bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic Differences between
Fibrobacter succinogenes
S85 and
Fibrobacter intestinalis
DR7, Identified by Suppression Subtractive Hybridization
Authors: Qi M. , Nelson K. E. , Daugherty S. C. , Nelson W. C. , Hance I. R. , Morrison M. , Forsberg C. W.
Published: December 2007

Fibrobacter
is a highly cellulolytic genus commonly found in the rumen of ruminant animals and cecum of monogastric animals. In this study, suppression subtractive hybridization was used to identify the genes present in
Fibrobacter succinogenes
S85 but absent from
F. intestinalis
DR7. A total of 1,082 subtractive clones were picked, plasmids were purified, and inserts were sequenced, and the clones lacking homology to
F. intestinalis
were confirmed by Southern hybridization. By comparison of the sequences of the clones to one another and to those of the
F. succinogenes
genome, 802 sequences or 955 putative genes, comprising approximately 409 kb of
F. succinogenes
genomic DNA, were identified that lack similarity to those of
F. intestinalis
chromosomal DNA. The functional groups of genes, including those involved in cell envelope structure and function, energy metabolism, and transport and binding, had the largest number of genes specific to
F. succinogenes
. Low-stringency Southern hybridization showed that at least 37 glycoside hydrolases are shared by both species. A cluster of genes responsible for heme, porphyrin, and cobalamin biosynthesis in
F. succinogenes
S85 was either missing from or not functional in
F. intestinalis
DR7, which explains the requirement of vitamin B
12
for the growth of the
F. intestinalis
species. Two gene clusters encoding NADH-ubiquinone oxidoreductase subunits probably shared by
Fibrobacter
genera appear to have an important role in energy metabolism.
Determination of Glycosyltransferase Specificities for the
Escherichia coli
O111 O Antigen by a Generic Approach
Authors: Stevenson Gordon , Dieckelmann Manuela , Reeves Peter R.
Published: December 2007

We describe a bacterial strain developed to facilitate the determination of glycosyltransferase (GT) specificities for O antigens of known structure and gene cluster sequence. For proof of principle for the approach, the strain was used to determine the specificity of the
Escherichia coli
O111 O-antigen GT genes.
ABotrytis cinereaEmopamil Binding Domain Protein, Required for Full Virulence, Belongs to a Eukaryotic Superfamily Which Has Expanded in Euascomycetes
Authors: Gioti A. , Pradier J. M. , Fournier E. , Le Pêcheur P. , Giraud C. , Debieu D. , Bach J. , Leroux P. , Levis C.
Published: December 2007

A previous transcriptomic analysis of 3,032 fungal genes identified theBotrytis cinerea PIE3(BcPIE3) gene to be up-regulated early in planta (A. Gioti, A. Simon, P. Le Pêcheur, C. Giraud, J. M. Pradier, M. Viaud, and C. Levis, J. Mol. Biol. 358:372-386, 2006). In the present study, BcPIE3was disrupted in order to determine its implication in pathogenicity. BcPIE3 was shown to be a virulence factor, since the ΔBcPIE3 mutant was blocked during the colonization of tomato and bean leaves, giving lesions reduced in size by at least 74%. Within the emopamil binding domain (EBD), BcPIE3 shows significant structural similarities to mammalian emopamil binding proteins (EBPs). Mammalian EBPs function as sterol isomerases, but an analysis of the sterol content and the results of growth inhibition experiments with the ΔBcPIE3 strain indicated that BcPIE3 is dispensable for ergosterol biosynthesis. The systematic identification of EBD-containing proteins included in public databases showed that these proteins constitute a protein superfamily present only in eukaryotes. Phylogenetic analysis showed that the ancestral EBD-encoding gene was duplicated in the common ancestor of animals and fungi after the split from plants. Finally, we present evidence that the EBP phylogenetic clade of this superfamily has further expanded exclusively in euascomycetes, especially inB. cinerea, which contains three copies of theEBPgene.
Role of Heme in the Antifungal Activity of the Azaoxoaporphine Alkaloid Sampangine
Authors: Agarwal Ameeta K. , Xu Tao , Jacob Melissa R. , Feng Qin , Lorenz Michael C. , Walker Larry A. , Clark Alice M.
Published: December 2007

