bluuebunny/crossref_metadata_2025_split huggingface dataset:
The influence of ribosome‐binding‐site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo
Authors: Vellanoweth Robert Luis , Rabinowitz Jesse C. 
Published: October 2006

SummaryA method is described to determine simultaneously the effect of any changes in the ribosome‐binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine‐Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7–9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two‐ to threefold. In E coli GUG was slightly better than (JUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine‐Dalgarno interaction in both organisms. B. subtilis v/as also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.
The autocatalytic processing of the subtilisin Carlsberg pro‐region is independent of the primary structure of the cleavage site
Authors: Egnell Per , Flock Jan‐Ingmar 
Published: October 2006

SummarySubtilisins are extracellular sery l‐proteases produced by bacilli (Markland and Emil, 1971). In addition to signal sequences, these proteases have N‐terminal extensions (pro‐regions) which have also been identified in several other proteases (Silen et al., 1988; Vasantha et al., 1984; Polhner et al., 1987; Henderson et al., 1987; Yanagida et al., 1986; Takagi et al., 1985). The pro‐region holds the pro‐protease associated with the membrane and release of the protease takes place as a result of pro‐region removal by autocatalytic processing (Egnell and Flock, 1991).In this report we describe the construction of four deletion‐mutations in the gene encoding subtilisin Carlsberg at the junction between the pro‐region and mature subtilisin Carlsberg. We found that the introduction of different deletions abolished the ability of subtilisin to undergo autocatalytic cleavage of the pro‐region in cis, whereas cleavage by exogenous subtilisin could still occur in trans. Point mutations were also introduced in positions ‐5 to +4 around the pro‐region and native subtilisin cleavage site. Processing of pro‐subtilisin with the point mutations showed that the autocatalytic cleavage and recognition of this junction of the subtilisin Carlsberg pro‐region is independent of the amino acid sequence around the cleavage site.
Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase
Authors: Bally Marc , Filloux Alain , Akrim Mohammed , Ball Geneviéve , Lazdunski Andree , Tommassen Jan 
Published: October 2006

SummaryThe xcp genes are required for the secretion of most extracellular proteins by Pseudomonas aeruginosa. The products of these genes are essential for the transport of exoproteins across the outer membrane after they have reached the periptasm via a signal sequence‐dependent pathway. To date, analysis of three xcp genes has suggested the conservation of this secretion pathway in many Gram‐negative bacteria. Furthermore, the xcpA gene was shown to be identical to pilD, which encodes a peptidase involved in the processing of fimbrial (pili) subunits, suggesting a connection between pili biogenesis and protein secretion. Here the nucleotide sequences of seven other xcp genes, designated xcpR to ‐X, are presented. The N termini of four of the encoded Xcp proteins display similarity to the N‐termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in vivo. Furthermore, two other proteins, XcpR and XcpS, show similarity to the PilB and PilC proteins required for fimbriae assembly. Since XcpR and PilB display a canonical nucleotide‐binding site, ATP hydrolysis may provide energy for both systems.
Further studies on Pseudomonas aeruginosa LasA: analysis of specificity
Authors: Peters J. E. , Park S. J. , Darzins A. , Freck L. C. , Saulnier J. M. , Wallach J. M. , Galloway D. R. 
Published: October 2006

SummaryFull elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA‐mediated enhancement of elastotysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave β‐casein. Preliminary analysis of β‐casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.
Biochemical characterization of Escherichia coli DNA helicase I
Authors: Dash Pramod K. , Traxler Beth A. , Panicker Mitradas M. , Hackney David D. , Minkley Edwin G. 
Published: October 2006

SummaryThe gene product of F tral is a bifunctional protein which nicks and unwinds the F plasmid during conjugal DNA transfer. Further biochemical characterization of the Tral protein reveals that it has a second, much lower, Km for ATP hydrolysis, in addition to that previously identified. Measurement of the single‐stranded DNA‐stimulated ATPase rate indicates that there is co‐operative interaction between the enzyme monomers for maximal activity. Furthermore, 18O‐exchange experiments indicate that Tral protein hydrolyses ATP with, at most, a low‐level reversal of the hydrolytic step during each turnover.
Many group A streptococcal strains express two different immunoglobulin‐binding proteins, encoded by closely linked genes: characterization of the proteins expressed by four strains of different M‐type
Authors: Stenberg Lars , O'Toole Paul , Lindahl Gunnar 
Published: October 2006

