bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional Pausing in the
Bacillus subtilis pyr
Operon In Vitro: a Role in Transcriptional Attenuation?
Authors: Zhang Hesheng , Switzer Robert L.
Published: August 2003

The genes encoding the enzymes of pyrimidine nucleotide biosynthesis (
pyr
genes) are regulated in
Bacillus subtilis
and many other bacterial species by transcriptional attenuation. When UMP or UTP is bound to the PyrR regulatory protein, it binds to
pyr
mRNA at specific sequences and secondary structures in the RNA. Binding to this site prevents formation of an antiterminator stem-loop in the RNA and permits formation of a downstream terminator, leading to reduced expression of the
pyr
genes lying downstream from the terminator. The functioning of several other transcriptional attenuation systems has been shown to involve transcriptional pausing; this observation stimulated us to use single-round transcription of
pyr
genes to test for formation of paused transcripts in vitro. Using templates with each of the three known
B. subtilis pyr
attenuation sites, we identified one major pause site in each in which the half-life of the paused transcript was increased four- to sixfold by NusA. In each case pausing at the NusA-stimulated site prevented formation of a complete antiterminator stem-loop, while it resulted in increased time for a PyrR binding loop to form and for PyrR to bind to this loop. Thus, the pausing detected in vitro is potentially capable of playing a role in establishing the correct timing for
pyr
attenuation in vivo. With two of three
pyr
templates the combination of NusA with PyrR markedly stimulated termination of transcription at the normal termination sites. This suggests that NusA, by stabilizing pausing, plays a role in termination of
pyr
transcription in vivo.
Dependence of
Helicobacter pylori
Urease Activity on the Nickel-Sequestering Ability of the UreE Accessory Protein
Authors: Benoit Stéphane , Maier Robert J.
Published: August 2003

The
Helicobacter pylori ureE
gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in
Escherichia coli
, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of
ureE
into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in
E. coli
and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type
H. pylori
and in
hypA
and
hypB
mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the
hypA
and
hypB
mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between
H. pylori
urease activity and the nickel-sequestering ability of the UreE accessory protein.
Regulation of Sialic Acid Catabolism by the DNA Binding Protein NanR in
Escherichia coli
Authors: Kalivoda Kathryn A. , Steenbergen Susan M. , Vimr Eric R. , Plumbridge Jacqueline
Published: August 2003

All
Escherichia coli
strains so far examined possess a chromosomally encoded
nanATEK-yhcH
operon for the catabolism of sialic acids. These unique nine-carbon sugars are synthesized primarily by higher eukaryotes and can be used as carbon, nitrogen, and energy sources by a variety of microbial pathogens or commensals. The gene
nanR
, located immediately upstream of the operon, encodes a protein of the FadR/GntR family that represses
nan
expression in
trans
. S1 analysis identified the
nan
transcriptional start, and DNA footprint analysis showed that NanR binds to a region of ∼30 bp covering the promoter region. Native (nondenaturing) polyacrylamide gel electrophoresis, mass spectrometry, and chemical cross-linking indicated that NanR forms homodimers in solution. The region protected by NanR contains three tandem repeats of the hexameric sequence GGTATA. Gel shift analysis with purified hexahistidine-tagged or native NanR detected three retarded complexes, suggesting that NanR binds sequentially to the three repeats. Artificial operators carrying different numbers of repeats formed the corresponding number of complexes. Among the sugars tested that were predicted to be products of the
nan
-encoded system, only the exogenous addition of sialic acid resulted in the dramatic induction of a chromosomal
nanA
-
lacZ
fusion or displaced NanR from its operator in vitro. Titration of NanR by the
nan
promoter region or artificial operators carrying different numbers of the GGTATA repeat on plasmids in this fusion strain supported the binding of the regulator to target DNA in vivo. Together, the results indicate that GGTATA is important for NanR binding, but the precise mechanism remains to be determined.
Mannitol-1-Phosphate Dehydrogenase (MtlD) Is Required for Mannitol and Glucitol Assimilation in
Bacillus subtilis
: Possible Cooperation of
mtl
and
gut
Operons
Authors: Watanabe Shouji , Hamano Miyuki , Kakeshita Hiroshi , Bunai Keigo , Tojo Shigeo , Yamaguchi Hirotake , Fujita Yasutaro , Wong Sui-Lam , Yamane Kunio
Published: August 2003

