bluuebunny/crossref_metadata_2025 huggingface dataset:
The Global Transcriptional Response ofBacillus subtilisto Peroxide Stress Is Coordinated by Three Transcription Factors
Authors: Helmann John D. , Wu Ming Fang Winston , Gaballa Ahmed , Kobel Phil A. , Morshedi Maud M. , Fawcett Paul , Paddon Chris
Published: December 2002

Bacillus subtilisexhibits a complex adaptive response to low levels of peroxides. We used global transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA population after exposure to either hydrogen peroxide (H2O2) ortert-butyl peroxide (t-buOOH). The peroxide stimulons could be largely accounted for by three regulons controlled by the PerR, σB, and OhrR transcription factors. Three members of the PerR regulon (katA,mrgA, andzosA) were strongly induced by H2O2and weakly induced by t-buOOH. The remaining members of the PerR regulon were only modestly up-regulated by peroxide treatment. Overall, the magnitude of peroxide induction of PerR regulon genes corresponded well with the extent of derepression in aperRmutant strain. The σBregulon was activated by 58 μM H2O2but not by 8 μM H2O2and was strongly activated by either t-buOOH or, in a control experiment,tert-butyl alcohol. Apart from the σBregulon there was a single gene,ohrA, that was strongly and rapidly induced by t-buOOH exposure. This gene, controlled by the peroxide-sensing repressor OhrR, was not induced by any of the other conditions tested.
The Crystal Structure of the Phosphorylation Domain in PhoP Reveals a Functional Tandem Association Mediated by an Asymmetric Interface
Authors: Birck Catherine , Chen Yinghua , Hulett F. Marion , Samama Jean-Pierre
Published: December 2002

PhoP fromBacillus subtilisbelongs to the OmpR subfamily of response regulators. It regulates the transcription of several operons and participates in a signal transduction network that controls adaptation of the bacteria to phosphate deficiency. The receiver domains of two members of this subfamily, PhoB fromEscherichia coliand DrrD fromThermotoga maritima, have been structurally characterized. These modules have similar overall folds but display remarkable differences in the conformation of the β4-α4 and α4 regions. The crystal structure of the receiver domain of PhoP (PhoPN) described in this paper illustrates yet another geometry in this region. Another major issue of the structure determination is the dimeric state of the protein and the novel mode of association between receiver domains. The protein-protein interface is provided by two different surfaces from each protomer, and the tandem unit formed through this asymmetric interface leaves free interaction surfaces. This design is well suited for further association of PhoP dimers to form oligomeric structures. The interprotein interface buries 970 Å2from solvent and mostly involves interactions between charged residues. As described in the accompanying paper, mutations of a single residue in one salt bridge shielded from solvent prevented dimerization of the unphosphorylated and phosphorylated response regulator and had drastic functional consequences. The three structurally documented members of the OmpR family (PhoB, DrrD, and PhoP) provide a framework to consider possible relationships between structural features and sequence signatures in critical regions of the receiver domains.
Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure
Authors: Chen Yinghua , Birck Catherine , Samama Jean-Pierre , Hulett F. Marion
Published: December 2002

Bacillus subtilisPhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Piis growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3′ of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60Kresulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113Ewas phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E∼P was deficient in DNA binding, contributing to the PhoPR113Ein vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.
LuxS-Based Signaling inStreptococcus gordonii: Autoinducer 2 Controls Carbohydrate Metabolism and Biofilm Formation withPorphyromonas gingivalis
Authors: McNab Roderick , Ford Suzannah K. , El-Sabaeny Azza , Barbieri Bruno , Cook Guy S. , Lamont Richard J.
Published: December 2002

Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identifiedluxS, a gene responsible for the synthesis of AI-2, inStreptococcus gordonii, a major component of the dental plaque biofilm.S. gordoniiconditioned medium induced bioluminescence in an AI-2 reporter strain ofVibrio harveyi. An isogenic mutant ofS. gordonii, generated by insertional inactivation of theluxSgene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence inV. harveyiand was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogenPorphyromonas gingivalis. Complementation of theluxSmutation inS. gordoniirestored normal biofilm formation with theluxS-deficientP. gingivalis. Differential display PCR demonstrated that the inactivation ofS. gordonii luxSdownregulated the expression of a number of genes, includinggtfG, encoding glucosyltransferase;fruA, encoding extracellular exo-β-d-fructosidase; andlacDencoding tagatose 1,6-diphosphate aldolase. However,S. gordoniicell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion betweenS. gordoniiandP. gingivalis, was unaffected by inactivation ofluxS. The results suggest thatS. gordoniiproduces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells ofP. gingivalisandS. gordonii.
Regulation of the
Escherichia coli rrnB
P2 Promoter
Authors: Murray Heath D. , Appleman J. Alex , Gourse Richard L.
Published: December 2002

