bluuebunny/crossref_metadata_2025 huggingface dataset:
Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo
Authors: Sussman Jacqueline K. , Simons Elizabeth L. , Simons Robert W.
Published: November 2003

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS10 transposase gene (tnp), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se, was not increased. Rather, these mutations appear to increase expression of the tnp′–′lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo: Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1–3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification. Importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3–5‐fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo, consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.
The coupling between ftsZ transcription and initiation of DNA replication is not mediated by the DnaA‐boxes upstream of ftsZ or by DnaA
Authors: Smith Richard W. P. , McAteer Sean , Masters Millicent
Published: November 2003

The DnaA protein of Escherichia coli is a multifunctional protein which, in addition to promoting initiation of replication, can regulate the initiation or termination of transcription of a variety of genes. It acts by binding to DNA at a defined sequence, termed a DnaA‐box. Three candidate DnaA‐boxes which occur within the essential cell‐division genes, ftsQ and ftsA, have been hypothesized to mediate the response of the downstream ftsZ gene to intracellular levels of DnaA, and thus to couple the processes of initiation and cell division. We show here that, although transcription from promoters upstream of ftsZ is increased when initiation of chromosome replication is blocked by DnaA inactivation, this response is not mediated by the DnaA‐boxes near these promoters, nor is it specific to DnaA. We show, furthermore, that mutational inactivation of the putative DnaA‐binding sites in the fts region of the chromosome does not lead to impaired growth or reduced survival of cells.
Protein folding in the cytoplasm of Escherichia coli: requirements for the DnaK‐DnaJ‐GrpE and GroEL‐GroES molecular chaperone machines
Authors: Thomas Jeffrey G. , Baneyx Francois
Published: November 2003

We have systematically investigated the influence of mutations in the σ32 heat‐shock transcription factor and the DnaK‐DnaJ‐GrpE and GroEL‐GroES molecular chaperone machines on the folding of preS2‐β‐galactosidase. This 120kDa fusion protein between the hepatitis B surface antigen preS2 sequence and β‐galactosidase was synthesized in a highly soluble and enzymatically active form in wild‐type Escherichia coli cells cultured at temperatures between 30°C and 42°C, but aggregated extensively in an rpoH165(Am) mutant. Proper folding was partially restored upon co‐overexpression of the dnaKJ operon, but not when the groE operon or dnaK alone were overproduced. The enzymatic activities in dnaK103, dnaJ259 and grpE280 mutants were 40–60% lower relative to a dnaK756 mutant or isogenic wild‐type cells at 30°C and 37°C. At 42°C, only 10–40% of the wild‐type activity was present in each of the early‐folding‐factor mutants. Although the synthesis levels of preS2‐β‐galactosidase were reduced in the dnaK103, dnaJ259 and grpE280 genetic backgrounds, aggregation was primarily responsible for the loss of activity when the cells were grown at 37°C or 42°C. By contrast, the groEL140, groES30 and groES619 mutations, which induced the aggregation of homodimeric ribulose bisphosphate carboxylase (Rubisco), did not affect the solubility of preS2‐β‐galactosidase at temperatures up to 42°C. Our results are discussed in terms of the current understanding of the E. coli protein‐folding cascade. The potential usefulness of heat‐shock protein mutants for the production of soluble proteins in an inclusion‐body form is addressed.
Multicopy fim B gene expression in Escherichia coli: binding to inverted repeats in vivo, effect on fimA gene transcription and DNA inversion
Authors: Dove Simon L. , Dorman Charles J.
Published: November 2003

Transcription of fimA, the Escherichia coli gene encoding the type 1 fimbrial subunit protein, is driven by a promoter carried on a 314bp segment of invertible DNA. We have discovered that overexpression of fimB, one of the genes required for inversion of this DNA element, results in transcriptional repression of fimA. Furthermore, under these conditions inversion ceases to be dependent on the integration host factor (IHF) or the leucine‐responsive regulatory protein (LRP), cofactors hitherto considered to be essential for inversion. Inversion will even occur (albeit at a very low level) in the absence of both cofactors. The interaction of the fimB gene product with the invertible element was studied in vivo in the presence of single‐ and multicopy fimB genes. Dimethyl sulphoxide (DMS)‐mediated methylation of DNA at the 9 bp inverted repeats, which flank the invertible element, was found to vary in the presence and absence of functional fimB. The DMS reactivity profile at the left‐hand inverted repeat was similar with single or multicopy fimB. The corresponding profile at the right‐hand inverted repeat varied with fimB copy number. As this repeat lies between the fimA promoter and open reading frame, FimB binding here is likely to modulate fimA transcription and vice versa.
Topology of diphtheria toxin in lipid vesicle membranes: a proteolysis study
Authors: Quertenmont Pierre , Wattiez Ruddy , Falmagne Paul , Ruysschaert Jean‐Marie , Cabiaux Veronique
Published: November 2003