We found that mannitol-1-phosphate dehydrogenase (MtlD), a component of the mannitol-specific phosphotransferase system, is required for glucitol assimilation in addition to GutR, GutB, and GutP in
Bacillus subtilis
. Northern hybridization of total RNA and microarray studies of RNA from cells cultured on glucose, mannitol, and glucitol indicated that mannitol as the sole carbon source induced hyperexpression of the
mtl
operon, whereas glucitol induced both
mtl
and
gut
operons. The
B. subtilis mtl
operon consists of
mtlA
(encoding enzyme IICBA
mt1
) and
mtlD
, and its transcriptional regulator gene,
mtlR
, is located 14.4 kb downstream from the
mtl
operon on the chromosome. The
mtlA
,
mtlD
, and
mtlR
mutants disrupted by the introduction of the pMUTin derivatives MTLAd, MTLDd, and MTLRd, respectively, could not grow normally on either mannitol or glucitol. However, the growth of MTLAd on glucitol was enhanced by IPTG (isopropyl-β-
d
-thiogalactopyranoside). This mutant has an IPTG-inducible promoter (P
spac
promoter) located in
mtlA
, and this site corresponds to the upstream region of
mtlD
. Insertion mutants of
mtlD
harboring the chloramphenicol resistance gene also could not grow on either mannitol or glucitol. In contrast, an insertion mutant of
mtlA
could grow on glucitol but not on mannitol in the presence or absence of IPTG. MtlR bound to the promoter region of the
mtl
operon but not to a DNA fragment containing the
gut
promoter region.
The Virulence Activator AphA Links Quorum Sensing to Pathogenesis and Physiology in
Vibrio cholerae
by Repressing the Expression of a Penicillin Amidase Gene on the Small Chromosome
Authors: Kovacikova Gabriela , Lin Wei , Skorupski Karen
Published: August 2003

Activation of the
tcpPH
promoter on the
Vibrio
pathogenicity island by AphA and AphB initiates the
Vibrio cholerae
virulence cascade and is regulated by quorum sensing through the repressive action of HapR on
aphA
expression. To further understand how the chromosomally encoded AphA protein activates
tcpPH
expression, site-directed mutagenesis was used to identify the base pairs critical for AphA binding and transcriptional activation. This analysis revealed a region of partial dyad symmetry, TATGCA-N6-TNCNNA, that is important for both of these activities. Searching the
V. cholerae
genome for this binding site permitted the identification of a second one upstream of a penicillin V amidase (PVA) gene on the small chromosome. AphA binds to and footprints this site, which overlaps the
pva
transcriptional start, consistent with its role as a repressor at this promoter. Since
aphA
expression is under quorum-sensing control, the response regulators LuxO and HapR also influence
pva
expression. Thus,
pva
is repressed at low cell density when AphA levels are high, and it is derepressed at high cell density when AphA levels are reduced. Penicillin amidases are thought to function as scavengers for phenylacetylated compounds in the nonparasitic environment. That AphA oppositely regulates the expression of
pva
from that of virulence, together with the observation that PVA does not play a role in virulence, suggests that these activities are coordinated to serve
V. cholerae
in different biological niches.
Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis
Authors: Grimek T. L. , Holden H. , Rayment I. , Escalante-Semerena J. C.
Published: August 2003

TheprpBgene ofSalmonella entericaserovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg2+ions; equimolar concentrations of Mn2+ions were a poor substitute for Mg2+(28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. TheKmof PrpB for 2-MIC was measured at 19 μM, with akcatof 105 s−1. Mutations in theprpBgene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpBK121A, PrpBH125A, and PrpBR122Kmutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases inkcatfor 2-MIC lyase activity, respectively. The PrpBD58Aand PrpBC123Aproteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg2+ion.
Allosteric Regulation of
Bacillus subtilis
NAD Kinase by Quinolinic Acid
Authors: Garavaglia Silvia , Galizzi Alessandro , Rizzi Menico
Published: August 2003

NADP is essential for biosynthetic pathways, energy, and signal transduction. In living organisms, NADP biosynthesis proceeds through the phosphorylation of NAD with a reaction catalyzed by NAD kinase. We expressed, purified, and characterized
Bacillus subtilis
NAD kinase. This enzyme represents a new member of the inorganic polyphosphate [poly(P)]/ATP NAD kinase subfamily, as it can use poly(P), ATP, or other nucleoside triphosphates as phosphoryl donors. NAD kinase showed marked positive cooperativity for the substrates ATP and poly(P) and was inhibited by its product, NADP, suggesting that the enzyme plays a major regulatory role in NADP biosynthesis. We discovered that quinolinic acid, a central metabolite in NAD(P) biosynthesis, behaved like a strong allosteric activator for the enzyme. Therefore, we propose that NAD kinase is a key enzyme for both NADP metabolism and quinolinic acid metabolism.
Autoinducer 2 Production byStreptococcusgordoniiDL1 and the Biofilm Phenotype of aluxSMutant Are Influenced by Nutritional Conditions
Authors: Blehert David S. , Palmer Robert J. , Xavier Joao B. , Almeida Jonas S. , Kolenbrander Paul E.
Published: August 2003

