bluuebunny/crossref_metadata_2025 huggingface dataset:
The “Gradient Effect” in Federal Laboratory‐Industry Technology Transfer Partnerships
Authors: Saavedra Pablo , Bozeman Barry
Published: April 2004

Based on data from 229 industry‐federal laboratory projects, our research analyzes combinations of technical roles and laboratory characteristics to determine their relation to projects’ effectiveness. The results show increased effectiveness when the combination of technical roles follows a “gradient.” That is, performance is enhanced when the company plays a different technical role than the laboratory, but a role that is only a step away on the basic‐applied‐development spectrum. Effectiveness diminishes when both the company and the federal laboratory play the same technical role or when the technical roles are far apart on the basic‐applied‐development spectrum. The results may have general implications for public policy partnerships, suggesting that although partnership effectiveness requires distinctive roles, partner roles should not be so different as to undermine possibilities for coordination and integration.
An Empirical Assessment of Devolution's Policy Impact
Authors: Kelleher Christine A. , Yackee Susan Webb
Published: April 2004

This article evaluates the influence of devolution on perceived and actual policy outcomes at the local level in the United States. Proponents of devolution often argue that the transfer of policymaking authority to a lower level of government allows subnational officials to more effectively address problems on the government's agenda. We provide one of the first empirical evaluations of this implicit assumption of devolution. We utilize data from 100 counties in one state, North Carolina, that were the recipients of additional welfare policymaking authority in 1997 to provide a preliminary assessment of devolution's consequences and policy impact. We then investigate the recipient governments’ ability to bring about positive change using three prominent indicators associated with welfare policy. Our findings provide evidence that devolution matters quite significantly to the perception of policymaking effectiveness; however, devolution's actual impact on policy outcomes is mixed.
Enzymatic Characterization of a Prokaryotic Urea Carboxylase
Authors: Kanamori Takeshi , Kanou Norihisa , Atomi Haruyuki , Imanaka Tadayuki
Published: April 2004

We identified the first prokaryotic urea carboxylase (UCA) from a member of the alpha subclass of the class
Proteobacteria
,
Oleomonas sagaranensis
. This enzyme (
O. sagaranensis
Uca) was composed of 1,171 amino acids, and its N-terminal region resembled the biotin carboxylase domains of various biotin-dependent carboxylases. The C-terminal region of the enzyme harbored the Met-Lys-Met motif found in biotin carboxyl carrier proteins. The primary structure of the enzyme was 45% identical to that of the urea carboxylase domain of urea amidolyase from
Saccharomyces cerevisiae. O. sagaranensis
Uca did not harbor the allophanate hydrolase domain found in the yeast enzyme, but a separate gene with structural similarity was found to be adjacent to the
uca
gene. Purified recombinant
O. sagaranensis
Uca displayed ATP-dependent carboxylase activity towards urea (
V
max
= 21.2 μmol mg
−1
min
−1
) but not towards acetyl coenzyme A (acetyl-CoA) and propionyl-CoA, indicating that the gene encoded a bona fide UCA and not an acetyl-CoA or propionyl-CoA carboxylase. The enzyme also exhibited high levels of activity towards acetamide and formamide. Kinetic parameters of the enzyme reaction were determined with ATP, urea, acetamide, and formamide.
O. sagaranensis
could grow on urea, acetamide, and formamide as sole nitrogen sources; moreover, ATP-dependent urea-degrading activity was found in cells grown with urea but not in cells grown with ammonia. The results suggest that the UCA of this organism may be involved in the assimilation of these compounds as nitrogen sources. Furthermore, orthologues of the
O. sagaranensis uca
gene were found to be widely distributed among
Bacteria
. This implies that there are two systems of urea degradation in
Bacteria
, a pathway catalyzed by the previously described ureases and the UCA-allophanate hydrolase pathway identified in this study.
Characterization of the Alternative Sigma Factor σ
54
and the Transcriptional Regulator FleQ of
Legionella pneumophila
, Which Are Both Involved in the Regulation Cascade of Flagellar Gene Expression
Authors: Jacobi Sebastian , Schade Rüdiger , Heuner Klaus
Published: April 2004

