bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of enterobacterial cephalosporinase production: the role of a membrane‐bound sensory transducer
Authors: Honoré N. , Nicolas M.‐H. , Cole S.T. 
Published: October 2006

SummaryIn clinical isolates of Enterobacter cloacae, resistance to the newer β‐lactam antibiotics often results from overproduction of a cephalosporinase encoded by the β‐lactam‐inducible ampC gene. Regulation of ampC is controlled by the divergently expressed activator gene, ampR, and a second unlinked locus. In this presentation we show that although Escherichia coli has IQst its ampR gene it has retained the second regulatory locus and that this comprises the bicistronic ampDE operon. Genetic and biochemical studies define the ampD gene as encoding a repressor for amp C transcription whereas the ampE gene product is a cytoplasmic membrane protein. Inactivation of the AmpD protein by mutation causes massive overproduction of cephalosporinase which, in E. cloacae, can terminate in therapeutic failure. In contrast, toss of AmpE results in a total block in induction, despite the presence of the activator, AmpR.
Functional organization and nucleotide sequence of virulence Region‐2 on the large virulence plasmid in Shigella flexneri 2a
Authors: Sasakawa C. , Adler B. , Tobe T. , Okada N. , Nagai S. , Komatsu K. , Yoshikawa M. 
Published: October 2006

SummaryThe 7kb virulence Region‐2 of the large (virulence) plasmid in Shigella flexneri 2a encodes several proteins required for invasion of intestinal epithelial cells. Insertion and deletion mutagenesis, DNA subcloning and SDS‐polyacrylamide gel electro‐phoresis of proteins synthesized in minicells demonstrated five genes in this region. They encode 24, 18, 62 (lpaB), 41 (lpaC) and 37 (lpaD)‐kiloDalton (kD) proteins. Complementation of Tn5‐induced mutations in Region‐2 with the above plasmid constructs indicated that Region‐2 consists of two operons and that the three lpa proteins are essential for the virulence phenotype. The transcriptional organization determined by Northern blotting, S1 nuclease protection and the effect of Tn5 insertions on expression of the lpa proteins revealed that Region‐2 has three promoters that transcribe RNAs of 4.0, 4.5 and 7.5kb. The 4.0 kb RNA was the transcript for the operon encoding the 24, 18 kD, lpaB and C proteins and the 4.5 kb RNA for the ipsD gene. In addition, the full‐length RNA of 7.5 kb which covers Region‐2 supplemented full expression of the lpa proteins. The 7663 nucleotides of Region‐2 were determined to confirm the five open reading frames encoding 23655, 17755, 62168, 41077 and 36660 Dalton proteins, respectively, and their regulatory sequences.
Conserved serine‐rich sequences in xylanase and cellulase from Pseudomonas fluorescens subspecies cellulosa: internal signal sequence and unusual protein processing
Authors: Hall J. , Hazlewood G. P. , Huskisson N. S. , Durrant A. J. , Gilbert H. J. 
Published: October 2006

SummaryThe complete nucleotide sequence of the xynA gene coding for a xylanase (XYLA) expressed by Pseudomonas fluorescens subspecies cellulosa, has been determined. The structural gene consists of an open reading frame of 1833 bp followed by a TAA stop codon. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N‐terminal analysis of purified forms of the xylanase. The signal peptide present at the N terminus of mature XYLA closely resembles signal peptides of other secreted proteins. Truncated forms of the xylanase gene, in which the sequence encoding the N‐terminal signal peptide had been deleted, still expressed an enzyme which was secreted in Escherichia coli. XYLA contains domains which are homologous to an endoglucanase expressed by the same organism. These structures include serine‐rich sequences. Bal31 deletions of xynA revealed the extent to which these conserved sequences, in XYLA, were essential for xylanase activity. Downstream of the TAA stop codon is a G + C‐rich region of dyad symmetry (δG = 24kcal) characteristic of E. coli Rho‐independent transcription terminators.
Isolation and characterization of functional Shiga toxin subunits and renatured holotoxin
Authors: Donohue‐Rolfe A. , Jacewicz M. , Keusch G. T. 
Published: October 2006

