bluuebunny/crossref_metadata_2025 huggingface dataset:
The cytoplasmic domain of FtsK protein is required for resolution of chromosome dimers
Authors: Steiner Walter , Liu Guowen , Donachie William D. , Kuempel Peter
Published: March 2003

Chromosome dimers, formed by homologous recombination between sister chromosomes, normally require cell division to be resolved into monomers by site‐specific recombination at the dif locus of Escherichia coli. We report here that it is not in fact cell division per se that is required for dimer resolution but the action of the cytoplasmic domain of FtsK, which is a bifunctional protein required both for cell division and for chromosome partition.
Yersinia enterocolitica type III secretion: an mRNA signal that couples translation and secretion of YopQ
Authors: Anderson Deborah M. , Schneewind Olaf
Published: March 2003

Pathogenic Yersinia species export Yop proteins via a type III machinery to escape their phagocytic killing during animal infections. Here, we reveal the type III export mechanism of YopQ. In the presence of calcium, when type III secretion was blocked, yopQ mRNA was not translated. The signal of YopQ sufficient for the secretion of translationally fused reporter proteins was contained within the first 10 codons of its open reading frame. Some frameshift mutations that completely altered the peptide sequence specified by this signal did not impair secretion of the reporter protein. Exchanging the upstream untranslated mRNA leader of yopQ for that of E. coli lacZ also did not affect secretion. However, removal of the first 15 codons abolished YopQ export. Pulse‐labelled YopE, but not YopQ, could be secreted after the polypeptide had been synthesized within the cytoplasm of Yersinia (post‐translational secretion). Thus, YopQ appears to be exported by a mechanism that couples yopQ mRNA translation with the type III secretion of the encoded polypeptide.
Quorum sensing inVibrio fischeri: elements of theluxIpromoter
Authors: Egland Kristi A. , Greenberg E. P.
Published: March 2003

Although cell density‐dependent regulation of the luminescence genes inVibrio fischeriis a model for quorum sensing in Gram‐negative bacteria, relatively little is known about the promoter of the luminescence operon. The luminescence operon is activated by the LuxR protein, which requires a diffusible acylhomoserine lactone signal. Theluxbox, a 20 bp inverted repeat, is located in theluxIpromoter region and is required for LuxR‐dependent induction of the luminescence genes. Using primer extension, we mapped the LuxR‐dependent transcriptional start site of theluxoperon to 19 bp upstream of theluxIstart codon. This indicates that theluxbox is centred at −42.5 bp from the start of transcription. To gain evidence about the location of the −10 sequence, we placed a consensus −35 hexamer at different locations relative to theluxItranscriptional start site and measured constitutive levels of luminescence in recombinantEscherichia coli. The strongest constitutive promoter contained a TATAGT hexamer 17 bp from the −35 consensus sequence and 6 bp from the transcriptional start site. We propose that this is the −10 hexamer. Also in recombinantE. coli, both half‐sites of theluxbox were required for LuxR‐dependent gene activation and for activation by an autoinducer‐independent, monomeric LuxR deletion protein. LuxR‐dependent activation of luminescence was eliminated when theluxbox was centred at −47.5, −52.5 and −62.5 with respect to theluxItranscriptional start site. Our evidence, taken together with other information, points to a model in which a LuxR dimer overlaps the −35 region of theluxIpromoter and functions as an ambidextrous activator with each LuxR subunit interacting with a different region of RNA polymerase.
Transcription initiation in Archaea: facts, factors and future aspects
Authors: Soppa Jörg
Published: March 2003

The basal apparatus for transcription initiation in Archaea is more closely related to the eukaryal than to the bacterial counterpart. The understanding of archaeal transcription initiation has been deepened by recent advances, which include genome sequencing, biochemical approaches and the structure determination of a protein DNA complex. Archaeal promoter elements, transcription factors, RNA polymerase and their interactions are discussed and compared with the eukaryal situation. It is emerging that transcription initiation is not uniform in Archaea. A minimal set of promoter elements and transcription factors is conserved, but the relative importance for transcription initiation can vary. Furthermore, additional basal transcription factors and promoter elements seem to be crucial in subgroups of Archaea. Finally, some aspects of global as well as gene‐specific transcriptional regulation are discussed.
The lipopolysaccharide outer core of Yersinia enterocolitica serotype O:3 is required for virulence and plays a role in outer membrane integrity
Authors: Skurnik Mikael , Venho Reija , Bengoechea José‐Antonio , Moriyón Ignacio
Published: March 2003

