bluuebunny/crossref_metadata_2025 huggingface dataset:
Chlamydial Type III Secretion System Is Encoded on Ten Operons Preceded by Sigma 70-Like Promoter Elements
Authors: Hefty P. Scott , Stephens Richard S.
Published: December 2006

Many gram-negative bacterial pathogens employ type III secretion systems for infectious processes. Chlamydiae are obligate intracellular bacteria that encode a conserved type III secretion system that is likely requisite for growth. Typically, genes encoding type III secretion systems are located in a single locus; however, for chlamydiae these genes are scattered throughout the genome. Little is known regarding the gene regulatory mechanisms for this essential virulence determinant. To facilitate identification ofcis-acting transcriptional regulatory elements, the operon structure was determined. This analysis revealed 10 operons that contained 37 genes associated with the type III secretion system. Linkage within these operons suggests a role in type III secretion for each of these genes, including 13 genes encoding proteins with unknown function. The transcriptional start site for each operon was determined. In conjunction with promoter activity assays, this analysis revealed that the type III secretion system operons encode σ70-like promoter elements. Transcriptional initiation by a sigma factor responsible for constitutive gene expression indicates that undefined activators or repressors regulate developmental stage-specific expression of chlamydial type III secretion system genes.
Transcriptional Regulation of the
yghJ-pppA-yghG-
gspCDEFGHIJKLM
Cluster, Encoding the Type II Secretion Pathway in Enterotoxigenic
Escherichia coli
Authors: Yang Ji , Baldi Deborah L. , Tauschek Marija , Strugnell Richard A. , Robins-Browne Roy M.
Published: December 2006

The gene cluster
gspCDEFGHIJKLM
codes for various structural components of the type II secretion pathway which is responsible for the secretion of heat-labile enterotoxin by enterotoxigenic
Escherichia coli
(ETEC). In this work, we used a variety of molecular approaches to elucidate the transcriptional organization of the ETEC type II secretion system and to unravel the mechanisms by which the expression of these genes is controlled. We showed that the
gspCDEFGHIJKLM
cluster and three other upstream genes,
yghJ
,
pppA
, and
yghG
, are cotranscribed and that a promoter located in the upstream region of
yghJ
plays a major role in the expression of this 14-gene transcriptional unit. Transcription of the
yghJ
promoter was repressed 168-fold upon a temperature downshift from 37°C to 22°C. This temperature-induced repression was mediated by the global regulatory proteins H-NS and StpA. Deletion mutagenesis showed that the promoter region encompassing positions −321 to +301 relative to the start site of transcription of
yghJ
was required for full repression. The
yghJ
promoter region is predicted to be highly curved and bound H-NS or StpA directly. The binding of H-NS or StpA blocked transcription initiation by inhibiting promoter open complex formation. Unraveling the mechanisms of regulation of type II secretion by ETEC enhances our understanding of the pathogenesis of ETEC and other pathogenic varieties of
E. coli
.
Molecular Evolution of the H-NS Protein: Interaction with Hha-Like Proteins Is Restricted toEnterobacteriaceae
Authors: Madrid Cristina , García Jesús , Pons Miquel , Juárez Antonio
Published: December 2006

We show here that chromosomalhha-like genes are restricted to theEnterobacteriaceae. The H-NS N-terminal domain of members of this family includes an unaltered seven-amino-acid sequence located between helixes 1 and 2, termed the Hha signature, that contains key residues for H-NS-Hha interaction.
Characterization of
lptA
and
lptB
, Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of
Escherichia coli
Authors: Sperandeo Paola , Cescutti Rachele , Villa Riccardo , Di Benedetto Cristiano , Candia Daniela , Dehò Gianni , Polissi Alessandra
Published: December 2006

The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two
Escherichia coli
essential genes,
yhbN
and
yhbG
, now renamed
lptA
and
lptB
, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the
lptA-lptB
operon is controlled by the extracytoplasmic σ factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of
E. coli
.
FoxB of
Pseudomonas aeruginosa
Functions in the Utilization of the Xenosiderophores Ferrichrome, Ferrioxamine B, and Schizokinen: Evidence for Transport Redundancy at the Inner Membrane
Authors: Ó Cuív Páraic , Keogh Damien , Clarke Paul , O'Connell Michael
Published: December 2006

Expression of the inner membrane protein FoxB (PA2465) of
Pseudomonas aeruginosa
in mutants of
Sinorhizobium meliloti
that are defective in the utilization of ferrichrome, ferrioxamine B, and schizokinen resulted in the restoration of siderophore utilization. Mutagenesis of
foxB
in
P. aeruginosa
did not abolish siderophore utilization, suggesting that the function is redundant.
Response of
Staphylococcus aureus
to Salicylate Challenge
Authors: Riordan James T. , Muthaiyan Arunachalam , Van Voorhies Wayne , Price Christopher T. , Graham James E. , Wilkinson Brian J. , Gustafson John E.
Published: December 2006