SummaryShiga toxin is a potent protein toxin produced by Shigella dysenteriae type I strains. In this report we present a procedure for the separation of functionally intact toxin A and B chains and for their reconstitution to form biologically active molecules. In agreement with the findings of others, the isolated A chain was shown to be a potent in vitro inhibitor of eukaryotic protein synthesis. The isolated B chain bound to HeLa cells and competitively inhibited the binding and cyto‐toxic activity of holotoxin. These findings show that the functional role of the B chain is to recognize cell surface functional receptors. By labelling the B sub‐unit alone, prior to renaturatlon of holotoxin, the polypeptide chains were shown to associate non‐covalently with a stoichiometry of one A chain and five B chains.
The role of the sporulation gene spollIE in the regulation of prespore‐specific gene expression in Bacillus subtilis
Authors: Foulger D. , Errington J. 
Published: October 2006

SummaryThe spollIG gene encodes a sigma factor that determines prespore‐specific gene expression during sporulation in Bacillus subtilis. Correct spatial and temporal expression of the spollIG gene depends on a number of other sporulation (spo) genes, but only one of these genes, spollIE, has a specific effect on spollIG expression and not on gene expression in the other differentiating cell, the mother cell. However, the spollIE gene is expressed predominantly before differentiation begins. Thus, its product must play an important role in sensing or determining the spatial localization of prespore‐specific gene expression in this system.
Osmotic regulation of porin expression: a role for DNA supercoiling
Authors: Graeme‐Cook K. A. , May G. , Bremer E. , Higgins C. F. 
Published: October 2006

SummaryThe OmpC and OmpF porins are major outer membrane proteins of Escherichia coli and Saimonella typhimurium. Their expression is affected by many environmental factors and by mutations in a variety of independent genes. The pair of regulatory proteins, OmpR and EnvZ, are required for normal porin expression. Despite intensive investigation, the mechanisms by which porin expression is regulated remain unclear. Mutations which alter supercoiling, as well as inhibitors of DNA gyrase, show that porin expression is extremely and specifically sensitive to the level of DNA supercoiling. Our data lead us to suggest that environmentally induced changes in DNA supercoiling may play a role in determining the level of porin expression. These findings have implications for current models of porin regulation.
Biochemical and antigenic characterization of the Mycobacterium tuberculosis 71 kD antigen, a member of the 70 kD heat‐shock protein family
Authors: Mehlert A. , Young D. B. 
Published: October 2006

SummaryA 71 kiloDalton antigen from Mycobacterium tuberculosis is recognized by antibodies and by T lymphocytes during infection (Britton et al., 1986a). Partial sequence analysis indicates a relationship between this antigen and the highly conserved family of 70‐kiloDalton heat shock proteins (hsp70) (Young et al., 1988). Biochemical and serological characterization of the protein confirms its membership of the hsp70 gene family, and metabolic labelling demonstrates that it is a major component of the mycobacterial response to heat stress. The role of stress proteins as antigens during infection is discussed.
The streptokinase gene of group A streptococci: cloning, expression in Escherichia coli, and sequence analysis
Authors: Huang T.‐T. , Malke H. , Ferretti J. J. 
Published: October 2006

SummaryThe gene specifying the group A streptokinase (ska) gene was cloned from an M type 49 strain of Streptococcus pyogenes and shown to express in Escherichia coli. The nucleotide sequence of the DNA fragment carrying ska was determined and compared to that of the group C streptokinase gene (skc). There is 90% sequence identity between the two genes, with highly conserved transcription and translation control regions. The deduced amino acid sequence of the group A streptokinase (SKA) contains the same number of amino acids as that of group C streptokinase, with 85% sequence identity between the two proteins. Among 440 amino acid residues specified by the coding sequence, there are 62 non‐identical residues with 45 conserved and 17 non‐conserved residues. The non‐identical residues are located in two major regions, spanning residues 174 to 244 and 270 to 290, with 40 and 10 amino acid changes, respectively. The sequence differences provide an explanation at the molecular level for the previous findings of immunological and chemical heterogeneity among streptokinases produced by pathogenic streptococci.
Molecular evolution of ubiquitous β‐lactamases towards extended‐spectrum enzymes active against newer β‐lactam antibiotics
Authors: Collatz E. , Labia R. , Gutmann L. 
Published: October 2006