TheluxSgene, present in many bacterial genera, encodes the autoinducer 2 (AI-2) synthase. AI-2 has been implicated in bacterial signaling, and this study investigated its role in biofilm formation byStreptococcusgordonii, an organism that colonizes human tooth enamel within the first few hours after professional cleaning. Northern blotting and primer extension analyses revealed thatS.gordoniiluxSis monocistronic. AI-2 production was dependent on nutritional conditions, and maximum AI-2 induction was detected whenS.gordoniiwas grown in the presence of serum and carbonate. In planktonic cultures, AI-2 production rose sharply during the transition from exponential to stationary phase, and the AI-2 concentration peaked approximately 4 h into stationary phase. AnS.gordoniiluxSmutant that did not produce AI-2 was constructed by homologous recombination. Complementation of the mutant by insertion of an intactluxSgene into the chromosome in tandem with the disrupted gene restored AI-2 production to a level similar to that of the wild-type strain. In planktonic culture, no growth differences were observed between the mutant and wild-type strains when five different media were used. However, when grown for 4 h as biofilms in 25% human saliva under flow, theluxSmutant formed tall microcolonies that differed from those formed by the wild-type and complemented mutant strains. Biofilms of theluxSmutant exhibited finger-like projections of cells that extended into the flow cell lumen. Thus, the inability to produce AI-2 is associated with altered microcolony architecture withinS.gordoniibiofilms formed in saliva during a time frame consistent with initial colonization of freshly cleaned enamel surfaces.
Probing Conservation of HAMP Linker Structure and Signal Transduction Mechanism through Analysis of Hybrid Sensor Kinases
Authors: Appleman J. Alex , Chen Li-Ling , Stewart Valley
Published: August 2003

The HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules. HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic α-helices (AS-1 and AS-2) joined by an unstructured connector. HAMP linkers are found in theEscherichia colinitrate- and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity). We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA. A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA-1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxA-NarX-1) responded to nitrate by increasing kinase activity. However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components. Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX. We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound.
A Novel IS Element, IS
621
, of the IS
110
/IS
492
Family Transposes to a Specific Site in Repetitive Extragenic Palindromic Sequences in
Escherichia coli
Authors: Choi Sunju , Ohta Shinya , Ohtsubo Eiichi
Published: August 2003

An
Escherichia coli
strain, ECOR28, was found to have insertions of an identical sequence (1,279 bp in length) at 10 loci in its genome. This insertion sequence (named IS
621
) has one large open reading frame encoding a putative protein that is 326 amino acids in length. A computer-aided homology search using the DNA sequence as the query revealed that IS
621
was homologous to the
piv
genes, encoding pilin gene invertase (PIV). A homology search using the amino acid sequence of the putative protein encoded by IS
621
as the query revealed that the protein also has partial homology to transposases encoded by the IS
110
/IS
492
family elements, which were known to have partial homology to PIV. This indicates that IS
621
belongs to the IS
110
/IS
492
family but is most closely related to the
piv
genes. In fact, a phylogenetic tree constructed on the basis of amino acid sequences of PIV proteins and transposases revealed that IS
621
belongs to the
piv
gene group, which is distinct from the IS
110
/IS
492
family elements, which form several groups. PIV proteins and transposases encoded by the IS
110
/IS
492
family elements, including IS
621
, have four acidic amino acid residues, which are conserved at positions in their N-terminal regions. These residues may constitute a tetrad D-E(or D)-D-D motif as the catalytic center. Interestingly, IS
621
was inserted at specific sites within repetitive extragenic palindromic (REP) sequences at 10 loci in the ECOR28 genome. IS
621
may not recognize the entire REP sequence in transposition, but it recognizes a 15-bp sequence conserved in the REP sequences around the target site. There are several elements belonging to the IS
110
/IS
492
family that also transpose to specific sites in the repeated sequences, as does IS
621
. IS
621
does not have terminal inverted repeats like most of the IS
110
/IS
492
family elements. The terminal sequences of IS
621
have homology with the 26-bp inverted repeat sequences of pilin gene inversion sites that are recognized and used for inversion of pilin genes by PIV. This suggests that IS
621
initiates transposition through recognition of their terminal regions and cleavage at the ends by a mechanism similar to that used for PIV to promote inversion at the pilin gene inversion sites.
Benzoate-Coenzyme A Ligase from
Thauera aromatica
: an Enzyme Acting in Anaerobic and Aerobic Pathways
Authors: Schühle Karola , Gescher Johannes , Feil Ulrich , Paul Michael , Jahn Martina , Schägger Hermann , Fuchs Georg
Published: August 2003

