bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of phenyllactate dehydratase and its initiator from Clostridium sporogenes
Authors: Dickert Sandra , Pierik Antonio J. , Buckel Wolfgang
Published: March 2003

The heterotrimeric phenyllactate dehydratase from Clostridium sporogenes,
FldABC, catalyses the reversible dehydration of (R)‐phenyllactate to (E)‐cinnamate
in two steps: (i) CoA‐transfer from the cofactor cinnamoyl‐CoA to phenyllactate to
yield phenyllactyl‐CoA and the product cinnamate mediated by FldA, a (R)‐phenyllactate
CoA‐transferase; followed by (ii) dehydration of phenyllactyl‐CoA to cinnamoyl‐CoA
mediated by heterodimeric FldBC, a phenyllactyl‐CoA dehydratase. Phenyllactate dehydratase
requires initiation by ATP, MgCl2 and a reducing agent such as dithionite
mediated by an extremely oxygen‐sensitive initiator protein (FldI) present in the
cell‐free extract. All four genes coding for these proteins were cloned and shown
to be clustered in the order fldAIBC, which shares over 95% sequence
identity of nucleotide and protein levels with a gene cluster detected in the genome
of the closely related Clostridium botulinum Hall strain A. FldA shows sequence
similarities to a new family of CoA‐transferases, which apparently do not form covalent
enzyme CoA‐ester intermediates. An N‐terminal Strep II‐Tag containing enzymatically
active FldI was overproduced and purified from Escherichia coli. FldI was
characterized as a homodimeric protein, which contains one [4Fe‐4S]1+/2+
cluster with an electron spin S= 3/2 in the reduced form. The amino
acid sequence as well as the chemical and EPR‐properties of the pure protein are
very similar to those of component A of 2‐hydroxyglutaryl‐CoA dehydratase from Acidaminococcus
fermentans (HgdC), which was able to replace FldI in the activation of phenyllactate
dehydratase. Only in the oxidized state, FldI and component A exhibit significant
ATPase activity, which appears to be essential for unidirectional electron transfer.
Both subunits of phenyllactyl‐CoA dehydratase (FldBC) show significant sequence similarities
to both subunits of 2‐hydroxyglutaryl‐CoA dehydratase (HgdAB). The fldAIBC
gene cluster resembles the hadAIBC gene cluster in the genome of Clostridium
difficile and the hadABC,I genes in C. botulinum. The four subunits
of these deduced 2‐hydroxyacid dehydratases (65–81%
amino acid sequence identity between the had genes) probably code for a 2‐hydroxyisocaproate
dehydratase involved in leucine fermentation. This enzyme could be the target for
metronidazole in the treatment of pseudomembranous enterocolitis caused by C.
difficile.
StoPK‐1, a serine/threonine protein kinase from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009, affects oxidative stress response
Authors: Neu John M. , MacMillan Susan V. , Nodwell Justin R. , Wright Gerard D.
Published: March 2003

The glycopeptide antibiotic‐producing bacterium, Streptomyces toyocaensis NRRL 15009, has proteins phosphorylated on Ser, Thr, Tyr and His, implying the presence of a battery of associated kinases. We have identified the Ser/Thr protein kinase gene fragments stoPK‐1, stoPK‐2, stoPK‐3 and stoPK‐4 from S. toyocaensis NRRL 15009 by a polymerase chain reaction (PCR) strategy using oligonucleotide primers based on eukaryotic Ser/Thr and Tyr kinase sequences. One of these (stoPK‐1) was subsequently cloned in its entirety from a 3.2 kb genomic BamHI fragment. stoPK‐1 encodes a 642‐amino‐acid protein with a predicted N‐terminal Ser/Thr kinase domain and a C‐terminal coiled‐coil region divided by a membrane‐spanning region. Expression of StoPK‐1 in Escherichia coli yielded a protein confined to the membrane fraction, which was found to be phosphorylated exclusively on Thr residues and could transfer phosphate to the model substrates myelin basic protein and histone H1. Both autophosphorylation and phosphoryl transfer could be inhibited by the flavanoid apigenin. Disruption of stoPK‐1 with the apramycin resistance gene in the S. toyo‐caensis chromosome resulted in changes in mycelial morphology and an increased sensitivity to the redox cycling agents paraquat and nitrofurantoin on glucose‐containing media. Supplying stoPK‐1 or the S. coelicolor homologue pkaF in trans could reverse this sensitivity, whereas a catalytically inactive mutant of stoPK‐1 could not, indicating that kinase activity is essential for this phenotype. This suggests a link between this membrane‐bound protein kinase in signalling pathways sensitive to oxidative stress and/or glucose metabolism. These results broaden the roles of Ser/Thr protein kinases in bacteria and underscore the diversity of signal transduction mechanisms available to respond to various stimuli.
Quantification of known components of the Escherichia coli TonB energy transduction system: TonB, ExbB, ExbD and FepA
Authors: Higgs Penelope I. , Larsen Ray A. , Postle Kathleen
Published: March 2003

The TonB‐dependent energy transduction system couples cytoplasmic membrane proton motive force to active transport of iron–siderophore complexes across the outer membrane in Gram‐negative bacteria. In Escherichia coli, the primary

players known in this process to date are: FepA, the TonB‐gated transporter for the

siderophore enterochelin; TonB, the energy‐transducing protein; and two cytoplasmic

membrane proteins with less defined roles, ExbB and ExbD. In this study, we report

the per cell numbers of TonB, ExbB, ExbD and FepA for cells grown under iron‐replete

and iron‐limited conditions. Under iron‐replete conditions, TonB and FepA were present

at 335 ± 78 and 504 ± 165 copies per cell respectively.