We cloned and analyzed
Legionella pneumophila
Corby homologs of
rpoN
(encoding σ
54
) and
fleQ
(encoding σ
54
activator protein). Two other genes (
fleR
and
pilR
) whose products have a σ
54
interaction domain were identified in the genome sequence of
L. pneumophila
. An
rpoN
mutant strain was nonflagellated and expressed very small amounts of the FlaA (flagellin) protein. Like the
rpoN
mutant, the
fleQ
mutant strain of
L. pneumophila
was also nonflagellated and expressed only small amounts of FlaA protein compared to the amounts expressed by the wild type. In this paper we show that the σ
54
factor and the FleQ protein are involved in regulation of flagellar gene operons in
L. pneumophila.
RpoN and FleQ positively regulate the transcription of FliM and FleN, both of which have a σ
54
-dependent promoter consensus sequence. However, they seemed to be dispensable for transcription of
flaA
,
fliA
, or
icmR
. Our results confirmed a recently described model of the flagellar gene regulation cascade in
L. pneumophila
(K. Heuner and M. Steinert, Int. J. Med. Microbiol.
293:
133-145, 2003). Flagellar gene regulation was found to be different from that of
Enterobacteriaceae
but seems to be comparable to that described for
Pseudomonas
or
Vibrio
spp.
The
bchU
Gene of
Chlorobium tepidum
Encodes the C-20 Methyltransferase in Bacteriochlorophyll
c
Biosynthesis
Authors: Maresca Julia A. , Maqueo Chew Aline Gomez , Ros Ponsatí Marta , Frigaard Niels-Ulrik , Ormerod John G. , Bryant Donald A.
Published: April 2004

Bacteriochlorophylls (BChls)
c
and
d
, two of the major light-harvesting pigments in photosynthetic green sulfur bacteria, differ only by the presence of a methyl group at the C-20 methine bridge position in BChl
c
. A gene potentially encoding the C-20 methyltransferase,
bchU
, was identified by comparative analysis of the
Chlorobium tepidum
and
Chloroflexus aurantiacus
genome sequences. Homologs of this gene were amplified and sequenced from
Chlorobium phaeobacteroides
strain 1549,
Chlorobium vibrioforme
strain 8327
d
, and
C. vibrioforme
strain 8327
c
, which produce BChls
e
,
d
, and
c
, respectively. A single nucleotide insertion in the
bchU
gene of
C. vibrioforme
strain 8327
d
was found to cause a premature, in-frame stop codon and thus the formation of a truncated, nonfunctional gene product. The spontaneous mutant of this strain that produces BChl
c
(strain 8327
c
) has a second frameshift mutation that restores the correct reading frame in
bchU
. The
bchU
gene was inactivated in
C. tepidum
, a BChl
c
-producing species, and the resulting mutant produced only BChl
d
. Growth rate measurements showed that BChl
c
- and
d
-producing strains of the same organism (
C. tepidum
or
C. vibrioforme
) have similar growth rates at high and intermediate light intensities but that strains producing BChl
c
grow faster than those with BChl
d
at low light intensities. Thus, the
bchU
gene encodes the C-20 methyltransferase for BChl
c
biosynthesis in
Chlorobium
species, and methylation at the C-20 position to produce BChl
c
rather than BChl
d
confers a significant competitive advantage to green sulfur bacteria living at limiting red and near-infrared light intensities.
Restricted Mobility of Cell Surface Proteins in the Polar Regions of
Escherichia coli
Authors: de Pedro Miguel A. , Grünfelder Christoph G. , Schwarz Heinz
Published: April 2004

The polar regions of the
Escherichia coli
murein sacculus are metabolically inert and stable in time. Because the sacculus and the outer membrane are tightly associated, we investigated whether polar inert murein could restrict the mobility of other cell envelope elements. Cells were covalently labeled with a fluorescent reagent, chased in dye-free medium, and observed by microscopy. Fluorescent material was more efficiently retained at the cell poles than at any other location. The boundary between high and low fluorescence intensity areas was rather sharp. Labeled material consisted mostly of cell envelope proteins, among them the free and murein-bound forms of Braun's lipoprotein. Our results indicate that the mobility of at least some cell envelope proteins is restrained at regions in correspondence with underlying areas of inert murein.
Identification and Characterization of a Linear-Plasmid-Encoded Factor H-Binding Protein (FhbA) of the Relapsing Fever Spirochete
Borrelia hermsii
Authors: Hovis Kelley M. , McDowell John V. , Griffin LaToya , Marconi Richard T.
Published: April 2004

In North America, tick-borne relapsing fever (TBRF) is caused by the spirochete species
Borrelia hermsii
,
Borrelia parkeri
, and
Borrelia turicatae
. We previously demonstrated that some isolates of
B. hermsii
and
B. parkeri
are capable of binding factor H and that cell-bound factor H can participate in the factor I-mediated cleavage of C3b. Isolates that bound factor H expressed a factor H-binding protein (FHBP) that we estimated to be approximately 19 to 20 kDa in size and thus, pending further characterization, temporarily designated FHBP19. Until this report, none of the FHBPs of the TBRF spirochetes had been characterized. Here we have recovered the gene encoding the FHBP of
B. hermsii
YOR from a lambda ZAP II library and determined its sequence. The gene encodes a full-length protein of 22.7 kDa, which after processing is predicted to be 20.5 kDa. This protein, which we redesignate factor H-binding protein A (FhbA), is unique to
B. hermsii
. Two-dimensional pulsed-field gel electrophoresis and hybridization analyses revealed that the
B. hermsii
gene encoding FhbA is a single genetic locus that maps to a linear plasmid of approximately 220 kb. The general properties of FhbA were also assessed. The protein was found to be surface exposed and lipidated. Analysis of the antibody response to FhbA in infected mice revealed that it is antigenic during infection, indicating expression during infection. The identification and characterization of FhbA provides further insight into the molecular mechanisms of pathogenesis of the relapsing fever spirochetes.
Host Restriction of
Salmonella enterica
Serotype Typhimurium Pigeon Isolates Does Not Correlate with Loss of Discrete Genes
Authors: Andrews-Polymenis Helene L. , Rabsch Wolfgang , Porwollik Steffen , McClelland Michael , Rosetti Carlos , Adams L. Garry , Bäumler Andreas J.
Published: April 2004