Sampangine, a plant-derived alkaloid found in the
Annonaceae
family, exhibits strong inhibitory activity against the opportunistic fungal pathogens
Candida albicans
,
Cryptococcus neoformans
, and
Aspergillus fumigatus
. In the present study, transcriptional profiling experiments coupled with analyses of mutants were performed in an effort to elucidate its mechanism of action. Using
Saccharomyces cerevisiae
as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low-oxygen conditions. Several additional lines of evidence obtained suggest that these responses could involve effects on heme. First, the
hem1
Δ mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells. In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine. Furthermore, spectral analyses of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells. Transcriptional profiling experiments were also performed with
C. albicans
to investigate the response of a pathogenic fungal species to sampangine. Taking into account the known differences in the physiological responses of
C. albicans
and
S. cerevisiae
to low oxygen, significant correlations were observed between the two transcription profiles, suggestive of heme-related defects. Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme.
Protein Kinase A, TOR, and Glucose Transport Control the Response to Nutrient Repletion in
Saccharomyces cerevisiae
Authors: Slattery Matthew G. , Liko Dritan , Heideman Warren
Published: December 2007

Nutrient repletion leads to substantial restructuring of the transcriptome in
Saccharomyces cerevisiae
. The expression levels of approximately one-third of all
S. cerevisiae
genes are altered at least twofold when a nutrient-depleted culture is transferred to fresh medium. Several nutrient-sensing pathways are known to play a role in this process, but the relative contribution that each pathway makes to the total response has not been determined. To better understand this, we used a chemical-genetic approach to block the protein kinase A (PKA), TOR (target of rapamycin), and glucose transport pathways, alone and in combination. Of the three pathways, we found that loss of PKA produced the largest effect on the transcriptional response; however, many genes required both PKA and TOR for proper nutrient regulation. Those genes that did not require PKA or TOR for nutrient regulation were dependent on glucose transport for either nutrient induction or repression. Therefore, loss of these three pathways is sufficient to prevent virtually the entire transcriptional response to fresh medium. In the absence of fresh medium, activation of the cyclic AMP/PKA pathway does not induce cellular growth; nevertheless, PKA activation induced a substantial fraction of the PKA-dependent genes. In contrast, the absence of fresh medium strongly limited gene repression by PKA. These results account for the signals needed to generate the transcriptional responses to glucose, including induction of growth genes required for protein synthesis and repression of stress genes, as well as the classical glucose repression and hexose transporter responses.
Blastocystis rattiContains Cysteine Proteases That Mediate Interleukin-8 Response from Human Intestinal Epithelial Cells in an NF-κB-Dependent Manner
Authors: Puthia Manoj K. , Lu Jia , Tan Kevin S. W.
Published: December 2007

Blastocystisis a ubiquitous enteric protozoan found in the intestinal tracts of humans and a wide range of animals. Evidence accumulated over the last decade suggests association ofBlastocystiswith gastrointestinal disorders involving diarrhea, abdominal pain, constipation, nausea, and fatigue. Clinical and experimental studies have associatedBlastocystiswith intestinal inflammation, and it has been shown thatBlastocystishas potential to modulate the host immune response.Blastocystisis also reported to be an opportunistic pathogen in immunosuppressed patients, especially those suffering from AIDS. However, nothing is known about the parasitic virulence factors and early events following host-parasite interactions. In the present study, we investigated the molecular mechanism by whichBlastocystisactivates interleukin-8 (IL-8) gene expression in human colonic epithelial T84 cells. We demonstrate for the first time that cysteine proteases ofBlastocystis rattiWR1, a zoonotic isolate, can activate IL-8 gene expression in human colonic epithelial cells. Furthermore, we show that NF-κB activation is involved in the production of IL-8. In addition, our findings show that treatment with the antiprotozoal drug metronidazole can avert IL-8 production induced byB. rattiWR1. We also show for the first time that the central vacuole ofBlastocystismay function as a reservoir for cysteine proteases. Our findings will contribute to an understanding of the pathobiology of a poorly studied parasite whose public health importance is increasingly recognized.
Cryptococcus neoformans
Capsular Polysaccharide and Exopolysaccharide Fractions Manifest Physical, Chemical, and Antigenic Differences
Authors: Frases Susana , Nimrichter Leonardo , Viana Nathan B. , Nakouzi Antonio , Casadevall Arturo
Published: December 2007