ExbB and ExbD, despite being encoded from the same operon, were not equimolar, being

present at 2463 ± 522 and 741 ± 105 copies respectively. The ratio of these proteins was calculated at one TonB:two ExbD:seven ExbB under all four growth conditions tested. In contrast, the TonB:FepA ratio varied with iron status and according to the method used for iron limitation. Differences in the method of iron limitation also resulted in significant differences in cell size, skewing the per cell copy numbers for all proteins.
Multiple mechanisms for the activation of human platelet aggregation by Staphylococcus aureus: roles for the clumping factors ClfA and ClfB, the serine–aspartate repeat protein SdrE and protein A
Authors: O´Brien Louise , Kerrigan Steven W. , Kaw Gideon , Hogan Michael , Penadés José , Litt David , Fitzgerald Desmond J. , Foster Timothy J. , Cox Dermot
Published: March 2003

The ability of Staphylococcus aureus cells to induce platelet aggregation
has long been recognized. However, despite several attempts to identify the mechanisms
involved in this interaction, the nature of the bacterial receptors required remains
poorly understood. Using genetic manipulation, this study for the first time provides
clear evidence that several S. aureus surface proteins participate in the
inter‐action with platelets. Mutants of S. aureus strain Newman lacking one
or more surface proteins were tested for their ability to stimulate platelet aggre‐gation.
This approach was complemented by the expression of a number of candidate proteins
in the non‐aggregating Gram‐positive bacterium Lacto‐coccus lactis. S.
aureus‐induced aggregation was monophasic and was dependent on the platelet receptor
GPIIb/IIIa. The fibrinogen‐binding proteins, clumping factors A and B and the serine‐aspartate
repeat protein SdrE could each induce aggregation when expressed in L. lactis.
Although protein A expressed in L. lactis was not capable of inducing aggregation
independently, it enhanced the aggregation response when expressed on the surface
of S. aureus. Thus, S. aureus has multiple mechanisms for stimulating platelet aggregation. Such functional redundancy suggests that this phenomenon may be important in the pathogenesis of invasive diseases such as infective endocarditis.
Co‐ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules
Authors: Kenny Brendan , Ellis Sarah , Leard Alan D. , Warawa Jonathan , Mellor Harry , Jepson Mark A.
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) is a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. A/E pathogens encode a type III secretion system to transfer effector proteins into host cells. The EPEC Tir effector protein acts as a receptor for the bacterial surface protein intimin and is involved in the formation of Cdc42‐independent, actin‐rich pedestal structures beneath the adhered bacteria. In this paper, we demonstrate that EPEC binding to HeLa cells also induces Tir‐independent, cytoskeletal rearrangement evidenced by the early, transient formation of filopodia‐like structures at sites of infection. Filopodia formation is dependent on expression of the EPEC Map effector molecule – a protein that targets mitochondria and induces their dysfunction. We show that Map‐induced filopodia formation is independent of mitochondrial targeting and is abolished by cellular expression of the Cdc42 inhibitory WASP‐CRIB domain, demonstrating that Map has at least two distinct functions in host cells. The transient nature of the filopodia is related to an ability of EPEC to downregulate Map‐induced cell signalling that, like pedestal formation, was dependent on both Tir and intimin proteins. The ability of Tir to downregulate filopodia was impaired by disrupting a putative GTPase‐activating protein (GAP) motif, suggesting that Tir may possess such a function, with its interaction with intimin triggering this activity. Furthermore, we also found that Map‐induced cell signalling inhibits pedestal formation, revealing that the cellular effects of Tir and Map must be co‐ordinately regu‐lated during infection. Possible implications of the multifunctional nature of EPEC effector molecules in pathogenesis are discussed.
Mystery of multidrug transporters: the answer can be simple
Authors: Neyfakh Alex A.
Published: March 2003

Multidrug efflux transporters, found in all living cells and protecting them from multiple structurally dissimilar hydrophobic toxins, have fascinated researchers for decades and presented a number of puzzling questions. These transporters demonstrate a remarkably broad substrate specificity, which seemingly contradicts established dogmas of biochemistry. Although sharing highly unusual properties, in some unexplained way, they have arisen multiple times in the evolution of several families of membrane proteins. Furthermore, the number of multidrug transporters encoded in each genome is so large that their role in cellular physiology has remained un‐certain. Recent advances in the structural analysis of a number of soluble multidrug‐recognizing proteins show that these proteins possess large hydrophobic binding sites and bind their substrates through a combination of a hydrophobic effect and electrostatic attraction, rather than by establishing a precise network of hydrogen bonds and other specific interactions characteristic of traditionally studied enzymes and receptors. Low‐resolution structural studies of multidrug transporters suggest that they possess similar large binding sites and may use similar simple principles of substrate recognition. This would explain not only their broad substrate specificity, but also their unusual evolutionary relationships and the apparent multiplicity in genomes of organisms of all evolutionary kingdoms. Although further structural studies will be needed to prove this hypothesis, it is already clear that the explanation of the puzzling phenomenon of multidrug efflux may not necessarily require any substantially new biochemical or biological principles.
Listeriolysin O secreted by Listeria monocytogenes induces NF‐κB signalling by activating the IκB kinase complex
Authors: Kayal Samer , Lilienbaum Alain , Join‐Lambert Olivier , Li Xiaoxia , Israël Alain , Berche Patrick
Published: March 2003