The seven rRNA operons in
Escherichia coli
each contain two promoters,
rrn
P1 and
rrn
P2. Most previous studies have focused on the
rrn
P1 promoters. Here we report a systematic analysis of the activity and regulation of the
rrnB
P2 promoter in order to define the intrinsic properties of
rrn
P2 promoters and to understand better their contributions to rRNA synthesis when they are in their natural setting downstream of
rrn
P1 promoters. In contrast to the conclusions reached in some previous studies, we find that
rrnB
P2 is regulated: it displays clear responses to amino acid availability (stringent control), rRNA gene dose (feedback control), and changes in growth rate (growth rate-dependent control). Stringent control of
rrnB
P2 requires the alarmone ppGpp, but growth rate-dependent control of
rrnB
P2 does not require ppGpp. The
rrnB
P2 core promoter sequence (−37 to +7) is sufficient to serve as the target for growth rate-dependent regulation.
Common Extracellular Sensory Domains in Transmembrane Receptors for Diverse Signal Transduction Pathways inBacteriaandArchaea
Authors: Zhulin Igor B. , Nikolskaya Anastasia N. , Galperin Michael Y.
Published: December 2002

Transmembrane receptors in microorganisms, such as sensory histidine kinases and methyl-accepting chemotaxis proteins, are molecular devices for monitoring environmental changes. We report here that sensory domain sharing is widespread among different classes of transmembrane receptors. We have identified two novel conserved extracellular sensory domains, named CHASE2 and CHASE3, that are found in at least four classes of transmembrane receptors: histidine kinases, adenylate cyclases, predicted diguanylate cyclases, and either serine/threonine protein kinases (CHASE2) or methyl-accepting chemotaxis proteins (CHASE3). Three other extracellular sensory domains were shared by at least two different classes of transmembrane receptors: histidine kinases and either diguanylate cyclases, adenylate cyclases, or phosphodiesterases. These observations suggest that microorganisms use similar conserved domains to sense similar environmental signals and transmit this information via different signal transduction pathways to different regulatory circuits: transcriptional regulation (histidine kinases), chemotaxis (methyl-accepting proteins), catabolite repression (adenylate cyclases), and modulation of enzyme activity (diguanylate cyclases and phosphodiesterases). The variety of signaling pathways using the CHASE-type domains indicates that these domains sense some critically important extracellular signals.
Evolutionary History of
hrgA
, Which Replaces the Restriction Gene
hpy
IIIR in the
hpy
III Locus of
Helicobacter pylori
Authors: Ando T. , Aras R. A. , Kusugami K. , Blaser M. J. , Wassenaar T. M.
Published: December 2002

A recently identified
Helicobacter pylori
gene,
hrgA
, was previously reported to be present in 70 (33%) of 208 strains examined (T. Ando, T. M. Wassenaar, R. M. Peek, R. A. Aras, A. I. Tschumi, L.-J. Van Doorn, K. Kusugami, and M. J. Blaser, Cancer Res.
62:
2385-2389, 2002). Sequence analysis of nine such strains indicated that in each strain
hrgA
replaced
hpy
IIIR, which encodes a restriction endonuclease and which, together with the gene for its cognate methyltransferase, constitutes the
hpy
III locus. As a consequence of either the
hrgA
insertion or independent mutations,
hpy
IIIM function was lost in 11 (5%) of the 208 strains examined, rendering chromosomal DNA sensitive to
Mbo
I digestion. The evolutionary history of the locus containing either
hpy
III or
hrgA
was reconstructed. By homologous recombination involving flanking sequences,
hrgA
and
hpy
IIIR can replace one another in the
hpy
III locus, and there is simultaneous replacement of several flanking genes. These findings, combined with the
hpy
IM
/iceA2
locus discovered previously, suggest that the two most strongly conserved methylase genes of
H. pylori
,
hpy
IIIM and
hpy
IM, are both preceded by alternative genes that compete for presence at their loci.
A Previously Unrecognized Step in Pentachlorophenol Degradation in
Sphingobium chlorophenolicum
Is Catalyzed by Tetrachlorobenzoquinone Reductase (PcpD)
Authors: Dai MingHua , Rogers Julie Bull , Warner Joseph R. , Copley Shelley D.
Published: December 2002

