bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Heat Shock Transcription Factor in
Saccharomyces cerevisiae
Oxidative Stress Response
Authors: Yamamoto Ayako , Ueda Junko , Yamamoto Noritaka , Hashikawa Naoya , Sakurai Hiroshi
Published: June 2007

The heat shock transcription factor Hsf1 of the yeast
Saccharomyces cerevisiae
regulates the transcription of a set of genes that contain heat shock elements (HSEs) in their promoters and function in diverse cellular processes, including protein folding. Here, we show that Hsf1 activates the transcription of various target genes when cells are treated with oxidizing reagents, including the superoxide anion generators menadione and KO
2
and the thiol oxidants diamide and 1-chloro-2,4-dinitrobenzene (CDNB). Similar to heat shock, the oxidizing reagents are potent inducers of both efficient HSE binding and extensive phosphorylation of Hsf1. The inducible phosphorylation of Hsf1 is regulated by the intramolecular domain-domain interactions and affects HSE structure-specific transcription. Unlike the heat shock, diamide, or CDNB response, menadione or KO
2
activation of Hsf1 is inhibited by cyclic-AMP-dependent protein kinase (PKA) activity, which negatively regulates the activator functions of other transcriptional regulators implicated in the oxidative stress response. These results demonstrate that Hsf1 is a member of the oxidative stress-responsive activators and that PKA is a general negative regulator in the superoxide anion response.
Development of a Gene Knockout System in
Candida parapsilosis
Reveals a Conserved Role for
BCR1
in Biofilm Formation
Authors: Ding Chen , Butler Geraldine
Published: June 2007

Candida parapsilosis
is an important cause of candidiasis, yet few molecular tools are available. We adapted a recyclable nourseothricin resistance marker gene (
SAT1
) originally developed for use with
C. albicans
in order to generate gene knockouts from
C. parapsilosis
. We first replaced the promoters driving expression of the FLP recombinase and the
SAT1
genes with the equivalent sequences from
C. parapsilosis
. We then used the cassette to generate a homozygous knockout of
C. parapsilosis URA3
. The
ura3
knockouts have altered colony morphologies. We also knocked out both alleles of an ortholog of
BCR1
, a gene encoding a transcription factor known to be required for biofilm development in
C. albicans
. We show that
C. parapsilosis BCR1
is necessary for biofilm development in
C. parapsilosis
and for expression of the cell wall protein encoded by
RBT1
. Our results suggest that there are significant similarities in the regulation of biofilms between the two species, despite the fact that
C. parapsilosis
does not generate true hyphae and that
BCR1
regulates the expression of many hypha-specific adhesins in
C. albicans
.
Lia1p, a Novel Protein Required during Nuclear Differentiation for Genome-Wide DNA Rearrangements in
Tetrahymena thermophila
Authors: Rexer Charles H. , Chalker Douglas L.
Published: June 2007

Extensive genome-wide rearrangements occur during somatic macronuclear development in
Tetrahymena thermophila
. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins' localization to differentiating somatic nuclei, and here we demonstrate that one,
LIA1
, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of
LIA1
(Δ
LIA1
) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the
Tetrahymena
genome.
GroEL Plays a Central Role in Stress-Induced Negative Regulation of Bacterial Conjugation by Promoting Proteolytic Degradation of the Activator Protein TraJ
Authors: Zahrl Doris , Wagner Andrea , Tscherner Michael , Koraimann Günther
Published: June 2007

Transcription of DNA transfer genes is a prerequisite for conjugative DNA transfer of F-like plasmids. Transfer gene expression is sensed by the donor cell and is regulated by a complex network of plasmid- and host-encoded factors. In this study we analyzed the effect of induction of the heat shock regulon on transfer gene expression and DNA transfer in
Escherichia coli
. Raising the growth temperature from 22°C to 43°C transiently reduced transfer gene expression to undetectable levels and reduced conjugative transfer by 2 to 3 orders of magnitude. In contrast, when host cells carried the temperature-sensitive
groEL44
allele, heat shock-mediated repression was alleviated. These data implied that the chaperonin GroEL was involved in negative regulation after heat shock. Investigation of the role of GroEL in this regulatory process revealed that, in
groEL
(Ts) cells, TraJ, the plasmid-encoded master activator of type IV secretion (T4S) system genes, was less susceptible to proteolysis and had a prolonged half-life compared to isogenic wild-type
E. coli
cells. This result suggested a direct role for GroEL in proteolysis of TraJ, down-regulation of T4S system gene expression, and conjugation after heat shock. Strong support for this novel role for GroEL in regulation of bacterial conjugation was the finding that GroEL specifically interacted with TraJ in vivo. Our results further suggested that in wild-type cells this interaction was followed by rapid degradation of TraJ whereas in
groEL
(Ts) cells TraJ remained trapped in the temperature-sensitive GroEL protein and thus was not amenable to proteolysis.
A Large Gene Cluster in
Burkholderia xenovorans
Encoding Abietane Diterpenoid Catabolism
Authors: Smith Daryl J. , Park Joonhong , Tiedje James M. , Mohn William W.
Published: June 2007