Listeriolysin O (LLO) is a pore‐forming cytolysin secreted by the pathogen Listeria
monocytogenes and is required for its intracellular survival. We recently demonstrated that in endothelial cells, LLO activates the NF‐κB signalling pathway. In this work, we studied the LLO‐induced molecular cascade of NF‐κB activation with a cellular model extensively used to analyse the signalling pathway of NF‐κB activation, i.e. the human embryonic kidney HEK‐293 cell line and its derivatives (transfectants or mutants). When the stably transfected derivative HEK‐293 cells expressing IL‐1RI were exposed to LLO, a strong NF‐κB activation was detected, contrasting with other cell lines (HEK‐293 wild type, HEK‐293.T and COS) expressing a very low level of IL‐1RI. Although a delayed kinetics of LLO‐dependent NF‐κB activation suggests an autocrine or paracrine IL‐1‐dependent pathway, we found that LLO‐dependent NF‐κB activation did not require the IL‐1 protein synthesis nor the interaction with the IL‐1RI specific receptor. Herein, we demonstrated that LLO‐dependent NF‐κB activation requires the activation of the IκB kinase β (IKKβ) subunit of IKK complex to phosphorylate and degrade cytoplasmic IκBα, a natural inhibitor of NF‐κB. The activation induced by LLO does not require the adapters MyD88 and IL‐1R‐associated kinase (IRAK). We suggested that LLO induces a distinct signalling pathway from that of IL‐1 and its receptor.
CsrA regulates glycogen biosynthesis by preventing translation of glgC in Escherichia coli
Authors: Baker Carol S. , Morozov Igor , Suzuki Kazushi , Romeo Tony , Babitzke Paul
Published: March 2003

The carbon storage regulatory system of Escherichia coli controls the expression
of genes involved in carbohydrate metabolism and cell motility. CsrA binding to glgCAP
transcripts inhibits glycogen metabolism by promoting glgCAP mRNA decay. CsrB
RNA functions as an antagonist of CsrA by sequestering this protein and preventing
its action. In this paper, we elucidate further the mechanism of CsrA‐mediated glgC
regulation. Results from gel shift assays demonstrate that several molecules of CsrA
can bind to each glgC transcript. RNA footprinting studies indicate that CsrA
binds to the glgCAP leader transcript at two positions. One of these sites
overlaps the glgC Shine–Dalgarno sequence, whereas the other CsrA target
is located further upstream in an RNA hairpin. Results from toeprint and cell‐free
translation experiments indicate that bound CsrA prevents ribosome binding to the
glgC Shine–Dalgarno sequence and that this reduces GlgC synthesis.
The effect of two deletions in the upstream binding site was examined. Both of these
deletions reduced, but did not eliminate, CsrA binding in vitro and CsrA‐dependent
regulation in vivo. Our findings establish that bound CsrA inhibits initiation
of glgC translation, thereby reducing glycogen biosynthesis. This inhibition
of translation probably contributes to destabilization of the glgC transcript that was observed previously.
CpsK of Streptococcus agalactiae exhibits α2,3‐sialyltransferase activity in Haemophilus ducreyi
Authors: Chaffin Donald O. , McKinnon Katherine , Rubens Craig E.
Published: March 2003

Streptococcus agalactiae
(GBS) is a major cause of serious newborn bacterial infections. Crucial to GBS evasion of host immunity is the production of a capsular polysaccharide (CPS) decorated with sialic acid, which inactivates the alternative complement pathway. The CPS operons of serotypes Ia and III GBS have been described, but the CPS sialyltransferase gene was not identified. We identified
cpsK
, an open reading frame in the CPS operon of most serotypes, which was homologous to the lipooligosaccharide (LOS) sialyltransferase gene,
lst
, of
Haemophilus ducreyi
. To determine if
cpsK
might encode a sialyltransferase, we complemented a
H. ducreyi lst
mutant with
cpsK
. CpsK was expressed in
H. ducreyi
and LOS was isolated and analysed for sialic acid content by SDS–PAGE and high‐performance liquid chromatography (HPLC). Sialo‐LOS was seen in the wild‐type,
cpsK‐
or
lst
‐complemented mutant strains, but not in the mutant without
cpsK
. Addition of Neu5Ac to the LOS was confirmed by mass spectro‐scopy. Lectin binding studies detected terminal Neu5Ac(
α
2
→
3)Gal
(β
1‐ on LOS produced by the wild‐type,
cpsK
or
lst
‐complemented mutant strain LOS, compared with the mutant alone. Our data charac‐terize the first sialyltransferase gene from a Gram‐ positive bacterium and provide compelling evidence that its product catalyses the
α
2,3 addition of Neu5Ac to
H. ducreyi
LOS and therefore the terminal side‐chain of GBS CPS. Phylogenetic studies further indicated that
lst
and
cpsK
are related but distinct from sialyltransferases of most other bacteria and, along with their similar codon usage bias and G
+
C content, suggests acquisition by lateral transfer from an ancestral low G
+ C organism.
Cooperation and competition in mismatch repair: very short‐patch repair and methyl‐directed mismatch repair in Escherichia coli
Authors: Bhagwat Ashok S. , Lieb Margaret
Published: March 2003

