bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cassette mutagenesis implicates a helix‐turn‐helix motif in promoter recognition by the novel RNA polymerase Sigma factor σ54
Authors: Coppard J. R. , Merrick M. J. 
Published: October 2006

SummaryCassette mutagenesis has been used to study the role of a helix‐turn‐helix (HTH) motif in the novel RNA polymerase sigma factor σ54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent‐exposed in the second helix, the first (Glu‐378) tolerated all substitutions, and some mutations of this residue increased expression from σ54‐dependent promoters. Certain substitutions in the third exposed residue (Ser‐382) produced a promoter‐specific phenotype and all substitutions in the fourth residue (Arg‐383) inactivated the protein, identifying this residue as being likely to be involved in base‐specific interactions with the promoter. In vivo foot‐printing indicated that the inactive HTH mutants of a54 were defective in interaction with both the ‐24 and ‐12 regions of the gln Ap2 promoter.
Analysis of an anaerobically induced promoter for the cobalamin biosynthetic genes in Salmonella typhimurium
Authors: Richter‐Dahlfors A. A. , Andersson D. I. 
Published: October 2006

SummaryWe have identified an anaerobicaily induced promoter for the cobalamin biosynthetic (cob) genes. In a plasmid the Cob promoter showed two of the three types of control of the intact chromosomal Cob operon: anaerobic induction and cAMP stimulation. Cobalamin repression was observed only in promoter fragments which included sequences far downstream of the transcription start site, suggesting that this control is post‐transcriptional. One anaerobically induced transcript was identified and its 5′ end was determined. Deletion mapping showed that 60 nucleotides upstream of the start site were sufficient for anaerobic synthesis of this transcript. Upstream of the transcription start site a putative σ70‐dependent ‐10 recognition sequence was identified; however, no consensus ‐35 region was observed.
Sequential activation of dual promoters by different Sigma factors maintains spoVJ expression during successive developmental stages of Bacillus subtilis
Authors: Foulger D. , Errington J. 
Published: October 2006

SummaryThe spoVJ gene of Bacillus subtilis encodes a 36 k Da protein and is expressed only in the mother cell. spoVJ has an interesting pattern of regulation during sporulation because it is expressed from sequentially activated promoters. These promoters, designated P1 and P2, are under the control of different sigma factors, σEand σK, which become active at separate times during sporulation. Removal of promoter P1, leaving promoter P2 active, resulted in about a 30‐minute delay in the formation of heat‐resistant spores and demonstrated that the expression of spoVJ from both promoters is essential for normal sporulation. A comparison is made between the sequences of the spoVJ promoters and the promoters of other genes dependent upon σE and σk.
Genetic analysis of the rfb region of Shigella flexneri encoding the Y serotype O‐antigen specificity
Authors: Macpherson D. F. , Morona R. , Beger D. W. , Cheah K.‐C. , Manning P. A. 
Published: October 2006

SummaryThe gene cluster (rfb region) which determines the biosynthesis of the Shigella flexneri O‐antigen of the Y serotype specificity was cloned from a S. flexneri serotype 2a strain. Two plasmids, pPM2212 and pPM2213, which conferred O‐antigen biosynthesis were generated from separate cosmid clones by deletion with Cla l. These plasmids expressed O‐antigen in Escherichia coli K12 like that of the parental strain, as assessed by reactions to antisera in colony and Western immunoblots, sensitivity to bacteriophage Sf6, and by silver staining of lipopolysaccharides separated by sodium dodecyl sulphate/polyacryl‐amide gel electrophoresis. These plasmids also mediated O‐antigen expression in an E. coli K12 rfb‐delete background, indicating that all the necessary genes have been cloned. A detailed restriction map of the region has been constructed and analysis of various subclones has allowed the limits of the coding region for O‐antigen biosynthesis to be defined to a maximum of 11kb. Expression of these plasmids demonstrates a novel phenotype associated with control of lipopolysaccharide chain length. The gene(s) responsible maps adjacent to, but separate from, those associated with the biosynthesis of the O‐antigen unit. Analysis of plasmid‐encoded proteins in minicells and maxicells has facilitated the construction of a physical map. Finally, plasmid pPM‐2212 was used to probe a collection of S. flexneri serotypes by Southern hybridization. With the exception of serotype 6, which appears to be unrelated, a similar pattern was found in all serotypes.
Anthrax toxin protective antigen: low‐pH‐induced hydrophobicity and channel formation in liposomes
Authors: Koehler T. M. , Collier R. J. 
Published: October 2006