The definitive phage types (DT) 2 and 99 of
Salmonella enterica
serotype Typhimurium are epidemiologically correlated with a host range restricted to pigeons, in contrast to phage types with broader host ranges such as epidemic cattle isolates (DT104 and DT204). To determine whether phage types with broad host range possess genetic islands absent from host-restricted phage types, we compared the genomes of four pigeon isolates to serotype Typhimurium strain LT2 using a DNA microarray. Three of the four isolates tested caused fluid accumulation in bovine ligated ileal loops, but they had reduced colonization of liver and spleen in susceptible BALB/c mice and were defective for intestinal persistence in
Salmonella
-resistant CBA mice. The genomes of the DT99 and DT2 isolates were extremely similar to the LT2 genome, with few notable differences on the level of complete individual genes. Two large groups of genes representing the Fels-1 and Fels-2 prophages were missing from the DT2 and DT99 phage types we analyzed. One of the DT99 isolates examined was lacking a third cluster of five chromosomal genes (STM1555 to -1559). Results of the microarray analysis were extended using Southern analysis to a collection of 75 serotype Typhimurium clinical isolates of 24 different phage types. This analysis revealed no correlation between the presence of Fels-1, Fels-2, or STM1555 to -1559 and the association of phage types with different host reservoirs. We conclude that serotype Typhimurium phage types with broad host range do not possess genetic islands influencing host restriction, which are absent from the host-restricted pigeon isolates.
Divergence and Redundancy of 16S rRNA Sequences in Genomes with Multiple
rrn
Operons
Authors: Acinas Silvia G. , Marcelino Luisa A. , Klepac-Ceraj Vanja , Polz Martin F.
Published: April 2004

The level of sequence heterogeneity among
rrn
operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related
rrn
operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of
rrn
copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but ∼40% of the organisms analyzed have either one or two operons. Among the
Archaea
, a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For
Archaea
, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single
rrn
operon may lower this correction factor to ∼2.5. Divergence among operons appears to be small overall for both
Bacteria
and
Archaea
, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and
Thermoanaerobacter tengcongensis
exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups.
Formation of SXT Tandem Arrays and SXT-R391 Hybrids
Authors: Burrus Vincent , Waldor Matthew K.
Published: April 2004

SXT is an integrative and conjugative element (ICE) isolated from
Vibrio cholerae
. This ∼100-kb ICE encodes resistance to multiple antibiotics and integrates site specifically into the chromosome. SXT excises from the chromosome to form a circular but nonreplicative extrachromosomal molecule that is required for its transfer. Here we found that a significant fraction of freshly isolated SXT exconjugants contained tandem SXT arrays. There was heterogeneity in the size of the SXT arrays detected in single exconjugant colonies. Some arrays consisted of more than five SXTs arranged in tandem. These extended arrays were unstable and did not persist during serial passages. The mechanism accounting for the generation of SXT arrays is unknown; however, array formation was not dependent upon
recA
and appeared to depend on conjugative transfer. While such arrays did not alter the transfer frequency of wild-type SXT, they partially complemented the transfer deficiency of a Δ
xis
SXT mutant, which is ordinarily unable to generate the extrachromosomal intermediate required for SXT transfer. Exconjugants derived from donor strains that harbored tandem arrays of SXT and R391, an SXT-related element, contained functional hybrid elements that arose from
recA
-independent recombination between the two ICEs. Thus, arrays of SXT-related elements promote the creation of novel ICEs.
The Membrane Fluidity Sensor DesK of
Bacillus subtilis
Controls the Signal Decay of Its Cognate Response Regulator
Authors: Albanesi Daniela , Mansilla María Cecilia , de Mendoza Diego
Published: April 2004

