Lee Ann McCue

Transcriptomic and Proteomic Characterization of the Fur Modulon in the Metal-Reducing Bacterium Shewanella oneidensis

The availability of the complete genome sequence for Shewanella oneidensis MR-1 has permitted a comprehensive characterization of the ferric uptake regulator (Fur) modulon in this dissimilatory metal-reducing bacterium. We have employed targeted gene mutagenesis, DNA microarrays, proteomic analysis using liquid chromatography-mass spectrometry, and computational motif discovery tools to define the S. oneidensis Fur regulon. Using this integrated approach, we identified nine probable operons (containing 24 genes) and 15 individual open reading frames (ORFs), either with unknown functions or encoding products annotated as transport or binding proteins, that are predicted to be direct targets of Fur-mediated repression. This study suggested, for the first time, possible roles for four operons and eight ORFs with unknown functions in iron metabolism or iron transport-related functions. Proteomic analysis clearly identified a number of transporters, binding proteins, and receptors related to iron uptake that were up-regulated in response to a fur deletion and verified the expression of nine genes originally annotated as pseudogenes. Comparison of the transcriptome and proteome data revealed strong correlation for genes shown to be undergoing large changes at the transcript level. A number of genes encoding components of the electron transport system were also differentially expressed in a fur deletion mutant. The gene omcA (SO1779), which encodes a decaheme cytochrome c, exhibited significant decreases in both mRNA and protein abundance in the fur mutant and possessed a strong candidate Fur-binding site in its upstream region, thus suggesting that omcA may be a direct target of Fur activation.

Characterization of Mycobacterium tuberculosis Rv3676 (CRPMt), a Cyclic AMP Receptor Protein-Like DNA Binding Protein

Little is known about cyclic AMP (cAMP) function in Mycobacterium tuberculosis, despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins. M. tuberculosis Rv3676, which we have designated CRP(Mt), is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRP(Mt)s interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRP(Mt) DNA motif that resembles the cAMP receptor protein (CRP) binding motif model for Escherichia coli. CRP(Mt) binding sites were identified in a total of 73 promoter regions regulating 114 genes in the M. tuberculosis genome, which are being explored as a regulon. Specific CRP(Mt) binding caused DNA bending, and the substitution of highly conserved nucleotides in the binding site resulted in a complete loss of binding to CRP(Mt). cAMP enhanced CRP(Mt)s ability to bind DNA and caused allosteric alterations in CRP(Mt) conformation. These results provide the first direct evidence for cAMP binding to a transcription factor in M. tuberculosis, suggesting a role for cAMP signal transduction in M. tuberculosis and implicating CRP(Mt) as a cAMP-responsive global regulator.

Identification of co-regulated genes through Bayesian clustering of predicted regulatory binding sites

The identification of co-regulated genes and their transcription-factor binding sites (TFBS) are key steps toward understanding transcription regulation. In addition to effective laboratory assays, various computational approaches for the detection of TFBS in promoter regions of coexpressed genes have been developed. The availability of complete genome sequences combined with the likelihood that transcription factors and their cognate sites are often conserved during evolution has led to the development of phylogenetic footprinting. The modus operandi of this technique is to search for conserved motifs upstream of orthologous genes from closely related species. The method can identify hundreds of TFBS without prior knowledge of co-regulation or coexpression. Because many of these predicted sites are likely to be bound by the same transcription factor, motifs with similar patterns can be put into clusters so as to infer the sets of co-regulated genes, that is, the regulons. This strategy utilizes only genome sequence information and is complementary to and confirmative of gene expression data generated by microarray experiments. However, the limited data available to characterize individual binding patterns, the variation in motif alignment, motif width, and base conservation, and the lack of knowledge of the number and sizes of regulons make this inference problem difficult. We have developed a Gibbs sampling-based Bayesian motif clustering (BMC) algorithm to address these challenges. Tests on simulated data sets show that BMC produces many fewer errors than hierarchical and K-means clustering methods. The application of BMC to hundreds of predicted γ-proteobacterial motifs correctly identified many experimentally reported regulons, inferred the existence of previously unreported members of these regulons, and suggested novel regulons.