In Escherichia coli and related enteric bacteria, repair of base‐base mismatches is performed by two overlapping biochemical processes, methyl‐directed mismatch repair (MMR) and very short‐patch (VSP) repair. While MMR repairs replication errors, VSP repair corrects to C•G mispairs created by 5‐methylcytosine deamination to T. The efficiency of the two pathways changes during the bacterial life cycle; MMR is more efficient during exponential growth and VSP repair is more efficient during the stationary phase. VSP repair and MMR share two proteins, MutS and MutL, and although the two repair pathways are not equally dependent on these proteins, their dual use creates a competition within the cells between the repair processes. The structural and biochemical data on the endonuclease that initiates VSP repair, Vsr, suggest that this protein plays a role similar to MutH (also an endonuclease) in MMR. Biochemical and genetic studies of the two repair pathways have helped eliminate certain models for MMR and put restrictions on models that can be developed regarding either repair process. We review here recent information about the biochemistry of both repair processes and describe the balancing act performed by cells to optimize the competing processes during different phases of the bacterial life cycle.
Gene expression profiling of Escherichia coli growth transitions: an expanded stringent response model
Authors: Chang Dong‐Eun , Smalley Darren J. , Conway Tyrrell
Published: March 2003

When conditions cause bacterial growth to stop, extensive reprogramming of physiology and gene expression allows for the cell's survival. We used whole‐genome DNA arrays to determine the system response in Escherichia coli cells experiencing transient growth arrest caused by glucose–lactose diauxie and H2O2 treatment, and also entry into stationary phase. The results show that growth‐arrested cells induce stringent control of several gene systems. The vast majority of genes encoding the transcription and translation apparatus immediately downregulate, followed by a global return to steady state when growth resumes. Approximately one‐half of the amino acid biosynthesis genes downregulate during growth arrest, with the notable exception of the his operon, which transiently upregulates in the diauxie experiment. Nucleotide biosynthesis downregulates, a result that is again consistent with the stringent response. Likewise, aerobic metabolism downregulates during growth arrest, and the results led us to suggest a model for stringent control of the ArcA regulon. The stationary phase stress response fully induces during growth arrest, whether transient or permanent, in a manner consistent with known mechanisms related to stringent control. Cells similarly induce the addiction module anti‐toxin and toxin genes during growth arrest; the latter are known to inhibit translation and DNA replication. The results indicate that in all aspects of the response cells do not distinguish between transient and potentially permanent growth arrest (stationary phase). We introduce an expanded model for the stringent response that integrates induction of stationary phase survival genes and inhibition of transcription, translation and DNA replication. Central to the model is the reprogramming of transcription by guanosine tetraphosphate (ppGpp), which provides for the cell's rapid response to growth arrest and, by virtue of its brief half‐life, the ability to quickly resume growth as changing conditions allow.
Bacterial conjugation: a two‐step mechanism for DNA transport
Authors: Llosa Matxalen , Gomis‐Rüth F. Xavier , Coll Miquel , Cruz Fernando de la
Published: March 2003

Bacterial conjugation is a promiscuous DNA transport mechanism. Conjugative plasmids transfer themselves between most bacteria, thus being one of the main causal agents of the spread of antibiotic resistance among pathogenic bacteria. Moreover, DNA can be transferred conjugatively into eukaryotic host cells. In this review, we aim to address several basic questions regarding the DNA transfer mechanism. Conjugation can be visualized as a DNA rolling‐circle replication (RCR) system linked to a type IV secretion system (T4SS), the latter being macromolecular transporters widely involved in pathogenic mechanisms. The scheme ‘replication + secretion’ suggests how the mechanism would work on the DNA substrate and at the bacterial membrane. But, how do these two parts come into contact? Furthermore, how is the DNA transported? T4SS are known to be involved in protein secretion in different organisms, but DNA is a very different macromolecule. The so‐called coupling proteins could be the answer to both questions by performing a dual role in conjugation: coupling the two main components of the machinery (RCR and T4SS) and actively mediating DNA transport. We postulate that the T4SS is responsible for transport of the pilot protein (the relaxase) to the recipient. The DNA that is covalently linked to it is initially transported in a passive manner, trailing on the relaxase. We speculate that the pilus appendage could work as a needle, thrusting the substrate proteins to cross one or several membrane barriers into the recipient cytoplasm. This is the first step in conjugation. The second step is the active pumping of the DNA to the recipient, using the already available T4SS transport conduit. It is proposed that this second step is catalysed by the coupling proteins. Our ‘shoot and pump’ model solves the protein–DNA transport paradox of T4SS.
Identification of amino acids in the Dr adhesin required for binding to decay‐accelerating factor
Authors: Loy Cristina P. Van , Sokurenko Evgeni V. , Samudrala Ram , Moseley Steve L.
Published: March 2003

Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dra blood‐group antigen present on the complement regulatory and signalling molecule, decay‐accelerating factor (DAF). One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen. Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined. We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF. The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression. The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen. Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen. Over half of the mutants obtained had substitutions within amino acids 63–81. Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF.
Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins
Authors: Pedersen Kim , Christensen Susanne K. , Gerdes Kenn
Published: March 2003

RelE and ChpAK (MazF) toxins of Escherichia coli have previously been described as proteins that mediate efficient cell killing. We show here that induction of relE or chpAK transcription does not confer cell killing but, instead, induces a static condition in which the cells are still viable but unable to proliferate. Later induction of transcription of the antitoxin genes relB or chpAI fully reversed the static condition induced by RelE and ChpAK respectively. We also provide a mechanistic explanation for these findings. Thus, induction of relE transcription severely inhibited translation, whereas induction of chpAK transcription inhibited both translation and replication. Hence, most likely, lack of colony formation is due to inhibition of translation in the case of relE and inhibition of translation and/or replication in the case of chpAK. Consistent with this proposal, later induction of transcription of the cognate antitoxin genes simultaneously reversed cell stasis and the inhibitory effects of RelE and ChpAK on macromolecular syntheses. These results preclude that RelE and ChpAK mediate cell killing during the conditions used here. In vivo and in vitro analyses of a mutant RelE protein supported that inhibition of colony formation was due to inhibition of translation.
Fe(III)‐mediated cellular toxicity
Authors: Chamnongpol Sangpen , Dodson Walter , Cromie Michael J. , Harris Z. Leah , Groisman Eduardo A.
Published: March 2003

Because it can undergo reversible changes in oxidation state, iron is an excellent biocatalyst but also a potentially deleterious metal. Iron‐mediated toxicity has been ascribed to Fe(II), which reacts with oxygen to generate free radicals that damage macromolecules and cause cell death. However, we now report that Fe(III) exhibits microbicidal activity towards strains of Salmonella enterica, Escherichia coli and Klebsiella pneumoniae defective in the Fe(III)‐responding PmrA/PmrB signal transduction system. Fe(III) bound to a pmrA Salmonella mutant more effectively than to the isogenic wild‐type strain and exerted its microbicidal activity even under anaerobic conditions. Moreover, Fe(III) permeabilized the outer membrane of the pmrA mutant, rendering it susceptible to vancomycin, which is normally non‐toxic to Gram‐negative species. On the other hand, Fe(III) did not affect the viability of a mutant defective in Fur, the major regulator of cytosolic iron homeostasis, which is hypersensitive to Fe(II)‐mediated toxicity. A functional pmrA gene was necessary for bacterial survival in soil. Our results indicate that Fe(III) exerts its microbicidal activity by a mechanism that is oxygen independent and different from that mediated by Fe(II).
Conflicting needs for a Salmonella hypervirulence gene in host and non‐host environments
Authors: Mouslim Chakib , Hilbert Friederike , Huang Henry , Groisman Eduardo A.
Published: March 2003

The Gram‐negative pathogen Salmonella enterica harbours a periplasmic D‐Ala‐D‐Ala dipeptidase (termed PcgL), which confers the ability to grow on D‐Ala‐D‐Ala as sole carbon source. We now demonstrate that inactivation of the pcgL gene renders Salmonella hypervirulent. This phenotype results from the accumulation of peptidoglycan‐derived D‐Ala‐D‐Ala in the pcgL mutant and not from an intrinsically faster growth rate. Synthetic D‐Ala‐D‐Ala (but not L‐Ala‐L‐Ala or D‐Ala) increased the number of wild‐type Salmonella in the liver and spleen of mice within 24 h of injection, suggesting that D‐Ala‐D‐Ala interferes with some aspect of innate immunity. However, the pcgL mutant was unable to grow on D‐Ala‐D‐Ala as sole carbon source and was defective for survival in nutrient‐poor conditions. We identified clinical isolates lacking D‐Ala‐D‐Ala dipeptidase activity and unable to grow on D‐Ala‐D‐Ala because of inactivation of the pcgL gene. Our data suggest that genes (such as pcgL) that, when mutated make pathogens more virulent, may be retained because their contribution to pathogen fitness in non‐host environments outweighs potential advantages of the hypervirulent vari‐ant in the infected host.
Characterization of Cah, a calcium‐binding and heat‐extractable autotransporter protein of enterohaemorrhagic Escherichia coli
Authors: Torres Alfredo G. , Perna Nicole T. , Burland Valerie , Ruknudin Abdul , Blattner Fred R. , Kaper James B.
Published: March 2003