The diphtheria toxin (DT) membrane topology was investigated by proteolysis experiments. Diphtheria toxin was incubated with asolectin liposomes at pH 5 in order to promote its membrane insertion, and the protein domains located outside the lipid vesicles were digested with proteinase K (which is a non‐specific protease). The protected peptides were separated by electrophoresis and identified by microsequence analysis. Their orientation with respect to the lipid bilayer and their accessibility to the aqueous phase were determined by attenuated total reflection Fourier‐transform infrared spectroscopy (ATR‐FTIR). These data, combined with those provided by proteolytic cleavage with a specific protease (endoproteinase Glu‐C), led us to propose a topological model of the N‐terminal part of the diphtheria toxin B fragment inserted into the lipid membrane. In this model, two a‐helices adopt a transmembrane orientation, with their axes parallel to the lipid acyl chains, while a third o‐helix could adopt a transmembrane topology only in a small proportion of DT molecules.
Transcriptional activation of Salmonella typhimurium invasion genes by a member of the phosphorylated response‐regulator superfamily
Authors: Johnston Christine , Pegues David A. , Hueck Christoph J. , Lee Catherine A. , Miller Samuel I.
Published: November 2003

The Salmonella typhimurium PhoP‐repressed locus prgHIJK encodes components of a sec‐independent type III secretion apparatus. This apparatus is composed of at least 17 proteins encoded on a 40kb pathogenicity island located at centisome 63 on the S. typhimurium chromosome. The secretion apparatus and some of its targets, SspB, SspC and SspD, are necessary for epithelial cell invasion. The transcription of many invasion genes, including prgHIJK, is co‐ordinately activated by HilA, a transcription factor encoded within the pathogenicity island. In this report we identify sirA, a gene located outside the pathogenicity island that is essential for induction of prgHIJK and hilA transcription. sirA encodes a 234‐amino‐acid protein that is essential for S. typhimurium Ssp (Salmonella secreted protein) secretion and invasion and is similar to response regulators of two‐component regulatory systems. sirA‐mutant phenotypes could be suppressed by two DNA clones from unlinked loci, designated sirB and sirC. These data suggest that SirA may be phosphorylated in response to S. typhimurium sensing a mammalian microenvironment. Furthermore, SirA phosphorylation is predicted to initiate a cascade of transcription‐factor synthesis which results in invasion‐gene transcription, Ssp secretion, and bacterial invasion of epithelia.
A putative helical domain in the MalK subunit of the ATP‐binding‐cassette transport system for maltose of Salmonella typhimurium (MalFGK2) is crucial for interaction with MalF and MalG. A study using the LacK protein of Agrobacterium radiobacter as a tool.
Authors: Wilken Sylke , Schmees Günter , Schneider Erwin
Published: November 2003

The ATP‐binding‐cassette (ABC) protein LacK of Agro‐bacterium radiobacter displays high sequence similarity to the MalK subunit of the Salmonella typhimurium maltose‐transport system (MalFGK2). We have used LacK as a tool to identify sites of interaction of MalK with the membrane‐integral components MalF and MalG. Small amounts of LacK, resulting from the expression of the plasmid‐borne lacK gene, proved to be sufficient for partial restoration of growth of a malK strain of S. typhimurium on maltose. LacK failed to substitute for MalK in regulating the expression of maltose‐inducible genes but the hybrid complex MalFGLacK2 was sensitive to inducer exclusion. The lacK gene also complemented a ugpC mutant of Escherichia coli to growth on sn‐glycerol‐3‐phosphate as the phosphate source. Partially purified LacK exhibited a spontaneous ATPase activity comparable to that of MalK. A MalK′–′LacK chimeric protein was isolated (by in vivo recombination) in which the N‐terminal 140 amino acids of MalK are fused to residues 141–363 of LacK. The protein substituted for MalK in maltose transport considerably better than LacK. Furthermore, random mutagenesis of the plasmid‐borne lacK gene yielded three clones that were superior to wild‐type lacK in complementing a malK mutation. Single mutations (V114M or L123F) substantially improved the growth of a malK strain on maltose, whereas a double mutation (L123F, S295N) resulted in growth and transport rates that were indistinguishable from those obtained with MalK. In contrast, the introduction of the single change S295N into LacK had no effect but combination with the V114M mutation led to a further twofold increase in transport activity. These results indicate that a putative helical domain in MalK, encompassing residues 89–140, is crucial for a functional, high‐affinity interaction with MalF and MalG.
Increased distal gene transcription by the elongation factor RfaH, a specialized homologue of NusG
Authors: Bailey Marc J. A. , Hughes Colin , Koronakis Vassilis
Published: November 2003

The Escherichia coli RfaH protein is required for the expression of operons directing synthesis and export of the toxin haemolysin, the lipopolysaccharide core, and the F‐factor sex pilus. Mutation of rfaH increases transcriptional polarity along all three operons. By demonstrating strong RfaH‐dependent suppression of transcription polarity in vitro, we have established RfaH as a novel transcriptional activator, and we reveal that RfaH is a homologue of the essential protein NusG that modulates general transcriptional pausing and termination in prokaryotes. Full transcription of the distal genes from an upstream promoter required RfaH and the 5′ cis‐acting ops element, both in vivo and in vitro. In vivo the requirement for the ops element was suppressed by overexpressing RfaH, and in vitro the presence of ops lowered the concentration of RfaH required to stimulate transcript elongation. We suggest that RfaH directs transcript elongation in an analogous way to NusG, but does so in a subset of bacterial operons primarily engaged in the production of extracellular components required for virulence and fertility.
Bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sipS gene for signal peptidase I
Authors: Bolhuis Albert , Sorokin Alexei , Azevedo Vasco , Ehrlich S. Dusko , Braun Peter G. , De Jong Anne , Venema Gerard , Bron Sierd , Maarten van Dijl Jan
Published: November 2003