Lipopolysaccharide (LPS) of Yersinia enterocolitica O:3 has an inner core linked to both the O‐antigen and to an outer core hexasaccharide that forms a branch. The biological role of the outer core was studied using polar and non‐polar mutants of the outer core biosynthetic operon. Analysis of O‐antigen‐ and outer core‐deficient strains suggested a critical role for the outer core in outer membrane properties relevant in resistance to antimicrobial peptides and permeability to hydrophobic agents, and indirectly relevant in resistance to killing by normal serum. Wild‐type bacteria but not outer core mutants killed intragastrically infected mice, and the intravenous lethal dose was ≈104‐fold higher for outer core mutants. After intragastric infection, outer core mutants colonized Peyer's patches and invaded mesenteric lymph nodes, spleen and liver, and induced protective immunity against wild‐type bacteria. In mice co‐infected intragastrically with an outer core mutant–wild type mixture, both strains colonized Peyer's patches similarly during the first 2 days, but the mutant was much less efficient in colonizing deeper organs and was cleared faster from Peyer's patches. The results demonstrate that outer core is required for Y. enterocolitica O:3 full virulence, and strongly suggest that it provides resistance against defence mechanisms (most probably those involving bactericidal peptides).
Pneumococcal licD2 gene is involved in phosphorylcholine metabolism
Authors: Zhang Jing‐Ren , Idanpaan‐Heikkila Ilona , Fischer Werner , Tuomanen Elaine I.
Published: March 2003

Phosphorylcholine is an important bioactive adduct to the teichoic acid (TA) and lipoteichoic acid (LTA) of the surface of Streptococcus pneumoniae. We have identified and characterized a genetic locus lic that is required for phosphorylcholine metabolism in S. pneumoniae. The pneumococcal lic locus consists of eight genes, licA, licB, licC and licD1, licD2 and three additional open reading frames. Pneumococcal licA, licB, licC, licD1 and licD2 have significant sequence similarity to licA, licB, licC and licD of Haemophilus influenzae. Mutation of licD2 led to decreased [3H]‐choline uptake, aberrant migration of LTA chains in SDS–PAGE gels, loss of several surface proteins, and a phase‐locked hypertransparent colony phenotype. Moreover, the licD2− mutant failed to undergo lysis after treatment with penicillin at high cell density and showed decreased transformation competence. Finally, the licD2− mutant demonstrated decreased adherence to the human type II alveolar cells, reduced nasopharyngeal colonization in infant rats, as well as significantly impaired virulence upon intraperitoneal challenge of CF1 mice. Identification of the lic genes in the pneumococcus will facilitate further characterization of the role of surface choline in microbial physiology and pathogenesis.
Genetic and functional characterization of the alpAB gene locus essential for the adhesion of Helicobacter pylori to human gastric tissue
Authors: Odenbreit Stefan , Till Markus , Hofreuter Dirk , Faller Gerhard , Haas Rainer
Published: March 2003

In this study, we isolated and characterized a chromosomal locus of Helicobacter pylori previously identified by transposon shuttle mutagenesis as being involved in the adhesion of the pathogen to gastric epithelial cells. Two closely homologous genes were identified, designated as alpA and alpB, encoding outer membrane (OM) proteins of 518 amino acids each. They are members of the outer membrane protein supergene family identified in the H. pylori 26695 complete genome sequence. AlpA carries a functional lipoprotein signal sequence. AlpB carries a putative standard N‐terminal signal sequence and shows a strong amino‐acid sequence identity to AlpA. Transposon insertion mutagenesis, immunoblotting and primer extension studies indicate that both genes are organized in an operon, but no obvious consensus promoter sequence was found upstream of the transcriptional start site. The C‐terminal portion of both proteins is predicted to form a porin‐like β‐barrel in the outer membrane, consisting of 14 transmembrane amphipathic β‐strands. Adhesion experiments with defined isogenic mutants indicate that both proteins are necessary for specific adherence of H. pylori to human gastric tissue. The pattern of AlpAB‐dependent adherence of H. pylori to the gastric epithelial surface shows a clear difference to the BabA2‐mediated adherence to Lewisb, suggesting that a different receptor is involved.
Single‐cell analysis of glycopeptide resistance gene expression in teicoplanin‐resistant mutants of a VanB‐type Enterococcus faecalis
Authors: Baptista Marina , Rodrigues Pierre , Depardieu Florence , Courvalin Patrice , Arthur Michel
Published: March 2003

The vanB gene cluster confers resistance to vancomycin but not to the related antibiotic teicoplanin, as the VanRBSB two‐component regulatory system triggers expression of the glycopeptide resistance genes only in response to vancomycin. The VanRB regulator activates promoters PRB and PYB for transcription of the regulatory (vanRBSB) and resistance (vanYBWHBBXB) genes respectively. The gfpmut1 gene encoding a green fluorescent protein was fused to PYB to analyse promoter activation in single cells by fluorescence microscopy and flow cytometry. Characterization of 17 teicoplanin‐resistant mutants indicated that amino acid substitutions on either side of the VanSB autophosphorylation site led to a constitutive phenotype. Substitutions in the membrane‐associated domain resulted in a gain of function, as they allowed induction by teicoplanin. A vanSB null mutant expressed gfpmut1 at various levels under non‐inducing conditions, and the majority of the bacteria were not fluorescent. Bacteria grown in the presence of vancomycin or teicoplanin were homogeneously fluorescent. The increase in the number of fluorescent bacteria resulted from induction in negative cells rather than from selection of a resistant subpopulation, indicating that VanRB was activated by cross‐talk. Transglycosylase inhibition was probably the stimulus for the heterologous kinase, as moenomycin was also an inducer.
Type IV pili and cell motility
Authors: Wall Daniel , Kaiser Dale
Published: March 2003