The human pathogenic fungus
Cryptococcus neoformans
has a large polysaccharide (PS) capsule and releases copious amounts of PS into cultures and infected tissues. The capsular PS is a major virulence factor that can elicit protective antibody responses. PS recovered from culture supernatants has historically provided an ample and convenient source of material for structural and immunological studies. Two major assumptions in such studies are that the structural features of the exopolysaccharide material faithfully mirror those of capsular PS and that the isolation methods do not change PS properties. However, a comparison of exopolysaccharide made by two isolation techniques with capsular PS stripped from cells with gamma radiation or dimethyl sulfoxide revealed significant differences in glycosyl composition, mass, size, charge, viscosity, circular-dichroism spectra, and reactivity with monoclonal antibodies. Our results strongly suggest that exopolysaccharides and capsular PS are structurally different. A noteworthy finding was that PS made by cetyltrimethylammonium bromide precipitation had a larger mass and a different conformation than PS isolated by concentration and filtration, suggesting that the method most commonly used to purify glucuronoxylomannan alters the PS. Hence, the method used to isolate PS can significantly influence the structural and antigenic properties of the product. Our findings have important implications for current views of the relationship between capsular PS and exopolysaccharides, for the generation of PS preparations suitable for immunological studies, and for the formulation of PS-based vaccines for the prevention of cryptococcosis.
Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in
Saccharomyces cerevisiae
Authors: Fei Weihua , Alfaro Gabriel , Muthusamy Baby-Periyanayaki , Klaassen Zachary , Graham Todd R. , Yang Hongyuan , Beh Christopher T.
Published: December 2007

The pandemic of lipid-related disease necessitates a determination of how cholesterol and other lipids are transported and stored within cells. The first step in this determination is the identification of the genes involved in these transport and storage processes. Using genome-wide screens, we identified 56 yeast (
Saccharomyces cerevisiae
) genes involved in sterol-lipid biosynthesis, intracellular trafficking, and/or neutral-lipid storage. Direct biochemical and cytological examination of mutant cells revealed an unanticipated link between secretory protein glycosylation and triacylglycerol (TAG)/steryl ester (SE) synthesis for the storage of lipids. Together with the analysis of other deletion mutants, these results suggested at least two distinct events for the biogenesis of lipid storage particles: a step affecting neutral-lipid synthesis, generating the lipid core of storage particles, and another step for particle assembly. In addition to the lipid storage mutants, we identified mutations that affect the localization of unesterified sterols, which are normally concentrated in the plasma membrane. These findings implicated phospholipase C and the protein phosphatase Ptc1p in the regulation of sterol distribution within cells. This study identified novel sterol-related genes that define several distinct processes maintaining sterol homeostasis.
Regulation of
Cryptococcus neoformans
Capsule Size Is Mediated at the Polymer Level
Authors: Yoneda Aki , Doering Tamara L.
Published: December 2007

The fungal pathogen
Cryptococcus neoformans
regulates its polysaccharide capsule depending on environmental stimuli. To investigate whether capsule polymers change under different growth conditions, we analyzed shed capsules at physiological concentrations without physical perturbation. Our results indicate that regulation of capsule size is mediated at the level of individual polysaccharide molecules.
Function of a Mycobacterial Major Facilitator Superfamily Pump Requires a Membrane-Associated Lipoprotein
Authors: Farrow Mary F. , Rubin Eric J.
Published: December 2007

The
lprG
-Rv1410c operon is critical for the survival of
Mycobacterium tuberculosis
during infection, but very little is known about the functions of its proteins. LprG is a lipoprotein, and Rv1410c encodes the major facilitator superfamily small molecule transporter P55. P55 likely exports small molecules outside of the bacterial cell, but the function of LprG is unclear. A deletion of the homologous operon in
Mycobacterium smegmatis
is more susceptible to ethidium bromide, and drug resistance is restored by the intact operon from
M. tuberculosis
. The multidrug resistance pump inhibitor reserpine inhibits resistance to ethidium bromide in both wild-type
M. smegmatis
and the complemented mutant, suggesting that P55-mediated transport is responsible for drug resistance and that ethidium bromide is a novel substrate for P55. In addition to hypersensitivity to ethidium bromide, cells that lack the
lprG
-Rv1410c operon display abnormal colony morphology and are defective for sliding motility, properties that suggest an alteration of cell wall composition. Strikingly, both ethidium bromide transport and normal cell surface properties require functional P55 and LprG, as neither alone is sufficient to restore function to the deletion mutant. Thus, P55 requires the cell surface lipoprotein for normal function.
RhaU of
Rhizobium leguminosarum
Is a Rhamnose Mutarotase
Authors: Richardson Jason S. , Carpena Xavi , Switala Jack , Perez-Luque Rosa , Donald Lynda J. , Loewen Peter C. , Oresnik Ivan J.
Published: December 2007