Bacillus subtilis contains three chromosomally encoded type I signal peptidases (SipS, SipT and SipU), which remove signal peptides from secretory precursor proteins. In the present study the biological function of SipS and the regulation of its synthesis were analysed. Unlike the type I signal peptidase of Escherichia coli, SipS was essential neither for protein secretion nor viability of the cell. However, in the absence of SipS the rate of processing of several preproteins was reduced, and four of the seven major secreted proteins of B. subtilis were hardly detectable in the growth medium. Surprisingly, the processing of Bacillus amyloliquefaciensα‐amylase and the secretion of at least two endogenous B. subtilis proteins was improved in the absence of SipS. These findings indicate that the substrate preference of SipS differs from that of SipT and SipU, and that SipS is an important factor determining the efficiency of protein secretion in B. subtilis. SipS is transcribed in a growth phase‐ and medium‐dependent manner. In minimal medium, the growth phase‐dependent transcription of sipS is controlled by the DegS–DegU two‐component regulatory system, indicating that the expression of sipS is regulated by the same factors that control the expression of most genes for secreted degradative enzymes. Our observations suggest that B. subtilis can modulate its capacity and specificity for protein secretion through the controlled expression of sipS.
The Myxococcus xanthuspilT locus is required for social gliding motility although pili are still produced
Authors: Wu Samuel S. , Wu Jie , Kaiser Dale
Published: November 2003

Social gliding motility in Myxococcus xanthus depends on the presence of Type IV pili. To begin to examine the role of pili in social motility, 17 mutants were identified which had lost social motility, but still expressed pili. Four of these mutants carry point mutations which mapped to a locus upstream of the recently identified pilS, pilR, and pilA genes. Sequencing of this locus revealed a gene with homology to pilT from Pseudomonas aeruginosa. Sequencing of the four point mutations revealed that they occurred within the M. xanthus pilT locus. A markerless deletion within M. xanthuspilT, similar to the four point mutations, disrupted social gliding behaviour but did not interfere with pilus formation or pilus‐dependent cell–cell agglutination. Using time‐lapse videomicroscopy, residual social motility was observed in dsp− strains (known to be deficient in fibril but not pilus production); this was not observed in a ΔpilTdsp− double mutant. Two genes flanking pilT were also sequenced, and found to have homology to pilB and pilC from P. aeruginosa. Markerless deletions within these genes caused both pilus and social‐motility defects. These results indicate that M. xanthuspilB and pilC are required for pilus biogenesis, while pilT is required for assembled pili to play their role in social motility. Thus, pilB, pilT, pilC, pilS, pilR and pilA form a contiguous cluster of pil genes required for social motility.
Two‐dimensional structure of the Opc invasin from Neisseria meningitidis
Authors: Merker Petra , Tommassen Jan , Kusecek Barica , Virji Mumtaz , Sesardic Dorothea , Achtman Mark
Published: November 2003

A two‐dimensional structural model was devised for the Opc outer membrane protein invasin which contains 10 transmembrane strands and five surface‐exposed loops. One continuous epitope recognized by three monoclonal antibodies was localized to the tip of loop 2 by synthetic peptides and site‐directed mutagenesis while a second, discontinuous epitope recognized by a fourth antibody was localized to loops 4 and 5 by insertion mutagenesis. These monoclonal antibodies are bactericidal and inhibit adhesion and invasion. Most of the T‐cell epitopes defined by Wiertz et al. (1996) were localized to the transmembrane strands. Oligonucleotides encoding a foreign epitope (∇) from Semliki Forest virus were inserted into BglII restriction sites created by site‐directed mutagenesis. The ∇ epitopes inserted in all five predicted loops were recognized on the cell surface of live Escherichia coli bacteria by a monoclonal antibody and are exposed while ∇ epitopes in the N‐terminus or three predicted turns were not. The results thus confirm important predictions of the model and define five permissive sites within surface‐exposed loops which can be used to insert foreign epitopes.
Amino acid‐dependent regulation of the Saccharomyces cerevisiaeGSH1 gene by hydrogen peroxide
Authors: Stephen Duncan W. S. , Jamieson Derek J.
Published: November 2003

The tripeptide γ‐l‐glutamyl‐l‐cysteinylglycine (glutathione) is one of the major antioxidant molecules of cells and plays a vital role in buffering the cell against reactive oxygen species and toxic electrophiles. In the yeast Saccharomyces cerevisiae, the first enzyme involved in glutathione biosynthesis, γ‐glutamylcysteine synthetase, is encoded by the GSH1 gene. This study shows that the regulation of the yeast GSH1 gene by oxidants and the heavy metal cadmium is at the level of transcription. We also demonstrate that the regulation of the GSH1 gene by H2O2 depends upon the presence of the amino acids glutamate, glutamine and lysine in the media. Moreover, regulation of the GSH1 gene by H2O2, although requiring the Yap1 protein, appears to be mediated by a mechanism distinct from that which regulates the Yap1‐dependent induction of genes encoding thioredoxin (TRX2) and a stress‐inducible HSP70 (SSA1) by H2O2.
An RNA polymerase α subunit mutant impairs N‐dependent transcriptional antiterminationin Escherichia coli
Authors: Obuchowski Michal , We¸grzyn Alicja , Szalewska‐Palasz Agnieszka , Thomas Mark S. , We¸grzyn Grzegorz
Published: November 2003