Abietane diterpenoids are defense compounds synthesized by trees that are abundant in natural environments and occur as significant pollutants from pulp and paper production.
Burkholderia xenovorans
LB400 has diverse catabolic capabilities and represents an important group of heterotrophic bacteria in soil environments. The genome sequence of LB400 revealed homologs of the
dit
genes of
Pseudomonas abietaniphila
BKME-9, which encode abietane diterpenoid degradation. LB400 grew on abietic acid (AbA), dehydroabietic acid (DhA), palustric acid (PaA), and 7-oxo-DhA. A Xeotron microarray set, with probes for 8450 of the estimated 9000 LB400 genes, was used to compare the transcriptomes of LB400 growing on DhA versus on succinate. On DhA, 97 genes were upregulated, 43 of which were within an 80-kb cluster located on the 1.47-Mbp megaplasmid of LB400. Upregulated genes in this cluster encode a permease, a ring-hydroxylating dioxygenase system (DitA), a ring-cleavage dioxygenase (DitC), a P450 monooxygenase (DitQ), and enzymes catalyzing beta-oxidation-type reactions. Disruption of the
ditA1
gene, encoding the alpha-subunit of DitA, abolished growth on these abietanes. Analyses of the metabolism of abietanes by cell suspensions of wild-type LB400 and the
ditA1
mutant indicate a convergent pathway, with 7-oxo-DhA as a common intermediate for ring hydroxylation by DitA. Also, 7-oxo-PaA was identified as a metabolite of both AbA and PaA. Sequence analysis indicates that genes encoding this pathway have been horizontally transferred among
Betaproteobacteria
and
Gammaproteobacteria
.
Two-Component Response Regulators ofVibrio fischeri: Identification, Mutagenesis, and Characterization
Authors: Hussa Elizabeth A. , O'Shea Therese M. , Darnell Cynthia L. , Ruby Edward G. , Visick Karen L.
Published: June 2007

Two-component signal transduction systems are utilized by prokaryotic and eukaryotic cells to sense and respond to environmental stimuli, both to maintain homeostasis and to rapidly adapt to changing conditions. Studies have begun to emerge that utilize a large-scale mutagenesis approach to analyzing these systems in prokaryotic organisms. Due to the recent availability of its genome sequence, such a global approach is now possible for the marine bioluminescent bacteriumVibrio fischeri, which exists either in a free-living state or as a mutualistic symbiont within a host organism such as the Hawaiian squid speciesEuprymna scolopes. In this work, we identified 40 putative two-component response regulators encoded within theV. fischerigenome. Based on the type of effector domain present, we classified six as NarL type, 13 as OmpR type, and six as NtrC type; the remaining 15 lacked a predicted DNA-binding domain. We subsequently mutated 35 of these genes via a vector integration approach and analyzed the resulting mutants for roles in bioluminescence, motility, and competitive colonization of squid. Through these assays, we identified three novel regulators ofV. fischeriluminescence and seven regulators that altered motility. Furthermore, we found 11 regulators with a previously undescribed effect on competitive colonization of the host squid. Interestingly, five of the newly characterized regulators each affected two or more of the phenotypes examined, strongly suggesting interconnectivity among systems. This work represents the first large-scale mutagenesis of a class of genes inV. fischeriusing a genomic approach and emphasizes the importance of two-component signal transduction in bacterium-host interactions.
Ler and H-NS, Regulators Controlling Expression of the Long Polar Fimbriae of
Escherichia coli
O157:H7
Authors: Torres Alfredo G. , López-Sánchez Guillermo N. , Milflores-Flores Lorena , Patel Shilpa D. , Rojas-López Maricarmen , Martínez de la Peña Claudia F. , Arenas-Hernández Margarita M. P. , Martínez-Laguna Ygnacio
Published: June 2007

Enterohemorrhagic
Escherichia coli
(EHEC) serotype O157:H7 colonizes the human intestine and is responsible for diarrheal outbreaks worldwide. Previously we showed that EHEC produces long polar fimbriae (LPF) and that maximum expression is observed during the exponential phase of growth at 37°C and pH 6.5. In this study, we analyzed the roles of several regulators in the expression of LPF using the β-galactosidase reporter system, and we found that H-NS functions as a transcriptional silencer while Ler functions as an antisilencer of LPF expression. Interestingly, deletion of the
hns
and
ler
genes in EHEC caused constitutive expression of the fusion reporter protein. Semiquantitative reverse transcription (RT)-PCR was also used to analyze LPF expression in the EHEC
ler
or
hns
mutant strain. The
hns
mutant exhibited an increase in
lpf
mRNA expression, while expression in the
ler
mutant was decreased, compared to that in the wild-type strain. Using primer extension analysis, we identified two potential transcriptional start sites within the regulatory region of
lpf
and located consensus hexamers of −10 (CAAGAT) and −35 (TTCAAA), which are commonly found in σ
70
-dependent promoters. Further, we determined whether H-NS and Ler interact directly with the
lpf
promoter region by using purified His-tagged proteins and electrophoretic mobility shift assays. Our data are the first to show direct binding interactions between the H-NS and Ler proteins within the regulatory sequence of the
lpf
operon. Based on the electrophoretic mobility shift assay, RT-PCR, primer extension, and β-galactosidase assay results, we concluded that the
E. coli
O157:H7
lpf
operon possesses a promoter dependent on σ
70
, that H-NS binds to the regulatory sequence of
lpfA
and “silences” the transcription of
lpf
, and that Ler binds to the regulatory sequence and inhibits the action of the H-NS protein.
The Quorum-Sensing Molecule Autoinducer 2 Regulates Motility and Flagellar Morphogenesis in
Helicobacter pylori
Authors: Rader Bethany A. , Campagna Shawn R. , Semmelhack Martin F. , Bassler Bonnie L. , Guillemin Karen
Published: June 2007