Of the nine genes comprising the
l
-rhamnose operon of
Rhizobium leguminosarum
,
rhaU
has not been assigned a function. The construction of a Δ
rhaU
strain revealed a growth phenotype that was slower than that of the wild-type strain, although the ultimate cell yields were equivalent. The transport of
l
-rhamnose into the cell and the rate of its phosphorylation were unaffected by the mutation. RhaU exhibits weak sequence similarity to the formerly hypothetical protein YiiL of
Escherichia coli
that has recently been characterized as an
l
-rhamnose mutarotase. To characterize RhaU further, a His-tagged variant of the protein was prepared and subjected to mass spectrometry analysis, confirming the subunit size and demonstrating its dimeric structure. After crystallization, the structure was refined to a 1.6-Å resolution to reveal a dimer in the asymmetric unit with a very similar structure to that of YiiL. Soaking a RhaU crystal with
l
-rhamnose resulted in the appearance of β-
l
-rhamnose in the active site.
Transcriptional Regulation of the
Clostridium cellulolyticum cip-cel
Operon: a Complex Mechanism Involving a Catabolite-Responsive Element
Authors: Abdou Laetitia , Boileau Céline , de Philip Pascale , Pagès Sandrine , Fiérobe Henri-Pierre , Tardif Chantal
Published: December 2007

The
cip-cel
cluster of genes plays an important role in the catabolism of the substrate cellulose by
Clostridium cellulolyticum
. It encodes several key components of the cellulosomes, including the scaffolding protein CipC and the major cellulase Cel48F. All the genes of this cluster display linked transcription, focusing attention on the promoter upstream from the first gene,
cipC
. We analyzed the regulation of the
cipC
promoter using a transcriptional fusion approach. A single promoter is located between nucleotides −671 and −643 with respect to the ATG start codon, and the large mRNA leader sequence is processed at position −194. A catabolite-responsive element (CRE) 414 nucleotides downstream from the transcriptional start site has been shown to be involved in regulating this operon by a carbon catabolite repression mechanism. This CRE is thought to bind a CcpA-like regulator complexed with a P-Ser-Crh-like protein. Sequences surrounding the promoter sequence may also be involved in direct (sequence-dependent DNA curvature) or indirect (unknown regulator binding) regulation.
Large-Scale Transposon Mutagenesis ofPhotobacterium profundumSS9 Reveals New Genetic Loci Important for Growth at Low Temperature and High Pressure
Authors: Lauro Federico M. , Tran Khiem , Vezzi Alessandro , Vitulo Nicola , Valle Giorgio , Bartlett Douglas H.
Published: December 2007

Microorganisms adapted to piezopsychrophilic growth dominate the majority of the biosphere that is at relatively constant low temperatures and high pressures, but the genetic bases for the adaptations are largely unknown. Here we report the use of transposon mutagenesis with the deep-sea bacteriumPhotobacterium profundumstrain SS9 to isolate dozens of mutant strains whose growth is impaired at low temperature and/or whose growth is altered as a function of hydrostatic pressure. In many cases the gene mutation-growth phenotype relationship was verified by complementation analysis. The largest fraction of loci associated with temperature sensitivity were involved in the biosynthesis of the cell envelope, in particular the biosynthesis of extracellular polysaccharide. The largest fraction of loci associated with pressure sensitivity were involved in chromosomal structure and function. Genes for ribosome assembly and function were found to be important for both low-temperature and high-pressure growth. Likewise, both adaptation to temperature and adaptation to pressure were affected by mutations in a number of sensory and regulatory loci, suggesting the importance of signal transduction mechanisms in adaptation to either physical parameter. These analyses were the first global analyses of genes conditionally required for low-temperature or high-pressure growth in a deep-sea microorganism.
Anaerobic Metabolism of Catechol by the Denitrifying Bacterium
Thauera aromatica
—a Result of Promiscuous Enzymes and Regulators?
Authors: Ding Bin , Schmeling Sirko , Fuchs Georg
Published: December 2007

The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium
Thauera aromatica
that was grown with CO
2
as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the
para
position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.
Precise Region and the Character of the Pathogenicity Island in Clinical
Vibrio parahaemolyticus
Strains
Authors: Sugiyama Tomohiko , Iida Tetsuya , Izutsu Kaori , Park Kwon-Sam , Honda Takeshi
Published: December 2007

In this study, we determined the borders of the pathogenicity island in
V. parahaemolyticus
RIMD2210633 (Vp-PAI). Vp-PAI has features in common with Tn
7
and other related elements at both terminal ends. Our findings indicate that the mobile element with a transposase which contains the DDE motif may have been involved in Vp-PAI formation.
Mutation at Different Sites in the
Nostoc punctiforme cyaC
Gene, Encoding the Multiple-Domain Enzyme Adenylate Cyclase, Results in Different Levels of Infection of the Host Plant
Blasia pusilla
Authors: Chapman Karen E. , Duggan Paula S. , Billington Natalie A. , Adams David G.
Published: December 2007