We show that the rpoA341 mutation in the gene encoding the α subunit of Escherichia coli RNA polymerase results in a decreased level of transcripts originating from the lytic promoters pL and pR of infecting λ phage. However, using lacZ fusions we demonstrate that initiation of transcription from both pL and pR is not impaired in the rpoA341 host. Rather, it is the level of the longer, antiterminated pL‐ and pR‐derived transcripts which is altered: the activity of β‐galactosidase in bacteria harbouring a source of N and a pL–nutL–tL1–tI–lacZ or pR–nutR–tR1–lacZ fusion is considerably lower in the rpoA341 mutant relative to the rpoA+ strain. In the absence of the antiterminator protein N no difference is observed in the level of longer pR‐derived transcripts between wild‐type (rpoA+) and mutant (rpoA341) hosts. Although synthesis of N appears to be similar in both phage‐infected rpoA+ and rpoA341 cells, overexpression of the N gene leads to restoration of wild‐type levels of the longer pL‐ and pR‐derived transcripts in the mutant host. While this mutation does not appear to affect vegetative phage growth in nus+ backgrounds, in combination with certain nus mutations it retards lytic development. Therefore, we conclude that the rpoA341 mutation specifically interferes with the function of the N‐antitermination complex, suggesting that the C‐terminal domain of the RNA polymerase α subunit may play an important role in N‐dependent transcriptional antitermination.
DNA sequence of recombinase‐binding sites can determine Xer site‐specific recombination outcome
Authors: Blake Jennifer A. R. , Ganguly Nivedita , Sherratt David J.
Published: November 2003

Xer site‐specific recombination functions in the stable inheritance of circular plasmids and bacterial chromosomes. Two related recombinases, XerC and XerD, mediate this recombination, which ‘undoes’ the potential damage of homologous recombination. Xer recombination on natural plasmid sites is preferentially intramolecular, converting plasmid multimers to monomers. In contrast, recombination at the Escherichia coli recombination site, dif, occurs both intermolecularly and intramolecularly, at least when dif is inserted into a multicopy plasmid. Here the DNA sequence features of a family of core recombination sites in which the XerC‐ and XerD‐binding sites, which are separated by 6 bp, were analysed in order to ascertain what determines whether recombination will be preferentially intramolecular, or will occur both within and between molecules. Sequence changes in either the XerC‐ or XerD‐binding site can alter the recombination outcome. Preferential intramolecular recombination between a pair of recombination sites requires additional accessory DNA sequences and accessory recombination proteins and is correlated with reduced affinities of recombinase binding to recombination core sites, reduced XerC‐mediated cleavage in vitro, and an apparent increased overall bending in recombinase–core‐site complexes.
Characterization of MexE–MexF–OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa
Authors: Ko¨hler Thilo , Michéa‐Hamzehpour Mehri , Henze Uta , Gotoh Naomasa , Kocjancic Curty Lasta , Pechère Jean‐Claude
Published: November 2003

Antibiotic‐resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB–oprM efflux operon. Antibiotic‐accumulation studies indicated that OprN was part of an energy‐dependent antibiotic‐efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB–oprM and mexCD–oprJ efflux operons of P. aeruginosa. The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug‐resistance phenotype in an OprN‐overexpressing strain, but did not affect the susceptibility profile of the wild‐type strain. Expression of the mexEF–oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild‐type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF–oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa.
RecR is a zinc metalloprotein from Bacillus subtilis 168
Authors: Ayora Silvia , Stiege Asita C. , Alonso Juan C.
Published: November 2003

The Bacillus subtilis RecR protein is required for DNA repair and recombination in vivo. In its N‐terminal portion, RecR possesses potential zinc‐ligand structures associated with the multicysteine (C4) superfamily. The number and arrangement of the cysteine residues is suggestive of RecR being a zinc‐finger protein. One of the four cysteines (Cys‐60) has been replaced by a Ser (C60S) or an Ala (C60A) residue to generate the recR60 and recR601 genes, respectively. B. subtilisrecR60, recR601 or ΔrecR1 (a null‐mutant allele) cells are 10‐, 134‐ and 144‐fold more sensitive to 10 mM methanesulphonate and 95‐, 900‐ and 1100‐fold more sensitive to the lethal effect of 100 μM 4‐nitroquinoline‐1‐oxide (4NQO) than the wild‐type strain, respectively. The RecR zinc‐ligand C4 motif does not seem to be accessible, because the protein is highly resistant to oxidation and moderately resistant to reduction. We have determined by different biochemical methods that RecR is a zinc metalloprotein whose cysteine residues have a structural and/or functional role.
The type IC hsd loci of the enterobacteria are flanked by DNA with high homology to the phage P1 genome: implications for the evolution and spread of DNA restriction systems
Authors: Tyndall Chiara , Lehnherr Hansjo¨rg , Sandmeier Ursula , Kulik Eva , Bickle Thomas A.
Published: November 2003