SummaryTo probe the role of the protective antigen (PA) component of anthrax toxin in toxin entry into animals cells, we examined the membrane channel‐forming properties and hydrophobicity of intact and trypsin‐cleaved forms of the protein at various pH values. At neutral pH neither form caused release of entrapped K+ from unilamellar lipid vesicles. At pH values below 6.0, however, K+ was rapidly released upon addition of either the nicked PA (PAN) or the 63kDa tryptic fragment of PA (PA63), which has been implicated in the toxin entry process. Under the same conditions intact PA exhibited only weak channel‐forming activity, and PA20, the complementary tryptic fragment, showed no such activity. Both PA and PA63 exhibited enhanced hydrophobicity at acidic pH values, but the enhancement was greater and the pH threshold higher with PA63. Our findings indicate that proteolytic removal of PA20 from intact PA enables the residual protein, Pasb63, to adopt a conformation at mildly acidic pH values that permits it to insert readily and form channels in membranes. Thus acidic conditions within endocytic vesicles may trigger membrane insertion of PA63, which in turn promotes translocation of ligated effector moieties, edema factor or lethal factor, across the vesicle membrane into the cytosol.
The pathogenic mechanisms of Shiga toxin and the Shiga‐like toxins
Authors: Tesh V. L. , O'Brien A. D. 
Published: October 2006

SummaryIt is now well documented that some enteric bacteria which cause diarrhoeal and/or dysenteric disease produce, at high levels, one or more of a family of protein toxins referred to as Shiga toxin and Shiga‐like toxins (SLTs; alternatively called verocytotoxins or VTs). Within the past few years, there have been considerable advancements made in our understanding of the biochemistry and molecular biology of Shiga toxin and SLTs. However, the precise role of the toxins in mediating colonic disease, as well as their contribution to the development of extra‐intestinal sequelae (e.g. the haemolytic uraemic syndrome and neurological disorders), remain less clear. In thisMicroReview, we will briefly summarize recent progress in Shiga toxin‐ and SLT‐related research and present evidence supporting the concept that these toxins contribute to pathogenesis by directly damaging vascular endothelial cells, thereby disrupting the homeostatic properties of these cells. We will also discuss data which suggest that toxin‐mediated damage in the kidney may not be limited to glomerular endothelial cells but may include tubular epithelial cells. Thus, the role of the toxins in renal disease may not be limited to the glomeruli, as was initially hypothesized when the association of infection with toxin‐producing strains and the development of acute renal failure was established.
The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes
Authors: Bhat K. S. , Gibbs C. P. , Barrera O. , Morrison S. G. , Jähnig F. , Stern A. , Kupsch E.‐M. , Meyer T. F. , Swanson J. 
Published: October 2006

SummaryVariants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non‐reciprocat inter‐opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter‐opa recombination events.
Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum
Authors: Rossbach S. , Hennecke H. 
Published: October 2006

SummaryA Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non‐fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyitransferase (SHMT; E.C. 2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one‐carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1 ‐metabolism are indispensable for the establishment of a fully effective, nitrogen‐fixing root nodule symbiosis.
Molecular characterization of the O‐acetyl transferase gene of converting bacteriophage SF6 that adds group antigen 6 to Shigella flexneri
Authors: Verma N. K. , Brandt J. M. , Verma D. J. , Lindberg A. A. 
Published: October 2006

SummaryBacteriophage SF6 antigenically converts Shigella flexneri serotype Y strains (‐;3,4) to type 3b carrying group antigen 6,3,4 by means of an O‐acetylation of the O‐antigenic polysaccharide chain. The gene for O‐acetyl transferase of bacteriophage SF6 has been cloned, identified and sequenced. The predicted O‐acetyl transferase protein encoded by this gene was found to consist of 333 amino acids, (37 185 daltons) and to have some similarity with the galactose‐1‐phosphate uridylyltransferase protein of Escherichia coli. The gene has been shown to function in a live vaccine strain of S. flexneri Y type (ΔaroD), making it a 3b type. The converted type 3b strain, SFL1104, was found to elicit significant protection against challenge by both wild‐type serotypes 3b and Y in a guinea‐pig keratoconjunctivitis model.
Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA
Authors: Berzal‐Herranz A. , Wagner E. G. H. , Diaz‐Orejas R. 
Published: October 2006