The
Bacillus subtilis
DesK/DesR two-component system regulates the expression of the
des
gene coding for the Δ5 acyl lipid desaturase. It is believed that a decrease in membrane lipid fluidity activates the DesK/DesR signal transduction cascade, which results in synthesis of the Δ5 acyl lipid desaturase and desaturation of membrane phospholipids. These newly synthesized unsaturated fatty acids then act as negative signals of
des
transcription, thus generating a regulatory metabolic loop that optimizes membrane fluidity. We previously suggested that DesK is a bifunctional enzyme with both kinase and phosphatase activities that could assume different signaling states in response to changes in the fluidity of membrane lipids. However, no direct experimental evidence supported this proposed model. In this study, we show that the C-terminal fragment of the DesK protein (DesKC) indeed acts as an autokinase. Addition of the response regulator DesR to phosphorylated DesKC resulted in rapid transfer of the phosphoryl group to DesR. Further, phosphorylated DesR can be dephosphorylated in the presence of DesKC, thus demonstrating that the sensor kinase has the ability to covalently modify DesR through both kinase and phosphatase activities. We also present evidence that DesKC might be locked in a kinase-dominant state in vivo and that its activities are not affected either in vivo or in vitro by unsaturated fatty acids. These findings provide the first direct evidence that the transmembrane segments of DesK are essential to sense changes in membrane fluidity and for regulating the ratio of kinase to phosphatase activities of the cytoplasmic C-terminal domain.
Chemotactic Signaling by an
Escherichia coli
CheA Mutant That Lacks the Binding Domain for Phosphoacceptor Partners
Authors: Jahreis Knut , Morrison Tom B. , Garzón Andrés , Parkinson John S.
Published: April 2004

CheA is a multidomain histidine kinase for chemotaxis in
Escherichia coli
. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheAΔP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheAΔP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheAΔP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheAΔP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.
Conditional Lethal Amber Mutations in Essential
Escherichia coli
Genes
Authors: Herring Christopher D. , Blattner Frederick R.
Published: April 2004

The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in
Escherichia coli
that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential
E. coli
genes. Amber stop codons were introduced as “tagalong” mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes
frr
,
gcpE, lpxC
,
map
,
murA
,
ppa
, and
rpsA
. We were unable to isolate an amber mutation in
ftsZ
. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in
gcpE
, which was bacteriostatic. The strain carrying an amber mutation in
murA
was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.
LuxS-Mediated Signaling inStreptococcus mutansIs Involved in Regulation of Acid and Oxidative Stress Tolerance and Biofilm Formation
Authors: Wen Zezhang T. , Burne Robert A.
Published: April 2004

LuxS-mediated quorum sensing has recently been shown to regulate important physiologic functions and virulence in a variety of bacteria. In this study, the role ofluxSofStreptococcus mutansin the regulation of traits crucial to pathogenesis was investigated. Reporter gene fusions showed that inactivation ofluxSresulted in a down-regulation of fructanase, a demonstrated virulence determinant, by more than 50%. The LuxS-deficient strain (TW26) showed increased sensitivity to acid killing but could still undergo acid adaptation. Northern hybridization revealed that the expression of RecA, SmnA (AP endonuclease), and Nth (endonuclease) were down-regulated in TW26, especially in early-exponential-phase cells. Other down-regulated genes includedffh(a signal recognition particle subunit) andbrpA(biofilm regulatory protein A). Interestingly, theluxSmutant showed an increase in survival rate in the presence of hydrogen peroxide (58.8 mM). TheluxSmutant formed less biofilm on hydroxylapatite disks, especially when grown in biofilm medium with sucrose, and the mutant biofilms appeared loose and hive-like, whereas the biofilms of the wild type were smooth and confluent. The mutant phenotypes were complemented by exposure to supernatants from wild-type cultures. Two loci,smu486andsmu487, were identified and predicted to encode a histidine kinase and a response regulator. The phenotypes of thesmu486 smu487mutant were, in almost all cases, similar to those of theluxSmutant, although our results suggest that this is not due to AI-2 signal transduction via Smu486 and Smu487. This study demonstrates thatluxS-dependent signaling plays critical roles in modulating key virulence properties ofS. mutans.
The Protease Lon and the RNA-Binding Protein Hfq Reduce Silencing of theEscherichia coli bglOperon by H-NS
Authors: Dole Sudhanshu , Klingen Yvonne , Nagarajavel V. , Schnetz Karin
Published: April 2004

The histone-like nucleoid structuring protein H-NS represses theEscherichia coli bgloperon at two levels. H-NS binds upstream of the promoter, represses transcription initiation, and binds downstream within the coding region of the first gene, where it induces polarity of transcription elongation. Inhnsmutants, silencing of thebgloperon is completely relieved. Various screens for mutants in which silencing ofbglis reduced have yielded mutations inhnsand in genes encoding the transcription factors LeuO and BglJ. In order to identify additional factors that regulatebgl, we performed a transposon mutagenesis screen for mutants in which silencing of the operon is strengthened. This screen yielded mutants with mutations incyaA,hfq,lon, andpgi, encoding adenylate cyclase, RNA-binding protein Hfq, protease Lon, and phosphoglucose isomerase, respectively. IncyaAmutants, the cyclic AMP receptor protein-dependent promoter is presumably inactive. The specific effect of thepgimutants onbglis low. Interestingly, in thehfqandlonmutants, the downstream silencing ofbglby H-NS (i.e., the induction of polarity) is more efficient, while the silencing of the promoter by H-NS is unaffected. Furthermore, in anhnsmutant, Hfq has no significant effect and the effect of Lon is reduced. These data provide evidence that the specific repression by H-NS can (directly or indirectly) be modulated and controlled by other pleiotropic regulators.
The
pgaABCD
Locus of
Escherichia coli
Promotes the Synthesis of a Polysaccharide Adhesin Required for Biofilm Formation
Authors: Wang Xin , Preston James F. , Romeo Tony
Published: April 2004