EcoR124I, EcoDXXI and EcoprrI are the known members of the type IC family of DNA restriction and modification systems. The first three are carried on large, conjugative plasmids, while EcoprrI is chromosomally encoded. The enzymes are coded by three genes, hsdR, hsdM and hsdS. Analysis of the DNA sequences upstream and downstream of the type IC hsd loci shows that all are highly homologous to each other and also to sequences present in the bacteriophage P1 genome. The upstream sequences include functional phd and doc genes, which encode an addiction system that stabilizes the P1 prophage state, and extend to and beyond pac, the site at which phage DNA packaging begins. Downstream of the hsd loci, P1 DNA sequences begin at exactly the same place for all of the systems. For EcoDXXI and EcoprrI the P1 homology extends for thousands of base pairs while for EcoR124I an IS1 insertion and an associated deletion have removed most of the P1‐homologous sequences. The significance of these results for the evolution of DNA restriction and modification systems is discussed.
Peptidoglycan structure of Salmonella typhimurium growing within cultured mammalian cells
Authors: Quintela José Carlos , De Pedro Miguel A. , Zo¨llner Peter , Allmaier Gu¨nter , Garcia‐del Portillo Francisco
Published: November 2003

The cell wall structure of Salmonella typhimurium has been studied for the first time during transit from free‐living to parasitic lifestyles. Peptidoglycan of S. typhimurium proliferating within human epithelial cells contains a high proportion of previously unidentified muropeptides (5–10‐fold higher than in extracellular bacteria). Amino acid and mass‐spectrometry analyses showed that these new components consist of dimeric cross‐linked muropeptides lacking one of the two disaccharide (N‐acetyl‐glucosamine‐β‐(1→4)‐N‐acetyl‐muramic acid) molecules. This unique structure suggests an active role for an N‐acetyl‐muramyl‐l‐alanine‐amidase in remodelling the peptidoglycan of intracellular S. typhimurium. Additional alterations observed included: (i) the absence of glycine‐containing muropeptides; (ii) the increase in the relative proportion of muropeptides cross‐linked by l(meso)‐diaminopimelyl‐d(meso)‐diaminopimelic acid (l–d) peptide bridges; and, (iii) the decrease in the global cross‐linkage of the macromolecule. The structural alterations observed in the peptidoglycan of intracellular bacteria do not produce loss of the cell envelope. These results show that intracellular residence of S. typhimurium within epithelial cells is accompanied by significant changes in the bacterial cell wall. Remodelling of peptidoglycan structure may constitute another sophisticated strategy of this pathogen for adapting to and colonizing the intracellular niche of eukaryotic cells.
The N‐terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step
Authors: Bouveret Emmanuelle , Rigal Alain , Lazdunski Claude , Bénédetti Hélène
Published: November 2003

Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli: the Tol system for group A colicins and the TonB system for group B colicins. The N‐terminal domain of colicins is involved in the translocation step. To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N‐terminal domains of colicin E3 (group A) and colicin B (group B). Producing cells became specifically tolerant to entire extracellular colicins of the same group. The periplasmic N‐terminal domains therefore compete with entire colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane. In vivo cross‐linking and co‐immunoprecipitation experiments in cells producing the colicin E3 N‐terminal domain demonstrated the existence of a 120 kDa complex containing the colicin domain and TolB. After in vitro cross‐linking experiments with these two purified proteins, a 120 kDa complex was also obtained. This suggests that the complex obtained in vivo contains exclusively TolB and the colicin E3 domain. The N‐terminal domain of a translocation‐defective colicin E3 mutant was found to no longer interact with TolB. Hence, this interaction must play an important role in colicin E3 translocation.
Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus
Authors: Thumm Günther , Götz Friedrich
Published: November 2003

Lysostaphin is an extracellular glycylglycine endopep‐tidase produced by Staphylococcus simulans biovar staphylolyticus ATCC1362 that lyses staphylococcal cells by hydrolysing the polyglycine interpeptide bridges of the peptidoglycan. Renewed analysis of the sequence of the lysostaphin gene (Iss), and the sequencing of the amino‐terminus of purified prolysostaphin and of mature lysostaphin revealed that lysostaphin is organized as a preproprotein of 493 amino acids (aa), with a signal peptide consisting of 36 aa, a propeptide of 211 aa from which 195 aa are organized in 15 tandem repeats of 13 aa length, and a mature protein of 246 aa. Prolysostaphin is processed in the culture supernatant of S. simulans biovar staphylolyticus by an extracellular cysteine protease. Although prolysostaphin was staphylolytically active, the mature lysostaphin was about 4.5‐fold more active. The controlled expression in Staphylococcus carnosus of Iss and Iss with deletions in the prepropeptide region indicated that the tandem repeats of the propeptide are not necessary for protein export or activation of Lss, but keep Lss in a less active state. Intracellular expressed pro‐ and mature lysostaphin exert staphy‐lolytic activity in cell‐free extracts, but do not affect growth of the corresponding clones. We characterized a lysostaphin immunity factor gene (lif) which is located in the opposite direction to Iss. The expression of lif in S. carnosus led to an increase in the serine/glycine ratio of the interpeptide bridges of peptidoglycan from 2 to 35%, suggesting that lysostaphin immunity depends on serine incorporation into the interpeptide bridge. If, in addition to lif, Iss is co‐expressed the serine/glycine ratio is further increased to 58%, suggesting that Lss selects for optimal serine incorporation. Lif shows similarity to FemA and FemB
A selenium‐dependent and a selenium‐independent formylmethanofuran dehydrogenase and their transcriptional regulation in the hyperthermophilic Methanopyrus kandleri
Authors: Vorholt Julia A. , Vaupel Martin , Thauer Rudolf K.
Published: November 2003