Growth of
Staphylococcus aureus
with the nonsteroidal anti-inflammatory salicylate reduces susceptibility of the organism to multiple antimicrobials. Transcriptome analysis revealed that growth of
S. aureus
with salicylate leads to the induction of genes involved with gluconate and formate metabolism and represses genes required for gluconeogenesis and glycolysis. In addition, salicylate induction upregulates two antibiotic target genes and downregulates a multidrug efflux pump gene repressor (
mgrA
) and
sarR
, which represses a gene (
sarA
) important for intrinsic antimicrobial resistance. We hypothesize that these salicylate-induced alterations jointly represent a unique mechanism that allows
S. aureus
to resist antimicrobial stress and toxicity.
Analysis of the Roles of FlgP and FlgQ in Flagellar Motility of
Campylobacter jejuni
Authors: Sommerlad Shawn M. , Hendrixson David R.
Published: December 2006

Flagellar motility is an important determinant of
Campylobacter jejuni
that is required for promoting interactions with various hosts to promote gastroenteritis in humans or commensal colonization of many animals. In a previous study, we identified a nonmotile mutant of
C. jejuni
81-176 with a transposon insertion in Cj1026c, but verification of the role of the encoded protein in motility was not determined. In this study, we have determined that Cj1026c and the gene immediately downstream, Cj1025c (here annotated as
flgP
and
flgQ
, respectively), are both required for motility of
C. jejuni
but not for flagellar biosynthesis. FlgP and FlgQ are not components of the transcriptional regulatory cascades to activate σ
28
- or σ
54
-dependent expression of flagellar genes. In addition, expression of
flgP
and
flgQ
is not largely dependent on σ
28
or σ
54
. Immunblot analyses revealed that the majority of FlgP in
C. jejuni
is associated with the outer membrane. However, in the absence of FlgQ, the amounts of FlgP in the outer membrane of
C. jejuni
are greatly reduced, suggesting that FlgQ may be required for localization or stability of FlgP at this location. This study provides insight into features of FlgP and FlgQ, two proteins with previously undefined functions that are required for the larger, multicomponent flagellar system of
C. jejuni
that is necessary for motility.
Glycosylation of
Pseudomonas aeruginosa
Strain Pa5196 Type IV Pilins with Mycobacterium-Like α-1,5-Linked
d
-Ara
f
Oligosaccharides
Authors: Voisin Sébastien , Kus Julianne V. , Houliston Scott , St-Michael Frank , Watson Dave , Cvitkovitch Dennis G. , Kelly John , Brisson Jean-Robert , Burrows Lori L.
Published: December 2006

Pseudomonas aeruginosa
is a gram-negative bacterium that uses polar type IV pili for adherence to various materials and for rapid colonization of surfaces via twitching motility. Within the
P. aeruginosa
species, five distinct alleles encoding variants of the structural subunit PilA varying in amino acid sequence, length, and presence of posttranslational modifications have been identified. In this work, a combination of mass spectrometry and nuclear magnetic resonance spectroscopy was used to identify a novel glycan modification on the pilins of the group IV strain Pa5196. Group IV pilins continued to be modified in a lipopolysaccharide (
wbpM
) mutant of Pa5196, showing that, unlike group I strains, the pilins of group IV are not modified with the O-antigen unit of the background strain. Instead, the pilin glycan was determined to be an unusual homo-oligomer of α-1,5-linked
d
-arabinofuranose (
d
-Ara
f
). This sugar is uncommon in prokaryotes, occurring mainly in the cell wall arabinogalactan and lipoarabinomannan (LAM) polymers of mycobacteria, including
Mycobacterium tuberculosis
and
Mycobacterium leprae
. Antibodies raised against
M. tuberculosis
LAM specifically identified the glycosylated pilins from Pa5196, confirming that the glycan is antigenically, as well as chemically, identical to those of
Mycobacterium. P. aeruginosa
Pa5196, a rapidly growing strain of low virulence that expresses large amounts of glycosylated type IV pilins on its surface, represents a genetically tractable model system for elucidation of alternate pathways for biosynthesis of
d
-Ara
f
and its polymerization into mycobacterium-like α-1,5-linked oligosaccharides.
Export of the Pseudopilin XcpT of the
Pseudomonas aeruginosa
Type II Secretion System via the Signal Recognition Particle-Sec Pathway
Authors: Arts Jorik , van Boxtel Ria , Filloux Alain , Tommassen Jan , Koster Margot
Published: December 2006