SummaryMost group A streptococcal strains are able to bind immunoglobulin (lg) in a non‐immune manner, and the majority of these strains bind both IgA and IgG. Using molecular cloning and immunochemical techniques, we have purified and characterized the Ig Fc‐receptors expressed by four such strains. Two of the strains express a novel type of receptor, designated protein Sir, which binds IgA and IgG of all subclasses, and therefore has broader reactivity than any Fc‐receptor previously described. The other two strains express protein Arp, a receptor that binds IgA of both subclasses, and also binds potyclonal IgG weakly. Characterization of the weak IgG‐binding ability of protein Arp shows that it binds only some monoclonal IgG proteins, in particular those of the IgG3 subclass. The four strains studied here were unexpectedly found to also express a second Ig‐receptor, called protein Mrp, encoded by a gene closely linked to the gene for the first receptor. The Mrp protein does not bind IgA, but it binds IgG molecules of the IgG1, IgG2 and tgG4 subclasses, and it also binds fibrinogen. Binding of fibrinogen has been reported to be a characteristic property of streptococcal M proteins, which suggests that the Mrp protein may be an M protein that also binds Ig. Taken together, all available evidence now indicates that most strains of group A streptococci express two different Ig‐binding proteins, encoded by closely linked genes.
Alielic exchange in Pseudomonas aeruginosa using novel ColE1‐type vectors and a family of cassettes containing a portable oriT and the counter‐selectable Bacillus subtilis sacB marker
Authors: Schweizer Herbert P. 
Published: October 2006

SummaryAn improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1‐type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter‐selectable marker, and a chloramphenicol‐resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid‐borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1‐type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose‐containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single‐ or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild‐type strain PA01 and should allow the exchange of any DNA segment into any non‐essential regions of the P. aeruginosa chromosome.
SecY variants that interfere with Escherichia coli protein export in the presence of normal secY
Authors: Shimoike Takashi , Akiyama Yoshinori , Baba Tadashi , Taura Tetsuya , Ito Koreaki 
Published: October 2006

SummaryAs an approach for studying how SecY, an integral membrane protein translocation factor of Escherichia coli, interacts with other protein molecules, we isolated a dominant negative mutation, secY‐d1, of the gene carried on a plasmid. The mutant plasmid severely inhibited export of maltose‐binding protein and less severely of OmpA, when introduced into sec+ cells. It inhibited growth of secY and secE mutant cells, but not of secA and secD mutant cells or wild‐type cells. The mutation deletes three amino acids that should be located at the interface of cytopiasmic domain 5 and transmembrane segment 9. We also found that some SecY‐PhoA fusion proteins that lacked carboxy‐terminal portions of SecY but retain a region from periplasmic domain 3 to transmembrane segment 7 were inhibitory to protein export. We suggest that these SecY variants are severely defective in catalytic function of SecY, which requires cytoplasmic domain 5 and its carboxy‐terminal side, but retain the ability to associate with other molecules of the protein export machinery, which requires the central portion of SecY; they probably exert the ‘dominant negative’ effects by competing with normal SecY for the formation of active Sec complex. These observations should provide a basis for further genetic analysis of the Sec protein complex in the membrane.
Translation initiation in Escherichia coli: sequences within the ribosome‐binding site
Authors: Ringquist Steven , Shinedling Sidney , Barrick Doug , Green Louis , Binkley Jonathan , Stormo Gary D. , Gold Larry 
Published: October 2006

SummaryThe translational roles of the Shine‐Dalgarno sequence, the initiation codon, the space between them, and the second codon have been studied. The Shine Dalgarno sequence UAAGGAGG initiated translation roughly four times more efficiently than did the shorter AAGGA sequence. Each Shine‐Dalgarno sequence required a minimum distance to the initiation codon in order to drive translation; spacing, however, could be rather long. Initiation at AUG was more efficient than at GUG or UUG at each spacing examined; initiation at GUG was only slightly better than UUG. Translation was also affected by residues 3′ to the initiation codon. The second codon can influence the rate of initiation, with the magnitude depending on the initiation codon. The data are consistent with a simple kinetic model in which a variety of rate constants contribute to the process of translation initiation.
Molecular characterization and regulation of the phosphoglycerate kinase gene from Trichoderma viride
Authors: Goldman G. H. , Geremia R. A. , Caplan A. B. , Vila S. B. , Villarroel R. , Van Montagu M. , Herrera‐Estrellal A. 
Published: October 2006