We have identified and characterized a protein of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 that shares homology with antigen 43 and AIDA‐I of E. coli. The gene encoding this protein consists of a 2850 bp open reading frame and was named cah for calcium binding antigen 43 homologue. The prototype EHEC strain EDL933 possesses identical duplicate copies of cah (cah1 and cah2), which showed 100% identity at the nucleotide level. We showed that E. coli K‐12 containing the recombinant cah gene produced two proteins, an ≈ 80 kDa outer membrane protein and a 43.0 kDa heat‐extractable protein. The Cah protein contains a predicted 52‐amino‐acid extended signal sequence found in several autotransporter proteins, and N‐terminal sequencing data indicated that the 43.0 kDa passenger protein was derived from cleavage of the signal sequence from alanine at position 53. Phenotypes such as autoaggregation and change in bacterial shape were observed when a recombinant plasmid containing the cah gene was introduced into a laboratory E. coli strain, and these phenotypes were eliminated upon mutation of the cah gene. The passenger domain contains six domains found in calcium‐binding proteins, and the recombinant Cah passenger protein bound 45Ca2+. In E. coli O157:H7, Cah is a heat‐extractable protein, the expression of which is induced in minimal essential media and under divalent ion‐depleting conditions; it also participates in the formation of biofilms. Our results provide insight into the expression, secretion and preliminary features of the calcium‐binding Cah autotransporter protein of EHEC O157:H7.
A Salmonella SipB‐derived polypeptide blocks the ‘trigger’ mechanism of bacterial entry into eukaryotic cells
Authors: Hayward Richard D. , Hume Peter J. , McGhie Emma J. , Koronakis Vassilis
Published: March 2003

Entry into non‐phagocytic mammalian cells by the invasive pathogens Salmonella and Shigella is triggered by the delivery of bacterial virulence effector proteins into the host cell. This is dependent upon Salmonella SipB or its Shigella homologue IpaB, which insert into the eukaryotic cell plasma membrane. Here we show that a SipB‐derived 166 residue α‐helical polypeptide is a potent inhibitor of SipB‐directed liposome fusion in vitro, preventing the membrane‐associated form of SipB from inserting deeply into the bilayer. This polypeptide blocks Salmonella entry into cultured mammalian cells at 10−10 M, and is a heterologous inhibitor of analogous IpaB activity and Shigella cell entry. These findings reveal a potential strategy to identify inhibitors of the ‘trigger’ mechanism underlying cell entry by these major invasive pathogens.
IHF and HU stimulate assembly of pre‐replication complexes at Escherichia coli oriC by two different mechanisms
Authors: Ryan Valorie T. , Grimwade Julia E. , Nievera Christian J. , Leonard Alan C.
Published: March 2003

Pre‐replication complexes (pre‐RC) assemble on replication origins and unwind DNA in the presence of chromatin proteins. As components of Escherichia coli pre‐RC, two histone‐like proteins HU and IHF (integration host factor), stimulate initiator DnaA‐catalysed unwinding of the chromosomal replication origin, oriC. Using in vivo footprint analysis just before DNA synthesis initiates, we detect IHF binding coincident with a shift of DnaA to weaker central oriC sites. Integration host factor redistributed pre‐bound DnaA to identical sites in vitro. HU did not redistribute DnaA, but suppressed binding specifically at I3. These results suggest that different pathways mediated by bacterial chromatin proteins exist to regulate pre‐RC assembly and unwind oriC.
Inducible bacteriocin production in Lactobacillus is regulated by differential expression of the pln operons and by two antagonizing response regulators, the activity of which is enhanced upon phosphorylation
Authors: Diep Dzung B. , Myhre Ronny , Johnsborg Ola , Aakra Ågot , Nes Ingolf F.
Published: March 2003

Expression of the five (pln) operons involved in the bacteriocin production of Lactobacillus plantarum C11 is regulated by a so‐called pheromone‐based signal‐transducing network, in which the peptide pheromone (PlnA) induces bacteriocin production through the action of a histidine protein kinase (PlnB) and two antagonizing response regulators (PlnC as an activator and PlnD as a negative regulator). All pln‐regulated promoters contain a conserved pair of direct repeats that serve as binding sites for PlnC and PlnD. In the present work, we show that the five PlnA‐responsive operons are differentially expressed with regard to both timing and strength, and that the pheromone triggers a strong autoactivating loop of the regulatory unit (plnABCD) during an early stage of induction that gradually leads to enhanced activa‐tion of the other operons. The transport operon (plnGHSTUV), which is involved in the secretion of the pheromone and bacteriocins, is also expressed relatively early upon induction, but is quickly turned off soon after peak expression. Further investigation of the various promoters revealed that, although subtle differences within the promoter regions could account for the observed differential regulation, the presence of a downstream promoter‐proximal se‐quence in one promoter was found to cause delayed peak activity. How phosphorylation regulates the activity of the pln response regulators was also accessed by direct mutagenesis at their phosphorylation sites. It was found that the two response regulators exert activity at two different levels: a low level when they are not phosphorylated and an elevated level when they are phosphorylated. The present data demonstrate that bacteriocin production in L. plantarum C11 is a highly regulated process, in which different regulatory mechanisms are applied to fine tune the timing and strength of expression of the five pln operons.
Conservation of a gene conversion mechanism in two distantly related paralogues of Anaplasma marginale
Authors: Meeus Patrick F. M. , Brayton Kelly A. , Palmer Guy H. , Barbet Anthony F.
Published: March 2003

