bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoregulation of the Escherichia coli replication initiator protein, DnaA, is indirect
Authors: Smith R. W. P. , McAteer Sean , Masters Millicent
Published: November 2003

The expression of dnaA is autoregulated, in that transcription of the gene increases when DnaA is inactivated (and initiation of replication prevented) and decreases when DnaA is supplied in excess. However, the inactivation of DnaA does not necessarily lead to increased DnaA production, as dnaA(7s; temperature sensitive) strains which are integratively suppressed by derivatives of the plasmid R1 do not show temperature‐induced derepression. Several possible explanations for this unanticipated behaviour were considered and ruled out. We suggest here that the completion of a critical step in initiation may prevent dnaA derepression: although DnaA would be required to complete this step at oriC, DnaA(Ts) would be sufficient at the R1 origin. Autoregulation of dnaA has been attributed to the binding of DnaA at a consensus binding site in the dnaA promoter region. We show here, using reporter systems, that this DnaA‐binding site is not required for the autoregu‐latory response. We find, further, that replacement of the chromosomal dnaA gene with one containing a mutated binding site causes no demonstrable pheno‐typic change: cells with the mutant gene show no disadvantage in competition with dnaA+ cells.
Functional importance of RNA interactions in selection of translation initiation codons
Authors: Sprengart Michael L. , Porter Alan G.
Published: November 2003

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the ‘correct’ mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5′ non‐coding regions of their mRNAs and ‘consensus sequences’ surrounding initiation codons have been shown to be strong determinants for initiation‐site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA–small ribosomal subunit–initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis‐acting mRNA elements and trans‐acting proteins may form specific ‘bridging’ interactions with ribosomes during translation initiation.
DnaA initiator—also a transcription factor
Authors: Messer Walter , Weigel Christoph
Published: November 2003

The replication‐initiator protein DnaA is ubiquitous in the eubacterial world. It binds to an asymmetric 9 bp consensus DNA sequence, the DnaA box. Besides its primary function as an initiator, it acts as a transcription factor that represses or activates several genes, or terminates transcription, depending on the location and arrangement of DnaA boxes.
spoIIQ, a forespore‐expressed gene required for engulfment in Bacillus subtilis
Authors: Londoño‐Vallejo J.‐Arturo , Fréhel Claude , Stragier Patrick
Published: November 2003

A crucial step in converting an actively growing Bacillus subtilis cell into a dormant spore is the formation of a cell within a cell. This unusual structure is created by a phagocytosis‐like process in which the larger mother cell progressively engulfs the adjacent smaller forespore. Only mutations blocking engulfment at an early stage and affecting genes expressed in the mother cell have been identified. Here we describe a new locus, spoIIQ, which is transcribed in the forespore and which encodes a membrane‐bound protein required at a late stage of engulfment. Immunofluorescence microscopy analysis have shown that SpoIIQ is initially targeted to the septum at the boundary between the two cells and then spreads around the entire membrane of the forespore. Septum targeting requires only the first 52 residues of SpoIIQ as well as unidentified forespore‐specific components. Electron‐microscopy studies of cells engineered to activate the mother‐cell program of gene expression independently of the forespore indicate that other as yet uncharacterized genes are involved in engulfment and that this morphological process is driven from both sides of the forespore envelope.
The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel‐dependent transcriptional regulation of hydrogenase
Authors: Olson Jonathan W. , Fu Changlin , Maier Robert J.
Published: November 2003

The HypB protein from Bradyrhizobium japonicum is a metal‐binding GTPase required for hydrogenase expression. In‐frame mutagenesis of hypB resulted in strains that were partially or completely deficient in hydrogenase expression, depending on the degree of disruption of the gene. Complete deletion of the gene yielded a strain (JHΔEg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules. Mutant strain JHΔ23H lacking only the N‐terminal histidine‐rich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity. The activity of strain JHΔ23H was low in comparison to the wild type in 10–50 nM nickel levels, but could be cured to nearly wild‐type levels by including 50 μM nickel during the derepression incubation. Studies on strains harbouring the hup promoter–lacZ fusion plasmid showed that the complete deletion of hypB nearly abolished hup promoter activity, whereas the histidine deletion mutant had 60% of the wild‐type promoter activity in 50 μM NiCl2. Further evidence that HypB is required for hup promoter‐binding activity was obtained from gel‐shift assays. HypB could not be detected by immunoblotting when the cells were cultured heterotrophically, but when there was a switch to microaerobic conditions (1% partial pressure O2, 10% partial pressure H2) HypB was detected, and its expression preceded hydrogenase synthesis by 3–6 h. 63Ni accumulation by whole cells showed that both of the mutant strains accumulate less nickel than the wild‐type strain at all time points tested during the derepression incubation. Wild‐type cultures that received nickel during the HypB expression‐specific period and were then washed and derepressed for hydrogenase without nickel had activities comparable to those cells that were derepressed for hydrogenase with nickel for the entire time period. In contrast to the wild type, strain JHΔ23H cultures supplied with nickel only during the HypB expression period achieved hydrogenase activities that were 30% of those cultures supplied with nickel for the entire hydrogenase derepression period. These results indicate that the loss of the metal‐binding area of HypB causes a decrease in the ability of the cells to sequester and store nickel for later use in one or more hydrogenase expression steps.
TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli
Authors: Letain Tracy E. , Postle Kathleen
Published: November 2003

