bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glycopeptide resistance mediated by enterococcal transposon Tn 1546 requires production of VanX for hydrolysis of D‐alanyl‐D‐alanine
Authors: Reynolds Peter E. , Depardieu Florence , Dutka‐Malen Sylvie , Arthur Michel , Courvalin Patrice 
Published: October 2006

SummaryCloning and nucleotide sequencing indicated that transposon Tn 1546 from Enterococcus faecium BM4147 encodes a 23365 Da protein, VanX, required for glycopeptide resistance. The vanX gene was located downstream from genes encoding the VanA ligase and the VanH dehydrogenase which synthesize the depsipeptide D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac). In the presence of ramoplanin, an Enterococcus faecalis JH2‐2 derivative producing VanH, VanA and VanX accumulated mainly UDP‐MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys‐D‐Ala‐D‐Lac (pentadepsipeptide) and small amounts of UDP‐MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys‐D‐Ala‐D‐Ala (pentapeptide) in the ratio 49:1. Insertional inactivation of vanX led to increased synthesis of pentapeptide with a resulting change in the ratio of pentadepsipeptide: pentapeptide to less than 1:1. Expression of vanX in E. faecalis and Escherichia coli resulted in production of a D,D‐dipeptidase that hydrolysed D‐Ala‐D‐Ala. Pentadepsipeptide, pentapeptide and D‐Ala‐D‐Lac were not substrates for the enzyme. These results establish that VanX is required for production of a D,D‐dipeptidase that hydrolyses D‐Ala‐D‐Ala, thereby preventing pentapeptide synthesis and subsequent binding of glycopeptides to D‐Ala‐D‐Ala‐containing peptidoglycan precursors at the cell surface.
Characterization of a gene, pilU, required for twitching motility but not phage sensitivity in Pseudomonas aeruginosa
Authors: Whitchurch Cynthia B. , Mattick John S. 
Published: October 2006

SummaryType 4 fimbriae (or pili) are associated with a form of bacterial surface translocation known as twitching motility. Fimbriae are also associated with sensitivity to certain bacteriophages such as PO4. Transposon mutagenesis was used to generate a library of Pseudomonas aeruginosa mutants which lack the spreading‐colony morphology characteristic of twitching motility. in four of these mutants the transposon was found to be located in the vicinity of the previously described pilT locus, but in only one case was it found to have inserted within the pilT coding sequence. Two twitching‐motility mutants originally isolated by Bradley, K2.2, and PAO2001.2, which have been widely used in studies of P. aeruginosa fimbrial structure and expression, were also shown to affect pilT and to comprise a small deletion and a frameshift mutation, respectively. The other three transposon mutations were found to have occurred within a new gene located directly downstream of pilT. This gene, termed pilU, encodes a 382‐amino‐acid protein closely related to PilT and to other members of a family of putative nucleotide‐binding proteins which are involved in the assembly of ceil surface‐associated complexes. Furthermore, the pilT and pilU genes appear to be independently expressed. Like pilT mutants, the pilU mutants were hyperfimbriate, but in neither case was this associated with an increase in transcription of the fimbrial subunit gene pilA. However, in contrast to pilT mutants, the pilU mutants had not also acquired resistance to infection by bacteriophage P04. A broader survey showed differential patterns of sensitivity to various fimbrial‐specific phages among the pilU mutants and other twitching‐motility mutants in the transposon library. The fact that twitching motility is not obligatorily associated with phage sensitivity suggests that the latter may not be directly dependent upon fimbrial function but rather may be a consequence of some common factor(s) involved in their assembly or export pathways.
Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity
Authors: Klimpel Kurt R. , Arora Naveen , Leppla Stephen H. 
Published: October 2006

SummaryComparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc‐binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site‐directed mutagenesis that substituted Ala for either of the residues (H‐686 and H‐690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E‐687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E‐720 and E‐721 with Ala had no effect on LF activity. LF bound 65Zn both in solution and on protein blots. The 65Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.
Positive selection for resistance to 2‐deoxyglucose gives rise, in Streptococcus salivarius, to seven classes of pleiotropic mutants, including ptsH and ptsl missense mutants
Authors: Gauthier Lucie , Thomas Suzanne , Gagnon Guy , Frenette Michel , Trahan Luc , Vadeboncoeur Christian 
Published: October 2006