The genome of the gastric pathogen
Helicobacter pylori
contains a homologue of the gene
luxS
, which has been shown to be responsible for production of the quorum-sensing signal autoinducer 2 (AI-2). We report here that deletion of the
luxS
gene in strain G27 resulted in decreased motility on soft agar plates, a defect that was complemented by a wild-type copy of the
luxS
gene and by the addition of cell-free supernatant containing AI-2. The flagella of the
luxS
mutant appeared normal; however, in genetic backgrounds lacking any of three flagellar regulators—the two-component sensor kinase
flgS
, the sigma factor σ
28
(also called
fliA
), and the anti-sigma factor
flgM
—loss of
luxS
altered flagellar morphology. In all cases, the double mutant phenotypes were restored to the
luxS
+
phenotype by the addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Furthermore, in all mutant backgrounds loss of
luxS
caused a decrease in transcript levels of the flagellar regulator
flhA
. Addition of DPD to
luxS
cells induced
flhA
transcription in a dose-dependent manner. Deletion of
flhA
in a wild-type or
luxS
mutant background resulted in identical loss of motility, flagella, and flagellar gene expression. These data demonstrate that AI-2 functions as a secreted signaling molecule upstream of FlhA and plays a critical role in global regulation of flagellar gene transcription in
H. pylori
.
UnstableEscherichia coliL Forms Revisited: Growth Requires Peptidoglycan Synthesis
Authors: Joseleau-Petit Danièle , Liébart Jean-Claude , Ayala Juan A. , D'Ari Richard
Published: June 2007

Growing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-typeEscherichia coliK-12 strain overnight to a growing L-form-like state by use of the β-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requiresd-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the β-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also required-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells ofE. coli—and possibly all L forms—have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.
BigR, a Transcriptional Repressor from Plant-Associated Bacteria, Regulates an Operon Implicated in Biofilm Growth
Authors: Barbosa Rosicler L. , Benedetti Celso E.
Published: June 2007

Xylella fastidiosa
is a plant pathogen that colonizes the xylem vessels, causing vascular occlusion due to bacterial biofilm growth. However, little is known about the molecular mechanisms driving biofilm formation in
Xylella
-plant interactions. Here we show that BigR (for “biofilm growth-associated repressor”) is a novel helix-turn-helix repressor that controls the transcription of an operon implicated in biofilm growth. This operon, which encodes BigR, membrane proteins, and an unusual beta-lactamase-like hydrolase (BLH), is restricted to a few plant-associated bacteria, and thus, we sought to understand its regulation and function in
X. fastidiosa
and
Agrobacterium tumefaciens
. BigR binds to a palindromic AT-rich element (the BigR box) in the
Xylella
and
Agrobacterium blh
promoters and strongly represses the transcription of the operon in these cells. The BigR box overlaps with two alternative −10 regions identified in the
blh
promoters, and mutations in this box significantly affected transcription, indicating that BigR competes with the RNA polymerase for the same promoter site. Although BigR is similar to members of the ArsR/SmtB family of regulators, our data suggest that, in contrast to the initial prediction, it does not act as a metal sensor. Increased activity of the BigR operon was observed in both
Xylella
and
Agrobacterium
biofilms. In addition, an
A. tumefaciens bigR
mutant showed constitutive expression of operon genes and increased biofilm formation on glass surfaces and tobacco roots, indicating that the operon may play a role in cell adherence or biofilm development.
Functional Characterization by Genetic Complementation of
aroB
-Encoded Dehydroquinate Synthase from
Mycobacterium tuberculosis
H37Rv and Its Heterologous Expression and Purification
Authors: de Mendonça Jordana Dutra , Ely Fernanda , Palma Mario Sergio , Frazzon Jeverson , Basso Luiz Augusto , Santos Diógenes Santiago
Published: June 2007

The recent recrudescence of
Mycobacterium tuberculosis
infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in
M. tuberculosis
, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The
aroB
-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-
d
-
arabino
-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the
aroB
gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The
Escherichia coli
AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant
M. tuberculosis
dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with
M. tuberculosis
dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.
Distribution of Megaplasmids inLactobacillus salivariusand Other Lactobacilli
Authors: Li Yin , Canchaya Carlos , Fang Fang , Raftis Emma , Ryan Kieran A. , van Pijkeren Jan-Peter , van Sinderen Douwe , O'Toole Paul W.
Published: June 2007