The energy source for active transport of iron–siderophore complexes and vitamin B12 across the outer membrane in Gram‐negative bacteria is the cytoplasmic membrane proton‐motive force (pmf). TonB protein is required in this process to transduce cytoplasmic membrane energy to the outer membrane. In this study, Escherichia coli TonB was found to be distributed in sucrose density gradients approximately equally between the cytoplasmic membrane and the outer membrane fractions, while two proteins with which it is known to interact, ExbB and ExbD, as well as the NADH oxidase activity characteristic of the cytoplasmic membrane, were localized in the cytoplasmic membrane fraction. Neither the N‐terminus of TonB nor the cytoplasmic membrane pmf, both of which are essential for TonB activity, were required for TonB to associate with the outer membrane. When the TonB C‐terminus was absent, TonB was found associated with the cytoplasmic membrane, suggesting that the C‐terminus was required for outer membrane association. When ExbB and ExbD, as well as their cross‐talk‐competent homologues TolQ and TolR, were absent, TonB was found associated with the outer membrane. TetA–TonB protein, which cannot interact with ExbB/D, was likewise found associated with the outer membrane. These results indicated that the role of ExbB/D in energy transduction is to bring TonB that has reached the outer membrane back to associate with the cytoplasmic membrane. Two possible explanations exist for the observations presented in this study. One possibility is that TonB transduces energy by shuttling between membranes, and, at some stages in the energy‐transduction cycle, is associated with either the cytoplasmic membrane or the outer membrane, but not with both at the same time. This hypothesis, together with the alternative interpretation that TonB remains localized in the cytoplasmic membrane and changes its affinity for the outer and cytoplasmic membrane during energy transduction, are incorporated with previous observations into two new models, consistent with the novel aspects of this system, that describe a mechanism for TonB‐dependent energy transduction.
Purification and characterization of the flagellar hook–basal body complex of Bacillus subtilis
Authors: Kubori Tomoko , Okumura Mitsumasa , Kobayashi Nobuhiro , Nakamura Dai , Iwakura Masahiro , Aizawa Shin‐Ichi
Published: November 2003

The flagellar hook–basal body (HBB) complex of the Gram‐positive bacterium Bacillus subtilis was purified and analysed by electron microscopy, gel electrophoresis, and amino acid sequencing of the major component proteins. The purified HBB complex consisted of the inner (M and S) rings, a rod and a hook. There were no outer (P and L) rings that are found in Gram‐negative bacteria. The hook was 15 nm in thickness and 70 nm in length, which is thinner and longer than the hook of Salmonella typhimurium. The hook protein had an apparent molecular mass of 29 kDa, and its N‐terminal sequence was identical to that of B. subtilis FlgG, which was previously reported as a rod protein. The sequence of the reported FlgG protein of B. subtilis is more closely related to that of FlgE (the hook protein) rather than FlgG (the rod protein) of S. typhimurium, in spite of the difference of the apparent molecular masses between the two hook proteins (29 kDa versus 42 kDa). The hook–basal body contained six major proteins (with apparent molecular masses of 82, 59, 35, 32, 29 and 20 kDa) and two minor proteins (23 kDa and 13 kDa), which consistently appeared from preparation to preparation. The N‐terminus of each of these proteins was sequenced. Comparison with protein databases revealed the following polypeptide–gene correspondences: 82 kDa, fliF; 59 kDa, flgK; 35 kDa, orfF; 32 kDa, yqhF; 23 kDa, orf3 of the flaA locus; 20 kDa, flgB and flgC; 13 kDa, not determined. The band at 20 kDa was a mixture of FlgB and FlgC, as revealed by two‐dimensional gel analysis. Characteristic features of B. subtilis HBB are discussed in comparison with those of S. typhimiurium.
Biochemistry and molecular genetics of cell‐wall lipid biosynthesis in mycobacteria
Authors: Kolattukudy P. E. , Fernandes Norvin D. , Azad A. K. , Fitzmaurice Ann Marie , Sirakova Tatiana D.
Published: November 2003

Tuberculosis and other mycobacterial infections are the most serious infectious diseases in terms of human fatalities. The high content of unique cell‐wall lipids helps these organisms to resist antimicrobial drugs and host defences. The biosynthesis of these lipids is discussed briefly. The recent advances in recombinant DNA technology have begun to help to elucidate the nature of some of the enzymes involved in this process and the genes that encode them. Gene disruption and other molecular genetic technologies are beginning to provide new approaches to test for the biological functions of these gene products and may lead to identification of new antimycobacterial drug targets.
Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion
Authors: Rockey D. D. , Grosenbach D. , Hruby D. E. , Peacock M. G. , Heinzen R. A. , Hackstadt T.
Published: November 2003