SummaryProduction of β‐lactamases, and of the plasmid‐encoded TEM‐and SHV‐type enzymes in particular, is the most common mechanism of resistance against β‐lactam antibiotics in Gram‐negative bacteria. The two ubiquitous types of enzyme have a large spectrum of activity and preferentially hydrolyse the penicillins as well as some first‐and second‐generation cephalosporins. Recently, point mutations in the corresponding genes have been observed, apparently selected for, in the clinical setting, by originally ‘β‐lactamase‐stable’ third‐generation cephalosporins or by mono‐bactams, which fall into the substrate range of the mutant or ‘extended‐spectrum’β‐lactamases. The point mutations are clustered in three areas, each adjacent to one of the seven evolutionarily conserved boxes described by Joris et al. (1988). The substituted amino acids at positions 102 (adjacent to the α‐3 helix), 162 (adjacent to the α‐7 helix) and 235,236 and 237 (on the β‐3 strand) are located in close proximity to the active‐site cavity and are thought to open up novel enzyme‐substrate interactions, involving, in particular, the oxyimino moieties of the newer β‐lactam compounds.
The molecular structure of the Corynebacterium glutamicum threonine synthase gene
Authors: Han K.‐S. , Archer J. A. C. , Sinskey A. J. 
Published: October 2006

SummaryThe minimal region encoding the Corynebacterium glutamicum threonine synthase structural gene and its promoter was mapped by deletion analysis and complementation of the C. glutamicum thrC allele to a 1.6kb region of the recombinant plasmid pFS80. The nucleotide sequence of this and flanking DNA was determined. The transcription and translation start points were identified by S1 mapping analysis and amino‐terminal protein sequencing, respectively. The thrC gene encodes a 54481‐Dalton polypeptide product. Translation of the thrC mRNA initiates only six nucleotides downstream from transcription. The length of the mRNA transcript is consistent with a single gene transcription unit. The C. glutamicum thrC gene is expressed independently of the other threonine‐specific genes hom and thrB.
Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences?
Authors: Fayet O. , Ramond P. , Polard P. , Prère M. F. , Chandler M. 
Published: October 2006

SummaryMembers of the IS3 family of insertion sequences are found in a wide range of bacteria. At least 10 members of this family carry two major open reading frames: a small upstream frame (0 phase), and a longer downstream frame In the –1 phase. The downstream frame shows significant similarity at the amino acid level. A highly conserved region of this frame also exhibits notable similarity with a region of the Integrase (endonuclease) domain of retroviruses. Although the overall transposition mechanism of the insertion sequence and retroviral elements is certainly different, the two groups may share additional common features, including a −1 frameshift resulting in the production of a fusion protein.
Characterization of FapR, a positive regulator of expression of the 987P operon in enterotoxigenic Escherichia Coli
Authors: Klaasen P. , de Graaf F. K. 
Published: October 2006

SummaryExpression of the 987P gene cluster is activated by the adjacent IS1 element of an STpa transposon. Nucleotide sequence analysis of the 987P‐DNA region contiguous with this IS1 element revealed the presence of an open reading frame designated fapR, encoding a basic protein of 260 amino acid residues with a molecular mass of 30349 Daltons. The gene product, FapR, possesses similarity to a number of positive regulators of gene expression: VirF, Rns, AppY and EnvY. Moreover, a 43‐amino‐acid residue sequence in the C‐terminal part of FapR is similar to the C‐terminal domain of AraC, RhaR, and RhaS. Expression of fapR is dependent on the adjacent IS1 element. The FapR protein appears to be required for activation of the silent promoter of the fimbrial subunit gene, fapC
Characterization of the type 1 fimbrial subunit gene (fimA) of Serratia marcescens
Authors: Nichols W. A. , Clegg S. , Brown M. R. 
Published: October 2006