The first step in the pentachlorophenol (PCP) degradation pathway in
Sphingobium chlorophenolicum
has been believed for more than a decade to be conversion of PCP to tetrachlorohydroquinone. We show here that PCP is actually converted to tetrachlorobenzoquinone, which is subsequently reduced to tetrachlorohydroquinone by PcpD, a protein that had previously been suggested to be a PCP hydroxylase reductase.
pcpD
is immediately downstream of
pcpB
, the gene encoding PCP hydroxylase (PCP monooxygenase). Expression of PcpD is induced in the presence of PCP. A mutant strain lacking functional PcpD has an impaired ability to remove PCP from the medium. In contrast, the mutant strain removes tetrachlorophenol from the medium at the same rate as does the wild-type strain. These data suggest that PcpD catalyzes a step necessary for degradation of PCP, but not for degradation of tetrachlorophenol. Based upon the known mechanisms of flavin monooxygenases such as PCP hydroxylase, hydroxylation of PCP should produce tetrachlorobenzoquinone, while hydroxylation of tetrachlorophenol should produce tetrachlorohydroquinone. Thus, we proposed and verified experimentally that PcpD is a tetrachlorobenzoquinone reductase that catalyzes the NADPH-dependent reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone.
ThecobYGene of the ArchaeonHalobacteriumsp. Strain NRC-1 Is Required for De Novo Cobamide Synthesis
Authors: Woodson J. D. , Peck R. F. , Krebs M. P. , Escalante-Semerena J. C.
Published: December 2002

Genetic and nutritional analyses of mutants of the extremely halophilic archaeonHalobacteriumsp. strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity. This activity was previously associated with thecobYgene of the methanogenic archaeonMethanobacterium thermoautotrophicumstrain ΔH, but no evidence was obtained to demonstrate the direct involvement of this protein in cobamide biosynthesis in archaea. Computer analysis of theHalobacteriumsp. strain NRC-1 ORF Vng1581C gene and thecobYgene ofM. thermoautotrophicumstrain ΔH showed the primary amino acid sequence of the proteins encoded by these two genes to be 35% identical and 48% similar. A strain ofHalobacteriumsp. strain NRC-1 carrying a null allele of thecobYgene was auxotrophic for cobinamide-GDP, a known intermediate of the late steps of cobamide biosynthesis. The auxotrophic requirement for cobinamide-GDP was corrected when a wild-type allele ofcobYwas introduced into the mutant strain, demonstrating that the lack ofcobYfunction was solely responsible for the observed block in cobamide biosynthesis in this archaeon. The data also show thatHalobacteriumsp. strain NRC-1 possesses a high-affinity transport system for corrinoids and that this archaeon can synthesize cobamides de novo under aerobic growth conditions. To the best of our knowledge this is the first genetic and nutritional analysis of cobalamin biosynthetic mutants in archaea.
Interdomain Linkers of Homologous Response Regulators Determine Their Mechanism of Action
Authors: Walthers Don , Tran Van K. , Kenney Linda J.
Published: December 2002

OmpR and PhoB are response regulators that contain an N-terminal phosphorylation domain and a C-terminal DNA binding effector domain connected by a flexible interdomain linker. Phosphorylation of the N terminus results in an increase in affinity for specific DNA and the subsequent regulation of gene expression. Despite their sequence and structural similarity, OmpR and PhoB employ different mechanisms to regulate their effector domains. Phosphorylation of OmpR in the N terminus stimulates the DNA binding affinity of the C terminus, whereas phosphorylation of the PhoB N terminus relieves inhibition of the C terminus, enabling it to bind to DNA. Chimeras between OmpR and PhoB containing either interdomain linker were constructed to explore the basis of the differences in their activation mechanisms. Our results indicate that effector domain regulation by either N terminus requires its cognate interdomain linker. In addition, our findings suggest that the isolated C terminus of OmpR is not sufficient for a productive interaction with RNA polymerase.
Quorum Sensing Controls Exopolysaccharide Production in
Sinorhizobium meliloti
Authors: Marketon Melanie M. , Glenn Sarah A. , Eberhard Anatol , González Juan E.
Published: December 2002

Sinorhizobium meliloti
is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by
S. meliloti
, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the
sinRI
locus of
S. meliloti
encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the
sinRI
locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene,
sinI
, abolished EPS II production as well as the expression of several genes in the
exp
operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a
sinI
mutant, indicating that the
sinRI
-specified AHLs are required for
exp
gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C
16:1
-HL), one of the previously identified
sinRI
-specified AHLs, specifically restored
exp
gene expression. Most importantly, the absence of symbiotically active EPS II in a
sinI
mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.
The
stm4066
Gene Product of
Salmonella enterica
Serovar Typhimurium Has Aminoimidazole Riboside (AIRs) Kinase Activity and Allows AIRs To Satisfy the Thiamine Requirement of
pur
Mutant Strains
Authors: Dougherty Michael , Downs Diana M.
Published: December 2002

