David W. Dyer

Molecular characterization of hpuAB, the haemoglobin-haptoglobin-utilization operon of Neisseria meningitidis

We previously identified HpuB, an 85 kDa Fe‐repressible protein required for utilization of Fe from, and binding to, haemoglobin and the haemoglobin–haptoglobin complex. The gene for hpuB was cloned from Neisseria meningitidis strain DNM2 and the predicted amino acid sequence indicates that HpuB is an outer membrane receptor belonging to the TonB family of high‐affinity transport proteins. A second open reading frame, predicted to encode a 34.8 kDa lipoprotein, was discovered 5′ to hpuB, and was designated hpuA. HpuA was identified in a total‐membrane‐protein preparation by construction of a mutant lacking HpuA. Acylation of HpuA was confirmed by [3H]‐palmitic acid labelling of meningococci. Consensus promoter sequences were not apparent 5′ to hpuB. The hpuA insertion mutation exerted a polar effect, abolishing expression of hpuB, suggesting that hpuA and hpuB are co‐transcribed. The 3.5 kb polycistronic hpuAB mRNA was identified and shown to be transcriptionally repressed by iron. The transcriptional start site was identified 33 nucleotides 5′ to the hpuA translational start site, appropriately positioned around consensus promoter and ferric uptake regulator (Fur)‐box sequences. The structure of this operon suggests that HpuA–HpuB is a two‐component receptor analogous to the bipartite transferrin receptor TbpB–TbpA.

Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group.

BackgroundThe Bacillus cereussensu lato group consists of six species (B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. weihenstephanensis). While classical microbial taxonomy proposed these organisms as distinct species, newer molecular phylogenies and comparative genome sequencing suggests that these organisms should be classified as a single species (thus, we will refer to these organisms collectively as the Bc species-group). How do we account for the underlying similarity of these phenotypically diverse microbes? It has been established for some time that the most rapidly evolving and evolutionarily flexible portions of the bacterial genome are regulatory sequences and transcriptional networks. Other studies have suggested that the sigma factor gene family of these organisms has diverged and expanded significantly relative to their ancestors; sigma factors are those portions of the bacterial transcriptional apparatus that control RNA polymerase recognition for promoter selection. Thus, examining sigma factor divergence in these organisms would concurrently examine both regulatory sequences and transcriptional networks important for divergence. We began this examination by comparison to the sigma factor gene set of B. subtilis.ResultsPhylogenetic analysis of the Bc species-group utilizing 157 single-copy genes of the family Bacillaceae suggests that several taxonomic revisions of the genus Bacillus should be considered. Within the Bc species-group there is little indication that the currently recognized species form related sub-groupings, suggesting that they are members of the same species. The sigma factor gene family encoded by the Bc species-group appears to be the result of a dynamic gene-duplication and gene-loss process that in previous analyses underestimated the true heterogeneity of the sigma factor content in the Bc species-group.ConclusionsExpansion of the sigma factor gene family appears to have preferentially occurred within the extracytoplasmic function (ECF) sigma factor genes, while the primary alternative (PA) sigma factor genes are, in general, highly conserved with those found in B. subtilis. Divergence of the sigma-controlled transcriptional regulons among various members of the Bc species-group likely has a major role in explaining the diversity of phenotypic characteristics seen in members of the Bc species-group.

IDENTIFICATION OF ALCA, A BORDETELLA BRONCHISEPTICA GENE NECESSARY FOR ALCALIGIN PRODUCTION

The alcA gene, essential for the production of the dihydroxamate siderophore, alcaligin, by Bordetella bronchiseptica, was cloned and sequenced. The alcA gene was identified on a 4.7-kb EcoRI genomic fragment adjacent to a Tn5lac transposon insertion that inactivated alcaligin production in strain MBORD846. Analysis of the alcA nucleotide sequence revealed a putative Fur-binding site, suggesting that expression of this gene is repressed by iron. The deduced amino-acid sequence of this open reading frame had significant homology with the Escherichia coli iucD gene product, an enzyme required for biosynthesis of the dihydroxamate siderophore aerobactin.