SummaryThe nucleotide sequence of a DNA fragment that contains the fimA gene, encoding the major fimbrial subunit, of Serratia marcescens IA506 and associated flanking sequences has been elucidated. In addition, the origin of transcription has been identified and is located 120 base pairs upstream of the fimA initiation codon. The predicted amino acid sequence of the FimA polypeptide exhibits some degree of sequence homology with the fimbrial subunits encoded by the fimA determinants of Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli and also to Smf2, the major structural component of mannose‐resistant (MR) fimbriae of S. marcescens. The Serratia adhesin that facilitates haemagglutination mediated by type 1 fimbriae is less susceptible to inhibition by D‐mannose than has been observed to be the case in other type 1 fimbrial adhesins. The molecule conferring this adherence specificity has been shown to be distinct from the fimA gene product and, therefore, is analogous to the fimbrial systems reported in other species.
Molecular characterization of immune inhibitor A, a secreted virulence protease from Bacillus thuringiensis
Authors: Lövgren A. , Zhang M. , Engström A. , Dalhammar G. , Landén R. 
Published: October 2006

SummaryThe gene for the secreted neutral metalloprotease, immune inhibitor A (InA), from Bacillus thuringiensis var. alesti has been cloned and sequenced. The deduced amino acid sequence has been confirmed by partial amino acid sequencing. The central part of the amino acid sequence showed similarity to the active site in thermolysin. Southern and Western blots show that InA‐related sequences are common among other B. thuringiensis subspecies. In Western blots, 17 out of 25 tested species gave a positive signal. Culture filtrates from subspecies expressing InA were toxic when injected in Trichoplusia ni larvae, whereas filtrate from a strain negative in Western blot had no effect when injected. The LD50 dose of purified InA protein injected in T. ni larvae was 12.5 ± 2.5 ng per mg of larval body weight.
Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae
Authors: Au‐Young J. , Robbins P. W. 
Published: October 2006

SummaryChitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH‐activity profiles showed that yeast and hyphae contain a protease‐dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin syntheses in C albicans.A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane‐bound zymogen capable of chitin biosynthesis in vitro was based on several criteria, (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]‐GlcNAc from the substrate, UDP[U‐14C]‐GlcNAc, into alkali‐insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH‐activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 6.0 is proposed.DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).
Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenylase components
Authors: Böhm R. , Sauter M. , Böck A. 
Published: October 2006

SummaryAn 8kb segment of DNA from the 58/59 min region of the E. coli chromosome, which complements the defect of a mutant devoid of hydrogenase 3 activity, has been sequenced. Eight open reading frames were identified which are arranged in a transcriptional unit; all open reading frames were transcribed and translated in vivo in a T7 promoter/polymerase system. Analysis of the amino acid sequences derived from the nucleic acid sequences revealed that one of them, open reading frame 5 (0RF5), exhibits significant sequence similarity to conserved regions of the large subunit from Ni/Fe hydrogenases. Two of the open reading frames (orf2, orf6) code for proteins apparently carrying iron‐sulphur clusters of the 4Fe/4S ferredoxin type. The product of one of the open reading frames, orf7, displays extensive sequence similarity with protein G from the chloroplast electron transport chain. ORF3 and ORF4, on the other hand, are extremely hydrophobic proteins with nine and six putative transmembrane helices, respectively. Over a limited hydrophilic sequence stretch, bordered by putative transmembrane areas, ORF3 and ORF4 exhibit homology with subunits 4 and 1 of mitochondrial and plastid NADH‐ubiquinol oxidoreductases, respectively. The operon described, therefore, appears to comprise genes for redox carriers linking formate oxidation to proton reduction and for a hydrogenase of hitherto unique composition.
Two precursors of the heat‐stable enterotoxin of Escherichia coli: evidence of extracellular processing
Authors: Rasheed J. K. , Guzmán‐Verduzco L.‐M. , Kupersztoch Y. M. 
Published: October 2006