Type IV pili (Tfp) mediate the movement of bacteria over surfaces without the use of flagella. These movements are known as social gliding in Myxococcus xanthus and twitching in organisms such as Pseudomonas aeruginosa and Neisseria gonorrhoeae. Tfp are localized polarly. Type IV pilins have a signature N‐terminal domain, which forms a coiled‐coil with other monomer units to polymerize a pilus fibre. At least 10 more proteins at the base of the fibre are conserved; they are related to the type II secretion system. Movements produced by Tfp range from short, jerky displacements to lengthy, smooth ones. Tfp also participate in cell–cell interactions, pathogenesis, biofilm formation, natural DNA uptake, auto‐aggregation of cells and development. What is the means by which Tfp bring about the movement of cells?
Autoaggregation of Lactobacillus reuteri is mediated by a putative DEAD‐box helicase
Authors: Roos Stefan , Lindgren Sven , Jonsson Hans
Published: March 2003

We have cloned and sequenced a gene from Lactobacillus reuteri that encodes a 56 kDa protein, which mediates autoaggregation of the bacteria. Using an antiserum raised against extracellular proteins from the pig intestinal isolate L. reuteri 1063, we screened a genomic λ library derived from the same strain. Affinity purification of recombinant protein from the isolated λ clones showed that one type of clone expressed a protein that efficiently aggregated the parental strain when added to the bacteria. Subcloning and introduction of the corresponding gene, here denoted aggHinto the L. reuteri type strain markedly enhanced aggregation. Furthermore, insertional inactivation of aggH in strain 1063 resulted in an autoaggregation‐deficient phenotype. Finally, an affinity‐purified and cleaved fusion of AggH protein and the maltose‐binding protein, MBP, strongly promoted aggregation of L. reuteri 1063, whereas the uncleaved fusion protein was inactive. Sequencing of aggH revealed that the corresponding protein has extensive sequence homology to the large family of ATP‐dependent DEAD‐box helicases. These results are intriguing in view of earlier data on the promotion of genetic exchange in Lactobacillus by aggregation.
The stationary‐phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth
Authors: Santos Jorge M. , Freire Patrick , Vicente Miguel , Arraiano Cecília M.
Published: March 2003

The Escherichia coli morphogene bolA causes round morphology when overexpressed. The expression of bolA is mainly regulated by a σs‐dependent gearbox promoter bolA1p. Such regulation results in increased relative levels of expression at slow growth rates, as seen with those attained at the onset of stationary phase. We demonstrate that bolA1p is also induced during early logarithmic growth in response to several forms of stress, and that this induction can be partially σs independent. Sudden carbon starvation results in a 17‐fold increase in mRNA levels derived from bolA1p 1 h after stress imposition. Increased osmolarity results in a more than 20‐fold increase after the same period. Considerable increases in bolA1p mRNA levels were also detected as a result of heat shock, acidic stress and oxidative stress, which has been shown to inhibit σs translation. The orders of magnitude of bolA1p induction in log phase due to sudden starvation, osmotic shock and oxidative stress surpass the levels reached in stationary phase. Under sudden carbon starvation and osmotic shock, the cells changed their morphology, resembling those cells in which bolA is overexpressed in stationary phase. Increased expression and morphological changes due to sudden carbon starvation and osmotic shock still occur when σS is not present in a rpoS− background. The results show that expression of bolA is not confined to stationary phase, but it can also play an important role in general stress response. We propose that bolA1p stress induction overrides the normal regulation imposed by growth rate, which is strictly the result of σS‐directed transcription.
Iron acquisition systems in the pathogenic Neisseria
Authors: Schryvers Anthony B. , Stojiljkovic Igor
Published: March 2003

Pathogenic neisseriae have a repertoire of high‐affinity iron uptake systems to facilitate acquisition of this essential element in the human host. They possess surface receptor proteins that directly bind the extracellular host iron‐binding proteins transferrin and lactoferrin. Alternatively, they have siderophore receptors capable of scavenging iron when exogenous siderophores are present. Released intracellular haem iron present in the form of haemoglobin, haemoglobin–haptoglobin or free haem can be used directly as a source of iron for growth through direct binding by specific surface receptors. Although these receptors may vary in complexity and composition, the key protein involved in the transport of iron (as iron, haem or iron‐siderophore) across the outer membrane is a TonB‐dependent receptor with an overall structure presumably similar to that determined recently for Escherichia coli FhuA or FepA. The receptors are potentially ideal vaccine targets in view of their critical role in survival in the host. Preliminary pilot studies indicate that transferrin receptor‐based vaccines may be protective in humans.
Co‐ordinating DNA replication with cell division in bacteria: a link between the early stages of a round of replication and mid‐cell Z ring assembly
Authors: Harry E. J. , Rodwell J. , Wake R. G.
Published: March 2003

