No abstract

LiaR is a ‘master regulator’ of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structural basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaRD191N increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. Crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons.

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30-50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs.

The cbb I region of Rhodopseudomonas palustris (Rp. palustris) contains the cbbLS genes encoding form I ribulose-1,5-bisphosphate (RuBP) carboxylase oxygenase (RubisCO) along with a divergently transcribed regulator gene, cbbR. Juxtaposed between cbbR and cbbLS are the cbbRRS genes, encoding an unusual three-protein two-component (CbbRRS) system that modulates the ability of CbbR to influence cbbLS expression. The nature of the metabolic signals that Rp. palustris CbbR perceives to regulate cbbLS transcription is not known. Thus, in this study, the CbbR binding region was first mapped within the cbbLS promoter by the use of gel mobility shift assays and DNase I footprinting. In addition, potential metabolic coinducers (metabolites) were tested for their ability to alter the cbbLS promoter binding properties of CbbR. Gel mobility shift assays and surface plasmon resonance analyses together indicated that biosynthetic intermediates such as RuBP, ATP, fructose 1,6-bisphosphate, and NADPH enhanced DNA binding by CbbR. These coinducers did not yield identical CbbR-dependent DNase I footprints, indicating that the coinducers caused significant changes in DNA structure. These in vitro studies suggest that cellular signals such as fluctuating metabolite concentrations are perceived by and transduced to the cbbLS promoter via the master regulator CbbR.

Summary Differential gene expression is a key strategy adopted by the Lyme disease spirochaete, Borrelia burgdorferi, for adaptation and survival in the mammalian host and the tick vector. Many B. burgdorferi surface lipoproteins fall into two distinct groups according to their expression patterns: one group primarily expressed in the tick and the other group primarily expressed in the mammal. Here, we show that the Fur homologue in this bacterium, also known as Borrelia oxidative stress regulator (BosR), is required for repression of outer surface protein A (OspA) and OspD in the mammal. Furthermore, BosR binds directly to sequences upstream of the ospAB operon and the ospD gene through recognition of palindromic motifs similar to those recognized by other Fur homologues but with a 1-bp variation in the spacer length. Putative BosR-binding sites have been identified upstream of 156 B. burgdorferi genes. Some of these genes share the same expression pattern as ospA and ospD. Most notably, 12 (67%) of the 18 genes previously identified in a genome-wide microarray study to be most significantly repressed in the mammal are among the putative BosR regulon. These data indicate that BosR may directly repress transcription of many genes that are down-regulated in the mammal.

The cyclic antimicrobial lipopeptide daptomycin triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, nonsusceptibility to daptomycin by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaRW73C). We have determined the structure of the activated Enterococcus faecium LiaR protein at 3.2 Å resolution and, in combination with solution studies, show that activation of LiaR induces formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to daptomycin by upregulation of the LiaFSR regulon. A comparison of the Enterococcus faecalis LiaR, Enterococcus faecium LiaR and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with daptomycin resistance suggest that physicochemical properties such as oligomerization state and DNA specificity, though tuned to the biology of each organism, share some features that could be targeted for new antimicrobials.

Pantoea stewartii subsp. stewartii is a bacterial pathogen of corn. Its pathogenicity depends on the translocation of effector proteins into host cells by the Hrp type III secretion system. We previously showed by genetic analysis that the HrpX sensor kinase and the HrpY response regulator are at the head of a complex cascade of regulators controlling hrp/hrc secretion and wts effector genes. This cascade also includes the HrpS response regulator and the HrpL alternative sigma factor. These regulators are shared among many important plant pathogens in the genera Pantoea, Erwinia, and Pseudomonas. In this study, we dissect the regulatory elements in the hrpS promoter region, using genetic and biochemical approaches, and show how it integrates various environmental signals, only some of which are dependent on phosphorylation of HrpY. Primer extension located the transcriptional start site of hrpS at a 70 promoter 601 bp upstream of the open reading frame. Electrophoretic mobility shift assays and DNase I footprinting analysis demonstrated that HrpY binds to conserved regulatory elements immediately adjacent to this promoter, and its binding affinity was increased by phosphorylation at D57. A consensus sequence for the two direct repeats bound by HrpY is proposed. Deletion analysis of the promoter region revealed that both the HrpY binding site and additional sequences farther upstream, including a putative integration host factor binding site, are required for hrpS expression. This finding suggests that other unknown regulatory proteins may act cooperatively with HrpY.The hrp/hrc genes of gram-negative, phytopathogenic bacteria encode type III secretion systems that deliver effector proteins into host cells. These proteins may elicit cell death or suppress host defenses. They are required for infection and colonization of plants by all biotrophic pathogens of the genera Pseudomonas, Ralstonia, Xanthomonas, Erwinia, and Pantoea (3). They also play a role in rapid establishment of necrotrophic, soft-rot bacteria in the genus Erwinia, where enzymatic tissue disintegration is delayed by hrp mutations (33). Pantoea stewartii subsp. stewartii (synonym, Erwinia stewartii) causes vascular wilting and leaf blight of sweet corn and maize. This pathogen carries a group I hrp gene cluster (1,7,8,11), similar to those in Pseudomonas, Erwinia, and other Pantoea species. The group I hrp gene clusters are regulated by the HrpS enhancer-binding protein and the HrpL extracytoplasmic function alternative sigma factor (12, 25, 36-39). P. stewartii hrp genes are also controlled by the HrpX/HrpY two-component regulatory system, which is also present in Pantoea agglomerans pv. gypsophilae, Erwinia amylovora, Erwinia carotovora subsp. carotovora, and Erwinia chrysanthemi (19,25,27,35,39). In erwinias and pantoeas, the hrpL-hrpXY-hrpS genes have the same map order, and the corresponding proteins share greater than 80% similarity at the amino acid level (25). However, in P. stewartii, a large remnant of an insertion sequence (IS) element i...

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.