Chlamydiae are obligate intracellular bacteria that replicate within a non‐acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher Mr forms are found in C. psittaci‐infected cells. This finding suggested that IncA may be post‐translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple‐banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of [32P]‐orthophosphate‐labelled infected HeLa cells with anti‐IncA antisera demonstrated that the two highest Mr IncA bands were phosphorylated. A vaccinia‐virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background. IncA in lysates of these cells migrated identically to that seen in C. psittaci‐infected cells, indicating the host cell was responsible for the phosphorylation of the protein. Microinjection of fluorescently labelled anti‐IncA antibodies into C. psittaci‐infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane. Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion.
Microbial pathogenesis in cystic fibrosis: co‐ordinate regulation of heat‐shock response and conversion to mucoidy in Pseudomonas aeruginosa
Authors: Schurr M. J. , Deretic V.
Published: November 2003

Conversion of Pseudomonas aeruginosa to the mucoid phenotype plays a major role in the pathogenesis of respiratory infections in cystic fibrosis (CF). One mechanism responsible for mucoidy is based on mutations that inactivate the anti‐σ factor, MucA, which normally inhibits the alternative sigma factor, AlgU. The loss of MucA allows AlgU to freely direct transcription of the genes responsible for the production of the exopolysaccharide alginate resulting in mucoid colony morphology. In Escherichia coli, a close homologue of AlgU, σE, directs transcription of several genes under conditions of extreme heat shock. Here we examined whether AlgU, besides its role in controlling alginate production, affects the heat‐shock response in P. aeruginosa. The P. aeruginosa rpoH gene encoding a homologue of the major heat‐shock sigma factor, σ32, was found to be transcribed by AlgU containing RNA polymerase from one of its promoters (P3) identified in this study. Transcription of rpoH from P3 was elevated upon exposure to extreme heat shock in an algU‐dependent manner. Importantly, the AlgU‐dependent promoter of rpoH was found to be activated in mucoid mucA mutants. In keeping with this observation, introduction of a wild‐type mucA gene abrogated AlgU‐dependent rpoH transcription in mucoid P. aeruginosa laboratory isolates and CF isolates. These results suggest that conversion to mucoidy and the heat‐shock response are co‐ordinately regulated in P. aeruginosa. The simultaneous activation of both systems in mucA mutants, selected in the lungs of CF patients, may have significance for the inflammatory processes characteristic of the establishment of chronic infection and ensuing clinical deterioration in CF.
Analysis of ssb mutations in vivo implicates SSB protein in two distinct pathways of SOS induction and in recombinational DNA repair
Authors: Carlini Leslie E. , Porter Ronald D.
Published: November 2003

Site‐directed mutations in the Escherichia coli ssb gene were tested for the ability to complement a chromosomal ssb deletion for viability, and only the ssb W54→G mutation failed to do so at the pSC101 copy level. Non‐aromatic amino acid substitutions for SSB Trp‐54 (ssb W54→L and ssb W54→S) produced the greatest effects on in vivo protein function including altered marker linkage subsequent to generalized transduction, extreme UV sensitivity, and a lack of ability to support SOS induction. Additionally, the ssb‐113 (ssb P176→S) mutation demonstrated the existence of both uvrA‐dependent and uvrA‐independent components of SOS induction. Although nucleotide excision repair appeared unaffected by alterations in the SSB protein, the mutational analysis suggests a direct role for SSB in recombinational repair.
Aminoglycoside 2′‐N‐acetyltransferase genes are universally present in mycobacteria: characterization of the aac(2 ′)‐Ic gene from Mycobacterium tuberculosis and the aac(2 ′)‐Id gene from Mycobacterium smegmatis
Authors: Aínsa José A. , Pérez Esther , Pelicic Vladimir , Berthet François‐Xavier , Gicquel Brigitte , Martín Carlos
Published: November 2003