The genome of Methanopyrus kandleri was found to harbour a gene, fwuB, predicted to encode the catalytic subunit of a tungsten formylmethanofuran dehydrogenase with an active site selenocysteine, and a second gene, fwcB, encoding a tungsten formylmethanofuran dehydrogenase with an active site cysteine. Northern blot and primer‐extension analysis revealed that both genes were differentially transcribed. During growth of the methanogen on medium supplemented with selenium only fwuB was transcribed, whereas transcription of both fwuB and fwcB was observed on selenium‐deprived medium. Growth of M. kandleri was stimulated by tungstate and selenite but not by molybdate. The findings indicate that the hyperthermophilic archaeon contains two tungsten isoenzymes of formylmethanofuran dehydrogenase, one of which is a novel selenium enzyme. They also indicate that the hyperthermophilic methanogen probably does not contain a molybdenum formylmethanofuran dehydrogenase which appears to be present only in thermophilic and mesophilic methanogens.
The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase
Authors: Jia Y.‐K. , Bécam A.‐M. , Herbert C. J.
Published: November 2003

We have identified a third citrate synthase gene in Saccharomyces cerevisiae which we have called CIT3Complementation of a citrate synthase‐deficient strain of Escherichia coli by lacZ ::CIT3 gene fusions demonstrated that the CIT3 gene encodes an active citrate synthase. The CIT3 gene seems to be regulated in the same way as CIT1, which encodes the mitochondrial isoform of citrate synthase. Deletion of the CIT3 gene in a Δcit1 background severely reduced growth on the respiratory substrate glycerol, whilst multiple copies of the CIT3 gene in a Δcit1 background significantly improved growth on acetate. In vitro import experiments showed that cit3p is transported into the mitochondria. Taken together, these data show that the CIT3 gene encodes a second mitochondrial isoform of citrate synthase.
DNA topology in hyperthermophilic archaea: reference states and their variation with growth phase, growth temperature, and temperature stresses
Authors: López‐García Purificación , Forterre Patrick
Published: November 2003

In order to address the dynamics of DNA topology in hyperthermophilic archaea, we analysed the topological state of several plasmids recently discovered in Thermococcales and Sulfolobales. All of these plasmids were from relaxed to highly positively super‐coiled in vitro, i.e. they exhibited a significant linking excess compared to the negatively supercoiled plasmids from mesophilic organisms (both Archaea and Bacteria). In the two archaeai orders, plasmid linking number (Lk) decreased as growth temperature was lowered from its optimal value, i.e. positively super‐coiled plasmids were relaxed whereas relaxed plasmids became negatively supercoiled. Growth temperatures above the optimum correlated with higher positive supercoiling in Sulfolobales (Lk increase) but with relaxation of positive supercoils in Thermococcus sp. GE31. The topological variation of plasmid DNA isolated from cells at different growth phases were found to be species specific in both archaeai orders. In contrast, the direction of topological variation under temperature stress was the same, i.e. a heat shock correlated with an increase in plasmid positive supercoiling, whilst a cold shock induced negative supercoiling. The kinetics of these effects were analysed in Sulfolobales. In both temperature upshift (from 80 to 85C) and downshift (from 80 to 65C), a transient sharp variation of Lk occurred first, and then DNA supercoiling progressively reached levels typical of steady‐state growth at the final temperature. These results indicate that DNA topology can change with physiological states and environmental modifications in hyperthermophilic archaea.
Surface proteins and a novel transcription factor regulate the expression of the S‐layer gene in Thermus thermophilus HB8
Authors: Fernández‐Herrero Luis Angel , Olabarría Garbiñe , Berenguer José
Published: November 2003

We have identified proteins that control the expression of slpA, the gene encoding the crystalline surface layer of Thermus thermophilus HB8. We cloned three genes from T. thermophilus that specifically repressed the expression of the slpA promoter in Escherichia coli. The proteins encoded by two of them (Rep6 and Rep29) bound in vitro to the slpA promoter, while that from the third (Rep54) bound specifically to the 5′‐untranslated region (5′UTR) of the slpA mRNA. Rep6 protein was identified as a C‐fragment from a Thermus cytoplasmic basic protein of 28 kDa, whose coding gene, slrA (for S‐layer regulator), was characterized. Surprisingly, Rep29 was identified as a C‐fragment of SlpM, an S‐layer‐like protein that is overexpressed in slpA mutants. Insertional inactivation of slrA and slpM demonstrated their in vivo function in the control of slpA transcription: SlrA acts as a repressor, and SlpM as an activator. Even more surprising was the identification of Rep54, the 5′UTR mRNA‐binding protein, as a C‐terminal fragment of the SlpA protein. This result, in addition to further in vivo evidence presented here, supports the existence of a translational autoregulation in slpA expression. The physiological meaning of overlapping transcriptional and translational controls of S‐layer expression, and its relationships with other systems, are discussed.
Characterization of the chemotaxis protein CheW from Rhodobacter sphaeroides and its effect on the behaviour of Escherichia coli
Authors: Hamblin Paul A. , Bourne Natalie A. , Armitage Judith P.
Published: November 2003