In the denitrifying member of the β-
Proteobacteria
Thauera aromatica
, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on
V
max
/

K
m

, the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.
Haemophilus influenzae
Rd Lacks a Stringently Conserved Fatty Acid Biosynthetic Enzyme and Thermal Control of Membrane Lipid Composition
Authors: Wang Haihong , Cronan John E.
Published: August 2003

The organization of the fatty acid synthetic genes of
Haemophilus influenzae
Rd is remarkably similar to that of the paradigm organism,
Escherichia coli
K-12, except that no homologue of the
E. coli fabF
gene is present. This finding is unexpected, since
fabF
is very widely distributed among bacteria and is thought to be the generic 3-ketoacyl-acyl carrier protein (ACP) synthase active on long-chain-length substrates. However,
H. influenzae
Rd contains a homologue of the
E. coli fabB
gene, which encodes a 3-ketoacyl-ACP synthase required for unsaturated fatty acid synthesis, and it seemed possible that the
H. influenzae
FabB homologue might have acquired the functions of FabF.
E. coli
mutants lacking
fabF
function are unable to regulate the compositions of membrane phospholipids in response to growth temperature. We report in vivo evidence that the enzyme encoded by the
H. influenzae fabB
gene has properties essentially identical to those of
E. coli
FabB and lacks FabF activity. Therefore,
H. influenzae
grows without FabF function. Moreover, as predicted from studies of the
E. coli fabF
mutants,
H. influenzae
is unable to change the fatty acid compositions of its membrane phospholipids with growth temperature. We also demonstrate that the
fabB
gene of
Vibrio cholerae
El Tor N16961 does not contain a frameshift mutation as was previously reported.
Mapping the MinE Site Involved in Interaction with the MinD Division Site Selection Protein of
Escherichia coli
Authors: Ma Lu-Yan , King Glenn , Rothfield Lawrence
Published: August 2003

Interactions between the MinD and MinE proteins are required for proper placement of the
Escherichia coli
division septum. The site within MinE that is required for interaction with MinD was mapped by studying the effects of site-directed
minE
mutations on MinD-MinE interactions in yeast two-hybrid and three-hybrid experiments. This confirmed that the MinE N-terminal domain is responsible for the interaction of MinE with MinD. Mutations that interfered with the interaction defined an extended surface on one face of the α-helical region of the MinE N-terminal domain, consistent with the idea that the MinE-MinD interaction involves formation of a coiled-coil structure by interaction with a complementary helical surface within MinD.
Phenotype MicroArray Analysis of
Escherichia coli
K-12 Mutants with Deletions of All Two-Component Systems
Authors: Zhou Lu , Lei Xiang-He , Bochner Barry R. , Wanner Barry L.
Published: August 2003

Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in
Escherichia coli
K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including
creBC
-regulated genes (
cbrA
and
cbrBC
),
phoBR
-regulated genes (
phoA
,
phoH
,
phnCDEFGHIJKLMNOP
,
psiE
, and
ugpBAECQ
),
csgD
,
luxS
, and
rpoS
. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed.
Genomic Profiling of Iron-Responsive Genes in
Salmonella enterica
Serovar Typhimurium by High-Throughput Screening of a Random Promoter Library
Authors: Bjarnason Jaime , Southward Carolyn M. , Surette Michael G.
Published: August 2003

The importance of iron to bacteria is shown by the presence of numerous iron-scavenging and transport systems and by many genes whose expression is tightly regulated by iron availability. We have taken a global approach to gene expression analysis of
Salmonella enterica
serovar Typhimurium in response to iron by combining efficient, high-throughput methods with sensitive, luminescent reporting of gene expression using a random promoter library. Real-time expression profiles of the library were generated under low- and high-iron conditions to identify iron-regulated promoters, including a number of previously identified genes. Our results indicate that approximately 7% of the genome may be regulated directly or indirectly by iron. Further analysis of these clones using a Fur titration assay revealed three separate classes of genes; two of these classes consist of Fur-regulated genes. A third class was Fur independent and included both negatively and positively iron-responsive genes. These may reflect new iron-dependent regulons. Iron-responsive genes included iron transporters, iron storage and mobility proteins, iron-containing proteins (redox proteins, oxidoreductases, and cytochromes), transcriptional regulators, and the energy transducer
tonB
. By identifying a wide variety of iron-responsive genes, we extend our understanding of the global effect of iron availability on gene expression in the bacterial cell.
Identification of a Regulated Alkaline Phosphatase, a Cell Surface-Associated Lipoprotein, inMycobacterium smegmatis
Authors: Kriakov Jordan , Lee Sun hee , Jacobs William R.
Published: August 2003