Type IV pilins and pseudopilins are found in various prokaryotic envelope protein complexes, including type IV pili and type II secretion machineries of gram-negative bacteria, competence systems of gram-positive bacteria, and flagella and sugar-binding structures in members of the archaeal kingdom. The precursors of these proteins have highly conserved N termini, consisting of a short, positively charged leader peptide, which is cleaved off by a dedicated peptidase during maturation, and a hydrophobic stretch of approximately 20 amino acid residues. Which pathway is involved in the inner membrane translocation of these proteins is unknown. We used XcpT, the major pseudopilin from the type II secretion machinery of
Pseudomonas aeruginosa
, as a model to study this process. Transport of an XcpT-PhoA hybrid was shown to occur in the absence of other Xcp components in
P. aeruginosa
and in
Escherichia coli
. Experiments with conditional
sec
mutants and reporter-protein fusions showed that this transport process involves the cotranslational signal recognition particle targeting route and is dependent on a functional Sec translocon.
Functional Analysis of the Carboxy-Terminal Region of
Bacillus subtilis
TnrA, a MerR Family Protein
Authors: Wray Lewis V. , Fisher Susan H.
Published: December 2006

The
Bacillus subtilis
TnrA transcription factor belongs to the MerR family of proteins and regulates gene expression during nitrogen-limited growth. When
B. subtilis
cells are grown with excess nitrogen, feedback-inhibited glutamine synthetase forms a protein-protein complex with TnrA that prevents TnrA from binding to DNA. The C-terminal region of TnrA is required for the interaction with glutamine synthetase. Alanine scanning mutagenesis of the C-terminal region of TnrA identified three classes of mutants that altered the regulation by glutamine synthetase. While expression of the TnrA-regulated
amtB
gene was expressed constitutively in the class I (M96A, Q100A, and A103G) and class II (L97A, L101A, and F105A) mutants, the class II mutants were unable to grow on minimal medium unless a complex mixture of amino acids was present. The class III
tnrA
mutants (R93A, G99A, N102A, H104A, and Y107A mutants) were partially defective in the regulation of TnrA activity. In vitro experiments showed that feedback-inhibited glutamine synthetase had a significantly reduced ability to inhibit the DNA-binding activity of several class I and class II mutant TnrA proteins. A coiled-coil homology model of the C-terminal region of TnrA is used to explain the properties of the class I and II mutant proteins. The C-terminal region of TnrA corresponds to a dimerization domain in other MerR family proteins. Surprisingly, gel filtration and cross-linking analysis showed that a truncated TnrA protein which contained only the N-terminal DNA binding domain was dimeric. The implications of these results for the structure of TnrA are discussed.
Conversion of Methionine to Cysteine in
Bacillus subtilis
and Its Regulation
Authors: Hullo Marie-Françoise , Auger Sandrine , Soutourina Olga , Barzu Octavian , Yvon Mireille , Danchin Antoine , Martin-Verstraete Isabelle
Published: December 2006

Bacillus subtilis
can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in
Escherichia coli
were tested. Both CysK and YrhA have an
O
-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine β-synthase activity using
O
-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine γ-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for
cysK
or
yrhB
grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an Δ
yrhA
mutant or a
luxS
mutant, inactivated for the
S
-ribosyl-homocysteinase step of the
S
-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a Δ
yrhA ΔcysK
or
cysE
mutant did not grow at all under the same conditions. The
yrhB
and
yrhA
genes form an operon together with
yrrT
,
mtnN
, and
yrhC
. The expression of the
yrrT
operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the
yrrT
promoter region in gel-shift experiments. The addition of
O
-acetyl-serine prevented the formation of this protein-DNA complex.
Complete Sequence Analysis of Novel Plasmids from Emetic and Periodontal
Bacillus cereus
Isolates Reveals a Common Evolutionary History among the
B. cereus
-Group Plasmids, Including
Bacillus anthracis
pXO1
Authors: Rasko David A. , Rosovitz M. J. , Økstad Ole Andreas , Fouts Derrick E. , Jiang Lingxia , Cer Regina Z. , Kolstø Anne-Brit , Gill Steven R. , Ravel Jacques
Published: December 2006

The plasmids of the members of the
Bacillus cereus
sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example,
Bacillus anthracis
contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines
Bacillus thuringiensis
isolates. In this study the plasmids from
B. cereus
isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical ∼272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one ∼270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these
B. cereus
plasmids revealed a high degree of sequence similarity to the
B. anthracis
pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified
B. cereus
plasmids. Screening of a population of
B. cereus
group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic
B. cereus
isolates in the same way that pXO1 and pXO2 define the
B. anthracis
species.
The Timing ofcotEExpression AffectsBacillus subtilisSpore Coat Morphology but Not Lysozyme Resistance
Authors: Costa Teresa , Serrano Mónica , Steil Leif , Völker Uwe , Moran Charles P. , Henriques Adriano O.
Published: December 2006