A family of acr-coregulated Mycobacterium tuberculosis genes shares a common DNA motif and requires Rv3133c (dosR or devR) for expression.

ABSTRACT Previous work has shown that the divergently transcribed Mycobacterium tuberculosis genes acr (hspX, Rv2031c) and acg (Rv2032) are induced under conditions of shallow standing culture and low oxygen and intracellularly within macrophages. We used a combination of computational and experimental methods to identify promoters for eight additional genes that are regulated in a similar manner and that comprise an acr-coregulated promoter (ACP) family. Transcriptional regulation of these ACP family members was evaluated by using a plasmid-based promoter-green fluorescent protein fusion system and flow cytometry. All promoters showed increased expression in shallow standing versus shaking cultures, in low- versus high-oxygen conditions, and intracellularly within macrophages versus extracellularly in tissue culture medium. However, there were quantitative differences in expression among promoters and among conditions for each promoter. A conserved 18-bp palindromic sequence motif was identified in all ACPs by Gibbs sampling-based computational analyses. Two such motifs overlap regions in the acr and acg promoters that were previously shown to be required for their expression. In addition, we found that 5% carbon dioxide was required for growth of Mycobacterium bovis BCG under microaerophilic (1.3% O2) culture conditions and fully prevented the growth cessation typically associated with rapid removal of oxygen. These findings are likely to be relevant to the in vivo environment and will contribute to our understanding of the pathogenesis of tuberculosis infection.

Identification and Characterization of Mycobacterial Proteins Differentially Expressed under Standing and Shaking Culture Conditions, Including Rv2623 from a Novel Class of Putative ATP-Binding Proteins

ABSTRACT The environmental signals that affect gene regulation inMycobacterium tuberculosis remain largely unknown despite their importance to tuberculosis pathogenesis. Other work has shown that several promoters, including acr (also known as hspX) (α-crystallin homolog), are upregulated in shallow standing cultures compared with constantly shaking cultures. Each of these promoters is also induced to a similar extent within macrophages. The present study used two-dimensional gel electrophoresis and mass spectrometry to further characterize differences in mycobacterial protein expression during growth under standing and shaking culture conditions. Metabolic labeling ofM. bovis BCG showed that at least 45 proteins were differentially expressed under standing and shaking culture conditions. Rv2623, CysA2-CysA3, Gap, and Acr were identified from each of four spots or gel bands that were specifically increased in bacteria from standing cultures. An additional standing-induced spot contained two comigrating proteins, GlcB and KatG. The greatest induction was observed with Rv2623, a 32-kDa protein of unknown function that was strongly expressed under standing conditions and absent in shaking cultures. Analysis using PROBE, a multiple sequence alignment and database mining tool, classified M. tuberculosis Rv2623 as a member of a novel class of ATP-binding proteins that may be involved in M. tuberculosiss response to environmental signals. These studies demonstrate the power of combined proteomic and computational approaches and demonstrate that subtle differences in bacterial culture conditions may have important implications for the study of gene expression in mycobacteria.

Identification of a novel class in the α/β hydrolase fold superfamily: The N‐myc differentiation‐related proteins

The α/β hydrolases constitute a large protein superfamily that mainly consists of enzymes that catalyze a diverse range of reactions. These proteins exhibit the α/β hydrolase fold, the essential features of which have recently been delineated: the presence of at least five parallel β‐strands, a catalytic triad in a specific order (nucleophile‐acid‐histidine), and a nucleophilic elbow. Because of the difficulties experimentally in identifying protein structures, we have used a Bayesian computational algorithm (PROBE) to identify the members of this superfamily based on distant sequence relationships. We found that the presence of five sequence motifs, which contain residues important for substrate binding and stabilization of the fold, are required for membership in this superfamily. The superfamily consists of at least 909 members, including the N‐myc downstream regulated proteins, which are believed to be involved in cell differentiation. Unlike most of the other superfamily members, the N‐myc downstream regulated proteins have never been proposed to possess the α/β hydrolase fold and do not appear to be hydrolases. Proteins 2002;47:163–168.