Although alkaline phosphatases are common in a wide variety of bacteria, there has been no prior evidence for alkaline phosphatases inMycobacterium smegmatis. Here we report that transposon insertions in thepstoperon, encoding homologues of an inorganic phosphate transporter, leads to constitutive expression of a protein with alkaline phosphatase activity. DNA sequence analysis revealed thatM. smegmatisdoes indeed have aphoAgene that shows high homology to otherphoAgenes. TheM. smegmatis phoAgene was shown to be induced by phosphate starvation and thus negatively regulated by thepstoperon. Interestingly, the putativeM. smegmatisPhoA has a hydrophobic N-terminal domain which resembles a lipoprotein signal sequence. TheM. smegmatisPhoA was demonstrated to be an exported protein associated with the cell surface. Furthermore, immunoprecipitation of PhoA from [14C]acetate-labeledM. smegmatiscell lysates demonstrated that this phosphatase is a lipoprotein.
Identification of Long Intergenic Repeat Sequences Associated with DNA Methylation Sites in
Caulobacter crescentus
and Other α-Proteobacteria
Authors: Chen Swaine L. , Shapiro Lucy
Published: August 2003

A systematic search for motifs associated with CcrM DNA methylation sites revealed four long (>100-bp) motifs (CIR sequences) present in up to 21 copies in
Caulobacter crescentus
. The CIR1 and CIR2 motifs exhibit a conserved inverted repeat organization, with a CcrM site in the center of one of the repeats.
Mutational Analysis of Cell Wall Biosynthesis inMycobacterium avium
Authors: Laurent Jean-Pierre , Hauge Kirsten , Burnside Kellie , Cangelosi Gerard
Published: August 2003

The cell wall of the environmental pathogenMycobacterium aviumis important to its virulence and intrinsic antimicrobial resistance. To identify genes involved in cell wall biosynthesis, “transposome” insertion libraries were screened for mutants with altered colony morphology on medium containing the lipoprotein stain Congo red. Nineteen such mutants were isolated and mapped, including 10 with insertions in a functional island of cell wall biosynthetic genes that spans approximately 40 kb of theM.aviumgenome.
The Product of the
fimI
Gene Is Necessary for
Escherichia coli
Type 1 Pilus Biosynthesis
Authors: Valenski Mary L. , Harris Sandra L. , Spears Patricia A. , Horton John R. , Orndorff Paul E.
Published: August 2003

Site-directed mutagenesis was employed to create lesions in
fimI
, a gene of uncertain function located in the chromosomal gene cluster (
fim
) involved in
Escherichia coli
type 1 pilus biosynthesis. Chromosomal
fimI
mutations produced a piliation-negative phenotype. Complementation analysis indicated that a
fimI
′
-kan
insertion mutation and a
fimI
frameshift mutation produced polarity-like effects not seen with an in-frame
fimI
deletion mutation. Minicell analysis associated
fimI
with a 16.4-kDa noncytoplasmic protein product (FimI). We conclude that FimI has a required role in normal pilus biosynthesis.
Regulated Expression of the
Escherichia coli dam
Gene
Authors: Calmann Melissa A. , Marinus M. G.
Published: August 2003

Regulated expression of the
Escherichia coli dam
gene has been achieved with the
araBAD
promoter lacking a ribosome binding site. Cultures of
dam
mutants containing plasmid pMQ430 show no detectable methylation in the absence of arabinose and complete methylation in its presence. Dam methyltransferase is a substrate for the Lon protease.
Escherichia coli
Cells Bearing a Ribosomal Ambiguity Mutation in
rpsD
Have a Mutator Phenotype That Correlates with Increased Mistranslation
Authors: Balashov Sergey , Humayun M. Zafri
Published: August 2003

Escherichia coli
cells bearing certain mutations in
rpsD
(coding for the 30S ribosomal protein S4) show a ribosomal ambiguity (Ram) phenotype characterized by increased translational error rates. Here we show that spontaneous mutagenesis increases in Ram cells bearing the
rpsD14
allele, suggesting that the recently described translational stress-induced mutagenesis pathway is activated in Ram cells.
Osteoarthritis
Authors: Haq I , Murphy E , Dacre J
Published: August 2003

Osteoarthritis is a chronic degenerative disorder characterised by cartilage loss. It is extremely prevalent in society and is a major cause of disability. It is important to treat osteoarthritis effectively using a multidisciplinary approach tailored to the patient’s needs. This paper reviews current thinking on the aetiology, pathogenesis, investigations, and management of osteoarthritis. The paper also discusses the challenges for developing good quality outcome measures for use in large scale multicentre clinical trials for new osteoarthritis treatments, especially disease modifying osteoarthritis drugs.
Pulmonary manifestations of sickle cell disease
Authors: Siddiqui A K , Ahmed S
Published: August 2003