SummaryThe RepA protein of plasmid R1 is rate‐limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini‐R1 derivatives and to lowered repA expression assayed in translational repA‐lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA‐mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication.
Molecular characterization of the gor gene encoding glutathione reductase from Pseudomonas aeruginosa: determinants of substrate specificity among pyridine nucleotide‐disulphide oxidoreductases
Authors: Perr A. C. F. , Bhriain N. Ni , Brown N. L. , Rouch D. A. 
Published: October 2006

SummaryIn order to investigate the basis of functional diversity among the pyridine nucleotide‐oxidoreductases the gor gene from Pseudomonas aeruginosa PAO, which encodes glutathione reductase, was analysed. The P. aeruginosa gor gene was identified by hybridization with a short DNA sequence from the gene encoding mercuric reductase in transposon Tn501. The gene was cloned, sequenced and overexpressed in Escherichia coli. Expression of the gene enabled rescue of an E. coli gor‐ mutant, confirming the identity of the cloned gene. The predicted sequence of the gene product showed homology with other members of the pyridine nucleotide‐disulphide oxidoreductase family, and allowed determination of positions that may be involved in substrate specificity. These predictions provided information on the relationship of sequence to function, independently of structural data used in previous studies.
Response of Escherichia coli cell membranes to induction of λc1857 prophage by heat shock
Authors: Kucharczyk K. , Laskowska E. , Taylor A. 
Published: October 2006

SummaryHeat shock induces protein aggregation in Escherichia coli and E. coli (λc1857). The aggregates (S fraction) appear 15 min post‐induction and are separable from membranes by sucrose density‐gradient centrifugation. The S fraction quickly disappears in wild type strains but persists in rpoH mutant with concomitant quick inner membrane destruction. We propose that: (1) the disappearance of the S fraction reflects a rpoH‐dependent processing, (2) the membrane destruction explains the lethality of the rpoH mutation at elevated temperatures; and (3) the protection of the inner membrane integrity is an important physiological function of the heat‐shock response. We assume that the S fraction of aggregated proteins represents the signal inducing the heat‐shock response.The prophage thermo‐induction results in an increase (35 min post‐induction) in the A fraction resembling that of the adhesion zones of the membranes. This fraction is greater than the corresponding fraction from uninduced cells. The increase is mediated by the λ late genes, since it is absent in the induced E.coli (λc1857 Oam21).Since heat shock is widely used for induction of the λ promoters in expression vectors it is possible that the formation of the protein aggregates (though transient in WT strains) and/or the fragility of membranes in rpoH mutants may be the cause of poor expression of cloned genes or may lead to mistaken localization of their expression products.
Multifunctional nature of P fimbriae of uropathogenic Escherichia coli: mutations in fsoE and feoF influence fimbrial binding to renal tubuli and immobilized fibronectin
Authors: Westerlund B. , van Die I. , Kramer C. , Kuusela P. , Holthöfer H. , Tarkkanen A.‐M. , Virkola R. , Riegman N. , Bergmans H. , Hoekstra W. , Korhonen T. K. 
Published: October 2006

SummaryP fimbriae of the F71 serotype of Escherichia coli are composed of a major subunit, FsoA, and of three minor proteins named FsoG, FsoE, and FsoF. FsoG is the Galα(1–4)Gal‐specific lectin. We assessed mutated recombinant strains each deficient in one fimbrial component for adhesion to frozen sections of rat cortical kidney and to fibronectin immobilized on glass. Rat kidney lacks the Galα(1–4)Gal‐containing glycolipids. The fsoG mutant strain was as adhesive to sections of rat kidney and to fibronectin‐coated glass as was the recombinant strain expressing the complete fso gene cluster. The fsoA mutant strain was highly adhesive to fibronectin and to kidney sections. In the rat kidney, the adhesion of these strains was predominantly localized to sites of basolateral membranes of tubuli. The fsoE and the fsoF mutant strains were slightly less adhesive to kidney structures and failed to adhere to fibronectin. The fsoE, fsoF double mutant strain adhered neither to fibronectin nor to kidney sections. None of the fso recombinant strains reacted with soluble fibronectin, suggesting that the interaction is dependent on the conformation of the fibronectin molecules. Recombinant strains expressing the F72, F8, F11, F13, and F14 serovariants of the P fimbria also showed adherence to immobilized fibronectin. The results show that in addition to binding to globoseries of glycolipids via the G protein, the P fimbriae of uropathogenic E. coli exhibit a tissue‐binding property influenced by fsoE and fsoF gene products and with affinity for basolateral membranes and fibronectin.
Involvement of the syrM and nodD3 genes of Rhizobium meliloti in nod gene activation and in optimal nodulation of the plant host
Authors: Kondorosi E. , Buiré M. , Cren M. , Iyer N. , Hoffmann B. , Kondorosi A. 
Published: October 2006