Anaplasmataceae, the causative agents of anaplasmosis and ehrlichiosis, persist in the bloodstream of their mammalian hosts, allowing acquisition and transmission by tick vectors. Anaplasma marginale establishes persistent infection characterized by sequential cycles of rickettsaemia in which new antigenic variants emerge. The two most immunodominant outer membrane proteins, MSP2 and MSP3, are paralogues, each encoded by a distinct family of related genes. This study demonstrates that, although the two gene families have diverged substantially, each has maintained a similar mechanism to generate structurally and antigenically polymorphic surface antigens. Like MSP2, MSP3 is expressed from a single locus in which variation of the expressed msp3 gene is generated by recombination using msp3 pseudogenes. Each of the msp3 pseudogenes encodes a unique central variable region (CVR) flanked by conserved 5′ and 3′ regions. Changes in the CVR of the expressed msp3, concomitant with invariance of the pseudogenes, indicate that expression site variation is generated using gene conversion. A. marginale thus maintains two large, separate systems within its small genome to generate antigenic variation of its surface proteins, while analogous structural elements indicate a common mechanism.
Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram‐positive bacteria
Authors: Chastanet Arnaud , Fert Juliette , Msadek Tarek
Published: March 2003

Multiple regulatory mechanisms for coping with stress co‐exist in low G+C Gram‐positive bacteria. Among these, the HrcA and CtsR repressors control distinct regulons in the model organism, Bacillus subtilis. We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus. Sequence analysis of the S. aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP‐dependent protease, as in B. subtilis, but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA. The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors. The S. aureus ctsR and hrcA genes were cloned under the control of the PxylA xylose‐inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B. subtilis as a heterologous host. Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S. aureus CtsR and HrcA proteins. ΔctsR, ΔhrcA and ΔctsRΔhrcA mutants of S. aureus were constructed, indicating that the two repressors are not redundant but, instead, act together synergistically to maintain low basal levels of expression of the dnaK and groESL operons in the absence of stress. This novel regulatory mode appears to be specific to Staphylococci.
The conserved cytoplasmic module of the transmembrane chemoreceptor McpC mediates carbohydrate chemotaxis in Bacillus subtilis
Authors: Kristich Christopher J. , Glekas George D. , Ordal George W.
Published: March 2003

Escherichia coli
cells use two distinct sensory circuits during chemotaxis towards carbohydrates. One circuit requires the phosphoenolpyruvate‐dependent phosphotransferase system (PTS) and is independent of any specific chemoreceptor, whereas the other uses a chemoreceptor‐dependent sensory mechanism analogous to that used during chemotaxis towards amino acids. Work on the carbohydrate chemotaxis sensory circuit of
Bacillus subtilis
reported in this article indicates that the
B. subtilis
circuit is different from either of those used by
E. coli
. Our chemotactic analysis of
B. subtilis
strains expressing various chimeric chemoreceptors indicates that the cytoplasmic, C‐terminal module of the chemoreceptor McpC acts as a sensory‐input element during carbohydrate chemotaxis
. Our results also indicate that PTS‐mediated carbohydrate transport, but not carbohydrate metabolism, is required for production of a chemotactic signal. We propose a model in which PTS‐transport‐induced chemotactic signals are transmitted to the C‐terminal module of McpC for control of chemotaxis towards PTS carbohydrates.
Fedotozine, a kappa‐opioid agonist, prevents spinal and supra‐spinal Fos expression induced by a noxious visceral stimulus in the rat
Authors: Bonaz  , Rivière  , Sinniger  , Pascaud  , Junien  , Fournet  , Feuerstein
Published: March 2003

Fedotozine, a kappa opioid agonist, reverses digestive ileus caused by acetic acid (AA)‐induced visceral pain in rats. The aims of this study were: to map, in conscious rats, central pathways activated by AA using Fos as a marker of neuronal activation;  to characterize primary afferent fibres involved in this activation; and  to investigate the effect of fedotozine on AA‐induced Fos expression. AA (0.6%; 10 mL kg–1) was injected i.p. in conscious rats either untreated; pretreated 14 days before with capsaicin; pretreated 20 min previously with fedotozine; or pretreated 2 h prior to fedotozine with the κ‐antagonist nor‐binaltorphimine (nor‐BNI). Controls received the vehicle alone. 60 min after injection of AA, rats were processed for Fos immunohistochemistry. Visceral pain was assessed by counting abdominal cramps. AA induced Fos in the thoraco‐lumbar spinal cord (laminae I, V, VII and X) and numerous brain structures such as the nucleus tractus solitarius, and paraventricular nucleus (PVN) of the hypothalamus, whereas almost no Fos labelling was observed in controls. Capsaicin pretreatment blocked AA‐induced Fos in all structures tested. Fedotozine significantly decreased AA‐induced abdominal cramps and Fos immunoreactivity in the spinal cord and PVN, this effect being reversed by nor‐BNI pretreatment.