SummaryWe have used the toxic non‐metabolizabie glucose/ mannose analogue 2‐deoxygiucose to isolate a comprehensive collection of mutants of the phosphoenoipyruvate:sugar phosphotransferase system from Streptococcus salivarius. To increase the range of possible mutations, we isolated spontaneous mutants on different media containing 2‐deoxyglucose and various metabolizable sugars, either lactose, meli‐biose, galactose or fructose. We found that the frequency at which 2‐deoxygiucose‐resistant mutants Were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 2‐deoxygiucose and was 15‐fold higher than the rate observed with the mixture melibiose and 2‐deoxygiucose, the combination that gave the lowest frequency. By combining results from: (i) Western biol analysis of IIIMan, a specific component of the phosphoenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and EI, the two general energy‐coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes. Class 1 was composed of strains devoid of IIIManL, a low‐molecular‐weight form of IIIManL (35200), class 2 was composed of strains exhibiting a reduced level of IIIManL, class 3 was composed of strains devoid of both forms of IIIMan (IIIManL as well as IIIManH, the high‐molecular‐weight form of IIIMan (38900)), class 4 was composed of mutants bearing a mutation in ptsH, the gene encoding HPr, class 5 was composed of mutants bearing a mutation in ptsl, the gene encoding EI, class 6 was composed of 2‐deoxygiucose‐resistant strains without any apparent defect in PTS components, and class 7 was composed of strains possessing both forms of IIIMan but abnormal levels of HPr and/or EI without any mutation in the ptsH and/or the ptsI genes. Preliminary characterization of representative strains of each class is reported.
Immunity proteins to pore‐forming colicins: structure‐function relationships
Authors: Espesset David , Piet Philippe , Lazdunski Claude , Géli Vincent 
Published: October 2006

SummaryColicin A and B immunity proteins (Cai and Cbi, respectively) are homologous integral membrane proteins that interact within the core of the lipid bilayer with hydrophobic transmembrane helices of the corresponding colicin channel. By using various approaches (exchange of hydrophilic loops between Cai and Cbi, construction of Cbi/Cai hybrids, production of Cai as two fragments), we studied the structure‐function relationships of Cai and Cbi. The results revealed unexpectedly high structural constraints for the function of these proteins. The periplasmic loops of Cai and Cbi did not carry the determinants for colicin recognition although most of these loops were required for Cai function; the cytoplasmic loop of Cai was found to be Involved in topology and function of Cai. The immunity function did not seem to be confined to a particular region of the immunity proteins.
The colicin A pore‐forming domain fused to mitochondrial intermembrane space sorting signals can be functionally inserted into the Escherichia coli plasma membrane by a mechanism that bypasses the Tol proteins
Authors: Espesset David , Corda Yves , Cunningham Kyle , Bénédetti Hélène , Lloubès Roland , Lazdunski Claude , Géli Vincent 
Published: October 2006

SummaryColicin A is a pore‐forming bacteriocin that depends upon the Tol proteins in order to be transported from its receptor at the outer membrane surface to its target, the inner membrane. The presequence of yeast mitochondria cytochrome c1 (pc1) as well as the first 167 amino acids of cytochrome b2 (pb2) were fused to the pore‐forming domain of colicin A (pfColA). Both hybrid proteins (pc1‐pfColA and pb2‐pfColA) were cytotoxic for Escherichia coli strains devoid of colicin A immunity protein whereas the pore‐forming domain without presequence had no lethal effect. The entire precursors and their processed forms were found entirely associated with the bacterial inner membrane and their cytotoxicities were related to their pore‐forming activities. The proteins were also shown to kill the tol bacterial strains, which are unable to transport colicins. In addition, we showed that both the cytochrome C1 presequence fused to the dihydrofolate reductase (pc1‐DHFR) and the cytochrome c, presequence moiety of pc1‐pfColA were translocated across inverted membrane vesicles. Our results indicated that: (i) pc1‐pfColA produced in the cell cytoplasm was able to assemble in the inner membrane by a mechanism independent of the tol genes; (ii) the inserted pore‐forming domain had a channel activity; and (ii) this channel activity was inhibited within the membrane by the immunity protein.
Decay of the IS 10 antisense RNA by 3′ exoribonucleases: evidence that RNase II stabilizes RNA‐OUT against PNPase attack
Authors: Pepe Cynthia M. , Masleša‐Galić Saša , Simons Robert W. 
Published: October 2006

SummaryRNA‐OUT, the 69‐nucleotide antisense RNA that regulates Tn 10/IS 10 transposition folds into a simple stem‐loop structure. The unusually high metabolic stability of RNA‐OUT is dependent, in part, on the integrity of its stem‐domain: mutations that disrupt stem‐domain structure (Class II mutations) render RNA‐OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA‐OUT. We show here that stem‐domain integrity determines RNA‐OUT's resistance to 3’exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3′ exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA‐OUT species, albeit with different efficiencies: RNA‐OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA‐OUT is threefold more stable in wild‐type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens RNPase attack on RNA‐OUT. We discuss how this might occur. We also show that wild‐type RNA‐OUT stability changes only twofold across the normal range of physiological growth temperatures (30–44°C) in wild‐type cells, which has important implications for IS 10 biology.
Cytochrome c biogenesis in bacteria: a possible pathway begins to emerge
Authors: Thöny‐Meyer Linda , Ritz Daniel , Hennecke Hauke 
Published: October 2006