The genus Mycobacterium comprises clinically important pathogens such as M. tuberculosis, which has re‐emerged as a major cause of morbidity and mortality world‐wide especially with the emergence of multidrug‐resistant strains. The use of fast‐growing species such as Mycobacterium smegmatis has allowed important advances to be made in the field of mycobacterial genetics and in the study of the mechanisms of resistance in mycobacteria. The isolation of an aminoglycoside‐resistance gene from Mycobacterium fortuitum has recently been described. The aac(2′)‐Ib gene is chromosomally encoded and is present in all isolates of M. fortuitum. The presence of this gene in other mycobacterial species is studied here and genes homologous to that of M. fortuitum have been found in all mycobacterial species studied. In this report, the cloning of the aac(2′)‐Ic gene from M. tuberculosis H37Rv and the aac(2′)‐Id gene from M. smegmatis mc2155 is described. Southern blot hybridizations have shown that both genes are present in all strains of this species studied to date. In addition, the putative aac(2′)‐Ie gene has been located in a recent release of the Mycobacterium leprae genome. The expression of the aac(2′)‐Ic and aac(2′)‐Id genes has been studied in M. smegmatis and only aac(2′)‐Id is correlated with aminoglycoside resistance. In order to elucidate the role of the aminoglycoside 2′‐N‐acetyltransferase genes in mycobacteria and to determine whether they are silent resistance genes or whether they have a secondary role in mycobacterial metabolism, the aac(2′)‐Id gene from M. smegmatis has been disrupted in the chromosome of M. smegmatis mc2155. The disruptant shows an increase in aminoglycoside susceptibility along with a slight increase in the susceptibility to lysozyme.
The Escherichia coli phage‐shock‐protein (psp) operon
Authors: Model Peter , Jovanovic Goran , Dworkin Jonathan
Published: November 2003

The phage‐shock‐protein (psp) operon helps to ensure survival of Escherichia coli in late stationary phase at alkaline pH, and protects the cell against dissipation of its proton‐motive force against challenge. It is strongly induced by filamentous phage pIV and its bacterial homologues, and by mutant porins that don’t localize properly, as well as by a number of other stresses. Transcription of the operon is dependent on σ54 and a constitutively active, autogenously controlled activator. psp‐operon expression is controlled by one negatively and several positively acting regulators, none of which is a DNA‐binding protein. The major product of the operon, PspA, may also serve as a negative regulator of an unusual porin, OmpG.
The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme
Authors: Shankar Sandeep , Hershberger C. Douglas , Chakrabarty A. M.
Published: November 2003

We report the purification and characterization of the enzyme nucleoside diphosphate kinase (Ndk) from Mycobacterium smegmatis. The N‐terminus of the enzyme was blocked but an internal sequence showed approx. 70% homology with the same enzymes from Pseudomonas aeruginosa and Escherichia coli. Immobilization of the mycobacterial nucleoside diphosphate kinase on a Sepharose 4B matrix and passing the total cell extract through it revealed four proteins (P70, P65, P60, and P50, respectively) of Mr 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of Mr 70 kDa and 50 kDa modulated the activity of Ndk directing it towards GTP synthesis, the 60 kDa protein channelled the specificity of Ndk entirely towards CTP synthesis. The 65 kDa protein modulated the specificity of Ndk directing it entirely towards UTP synthesis. The specificity for such mycobacterial proteins towards NTP synthesis is retained when they are complexed with P. aeruginosa Ndk. We further demonstrate that the P70 protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk and alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intracellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor pool for cell wall biosynthesis as well as RNA/DNA synthesis.
Transcriptional regulation of the Yersinia pseudotuberculosis pH 6 antigen adhesin by two envelope‐associated components
Authors: Yang Youxu , Isberg Ralph R.
Published: November 2003

The Yersinia pseudotuberculosis pH 6 antigen mediates haemagglutination and adhesion to cultured mammalian cells. The synthesis of pH 6 antigen requires the products of the psaEFABC genes in both Yersinia pseudotuberculosis and Escherichia coli. In‐frame deletion mutations of psaE and psaF caused defective haemagglutination. In contrast, we showed that the psaABC genes were sufficient for haemagglutination if they were expressed by a heterologous promoter. Environmental regulation of pH 6 antigen by temperature and pH occurs via regulation of the major pilus protein PsaA at the transcriptional level. Northern blot analyses indicate that the psaA transcript was absent in either psaE or psaF mutant strains. Primer extension analyses indicate that, in Y. pseudotuberculosis, the transcription of the psaE and psaF genes is constitutive. Alkaline phosphatase fusion studies confirm the topology prediction that PsaE and PsaF are both inner‐membrane‐associated proteins. PsaE consists of an N‐terminal cytoplasmic domain, containing sequence similarity to transcriptional regulators found in two‐component systems as well as to the Salmonella typhimurium HilA protein, with a C‐terminal domain that is periplasmically localized. PsaF is predicted to be oriented with most of the protein in the periplasm, the hydrophobic N‐terminus being either integrated in the inner membrane or cleaved as a signal peptide.
Molecular characterization of the plasmid‐encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis
Authors: Kranenburg Richard van , Marugg Joey D. , Van Swam Iris I. , Willem Norwin J. , De Vos Willem M.
Published: November 2003