In bacteria the biosynthetic pathways for purine mononucleotides and the hydroxymethyl pyrimidine moiety of thiamine share five reactions that result in the formation of aminoimidazole ribotide, the last metabolite common to both pathways. Here we describe the characterization of a
Salmonella enterica
mutant strain that has gained the ability to efficiently use exogenous aminoimidazole riboside (AIRs) as a source of thiamine. The lesion responsible for this phenotype is a null mutation in a transcriptional regulator of the GntR family (encoded by
stm4068
). Lack of this protein derepressed transcription of an associated operon (
stm4065-4067
) that encoded a predicted kinase. The
stm4066
gene product was purified and shown to have AIRs kinase activity in vitro. This activity was consistent with the model presented to explain the phenotype caused by the original mutation. This mutation provides a genetic means to isolate the synthesis of the hydroxymethyl pyrimidine moiety of thiamine from the pathway for purine mononucleotide biosynthesis and thus facilitate in vivo analyses.
Forespore-Specific Expression ofBacillus subtilis yqfS, Which Encodes Type IV Apurinic/Apyrimidinic Endonuclease, a Component of the Base Excision Repair Pathway
Authors: Urtiz-Estrada Norma , Salas-Pacheco José M. , Yasbin Ronald E. , Pedraza-Reyes Mario
Published: December 2002

The temporal and spatial expression of theyqfSgene ofBacillus subtilis, which encodes a type IV apurinic/apyrimidinic endonuclease, was studied. A reporter gene fusion to theyqfSopening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment. In agreement with these results,yqfSmRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation. The expression pattern of theyqfS-lacZ fusion suggested thatyqfSmay be an additional member of the EσGregulon. A primer extension product mapped the transcriptional start site ofyqfS, 54 to 55 bp upstream of translation start codon ofyqfS. Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptionalyqfSstart site revealed the presence of a sequence with good homology to promoters preceding genes of the σGregulon. AlthoughyqfSexpression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.
Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in
Streptococcus pneumoniae
Authors: Luo Ping , Morrison Donald A.
Published: December 2002

Natural transformation in
Streptococcus pneumoniae
is regulated by a quorum-sensing system that acts through accumulation and sensing of a peptide pheromone (competence-stimulating peptide [CSP]) to control many competence-specific genes acting in DNA uptake, processing, and integration. The period of competence induced by CSP lasts only 15 min (quarter-height peak width). The recently identified regulator ComX is required for the CSP-dependent expression of many competence-specific genes that share an unusual consensus sequence (TACGAATA) at their promoter regions. To test the hypothesis that this regulator acts as a transient alternative sigma factor, ComX was purified from an
Escherichia coli
overexpression strain and core RNA polymerase was purified from a
comX
-deficient
S. pneumoniae
strain. The reconstituted ComX-polymerase holoenzyme produced transcripts for the competence-specific genes
ssbB
,
cinA
,
cglA
,
celA
, and
dalA
and was inhibited by anti-ComX antibody, but not by anti-σ
70
antibody. Western blotting using antibodies specific for ComX, σ
70
, and poly-His revealed a transient presence of ComX for a period of 15 to 20 min after CSP treatment, while RNA polymerase remained at a constant level and σ
A
remained between 60 and 125% of its normal level. ComX reached a molar ratio to RNA polymerase of at least 1.5. We conclude that ComX is unstable and acts as a competence-specific sigma factor.
Binding of σAand σBto Core RNA Polymerase after Environmental Stress inBacillus subtilis
Authors: Rollenhagen Claudia , Antelmann Haike , Kirstein Janine , Delumeau Olivier , Hecker Michael , Yudkin Michael D.
Published: December 2002

InBacillus subtilis, the alternative sigma factor σBis activated in response to environmental stress or energy depletion. The general stress regulon under the control of σBprovides the cell with multiple stress resistance. Experiments were designed to determine how activated σBreplaces σAas a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the σA-dependent stress geneclpEunder σB-inducing conditions showed that expression was higher in asigBmutant background than in the wild type. The relative affinities of σAand σBfor binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance. The results showed that the affinity of σBfor E was 60-fold lower than that of σA. Western blot analyses with antibodies against σA, σB, and E showed that, after exposure to ethanol stress, the concentration of σBwas only twofold higher than those of σAand E. Thus, the concentration of σBafter stress is not high enough to compensate for its relatively low affinity for E, and it seems that additional mechanisms must be invoked to account for the binding of σBto E after stress.
Transcriptional Regulation and Signature Patterns Revealed by Microarray Analyses of
Streptococcus pneumoniae
R6 Challenged with Sublethal Concentrations of Translation Inhibitors
Authors: Ng Wai-Leung , Kazmierczak Krystyna M. , Robertson Gregory T. , Gilmour Raymond , Winkler Malcolm E.
Published: December 2002