The genome ofLactobacillus salivariusUCC118 includes a 242-kb megaplasmid, pMP118. We now show that 33 strains ofL. salivariusisolated from humans and animals all harbor a megaplasmid, which hybridized with therepAandrepEreplication origin probes of pMP118. Linear megaplasmids that did not hybridize with the pMP118repAprobe were also found in some strains ofL. salivarius, showing for the first time that a lactic acid bacterium has multiple megaplasmids. Phylogenetic analysis of therepEandgroELsequences of 28L. salivariusstrains suggested similar evolutionary paths for the chromosome and megaplasmid. Although the replication origin of circular megaplasmids inL. salivariuswas highly conserved, genotypic and phenotypic comparisons revealed significant variation between megaplasmid-encoded traits. Furthermore, megaplasmids of sizes ranging from 120 kb to 490 kb were present in seven strains belonging to six otherLactobacillusspecies from among 91 strains and 47 species tested. The discovery of the widespread presence of megaplasmids inL. salivarius, and restricted carriage by otherLactobacillusspecies, provides an opportunity to study the contribution of large extrachromosomal replicons to the biology ofLactobacillus.
Cyclic AMP Directly Activates NasP, an
N
-Acyl Amino Acid Antibiotic Biosynthetic Enzyme Cloned from an Uncultured β-Proteobacterium
Authors: Clardy Jon , Brady Sean F.
Published: June 2007

The cyclic AMP (cAMP)-dependent biosynthesis of
N
-acylphenylalanine antibiotics by NasP, an environmental DNA-derived
N
-acyl amino acid synthase, is controlled by an NasP-associated cyclic nucleotide-binding domain and is independent of the global cAMP signal transducer, cAMP receptor protein. A 16S rRNA gene sequence found on the same environmental DNA cosmid as NasP is most closely related to 16S sequences from β-proteobacteria.
New
Streptococcus pneumoniae
Clones in Deceased Wild Chimpanzees
Authors: Chi Fang , Leider Michaela , Leendertz Fabian , Bergmann Carina , Boesch Christophe , Schenk Svenja , Pauli Georg , Ellerbrok Heinz , Hakenbeck Regine
Published: June 2007

In wild chimpanzees in the Taï National Park, Côte d'Ivoire, sudden deaths which were preceded by respiratory problems had been observed since 1999. Two new clones of
Streptococcus pneumoniae
were identified in deceased apes on the basis of multilocus sequence typing analysis and
ply
,
lytA
, and
pbp2x
sequences. The findings suggest that virulent
S. pneumoniae
occurs in populations of wild chimpanzees with the potential to cause infections similar to those observed in humans.
Bacteriochlorophyllide
c
C-8
2
and C-12
1
Methyltransferases Are Essential for Adaptation to Low Light in
Chlorobaculum tepidum
Authors: Chew Aline Gomez Maqueo , Frigaard Niels-Ulrik , Bryant Donald A.
Published: June 2007

Bacteriochlorophyll (BChl)
c
is the major photosynthetic pigment in the green sulfur bacterium
Chlorobaculum tepidum
, in which it forms protein-independent aggregates that function in light harvesting. BChls
c
,
d
, and
e
are found only in chlorosome-producing bacteria and are unique among chlorophylls because of methylations that occur at the C-8
2
and C-12
1
carbons. Two genes required for these methylation reactions were identified and designated
bchQ
(CT1777) and
bchR
(CT1320). BchQ and BchR are members of the radical
S
-adenosylmethionine (SAM) protein superfamily; each has sequence motifs to ligate a [4Fe-4S] cluster, and we propose that they catalyze the methyl group transfers.
bchQ
,
bchR
, and
bchQ bchR
mutants of
C. tepidum
were constructed and characterized. The
bchQ
mutant produced BChl
c
that was not methylated at C-8
2
, the
bchR
mutant produced BChl
c
that was not methylated at C-12
1
, and the double mutant produced [8-ethyl, 12-methyl]-BChl
c
that lacked methylation at both the C-8
2
and C-12
1
positions. Compared to the wild type, the Q
y
absorption bands for BChl
c
in the mutant cells were narrower and blue shifted to various extents. All three mutants grew slower and had a lower cellular BChl
c
content than the wild type, an effect that was especially pronounced at low light intensities. These observations show that the C-8
2
and C-12
1
methylations of BChl
c
play important roles in the adaptation of
C. tepidum
to low light intensity. The data additionally suggest that these methylations also directly or indirectly affect the regulation of the BChl
c
biosynthetic pathway.
Genetic Structure of the
nadA
and
nadB
Antivirulence Loci in
Shigella
spp
Authors: Prunier Anne-Laure , Schuch Raymond , Fernández Reinaldo E. , Maurelli Anthony T.
Published: June 2007

Comparison of
nadA
and
nadB
in 14
Shigella
strains and enteroinvasive
Escherichia coli
versus
E. coli
showed that at least one locus is altered in all strains. These observations explain the characteristic nicotinic acid auxotrophy of
Shigella
organisms and are consistent with the previously identified antivirulence nature of these genes for these pathogens.
A Novel Exclusion Mechanism for Carbon Resource Partitioning in
Aggregatibacter actinomycetemcomitans
Authors: Brown Stacie A. , Whiteley Marvin
Published: June 2007