Spores of a thymine‐requiring strain of Bacillus subtilis 168, which is also temperature sensitive for the initiation of chromosome replication, were germinated and allowed to grow out at the permissive temperature in a minimal medium containing no added thymine. Under these conditions, there was no or very limited progression into the elongation phase of the first round of replication. In a significant proportion of the outgrown cells, a Z ring formed precisely at mid‐cell and over the centrally positioned nucleoid, leading eventually to the formation of a mature division septum. When initiation of the first round of replication was blocked through a temperature shift and with thymine present, the Z ring was positioned acentrally. The central Z ring that formed in the absence of thymine was blocked by the presence of a DNA polymerase III inhibitor. It is concluded that the very early stages of a round of replication (initiation plus possibly limited progression into the elongation phase) play a key role in the precise positioning of the Z ring at mid‐cell and between replicating daughter chromosomes.
The aerobactin iron transport system genes in Shigella flexneri are present within a pathogenicity island
Authors: Vokes Steven A. , Reeves Stephanie A. , Torres Alfredo G. , Payne Shelley M.
Published: March 2003

Genes encoding the synthesis and transport of aerobactin, a hydroxamate siderophore associated with increased virulence of enteric bacteria, were mapped within a pathogenicity island in Shigella flexneri. The island, designated SHI‐2 for Shigella pathogenicity island 2, was located downstream of selC, the site of insertion of pathogenicity islands in several other enteric pathogens. DNA sequence analysis revealed the presence of multiple insertion sequences upstream and downstream of the aerobactin genes and an integrase gene that was nearly identical to an int gene found in Escherichia coli O157:H7. SHI‐2 sequences adjacent to selC were similar to sequences at the junction between selC and pathogenicity islands found in E. coli O157:H7 and in enteropathogenic E. coli, but the junctions between the island and downstream yic genes were variable. SHI‐2 also encoded immunity to the normally plasmid‐encoded colicins I and V, suggesting a common origin for the aerobactin genes in both S. flexneri and E. coli pColV. Polymerase chain reaction and Southern hybridization data indicate that SHI‐2 is present in the same location in Shigella sonnei, but the aerobactin genes are not located within SHI‐2 in Shigella boydii or enteroinvasive E. coli. Shigella dysenteriae type 1 strains do not produce aerobactin but do contain sequences downstream of selC that are homologous to SHI‐2. The presence of the aerobactin genes on plasmids in E. coli pColV and Salmonella, on a pathogenicity island in S. flexneri and S. sonnei and in a different chromosomal location in S. boydii and some E. coli suggests that these virulence‐enhancing genes are mobile, and they may constitute an island within an island in S. flexneri.
The C‐terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo
Authors: Lopez Pascal J. , Marchand Isabelle , Joyce Susan A. , Dreyfus Marc
Published: March 2003

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C‐terminal half of this 1061‐residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a ‘degradosome’, only the N‐terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N‐terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C‐terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.
The Per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)‐encoded regulator (Ler)
Authors: Mellies Jay L. , Elliott Simon J. , Sperandio Vanessa , Donnenberg Michael S. , Kaper James B.
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) is the prototype organism of a group of pathogenic Gram‐negative bacteria that cause attaching and effacing (AE) intestinal lesions. All EPEC genes necessary for the AE phenotype are encoded within a 35.6 kb pathogenicity island termed the locus of enterocyte effacement (LEE). The LEE encodes 41 predicted open reading frames (ORFs), including components of a type III secretion apparatus and secreted molecules involved in the disruption of the host cell cytoskeleton. To initiate our studies on regulation of genes within the LEE, we determined the genetic organization of the LEE, defining transcriptional units and mapping transcriptional start points. We found that components of the type III secretion system are transcribed from three polycistronic operons designated LEE1, LEE2 and LEE3. The secreted Esp molecules are part of a fourth polycistronic operon designated LEE4. Using reporter gene fusion assays, we found that the previously described plasmid‐encoded regulator (Per) activated operons LEE1, LEE2 and LEE3, and modestly increased the expression of LEE4 in EPEC. Using single‐copy lacZ fusions in K‐12‐derived strains, we determined that Per only directly activated the LEE1::lacZ fusion, and did not directly activate the other operons. Orf1 of the LEE1 operon activated the expression of single‐copy LEE2::lacZ and LEE3::lacZ fusions in trans and modestly increased the expression of LEE4::lacZ in K‐12 strains. Orf1 was therefore designated Ler, for LEE‐encoded regulator. Thus, the four polycistronic operons of the LEE that encode type III secretion components and secreted molecules are now included in the Per regulon, where Ler participates in this novel regulatory cascade in EPEC.
Dynamics of multigene expression during catabolic adaptation of Ralstonia eutropha JMP134 (pJP4) to the herbicide 2,4‐dichlorophenoxyacetate
Authors: Leveau Johan H. J. , König Franziska , Füchslin Hanspeter , Werlen Christoph , Van Der Meer Jan Roelof
Published: March 2003