The inflammatory and normal transcriptome of mouse bladder detrusor and mucosa

BackgroundAn organ such as the bladder consists of complex, interacting set of tissues and cells. Inflammation has been implicated in every major disease of the bladder, including cancer, interstitial cystitis, and infection. However, scanty is the information about individual detrusor and urothelium transcriptomes in response to inflammation. Here, we used suppression subtractive hybridizations (SSH) to determine bladder tissue- and disease-specific genes and transcriptional regulatory elements (TRE)s. Unique TREs and genes were assembled into putative networks.ResultsIt was found that the control bladder mucosa presented regulatory elements driving genes such as myosin light chain phosphatase and calponin 1 that influence the smooth muscle phenotype. In the control detrusor network the Pax-3 TRE was significantly over-represented. During development, the Pax-3 transcription factor (TF) maintains progenitor cells in an undifferentiated state whereas, during inflammation, Pax-3 was suppressed and genes involved in neuronal development (synapsin I) were up-regulated. Therefore, during inflammation, an increased maturation of neural progenitor cells in the muscle may underlie detrusor instability. NF-κB was specifically over-represented in the inflamed mucosa regulatory network. When the inflamed detrusor was compared to control, two major pathways were found, one encoding synapsin I, a neuron-specific phosphoprotein, and the other an important apoptotic protein, siva. In response to LPS-induced inflammation, the liver X receptor was over-represented in both mucosa and detrusor regulatory networks confirming a role for this nuclear receptor in LPS-induced gene expression.ConclusionA new approach for understanding bladder muscle-urothelium interaction was developed by assembling SSH, real time PCR, and TRE analysis results into regulatory networks. Interestingly, some of the TREs and their downstream transcripts originally involved in organogenesis and oncogenesis were also activated during inflammation. The latter represents an additional link between inflammation and cancer. The regulatory networks represent key targets for development of novel drugs targeting bladder diseases.

The Mechanism of Bacillus anthracis Intracellular Germination Requires Multiple and Highly Diverse Genetic Loci

ABSTRACT In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells.

Divergence of the SigB regulon and pathogenesis of the Bacillus cereus sensu lato group

BackgroundThe Bacillus cereus sensu lato group currently includes seven species (B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus) that recent phylogenetic and phylogenomic analyses suggest are likely a single species, despite their varied phenotypes. Although horizontal gene transfer and insertion-deletion events are clearly important for promoting divergence among these genomes, recent studies have demonstrated that a major basis for phenotypic diversity in these organisms may be differential regulation of the highly similar gene content shared by these organisms. To explore this hypothesis, we used an in silico approach to evaluate the relationship of pathogenic potential and the divergence of the SigB-dependent general stress response within the B. cereus sensu lato group, since SigB has been demonstrated to support pathogenesis in Bacillus, Listeria and Staphylococcus species.ResultsDuring the divergence of these organisms from a common “SigB-less” ancestor, the placement of SigB promoters at varied locations in the B. cereus sensu lato genomes predict alternative structures for the SigB regulon in different organisms. Predicted promoter changes suggesting differential transcriptional control of a common gene pool predominate over evidence of indels or horizontal gene transfer for explaining SigB regulon divergence.ConclusionsFour lineages of the SigB regulon have arisen that encompass different gene contents and suggest different strategies for supporting pathogenesis. This is consistent with the hypothesis that divergence within the B. cereus sensu lato group rests in part on alternative strategies for regulation of a common gene pool.