The bacterium
Aggregatibacter actinomycetemcomitans
is a common commensal of the human oral cavity and the putative causative agent of the disease localized aggressive periodontitis.
A. actinomycetemcomitans
is a slow-growing bacterium that possesses limited metabolic machinery for carbon utilization. This likely impacts its ability to colonize the oral cavity, where growth and community composition is mediated by carbon availability. We present evidence that in the presence of the in vivo relevant carbon substrates glucose, fructose, and lactate
A. actinomycetemcomitans
preferentially metabolizes lactate. This preference for lactate exists despite the fact that
A. actinomycetemcomitans
grows faster and obtains higher cell yields during growth with carbohydrates. The preference for lactate is mediated by a novel exclusion mechanism in which metabolism of lactate inhibits carbohydrate uptake. Coculture studies reveal that
A. actinomycetemcomitans
utilizes lactate produced by the oral bacterium
Streptococcus gordonii
, suggesting the potential for cross-feeding in the oral cavity.
SadC Reciprocally Influences Biofilm Formation and Swarming Motility via Modulation of Exopolysaccharide Production and Flagellar Function
Authors: Merritt Judith H. , Brothers Kimberly M. , Kuchma Sherry L. , O'Toole George A.
Published: June 2007

Pseudomonas aeruginosahas served as an important organism in the study of biofilm formation; however, we still lack an understanding of the mechanisms by which this microbe transitions to a surface lifestyle. A recent study of the early stages of biofilm formation implicated the control of flagellar reversals and production of an exopolysaccharide (EPS) as factors in the establishment of a stable association with the substratum and swarming motility. Here we present evidence that SadC (PA4332), an inner membrane-localized diguanylate cyclase, plays a role in controlling these cellular functions. Deletion of thesadCgene results in a strain that is defective in biofilm formation and a hyperswarmer, while multicopy expression of this gene promotes sessility. A ΔsadCmutant was additionally found to be deficient in EPS production and display altered reversal behavior while swimming in high-viscosity medium, two behaviors proposed to influence biofilm formation and swarming motility. Epistasis analysis suggests that thesadCgene is part of a genetic pathway that allows for the concomitant regulation of these aspects ofP. aeruginosasurface behavior. We propose that SadC and the phosphodiesterase BifA (S. L. Kuchma et al., J. Bacteriol. 189:8165-8178, 2007), via modulating levels of the signaling molecule cyclic-di-GMP, coregulate swarming motility and biofilm formation asP. aeruginosatransitions from a planktonic to a surface-associated lifestyle.
BifA, a Cyclic-Di-GMP Phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility byPseudomonas aeruginosaPA14
Authors: Kuchma Sherry L. , Brothers Kimberly M. , Merritt Judith H. , Liberati Nicole T. , Ausubel Frederick M. , O'Toole George A.
Published: June 2007

The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors inPseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this genebifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifAmutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by thepellocus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifAmutant but does not contribute to the observed swarming defect. The ΔbifAmutation also results in decreased flagellar reversals. Based on epistasis studies with the previously describedsadBgene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.
EbpR Is Important for Biofilm Formation by Activating Expression of the Endocarditis and Biofilm-Associated Pilus Operon (
ebpABC
) of
Enterococcus faecalis
OG1RF
Authors: Bourgogne Agathe , Singh Kavindra V. , Fox Kristina A. , Pflughoeft Kathryn J. , Murray Barbara E. , Garsin Danielle A.
Published: June 2007

We identify ef1090 (renamed
ebpR
) and show its importance for the transcriptional regulation of expression of the
Enterococcus faecalis
pilus operon,
ebpABC
. An
ebpR
deletion (Δ
ebpR
) mutant was found to have reduced
ebpABC
expression with loss of pilus production and a defect in primary adherence with, as a consequence, reduced biofilm formation.
The Antirepressor Needed for Induction of Linear Plasmid-Prophage N15 Belongs to the SOS Regulon
Authors: Mardanov Andrey V. , Ravin Nikolai V.
Published: June 2007

The physiological conditions and molecular interactions that control phage production have been studied in only a few families of temperate phages. We investigated the mechanisms that regulate activation of lytic development in lysogens of coliphage N15, a prophage that is not integrated into the host chromosome but exists as a linear plasmid with covalently closed ends. We identified the N15 antirepressor gene,antC, and showed that its product binds to and acts against the main phage repressor, CB. LexA binds to and represses the promoter ofantC. Mitomycin C-stimulated N15 induction required RecA-dependent autocleavage of LexA and expression of AntC protein. Thus, a cellular repressor whose activity is regulated by DNA damage controls N15 prophage induction.
Expression ofhurP, a Gene Encoding a Prospective Site 2 Protease, Is Essential for Heme-Dependent Induction ofbhuRinBordetella bronchiseptica
Authors: King-Lyons Natalie D. , Smith Kelsy F. , Connell Terry D.
Published: June 2007