Production of a polysaccharide matrix is a hallmark of bacterial biofilms, but the composition of matrix polysaccharides and their functions are not widely understood. Previous studies of the regulation of
Escherichia coli
biofilm formation suggested the involvement of an unknown adhesin. We now establish that the
pgaABCD
(formerly
ycdSRQP
) locus affects biofilm development by promoting abiotic surface binding and intercellular adhesion. All of the
pga
genes are required for optimal biofilm formation under a variety of growth conditions. A
pga
-dependent cell-bound polysaccharide was isolated and determined by nuclear magnetic resonance analyses to consist of unbranched β-1,6-
N
-acetyl-
d
-glucosamine, a polymer previously unknown from the gram-negative bacteria but involved in adhesion by staphylococci. The
pga
genes are predicted to encode envelope proteins involved in synthesis, translocation, and possibly surface docking of this polysaccharide. As predicted, if poly-β-1,6-GlcNAc (PGA) mediates cohesion, metaperiodate caused biofilm dispersal and the release of intact cells, whereas treatment with protease or other lytic enzymes had no effect. The
pgaABCD
operon exhibits features of a horizontally transferred locus and is present in a variety of eubacteria. Therefore, we propose that PGA serves as an adhesin that stabilizes biofilms of
E. coli
and other bacteria.
Involvement of Error-Prone DNA Polymerase IV in Stationary-Phase Mutagenesis in
Pseudomonas putida
Authors: Tegova Radi , Tover Andres , Tarassova Kairi , Tark Mariliis , Kivisaar Maia
Published: April 2004

In this work we studied involvement of DNA polymerase IV (Pol IV) (encoded by the
dinB
gene) in stationary-phase mutagenesis in
Pseudomonas putida
. For this purpose we constructed a novel set of assay systems that allowed detection of different types of mutations (e.g., 1-bp deletions and different base substitutions) separately. A significant effect of Pol IV became apparent when the frequency of accumulation of 1-bp deletion mutations was compared in the
P. putida
wild-type strain and its Pol IV-defective
dinB
knockout derivative. Pol IV-dependent mutagenesis caused a remarkable increase (approximately 10-fold) in the frequency of accumulation of 1-bp deletion mutations on selective plates in wild-type
P. putida
populations starved for more than 1 week. No effect of Pol IV on the frequency of accumulation of base substitution mutations in starving
P. putida
cells was observed. The occurrence of 1-bp deletions in
P. putida
cells did not require a functional RecA protein. RecA independence of Pol IV-associated mutagenesis was also supported by data showing that transcription from the promoter of the
P. putida dinB
gene was not significantly influenced by the DNA damage-inducing agent mitomycin C. Therefore, we hypothesize that mechanisms different from the classical RecA-dependent SOS response could elevate Pol IV-dependent mutagenesis in starving
P. putida
cells.
Physiologic and Proteomic Evidence for a Role of Nitric Oxide in Biofilm Formation by
Nitrosomonas europaea
and Other Ammonia Oxidizers
Authors: Schmidt Ingo , Steenbakkers Peter J. M. , op den Camp Huub J. M. , Schmidt Katrin , Jetten Mike S. M.
Published: April 2004

NO, a free radical gas, is the signal for
Nitrosomonas europaea
cells to switch between different growth modes. At an NO concentration of more than 30 ppm, biofilm formation by
N. europaea
was induced. NO concentrations below 5 ppm led to a reversal of the biofilm formation, and the numbers of motile and planktonic (motile-planktonic) cells increased. In a proteomics approach, the proteins expressed by
N. europaea
were identified. Comparison studies of the protein patterns of motile-planktonic and attached (biofilm) cells revealed several clear differences. Eleven proteins were found to be up or down regulated. Concentrations of other compounds such as ammonium, nitrite, and oxygen as well as different temperatures and pH values had no significant effect on the growth mode of and the proteins expressed by
N. europaea
.
RamB, a Novel Transcriptional Regulator of Genes Involved in Acetate Metabolism of
Corynebacterium glutamicum
Authors: Gerstmeir Robert , Cramer Annette , Dangel Petra , Schaffer Steffen , Eikmanns Bernhard J.
Published: April 2004