Ralstonia eutropha JMP134 carries a 22 kb DNA region on plasmid pJP4 necessary for the degradation of 2,4‐D (2,4‐dichlorophenoxyacetate). In this study, expression of the 2,4‐D pathway genes (designated tfd ) upon exposure to different concentrations of 2,4‐D was measured at a detailed timescale in chemostat‐grown R. eutropha cultures. A sharp increase in mRNA levels for tfdA, tfdCDEF‐B, tfdDIICIIEIIFII‐BII and tfdK was detected between 2 and 13 min after exposure to 2,4‐D. This response time was not dependent on the 2,4‐D concentration. The genes tfdA, tfdCD and tfdDIICII were expressed immediately upon induction, whereas tfdB, tfdBII and tfdK mRNAs could be detected only around 10 min later. The number of tfd mRNA transcripts per cell was estimated to be around 200–500 during maximal expression, after which they decreased to between 1 and 30 depending on the 2,4‐D concentration used for induction. Unlike the mRNAs, the specific activity of the 2,4‐D pathway enzyme chlorocatechol 1,2‐dioxygenase did not increase sharply but accumulated to a steady‐state plateau, which was dependent on the 2,4‐D concentration in the medium. At 1 mM 2,4‐D, several oscillations in mRNA levels were observed before steady‐state expression was reached, which was caused by transient accumulation of the first pathway intermediate, 2,4‐dichlorophenol, to toxic concentrations. Expression of tfdR and tfdS, the (identical) regulatory genes for the tfd pathway remained low and essentially unchanged during the entire adaptation phase.
The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor
Authors: Kaspar Sibylle , Perozzo Remo , Reinelt Stefan , Meyer Margareta , Pfister Karin , Scapozza Leonardo , Bott Michael
Published: March 2003

The two‐component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane‐bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE–CitAC) composed of the maltose‐binding protein and the CitA kinase domain (amino acids 327–547) showed constitutive autokinase activity and transferred the γ‐phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H‐350 and D‐56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB–D56N formed a stable complex with MalE–CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45–176) was overproduced as a soluble, cytoplasmic protein with a C‐terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri‐ and dicarboxylates tested, with high affinity (KD ≈ 5 μM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (ΔH = −76.3 kJ mol−1), whereas the entropy change was opposed (−TΔS = + 46.3 kJ mol−1). The pH dependency of the binding reaction indicated that the dianionic form H‐citrate2− is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg–citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.
The N‐ and C‐terminal domains of MecA recognize different partners in the competence molecular switch
Authors: Persuh M. , Turgay K. , Mandic‐Mulec I. , Dubnau D.
Published: March 2003

ComK is a transcription factor required for the expression of competence genes in Bacillus subtilis. Binding to MecA targets ComK for degradation by the ClpCP protease. MecA therefore acts as an adapter protein recruiting a regulatory protein for proteolysis. However, when ComS is synthesized, ComK is released from binding by MecA and thereby protected from degradation. MecA binds to three protein partners during these processes: ComK, ClpC and ComS. Using limited proteolysis, we have defined N‐ and C‐terminal structural domains of MecA and evaluated the interactions of these domains with the protein partners of MecA. Using surface plasmon resonance, we have determined that the N‐terminal domain of MecA interacts with ComK and ComS and the C‐terminal domain with ClpC. MecA is shown to exist as a dimer with dimerization sites on both the N‐ and C‐terminal domains. The C‐terminal domain stimulates the ATPase activity of ClpC and is degraded by the ClpCP protease, while the N‐terminal domain is inactive in both of these assays. In vivo data were consistent with these findings, as comG–lacZ expression was decreased in a strain overproducing the N‐terminal domain, indicating reduced ComK activity. We propose a model in which binding of ClpC to the C‐terminal domain of MecA induces a conformational change enabling the N‐terminal domain to bind ComK with enhanced affinity. MecA is widespread among Gram‐positive organisms and may act generally as an adapter protein, targeting proteins for regulated degradation.
MicroReview: Functional similarities among two‐component sensors and methyl‐accepting chemotaxis proteins suggest a role for linker region amphipathic helices in transmembrane signal transduction
Authors: Williams Stanly B. , Stewart Valley
Published: March 2003