Pulmonary complications account for significant morbidity and mortality in patients with sickle cell disease. Clinical lung involvement manifests in two major forms: the acute chest syndrome and sickle cell chronic lung disease. Acute chest syndrome is characterised by fever, chest pain, and appearance of a new infiltrate on chest radiograph. Sickle cell chronic lung disease, on the other hand, manifests as radiographic interstitial abnormalities, impaired pulmonary function, and, in its most severe form, by the evidence of pulmonary hypertension. Progress has been made in understanding the pathophysiology and management of these complications. In this review the current knowledge of the mechanism, diagnosis, and treatment of pulmonary complications of sickle cell disease are discussed.
Autonomic neuropathy in patients with hepatic cirrhosis
Authors: Bajaj B K , Agarwal M P , Ram B Krishna
Published: August 2003

Background

Autonomic neuropathy has been reported in patients with alcoholic liver disease but information on its occurrence in patients with non-alcoholic liver disease is contradictory.

Aim

To assess autonomic functions in patients with alcoholic and non-alcoholic liver disease.

Study design

Autonomic function using five standard tests was examined in 20 cirrhotics (10 alcoholics and 10 non-alcoholics) and 20 age and sex matched controls. The extent of autonomic dysfunction was determined in the patients and a comparison between the characteristics of patients with and without autonomic neuropathy was made.

Results

Sixteen (80%) of the cirrhotic subjects were found to have evidence of autonomic neuropathy. Of these, three (15%) patients had early parasympathetic damage, five (25%) had definite parasympathetic damage, and eight (40%) had combined (that is, both parasympathetic and sympathetic) damage. Nine (90%) of the alcoholics and seven (70%) of the non-alcoholics had autonomic dysfunction. Only one patient belonging to the alcoholic group had clinical evidence of peripheral neuropathy. Moreover, there was no significant association between subjective symptoms of autonomic neuropathy and objective evidence of autonomic damage as assessed by autonomic function tests. Autonomic dysfunction was significantly more frequent in advanced liver disease compared with early liver damage. Nine (75%) out of 12 cirrhotic subjects belonging to Child class B and six (85.7%) of the seven patients belonging to Child class C had autonomic neuropathy.

Conclusion

This study shows that autonomic neuropathy is common in cirrhotic subjects, that it is found with comparable frequency in alcoholics and non-alcoholics, and that it increases in severity with increase in extent of liver damage, suggesting that liver damage contributes to the neurological deficit.
Metabolic alkalosis and myoclonus
Authors: Simons P , Nadra I , McNally P G
Published: August 2003

This is the first case reported of vomiting-induced metabolic alkalosis associated with myoclonus. The report describes an unusual presentation of myoclonus secondary to acid-base disturbance caused by recreational drug-induced vomiting. The severe derangement of hyponatraemia, hypokalaemia, and alkalosis appears to have been reasonably well tolerated due to the gradual onset and relatively long history. The causes, mechanism, and management of metabolic alkalosis are discussed.
The Medium-Frequency Range in Computational Acoustics: Practical and Numerical Aspects
Authors: Ihlenburg Frank
Published: August 2003

The upper frequency limit of vibroacoustic calculations with finite element methods is usually concluded from resolution rules for the minimal wavelength encountered in the problem. Here, we derive general resolution rules that account for the pollution effect in finite element solutions of time-harmonic equations. These rules are given for the Helmholtz equation and the Bernoulli beam equation. The latter are based on an analysis of numerical dispersion for finite difference solutions. The theoretical results are given in the broader context of industrial vibroacoustic computations in the medium-frequency range. The governing equations of deterministic vibroacoustic computations and statistical energy analysis are reviewed with the goal to indicate, respectively, upper and lower frequency bounds for the applicability of either model. From the discussion of priorities in industrial application, open questions for theoretical investigations are deduced.
Using Plane Waves as Base Functions for Solving Time Harmonic Equations with the Ultra Weak Variational Formulation
Authors: Cessenat Olivier , Després Bruno
Published: August 2003

This article deals with the use of the Ultra Weak Variational Formulation to solve Helmholtz equation and time harmonic Maxwell equations.

The method, issued from domain decomposition techniques, lies in partitioning the domain into subdomains with the use of adapted interface conditions. Going further than in domain decomposition, we make so that the problem degenerates into an interface problem only.