The synthesis of structural components and morphogenetic factors required for the assembly of theBacillus subtilisspore coat is governed by a mother cell-specific transcriptional cascade. The first two temporal classes of gene expression, which involve RNA polymerase sigma σEfactor and the ancillary regulators GerR and SpoIIID, are deployed prior to engulfment of the prespore by the mother cell. The two last classes rely on σK, whose activation follows engulfment completion, and GerE. ThecotEgene codes for a morphogenetic protein essential for the assembly of the outer coat layer and spore resistance to lysozyme.cotEis expressed first from a σE-dependent promoter and, in a second stage, from a promoter that additionally requires SpoIIID and that remains active under σKcontrol. CotE localizes prior to engulfment completion close to the surface of the developing spore, but formation of the outer coat is a late, σK-controlled event. We have transplantedcotEto progressively later classes of mother cell gene expression. This created an early class of mutants in whichcotEis expressed prior to engulfment completion and a late class in which expression ofcotEfollows the complete engulfment of the prespore. Mutants of the early class assemble a nearly normal outer coat structure, whereas mutants of the late class do not. Hence, the early expression of CotE is essential for outer coat assembly. Surprisingly, however, all mutants were fully resistant to lysozyme. The results suggest that CotE has genetically separable functions in spore resistance to lysozyme and spore outer coat assembly.
High-Affinity Binding of the Staphylococcal HarA Protein to Haptoglobin and Hemoglobin Involves a Domain with an Antiparallel Eight-Stranded β-Barrel Fold
Authors: Dryla Agnieszka , Hoffmann Bernd , Gelbmann Dieter , Giefing Carmen , Hanner Markus , Meinke Andreas , Anderson Annaliesa S. , Koppensteiner Walter , Konrat Robert , von Gabain Alexander , Nagy Eszter
Published: December 2006

Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for “near transporter”) present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione
S
-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (

K
D

) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel β-barrel with the strand order (-β1
↓
-β2
↑
-β3
↓
-β6
↑
-β5
↓
-β4
↑
-β7
↓
-β8
↑
), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.
Mutations in the
Yersinia pseudotuberculosis
Type III Secretion System Needle Protein, YscF, That Specifically Abrogate Effector Translocation into Host Cells
Authors: Davis Alison J. , Mecsas Joan
Published: December 2006

The trafficking of effectors, termed Yops, from
Yersinia
spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD)
yscF
mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two
yscF
TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I
yscF
TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II
yscF
TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface.
The
l
-Tartrate/Succinate Antiporter TtdT (YgjE) of
l
-Tartrate Fermentation in
Escherichia coli
Authors: Kim Ok Bin , Unden Gottfried
Published: December 2006

Escherichia coli
ferments
l
-tartrate under anaerobic conditions in the presence of an additional electron donor to succinate. The carrier for
l
-tartrate uptake and succinate export and its relation to the general C
4
-dicarboxylate carriers DcuA, DcuB, and DcuC were studied. The secondary carrier TtdT, encoded by the
ttdT
(previously called
ygjE
) gene, is required for the uptake of
l
-tartrate. The
ttdT
gene is located downstream of the
ttdA
and
ttdB
genes, encoding the
l
-tartrate dehydratase TtdAB. Analysis of mRNA by reverse transcription-PCR showed that
ttdA
,
ttdB
, and
ttdT
are cotranscribed. Deletion of
ttdT
abolished growth by
l
-tartrate and degradation of
l
-tartrate completely. Bacteria containing TtdT catalyze
l
-tartrate or succinate uptake and specific heterologous
l
-tartrate/succinate antiporting.
d
-Tartrate is not a substrate for TtdT. TtdT operates preferentially in the direction of tartrate uptake and succinate excretion. The Dcu carriers do not support anaerobic growth on
l
-tartrate or
l
-tartrate transport. TtdT is related in sequence and function to CitT, which catalyzes heterologous citrate/succinate antiporting in citrate fermentation.
Rare Group I Intron with Insertion Sequence Element in a Bacterial Ribonucleotide Reductase Gene
Authors: Meng Qing , Zhang Yi , Liu Xiang-Qin
Published: December 2006