SummaryCytochrome c biogenesis describes the posttranslational pathway for the conversion of pre‐apocytochrome c into the mature holocytochrome c. It involves an unknown number of consecutive biochemical steps, including translocation of the precursor polypeptide and haem into the periplasm and the covalent linkage between these two molecules. Genetic and molecular analysis of several bacterial mutants suggest that at least eight genes contribute to this process. In this review we summarize the present knowledge of the cytochrome c maturation pathway in bacteria and propose a model in which certain genes and their products are attributed to specific functions.
The mechanism of transcription termination by RNA poiymerase I
Authors: Reeder Ronald H. , Lang Walter 
Published: October 2006

SummaryEukaryotic ribosomal gene transcription units are bordered at their 3’ends by short DNA sequences which specify site‐specific termination by RNA polymerase I (poll). Poll terminators from yeast through to mammals appear to follow similar rules: they contain a site for a sequence‐specific DNA‐binding protein; they function only in one orientation; 3’ends are formed upstream of the binding site; and 5’flanking sequences influence the position and efficiency of 3’end formation. Recent progress in understanding the mechanism of RNA chain elongation by other polymerases suggests a model for poll termination in which termination is seen as one of the several outcomes possible when a polymerase encounters a pause site.
Mutations that affect regulation of the korB gene of Streptomyces lividans plasmid plJ101 alter plasmid transmission
Authors: Tai Julie Tsu‐Ning , Cohen Stanley N. 
Published: October 2006

SummaryUsing the catechol dehydrogenase gene as a reporter, we isolated random mutations in the plJ101 korB gene operator/promoter (OP) region that affect korB expression and regulation. We mapped these mutations to inverted repeat sequences within the promoter and studied their effects on binding of the KorB repressor protein to the OP, on expression of the korB gene, and on plasmid transmission during mating. Additionally, we investigated the biological effects of KorB binding to a locus (sti, for strong Incompatibility) adjacent to the korB OP and implicated in plJ101 replication. Our results identify sites that influence the synthesis and autoregulation of KorB; they also show that interaction of KorB with sti affects repression of korB and transmission of plasmids to spores of recipients.
Regulation of transcription of the cell gene in Agaricus bisporus
Authors: Yagüe Ernesto , Wood David A. , Thurston Christopher F. 
Published: October 2006

SummaryNorthern analysis showed that accumulation of Agaricus bisporus cell mRNA was regulated by two independent mechanisms: (i) induction by cellulose; and (ii) repression by glucose and other sugars, Isolated A. bisporus nuclei were transcriptionally active. Nuclei Isolated from cellulose‐grown mycelium synthesized six times more cell mRNA than nuclei from glucose‐grown mycelium. The start point of transcription (tsp) was identified by primer extension and S1 nuclease analysis. Putative glucose‐, and cAMP‐responsive elements as well as regions with homology to promoter regions of other fungal cellulase genes were detected both upstream and downstream from the tsp of the cel1 gene.
Insertion of a novel DNA sequence, IS 1186, upstream of the silent carbapenemase gene cfiA, promotes expression of carbapenem resistance in clinical isolates of Bacteroides fragilis
Authors: Podglajen Isabelle , Breuil Jacques , Collatz Ekkehard 
Published: October 2006

SummaryA small number of isolates of Bacteroides fragilis, an anaerobic pathogen of the human intestinal flora, carries a copy (or copies) of the carbapenem‐resistance gene, cfiA, which may be silent or expressed. We have studied the mechanism of activation of the frequently silent gene in in vitro‐selected mutants and in clinical isolates. In both types of strains, activation was observed as the consequence of the insertion, at several possible sites, of a novel 1.3 kb insertion sequence, IS1186, immediately upstream of the carbapenemase gene. IS1186 has two open reading frames, on opposite strands, with coding capacities for a 41.2kDa (ORF1) and a 22.5 kDa (ORF2) protein. The 41.2kDa protein has homology with some proteins predicted from open reading frames of IS elements or DNA direct repeats of aerobic, but not anaerobic, Gram‐negative bacteria. Upon insertion, transcription of cfiA was found to be driven from a promoter identified on the right end of IS1186. In one instance, insertion occurred into the putative ribosome‐binding site of cfiA, leaving intact the tetranucleotide AGAA which is concluded to be a fully functional ribosome‐binding site. Between 3 and 14 copies of IS1186 were detected per genome and the element was found, within the species B. fragilis, almost exclusively in the subgroup carrying the cfiA gene.
The concentrations of stable RNA and ribosomes in Rickettsia prowazekii
Authors: Pang Huiling , Winkler Herbert H. 
Published: October 2006