Sequence Variation in the Herpes Simplex Virus U S 1 Ocular Virulence Determinant

PURPOSE The herpes simplex virus type 1 (HSV-1) U(S)1 gene encodes host-range and ocular virulence determinants. Mutations in U(S)1 affecting virulence are known in strain OD4, but the genomic variation across several strains is not known. The goal was to determine the degree of sequence variation in the gene from several ocular HSV isolates. METHODS The U(S)1 gene from six ocular HSV-1 isolates, as well as strains KOS and F, were sequenced, and bioinformatics analyses were applied to the data. RESULTS Strains 17, F, CJ394, and CJ311 had identical amino acid sequences. With the other strains, most of the variability was concentrated in the amino-terminal third of the protein. MEME analysis identified a 63-residue core sequence (motif 1) present in all α-herpesvirus U(S)1 homologs that were located in a region identified as structured. Ten amino acids were absolutely conserved in all the α-herpesvirus U(S)1 homologs and were all located in the central core. Consensus-binding motifs for cyclin-dependent kinases and pocket proteins were also identified. CONCLUSIONS These results suggest that significant sequence variation exists in the U(S)1 gene, that the α22 protein contains a conserved central core region with structurally variable regions at the amino- and carboxyl termini, that 10 amino acids are conserved in α-herpes U(S)1 homologs, and that additional host proteins may interact with the HSV-1 U(S)1 and U(S)1.5 proteins. This information will be valuable in designing further studies on structure-function relationships and on the role these play in host-range determination and keratitis.

Iron-regulated small RNA expression as Neisseria gonorrhoeae FA 1090 transitions into stationary phase growth

BackgroundFor most pathogens, iron (Fe) homeostasis is crucial for maintenance within the host and the ability to cause disease. The primary transcriptional regulator that controls intracellular Fe levels is the Fur (ferric uptake regulator) protein, which exerts its action on transcription by binding to a promoter-proximal sequence termed the Fur box. Fur-regulated transcriptional responses are often fine-tuned at the post-transcriptional level through the action of small regulatory RNAs (sRNAs). Consequently, identifying sRNAs contributing to the control of Fe homeostasis is important for understanding the Fur-controlled bacterial Fe-response network.ResultsIn this study, we sequenced size-selected directional libraries representing sRNA samples from Neisseria gonorrhoeae strain FA 1090, and examined the Fe- and temporal regulation of these sRNAs. RNA-seq data for all time points identified a pool of at least 340 potential sRNAs. Differential analysis demonstrated that expression appeared to be regulated by Fe availability for at least fifteen of these sRNAs. Fourteen sRNAs were induced in high Fe conditions, consisting of both cis and trans sRNAs, some of which are predicted to control expression of a known virulence factor, and one SAM riboswitch. An additional putative cis-acting sRNA was repressed by Fe availability. In the pathogenic Neisseria species, one sRNA that contributes to Fe-regulated post-transcriptional control is the Fur-repressible sRNA NrrF. The expression of five Fe-induced sRNAs appeared to be at least partially controlled by NrrF, while the remainder was expressed independently of NrrF. The expression of the 14 Fe-induced sRNAs also exhibited temporal control, as their expression levels increased dramatically as the bacteria entered stationary phase.ConclusionsHere we report the temporal expression of Fe-regulated sRNAs in N. gonorrhoeae FA 1090 with several appearing to be controlled by the Fe-repressible sRNA NrrF. Temporal regulation of these sRNAs suggests a regulatory role in controlling functions necessary for survival, and may be important for phenotypes often associated with altered growth rates, such as biofilm formation or intracellular survival. Future functional studies will be needed to understand how these regulatory sRNAs contribute to gonococcal biology and pathogenesis.

Genome Sequence of Bacillus Thuringiensis Subsp. Kurstaki Strain HD-1

ABSTRACT We report here the complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD-1, which serves as the primary U.S. reference standard for all commercial insecticidal formulations of B. thuringiensis manufactured around the world.

Genome Sequence of SCB34, a Sequence Type 131 Multidrug-Resistant Escherichia coli Isolate Causing Neonatal Early-Onset Sepsis

ABSTRACT SCB34 is a sequence type 131, highly invasive, multidrug-resistant Escherichia coli isolate that produced neonatal bacteremia. Whole-genome sequencing was performed using a 250-bp library on the Illumina MiSeq platform; 5,910,264 reads were assembled de novo using the A5 assembly pipeline. The total contig length was 5,227,742 bp; the RAST server was used for annotation.