Lactococcus lactis strain NIZO B40 produces an extracellular phosphopolysaccharide containing galactose, glucose, and rhamnose. A 40 kb plasmid encoding exopolysaccharide production was isolated through conjugal transfer of total plasmid DNA from strain NIZO B40 to the plasmid‐free L. lactis model strain MG1614 and subsequent plasmid curing. A 12 kb region containing 14 genes with the order epsRXABCDEFGHIJKL was identified downstream of an iso‐IS982 element. The predicted gene products of epsABCDEFGHIJK show sequence homologies with gene products involved in exopolysaccharide, capsular polysaccharide, lipopolysaccharide, or teichoic acid biosynthesis of other bacteria. Transcriptional analysis of the eps gene cluster revealed that the gene cluster is transcribed as a single 12 kb mRNA. The transcription start site of the promoter was mapped upstream of the first gene epsR. The involvement of epsD in exopolysaccharide (EPS) biosynthesis was demonstrated through a single gene disruption rendering an exopolysaccharide‐deficient phenotype. Heterologous expression of epsD in Escherichia coli showed that its gene product is a glucosyltransferase linking the first sugar of the repeating unit to the lipid carrier.
Structural basis for differential receptor binding of cholera and Escherichia coli heat‐labile toxins: influence of heterologous amino acid substitutions in the cholera B‐subunit
Authors: Bäckström Malin , Shahabi Vafa , Johansson Susanne , Teneberg Susann , Kjellberg Anders , Miller‐Podraza Halina , Holmgren Jan , Lebens Michael
Published: November 2003

The closely related B‐subunits of cholera toxin (CTB) and Escherichia coli heat‐labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins. A number of mutant CT B‐subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB. These were used to investigate the influence of specific residues on receptor‐binding specificity. A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non‐GM1 intestinal glycosphingolipids. In contrast, when LTB amino acid substitutions in the 1–25 region were combined with those in the 75–83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed. In addition, a mutant LTB with a single Gly‐33→Asp substitution that completely lacked affinity for both GM1 and non‐GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides. We conclude that the extra, non‐GM1 receptors for LTB consist of both sialylated and non‐sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.
Modulation of the Escherichia coliσE (RpoE) heat‐shock transcription‐factor activity by the RseA, RseB and RseC proteins
Authors: Missiakas Dominique , Mayer Matthias P. , Lemaire Marc , Georgopoulos Costa , Raina Satish
Published: November 2003

The σE (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities. The σE regulon is induced upon misfolding of proteins in the periplasm or the outer membrane. Similar to other alternative sigma factors, the activity of σE is tightly regulated in E. coli. We have previously shown that σE is positively autoregulated at the transcriptional level. DNA sequencing, coupled with transcriptional analyses, have shown that σE is encoded by the first gene of a four‐gene operon. The second gene of this operon, rseA, encodes an anti‐σE activity. This was demonstrated at both the genetic and biochemical levels. For example, mutations in rseA constitutively increase σE activity. Consistent with this, overproduction of RseA leads to an inhibitory effect on σE activity. Topological analysis of RseA suggests the existence of one transmembrane domain, with the N‐terminal part localized in the cytoplasm. Overproduction of this N‐terminal domain alone was shown to inhibit σE activity. These observations were confirmed in vitro, because either purified RseA or only its purified N‐terminal domain inhibited transcription from EσE‐dependent promoters. Furthermore, RseA and σE co‐purify, and can be co‐immunoprecipitated, and chemically cross‐linked. The σE activity is further modulated by the products of the remaining genes in this operon, rseB and rseC. RseB is a periplasmic protein, which negatively regulates σE activity and specifically interacts with the C‐terminal periplasmic domain of RseA. In contrast, RseC is an inner membrane protein that positively modulates σE activity. Most of these protein–protein interactions were verified in vivo using the yeast two‐hybrid system.
The σE‐mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of σE
Authors: De Las Peñas Alejandro , Connolly Lynn , Gross Carol A.
Published: November 2003

The extracytoplasmic stress response in Escherichia coli is controlled by the alternative sigma factor, σE. σE activity is uniquely induced by the accumulation of outer membrane protein precursors in the periplasmic space, and leads to the increased production of several proteins, including the periplasmic protease DegP, that are thought to be required for maintaining cellular integrity under stress conditions. Genetic and biochemical experiments show that σE activity is under the control of three genes, rseABC (for regulator of sigma E), encoded immediately downstream of the sigma factor. Deletion of rseA leads to a 25‐fold induction of σE activity. RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits σE‐directed transcription in vitroindicating that RseA acts as an anti‐sigma factor. Deletion of rseB leads to a slight induction of σE, indicating that RseB is also a negative regulator of σE. RseB is a periplasmic protein and was found to co‐purify with the periplasmic domain of RseA, indicating that RseB probably exerts negative activity on σE through RseA. Deletion of rseC, in contrast, has no effect on σE activity under steady‐state conditions. Under induction conditions, strains lacking RseB and/or C show wild‐type induction of σE activity, indicating either the presence of multiple pathways regulating σE activity, or the ability of RseA alone to both sense and transmit information to σE.
Modulator protein RsbR regulates environmental signalling in the general stress pathway of Bacillus subtilis
Authors: Akbar Samina , Kang Choong Min , Gaidenko Tatiana A. , Price Chester W.
Published: November 2003

