bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryThe 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

SummaryThe 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

SummaryThe 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

SummaryEnteropathogenic 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

SummaryMultidrug 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

SummaryListeriolysin 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

SummaryThe 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

Summary 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

SummaryIn 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

SummaryWhen 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

SummaryBacterial 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

SummaryMembers 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

SummaryRelE 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.