The filamentous cyanobacterium
Nostoc punctiforme
forms symbioses with plants. Disruption of the catalytic domain of the
N. punctiforme
adenylate cyclase (CyaC) significantly increased symbiotic competence, whereas reduced infectivity was observed in a mutant with a disruption close to the N terminus of CyaC. The total cellular cyclic AMP levels were significantly reduced in both mutants.
Localization and Interactions of Teichoic Acid Synthetic Enzymes inBacillus subtilis
Authors: Formstone Alex , Carballido-López Rut , Noirot Philippe , Errington Jeffery , Scheffers Dirk-Jan
Published: December 2007

The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. InBacillus subtilis168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis inB. subtilis: TagB, -F, -G, -H, and -O. All of these localized similarly to the inner side of the cytoplasmic membrane, in a pattern strikingly similar to that displayed by probes of nascent PG. Helix-like localization patterns are often attributable to the morphogenic cytoskeletal proteins of the MreB family. However, localization of the Tag proteins did not appear to be substantially affected by single disruption of any of the three MreB homologues ofB. subtilis. Bacterial and yeast two-hybrid experiments revealed a complex network of interactions involving TagA, -B, -E, -F, -G, -H, and -O and the cell shape determinants MreC and MreD (encoded by themreBCDoperon and presumably involved in the spatial organization of PG synthesis). Taken together, our results suggest that, inB. subtilisat least, the synthesis and export of WTA precursors are mediated by a large multienzyme complex that may be associated with the PG-synthesizing machinery.
Role of Accessory DNA Polymerases in DNA Replication in
Escherichia coli
: Analysis of the
dnaX36
Mutator Mutant
Authors: Gawel Damian , Pham Phuong T. , Fijalkowska Iwona J. , Jonczyk Piotr , Schaaper Roel M.
Published: December 2007

The
dnaX36
(TS) mutant of
Escherichia coli
confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (−1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various
E. coli
DNA polymerases. The
dnaX
gene encodes the τ subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The
dnaX36
defect resides in the C-terminal domain V of τ, essential for interaction of τ with the α (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered α-τ interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of
dnaX
36 with
mutM, mutY
, and
recA
defects, support our assumption that
dnaX36
-mediated mutations originate as errors of replication rather than DNA damage-related events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (
dinB
) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of τ subunit, in securing a high fidelity of replication.
Glycerate 2-Kinase of
Thermotoga maritima
and Genomic Reconstruction of Related Metabolic Pathways
Authors: Yang Chen , Rodionov Dmitry A. , Rodionova Irina A. , Li Xiaoqing , Osterman Andrei L.
Published: December 2007

Members of a novel glycerate-2-kinase (GK-II) family were tentatively identified in a broad range of species, including eukaryotes and archaea and many bacteria that lack a canonical enzyme of the GarK (GK-I) family. The recently reported three-dimensional structure of GK-II from
Thermotoga maritima
(TM1585; PDB code 2b8n) revealed a new fold distinct from other known kinase families. Here, we verified the enzymatic activity of TM1585, assessed its kinetic characteristics, and used directed mutagenesis to confirm the essential role of the two active-site residues Lys-47 and Arg-325. The main objective of this study was to apply comparative genomics for the reconstruction of metabolic pathways associated with GK-II in all bacteria and, in particular, in
T. maritima
. Comparative analyses of ∼400 bacterial genomes revealed a remarkable variety of pathways that lead to GK-II-driven utilization of glycerate via a glycolysis/gluconeogenesis route. In the case of
T. maritima
, a three-step serine degradation pathway was inferred based on the tentative identification of two additional enzymes, serine-pyruvate aminotransferase and hydroxypyruvate reductase (TM1400 and TM1401, respectively), that convert serine to glycerate via hydroxypyruvate. Both enzymatic activities were experimentally verified, and the entire pathway was validated by its in vitro reconstitution.
A Common Virulence Plasmid in Biotype 2
Vibrio vulnificus
and Its Dissemination Aided by a Conjugal Plasmid
Authors: Lee Chung-Te , Amaro Carmen , Wu Keh-Ming , Valiente Esmeralda , Chang Yi-Feng , Tsai Shih-Feng , Chang Chuan-Hsiung , Hor Lien-I
Published: December 2007