Bacillus subtilis responds to signals of environmental and metabolic stress by inducing over 40 general stress genes under the control of the σB transcription factor. σB activity is regulated post‐translationally by a multicomponent network composed of two coupled partner‐switching modules, RsbX‐RsbS‐RsbT and RsbU‐RsbV‐RsbW, each containing a serine phosphatase (X or U), an antagonist protein (S or V), and a switch protein/serine kinase (T or W). The upstream module (X‐S‐T) is required to transmit signals of environmental stress. In contrast, the downstream module (U‐V‐W) is required to transmit signals of energy stress as well as the environmental signals conveyed to it from the upstream module. Until now the function of the rsbR gene product was unknown. RsbR shares significant sequence similarity with the RsbS and RsbV antagonist proteins whose phosphorylation states control key protein–protein interactions within their respective modules. Here we present evidence that RsbR is associated with RsbS in the upstream, environmental‐sensing module. To investigate RsbR function, we constructed deletion and point mutations within rsbR and tested their effects on expression of σB‐dependent reporter fusions, both singly and in combination with other rsb mutations. To determine the possible interaction of RsbR with other Rsb proteins, we tested the ability of wild‐type or mutant RsbR to activate transcription in the yeast two‐hybrid system in conjunction with other Rsb regulators. On the basis of this genetic analysis, we conclude that RsbR is a positive regulator which modulates σB activity in response to salt and heat stress. Our data further suggest that: (i) RsbR influences the antagonist function of RsbS by direct protein–protein interaction; and (ii) this interaction with RsbS is likely controlled by the phosphorylation state of RsbR.
Action at a distance for glp repressor control of glpTQ transcription in Escherichia coli K‐12
Authors: Yang Bing , Gerhardt Susan G. , Larson Timothy J.
Published: November 2003

The adjacent, divergently transcribed glpACB and glpTQ operons of Escherichia coli encode the anaerobic glycerol 3‐phosphate dehydrogenase and glycerol 3‐phosphate transporter/phosphodiesterase, respectively. These operons are negatively controlled by glp repressor binding to operators that overlap the glpA promoter elements. Using DNase I footprinting, three additional operators (OT1–3) were identified at positions +307 to +359 within the glpT coding region. To assess a potential regulatory role for these remote operators in vivo, a glpT–lacZ transcriptional fusion containing all of the glpA and glpT operators was constructed. The response of this fusion to the glp repressor was compared to fusion constructs in which OT1 and OT3 were inactivated, either by deletion or by site‐directed mutagenesis. It was found that repression of glpT conferred by binding of glp repressor to glpA operators was increased about three‐ to fourfold upon introduction of the remote glpT operators. In addition, two integration host factor (IHF) binding sites were identified downstream of the glpT transcriptional start site at positions +15 to +51 and +193 to +227. A regulatory role for IHF was demonstrated by showing that repression of glpT mediated by GlpR was decreased about twofold in strains deficient in IHF and that mutations in IHF1 and/or IHF2 decreased repression about two‐ to threefold. The effect of IHF was apparent only when the remote operators were present. All of the results are consistent with a model of repression involving GlpR binding simultaneously to the glpA and remote glpT operators, with intervening DNA forming a loop.
A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule
Authors: Barber C. E. , Tang J. L. , Feng J. X. , Pan M. Q. , Wilson T. J. G. , Slater H. , Dow J. M. , Williams P. , Daniels M. J.
Published: November 2003

Mutations in the seven clustered rpf genes cause downregulated synthesis of extracellular enzymes and reduced virulence of Xanthomonas campestris pathovar campestris (Xcc). The phenotype of mutants in one of the genes, rpfF, can be restored by a diffusible extracellular factor (DSF) produced by all Xcc strains tested, apart from rpfF and rpfB mutants. DSF accumulates in early stationary phase (when synthesis of enzymes is maximal), but levels decline subsequently. Addition of DSF to exponentially‐growing wild‐type bacteria does not cause precocious enzyme synthesis. rpfB and rpfF are expressed throughout growth, but the rate increases in early stationary phase. RpfB is predicted to be a long‐chain fatty acyl CoA ligase, and RpfF shows some relatedness to enoyl CoA hydratases. The properties of DSF suggest that it may be a fatty‐acid derivative, and certain lipid preparations possess DSF activity at higher concentrations. These include lipid extracts and acid‐hydrolysed lipopolysaccharide and lipid A from Xcc, and purified dodecanoic and hydroxydodecanoic acid. DSF production is confined to certain xanthomonads. We propose a model for the DSF system, which represents a novel mechanism for regulating virulence factor synthesis in response to physiological or environmental changes.
Non‐invasive Salmonella typhimurium mutants are avirulent because of an inability to enter and destroy M cells of ileal Peyer's patches
Authors: Penheiter Kristi L. , Mathur Nitin , Giles Dobie , Fahlen Thomas , Jones Bradley D.
Published: November 2003