Signal‐responsive components of transmembrane signal‐transducing regulatory systems include methyl‐accepting chemotaxis proteins and membrane‐bound, two‐component histidine kinases. Prokaryotes use these regulatory networks to channel environmental cues into adaptive responses. A typical network is highly discriminating, using a specific phosphoryl relay that connects particular signals to appropriate responses. Current understanding of transmembrane signal transduction includes periplasmic signal binding with the subsequent conformational changes being transduced, via transmembrane helix movements, into the sensory protein's cytoplasmic domain. These induced conformational changes bias the protein's regulatory function. Although the mutational analyses reviewed here identify a role for the linker region in transmembrane signal transduction, no specific mechanism of linker function has yet been described. We propose a speculative, mechanistic model for linker function based on interactions between two putative amphipathic helices. The model attempts to explain both mutant phenotypes and hybrid sensor data, while accounting for recognized features of amphipathic helices.
Regulated expression of the Shiga toxin B gene induces apoptosis in mammalian fibroblastic cells
Authors: Nakagawa Ichiro , Nakata Masanobu , Kawabata Shigetada , Hamada Shigeyuki
Published: March 2003

Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli may induce colonic ulceration, bloody diarrhoea and acute renal failure. The A subunit (StxA) is known to inhibit protein synthesis, whereas the B subunits (StxB) bind to Gb3 on the cell surface. However, the mechanisms by which Stxs kill target cells remain unclear. Stx1A or Stx1B genes were introduced into pcDNA3.1 vectors and transfected into NIH3T3 and HeLa cells. The Stx1B gene‐transfected cells became apoptotic with accompanying DNA fragmentation, whereas the Stx1A gene‐transfected cells were found to be necrotic and no DNA fragmentation occurred. The HeLa/C4 cells integrated with the Stx1B gene with a tetracycline‐inducible promoter eventually produced cytoplasmic Stx1B, leading to DNA fragmentation on the addition of doxycycline. These apoptotic changes were abrogated by pretreatment with Z‐VAD‐fmk. These results suggest that the transfected Stx1B gene induces apoptosis by activating the caspase cascade after Stx1B expression in the cytoplasm.
PrfA, the transcriptional activator of virulence genes, is upregulated during interaction of Listeria monocytogenes with mammalian cells and in eukaryotic cell extracts
Authors: Renzoni Adriana , Cossart Pascale , Dramsi Shaynoor
Published: March 2003

Most virulence genes of Listeria monocytogenes are activated by the transcriptional regulator PrfA. Previous studies have shown that environmental parameters, such as temperature, pH, stress conditions and medium composition, affect the expression of PrfA and PrfA‐dependent proteins. In this report, we demonstrate a threefold increase in PrfA protein synthesis during infection of mammalian cells, which correlates with the increased activity of the plcA promoter, the major prfA promoter. Increased PrfA synthesis begins when L. monocytogenes adheres to host cells. In addition, we show that the observed induction of PrfA during the interaction of L. monocytogenes with mammalian cells can be reproduced in vitro using total cell extracts. Our data suggest a role for host proteinase K‐sensitive protein(s) in PrfA upregulation.
The Brucella abortus host factor I (HF‐I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice
Authors: Robertson Gregory T. , Roop R. Martin
Published: March 2003

Brucella abortus is a facultative intracellular pathogen that causes abortion and infertility in domestic animals and a severe debilitating febrile illness in humans. The mechanisms that this highly successful intracellular pathogen uses to adapt to, and survive within, the harsh intracellular environment of the host macrophage are presently unknown. Maintenance of the stationary phase growth state has been proposed to be critical for the virulence of several mammalian pathogens, but analysis of this relationship for the brucellae has not been undertaken. In order to evaluate this relationship, we examined the in vitro and in vivo characteristics of an isogenic hfq mutant constructed from virulent Brucella abortus 2308. In Escherichia coli, the hfq gene product is an RNA‐binding protein that participates in the regulation of stationary phase stress resistance, at least partly by enhancing translation of the stationary phase‐specific sigma factor RpoS. As expected, the Brucella abortus hfq mutant, designated Hfq3, showed increased sensitivity to H2O2, and decreased survival under acidic conditions (pH 4.0), during stationary phase growth compared with 2308. Hfq3 was also less able to withstand prolonged starvation than 2308. The Brucella abortus hfq mutant, unlike its parental strain 2308, fails to replicate in cultured murine macrophages, and is rapidly cleared from the spleens and livers of experimentally infected BALB/c mice. These findings suggest that the Brucella abortus hfq gene product makes an essential contribution to pathogenesis in mice, probably by allowing the brucellae to adapt appropriately to the harsh environmental conditions encountered within the host macrophage.
A response regulator‐like protein that functions at an intermediate stage of sporulation in Streptomyces coelicolor A3(2)
Authors: Aínsa José A. , Parry Huw D. , Chater Keith F.
Published: March 2003

whiI is one of several loci originally described as essential for sporulation in Streptomyces coelicolor A3(2). We have characterized whiI at the molecular level. It encodes an atypical member of the response regulator family of proteins, lacking at least two of the residues strongly conserved in the conventional phosphorylation pocket. It is not adjacent to a potential sensor kinase gene. Fifteen mutant alleles of whiI were sequenced, revealing, among others, six mutations affecting conserved amino acids, several frameshift mutations and one mutation in the promoter. The whiI promoter is specifically transcribed by the sporulation‐specific σWhiG‐containing form of RNA polymerase. Transcription of whiI is temporally controlled, reaching a maximum level coincident with the formation of spores. Further transcriptional studies suggested that WhiI is involved directly or indirectly in repressing its own expression and that of another σWhiG‐dependent sporulation‐specific regulatory gene, whiH.
A trans‐envelope protein complex needed for filamentous phage assembly and export
Authors: Feng Jian‐nong , Model Peter , Russel Marjorie
Published: March 2003