Strains of
Vibrio vulnificus
, a marine bacterial species pathogenic for humans and eels, are divided into three biotypes, and those virulent for eels are classified as biotype 2. All biotype 2 strains possess one or more plasmids, which have been shown to harbor the biotype 2-specific DNA sequences. In this study we determined the DNA sequences of three biotype 2 plasmids: pR99 (68.4 kbp) in strain CECT4999 and pC4602-1 (56.6 kb) and pC4602-2 (66.9 kb) in strain CECT4602. Plasmid pC4602-2 showed 92% sequence identity with pR99. Curing of pR99 from strain CECT4999 resulted in loss of resistance to eel serum and virulence for eels but had no effect on the virulence for mice, an animal model, and resistance to human serum. Plasmids pC4602-2 and pR99 could be transferred to the plasmid-cured strain by conjugation in the presence of pC4602-1, which was self-transmissible, and acquisition of pC4602-2 restored the virulence of the cured strain for eels. Therefore, both pR99 and pC4602-2 were virulence plasmids for eels but not mice. A gene in pR99, which encoded a novel protein and had an equivalent in pC4602-2, was further shown to be essential, but not sufficient, for the resistance to eel serum and virulence for eels. There was evidence showing that pC4602-2 may form a cointegrate with pC4602-1. An investigation of six other biotype 2 strains for the presence of various plasmid markers revealed that they all harbored the virulence plasmid and four of them possessed the conjugal plasmid in addition.
Genetic Evidence for the Actin Homolog Gene
mreBH
and the Bacitracin Resistance Gene
bcrC
as Targets of the Alternative Sigma Factor SigI of
Bacillus subtilis
Authors: Tseng Chi-Ling , Shaw Gwo-Chyuan
Published: December 2007

The
Bacillus subtilis sigI
gene, which is a member of the class VI heat shock genes of the
B. subtilis
heat shock stimulon, encodes an alternative sigma factor whose regulon is poorly defined. In this study, by using a binary vector system, we showed that
B. subtilis
SigI could drive expression of a transcriptional fusion between the
sigI
regulatory region from
Bacillus licheniformis, Bacillus
sp. strain NRRL B-14911,
B. subtilis
, or
Bacillus thuringiensis
and the
xylE
reporter gene in
B. subtilis
. The transcriptional initiation sites of these fusions in
B. subtilis
were mapped by primer extension analyses. A putative consensus promoter sequence probably recognized by the
B. subtilis
SigI was thus deduced. Using a consensus sequence-based search procedure, we found putative σ
I
promoters preceding the actin homolog gene
mreBH
and the bacitracin resistance gene
bcrC
of
B. subtilis
. Overexpression of the
B. subtilis sigI
gene could specifically stimulate expression of both an
mreBH
promoter region-
bgaB
fusion and a
bcrC
promoter region-
bgaB
fusion. Expression of these two fusions at the
amyE
locus of the
B. subtilis
chromosome was heat inducible and SigI dependent as revealed by
sigI
gene disruption experiments. Primer extension analysis showed that the identified
mreBH
and
bcrC
transcriptional start sites were at appropriate distances from their σ
I
promoter elements. This further supports the notion that SigI can directly regulate
mreBH
and
bcrC
expression. Taken together, these results strongly suggest that
mreBH
and
bcrC
are new members of the SigI regulon.
Transcriptome Analysis of Sorbic Acid-Stressed
Bacillus subtilis
Reveals a Nutrient Limitation Response and Indicates Plasma Membrane Remodeling
Authors: Beek Alex Ter , Keijser Bart J. F. , Boorsma Andre , Zakrzewska Anna , Orij Rick , Smits Gertien J. , Brul Stanley
Published: December 2007

The weak organic acid sorbic acid is a commonly used food preservative, as it inhibits the growth of bacteria, yeasts, and molds. We have used genome-wide transcriptional profiling of
Bacillus subtilis
cells during mild sorbic acid stress to reveal the growth-inhibitory activity of this preservative and to identify potential resistance mechanisms. Our analysis demonstrated that sorbic acid-stressed cells induce responses normally seen upon nutrient limitation. This is indicated by the strong derepression of the CcpA, CodY, and Fur regulon and the induction of tricarboxylic acid cycle genes, SigL- and SigH-mediated genes, and the stringent response. Intriguingly, these conditions did not lead to the activation of sporulation, competence, or the general stress response. The fatty acid biosynthesis (
fab
) genes and BkdR-regulated genes are upregulated, which may indicate plasma membrane remodeling. This was further supported by the reduced sensitivity toward the
fab
inhibitor cerulenin upon sorbic acid stress. We are the first to present a comprehensive analysis of the transcriptional response of
B. subtilis
to sorbic acid stress.
Distinct Roles for Two CYP226 Family Cytochromes P450 in Abietane Diterpenoid Catabolism by
Burkholderia xenovorans
LB400
Authors: Smith Daryl J. , Patrauchan Marianna A. , Florizone Christine , Eltis Lindsay D. , Mohn William W.
Published: December 2007