The adaptation of
Corynebacterium glutamicum
to acetate as a carbon and energy source involves transcriptional regulation of the
pta-ack
operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the
aceA
and
aceB
genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as
cis
-regulatory elements for expression of the
pta-ack
operon and the
aceA
and
aceB
genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the
pta-ack
operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the
pta-ack
and the
aceA/aceB
promoter/operator regions. Directed deletion of the
ramB
gene in the genome of
C. glutamicum
resulted in mutant strain RG1. Whereas the wild type of
C. glutamicum
showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional
cat
fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of
C. glutamicum
.
Haloviruses HF1 and HF2: Evidence for a Recent and Large Recombination Event
Authors: Tang Sen-Lin , Nuttall Stewart , Dyall-Smith Mike
Published: April 2004

Haloviruses HF1 and HF2 were isolated from the same saltern pond and are adapted to hypersaline conditions, where they infect a broad range of haloarchaeal species. The HF2 genome has previously been reported. The complete sequence of the HF1 genome has now been determined, mainly by PCR and primer walking. It was 75,898 bp in length and was 94.4% identical to the HF2 genome but about 1.8 kb shorter. A total of 117 open reading frames and five tRNA-like genes were predicted, and their database matches and characteristics were similar to those found in HF2. A comparison of the predicted restriction digest patterns based on nucleotide sequence with the observed restriction digest patterns of viral DNA showed that, unlike the case for HF2, some packaged HF1 DNA had cohesive termini. Except for a single base change, HF1 and HF2 were identical in sequence over the first 48 kb, a region that includes the early and middle genes. The remaining 28 kb of HF1 showed many differences from HF2, and the similarity of the two genomes over this late gene region was 87%. The abrupt shift in sequence similarity around 48 kb suggests a recent recombination event between either HF1 or HF2 and another HF-like halovirus that has swapped most of the right-end 28 kb. This example indicates there is a high level of recombination among viruses that live in this extreme environment.
Highly Divergent RfaH Orthologs from Pathogenic Proteobacteria Can Substitute for
Escherichia coli
RfaH both In Vivo and In Vitro
Authors: Carter Heather D. , Svetlov Vladimir , Artsimovitch Irina
Published: April 2004

The transcriptional enhancer protein RfaH positively regulates production of virulence factors in
Escherichia coli
and
Salmonella enterica
serovar Typhimurium via a
cis
element,
ops
. Genes coding for RfaH orthologs were identified in conceptually translated genomes of bacterial pathogens, including
Vibrio
and
Yersinia
spp. We cloned the
rfaH
genes from
Vibrio cholerae, Yersinia enterocolitica, S. enterica
serovar Typhimurium, and
Klebsiella pneumoniae
into
E. coli
expression vectors. Purified RfaH orthologs, including the most divergent one from
V. cholerae
, were readily recruited to the
E. coli
transcription elongation complex. Postrecruitment stimulation of transcript elongation appeared to vary with the degree of similarity to
E. coli
RfaH.
V. cholerae
RfaH was particularly defective in reducing downstream pausing and termination; this defect was substantially alleviated by an increase in its concentration. When overexpressed episomally, all of the
rfaH
genes complemented the disruption of the chromosomal copy of the
E. coli
gene. Thus, despite the apparently accelerated divergent evolution of the RfaH proteins, the mechanism of their action is conserved well enough to make them transcriptionally active in the
E. coli
system.
Genome of Staphylococcal Phage K: a New Lineage of
Myoviridae
Infecting Gram-Positive Bacteria with a Low G+C Content
Authors: O'Flaherty S. , Coffey A. , Edwards R. , Meaney W. , Fitzgerald G. F. , Ross R. P.
Published: April 2004

Phage K is a polyvalent phage of the
Myoviridae
family which is active against a wide range of staphylococci. Phage genome sequencing revealed a linear DNA genome of 127,395 bp, which carries 118 putative open reading frames. The genome is organized in a modular form, encoding modules for lysis, structural proteins, DNA replication, and transcription. Interestingly, the structural module shows high homology to the structural module from
Listeria
phage A511, suggesting intergenus horizontal transfer. In addition, phage K exhibits the potential to encode proteins necessary for its own replisome, including DNA ligase, primase, helicase, polymerase, RNase H, and DNA binding proteins. Phage K has a complete absence of GATC sites, making it insensitive to restriction enzymes which cleave this sequence. Three introns (
lys
-I1,
pol
-I2, and
pol
-I3) encoding putative endonucleases were located in the genome. Two of these (
pol
-I2 and
pol
-I3) were found to interrupt the DNA polymerase gene, while the other (
lys
-I1) interrupts the lysin gene. Two of the introns encode putative proteins with homology to HNH endonucleases, whereas the other encodes a 270-amino-acid protein which contains two zinc fingers (CX
2
CX
22
CX
2
C and CX
2
CX
23
CX
2
C). The availability of the genome of this highly virulent phage, which is active against infective staphylococci, should provide new insights into the biology and evolution of large broad-spectrum polyvalent phages.
Crystal Structure of the Response Regulator 02 Receiver Domain, the Essential YycF Two-Component System ofStreptococcus pneumoniaein both Complexed and Native States
Authors: Bent Colin J. , Isaacs Neil W. , Mitchell Timothy J. , Riboldi-Tunnicliffe Alan
Published: April 2004