A rare group I intron in a cyanobacterial ribonucleotide reductase gene has been characterized. It contains a mobile insertion sequence element not required for RNA splicing. Ribonucleotide reductase genes were found to be hot spots for all three types of self-splicing intervening sequences, including group I and II introns and inteins.
CapA, an Autotransporter Protein ofCampylobacter jejuni, Mediates Association with Human Epithelial Cells and Colonization of the Chicken Gut
Authors: Ashgar Sami S. A. , Oldfield Neil J. , Wooldridge Karl G. , Jones Michael A. , Irving Greg J. , Turner David P. J. , Ala'Aldeen Dlawer A. A.
Published: December 2006

Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence ofCampylobacter jejuniNCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. AcapAinsertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization byCampylobacter. In view of this demonstrated role, we propose that CapA stands forCampylobacteradhesion protein A.
Genetic Analysis of a Novel Pathway for
d
-Xylose Metabolism in
Caulobacter crescentus
Authors: Stephens Craig , Christen Beat , Fuchs Thomas , Sundaram Vidyodhaya , Watanabe Kelly , Jenal Urs
Published: December 2006

Genetic data suggest that the oligotrophic freshwater bacterium
Caulobacter crescentus
metabolizes
d
-xylose through a pathway yielding α-ketoglutarate, comparable to the recently described
l
-arabinose degradation pathway of
Azospirillum brasilense
. Enzymes of the
C. crescentus
pathway, including an NAD
+
-dependent xylose dehydrogenase, are encoded in the xylose-inducible
xylXABCD
operon (CC0823-CC0819).
Evolution of
Mycobacterium ulcerans
and Other Mycolactone-Producing Mycobacteria from a Common
Mycobacterium marinum
Progenitor
Authors: Yip Marcus J. , Porter Jessica L. , Fyfe Janet A. M. , Lavender Caroline J. , Portaels Françoise , Rhodes Martha , Kator Howard , Colorni Angelo , Jenkin Grant A. , Stinear Tim
Published: December 2006

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with
Mycobacterium ulcerans
, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the
M. ulcerans
pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both
M. ulcerans
and
Mycobacterium marinum
. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common
M. marinum
progenitor to form a genetically cohesive group among a more diverse assemblage of
M. marinum
strains. Like
M. ulcerans
, the fish and frog MPM show multiple copies of the insertion sequence IS
2404
. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.
Bacillus subtilisGene Cluster Involved in Calcium Carbonate Biomineralization
Authors: Barabesi Chiara , Galizzi Alessandro , Mastromei Giorgio , Rossi Mila , Tamburini Elena , Perito Brunella
Published: December 2006

Calcium carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological implications. Nevertheless, little is known of the molecular mechanisms by which bacteria foster calcium carbonate mineralization. In our laboratory, we are studying calcite formation byBacillus subtilis, in order to identify genes involved in the biomineralization process. A previous screening of UV mutants and of more than one thousand mutants obtained from the EuropeanB. subtilisFunctional Analysis project allowed us to isolate strains altered in the precipitation phenotype. Starting from these results, we focused our attention on a cluster of five genes (lcfA,ysiA,ysiB,etfB, andetfA) called thelcfAoperon. By insertional mutagenesis, mutant strains carrying each of the five genes were produced. All of them, with the exception of the strain carrying the mutatedlcfAoperon, were unable to form calcite crystals. By placing transcription under IPTG (isopropyl-β-d-thiogalactopyranoside) control, the last gene,etfA, was identified as essential for the precipitation process. To verify cotranscription in thelcfAoperon, reverse transcription-PCR experiments were performed and overlapping retrocotranscripts were found comprising three adjacent genes. The genes have putative functions linked to fatty acid metabolism. A link between calcium precipitation and fatty acid metabolism is suggested.
Linker Regions of the RhaS and RhaR Proteins
Authors: Kolin Ana , Jevtic Visnja , Swint-Kruse Liskin , Egan Susan M.
Published: December 2006

Substitutions within the interdomain linkers of the AraC/XylS family proteins RhaS and RhaR were tested to determine whether side chain identity or linker structure was required for function. Neither was found crucial, suggesting that the linkers do not play a direct role in activation, but rather simply connect the two domains.
“Cold-Sensitive” Mutants of the Lac Repressor
Authors: Barker Andrew , Oehler Stefan , Müller-Hill Benno
Published: December 2006

Thirteen of more than 4,000 single-amino-acid-replacement mutants of the Lac repressor, generated by suppression of amber nonsense mutants, were characterized as having a cold-sensitive phenotype. However, when expressed as missense mutations, none of the replacements cause cold sensitivity, implicating the suppression mechanism as being responsible for this phenotype.
Glucose Uptake Pathway-Specific Regulation of Synthesis of Neotrehalosadiamine, a Novel Autoinducer Produced in
Bacillus subtilis
Authors: Inaoka Takashi , Ochi Kozo
Published: December 2006