The effects of sublethal concentrations of four different classes of translation inhibitors (puromycin, tetracycline, chloramphenicol, and erythromycin) on global transcription patterns of
Streptococcus pneumoniae
R6 were determined by microarray analyses. Consistent with the general mode of action of these inhibitors, relative transcript levels of genes that encode ribosomal proteins and translation factors or that mediate tRNA charging and amino acid biosynthesis increased or decreased, respectively. Transcription of the heat shock regulon was induced only by puromycin or streptomycin treatment, which lead to truncation or mistranslation, respectively, but not by other antibiotics that block translation, transcription, or amino acid charging of tRNA. In contrast, relative transcript amounts of certain genes involved in transport, cellular processes, energy metabolism, and purine nucleotide (
pur
) biosynthesis were changed by different translation inhibitors. In particular, transcript amounts from a
pur
gene cluster and from purine uptake and salvage genes were significantly elevated by several translation inhibitors, but not by antibiotics that target other cellular processes. Northern blotting confirmed increased transcript amounts from part of the
pur
gene cluster in cells challenged by translation inhibitors and revealed the presence of a 10-kb transcript. Purine metabolism genes were negatively regulated by a homologue of the PurR regulatory protein, and full derepression in a Δ
purR
mutant depended on optimal translation. Unexpectedly, hierarchical clustering of the microarray data distinguished among the global transcription patterns caused by antibiotics that inhibit different steps in the translation cycle. Together, these results show that there is extensive control of transcript amounts by translation in
S. pneumoniae
, especially for de novo purine nucleotide biosynthesis. In addition, these global transcription patterns form a signature that can be used to classify the mode of action and potential mechanism of new translation inhibitors.
Choline Starvation Induces the Gene
licD2
in
Streptococcus pneumoniae
Authors: Desai Bhushan V. , Reiter Harvard , Morrison Donald A.
Published: December 2002

Mutant strains of
Streptococcus pneumoniae
were constructed to monitor the regulation of three dispersed genes known or predicted to act in choline metabolism. One gene (
licD2
) was regulated in response to choline deprivation over a 30-fold range. The other two (SP1860 and
licC
) responded little if at all to the same challenge.
Membrane Topology of the ZntB Efflux System of
Salmonella enterica
Serovar Typhimurium
Authors: Caldwell Andreia M. , Smith Ronald L.
Published: December 2002

The membrane topology of the ZntB Zn
2+
transport protein of
Salmonella enterica
serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to
blaM
or
lacZ
cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.
The
Pseudomonas aeruginosa rhlAB
Operon Is Not Expressed during the Logarithmic Phase of Growth Even in the Presence of Its Activator RhlR and the Autoinducer
N
-Butyryl-Homoserine Lactone
Authors: Medina Gerardo , Juárez Katy , Soberón-Chávez Gloria
Published: December 2002

The
Pseudomonas aeruginosa rhlAB
operon encodes the enzyme rhamnosyltransferase 1, which produces the biosurfactant mono-rhamnolipid;
rhlAB
induction is dependent on the quorum-sensing transcription activator RhlR complexed with the autoinducer
N
-butyryl-homoserine lactone (C
4
-HSL). In this work we studied
rhlAB
induction in a
P. aeruginosa
and
Escherichia coli
background. We found that, in both bacteria, its expression is not induced during the logarithmic phase of growth even in the presence of RhlR and C
4
-HSL. Additionally, we found that
rhlAB
expression is partially σ
s
dependent.
Identification of a Helix-Turn-Helix Motif of
Bacillus thermoglucosidasius
HrcA Essential for Binding to the CIRCE Element and Thermostability of the HrcA-CIRCE Complex, Indicating a Role as a Thermosensor
Authors: Hitomi Masafumi , Nishimura Hiroshi , Tsujimoto Yoshiyuki , Matsui Hiroshi , Watanabe Kunihiko
Published: December 2002

In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the
groE
and
dnaK
operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile
Bacillus thermoglucosidasius
KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in
B. thermoglucosidasius
HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from
Bacillus subtilis
and
B. thermoglucosidasius
, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of
B. thermoglucosidasius
and
B. subtilis
and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.
Characterization of the 4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase Gene and Operon Structure of the Protocatechuate 4,5-Cleavage Pathway Genes in
Sphingomonas paucimobilis
SYK-6
Authors: Hara Hirofumi , Masai Eiji , Miyauchi Keisuke , Katayama Yoshihiro , Fukuda Masao
Published: December 2002