Identification of a Mycobacterium tuberculosis Putative Classical Nitroreductase Gene Whose Expression Is Coregulated with That of the acr Gene within Macrophages, in Standing versus Shaking Cultures, and under Low Oxygen Conditions

ABSTRACT Tuberculosis remains a leading killer worldwide, and new approaches for its treatment and prevention are urgently needed. This effort will benefit greatly from a better understanding of gene regulation in Mycobacterium tuberculosis, particularly with respect to this pathogens response to its host environment. We examined the behavior of two promoters from the divergently transcribed M. tuberculosis genes acr/hspX/Rv2031c (α-crystallin homolog) and Rv2032/acg (acr-coregulated gene) by using a promoter-GFP fusion assay in Mycobacterium bovis BCG. We found that Rv2032 is a novel macrophage-induced gene whose expression is coregulated with that of acr. Relative levels of intracellular induction for both promoters were significantly affected by shallow standing versus shaking bacterial culture conditions prior to macrophage infection, and both promoters were strongly induced under low oxygen conditions. Deletion analyses showed that DNA sequences within a 43-bp region were required for expression of these promoters under all conditions. Multiple sequence alignment and database searches performed with PROBE indicated that Rv2032 is one of eight M. tuberculosis genes of previously unknown function that belong to an unusual superfamily of classical nitroreductases, which may have a role for bacteria within the host environment. These findings show that mycobacterial culture conditions can greatly influence the results and interpretation of subsequent gene regulation experiments. We propose that these differences might be exploited for dissection of the regulatory factors that affect mycobacterial gene expression within the host.

The Gibbs Centroid Sampler

The Gibbs Centroid Sampler is a software package designed for locating conserved elements in biopolymer sequences. The Gibbs Centroid Sampler reports a centroid alignment, i.e. an alignment that has the minimum total distance to the set of samples chosen from the a posteriori probability distribution of transcription factor binding-site alignments. In so doing, it garners information from the full ensemble of solutions, rather than only the single most probable point that is the target of many motif-finding algorithms, including its predecessor, the Gibbs Recursive Sampler. Centroid estimators have been shown to yield substantial improvements, in both sensitivity and positive predictive values, to the prediction of RNA secondary structure and motif finding. The Gibbs Centroid Sampler, along with interactive tutorials, an online user manual, and information on downloading the software, is available at: http://bayesweb.wadsworth.org/gibbs/gibbs.html.

Geoarchaeota: a new candidate phylum in the Archaea from high-temperature acidic iron mats in Yellowstone National Park

Geothermal systems in Yellowstone National Park (YNP) provide an outstanding opportunity to understand the origin and evolution of metabolic processes necessary for life in extreme environments including low pH, high temperature, low oxygen and elevated concentrations of reduced iron. Previous phylogenetic studies of acidic ferric iron mats from YNP have revealed considerable diversity of uncultivated and undescribed archaea. The goal of this study was to obtain replicate de novo genome assemblies for a dominant archaeal population inhabiting acidic iron-oxide mats in YNP. Detailed analysis of conserved ribosomal and informational processing genes indicates that the replicate assemblies represent a new candidate phylum within the domain Archaea referred to here as ‘Geoarchaeota’ or ‘novel archaeal group 1 (NAG1)’. The NAG1 organisms contain pathways necessary for the catabolism of peptides and complex carbohydrates as well as a bacterial-like Form I carbon monoxide dehydrogenase complex likely used for energy conservation. Moreover, this novel population contains genes involved in the metabolism of oxygen including a Type A heme copper oxidase, a bd-type terminal oxidase and a putative oxygen-sensing protoglobin. NAG1 has a variety of unique bacterial-like cofactor biosynthesis and transport genes and a Type3-like CRISPR system. Discovery of NAG1 is critical to our understanding of microbial community structure and function in extant thermophilic iron-oxide mats of YNP, and will provide insight regarding the evolution of Archaea in early Earth environments that may have important analogs active in YNP today.

Comparative Bacterial Proteomics: Analysis of the Core Genome Concept

While comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry, experimental validation of the existence of this core genome requires extensive measurement and is typically not undertaken. Enabled by an extensive proteome database developed over six years, we have experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. Although genomic studies can establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits.