Copyright (c) 2012 All rights reserved. http://works.bepress.com/brian_akerley Recent documents in Brian J. Akerley en-us Fri, 23 Nov 2012 06:17:01 PST 3600 http://works.bepress.com/brian_akerley/38 http://works.bepress.com/brian_akerley/38 Fri, 10 Aug 2012 11:15:00 PDT Haemophilus influenzae is a Gram-negative bacterium that has no identified natural niche outside of the human host. It primarily colonizes the nasopharyngeal mucosa in an asymp- tomatic mode, but has the ability to disseminate to other anatomical sites to cause otitis media, upper, and lower respiratory tract infections, septicemia, and meningitis. To persist in diverse environments the bacterium must exploit and utilize the nutrients and other resources available in these sites for optimal growth/survival. Recent evidence suggests that regulatory factors that direct such adaptations also control virulence determinants required to resist and evade immune clearance mechanisms. In this review, we describe the recent application of whole-genome approaches that together provide insight into distinct survival mechanisms of H. influenzae in the context of different sites of pathogenesis.

]]> Sandy M. Wong et al. Iron *Bacterial Infections Hydrogen Peroxide Oxidative Stress Carbohydrate Metabolism Gene Expression Profiling Lung Lipopolysaccharides Models, Biological Gene Expression Regulation, Bacterial Haemophilus influenzae type b Haemophilus Infections Haemophilus influenzae Immune Evasion High-Throughput Nucleotide Sequencing Genome, Bacterial http://works.bepress.com/brian_akerley/33 http://works.bepress.com/brian_akerley/33 Wed, 29 Feb 2012 13:02:37 PST Signaling mechanisms used by Haemophilus influenzae to adapt to conditions it encounters during stages of infection and pathogenesis are not well understood. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and contributes to resistance to bactericidal effects of serum and to bloodstream infection by H. influenzae. We show that ArcA of nontypeable H. influenzae (NTHI) activates expression of a glycosyltransferase gene, lic2B. Structural comparison of the lipooligosaccharide (LOS) of a lic2B mutant to that of the wild-type strain NT127 revealed that lic2B is required for addition of a galactose residue to the LOS outer core. The lic2B gene was crucial for optimal survival of NTHI in a mouse model of bacteremia and for evasion of serum complement. The results demonstrate that ArcA, which controls cellular metabolism in response to environmental reduction and oxidation (redox) conditions, also coordinately controls genes that are critical for immune evasion, providing evidence that NTHI integrates redox signals to regulate specific countermeasures against host defense.

]]> Sandy M. S. Wong et al. Animals Bacterial Proteins Blotting, Western Cell Separation Complement System Proteins Flow Cytometry Gene Expression Gene Expression Profiling Gene Expression Regulation, Bacterial Haemophilus Infections Haemophilus influenzae Humans Immune Evasion Lipopolysaccharides Mice Oxidation-Reduction Reverse Transcriptase Polymerase Chain Reaction Signal Transduction http://works.bepress.com/brian_akerley/32 http://works.bepress.com/brian_akerley/32 Wed, 29 Feb 2012 13:02:30 PST The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets.

]]> Jennifer E. Griffin et al. Animals Cholesterol Gene Expression Profiling Gene Expression Regulation, Bacterial Genes, Bacterial Mice Mutagenesis Mycobacterium tuberculosis Tuberculosis Virulence http://works.bepress.com/brian_akerley/31 http://works.bepress.com/brian_akerley/31 Wed, 29 Feb 2012 13:02:25 PST Nontypeable Haemophilus influenzae (NTHI) is a Gram-negative bacterial pathogen that causes upper and lower respiratory infections. Factors required for pulmonary infection by NTHI are not well understood. Previously, using high-throughput insertion tracking by deep sequencing (HITS), putative lung colonization factors were identified. Also, previous research indicates that secreted disulfide-dependent factors are important for virulence of H. influenzae. In the present study, HITS data were compared with an informatics-based list of putative substrates of the periplasmic oxidoreductase DsbA to find and characterize secreted virulence factors. This analysis resulted in identification of the "zinc binding essential for virulence" (zev) locus consisting of zevA (HI1249) and zevB (HI1248). NTHI mutants of zevA and zevB grew normally in rich medium but were defective for colonization in a mouse lung model. Mutants also exhibited severe growth defects in medium containing EDTA and were rescued by supplementation with zinc. Additionally, purified recombinant ZevA was found to bind to zinc with high affinity. Together, these data demonstrate that zevAB is a novel virulence factor important for zinc utilization of H. influenzae under conditions where zinc is limiting. Furthermore, evidence presented here suggests that zinc limitation is likely an important mechanism for host defense against pathogens during lung infection.

]]> Charles V. Rosadini et al. Animals Culture Media Female Gene Knockout Techniques Haemophilus Infections Haemophilus influenzae Lung Mice Mice, Inbred C57BL Microbial Viability Mutagenesis, Insertional Pneumonia, Bacterial Protein Binding Virulence Virulence Factors Zinc http://works.bepress.com/brian_akerley/30 http://works.bepress.com/brian_akerley/30 Wed, 29 Feb 2012 13:02:19 PST Whole-genome techniques toward identification of microbial genes required for their survival and growth during infection have been useful for studies of bacterial pathogenesis. The advent of massively parallel sequencing platforms has created the opportunity to markedly accelerate such genome-scale analyses and achieve unprecedented sensitivity, resolution, and quantification. This chapter provides an overview of a genome-scale methodology that combines high-density transposon mutagenesis with a mariner transposon and deep sequencing to identify genes that are needed for survival in experimental models of pathogenesis. Application of this approach to a model pathogen, Haemophilus influenzae, has provided a comprehensive analysis of the relative role of each gene of this human respiratory pathogen in a murine pulmonary model. The method is readily adaptable to nearly any organism amenable to transposon mutagenesis.

]]> Sandy M. S. Wong et al. Mutagenesis, Insertional Bacteria DNA Transposable Elements Animals DNA Primers Chromosomes, Bacterial Sequence Analysis, DNA Haemophilus influenzae DNA, Bacterial Biotin Polyadenylation Reproducibility of Results High-Throughput Nucleotide Sequencing Genome, Bacterial Biotinylation http://works.bepress.com/brian_akerley/29 http://works.bepress.com/brian_akerley/29 Mon, 08 Feb 2010 06:48:20 PST The intermediary metabolism of Haemophilus influenzae strain Rd KW20 was studied by a combination of protein expression analysis using a recently developed direct proteomics approach, mutational analysis, and mathematical modeling. Special emphasis was placed on carbon utilization, sugar fermentation, TCA cycle, and electron transport of H. influenzae cells grown microaerobically and anaerobically in a rich medium. The data indicate that several H. influenzae metabolic proteins similar to Escherichia coli proteins, known to be regulated by low concentrations of oxygen, were well expressed in both growth conditions in H. influenzae. An in silico model of the H. influenzae metabolic network was used to study the effects of selective deletion of certain enzymatic steps. This allowed us to define proteins predicted to be essential or non-essential for cell growth and to address numerous unresolved questions about intermediary metabolism of H. influenzae. Comparison of data from in vivo protein expression with the protein list associated with a genome-scale metabolic model showed significant coverage of the known metabolic proteome. This study demonstrates the significance of an integrated approach to the characterization of H. influenzae metabolism.

]]> Anu Raghunathan et al. Biochemistry Carbohydrate Metabolism Carbon Cell Division Culture Media DNA Mutational Analysis Electron Transport Electrons Escherichia coli Fermentation Haemophilus influenzae Models, Biological Models, Theoretical Oxygen Protein Array Analysis Proteome Proteomics Tricarboxylic Acids http://works.bepress.com/brian_akerley/28 http://works.bepress.com/brian_akerley/28 Mon, 08 Feb 2010 06:48:19 PST Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis.

]]> Jeffrey D. Gawronski et al. http://works.bepress.com/brian_akerley/27 http://works.bepress.com/brian_akerley/27 Mon, 08 Feb 2010 06:48:18 PST Nontypeable Haemophilus influenzae is an obligate human parasite that often causes middle ear infections in children and exacerbates chronic obstructive pulmonary disorder, the fourth leading cause of death in the United States. There are no effective vaccines available for this strain. The lipoprotein YraM (gene HI1655) was identified as essential for the growth and viability of H. influenzae but its function is unknown. Sequence comparisons showed that YraM is a fusion of two protein modules. We grew crystals of the carboxyl-terminal module of YraM comprising residues 257-573 (YraM-C), phased the diffraction data by the multiwavelength anomalous diffraction technique, and refined the model to a crystallographic R-factor of 0.16 (R(free) = 0.19) with data to 1.35 A resolution. The two-domain structure of YraM-C adopts a fold similar to that observed for the open, unliganded forms of several periplasmic binding proteins (PBPs) involved in bacterial active transport. Sequence alignments of YraM homologues from other Gram-negative species showed that the most conserved residues of YraM-C cluster between the two domains in the location where other PBPs bind their cognate ligand. Modeling of YraM-C into a closed conformation similar to the leucine-bound form of the Leu/Ile/Val-binding protein (LIVBP) shows a putative binding pocket larger than the leucine-binding site in LIVBP. The pocket has both polar and nonpolar surfaces, with the latter located in the same area where a leucine side chain binds to LIVBP. We discuss possible biological functions of YraM considering its predicted location in the outer membrane, a novel place for such a binding protein.

]]> J. Vijayalakshmi et al. Amino Acid Sequence Binding Sites Conserved Sequence Crystallography, X-Ray Haemophilus influenzae Lipoproteins Molecular Sequence Data Protein Conformation Sequence Alignment http://works.bepress.com/brian_akerley/26 http://works.bepress.com/brian_akerley/26 Mon, 08 Feb 2010 06:48:17 PST Haemophilus influenzae efficiently colonizes and persists at the human nasopharyngeal mucosa, causing disease when it spreads to other sites. Nitric oxide (NO) represents a major antimicrobial defense deployed by host cells in locations colonized by H. influenzae during pathogenesis that are likely to vary in oxygen levels. Formate-dependent nitrite reductase regulator (FNR) is an oxygen-sensitive regulator in several bacterial pathogens. We report that fnr of H. influenzae is required for anaerobic defense against exposure to NO donors and to resist NO-dependent effects of gamma interferon (IFN-gamma)-activated murine bone marrow-derived macrophages. To understand the mechanism of resistance, we investigated the role of FNR-regulated genes in defense against NO sources. Expression analysis revealed FNR-dependent activation of nrfA, dmsA, napA, and ytfE. Nonpolar deletion mutants of nrfA and ytfE exhibited sensitivity to NO donors, and the ytfE gene was more critical for survival. Compared to the wild-type strain, the ytfE mutant exhibited decreased survival when exposed to macrophages, a defect that was more pronounced after prior stimulation of macrophages with IFN-gamma or lipopolysaccharide. Complementation restored survival of the mutant to the level in the parental strain. Increased sensitivity of the ytfE mutant relative to that of the parent was abrogated by treatment of macrophages with a NO synthase inhibitor, implicating YtfE in resistance to a NO-dependent pathway. These results identify a requirement for FNR in positive control of ytfE and indicate a critical role for ytfE in resistance of H. influenzae to reactive nitrogen species and the antibacterial effects of macrophages.

]]> Jane Colleen Harrington et al. Animals Bacterial Proteins Gene Deletion Gene Expression Profiling *Gene Expression Regulation Genetic Complementation Test Haemophilus influenzae Humans Macrophages Mice Microbial Viability Nitric Oxide Transcription Factors Virulence Factors http://works.bepress.com/brian_akerley/25 http://works.bepress.com/brian_akerley/25 Mon, 08 Feb 2010 06:48:16 PST Developments in high-throughput analysis tools coupled with integrative computational techniques have enabled biological studies to reach new levels. The ability to correlate large volumes of diverse data types into cohesive models of organism function has spawned a new systematic approach to biological investigation. The creation of a new consortium has been proposed to investigate a single organism utilizing these comprehensive approaches. The Haemophilus influenzae Consortium (HIC) would be comprised of five laboratories, each providing separate and complementary areas of expertise in the study of Haemophilus influenzae (HI). The 5-year study proposes to develop coherent models of HI, both as a stand-alone organism, and more importantly, as a human pathogen. Studies in growth condition specificity followed by genomic, metabolic, and proteomic experimentation will be combined and integrated through computational and experimental analyses to form dynamic and predictive models of HI and its responses. Data from the HIC will allow greater understanding of cellular behavior, pathogen-host interactions, bacterial infection, and provide future scientific endeavors with a template for studies of other pathogens.

]]> Eugene Kolker et al. Bacterial Adhesion *Bacterial Infections *Bacterial Physiological Phenomena *Genome, Bacterial Haemophilus influenzae Humans Oligonucleotide Array Sequence Analysis Research Software http://works.bepress.com/brian_akerley/23 http://works.bepress.com/brian_akerley/23 Mon, 08 Feb 2010 06:48:14 PST Mariner-family transposable elements are active in a wide variety of organisms and are becoming increasingly important genetic tools in species lacking sophisticated genetics. The Himar1 element, isolated from the horn fly, Haematobia irritans, is active in Escherichia coli when expressed appropriately. We used this fact to devise a genetic screen for hyperactive mutants of Himar1 transposase that enhance overall transposition from approximately 4- to 50-fold as measured in an E. coli assay. Purified mutant transposases retain their hyperactivity, although to a lesser degree, in an in vitro transposition assay. Mutants like those described herein should enable sophisticated analysis of the biochemistry of mariner transposition and should improve the use of these elements as genetic tools, both in vivo and in vitro.

]]> David J. Lampe et al. Animals DNA *DNA Transposable Elements Muscidae Mutation Transposases http://works.bepress.com/brian_akerley/24 http://works.bepress.com/brian_akerley/24 Mon, 08 Feb 2010 06:48:14 PST The CD4R has been shown to exert variable effects on T cell activation responses. Depending on the manner of ligation, the CD4R has been demonstrated to have positive as well as negative effects on the generation of [Ca2+]i flux by the CD3R. Coaggregation of CD3 with CD4 enhanced Ca2+ flux while their independent ligation and aggregation diminished this response. To further elucidate these paradoxical CD4 effects, we studied induction of a microtubule-associated protein 2 kinase (MAP-2K) activity during ligation of the CD3R. Lymphoid MAP-2K activation by CD3 is an evanescent event that is dependent on phosphorylation of 43-kDa MAP-2K via a pathway that involves protein kinase C. Coaggregation of CD4 and CD3 with cross-linking antibodies and avidin enhanced the CD3-mediated MAP-2K response almost twofold. In contrast, independent ligation and cross-linking of CD4 reduced the CD3-induced MAP-2K response by approximately 50%. An important requirement for this inhibitory effect was that CD4 be ligated before stimulation with anti-CD3. The negative effect of anti-CD4 mAb was specific as other mAb failed to simulate this event. The PMA-induced MAP-2K response was not inhibited by anti-CD4. Intact 32P-labeled Jurkat and normal human T cells demonstrated the appearance of a single 43-kDa tyrosine phosphoprotein during stimulation with PMA and anti-CD3. When these crude cellular extracts were extensively fractionated across DEAE- and hydrophobic columns, MAP-2K was resolved into two peaks of activity, each containing a single tyrosine phosphoprotein around 43 kDa. In addition to tyrosine-specific labeling, mitogenic stimulation of normal human T cells also induced threonine-specific labeling of MAP-2K. These results imply that activation of lymphoid MAP-2K is a dual process requiring at least two independent kinases for optimal activity. Inasmuch as CD3 activates protein kinase C and CD4 is associated with a tyrosine kinase, pp56lck, we suggest that their coaggregation may create the conditions whereby MAP-2K may be activated by dual phosphorylation. Independent aggregation of these receptors may lead to physical separation and breakdown of this interactive mechanism.

]]> Andre E. Nel et al. Adult Antigens, CD3 Antigens, CD4 Antigens, Differentiation, T-Lymphocyte Calcium Calcium-Calmodulin-Dependent Protein Kinases Enzyme Activation Humans Phosphorylation Protein Kinases Receptors, Antigen, T-Cell Tetradecanoylphorbol Acetate Tyrosine http://works.bepress.com/brian_akerley/22 http://works.bepress.com/brian_akerley/22 Mon, 08 Feb 2010 06:48:13 PST Brian J. Akerley et al. Virulence Humans Mutagenesis, Insertional Bacteria DNA Footprinting Bacteriological Techniques DNA Transposable Elements Chromosome Mapping Mutation Phenotype http://works.bepress.com/brian_akerley/21 http://works.bepress.com/brian_akerley/21 Mon, 08 Feb 2010 06:48:12 PST Citation: In Rino Rappuoli, Vincenzo Scarlato, and Beatrice Aricò, Signal Transduction and Bacterial Virulence. Springer-Verlag, 1995. ISBN 3540592172, 9783540592174.

]]> Peggy A. Cotter et al. Bacterial Proteins Trans-Activators Bordetella Signal Transduction http://works.bepress.com/brian_akerley/20 http://works.bepress.com/brian_akerley/20 Mon, 08 Feb 2010 06:48:12 PST Ligation of CD3/TCR on T-cells induces transient activation of lymphoid MAP-2 kinase (MAP-2K), a 43 kDa serine kinase which itself is a substrate of an unidentified tyrosine kinase (pp43). The reversibility of the MAP-2K response agrees with removal of tyrosine phosphates from pp43. Since both activity as well as tyrosine phosphorylation of MAP-2K could be prolonged by Na3VO4, a phosphotyrosine phosphatase inhibitor, we studied the effect of the common CD45 isoform, which is a member of the CD45 phosphatase family, on MAP-2K activity in vivo and in vitro. We demonstrate the ability of purified CD45 phosphatase to remove tyrosine phosphates from partially purified lymphoid MAP-2K. Utilizing the approach of heterologous receptor aggregation, we also showed that CD45 could inhibit the induction of MAP-2K activity in intact Jurkat cells during CD3 or CD3 + CD4 stimulation. We therefore suggest that this phosphatase may control the activity of lymphoid MAP-2K in vivo.

]]> Shimon Pollack et al. Antigens, CD Antigens, CD3 Antigens, CD45 Antigens, Differentiation Antigens, Differentiation, T-Lymphocyte Calcium Calcium-Calmodulin-Dependent Protein Kinases Cell Line Enzyme Activation Histocompatibility Antigens Humans Kinetics Microtubules Phosphoprotein Phosphatases Phosphorylation Phosphotyrosine Protein Kinases Receptors, Antigen, T-Cell T-Lymphocytes Tyrosine http://works.bepress.com/brian_akerley/19 http://works.bepress.com/brian_akerley/19 Mon, 08 Feb 2010 06:48:11 PST A high-density transposon mutagenesis strategy was applied to the Haemophilus influenzae genome to identify genes required for growth or viability. This analysis detected putative essential roles for the products of 259 ORFs of unknown function. Comparisons between complete genomes defined a subset of these proteins in H. influenzae having homologs in Mycobacterium tuberculosis that are absent in Saccharomyces cerevisiae, a distribution pattern that favors their use in development of antimicrobial therapeutics. Three genes within this set are essential for viability in other bacteria. Interfacing the set of essential gene products in H. influenzae with the distribution of homologs in other microorganisms can detect components of unrecognized cellular pathways essential in diverse bacteria. This genome-scale phenotypic analysis identifies potential roles for a large set of genes of unknown function.

]]> Brian J. Akerley et al. Base Sequence Chromosome Mapping Chromosomes, Bacterial Genes, Bacterial *Genome, Bacterial Haemophilus influenzae Mutagenesis, Insertional Open Reading Frames Phenotype Plasmids http://works.bepress.com/brian_akerley/18 http://works.bepress.com/brian_akerley/18 Mon, 08 Feb 2010 06:48:10 PST In response to environmental signals in the host, bacterial pathogens express factors required during infection and repress those that interfere with specific stages of this process. Signalling pathways controlling virulence factors of the human respiratory pathogen, Haemophilus influenzae, are predominantly unknown. The lipooligosaccharide (LOS) outer core represents a prototypical virulence trait of H. influenzae that enhances virulence but also provides targets for innate and adaptive immunity. We report regulation of the display of the virulence-associated phosphorylcholine (PC) epitope on the LOS in response to environmental conditions. PC display is optimal under microaerobic conditions and markedly decreased under conditions of high culture aeration. Gene expression analysis using a DNA microarray was performed to begin to define the metabolic state of the cell under these conditions and to identify genes potentially involved in PC epitope modulation. Global gene expression profiling detected changes in redox responsive genes and in genes of carbohydrate metabolism. The effects on carbohydrate metabolism led us to examine the role of the putative H. influenzae homologue of csrA, a regulator of glycolysis and gluconeogenesis in Escherichia coli. A mutant containing an in-frame deletion of the H. influenzae csrA gene showed increased PC epitope levels under aerobic conditions. Furthermore, deletion of csrA elevated mRNA expression of galU, an essential virulence gene that is critical in generating sugar precursors needed for polysaccharide formation and LOS outer core synthesis. Growth conditions predicted to alter the redox state of the culture modulated the PC epitope and galU expression as well. The results are consistent with a multifactorial mechanism of control of LOS-PC epitope display involving csrA and environmental signals that coordinately regulate biosynthetic and metabolic genes controlling the LOS structure.