Expression of thehurIR bhuRSTUVheme utilization locus inBordetella bronchisepticais coordinately controlled by the global iron-dependent regulator Fur and the extracytoplasmic function sigma factor HurI. Activation of HurI requires transduction of a heme-dependent signal via HurI, HurR, and BhuR, a three-component heme-dependent regulatory system. In silico searches of theB. bronchisepticagenome to identify other genes that encode additional participants in this heme-dependent regulatory cascade revealedhurP, an open reading frame encoding a polypeptide with homology to (i) RseP, a site 2 protease (S2P) ofEscherichia colirequired for modifying the cytoplasmic membrane protein RseA, and (ii) YaeL, an S2P ofVibrio choleraerequired for modification of the cytoplasmic membrane protein TcpP. A mutant ofB. bronchisepticadefective forhurPwas incapable of regulating expression of BhuR in a heme-dependent manner. Furthermore, thehurPmutant was unable to utilize hemin as a sole source of nutrient Fe. These defects in hemin utilization and heme-dependent induction of BhuR were restored when recombinanthurP(or recombinantrseP) was introduced into the mutant. Introduction ofhurPinto ayaeLmutant ofV. choleraealso complemented its S2P defect. These data provided strong evidence that protease activity and cleavage site recognition was conserved in HurP, RseP, and YaeL. The data are consistent with a model in which HurP functionally modifies HurR, a sigma factor regulator that is essential for heme-dependent induction ofbhuR.
Dual Role of OhrR as a Repressor and an Activator in Response to Organic Hydroperoxides inStreptomyces coelicolor
Authors: Oh So-Young , Shin Jung-Ho , Roe Jung-Hye
Published: June 2007

Organic hydroperoxide resistance in bacteria is achieved primarily through reducing oxidized membrane lipids. The soil-inhabiting aerobic bacteriumStreptomyces coelicolorcontains three paralogous genes for organic hydroperoxide resistance:ohrA,ohrB, andohrC. TheohrAgene is transcribed divergently fromohrR, which encodes a putative regulator of MarR family. Both theohrAandohrRgenes were induced highly by various organic hydroperoxides. TheohrAgene was induced through removal of repression by OhrR, whereas theohrRgene was induced through activation by OhrR. Reduced OhrR bound to theohrA-ohrRintergenic region, which contains a central (primary) and two adjacent (secondary) inverted-repeat motifs that overlap with promoter elements. Organic peroxide decreased the binding affinity of OhrR for the primary site, with a concomitant decrease in cooperative binding to the adjacent secondary sites. The single cysteine C28 in OhrR was involved in sensing oxidants, as determined by substitution mutagenesis. The C28S mutant of OhrR bound to the intergenic region without any change in binding affinity in response to organic peroxides. These results lead us to propose a model for the dual action of OhrR as a repressor and an activator inS. coelicolor. Under reduced conditions, OhrR binds cooperatively to the intergenic region, repressing transcription from both genes. Upon oxidation, the binding affinity of OhrR decreases, with a concomitant loss of cooperative binding, which allows RNA polymerase to bind to both theohrAandohrRpromoters. The loosely bound oxidized OhrR can further activate transcription from theohrRpromoter.
Transcriptional Analysis of the Genetic Element pSSVx: Differential and Temporal Regulation of Gene Expression Reveals Correlation between Transcription and Replication
Authors: Contursi Patrizia , Cannio Raffaele , Prato Santina , She Qunxin , Rossi Mosè , Bartolucci Simonetta
Published: June 2007

pSSVx fromSulfolobus islandicusstrain REY15/4 is a hybrid between a plasmid and a fusellovirus. A systematic study performed by a combination of Northern blot analysis, primer extension, and reverse transcriptase PCR revealed the presence of nine major transcripts whose expression was differentially and temporally regulated over the growth cycle ofS. islandicus. The map positions of the RNAs as well as the clockwise and the anticlockwise directions of their transcription were determined. Some genes were clustered and appeared to be transcribed as polycistronic messengers, among which one long transcriptional unit comprised the genes for the plasmid copy number control protein ORF60 (CopG), ORF91, and the replication protein ORF892 (RepA). We propose that a termination readthrough mechanism might be responsible for the formation of more than one RNA species from a single 5′ end and therefore that the nine different RNAs corresponded to only seven different transcriptional starts. Three transcripts, ORF76 and two antisense RNAs, countertranscribed RNA1 (ctRNA1) and ctRNA2, were found to be specifically expressed during (and hence correlated to) the phase in which the pSSVx copy number is kept under stringent control, as they were completely switched off upon the onset of the induction of replication.
Natural Merodiploidy of thelux-ribOperon ofPhotobacterium leiognathifrom Coastal Waters of Honshu, Japan
Authors: Ast Jennifer C. , Urbanczyk Henryk , Dunlap Paul V.
Published: June 2007