Salmonella typhimurium initiates infection of a host by invading M cells of Peyer’s patches within the small intestine. The ability of the bacteria to invade mammalian cells has been shown to be regulated by environmental conditions, including oxygen concentrations, osmolarity, and growth phase. We have previously created oxygen‐regulated Tn5lacZY S. typhimurium mutants that are defective in invasion. We have now identified the invasion genes disrupted by eight of the transposon insertions. These genes encode transcriptional regulators (hilA and invF ), type III secretory components (orgA, invG and spaR) and secreted proteins (invC and invD). Examination of the protein‐secretion profiles of the non‐invasive mutants indicated that each of the mutants was defective in secretion of between one and six proteins. We have also demonstrated that the loss of tissue culture cell invasiveness corresponds to an inability to invade and destroy M cells of Peyer's patches in a murine ligated loop model. Virulence studies, performed in mice, demonstrated that these defects significantly reduced the ability of the mutants to cause murine typhoid fever by an oral route of infection. Virulence by an intraperitoneal route of infection was unaffected. The data indicate that in vitro invasiveness, invasion‐protein secretion, and M‐cell invasion are critical indicators of S. typhimurium virulence.
A C‐terminal di‐leucine motif and nearby sequences are required for NH4+‐induced inactivation and degradation of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae
Authors: Hein Claudine , André Bruno
Published: November 2003

The general amino acid permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source. Adding NH4+ to the medium triggers inactivation and degradation of the permease via a regulatory process involving Npi1p/Rsp5p, a ubiquitin–protein ligase. In this study, we describe several mutations affecting the C‐terminal region of Gap1p that render the permease resistant to NH4+‐induced inactivation. An in vivo isolated mutation (gap1pgr ) causes a single Glu→Lys substitution in an amino acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast α‐factor receptor, Ste2p. Another replacement, substitution of two alanines for a di‐leucine motif, likewise protects the Gap1 permease against NH4+‐induced inactivation. In mammalian cells, such a motif is involved in the internalization of several cell‐surface proteins. These data provide the first indication that a di‐leucine motif influences the function of a plasma membrane protein in yeast. Mutagenesis of a putative phosphorylation site upstream from the di‐leucine motif altered neither the activity nor the regulation of the permease. In contrast, deletion of the last eleven amino acids of Gap1p, a region conserved in other amino acid permeases, conferred resistance to NH4+ inactivation. Although the C‐terminal region of Gap1p plays an important role in nitrogen control of activity, it was not sufficient to confer this regulation to two NH4+‐insensitive permeases, namely the arginine (Can1p) and uracil (Fur4p) permeases.
The invasion‐associated type III system of Salmonella typhimurium directs the translocation of Sip proteins into the host cell
Authors: Collazo Carmen M. , Galán Jorge E.
Published: November 2003

The ability of Salmonella typhimurium to interact with host cells is largely dependent on the function of a type III protein‐secretion system encoded at centisome 63 of its chromosome. We have shown here that two targets of this protein‐secretion system, SipB and SipC, are translocated into cultured intestinal Henle‐407 cells. Translocation required the function of the type III secretion apparatus, as an S. typhimurium strain carrying a mutation in invA, which encodes an essential component of this system, failed to translocate the Sip proteins. Null mutations in the genes encoding SipB, SipC or SipD, prevented protein translocation, indicating that these proteins are involved in the translocation process. In contrast, mutations in sipA and sptP, which also encode secreted proteins, did not interfere with the translocation of SipC, indicating that only a subset of targets of the type III secretion system act as translocases. Externally or internally localized bacteria could direct protein translocation into Henle‐407 cells as this process occurred in the presence of cytochalasin D at a concentration that prevented bacterial entry, or in the presence of gentamicin added shortly after bacterial internalization at a concentration that killed extracellular Salmonella. These results indicate that protein translocation into host cells may be a universal function of all type III secretion systems.
A mutation in the Bordetella bronchiseptica bvgS gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg‐regulated antigens
Authors: Cotter Peggy A. , Miller Jeff F.
Published: November 2003