In contrast to the situation in enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants. A chemotaxis operon has been identified containing homologues of the enteric cheA, cheW, cheR genes and two homologues of the cheY gene. However, mutations in these genes have only minor effects on chemotaxis. In enteric species, CheW transmits sensory information from the chemoreceptors to the histidine protein kinase, CheA. Expression of R. sphaeroidescheW in Escherichia coli showed concentration‐dependent inhibition of wild‐type behaviour, increasing counter‐clockwise rotation and thus smooth swimming — a phenotype also seen when E. coli cheW is overexpressed in E. coli. In contrast, overexpression of R. sphaeroidescheW in wild‐type R. sphaeroides inhibited motility completely, the equivalent of inducing tumbly motility in E. coli. Expression of R. sphaeroides cheW in an E. coliΔcheW chemotaxis mutant complemented this mutation, confirming that CheW is involved in chemosensory signal transduction. However, unlike E. coliΔcheW mutants, in‐frame deletion of R. sphaeroides cheW did not affect either swimming behaviour or chemotaxis to weak organic acids, although the responses to sugars were enhanced. Therefore, although CheW may act as a signal‐transduction protein in R. sphaeroides, it may have an unusual role in controlling the rotation of the flagellar motor. Furthermore, the ability of a ΔcheW mutant to swim normally and show wild‐type responses to weak acids supports the existence of additional chemosensory signal‐transduction pathways.
Pheromone cCF10 and plasmid pCF10‐encoded regulatory molecules act post‐transcriptionally to activate expression of downstream conjugation functions
Authors: Bensing Barbara A. , Manias Dawn A. , Dunny Gary M.
Published: November 2003

Expression of aggregation protein Asc10 from the prgB gene of conjugative plasmid pCF10 in Enterococcus faecalis is induced by the peptide pheromone cCF10. Genes required for Asc10 production, prgQ and prgS, lie 3–5 kb upstream, but can function at much greater distances. The prgQ transcripts encode a pheromone inhibitor peptide (iCF10) at the extreme 5′ end. Neither production of this peptide nor translation of the 5′ end of prgQ transcripts was found to be necessary for prgB expression. Pheromone cCF10 is required to activate prgB expression, even in the absence of iCF10 production, and does not affect initiation of transcription. The prgS gene encodes a 10.5 kDa protein that appears to be required for translation of prgB, and a non‐coding RNA at the 3′ end of prgS may be required for readthrough of transcription to prgB from the prgQ promoter. Although the entire positive control region is transcribed constitutively from the prgQ promoter, translation of PrgS and transcriptional readthrough to prgB occur only after induction with pheromone. The combined data are consistent with a model in which the positive regulatory molecules and pheromone cCF10 activate prgB expression post‐transcriptionally.
Pheromone‐inducible expression of an aggregation protein in Enterococcus faecalis requires interaction of a plasmid‐encoded RNA with components of the ribosome
Authors: Bensing Barbara A. , Dunny Gary M.
Published: November 2003

Transfer of the conjugative plasmid pCF10 from Enterococcus faecalis donor strains is induced by a peptide pheromone (cCF10) secreted by recipient cells. High‐efficiency transfer requires expression of an aggregation protein (Asc10) encoded by the prgB gene and positively regulated by genes in a region 3–5 kb upstream, containing prgQ‐R‐S. Transcriptional fusion data reported here support the results of recent molecular analysis of the 5′ ends of prgB transcripts which indicated that prgB transcription occurs by readthrough from the prgQ promoter. A 530‐nucleotide prgQ‐encoded RNA molecule (Ql) with rRNA‐like domains is required for Asc10 production. Ql and cCF10 were found to interact with the L6 and S5 ribosomal proteins, respectively. Mutational analysis of Ql indicates that this RNA may also directly interact with 16S RNA. Ql is present in ribosomes translating the prgB message, and pheromone cCF10 may affect the association of this RNA with translation complexes. Results suggest that the positive regulatory molecules act post‐transcriptionally on the polycistronic message and modify a ribosome population to enhance pheromone‐induced translation of prgB.
Getting greasy: how transmembrane polypeptide segments integrate into the lipid bilayer
Authors: Von Heijne Gunnar
Published: November 2003

Many integral membrane proteins use the same translocation machinery for membrane insertion as secretory proteins use to get across the membrane. This requires that transmembrane segments can be discriminated from other parts of the protein during membrane translocation, and further requires that the transmembrane segments can be moved laterally out of the translocation channel into the surrounding lipid. The molecular basis for this remarkable intramembraneous sorting event is a major focus of current studies of membrane protein biogenesis.
A gene required for nuclear migration in Neurospora crassa codes for a protein with cysteine‐rich, LIM/RING‐like domains
Authors: Vierula P. J. , Mais J. M.
Published: November 2003