The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for degradation of low-molecular-weight products derived from lignin by
Sphingomonas paucimobilis
SYK-6. In the 10.5-kb
Eco
RI fragment carrying the genes for PCA 4,5-dioxygenase (
ligAB
), 2-pyrone-4,6-dicarboxylate hydrolase (
ligI
), 4-oxalomesaconate hydratase (
ligJ
), and a part of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (
ligC
), we found the
ligK
gene, which encodes 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase. The
ligK
gene was located 1,183 bp upstream of
ligI
and transcribed in the same direction as
ligI
. We also found the
ligR
gene encoding a LysR-type transcriptional activator, which was located 174 bp upstream of
ligK
. The
ligK
gene consists of a 684-bp open reading frame encoding a polypeptide with a molecular mass of 24,131 Da. The deduced amino acid sequence of
ligK
showed 57 to 88% identity with those of the corresponding genes recently reported in
Sphingomonas
sp. strain LB126,
Comamonas testosteroni
BR6020,
Arthrobacter keyseri
12B, and
Pseudomonas ochraceae
NGJ1. The
ligK
gene was expressed in
Escherichia coli
, and the gene product (LigK) was purified to near homogeneity. Electrospray-ionization mass spectrometry indicated that LigK catalyzes not only the conversion of CHA to pyruvate and oxaloacetate but also that of oxaloacetate to pyruvate and CO
2
. LigK is a hexamer, and its isoelectric point is 5.1. The

K
m

for CHA and oxaloacetate are 11.2 and 136 μM, respectively. Inactivation of
ligK
in
S. paucimobilis
SYK-6 resulted in the growth deficiency of vanillate and syringate, indicating that
ligK
encodes the essential CHA aldolase for catabolism of these compounds. Reverse transcription-PCR analysis revealed that the PCA 4,5-cleavage pathway genes of
S. paucimobilis
SYK-6 consisted of four transcriptional units, including the
ligK-orf1-ligI-lsdA
cluster, the
ligJAB
cluster, and the monocistronic
ligR
and
ligC
genes.
A Bacitracin-Resistant
Bacillus subtilis
Gene Encodes a Homologue of the Membrane-Spanning Subunit of the
Bacillus licheniformis
ABC Transporter
Authors: Ohki Reiko , Tateno Kozue , Okada Youji , Okajima Haruo , Asai Kei , Sadaie Yoshito , Murata Makiko , Aiso Toshiko
Published: December 2002

Bacitracin is a peptide antibiotic nonribosomally produced by
Bacillus licheniformis
. The
bcrABC
genes which confer bacitracin resistance to the bacitracin producer encode ATP binding cassette (ABC) transporter proteins, which are hypothesized to pump out bacitracin from the cells.
Bacillus subtilis
168, which has no bacitracin synthesizing operon, has several genes homologous to
bcrABC
. It was found that the disruption of
ywoA
, a gene homologous to
bcrC
, resulted in hypersensitivity to bacitracin. Resistance to other drugs such as surfactin, iturin A, vancomycin, tunicamycin, gramicidin D, valinomycin and several cationic dyes were not changed in the
ywoA
disruptant. Spontaneous bacitracin-resistant mutants (Bcr-1 and -2) isolated in the presence of bacitracin have a single base substitution from A to G in the ribosome binding region. Northern hybridization analysis and determination of the expression of
ywoA-LacZ
transcriptional fusion gene revealed that the transcription of the
ywoA
gene was dependent on extracytoplasmic function (ECF) σ factors σ
M
and σ
X
. Preincubation of wild-type cells in the presence of a low concentration of bacitracin induced increased resistance to bacitracin about two- to threefold, although the mechanism of this induction has not yet been elucidated. It has been reported that a commercially available bacitracin is a mixture of several components and also contains impurity. Bacitracin A was purified by reverse phase high-performance liquid chromatography (HPLC). Similar results were obtained with bacitracin A as those with crude bacitracin, indicating that contaminating substances were not responsible for the results obtained in this study.
TheStreptococcus pneumoniae ciaRegulon: CiaR Target Sites and Transcription Profile Analysis
Authors: Mascher Thorsten , Zähner Dorothea , Merai Michelle , Balmelle Nadège , de Saizieu Antoine B. , Hakenbeck Regine
Published: December 2002

TheciaR-ciaHsystem is one of 13 two-component signal-transducing systems of the human pathogenStreptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by theciasystem, theciaregulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing theS. pneumoniaegenome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimalciaregulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and thehtrA-spo0Jregion. In addition, the transcription profile ofcialoss-of-function mutants and those with an apparent activatedciasystem representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed thecia-dependent expression of seven putative target loci and revealed three additionalcia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, nocia-dependent expression could be detected. Furthermore, the competence regulon, including thecomCDEoperon required for induction of competence, was completely repressed by theciasystem.
Function of Oxygen Resistance Proteins in the Anaerobic, Sulfate-Reducing Bacterium
Desulfovibrio vulgaris
Hildenborough
Authors: Fournier Marjorie , Zhang Yi , Wildschut Janine D. , Dolla Alain , Voordouw Johanna K. , Schriemer David C. , Voordouw Gerrit
Published: December 2002