The new formulation is equivalent to the weak formulation. The discretization process is a Galerkin one. A possible advantage of the UWVF applied to wave equations is that we use the physical approach that consists in approximating the solution with plane waves. The formulation allows to use a very large mesh as compared to the frequency, on the contrary to the Finite Element Method when applied to time harmonic equations. Furthermore, the convergence analysis shows the method is a high order one: the order evolves as the square root of the number of degrees of freedom.
Use of Wave Boundary Elements for Acoustic Computations
Authors: Perrey-Debain Emmanuel , Trevelyan Jon , Bettess Peter
Published: August 2003

Discrete methods of numerical analysis have been used successfully for decades for the solution of problems involving wave diffraction, etc. However, these methods, including the finite element and boundary element methods, can require a prohibitively large number of elements as the wavelength becomes progressively shorter. In this work, a new type of interpolation for the acoustic field is described in which the usual conventional shape functions are modified by the inclusion of a set of plane waves propagating in multiple directions. Including such a plane wave basis in a boundary element formulation has been found in the current work to be highly successful. Results are shown for a variety of classical scattering problems, and also for scattering from nonconvex obstacles. Notable results include a conclusion that, using this new formulation, only approximately 2.5 degrees of freedom per wavelength are required. Compared with the 8 to 10 degrees of freedom normally required for conventional boundary (and finite) elements, this shows the marked improvement in storage requirement. Moreover, the new formulation is shown to be extremely accurate. It is estimated that for 2D Helmholtz problems, and for a given computational resource, the frequency range allowed by this method is extended by a factor of three over conventional direct collocation Boundary Element Method. Recent successful developments of the current method for plane elastodynamics problems are also briefly outlined.
Analysis of Medium-Frequency Vibrations in a Frequency Range
Authors: Ladevèze P. , Rouch P. , Riou H. , Bohineust X.
Published: August 2003

A new approach called the ''Variational Theory of Complex Rays'' (VTCR) is being developed in order to calculate the vibrations of slightly damped elastic structures in the medium-frequency range. Here, the emphasis is put on the extension of this theory to analysis across a range of frequencies. Numerical examples show the capability of the VTCR to predict the vibrational response of a structure in a frequency range.
Preparation of a Nickel-Nickel Oxide Composite by Hot Isostatic Pressing and Its Application for Anodes Used in Electrolytic Production of Nitrogen Trifluoride
Authors: Tasaka Akimasa , Suzuki Yasuhiro , Oshida Atsushi , Mimoto Atsuhisa , Hieda Taro , Tachikawa Toshiyasu , Takao Kazuchika , Takemura Hideaki , Yamaguchi Osamu
Published: August 2003

The nickel-nickel oxide

[
Ni
­

NiO

1
+
x

(
0
≦
x
<
0.5
)
]

composites prepared from a mixture of Ni and

LiNiO

2

or NiO powders at 900°C under 2000 atm for 2 h by hot isostatic pressing were employed as the anode for electrolytic production of

NF

3

.

In electrolysis of a molten

NH

4

F
⋅
2
HF

with and without LiF at 100°C and at 25 mA/cm2, the anode gas generated at the

Ni
­

NiO

1
+
x

composite anode was composed of

N

2

,

O

2

,

NF

3

,

N

2

F

2

,

N

2

F

4

,

and

N

2

O
,

and its composition was almost the same as that at the Ni sheet anode. The current efficiency for

NF

3

formation on the Ni-NiO composite anode from mixture of NiO and Ni powders was high compared with that on the

Ni
­

NiO

1
+
x

composite anode from the mixture of

LiNiO

2

and Ni powders. The best current efficiency for

NF

3

formation was ca. 53% on the Ni 5 mol % NiO composite anode, and it was almost the same as that of the Ni sheet anode. The addition of LiF in a molten

NH

4

F
⋅
2
HF

increased it, presumably because of deposition of

Li

2

NiF

6

on the anode. On the other hand, the anode consumption of the Ni-NiO composite was much smaller compared with that of the Ni sheet electrode. Also, the oxygen content in the oxidized layer formed on the Ni-NiO composite anode was high compared with that on the Ni sheet anode. The scanning electron microscope observation revealed that the surface of the Ni-NiO composite anode was covered with the compact film having some defects. From these results, it is concluded that the Ni-NiO composite anode is favorable for electrolytic production of

NF

3

,

and that the oxidized layer on the anode has a high resistance to corrosion, because of the compact film containing a higher content of oxygen formed on the anode. © 2003 The Electrochemical Society. All rights reserved.
Suicide in ethnic minority groups
Authors: McKenzie Kwame , Serfaty Marc , Crawford Michael
Published: August 2003

Suicide is a complex phenomenon with numerous influences, including the individual's personality, biology, culture and social environment, as well as the macro-economic and political context.
Evidence-based mental health policy: Acritical appraisal
Authors: Cooper Brian
Published: August 2003

Background

Arguments for and against evidence-based psychiatry have mostly centred on its value for clinical practice and teaching. Now, however, use of the same paradigm in evaluating health care has generated new problems.