A variety of bacterial cellular responses to environmental signals are mediated by two-component signal transduction systems comprising a membrane-associated histidine protein kinase and a cytoplasmic response regulator (RR), which interpret specific stimuli and produce a measured physiological response. In RR activation, transient phosphorylation of a highly conserved aspartic acid residue drives the conformation changes needed for full activation of the protein. Sequence homology reveals that RR02 fromStreptococcus pneumoniaebelongs to the OmpR subfamily of RRs. The structures of the receiver domains from four members of this family, DrrB and DrrD fromThermotoga maritima, PhoB fromEscherichia coli, and PhoP fromBacillus subtilis, have been elucidated. These domains are globally very similar in that they are composed of a doubly wound α5β5; however, they differ remarkably in the fine detail of the β4-α4 and α4 regions. The structures presented here reveal a further difference of the geometry in this region. RR02 is has been shown to be the essential RR in the gram-positive bacteriumS. pneumoniaeR. Lange, C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W. Keck, and K. E. Amrein, Gene 237:223-234, 1999; J. P. Throup, K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham, Mol. Microbiol. 35:566-576, 2000). RR02 functions as part of a phosphotransfer system that ultimately controls the levels of competence within the bacteria. Here we report the native structure of the receiver domain of RR02 from serotype 4S. pneumoniae(as well as acetate- and phosphate-bound forms) at different pH levels. Two native structures at 2.3 Å, phased by single-wavelength anomalous diffraction (xenon SAD), and 1.85 Å and a third structure at pH 5.9 revealed the presence of a phosphate ion outside the active site. The fourth structure revealed the presence of an acetate molecule in the active site.
Biofilm Formation in
Pseudomonas aeruginosa
: Fimbrial
cup
Gene Clusters Are Controlled by the Transcriptional Regulator MvaT
Authors: Vallet Isabelle , Diggle Stephen P. , Stacey Rachael E. , Cámara Miguel , Ventre Isabelle , Lory Stephen , Lazdunski Andrée , Williams Paul , Filloux Alain
Published: April 2004

Pseudomonas aeruginosa
is an opportunistic bacterial pathogen which poses a major threat to long-term-hospitalized patients and individuals with cystic fibrosis. The capacity of
P. aeruginosa
to form biofilms is an important requirement for chronic colonization of human tissues and for persistence in implanted medical devices. Various stages of biofilm formation by this organism are mediated by extracellular appendages, such as type IV pili and flagella. Recently, we identified three
P. aeruginosa
gene clusters that were termed
cup
(chaperone-usher pathway) based on their sequence relatedness to the chaperone-usher fimbrial assembly pathway in other bacteria. The
cupA
gene cluster, but not the
cupB
or
cupC
cluster, is required for biofilm formation on abiotic surfaces. In this study, we identified a gene (
mvaT
) encoding a negative regulator of
cupA
expression. Such regulatory control was confirmed by several approaches, including
lacZ
transcriptional fusions, Northern blotting, and transcriptional profiling using DNA microarrays. MvaT also represses the expression of the
cupB
and
cupC
genes, although the extent of the regulatory effect is not as pronounced as with
cupA
. Consistent with this finding,
mvaT
mutants exhibit enhanced biofilm formation. Although the
P. aeruginosa
genome contains a highly homologous gene,
mvaU
, the repression of
cupA
genes is MvaT specific. Thus, MvaT appears to be an important regulatory component within a complex network that controls biofilm formation and maturation in
P. aeruginosa
.
Polymerases Leave Fingerprints: Analysis of the Mutational Spectrum in
Escherichia coli rpoB
To Assess the Role of Polymerase IV in Spontaneous Mutation
Authors: Wolff Erika , Kim Mandy , Hu Kaibin , Yang Hanjing , Miller Jeffrey H.
Published: April 2004

We compared the distribution of mutations in
rpoB
that lead to rifampin resistance in strains with differing levels of polymerase IV (Pol IV), including strains with deletions of the Pol IV-encoding
dinB
gene, strains with a chromosomal copy of
dinB
, strains with the F′128 plasmid, and strains with plasmid amplification of either the
dinB
operon (
dinB-yafNOP
) or the
dinB
gene alone. This analysis identifies several hot spots specific to Pol IV which are virtually absent from the normal spontaneous spectrum, indicating that Pol IV does not contribute significantly to mutations occurring during exponential growth in liquid culture.
Correlation between Osmolarity and Luminescence of Symbiotic
Vibrio fischeri
Strain ES114
Authors: Stabb Eric V. , Butler Melissa S. , Adin Dawn M.
Published: April 2004