Two mutant strains of
Desulfovibrio vulgaris
Hildenborough lacking either the
sod
gene for periplasmic superoxide dismutase or the
rbr
gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H
2
O
2
) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a
sor
mutant lacking the gene for the cytoplasmic superoxide reductase. The
sor
mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the
sod
mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the
rbr
mutant, indicating that H
2
O
2
resistance may also be conferred by two other
rbr
genes in the
D. vulgaris
genome. Inhibition of Sod activity by azide and H
2
O
2
, but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of
D. vulgaris
. Thus, Sor plays a key role in oxygen defense of
D. vulgaris
under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.
The SecB Chaperone Is Bifunctional in
Serratia marcescens
: SecB Is Involved in the Sec Pathway and Required for HasA Secretion by the ABC Transporter
Authors: Sapriel Guillaume , Wandersman Cécile , Delepelaire Philippe
Published: December 2002

HasA is the secreted hemophore of the heme acquisition system (Has) of
Serratia marcescens
. It is secreted by a specific ABC transporter apparatus composed of three proteins: HasD, an inner membrane ABC protein; HasE, another inner membrane protein; and HasF, a TolC homolog. Except for HasF, the structural genes of the Has system are encoded by an iron-regulated operon. In previous studies, this secretion system has been reconstituted in
Escherichia coli
, where it requires the presence of the SecB chaperone, the Sec pathway-dedicated chaperone. We cloned and inactivated the
secB
gene from
S. marcescens
. We show that
S. marcescens
SecB is 93% identical to
E. coli
SecB and complements the secretion defects of a
secB
mutant of
E. coli
for both the Sec and ABC pathways of HasA secretion. In
S. marcescens
, SecB inactivation affects translocation by the Sec pathway and abolishes HasA secretion. This demonstrates that
S. marcescens
SecB is the genuine chaperone for HasA secretion in
S. marcescens
. These results also demonstrate that
S. marcescens
SecB is bifunctional, as it is involved in two separate secretion pathways. We investigated the effects of
secB
point mutations in the reconstituted HasA secretion pathway by comparing the translocation of a Sec substrate in various mutants. Two different patterns of SecB residue effects were observed, suggesting that SecB functions may differ for the Sec and ABC pathways.
Mutational Analysis of a Conserved Signal-Transducing Element: the HAMP Linker of theEscherichia coliNitrate Sensor NarX
Authors: Appleman J. Alex , Stewart Valley
Published: December 2002

The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic α-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in theEscherichia colinitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned thenarXHAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for thenarX+strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic α-helices have different roles in signal transduction.
Lack of the ApbC or ApbE Protein Results in a Defect in Fe-S Cluster Metabolism in
Salmonella enterica
Serovar Typhimurium
Authors: Skovran Elizabeth , Downs Diana M.
Published: December 2002

The
isc
genes function in the assembly of Fe-S clusters and are conserved in many prokaryotic and eukaryotic organisms. In most bacteria studied, the
isc
operon can be deleted without loss of cell viability, indicating that additional systems for Fe-S cluster assembly must exist. Several laboratories have described nutritional and biochemical defects resulting from mutations in the
isc
operon. Here we demonstrate that null mutations in two genes of unknown function,
apbC
and
apbE
, result in similar cellular deficiencies. Exogenous ferric chloride suppressed these deficiencies in the
apbC
and
apbE
mutants, distinguishing them from previously described
isc
mutants. The deficiencies caused by the
apbC
and
isc
mutations were additive, which is consistent with Isc and ApbC's having redundant functions or with Isc and ApbC's functioning in different areas of Fe-S cluster metabolism (e.g., Fe-S cluster assembly and Fe-S cluster repair). Both the ApbC and ApbE proteins are similar in sequence to proteins that function in metal cofactor assembly. Like the enzymes with sequence similarity to ApbC, purified ApbC protein was able to hydrolyze ATP. The data herein are consistent with the hypothesis that the ApbC and ApbE proteins function in Fe-S cluster metabolism in vivo.
Separation of ortho Inductive, Resonance and Steric Terms in Alkaline Hydrolysis of Substituted Phenyl Benzoates and Phenyl Tosylates
Authors: Nummert Vilve , Piirsalu Mare
Published: December 2002