Sequence analysis of the bacterial luminescence (lux) genes has proven effective in helping resolve evolutionary relationships among luminous bacteria. Phylogenetic analysis usingluxgenes, however, is based on the assumptions that theluxgenes are present as single copies on the bacterial chromosome and are vertically inherited. We report here that certain strains ofPhotobacterium leiognathicarry multiple phylogenetically distinct copies of the entire operon that codes for luminescence and riboflavin synthesis genes,luxCDABEG-ribEBHA. Merodiploidlux-ribstrains ofP. leiognathiwere detected during sequence analysis ofluxA. To define the gene content, organization, and sequence of eachlux-riboperon, we constructed a fosmid library of genomic DNA from a representative merodiploid strain,lnuch.13.1. Sequence analysis of fosmid clones and genomic analysis oflnuch.13.1 defined two complete, physically separate, and apparently functional operons, designatedlux-rib1andlux-rib2.P. leiognathistrainslelon.2.1 andlnuch.21.1 were also found to carrylux-rib1andlux-rib2, whereas ATCC 25521Tapparently carries onlylux-rib1. Inlnuch.13.1,lelon.2.1,lnuch.21.1, and ATCC 25521T,lux-rib1is flanked upstream bylumQandputAand downstream by a gene for a hypothetical multidrug efflux pump. In contrast, transposase genes flanklux-rib2oflnuch.13.1, and the chromosomal location oflux-rib2apparently differs inlnuch.13.1,lelon.2.1, andlnuch.21.1. Phylogenetic analysis demonstrated thatlux-rib1andlux-rib2are more closely related to each other than either one is to theluxandribgenes of other bacterial species, which rules out interspecies lateral gene transfer as the origin oflux-rib2inP. leiognathi;lux-rib2apparently arose within a previously unsampled or extinctP. leiognathilineage. Analysis of 170 additional strains ofP. leiognathi, for a total of 174 strains examined from coastal waters of Japan, Taiwan, the Philippine Islands, and Thailand, identified 106 strains that carry only a singlelux-riboperon and 68 that carry multiplelux-riboperons. Strains bearing a singlelux-riboperon were obtained throughout the geographic sampling range, whereaslux-ribmerodiploid strains were found only in coastal waters of central Honshu. This is the first report of merodiploidy ofluxorribgenes in a luminous bacterium and the first indication that a natural merodiploid state in bacteria can correlate with geography.
Membrane Sequestration of PII Proteins and Nitrogenase Regulation in the Photosynthetic Bacterium
Rhodobacter capsulatus
Authors: Tremblay Pier-Luc , Drepper Thomas , Masepohl Bernd , Hallenbeck Patrick C.
Published: June 2007

Both
Rhodobacter capsulatus
PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the
R. capsulatus
membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/demodification of one PII homolog is aberrant in the absence of the other. In a
glnK
mutant, more GlnB was found to be membrane associated under these conditions. In a
glnB
mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in
R. capsulatus
.
Sortase C-Mediated Anchoring of BasI to the Cell Wall Envelope of
Bacillus anthracis
Authors: Marraffini Luciano A. , Schneewind Olaf
Published: June 2007

Vegetative forms of
Bacillus anthracis
replicate in tissues of an infected host and precipitate lethal anthrax disease. Upon host death, bacilli form dormant spores that contaminate the environment, thereby gaining entry into new hosts where spores germinate and once again replicate as vegetative forms. We show here that sortase C, an enzyme that is required for the formation of infectious spores, anchors BasI polypeptide to the envelope of predivisional sporulating bacilli. BasI anchoring to the cell wall requires the active site cysteine of sortase C and an LPNTA motif sorting signal at the C-terminal end of the BasI precursor. The LPNTA motif of BasI is cleaved between the threonine (T) and the alanine (A) residue; the C-terminal carboxyl group of threonine is subsequently amide linked to the side chain amino group of diaminopimelic acid within the wall peptides of
B. anthracis
peptidoglycan.
Assembly of the MexAB-OprM Multidrug Pump of
Pseudomonas aeruginosa
: Component Interactions Defined by the Study of Pump Mutant Suppressors
Authors: Nehme Dominic , Poole Keith
Published: June 2007

In an effort to identify key domains of the Pseudo
monas aeruginosa
MexAB-OprM drug efflux system involved in component interactions, extragenic suppressors of various inactivating mutations in individual pump constituents were isolated and studied. The multidrug hypersusceptibility of
P. aeruginosa
expressing MexB with a mutation in a region of the protein implicated in oligomerization (G220S) was suppressed by mutations in the α/β domain of MexA. MexB(G220S) showed a reduced ability to bind MexA in vivo while representative MexA suppressors (V66M and V259F) restored the MexA-MexB interaction. Interestingly, these suppressors also restored resistance in
P. aeruginosa
expressing OprM proteins with mutations at the proximal (periplasmic) tip of OprM that is predicted to interact with MexB, suggesting that these suppressors generally overcame defects in MexA-MexB and MexB-OprM interaction. The multidrug hypersusceptibility arising from a mutation in the helical hairpin of MexA implicated in OprM interaction (V129M) was suppressed by mutations (T198I and F439I) in the periplasmic α-helical barrel of OprM. Again, the MexA mutation compromised an in vivo interaction with OprM that was restored by the T198I and F439I substitutions in OprM, consistent with the hairpin domain mediating MexA binding to this region of OprM. Interestingly, these OprM suppressor mutations restored multidrug resistance in
P. aeruginosa
expressing MexB(G220S). Finally, the
oprM
(
T198I
) suppressor mutation enhanced the yields of all three constituents of a MexA-MexB-OprM(T198I) pump as detected in whole-cell extracts. These data highlight the importance of MexA and interactions with this adapter in promoting MexAB-OprM pump assembly and in stabilizing the pump complex.
ClpP Modulates the Activity of theBacillus subtilisStress Response Transcription Factor, σB
Authors: Reeves Adam , Gerth Ulf , Völker Uwe , Haldenwang W. G.
Published: June 2007