SummaryThe obligate Intracellular parasite, Rickettsia prowazekii, is a slow‐growing bacterium with a doubling time of about 10h. In the present study, DNA and RNA were obtained from the rickettsiae by two independent methods, i.e. simultaneous isolation of DNA and RNA from the same sample by phenol:chloroform extraction and CsCI gradient centrifugation. In addition, ribosomal RNA was obtained by sedimentation of partially purified ribosomes from the rickettsiae. The results demonstrated that, after correction for the cell volumes, the concentrations of stable RNA and ribosomes in R prowazekii, a slow‐growing organism, were about 62fg μm−3 and 17000 per μm3, respectively, which were very simitar (66fg μm−3 and 21 000 per μm3) to those in Escherichia coli with a generation time of 40min. However, on a per cell basis, R. prowazekii had 5.6 fg of RNA and 1500 ribosomes per cell, which was only about 8% of the amount of both stable RNA (71.2 fg) and ribosomes (24000) per cell as was found in E. coli. These results indicated that R. prowazekii possesses a ribosome concentration greater than might have been predicted from its slow growth rate. This high concentration of ribosomes could be due to a large population of non‐functioning ribosomes, a low efficiency of amino acid production, or a high rate of protein turnover. However, this study also demonstrated that the rickettsiae have very limited protein turnover. Knowledge of the kinetics and control mechanisms for protein synthesis in R. prowazekii remains to be established to determine the logic of the extra rickettsial ribosomes.
Phosphorolytic error correction during transcription
Authors: Libby Randell T. , Gallant Jonathan A. 
Published: October 2006

Summary Escherichia coli DNA‐directed RNA polymerase is shown to contain a novel phosphorolytic error correction activity which removes erroneous nucleotides, as rNDPs, from the 3′‐end of the growing transcript. The activity we describe is biochemically similar to poly‐nucleotide phosphorylase (PNP), yet in contrast to PNP is activated by Mn2+. We demonstrate that the activity, which is mediated by Pi, is dependent on the presence of an incorrectly incorporated nucleotide at the leading 3′‐end of the transcript. The correction activity we describe exhibits a 4 × 104‐fold preference for the excision of incorrect nucleotides from the transcript. These findings suggest the possibility that RNA phosphorolysis may play a critical role in the process of transcriptionai proofreading.
Analysis of the multimer resolution system encoded by the parCBA operon of broad‐host‐range plasmid RP4
Authors: Eberl Leo , Kristensen Claus S. , Givskov Michael , Grohmann Elisabeth , Gerlitz Martin , Schwab Helmut 
Published: October 2006

SummaryThe broad‐host‐range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram‐negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site‐specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site‐specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis‐acting elements with the same orientation in an in vitro resolution assay. ParA‐mediated, site‐specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA‐binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis‐acting region. Both the biochemical properties of the ParA protein and the organization of the cis‐acting recombination site revealed a high degree of similarity to the site‐specific recombination systems of Tn3‐llke transposable elements suggesting an evolutionary retationship.
Protein H — a surface protein of Streptococcus pyogenes with separate binding sites for lgG and albumin
Authors: Frick Inga‐Maria , Åkesson Per , Cooney Jakki , Sjöbring Ulf , Schmidt Karl‐Hermann , Gomi Hideyuki , Hattori Shizuo , Tagawa Chiaki , Kishimoto Fumitaka , Björck Lars 
Published: October 2006

SummaryProtein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (lgGFc) region of immunoglobulin (lg) G. In absorption experiments with human plasma, protein H–sepharose could absorb not only lgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 × 109M−1, which is higher than the affinity between lgG and protein H (Ka= 1.6 × 109 M−1). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein–protein interaction assays, the binding of albumin was mapped to three repeats (C1–C3) in the C‐terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin‐ and lgGFc‐binding bacterial surface protein. Aiso lgGFc‐binding could be mapped with the protein H fragments and the region was found N‐terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized lgG or lgGFc. This sequence was not found in previously described lgGFc‐binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify lgGFc‐ and albumin binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.
Transposition of IST2 in Thiobacillus ferrooxidans
Authors: Cádiz Reginaldo , Gaete Leonardo , Jedlicki Eugenia , Yates James , Holmes David S. , Orellana Omar 
Published: October 2006

SummaryThe genome of Thiobacillus ferrooxidans contains at least two different repetitive DNA elements. One of these elements, termed IS T2 has been sequenced and shown to exhibit the characteristics of a typical prokaryotic insertion sequence. Furthermore, preliminary evidence has implicated IS T2 in genomic rearrangements, although the mechanism of rearrangement, whether by transposition or recombination, has not been established. In this report we provide evidence from detailed restriction enzyme analyses and DNA sequencing data that support a model of transposition, consistent with the notion that IS T2 is a mobile insertion sequence.