The 80-kb
dit
cluster of
Burkholderia xenovorans
LB400 encodes the catabolism of abietane diterpenoids. This cluster includes
ditQ
and
ditU
, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16
dit
-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that
ditQ
was required for growth on DhA,
ditU
was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 ± 0.01 μM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.
Staphylococcus aureusCodY Negatively Regulates Virulence Gene Expression
Authors: Majerczyk Charlotte D. , Sadykov Marat R. , Luong Thanh T. , Lee Chia , Somerville Greg A. , Sonenshein Abraham L.
Published: December 2007

CodY is a global regulatory protein that was first discovered inBacillus subtilis, where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G+C gram-positive bacteria, includingStaphylococcus aureus. The introduction of acodY-null mutation into twoS. aureusclinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin (hla), the accessory gene regulator (agr) (RNAIIandRNAIII/hld), and the operon responsible for the production of PIA (icaADBC). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors ofS. aureus.
The AraC-Like Transcriptional Regulator DhbR Is Required for Maximum Expression of the 2,3-Dihydroxybenzoic Acid Biosynthesis Genes inBrucella abortus2308 in Response to Iron Deprivation
Authors: Anderson Eric S. , Paulley James T. , Roop R. Martin
Published: December 2007

Phenotypic evaluation of isogenic mutants derived fromBrucella abortus2308 indicates that the AlcR homolog DhbR (2,3-dihydroxybenzoic acid [2,3-DHBA] biosynthesisregulator) modulates the expression of the genes involved in 2,3-DHBA production, employing 2,3-DHBA or brucebactin as a coinducer.
Reinvestigation of the Steady-State Kinetics and Physiological Function of the Soluble NiFe-Hydrogenase I of
Pyrococcus furiosus
Authors: van Haaster Daan J. , Silva Pedro J. , Hagedoorn Peter-Leon , Jongejan Jaap A. , Hagen Wilfred R.
Published: December 2007

Pyrococcus furiosus
has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H
2
evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H
2
production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low
K

m

for H
2
(∼20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the
K

m

for NADP
+
is ∼37 μM, we concluded that the soluble hydrogenase from
P. furiosus
is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.
The Intracellular Pathogen
Rhodococcus equi
Produces a Catecholate Siderophore Required for Saprophytic Growth
Authors: Miranda-CasoLuengo Raúl , Prescott John F. , Vázquez-Boland José A. , Meijer Wim G.
Published: December 2007

Little is known about the iron acquisition systems of the soilborne facultative intracellular pathogen
Rhodococcus equi
. We previously reported that expression of
iupABC
, encoding a putative siderophore ABC transporter system, is iron regulated and required for growth at low iron concentrations. Here we show that disruption of
iupA
leads to the concomitant accumulation of catecholates and a chromophore with absorption maxima at 341 and 528 nm during growth under iron-replete conditions. In contrast, the wild-type strain produces these compounds only in iron-depleted medium. Disruption of
iupU
and
iupS
, encoding nonribosomal peptide synthetases, prevented growth of the corresponding
R. equi
SID1 and SID3 mutants at low iron concentrations. However, only
R. equi
SID3 did not produce the chromophore produced by the wild-type strain during growth at low iron concentrations. The phenotype of
R. equi
SID3, but not that of
R. equi
SID1, could be rescued by coculture with the wild type, allowing growth at low iron concentrations. This strongly suggests that the product of the
iupS
gene is responsible for the synthesis of a diffusible compound required for growth at low iron concentrations. Transcription of
iupU
was constitutive, but that of
iupS
was iron regulated, with an induction of 3 orders of magnitude during growth in iron-depleted compared to iron-replete medium. Neither mutant was attenuated in vivo in a mouse infection model, indicating that the
iupU
- and
iupS
-encoded iron acquisition systems are primarily involved in iron uptake during saprophytic life.
In Situ Growth Rates and Biofilm Development of
Pseudomonas aeruginosa
Populations in Chronic Lung Infections
Authors: Yang Lei , Haagensen Janus A. J. , Jelsbak Lars , Johansen Helle Krogh , Sternberg Claus , Høiby Niels , Molin Søren
Published: December 2007