Neotrehalosadiamine (3,3′-diamino-3,3′-dideoxy-α,β-trehalose; NTD) is an amino-sugar antibiotic produced by several
Bacillus
species that functions as an autoinducer by activating its own biosynthetic operon,
ntdABC
. We previously reported that the introduction of a certain
rpoB
mutation (
rpoB5
) into
Bacillus subtilis
enables the cells to overproduce NTD.
B. subtilis
mini-Tn
10
transposant libraries have been screened for genes that affect NTD production. Inactivation of
ccpA
, which encodes a major transcriptional regulator of carbon catabolite regulation, markedly reduced NTD production. By contrast, inactivation of
glcP
, which is situated just downstream of
ntdABC
and encodes a glucose/mannose:H
+
symport permease, stimulated NTD production. Overexpression of
glcP
led to the repression of
ntdABC
expression (and thus NTD production) in response to GlcP-mediated glucose uptake. These results suggest that CcpA-mediated catabolite activation of
ntdABC
expression occurs in response to the increase of the in vivo concentration of fructose-1,6-bisphosphate via glucose-6-phosphate and that GlcP-mediated glucose repression of
ntdABC
expression occurs in association with the increase of the in vivo concentration of unphosphorylated glucose. In addition, Northern analysis showed that
glcP
is transcribed from the
ntdABC
promoter through transcription readthrough at the
ntdABC
transcription terminator site, which enables NTD to function as a modulator of glucose uptake through the stimulation of
ntdABC-glcP
transcription, even in wild-type (
rpoB
+
) cells. A trace amount (0.5 to 3 μg/ml) of NTD was sufficient to ensure expression of
glcP
, thus demonstrating the physiological role of “antibiotic” in the producing bacteria by functioning as an autoinducer for glucose uptake modulation.
Genome Analyses of Three Strains of
Rhodobacter sphaeroides
: Evidence of Rapid Evolution of Chromosome II
Authors: Choudhary M. , Zanhua Xie , Fu Y. X. , Kaplan S.
Published: December 2006

Three strains of
Rhodobacter sphaeroides
of diverse origin have been under investigation in our laboratory for their genome complexities, including the presence of multiple chromosomes and the distribution of essential genes within their genomes. The genome of
R. sphaeroides
2.4.1 has been completely sequenced and fully annotated, and now two additional strains (ATCC 17019 and ATCC 17025) of
R. sphaeroides
have been sequenced. Thus, genome comparisons have become a useful approach in determining the evolutionary relationships among different strains of
R. sphaeroides
. In this study, the concatenated chromosomal sequences from the three strains of
R. sphaeroides
were aligned, using Mauve, to examine the extent of shared DNA regions and the degree of relatedness among their chromosome-specific DNA sequences. In addition, the exact intra- and interchromosomal DNA duplications were analyzed using Mummer. Genome analyses employing these two independent approaches revealed that strain ATCC 17025 diverged considerably from the other two strains, 2.4.1 and ATCC 17029, and that the two latter strains are more closely related to one another. Results further demonstrated that chromosome II (CII)-specific DNA sequences of
R. sphaeroides
have rapidly evolved, while CI-specific DNA sequences have remained highly conserved. Aside from the size variation of CII of
R. sphaeroides
, variation in sequence lengths of the CII-shared DNA regions and their high sequence divergence among strains of
R. sphaeroides
suggest the involvement of CII in the evolution of strain-specific genomic rearrangements, perhaps requiring strains to adapt in specialized niches.
Characterization of the Transcriptional Activity of the Cryptic Plasmid pRN1 fromSulfolobus islandicusREN1H1 and Regulation of Its Replication Operon
Authors: Berkner Silvia , Lipps Georg
Published: December 2006

The plasmid pRN1 fromSulfolobus islandicusREN1H1 belongs to the crenarchaeal plasmid family pRN. The plasmids in this family encode three conserved proteins that participate in plasmid replication and copy number regulation, as suggested by biochemical characterization of the recombinant proteins. In order to deepen our understanding of the molecular biology of these plasmids, we investigated the transcriptional activity of the model plasmid pRN1. We detected five major transcripts present at about 2 to 15 copies per cell. One long transcriptional unit comprises the genes for the plasmid-copy-number control protein Orf56/CopG and the replication protein Orf904. A second transcript with a long 3′-untranslated region codes for the DNA binding protein Orf80. For both transcripts, we identified countertranscripts which could play a regulatory role. The function of the fifth transcript is unclear. For the five transcripts, we determined the start site, the transcript end, the stability, and the abundance in different growth phases. Reporter gene experiments demonstrated that the copy number control protein Orf56 represses transcription of theorf56-orf904cotranscript in vivo.
Functional Analysis of PilT from the Toxic Cyanobacterium
Microcystis aeruginosa
PCC 7806
Authors: Nakasugi Kenlee , Alexova Ralitza , Svenson Charles J. , Neilan Brett A.
Published: December 2006