AA induces Fos in the spinal cord and numerous brain nucuei, some of which are involved in the control of digestive motility in rats. This effect is mediated through capsaicin‐sensitive afferent fibres and prevented by fedotozine most likely through a peripheral action on visceral afferents.
A scintigraphic study of oropharyngeal swallowing dynamics in Chagas’ disease
Authors: Nobre E Souza  , Dantas  , De Oliveira  , Braga
Published: March 2003

We studied oropharyngeal function in patients with Chagas’ disease and oesophageal disease. Twenty‐four patients with Chagas’ disease, 17 of them with oesophageal disease, were submitted to scintigraphic oropharyngeal transit. A single swallow of a 10‐mL bolus of water labelled with 40 MBq 99m Tc‐phytate, followed by image acquisition at 20 images s–1 yielded time‐activity curves in which oropharyngeal dynamics was evaluated. Thirteen normal volunteers were used as controls. Oral transit was longer for chagasics with oesophageal disease than for controls (P=0.004) or for chagasics with a normal oesophagus (P=0.028). Oesophageal filling rate, measured by the slope of the ascending aspect of the oesophageal radioactivity curve, was lower for chagasics with oesophageal disease than for controls (P=0.037). Also, maximum oesophageal filling rate was lower (P=0.021) and occurred later (P=0.003) for chagasics with oesophageal disease than for controls. Pharyngeal clearance was positively correlated with oral transit (r=0.603, P=0.002). We conclude that chagasic oesophageal disease is associated with oral transit delay and a slower oesophageal filling rate.
Neuropeptide Y in the paraventricular nucleus of the hypothalamus stimulates colonic transit by peripheral cholinergic and central CRF pathways
Authors: Mönnikes  , Tebbe  , Bauer  , Grote  , Arnold
Published: March 2003

There is evidence suggesting that neuropeptide Y (NPY) as well as corticotropin‐releasing factor (CRF) in the paraventricular nucleus of the hypothalamus (PVN) are involved in the CNS regulation of gastrointestinal (GI) function. We studied the effects of NPY or Y1‐and Y2‐receptor agonists microinjected into the PVN on colonic transit. Microinjection of NPY into the PVN at doses of 0.15–1.5 μg decreased the colonic transit time of conscious rats up to 49%. Pretreatment with the peripherally acting cholinergic antagonist atropine methyl nitrate (0.1 mg kg−1 i.p.) blocked the NPY into PVN‐induced effect on colonic motor function.The agonist of the Y1‐receptor, NPY(Leu31, Pro34), as well as the Y2‐receptor agonist, NPY(13–36), dose‐dependently decreased colonic transit time when microinjected into the PVN (0.05, 0.15 and 0.5 μg). However, the Y1‐receptor agonist was more effective. Intracerebroventricular (ICV) application of the CRF‐receptor antagonist, α‐helical‐CRF9–41 (50 μg/rat), blocked the NPY effect in the PVN on colonic motor function. In conclusion, stimulation of colonic transit by NPY acting in the PVN was observed. The PVN is more sensitive to agonists acting on the Y1‐ than on the Y2‐receptor to mediate stimulation of propulsive colonic motility. The effect of NPY in the PVN on colonic motor function depends on central CRF and peripheral cholinergic pathways.
A nutrient drink test to assess maximum tolerated volume and postprandial symptoms: effects of gender, body mass index and age in health
Authors: Chial H. J. , Camilleri C. , Delgado‐Aros S. , Burton D. , Thomforde G. , Ferber I. , Camilleri M.
Published: March 2003

Abstract
 
To assess the effects of age, gender and body mass index on the maximum tolerated volume of a nutrient drink and postprandial symptoms in health. Healthy adolescents (15 M, 15 F, aged 13–17 years) and adults (15 M, 25 F, aged 19–51 years) ingested Ensure®(1 kcal mL−1) at a rate of 30 mL min−1. The maximum tolerated volume was recorded. Thirty minutes later, bloating, fullness, nausea and pain were rated using visual analogue scales. The Mann–Whitney test was used for comparisons between groups using body mass index and maximum tolerated volume as covariates. Age‐related differences in maximum tolerated volume were noted between adolescents and adults, and were observed in both genders. Adults had higher scores for bloating and pain, and lower scores for fullness. Gender‐related differences in maximum tolerated volume were noted in the group as a whole, and separately for adolescents and adults. Females had higher scores for nausea and pain. Gender and age‐related differences in the maximum tolerated volume of a nutrient drink and postprandial symptoms should be considered in future studies of upper gastrointestinal symptoms in disease. Body mass index does not appear to influence maximum tolerated volume beyond its association with age and gender.
In search of objective manometric criteria for colonic high‐amplitude propagated pressure waves
Authors: De Schryver A. M. P. , Samsom M. , Smout A. J. P. M.
Published: March 2003

Abstract
 The aims of this study were to explore all characteristics of high‐amplitude propagated contractions (HAPCs) that would allow them to be distinguished from nonHAPC colonic pressure waves, and to develop computer algorithms for automated HAPC detection. Colonic manometry recordings obtained from 24 healthy volunteers were used. Automated analysis was performed to detect propagated pressure waves and to determine their amplitude, duration and area under the curve (AUC). For each of these variables distribution plots were made. Automated HAPC counts were compared to visual counts by experienced investigators. Distribution plots of 141093 colonic pressure waves lacked a bimodal pattern, as was also the case for propagated contractions (n = 8758). With increasing high‐amplitude thresholds for HAPC detection, a gradual decrease in the automatically detected HAPC number was observed. These findings precluded determination of a threshold. Taking visually detected HAPCs as reference, amplitude thresholds of 100 mmHg in two channels, and 80 mmHg in one channel yielded the highest sensitivity (92%). In conclusion, objective criteria to distinguish HAPCs from other propagated pressure waves on the basis of their amplitude, duration or AUC do not exist. Automated detection of HAPCs using empirically derived criteria leads to an acceptable degree of correlation with visually detected HAPCs.