The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible
Pseudomonas aeruginosa
clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting
P. aeruginosa
cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of
P. aeruginosa
in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of
P. aeruginosa
during chronic infections in CF patients in particular and on long-term infections in general.
Pseudomonas syringaeBetT Is a Low-Affinity Choline Transporter That Is Responsible for Superior Osmoprotection by Choline over Glycine Betaine
Authors: Chen Chiliang , Beattie Gwyn A.
Published: December 2007

The plant pathogenPseudomonas syringaederives better osmoprotection from choline than from glycine betaine, unlike most bacteria that have been characterized. In this report, we identified a betaine/carnitine/choline family transporter (BCCT) inP. syringaepv. tomato strain DC3000 that mediates the transport of choline and acetylcholine. This transporter has a particularly low affinity (Kmof 876 μM) and high capacity (Vmaxof 80 nmol/min/mg of protein) for choline transport relative to other known BCCTs. Although BetT activity increased in response to hyperosmolarity, BetT mediated significant uptake under low-osmolarity conditions, suggesting a role in transport for both osmoprotection and catabolism. Growth studies with mutants deficient in BetT and other choline transporters demonstrated that BetT was responsible for the superior osmoprotection conferred toP. syringaeby choline over glycine betaine when these compounds were provided at high concentrations (>100 μM). These results suggest thatP. syringaehas evolved to survive in relatively choline-rich habitats, a prediction that is supported by the common association ofP. syringaewith plants and the widespread production of choline, but genus- and species-specific production of glycine betaine, by plants. Among the three putative BCCT family transporters inPseudomonas aeruginosaand six inPseudomonas putida, different transporters were predicted to function based on similarity toEscherichia coliBetT than toP. syringaeBetT. FunctionalP. putidaandP. aeruginosatransporters were identified, and their possession of a long C-terminal tail suggested an osmoregulatory function for this tail; this function was confirmed forP. syringaeBetT using deletion derivatives.
The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from
Streptomyces davawensis
Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin
Authors: Grill Simon , Busenbender Simone , Pfeiffer Matthias , Köhler Uwe , Mack Matthias
Published: December 2007

Streptomyces davawensi
s synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5′-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent
Sus scrofa
d
-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in
S. davawensi
s, which would not allow the formation of toxic RoFMN/RoFAD. The gene
ribC
from
S. davawensi
s was cloned. RibC from
S. davawensis
was overproduced in
Escherichia coli
and purified. Analysis of the flavokinase activity of RibC revealed that the
S. davawensis
enzyme is not riboflavin specific (roseoflavin,
k
cat
/

K
m

= 1.7 10
−2
μM
−1
s
−1
; riboflavin,
k
cat
/

K
m

= 7.5 10
−3
μM
−1
s
−1
). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium
Bacillus subtilis
(roseoflavin,
k
cat
/

K
m

= 1.3 10
−2
μM
−1
s
−1
; riboflavin,
k
cat
/

K
m

= 1.3 10
−2
μM
−1
s
−1
). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of
S. davawensis ribC
did not confer roseoflavin resistance to a
ribC
-defective
B. subtilis
strain.
The Target for the
Pseudomonas putida
Crc Global Regulator in the Benzoate Degradation Pathway Is the BenR Transcriptional Regulator
Authors: Moreno Renata , Rojo Fernando
Published: December 2007

Crc protein is a global regulator involved in catabolite repression control of several pathways for the assimilation of carbon sources in pseudomonads when other preferred substrates are present. In
Pseudomonas putida
cells growing exponentially in a complete medium containing benzoate, Crc strongly inhibits the expression of the benzoate degradation genes. These genes are organized into several transcriptional units. We show that Crc directly inhibits the expression of the peripheral genes that transform benzoate into catechol (the
ben
genes) but that its effect on genes corresponding to further steps of the pathway (the
cat
and
pca
genes of the central catechol and β-ketoadipate pathways) is indirect, since these genes are not induced because the degradation intermediates, which act as inducers, are not produced. Crc inhibits the translation of target genes by binding to mRNA. The expression of the
ben
,
cat
, and
pca
genes requires the BenR, CatR, and PcaR transcriptional activators, respectively. Crc significantly reduced
benABCD
mRNA levels but did not affect those of
benR
. Crc bound to the 5′ end of
benR
mRNA but not to equivalent regions of
catR
and
pcaR
mRNAs. A translational fusion of the
benR
and
lacZ
genes was sensitive to Crc, but a transcriptional fusion was not. We propose that Crc acts by reducing the translation of
benR
mRNA, decreasing BenR levels below those required for the full expression of the
benABCD
genes. This strategy provides great metabolic flexibility, allowing the hierarchical assimilation of different structurally related compounds that share a common central pathway by selectively regulating the entry of each substrate into the central pathway.