv. tomato str. DC3000

RNA-Seq has provided valuable insights into global gene expression in a wide variety of organisms. Using a modified RNA-Seq approach and Illuminas high-throughput sequencing technology, we globally identified 5′-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5′-ends obtained by this method were consistent with results obtained using global RNA-Seq and 5′RACE. As expected, many 5′-ends were positioned a short distance upstream of annotated genes. We also captured 5′-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, suggesting additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5′-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels.

The yydFGHIJ Operon of Bacillus subtilis Encodes a Peptide That Induces the LiaRS Two-Component System

The Bacillus subtilis LiaRS two-component system (TCS) responds to perturbations of the cell envelope induced by lipid II-interacting antibiotics, such as vancomycin, ramoplanin, nisin, and bacitracin. Here, we characterize Tn7-generated mutations that induce the liaRS TCS. In addition to insertions in liaF, a known negative regulator of the LiaRS TCS, we identified two disruptions in the last two genes of the yydFGHIJ operon. This operon is predicted to encode a 49-amino-acid peptide (YydF), a modification enzyme (YydG), a membrane-embedded protease (YydH), and an ATP-binding cassette (ABC) transporter (YydIJ). Genome sequence comparisons suggest that the yydFGHIJ operon may have been acquired by horizontal transfer. Inactivation of the YydIJ transporter resulted in increased expression from the LiaR-dependent P(liaI) promoter only in the presence of the yydFGH genes. Cells harboring the complete yydFGHIJ operon induced LiaR activity in cocultured cells lacking either this transporter or the complete operon. These results suggest that this operon is involved in the synthesis and export of a modified peptide (YydF*) that elicits cell envelope stress sensed by the LiaRS TCS.

An Extracytoplasmic Function Sigma Factor-Mediated Cell Surface Signaling System in Pseudomonas syringae pv. tomato DC3000 Regulates Gene Expression in Response to Heterologous Siderophores

The diversity of regulatory systems encoded by bacteria provides an indication of the variety of stresses and interactions that these organisms encounter in nature. We have been investigating how the plant pathogen Pseudomonas syringae pv. tomato DC3000 responds to iron limitation and have focused on the iron starvation (IS) sigma factors to identify regulon members and to explore the mechanistic details of genetic control for this class of regulators. In the study described in this report, we used chromatin immunoprecipitation paired with high-throughput sequencing (ChIP-Seq) to screen the genome for locations associated with binding of the P. syringae IS sigma factor PSPTO_1203. We used multiple methods to demonstrate differential regulation of two genes identified in the ChIP-Seq screen and characterize the promoter elements that facilitate PSPTO_1203-dependent regulation. The genes regulated by PSPTO_1203 encode a TonB-dependent transducer (PSPTO_1206) and a cytoplasmic membrane protein (PSPTO_2145), which is located in the P. syringae pyoverdine cluster. Additionally, we identified siderophores that induce the activity of PSPTO_1203 and used this information to investigate the functional components of the signal transduction cascade.

CrcZ and CrcX regulate carbon source utilization in Pseudomonas syringae pathovar tomato strain DC3000

Small non-coding RNAs (ncRNAs) are important components of many regulatory pathways in bacteria and play key roles in regulating factors important for virulence. Carbon catabolite repression control is modulated by small RNAs (crcZ or crcZ and crcY) in Pseudomonas aeruginosa and Pseudomonas putida. In this study, we demonstrate that expression of crcZ and crcX (formerly designated psr1 and psr2, respectively) is dependent upon RpoN together with the two-component system CbrAB, and is influenced by the carbon source present in the medium in the model plant pathogen Pseudomonas syringae pv tomato DC3000. The distribution of the members of the Crc ncRNA family was also determined by screening available genomic sequences of the Pseudomonads. Interestingly, variable numbers of the Crc family members exist in Pseudomonas genomes. The ncRNAs are comprised of three main subfamilies, named CrcZ, CrcX and CrcY. Most importantly the CrcX subfamily appears to be unique to all P. syringae strains sequenced to date.

Analysis of the small RNA P16/RgsA in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000.

Bacteria contain small non-coding RNAs (ncRNAs) that are responsible for altering transcription, translation or mRNA stability. ncRNAs are important because they regulate virulence factors and susceptibility to various stresses. Here, the regulation of a recently described ncRNA of Pseudomonas syringae pv. tomato DC3000, P16, was investigated. We determined that RpoS regulates the expression of P16. We found that deletion of P16 results in increased sensitivity to hydrogen peroxide compared to the wild-type strain, suggesting that P16 plays a role in the bacteria’s susceptibility to oxidative stress. Additionally the P16 mutant displayed enhanced resistance to heat stress. Our findings provide new information on the regulation and role of this ncRNA in P. syringae.