The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing
Microcystis aeruginosa
PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes
pilA
,
pilB
,
pilC
, and
pilT
were shown to be expressed in
M. aeruginosa
PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 ± 1.8 nmol P
i
min
−1
mg protein
−1
, with a requirement for Mg
2+
. Heterologous expression indicated that it could complement the
pilT
mutant of
Pseudomonas aeruginosa
, but not that of the cyanobacterium
Synechocystis
sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the
M. aeruginosa
PCC 7806 PilT (7806 PilT) and the
Synechocystis
sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the
pilT
gene in toxic and nontoxic strains of
Microcystis
was also performed.
In Vitro Analysis of Rates and Spectra of Mutations in a Polymorphic Region of the Rv0746 PE_PGRS Gene of
Mycobacterium tuberculosis
Authors: Machowski Edith E. , Barichievy Samantha , Springer Burkhard , Durbach Steven I. , Mizrahi Valerie
Published: December 2006

An assay modeled on a known polymorphism in the PE_PGRS9 gene of
Mycobacterium tuberculosis
was designed to assess the mutability of a sequence containing interspersed PGRS repeats. Application of the assay in
Mycobacterium smegmatis
revealed sequence plasticity: in addition to recapitulating the mutation on which it was based, other mutations likely mediated by replication slippage between PGRS repeats were detected. However, the mutation rates argued against marked hypermutability of such sequences in mycobacteria.
Physiologic Effects of Forced Down-Regulation of
dnaK
and
groEL
Expression in
Streptococcus mutans
Authors: Lemos José A. , Luzardo Yaima , Burne Robert A.
Published: December 2006

Strains of
Streptococcus mutans
lacking DnaK or GroEL appear not to be isolable. To better distinguish the roles played by these chaperones/chaperonins in the physiology of
S. mutans
, we created a knockdown strategy to lower the levels of DnaK by over 95% in strain SM12 and the level of GroEL about 80% in strain SM13. Interestingly, GroEL levels were approximately twofold higher in SM12 than in the parent strain, but the levels of DnaK were not altered in the GroEL knockdown strain. Both SM12 and SM13 grew slower than the parent strain, had a strong tendency to aggregate in broth culture, and showed major changes in their proteomes. Compared with the wild-type strain, SM12 and SM13 had impaired biofilm-forming capacities when grown in the presence of glucose. The SM12 strain was impaired in its capacity to grow at 44°C or at pH 5.0 and was more susceptible to H
2
O
2
, whereas SM13 behaved like the wild-type strain under these conditions. Phenotypical reversions were noted for both mutants when cells were grown in continuous culture at a low pH, suggesting the occurrence of compensatory mutations. These results demonstrate that DnaK and GroEL differentially affect the expression of key virulence traits, including biofilm formation and acid tolerance, and support that these chaperones have evolved to accommodate unique roles in the context of this organism and its niche.
The significance of substance P in physiological and malignant haematopoiesis: Figure 1
Authors: Nowicki Michal , Ostalska-Nowicka Danuta , Kondraciuk Beata , Miskowiak Bogdan
Published: December 2006

The role of substance P (SP) in physiological haematopoiesis is well established. However, it also seems to be important in the neoplastic transformation of bone marrow, leading to the development of acute leukaemia in children, and also metastases to bone marrow of solid tumours (particularly neuroblastoma and breast cancer) in early stages of these diseases. This review summarises the available data on SP involvement in both processes. In the future, SP antagonists may be used as anti-neoplastic drugs, for example by direct or indirect blocking of tumour cell proliferation through inhibition of growth factor production and interleukin-1b synthesis.
SequentialWT1andCTNNB1mutations and alterations of β-catenin localisation in intralobar nephrogenic rests and associated Wilms tumours: two case studies
Authors: Fukuzawa Ryuji , Heathcott Rosemary W , More Helen E , Reeve Anthony E
Published: December 2006

Background:Intralobar nephrogenic rests (ILNRs) are precursor lesions for Wilms tumours and are associated withWT1gene mutations. ILNR-associated Wilms tumours have a co-clustering ofWT1and β-catenin (CTNNB1) mutations and unique histological features characterised by a stromal-predominant histology.

Aim:To determine the order in whichWT1andCTNNB1mutations occur to understand the ILNR–Wilms tumour sequence.

Methods:Of nine Wilms tumours withWT1andCTNNB1mutations, three ILNRs lesions in two Wilms tumours were available for analysis ofWT1andCTNNB1mutations using microdissection. Immunohistochemistry was also performed to investigate how the mutations in β-catenin alter the localisation in Wilms tumour development.