SummaryWe have isolated the phosphoglycerate kinase gene (pgk) of Trichoderma viride and characterized its expression. Comparison of genomic and cDNA clones allowed the correct deduction of the intron boundaries and the 3′‐end cleavage site of this gene. Primer extension analysis showed that transcription initiated at three start points between ‐296 and ‐298bp upstream of the translational start codon. The promoter sequence contained a number of cis‐acting sequences commonly found in eukaryotic promoters. The pgk transcript analysis of T. viride grown on defined carbon sources showed that neither rate nor growth phase greatly affects pgk expression. By contrast, when Trichoderma spp. were grown in the presence of cell walls of a phytopathogenic fungus as carbon source, pgk messenger levels dropped markedly. This suggests that pgk mRNA accumulation is specifically repressed in the simulated mycoparasitic state.
The binding of Cellulomonas fimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N‐terminal repeats
Authors: Coutinho J. B. , Gilkes N. R. , Warren R. A. J. , Kilburn D. G. , Miller R. C. 
Published: October 2006

SummaryEndoglucanase C (CenC) from Celluiomonas fimi binds to cellulose and to Sephadex. The enzyme has two contiguous 150‐amino‐acid repeats (N1 and N2) at its N‐terminus and two unrelated contiguous 100‐amino‐acjd repeats (C1 and C2) at its C‐terminus. Polypeptides corresponding to N1, N1N2, C1, and C1C2 were produced by expression of appropriate cenC gene fragments in Escherichia coli. N1N2, but not N1 alone, binds to Sephadex; both polypeptides bind to Avicel, (a heterogeneous cellulose preparation containing both crystalline and non‐crystalline components). Neither C1 nor C1C2 binds to Avicel or Sephadex. N1N2 and N1 bind to regenerated (amorphous') cellulose but not to bacterial crystalline cellulose; the cellulose‐binding domain of C. fimi exoglucanase Cex binds to both of these forms of cellulose. Amino acid sequence comparison reveals that N1 and N2 are distantly related to the cellulose‐binding domains of Cex and C. fimi endoglucanases A and B.
Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic machanism
Authors: Aboagye‐Kwarteng T. , Smith K. , Fairlamb A. H. 
Published: October 2006

SummaryTrypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.
Use of a cis‐acting mutation to study the role of FLP‐mediated recombination in the maintenance of native yeast 2μm plasmids
Authors: Morrissey Julie A. , Cashmore Annette M. 
Published: October 2006

SummaryThe 2μm plasmid encodes a mechanism that ensures the partitioning of the plasmid at cell division. Little is known about the detailed mechanism of this partitioning system; for example, is there equal or unequal distribution of the plasmid molecules at mitosis? The plasmid also encodes a site‐specific recombination system that is thought to be involved in plasmid copy‐number amplification, although to date there has been no direct evidence that the recombination process itself is important for maintenance. We have identified a natural 2μm variant that has a cis‐acting mutation in the FLP‐mediated recombination system. We show that this plasmid is unable to amplify in vivo. Our results demonstrate that the average copy number per cell is not affected for the mutant but there is a large clonal variation. This is a direct demonstration that plasmid partitioning results in an unequal distribution of plasmids and that FLP‐mediated amplification compensates for this and therefore has an important role in maintenance.
Molecular and symbiotic characterization of exopolysaccharide‐deficient mutants of Rhizobium tropici strain CIAT899
Authors: Milner Jocelyn L. , Araujo Ricardo S. , Handelsman Jo 
Published: October 2006

SummaryWe studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non‐mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS‐deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS‐deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.
A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an anti‐sigma factor inhibits the activity of the flagellum‐specific Sigma factor, σF
Authors: Ohnishi K. , Kutsukake K. , Suzuki H. , Lino T. 
Published: October 2006