The general stress regulon ofBacillus subtilisis controlled by the activity state of σB, a transcription factor that is switched on following exposure to either physical or nutritional stress. ClpP is the proteolytic component of an ATP-dependent protease that is essential for the proper regulation of multiple adaptive responses inB. subtilis. Among the proteins whose abundance increases in ClpP−B. subtilisare several known to depend on σBfor their expression. In the current work we examine the relationship of ClpP to the activity of σB. The data reveal that the loss of ClpP in otherwise wild-typeB. subtilisresults in a small increase in σBactivity during growth and a marked enhancement of σBactivity following its induction by either physical or nutritional stress. It appears to be the persistence of σB's activity rather than its induction that is principally affected by the loss of ClpP. σB-dependent reporter gene activity rose in parallel in ClpP+and ClpP−B. subtilisstrains but failed to display its normal transience in the ClpP−strain. The putative ClpP targets are likely to be stress generated and novel. Enhanced σBactivity in ClpP−B. subtiliswas triggered by physical stress but not by the induced synthesis of the physical stress pathway's positive regulator (RsbT). In addition, Western blot analyses failed to detect differences in the levels of the principal known σBregulators in ClpP+and ClpP−B. subtilisstrains. The data suggest a model in which ClpP facilitates the turnover of stress-generated factors, which persist in ClpP's absence to stimulate ongoing σBactivity.
Spore Coat Architecture of
Clostridium novyi
NT Spores
Authors: Plomp Marco , McCaffery J. Michael , Cheong Ian , Huang Xin , Bettegowda Chetan , Kinzler Kenneth W. , Zhou Shibin , Vogelstein Bert , Malkin Alexander J.
Published: June 2007

Spores of the anaerobic bacterium
Clostridium novyi
NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Toward this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of both dormant and germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled, and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers, as well as the underlying spore coat and undercoat layers, sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of
C. novyi
NT, these studies document the presence of proteinaceous growth spirals in a biological organism.
The
Bordetella
Bps Polysaccharide Is Critical for Biofilm Development in the Mouse Respiratory Tract
Authors: Sloan Gina Parise , Love Cheraton F. , Sukumar Neelima , Mishra Meenu , Deora Rajendar
Published: June 2007

Bordetellae are respiratory pathogens that infect both humans and animals.
Bordetella bronchiseptica
establishes asymptomatic and long-term to life-long infections of animal nasopharynges. While the human pathogen
Bordetella pertussis
is the etiological agent of the acute disease whooping cough in infants and young children, it is now being increasingly isolated from the nasopharynges of vaccinated adolescents and adults who sometimes show milder symptoms, such as prolonged cough illness. Although it has been shown that
Bordetella
can form biofilms in vitro, nothing is known about its biofilm mode of existence in mammalian hosts. Using indirect immunofluorescence and scanning electron microscopy, we examined nasal tissues from mice infected with
B. bronchiseptica
. Our results demonstrate that a wild-type strain formed robust biofilms that were adherent to the nasal epithelium and displayed architectural attributes characteristic of a number of bacterial biofilms formed on inert surfaces. We have previously shown that the
Bordetella
Bps polysaccharide encoded by the
bpsABCD
locus is critical for the stability and maintenance of three-dimensional structures of biofilms. We show here that Bps is essential for the formation of efficient nasal biofilms and is required for the colonization of the nose. Our results document a biofilm lifestyle for
Bordetella
in mammalian respiratory tracts and highlight the essential role of the Bps polysaccharide in this process and in persistence of the nares.
Impact of Hepatitis C Virus (HCV) Genotypes on Quantification of HCV RNA in Serum by COBAS AmpliPrep/COBAS TaqMan HCV Test, Abbott Antiretroviral Therapy HCV RealTime Assay, and VERSANT HCV RNA Assay
Authors: Tuaillon Edouard , Mondain Anne-Marie , Ottomani Laure , Roudière Laurent , Perney Pascal , Picot Marie-Christine , Séguret Fabienne , Blanc François , Larrey Dominique , Van de Perre Philippe , Ducos Jacques
Published: June 2007

The VERSANT HCV RNA 3.0 (bDNA), COBAS AmpliPrep/COBAS TaqMan HCV, and Abbott ART HCV RealTime assays were compared for hepatitis C virus RNA quantification in 158 clinical specimens (genotypes 1 to 5). RNA values differed significantly between methods (
P
< 0.0001), and mean titer differences ranged from 0.01 to 0.50 log
10
IU/ml depending on the genotypes.