Aims

To outline the development of evidence-based health care; to summarise the main critiques of this approach; to review the evidence now beingemployed to evaluate mental health care; and to consider how the evidence base might be improved.

Method

The following sources were monitored: pub ications on evidence-based psychiatry and health care since 1990; reports of randomised trials and meta-analytic reviews to the end of 2002; and official British publications on mental health policy.

Results

Although evidence-based health care is now being promulgated as a rational basis for mental health planning in Britain, its contributions to service evaluation have been distinctly modest. Only 10% of clinical trials and meta-analyses have been focused on effectiveness of services, and many reviews proved inconclusive.

Conclusions

The current evidence-based approach is overly reliant on meta-analytic reviews, and is more applicable to specific treatments than to the care agencies that control theirdelivery. A much broader evidence base is called for, extending to studies in primary health care and the evaluation of preventive techniques.
Depression status, medical comorbidity and resource costs
Authors: Chisholm Daniel , Diehr Paula , Knapp Martin , Patrick Donald , Treglia Michael , Simon Gregory
Published: August 2003

Background

Despite the burden of depression, there remain few data on its economic consequences in an international context.

Aims

To explore the relationship between depression status (with and without medical comorbidity), work loss and health care costs, using cross-sectional data from a multi-national study of depression in primarycare.

Method

Primarycare attendees were screened for depression. Those meeting eligibility criteria were categorised according to DSM – IV criteria for major depressive disorder and comorbid status. Unit costs were attached to self-reported days absent from work and uptake of health care services.

Results

Medical comorbidity was associated with a 17–46% increase in health care costs in five of the six sites, but a clear positive association between costs and clinical depression status was identified in only one site.

Conclusions

The economic consequences of depression are influenced to a greater (and considerable) extent by the presence of medical comorbidity than by symptom severity alone.
Clozapine v. conventional antipsychotic drugs for treatment-resistant schizophrenia: A re-examination
Authors: Moncrieff Joanna
Published: August 2003

Background

Although there is a consensus that clozapine is more effective than conventional antipsychotic drugs for treatment-resistant schizophrenia, there is great heterogeneity among results of relevant trials.

Aims

To re-evaluate the evidence comparing clozapine with conventional antipsychotics and to investigate sources of heterogeneity.

Method

Individual studies were inspected with assessment of clinical relevance of results. Meta-regression analysis was performed to investigate sources of heterogeneity.

Results

Ten trials were examined. Recent large-scale studies have not found a substantial advantage for clozapine, especially in terms of a clinically relevant effect. Meta-regression showed that shorter study duration, financial support from a drug company and higher baseline symptom score consistently predicted greater advantage of clozapine.

Conclusions

It may be inappropriate to combine studies in meta-analysis, given the degree of heterogeneity between their findings. The benefits of clozapine compared with conventional treatment may not be substantial.
Consent in psychiatry – an ethical review
Authors: Lepping Peter
Published: August 2003

The aim of this article is to give a historical overview of the concept of consent to treatment and its development – one of the most important ethical and legal issues in recent years. Through the discussion of a number of high-profile examples, it deliberates the ethical and legal aspects of consent to treatment today and their implications for psychiatrists' clinical work.
Commentary (on: Calton & Arcelus, Adolescent units: a need for change?)
Authors: Gowers Simon G.
Published: August 2003

It is often said that you can't hold back progress. For those who work with troubled teenagers, much therapeutic time is taken up helping them cope with the daunting developmental challenges of adolescence. A 14-year-old boy with anorexia nervosa told me recently that life felt like an escalator that was taking him inexorably upwards and onwards. ‘On an escalator, you can't usually see what's beyond the top, as it's out of sight,’ he told me, adding: ‘At least on an escalator there's a big red button you can press in an emergency and stop it – and then someone will come and help you.’ Not though in life, and evidently not in the NHS!
Adolescent units: a need for change?
Authors: Calton Tim , Arcelus Jon
Published: August 2003

Aims and Method

To describe the characteristics and diagnoses of patients admitted to a general adolescent psychiatric in-patient unit. We describe the age, gender and psychiatric diagnosis of the patient, as well as whether the patient exhibited violent behaviour in the ward, whether he/she needed to be transferred to a different service and whether he/she was admitted under a section of the Mental Health Act 1983.

Results

Patients were evenly distributed in terms of gender, with most being 14–16 years old. Diagnoses were varied with adjustment disorder predominating, but could be separated into four main groups. Levels of violence were high, being associated with detention under the Mental Health Act 1983, and often resulted in transfer to another service.

Clinical Implications

The needs of certain adolescents admitted to a general-purpose adolescent unit may not be best met in this environment. Current services must change to meet the needs of their patients. There may be a need for greater specialisation.