The second-order rate constants k2 (l mol-1 s-1) for the alkaline hydrolysis of meta-, para- and ortho-substituted phenyl benzoates C6H5CO2C6H4-X and phenyl tosylates 4-CH3-C6H4SO2OC6H4-X in aqueous 5.3 M NaClO4 have been measured spectrophotometrically at various temperatures. The log k values at a single temperature were analysed according to the equations log km,p = log k0 + (ρ)°m,pσ°, log kortho = log k0+ (ρI)orthoσI + (ρR)orthoσR° + δorthoυ and log km,p,ortho = log k0+ (ρI)orthoσI + (ρI)metaσI + (ρI)paraσI + (ρR)orthoσR° + (ρR)metaσR° + (ρR)paraσR° + δorthoυ. In the case of various temperatures, the equation for data processing involved the additional c1(1/T) term and the cross term cm,p(1/T)σ° or c2(1/T)σI and c3(1/T)σR° different for ortho-, meta- and para-substituted derivatives. As the measure of the steric influence from ortho position, the Charton υ values were used. In the case of a single temperature, the sensitivity to the inductive effect of ortho substituents was found to be about 1.7 times (in water 1.5 times) stronger than that of para and meta substituents in both reaction series studied. The variation of the ortho inductive influence with temperature appeared to be more than twice larger than that for para substituents. Compared to water, in aqueous 5.3 M NaClO4 the inductive effect from ortho position was nearly unchanged while the para inductive effect was found to be about 0.13 units of ρI smaller in the case of both reaction series studied, though the polar effects in these reaction series differ about two-fold. Due to different variation of the ortho and para inductive effects with solvent and temperature, the relative increase in the ortho effect was observed when going from water to aqueous 5.3 M NaClO4.
Improving the utility of prostate specific antigen (PSA) in the diagnosis of prostate cancer: the use of PSA derivatives and novel markers
Authors: Jain S , Bhojwani A G , Mellon J K
Published: December 2002

Prostate specific antigen (PSA) testing is now a routine part of the investigation of men with suspected prostate cancer. While a very useful test it still has its problems, in particular its lack of specificity means abnormal results are often caused by benign disease. This review describes the current problems with PSA testing in prostate cancer diagnosis and highlights potential ways in which these may be reduced.
The NHS complaints system
Authors: Mayberry M K
Published: December 2002

This paper reviews the methods by which complaints are currently handled within the NHS.
Epidemiology of perforated colonic diverticular disease
Authors: Morris C R , Harvey I M , Stebbings W S L , Speakman C T M , Kennedy H J , Hart A R
Published: December 2002

Perforated colonic diverticular disease results in considerable mortality and morbidity. This review appraises existing evidence on the epidemiology and mechanisms of perforation, highlights areas of further study, and suggests an epidemiological approach towards preventing the condition. Computerised searches were used to identify published articles relating to the epidemiology, pathophysiology, and clinical features of perforated colonic diverticular disease. Several drug and dietary exposures have potential biological mechanisms for causing perforation. Of these only non-steroidal anti-inflammatory drugs have been consistently identified as risk factors in aetiological studies. The causes of perforated colonic diverticular disease remain largely unknown. Further aetiological studies, looking specifically at perforation, are required to investigate whether cause-effect relationships exist for both drug and dietary exposures. The identification of risk factors for perforation would allow primary public health prevention, secondary risk factor modification, and early prophylactic surgery to be aimed at people at high risk.
Defensive practice among psychiatrists: a questionnaire survey
Authors: Passmore K , Leung W-C
Published: December 2002

Objective

There has been little research on the prevalence of defensive practice within hospital settings. The aim of this report was to examine the extent of defensiveness among psychiatrists and to examine the relationship between defensiveness and seniority, as well as the effect of previous experiences on the level of defensiveness.

Design

A postal questionnaire survey on defensive practice.

Setting

Northern Region of England.

Subjects

154 psychiatrists in the region.

Results

96 responses were received from 48 equivalent consultants, 18 specialist registrars, and 23 equivalent senior house officers. Overall, 75% of those who replied had taken defensive actions within the past month. In particular, 21% had admitted patients overcautiously and 29% had placed patients on higher levels of observations. Junior psychiatrists were particularly prone to practise defensively. Important contributing factors included previous experience of complaints (against colleague or self), critical incidents, and legal claims.

Conclusion

Almost three quarters of the psychiatrists who responded had practised defensively within the past month. The higher propensity of junior trainees to practise defensively may be attributable to their lack of confidence and experience. Experience of complaints (colleague or self) and critical incidents were important factors for defensive practice. Better and more structured training might reduce the high level of defensive practice and the way complaints and investigations are handled should be improved to maintain a truly “no blame” environment conducive to learning from past experience.