SummaryWe have studied the molecular mechanism of the negative regulation by flgM of the late operons of the flagellar regulon of Salmonella typhimurium. A 7.8 kDa protein that was identified as the flgM gene product was purified to homogeneity; its amino‐terminal sequence was identical to the deduced sequence except for the lack of the initiating methionine. The purified FlgM repressed transcription from the fliC promoter, one that is activated by the sigma factor, FliA (σF). No DNA‐binding activity was detected in FlgM. Chemical cross‐linking experiments showed that the purified FlgM bound to σF and disturbed Its ability to form a complex with RNA polymerase core enzyme. These results indicate that FlgM is a novel type of negative regulator that probably inactivates the flagellum‐specific sigma factor through direct interaction, i.e. it is an anti‐sigma factor.
A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. I. Molecular cloning, transposon mutagenesis and sequence analysis of the gene
Authors: Coomber S. A. , Jones R. M. , Jordan P. M. , Hunter C. N. 
Published: October 2006

SummaryA mutant of Rhodobacter sphaeroides, N1, has been isolated which is incapable of photosynthetic growth and, instead of synthesizing bacteriochlorophyll, N1 excretes coproporphyrin III into the growth medium. Using conjugative gene transfer, several clones were isolated from a R. sphaeroides gene library which restored normal pigment synthesis and photosynthetic growth to N1. Using transposon Tn5 mutagenesis, the gene was located to a 1.05 kb Eco RI fragment. Sequence and transcription analysis defined the position and expression of an open reading frame of approximately 920 bp, which is proposed as the anaerobic coproporphyrinogen III oxidase dedicated to bacteriochlorophyll biosynthesis.
A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. II. Analysis of a region of the genome encoding hemF and the puc operon
Authors: Gibson L.C.D. , McGlynn P. , Chaudhri M. , Hunter C.N. 
Published: October 2006

SummaryThe puc operon of Rhodobacter sphaeroides encoding polypeptides of the major light‐harvesting complex, LH2, has been found to be linked to hemF, a gene encoding a putative anaerobic coproporphyrinogen III oxidase. The puc‐hemF region of the R. sphaeroides genome has been investigated by insertional mutagenesis, complementation analysis of these insertional mutants and DNA sequencing. A third gene, designated pucC, has been found immediately downstream of pucA and has been shown to be essential for LH2 expression. pucC is cotranscribed with pucB and pucA; however, hemF and the pucBAC operon were found not to be transcriptionally linked. Ultrastructural studies indicated that the morphology of the intracytoplasmic membrane may depend upon expression of pucC as well as pucBA.
Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein in Eschericha coli
Authors: Nyström Thomas , Neidhardt Frederick C. 
Published: October 2006

SummaryThe response of non‐differentiating bacteria to nutrient starvation is complex and includes the sequential synthesis of starvation‐inducible proteins. Although starvation for different individual nutrients generally provokes unique and individual patterns of protein expression, some starvation stimulons share member proteins. Two‐dimensional polyacrylamide gel electrophoresis revealed that the synthesis of a small (13.5 kDa) cytoplasmic protein in Escherichia coli was greatly increased during growth inhibition caused by the exhaustion of any of a variety of nutrients (carbon, nitrogen, phosphate, sulphate, required amino acid) or by the presence of a variety of toxic agents including heavy metals, oxidants, acids and antibiotics. To determine further the mode of regulation of the protein designated UspA (üniversal stress pUrotein A) we cloned the gene encoding the protein by the technique of reverse genetics. We isolated the protein from a preparative two‐dimensional polyacrylamide gel, determined its N‐terminal amino acid sequence, and used this sequence to construct a degenerate oligonucleotide probe. Two phages of the Kohara library were found to contain the gene which then was subcloned from the DNA in the overlapping region of these two clones. The amino acid sequence, deduced from the nucleotide sequence of the uspA gene, shows no significant homology with any other known protein. The uspA gene maps at 77 min on the E. coli W3110 chromosome, and is transcribed in a clockwise direction. The increase in the level of UspA during growth arrest was found to be primarily a result of transcriptional activation of the corresponding gene. The induction was independent of the RelA/SpoT, RpoH, KatF, OmpR, AppY, Lrp, PhoB and H‐NS proteins during stress conditions that are known to induce or activate these global regulators. The ‐10 and ‐35 regions upstream of the transcriptional start site of the uspA gene are characteristic of a σ70‐dependent promoter.