The ro mutants of Neurospora crassa are defective in nuclear migration and hyphal morphogenesis. Several of the ro loci have recently been shown to encode components of the dynein/dynactin motor complex. Here we report on the cloning and characterization of the ro‐2 gene which codes for a novel 80 kDa protein that has two Cys‐rich motifs which resemble zinc‐binding LIM or RING domains thought to mediate protein–protein interactions. RO2 also contains several potential binding sites for Src homology 3 (SH3) domains. The ro‐2B20 allele has a frameshift mutation within one of the Cys‐rich domains which eliminates the C‐terminal half of the open reading frame (ORF). Disruption of the ro‐2 locus by repeat‐induced point (RIP) mutation gave rise to progeny which have a nuclear migration defect, but which are also blocked in conidiation. The ability to assemble cytoplasmic microtubules and actin is maintained in ro‐2 mutants, although subapical actin patches are more prominent. Based on these observations, the RO2 gene product is proposed to play a role in mediating interactions between components of the dynein/dynactin motor complex or in linking this complex to the nucleus or cytoskeleton.
Transcriptional regulation of the macrophage‐induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant
Authors: Kwaik Yousef Abu , Gao Lian‐Yong , Harb Omar S. , Stone Barbara J.
Published: November 2003

Expression of the global stress protein gene (gspA) is induced during the intracellular infection of macrophages and upon exposure of Legionella pneumophila to in vitro stress stimuli. Transcription of gspA is regulated by two promoters, one of which is regulated by the σ32 heat‐shock transcription factor. We utilized a gspA promoter fusion to a promoterless lacZ to probe the phagososmal ‘microenvironment’ for the kinetics of exposure of intracellular L. pneumophila to stress stimuli. Expression through the gspA promoter was constitutively induced by approx. 16‐fold throughout the intracellular infection, and occurred predominantly through the σ32‐regulated promoter. Expression of the gspA promoter was induced approx. 4.5‐fold, 5‐, 11‐ and 9‐fold upon exposure of L. pneumophila to heat shock, oxidative stress, acid shock, and osmotic shock, respectively. An isogenic insertion mutant of L. pneumophila in gspA (strain AA224) was constructed by allelic exchange in the wild‐type strain AA200. Compared to in vitro‐grown wild‐type strain AA200, AA224 was more susceptible to all four in vitro stress stimuli. The wild‐type phenotypes were restored to strain AA224 by complementation with a plasmid containing wild‐type gspA. There was no difference between the wild‐type strain and the gspA mutant in cytopathogenicity to U937 cells or in their kinetics of intracellular replication within macrophages and amoebae. However, compared to in vitro‐grown bacteria, macrophage‐grown and amoebae‐grown AA200 and AA224 showed an equal and dramatic increase in resistance to in vitro stress stimuli. Our data showed that regardless of the capacity of L. pneumophila to subvert the microbicidal mechanisms of the macrophage, intracellular L. pneumophila is exposed to a high level of stress stimuli throughout the intracellular infection. Although the GspA protein is required for protection of the bacteria against in vitro stress stimuli, and is induced during intracellular multiplication, the loss of its function is probably compensated for by other macrophage‐induced and stress‐induced proteins within the intracellular environment.
Polypeptide chain release factors
Authors: Buckingham Richard H. , Grentzmann Guido , Kisselev Lev
Published: November 2003

Newly synthesized polypeptide chains are released from peptidyl‐tRNA when the ribosome encounters a stop signal on mRNA. Extra‐ribosomal proteins (release factors) play an essential role in this process. Although the termination process was first discovered in the late 1960s, much of the mechanism has remained obscure. However, important steps have recently been made in both prokaryotic and eukaryotic organisms in unlocking the secrets of this vital stage in protein synthesis. In this review we summarize these advances and focus attention on the remaining areas of uncertainty, particularly with respect to the models that have been proposed for the action of the GTP‐hydrolysing termination factors in prokaryotes and eukaryotes, i.e. RF3 and eRF3.
Involvement of the ribulosephosphate epimerase gene in the dnaA and dnaR functions for initiation of chromosome replication in Escherichia coli
Authors: Sakakibara Yoshimasa
Published: November 2003

Initiation of replication of the Escherichia coli chromosome is rendered temperature‐sensitive by the dnaR130 mutation in the prs gene that encodes phosphoribosylpyrophosphate synthetase. The thermosensitivity of the dnaR mutant is suppressed by a spontaneous mutation in rpe, the gene encoding ribulosephosphate epimerase. Disruption of the rpe gene reverses the thermosensitive growth of the dnaR mutant. The rpe‐disrupted dnaR mutant exhibits extensive DNA synthesis at low and high temperatures, as does the dnaR +rpe disruptant and the dnaR +rpe+ strain. The thermoresistant DNA synthesis in the rpe dnaR double mutant is dnaA dependent, because the dnaA167 mutation renders the synthesis thermosensitive. The rpe‐knockout mutation abolishes the ability of the dnaA115 allele to complement the defect of the dnaA167 mutant with or without the dnaR mutation and diminishes the dnaR‐complementing ability of the dnaR224 allele. These results show that the rpe product is involved in the functions of the products of both dnaA and dnaR for initiation of replication of the bacterial chromosome.