The Bordetella BvgAS signal‐transduction system has traditionally been viewed as mediating a transition between two distinct phenotypic phases: the Bvg+ phase, characterized by the expression of adhesins and toxins, and the Bvg− phase, characterized by motility in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis. In B. bronchiseptica, the Bvg+ phase is necessary and sufficient for respiratory tract colonization whereas the Bvg− phase is required for growth under nutrient‐limiting conditions. This report describes the characterization of a mutant that is locked in a Bvg‐intermediate (Bvgi) phase. The mutation conferring this phenotype, designated bvgS‐I1, results in a threonine‐to‐methionine substitution near the primary site of phosphorylation in BvgS. Compared to its Bvg+‐phase‐locked parent, the Bvgi mutant displays increased resistance to nutrient limitation and reduced virulence. Molecular analyses indicate that the mutant has lost the ability to express a subset of Bvg+‐phase factors and has gained the ability to express factors unique to the Bvgi phase. Although identified by mutation, this work indicates that the Bvgi phase is expressed by wild‐type B. bronchiseptica in response to certain (semi‐modulating) environmental conditions. The identification of Bvgi‐specific antigens suggests the existence of a new class of Bvg‐regulated genes. We hypothesize that BvgAS is capable of mediating the expression of a spectrum of phenotypic phases in response to the various environments encountered as Bordetella travels within and between mammalian hosts.
Two independent type III secretion mechanisms for YopE in Yersinia enterocolitica
Authors: Cheng Luisa W. , Anderson Deborah M. , Schneewind Olaf
Published: November 2003

Pathogenic Yersinia species escape the infected host’s defense mechanisms by targeting cytotoxic Yop proteins into the cytoplasm of macrophages via a type III secretion pathway. Two separate secretion signals contained in YopE were identified, each of which were sufficient but not necessary for the secretion of reporter molecules. One signal is located within the coding sequence of the first 15 amino acids and is sufficient for the secretion of fusion proteins but not required for YopE secretion. The second signal is located downstream at residues 15–100 of YopE and is only recognized by the type III machinery when it is bound to SycE. We propose the existence of two independent mechanisms that allow for the secretion of Yop proteins.
Structural analysis of the subunits of the trehalose‐6‐phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock
Authors: Reinders Anke , Bürckert Niels , Hohmann Stefan , Thevelein Johan M. , Boller Thomas , Wiemken Andres , De Virgilio Claudio
Published: November 2003

Synthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose‐6‐phosphate (Tre6P) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1, TPS2, and TSL1. Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6P synthase (Tps1) and Tre6P phosphatase (Tps2), while Tsl1 was suggested to have regulatory functions. In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits. Two‐hybrid analyses of the in vivo interactions of Tps1, Tps2, Tsl1, and Tps3, a protein with high homology to Tsl1, revealed that both Tsl1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other. In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1, TPS2, TSL1, and TPS3 in all possible combinations. These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that Tsl1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6P synthase/phosphatase complex in exponentially growing, heat‐shocked cells.
EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V
Authors: Brunder Werner , Schmidt Herbert , Karch Helge
Published: November 2003

In this study, we identified and characterized a novel secreted protein, the extracellular serine protease EspP, which is encoded by the large plasmid of enterohaemorrhagic Escherichia coli (EHEC) O157:H7. The corresponding espP gene consists of a 3900 bp open reading frame that is able to encode a 1300‐amino‐acid protein. EspP is synthesized as a large precursor which is then processed at the N‐ and C‐termini during secretion. It can be grouped into the autotransporter protein family. The deduced amino acid sequence of EspP showed homology to several secreted or surface‐exposed proteins of pathogenic bacteria, in particular EspC of enteropathogenic E. coli and IgA1 proteases from Neisseria spp. and Haemophilus influenzae. Hybridization experiments and immunoblot analysis of clinical EHEC isolates showed that EspP is widespread among EHEC of the serogroup O157 and that it also exists in serogroup O26. A specific immune response against EspP was detected in sera from patients suffering from EHEC infections. Functional analysis showed that EspP is a protease capable of cleaving pepsin A and human coagulation factor V. Degradation of factor V could contribute to the mucosal haemorrhage observed in patients with haemorrhagic colitis.
Regulation, replication, and integration functions of the Vibrio cholerae CTXφ are encoded by region RS2
Authors: Waldor Matthew K. , Rubin Eric J. , Pearson Gregory D. N. , Kimsey Harvey , Mekalanos John J.
Published: November 2003

CTXφ is a filamentous phage that encodes cholera toxin, one of the principal virulence factors of Vibrio cholerae. CTXφ is unusual among filamentous phages because it can either replicate as a plasmid or integrate into the V. cholerae chromosome at a specific site. The CTXφ genome has two regions, the ‘core’ and RS2. Integrated CTXφ is frequently flanked by an element known as RS1 which is related to RS2. The nucleotide sequences of RS2 and RS1 were determined. These related elements contain three nearly identical open reading frames (ORFs), which in RS2 were designated rstR, rstA2 and rstB2. RS1 contains an additional ORF designated rstC. Functional analyses indicate that rstA2 is required for CTXφ replication and rstB2 is required for CTXφ integration. The amino terminus of RstR is similar to the amino termini of other phage‐encoded repressors, and RstR represses the expression of rstA2. Although genes with related functions are clustered in the genome of CTXφ in a way similar to those for other filamentous phages, the CTXφ RS2‐encoded gene products mediating replication, integration and repression appear to be novel.