SummaryExpression of the gene of the methanol‐soluble, heat‐stable enterotoxin of Escherichia coli (STA) allowed the identification by SDS‐PAGE of a cell‐associated 7500 Dalton STA‐related peptide; when similar experiments were performed with a phosphate buffer SDS‐PAGE system, an additional Mr9800 band became apparent. The 9800 Dalton form, pre‐pro‐STA, accumulated as an intracellular species when the experiments were performed in the presence of the proton ionophore CCCP (carbonylcyanide m‐chlorophenylhydrazone); by pulse‐chase experiments, it was shown that pre‐pro‐STA became a periplasmic Mr 7500 pro‐STA and this form was chased to the culture supernatant; periplasmic and extracellular pro‐STA showed the same electrophoretic mobility. A short time after the pulse, pro‐STA was converted extracellularly to mature STA (Mr 4500). It is proposed that STA is synthesized as pre‐pro‐STA, a 72‐amino‐acid peptide that is subsequently cleaved between amino acids 19 and 20as it is translocated across the inner membrane. The resulting 53‐amino‐acid pro‐STA is first detected in the periplasm and is then secreted to the culture supernatant. Pro‐STA is cleaved extracellularly to yield mature STA (Mr 4500).
A 59 kiloDalton outer membrane protein of Salmonella typhimurium protects against oxidative intraleukocytic killing due to human neutrophils
Authors: Stinavage P. S. , Martin L. E. , Spitznagel J. K. 
Published: October 2006

SummaryWe have isolated a Salmonella typhimurium (ST) mutant, JKS400, deficient in the production of a surface‐exposed outer membrane protein (Omp) and phenotypically hypersensitive to the oxidative antimicrobial mechanism of polymorphonuclear leukocytes (PMNs). This Omp migrated at approximately 59 kiloDaltons (kD) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). We found with P22 transduction that the capacities to produce the protein and to exert wild‐type resistance to oxidative killing were tightly linked.Transduction of JKS400 with a P22(HT)Ht bacteriophage grown on a Tn10 insertion library in LT2 yielded tetracycline‐resistant isolates that had been returned to wild‐type protein production. Further experiments showed that restoration of protein production was accompanied by restoration of the parental resistance phenotype to killing by PMNs and by restoration to wild‐type resistance to H2O2. The map position of the Tn10 was determined to be at 96 minutes in the Salmonella chromosome.This protein appears to behave as a virulence factor, promoting the capacity of Salmonella typhimurium LT2 to survive oxygen‐dependent killing mechanisms in neutrophils.
Effect of positive redox potentials (>+400mV) on the expression of anaerobic respiratory enzymes in Escherichia coli
Authors: Unden G. , Trageser M. , Duchêne A. 
Published: October 2006

SummaryThe expression of fumarate reductase and other enzymes of anaerobic respiration in Escherichia coli was studied as a function of the redox potential (E>h) in the medium. Redox potentials up to +300mV allowed full expression of fumarate reductase (frd) genes. Higher values resulted in decreased expression. The relationship between Eh and expression of frd could be approximated by the Nernst equation, assuming a redox couple with a midpoint potential E0′=+400mV to 440 mV. At Eh values >+510mV (generated anaerobically by hexacyanoferrate(III)) the degree of repression was the same as that obtained by O2. Hexacyanoferrate(III) also caused decreased activities of dimethylsulphoxide (DMSO), nitrite and nitrate reductases. Since expression of these enzymes depends on FNR, the gene activator of anaerobic respiratory genes, it is suggested that the function of FNR is controlled by a redox couple of E0′=+400mV to 440mV.
Aging and senescence of the budding yeast Saccharomyces cerevisiae
Authors: Jazwinski S. M. 
Published: October 2006

SUMMARYThe budding yeast Saccharomyces cerevisiae has a limited life span, defined by the number of times an individual cell divides. Longevity in this organism involves a genetic component. Several morphological and physiological changes are associated with yeast aging and senescence. One of these, an increase in generation time with age, provides a ‘biomarker’ for the aging process. This increase in generation time has revealed the operation of a ‘ senescence factor(s)’, which is likely to be a product of age‐specific gene expression. The Cell Spiral Model indicates coordination of successive cell cycles to be inherent in the determination of life span. It is proposed that life expectancy depends on the function of a stochastic trigger during aging that sets in motion a programme leading to cell senescence and death.