]]> Sandy M. S. Wong et al. Aerobiosis Bacterial Proteins Epitopes Escherichia coli Proteins Gene Expression Profiling *Gene Expression Regulation, Bacterial Haemophilus influenzae Humans Lipopolysaccharides Oligonucleotide Array Sequence Analysis Oxygen Phosphorylcholine RNA-Binding Proteins Repressor Proteins UTP-Glucose-1-Phosphate Uridylyltransferase http://works.bepress.com/brian_akerley/17 http://works.bepress.com/brian_akerley/17 Mon, 08 Feb 2010 06:48:09 PST Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner-based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of approximately 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon-generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of sigma28 and sigma54 for motility. In addition, we show that sigma28 is involved in the transcription of flaA and that sigma54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni.

]]> David R. Hendrixson et al. Amino Acid Sequence Bacterial Proteins Campylobacter jejuni *DNA Transposable Elements Molecular Sequence Data Mutagenesis Reverse Transcriptase Polymerase Chain Reaction Sequence Homology, Amino Acid Transcription, Genetic http://works.bepress.com/brian_akerley/16 http://works.bepress.com/brian_akerley/16 Mon, 08 Feb 2010 06:48:08 PST The human respiratory pathogen Haemophilus influenzae, a Gram-negative bacterium, is the first free-living organism to have its complete genome sequenced, providing the opportunity to apply genomic-scale approaches to study gene function. This chapter provides an overview of a highly efficient, in vitro mariner transposon-based method that exploits the natural transformation feature of this organism for the identification of essential genes. In addition, we describe strategies for conditional expression systems that would facilitate further analysis of this class of genes. Finally, we outline a method based on the approach used in H. influenzae for identifying essential genes that can be applied to other bacteria that are not naturally transformable.

]]> Sandy M. S. Wong et al. DNA Transposable Elements DNA, Bacterial DNA-Binding Proteins *Genes, Bacterial *Genes, Essential *Genome, Bacterial Haemophilus influenzae Mutagenesis, Insertional Transposases http://works.bepress.com/brian_akerley/15 http://works.bepress.com/brian_akerley/15 Mon, 08 Feb 2010 06:48:07 PST Complete bacterial genomic DNA sequences have allowed application of genome-scale strategies to identify essential genes. Efficient generation of conditional loss of function mutations provides a means of characterization of this class of genes. Promoter systems conferring tight regulation are particularly applicable to generating such mutations and we sought to apply this approach for the analysis of an essential gene of Haemophilus influenzae. Therefore, we developed the use of a D-xylose-inducible promoter for verification of an essential role in growth for yraM, which encodes a putative periplasmic lipoprotein, in both H. influenzae Rd and virulent type b strains. This promoter was sufficiently tightly regulated to generate conditionally viable strains by inducible expression of YraM. A second approach was used to further characterize YraM. Natural transformation and an ordered mutant collection spanning the H. influenzae genome provide the means to target any gene of interest for mutagenesis and temperature-sensitive (TS) mutant isolation. This strategy was applied to generate a conditionally lethal allele of yraM. The resulting TS mutation was directly mapped to a single amino acid substitution within a motif conserved in all putative YraM orthologs and this mutation was shown to be sufficient to confer the TS phenotype.

]]> Sandy M. S. Wong et al. Bacterial Proteins Cell Division DNA Transposable Elements Gene Deletion Gene Expression Regulation, Bacterial Genetic Complementation Test Genetic Markers Genomics Haemophilus influenzae Mutagenesis, Insertional Mutagenesis, Site-Directed Mutation Promoter Regions, Genetic Temperature Time Factors Xylose