Vibrio fischeri
isolates from
Euprymna scolopes
are dim in culture but bright in the host. We found the luminescence of
V. fischeri
to be correlated with external osmolarity both in culture and in this symbiosis. Luminescence enhancement by osmolarity was independent of the
lux
promoter and unaffected by autoinducers or the level of
lux
expression, but the addition of an aldehyde substrate for luciferase raised the luminescence of cells grown at high and low osmolarities to the same high level.
V. fischeri
culture media have lower osmolarities than are typical in seawater or in cephalopods, partially accounting for the bacterium's low light output in culture.
Aircraft noise around a large international airport and its impact on general health and medication use
Authors: Franssen E A M , van Wiechen C M A G , Nagelkerke N J D , Lebret E
Published: April 2004

Aims:To assess the prevalence of general health status, use of sleep medication, and use of medication for cardiovascular diseases, and to study their relation to aircraft noise exposure.

Methods:These health indicators were measured by a cross-sectional survey among 11 812 respondents living within a radius of 25 km around Schiphol airport (Amsterdam).

Results:Adjusted odds ratios ranged from 1.02 to 2.34 per 10 dB(A) increase in Lden. The associations were statistically significant for all indicators, except for use of prescribed sleep medication or sedatives and frequent use of this medication. None of the health indicators were associated with aircraft noise exposure during the night, but use of non-prescribed sleep medication or sedatives was associated with aircraft noise exposure during the late evening (OR = 1.72). Vitality related health complaints such as tiredness and headache were associated with aircraft noise, whereas most other physical complaints were not. Odds ratios for the vitality related complaints ranged from 1.16 to 1.47 per 10 dB(A) increase in Lden. A small fraction of the prevalence of poor self rated health (0.13), medication for cardiovascular diseases or increased blood pressure (0.08), and sleep medication or sedatives (0.22) could be attributed to aircraft noise. Although the attributable fraction was highest in the governmentally noise regulated area, aircraft noise had more impact in the non-regulated area, due to the larger population.

Conclusions:Results suggest associations between community exposure to aircraft noise and the health indicators poor general health status, use of sleep medication, and use of medication for cardiovascular diseases.
Quantifying the advantages and disadvantages of pre-placement genetic screening
Authors: Palmer K T , Poole J , Rawbone R G , Coggon D
Published: April 2004

Background:Tests of genotype may enable workers at unusual risk of future ill-health to be identified. Using them to select for employment, however, entails gains and losses to employers and employees. Ensuring a fair balance between the rights and obligations of each group requires a value judgement, but the advantages and disadvantages to interested parties must first be quantified in a meaningful way.

Method and Results:The purposes of pre-employment screening are reviewed, and several simple measures relevant to the separate interests of employers and job applicants proposed—number screened to prevent a single adverse outcome; number excluded to prevent a case; expected incidence of the adverse outcome in those excluded; and preventable fraction. The derivation of these measures is illustrated, and the factors that influence them (the prevalence of the prognostic trait, the relative risk that it carries for an adverse outcome, and the overall incidence of disease) are related algebraically and graphically, to aid judgement on the utility of screening under different circumstances.

Conclusions:In sensitive areas such as genetic testing the onus should be on the employer to justify plans for pre-placement screening. Several quantitative measures can be used to inform the ethical and economic debate about screening and to evaluate alternative strategies for prevention.
Increased inflammation and altered macrophage chemotactic responses caused by two ultrafine particle types
Authors: Renwick L C , Brown D , Clouter A , Donaldson K
Published: April 2004

Background:Ultrafine particles have been hypothesised to be an important contributing factor in the toxicity and adverse health effects of particulate air pollution (PM10) and nanoparticles are used increasingly in industrial processes.

Aims:To compare the ability of ultrafine and fine particles of titanium dioxide and carbon black to induce inflammation, cause epithelial injury, and affect the alveolar macrophage clearance functions of phagocytosis and chemotaxis in vivo.

Methods:Rats were instilled with fine and ultrafine carbon black and titanium dioxide. Inflammation was quantified by bronchoalveolar lavage; the ability of the macrophages to phagoytose indictor fluorescent beads and to migrate towards aC5a were determined.

Results:Ultrafine particles induced more PMN recruitment, epithelial damage, and cytotoxicity than their fine counterparts, exposed at equal mass. Both ultrafine and fine particles significantly impaired the phagocytic ability of alveolar macrophages. Only ultrafine particle treatment significantly enhanced the sensitivity of alveolar macrophages to chemotact towards C5a.

Conclusions:Ultrafine particles of two very different materials induced inflammation and epithelial damage to a greater extent than their fine counterparts. In general, the effect of ultrafine carbon black was greater than ultrafine titanium dioxide, suggesting that there are differences in the likely harmfulness of different types of ultrafine particle. Epithelial injury and toxicity were associated with the development of inflammation after exposure to ultrafines. Increased sensitivity to a C5a chemotactic gradient could make the ultrafine exposed macrophages more likely to be retained in the lungs, so allowing dose to accumulate.