SummaryWe identified and sequenced the regulatory syrM and nodD3 genes of Rhizobium meliloti 41. Both genes were shown to contribute to optimal nodulation of alfalfa. In R. meliloti strains carrying syrM and nodD3 on plasmid, the nod genes are expressed constitutively, resulting in host‐range extension to siratro. This is due to the presence of multiple syrM copies, suggesting that SyrM participates directly in nod gene activation. NodD3 activates nod genes in conjunction with flavonoids and enhances syrM expression, which is controlled also by its own product, NodD2, and two putative trans‐acting factors. nodD3 is regulated by SyrM, NodD1, NodD3, the repressor NoIR, and two putative factors.
Genetic and biochemical analysis of the aspartokinase from Corynebacterium glutamicum
Authors: Kalinowski J. , Cremer. J. , Bachmann B. , Eggeling L. , Sahm H. , Pühler A. 
Published: October 2006

SummaryThe lysC/asd gene cluster of Corynebacterium glutamicum ATCC 13032 was cloned and sequenced. The lysC locus coding for aspartokinase consists of two in‐frame overlapping genes, lysCα encoding a protein of 421 amino acids (Mr 44300) and lysCβ encoding a protein of 172 amino acids (Mr 18600). The C. glutamicum aspartokinase was purified and found to contain two proteins of Mr 47000 and Mr 18000.A C. glutamicum mutant expressing a feedback‐resistant aspartokinase was shown to be changed in a single base pair of the lysCβ gene, leading to an amino acid exchange in the β‐subunit of the aspartokinase. In addition, the identified mutation was found to be responsible for the enhanced expression of the asd gene located downstream of lysC.
Expression of Salmonella typhi and Escherichia coli OmpC is influenced differently by medium osmolarity; dependence on Escherichia coli OmpR
Authors: Puente J. L. , Verdugo‐Rodríguez A. , Calva E. 
Published: October 2006

SummaryOmpC, a major outer‐membrane protein, is highly expressed when Salmonella typhi is grown in nutrient broth (NB) of either low (NB + 0% sucrose) or high (NB + 20% sucrose) osmolarity. This contrasts with the expression of Escherichia coli OmpC, which is inhibited in low osmolarity and enhanced in high osmolarity, as has been described previously (van Alphen and Lugtenberg, 1977; Verhoef et al., 1979; Kawaji et al., 1979). Nevertheless, expression of S. typhi OmpC is dependent on the E. coli OmpR transcriptional activator. These findings suggest differences between the mechanisms of osmoregulation of gene expression in both bacteria, although common effectors appear to be shared.
Filnmentous phage assembly
Authors: Russel M. 
Published: October 2006

SummaryFilamentous phages present a genetically well‐defined system for studying the ordered membrane assembly of five different phage‐encoded proteins around the circular single‐stranded DNA phage genome. Assembly occurs at high efficiency in vivo, catalysed by two phage‐encoded membrane proteins and al least one host protein, thioredoxin. This review presents a description of the virion and its cytoplasmic precursor and summarizes the results of genetic and biochemical experiments that are beginning to elucidate the rote of the three morphogenetic proteins. The recent discovery of bacterial transport proteins with homology to a phage morphogenetic protein located in the outer membrane suggests the existence of a common mechanism for moving complex macromolecules across bacterial membranes.
Analysis of structure and function of the B subunit of cholera toxin by the use of site‐directed mutagenesis
Authors: Jobling M. G. , Holmes R. K. 
Published: October 2006

SummaryOligonucleotide‐directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT‐B) altered at residues Cys‐9, Gly‐33, Lys‐34, Arg‐35, Cys‐86 and Trp‐88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT‐B poly‐peptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT‐B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly‐33 and negatively or positively charged residues for Trp‐88. Substitution of lysine or arginine for Gly‐33 did not affect immunoreactivity or GM1 ‐binding activity of CT‐B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg‐35 interfered with formation of holotoxin, but none of the observed substitutions for Lys‐34 or Arg‐35 affected binding of CT‐B to GM1. The Cys‐9, Cys‐86 and Trp‐88 residues were important for establishing or maintaining the native conformation of CT‐B or protecting the CT‐B polypeptide from rapid degradation in vivo.