Assembly and export of filamentous phage requires four non‐capsid proteins: the outer membrane protein, pIV; the inner membrane proteins, pI and pXI; and a cytoplasmic host factor, thioredoxin. Chemical cross‐linking of intact cells demonstrates a trans‐membrane complex containing pI and pIV. Formation of the complex protects pI from proteolytic cleavage by an endogenous protease. This protection also requires pXI, which is identical to the C‐terminal portion of pI. This indicates that pXI, which is required for phage assembly in its own right, is also part of the complex. This complex forms in the absence of any other phage proteins or the DNA substrate; hence, it represents the first preinitiation step of phage morphogenesis. On the basis of protease protection data, we propose that the preinitiation complex is converted to an initiation complex by binding phage DNA, thioredoxin and the initiating minor coat protein(s).
Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans
Authors: Sonneborn Anja , Bockmühl Dirk P. , Gerads Michaela , Kurpanek Katharina , Sanglard Dominique , Ernst Joachim F.
Published: March 2003

External signals induce the switch from a yeast to a hyphal growth form in the fungal pathogen Candida albicans. We demonstrate here that the catalytic subunit of a protein kinase A (PKA) isoform encoded by TPK2 is required for internal signalling leading to hyphal differentiation. TPK2 complements the growth defect of a Saccharomyces cerevisiae tpk1‐3 mutant and Tpk2p is able to phosphorylate an established PKA‐acceptor peptide (kemptide). Deletion of TPK2 blocks morphogenesis and partially reduces virulence, whereas TPK2 overexpression induces hyphal formation and stimulates agar invasion. The defective tpk2 phenotype is suppressed by overproduction of known signalling components, including Efg1p and Cek1p, whereas TPK2 overexpression reconstitutes the cek1 but not the efg1 phenotype. The results indicate that PKA activity of Tpk2p is an important contributing factor in regulating dimorphism of C. albicans.
Induction of neutral trehalase Nth1 by heat and osmotic stress is controlled by STRE elements and Msn2/Msn4 transcription factors: variations of PKA effect during stress and growth
Authors: Zähringer Harald , Thevelein Johan M. , Nwaka Solomon
Published: March 2003

Saccharomyces cerevisiae neutral trehalase, encoded by NTH1, controls trehalose hydrolysis in response to multiple stress conditions, including nutrient limitation. The presence of three stress responsive elements (STREs, CCCCT) in the NTH1 promoter suggested that the transcriptional activator proteins Msn2 and Msn4, as well as the cAMP‐dependent protein kinase (PKA), control the stress‐induced expression of Nth1. Here, we give direct evidence that Msn2/Msn4 and the STREs control the heat‐, osmotic stress‐ and diauxic shift‐dependent induction of Nth1. Disruption of MSN2 and MSN4 abolishes or significantly reduces the heat‐ and NaCl‐induced increases in Nth1 activity and transcription. Stress‐induced increases in activity of a lacZ reporter gene put under control of the NTH1 promoter is nearly absent in the double mutant. In all instances, basal expression is also reduced by about 50%. The trehalose concentration in the msn2 msn4 double mutant increases less during heat stress and drops more slowly during recovery than in wild‐type cells. This shows that Msn2/Msn4‐controlled expression of enzymes of trehalose synthesis and hydrolysis help to maintain trehalose concentration during stress. However, the Msn2/Msn4‐independent mechanism exists for heat control of trehalose metabolism. Site‐directed mutagenesis of the three STREs (CCCCT changed to CATCT) in NTH1 promoter fused to a reporter gene indicates that the relative proximity of STREs to each other is important for the function of NTH1. Elimination of the three STREs abolishes the stress‐induced responses and reduces basal expression by 30%. Contrary to most STRE‐regulated genes, the PKA effect on the induction of NTH1 by heat and sodium chloride is variable. During diauxic growth, NTH1 promoter‐controlled reporter activity strongly increases, as opposed to the previously observed decrease in Nth1 activity, suggesting a tight but opposite control of the enzyme at the transcriptional and post‐translational levels. Apparently, inactive trehalase is accumulated concomitant with the accumulation of trehalose. These results might help to elucidate the general connection between control by STREs, Msn2/Msn4 and PKA and, in particular, how these components play a role in control of trehalose metabolism.
Acid‐ and multistress‐resistant mutants of Lactococcus lactis : identification of intracellular stress signals
Authors: Rallu Fabien , Gruss Alexandra , Ehrlich S. Dusko , Maguin Emmanuelle
Published: March 2003

Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication. Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH. In order to characterize the genes involved in acid tolerance responses, we selected acid‐resistant insertional mutants of the L. lactis strain MG1363. Twenty‐one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism. None of these genes was identified previously as involved in lactococcal acid tolerance. The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress‐resistant strains. Our results reveal that L. lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance. In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction. Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.
Role of penicillin‐binding protein PBP 2B in assembly and functioning of the division machinery of Bacillus subtilis
Authors: Daniel Richard A. , Harry Elizabeth J. , Errington Jeff
Published: March 2003

We have characterized the role of the penicillin‐binding protein PBP 2B in cell division of Bacillus subtilis. We have shown that depletion of the protein results in an arrest in division, but that this arrest is slow, probably because the protein is relatively stable. PBP 2B‐depleted filaments contained, at about their mid‐points, structures resembling partially formed septa, into which most, if not all, of the division proteins had assembled. Although clearly deficient in wall material, membrane invagination seemed to continue, indicating that membrane and wall ingrowth can be uncoupled. At other potential division sites along the filaments, no visible ingrowths were observed, although FtsZ rings assembled at regular intervals. Thus, PBP 2B is apparently required for both the initiation of division and continued septal ingrowth. Immunofluorescence microscopy showed that the protein is recruited to the division site. The pattern of localization suggested that this recruitment occurs continually during septal ingrowth. During sporulation, PBP 2B was present transiently in the asymmetrical septum of sporulating cells, and its availability may play a role in the regulation of sporulation septation.
Shotgun DNA microarrays and stage‐specific gene expression in Plasmodium falciparum malaria
Authors: Hayward Rhian E. , DeRisi Joseph L. , Alfadhli Suad , Kaslow David C. , Brown Patrick O. , Rathod Pradipsinh K.
Published: March 2003

Malaria infects over 200 million individuals and kills 2 million young children every year. Understanding the biology of malarial parasites will be facilitated by DNA microarray technology, which can track global changes in gene expression under different physiological conditions. However, genomes of Plasmodium sp. (and many other important pathogenic organisms) remain to be fully sequenced so, currently, it is not possible to construct gene‐specific microarrays representing complete malarial genomes. In this study, 3648 random inserts from a Plasmodium falciparum mung bean nuclease genomic library were used to construct a shotgun DNA microarray. Through differential hybridization and sequencing of relevant clones, large differences in gene expression were identified between the blood stage trophozoite form of the malarial parasite and the sexual stage gametocyte form. The present study lengthens our list of stage‐specific transcripts in malaria by at least an order of magnitude above all previous studies combined. The results offer an unprecedented number of leads for developing transmission blocking agents and for developing vaccines directed at blood stage antigens. A significant fraction of the stage‐selective transcripts had no sequence homologues in the current genome data bases, thereby underscoring the importance of the shotgun approach. The malarial shotgun microarray will be useful for unravelling additional important aspects of malaria biology and the general approach may be applied to any organism, regardless of how much of its genome is sequenced.
BasT, a membrane‐bound transducer protein for amino acid detection in Halobacterium salinarum
Authors: Kokoeva Maia V. , Oesterhelt Dieter
Published: March 2003

Halophilic archaea, such as eubacteria, use methyl‐accepting chemotaxis proteins (MCPs) to sense their environment. We show here that BasT is a halobacterial transducer protein (Htp) responsible for chemotaxis towards five attractant amino acids. The C‐terminus of the protein exhibits the highly conserved regions that are diagnostic for MCPs: the signalling domain for communication with the histidine kinase and the methylation sites that interact with the methylation/demethylation enzymes for adaptation. Hydropathy analysis predicts an enterobacterial‐type transducer protein topology for BasT, with an extracellular putative ligand‐binding domain flanked by two transmembrane helices and a cytoplasmic domain. BasT‐inactivated mutant cells are missing a membrane protein radiolabelled with l‐[methyl‐3H]‐methionine in wild‐type cells, confirming that BasT is methylatable and membrane bound. Behavioural analysis of the basT mutant cells by capillary and chemical‐in‐plug assays demonstrates complete loss of chemotactic responses towards five (leucine, isoleucine, valine, methionine and cysteine) of the six attractant amino acids for Halobacterium salinarum, whereas they still respond to arginine. The volatile methyl group production assays also corroborate these findings and confirm that BasT signalling induces methyl group turnover. Our data identify BasT as the chemotaxis transducer protein for the branched chain amino acids leucine, isoleucine and valine as well as for methionine and cysteine. Thus, BasT and the arginine sensor Car cover the entire spectrum of chemotactic responses towards attractant amino acids in H. salinarum.