Results:WT1mutations were present in the ILNRs, howeverCTNNB1mutations were absent. Immunohistochemistry for WT1 confirmed inactivation of WT1 in both ILNRs and Wilms tumours. Both the ILNRs and the associated Wilms tumours had similar immunostaining patterns for β-catenin in the blastemal and epithelial components. Although rhabdomyoblasts were not included in ILNRs, the associated Wilms tumours showed rhabdomyogenic differentiation with a positive β-catenin nuclear staining.

Conclusions:The results suggest thatCTNNB1mutation is a later event in Wilms tumourigenesis.CTNNB1mutations might be associated with rhabdomyogenesis.
Best practice in primary care pathology: review 8
Authors: Smellie W S A , Hampton K K , Bowlees R , Martin S C , Shaw N , Hoffman J , Ng J P , Mackenzie S M , van Heyningen C
Published: December 2006

This eighth best practice review examines four series of common primary care questions in laboratory medicine: (i) sodium abnormalities; (ii) faecal occult blood testing; (iii) warfarin management; and (iv) sputum cytology in diagnosis of bronchopulmonary malignancy. The review is presented in question–answer format, referenced for each question series. The recommendations represent a précis of guidance found using a standardised literature search of national and international guidance notes, consensus statements, health policy documents and evidence-based medicine reviews, supplemented by Medline Embase searches to identify relevant primary research documents. They are not standards but form a guide to be set in the clinical context. Most are consensus rather than evidence-based. They will be updated periodically to take account of new information.
Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition prevents endothelin A receptor antagonism in rat aorta
Authors: Tosun M. , Erac Y. , Selli C. , Karakaya N.
Published: December 2006

This study tested whether sarcoplasmic-endoplasmic reticulum Ca2+-ATPase regulates the ability of endothelin receptor antagonist to inhibit the endothelin-1 constriction. The endothelin A receptor antagonist BQ-123 (1 μM) completely relaxed constriction to 10 nM endothelin-1 in endothelium-denuded rat aorta. Challenge with cyclopiazonic acid (10 μM), a sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibitor, during the plateau of endothelin-1 constriction enhanced the constriction by ∼30%. BQ-123 relaxed the endothelin-1 plus cyclopiazonic acid constriction by only ∼10%. In contrast, prazosin (1 μM), an α-adrenergic receptor antagonist, still completely relaxed the 0.3 μM phenylephrine constriction in the presence of cyclopiazonic acid. Verapamil relaxed the endothelin-1 plus cyclopiazonic acid constriction by ∼30%, whereas Ni2+and 2-aminoethoxydiphenyl borate, nonselective cation channel and store-operated channel blockers, respectively, completely relaxed the constriction. These results suggest that lowered sarcoplasmic-endoplasmic reticulum Ca2+-ATPase activity selectively decreases the ability of endothelin receptor antagonist to inhibit the endothelin A receptor. The decreased antagonism may be related to the opening of store-operated channels and subsequent greater internalization of endothelin A receptor.
Real-time imaging of mechanically injured femoral artery in mice reveals a biphasic pattern of leukocyte accumulation
Authors: Osaka Mizuko , Hagita Sumihiko , Haraguchi Mihoko , Kajimura Mayumi , Suematsu Makoto , Yoshida Masayuki
Published: December 2006

Wire injury of an artery has been recognized as a standard model of vascular inflammation and atherosclerosis; however, the mechanism of leukocyte recruitment has not been studied in this model. In this study, we documented the recruitment of leukocytes to the murine femoral artery after a wire injury. A transluminal mechanical injury was generated by insertion of a wire into the femoral artery of male C57BL/6J mice. The mice were anesthetized and ventilated after tracheotomy and protected from hypothermia by a warming lamp. Body temperature and blood pH did not significantly change during the experiment. The interaction between rhodamine 6G-labeled leukocytes and the injured femoral artery was monitored using an epifluorescent microscope, and the images were evaluated using a computer-assisted image analysis program. In the absence of injury, virtually no leukocyte adhesion was observed. In contrast, the number of adherent leukocytes increased 4 and 24 h after injury and declined 72 h after injury. The rolling flux of leukocytes increased 4 h after injury and remained high up to 7 days, but it was faster 72 h after injury. We identified another peak of leukocyte adhesion 7 days after injury. Injection of anti-P-selectin antibody significantly reduced leukocyte adhesion at the early and later phases. In conclusion, we have established a novel experimental system for direct observation of leukocyte recruitment to the injured femoral artery. Our system revealed a previously undetected, unique profile of leukocyte recruitment during vascular injury.