bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Comparison 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

We 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

Colicin 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

Colicin 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

RNA‐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

Cytochrome 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

Eukaryotic 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

Using 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

Northern 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

A 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

The 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

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

The 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

Protein 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

The 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.
Microevolution within a clonal population of pathogenic bacteria: recombination, gene duplication and horizontal genetic exchange in the opa gene family of Neisseria meningitidis
Authors: Hobbs Marcia M. , Seiler Andrea , Achtman Mark , Cannon Janne G.
Published: October 2006

Opacity (Opa) proteins are a family of antigenically variable outer‐membrane proteins of Neisseria meningitidis. Even among clonally related epidemic meningococcal isolates, there is greater variation of Opa protein expression than can be accounted for by the opa gene repertoire of any individual strain. We characterized the opa genes of eight closely related Isolates of serogroup A N. meningitidis (subgroup IV‐1) from a recent meningitis epidemic in West Africa. DNA sequence analysis and Southern blot experiments indicated that changes occurred in the opa genes of these bacteria as they spread through the human population, over a relatively short period of time. Such changes in one or a few loci within a clonal population are referred to as microevolution. The distribution of sequences present in hypervariable (HV) regions of the opa genes suggests that duplication of all or part of opa genes into other opa loci changed the repertoire of Opa proteins that could be expressed. Additional variability in this gene family appears to have been introduced by horizontal exchange of opa sequences from other meningococcal strains and from Neisseria gonorrhoeae. These results indicate that processes of recombination and genetic exchange contributed to variability in major surface antigens of this clonal population of pathogenic bacteria.
The cis‐effect of a nascent peptide on its translating ribosome: influence of the cat‐86 leader pentapeptide on translation termination at leader codon 6
Authors: Rogers Elizabeth J. , Lovett Paul S.
Published: October 2006

Inducible cat genes from Gram‐positive bacteria are regulated by translation attenuation. The inducer chloramphenicol stalls a ribosome at a specific site in the leader of cat transcripts; this destabillzes a downstream stem‐loop structure that normally sequesters the ribosome‐binding site for the cat structural gene. The five‐amino‐acid peptide MVKTD that is synthesized when a ribosome has translated to the leader induction site is an inhibitor of peptidyl transferase In vitro. Thus, the peptide may be the in vivo determinant of the site of ribosome stalling. Here we provide evidence that the leader pentapeptide can exert a cis‐effect on its translating ribosome In vivo. Converting leader codon 6 to the ochre codon results in expression of cat‐86 in the absence of Inducer. We term this autoinduction. Autoinduction is abolished by mutations that change the amino‐acid sequence of the leader peptide but have no, or little, effect on the sequence of nucleotides at the leader stall site. In contrast, four nucleotide changes within the leader site occupied by the stalled ribosome that result in synonymous codon replacements do not diminish autoinduction. Our evidence indicates that the cat‐86 leader pentapeptide can alter the function of its translating ribosome.
Thermoregulation in Yersinia enterocolitica is coincident with changes in DNA supercoiling
Authors: Rohde John R. , Fox James M. , Minnich Scott A.
Published: October 2006

Yersinia enterocolitica is a facultative intracellular parasite, displaying the ability to grow saprophytically or invade and persist intracellularly in the mammalian reticuloendothelial system. The transition between such diverse environments requires the co‐ordinated regulation of specific sets of genes on both the chromosome and virulence plasmid. Temperature has a profound pleiotropic effect on gene expression and phenotypically promotes alterations in cell morphology, outer‐membrane protein synthesis, urease production, lipopolysaccharide synthesis, motility, and synthesis of genes involved in invasion of euKaryotic host cells. By examining thermoregulated flagella biosynthesis, we have determined that motility is repressed at 25° C (permissive temperature) with subinhibitory concentrations of novobiocin. These conditions also induce virulence gene expression suggesting novobiocin addition stimulates, at least partially, a high‐temperature environment. Furthermore, temperature‐shift experiments, using Y. enterocolitica containing pACYC184 as a reporter plasmid, indicate that thermo‐induced alterations of DNA supercoiling coincide with temperature‐induced phenotypic changes. A class of putative DNA gyrase mutant (novobiocin resistant) likewise demonstrates the 37° C phenotype when cultured at 25°C; it is non‐motile, urease negative, calcium growth dependent, and positive for Yop expression. These results support a model implicating DNA topology as a contributing factor of Y. enterocolitica thermoregulation.
Characterization of the autophosphorylation of Era, an essential Escherichia coli GTPase
Authors: Sood Poonam , Lerner Claude G. , Shimamoto Toshi , Lu Qing , Inouye Masayori
Published: October 2006

Era is an essential protein in Escherichia coli which binds both GTP and GDP and has an intrinsic GTPase activity. Studies on the role of GTP/GDP binding and GTPase activity in an attempt to understand its function lead to the observation that Era is autophosphorylated. The autophosphorylated reaction is specific for GTP and cannot use ATP as a phosphoryl group donor. The reaction velocity is of first order with respect to protein concentration, suggesting an intramolecular mechanism. Autophosphorylation occurs at serine and threonine residues. The major phosphorylated tryptic peptide isolated after autophosphorylation has been identified as ISITSR, from residue 33 to 38. The peptide contains the site of phosphorylation and two potential sites for serine and threonine phosphorylation. Subsequently, both the threonine residue at position 36 and the serine residue at position 37 were altered to alanine. The double mutant Era, but not individual single mutants, was unable to functionally complement the growth of an E. coli strain which cannot produce wild‐type Era protein at high temperature. This suggests that either threonine 36 or serine 37 has to exist for the function of Era In vivo. phosphorylation of Era was also examined by two‐dimensional gel electrophoresis. Era has been previously assigned two distinct positions having two different X‐Y co‐ordinates: one of the spots (H032.0) was identified as phosphorylated Era, indicating that a substantial portion of Era in the cell is indeed phosphorylated. Therefore, Era autophosphorylation is likely to play an important physiological role in the cell. The sequence encoding the C‐terminus previously published had a missing C between A900 and GgO1. As a resuit of the frameshift, Era consists of 301 residues, 15 fewer than originaiiy reported.
Differential expression of multiple exo‐cellobiohydrolase I‐like genes in the lignin‐degrading fungus Phanerochaete chrysosporium
Authors: Sims Paul F. G. , Soares‐Felipe M. Sueli , Wang Qi , Gent Manda E. , Tempelaars Corinne , Broda Paul
Published: October 2006

The genome of Phanerochaete chrysosporium strain ME446 contains multiple, non‐allelic, cellobiohydrolase I (CSHI)‐like sequences, at least two of which are expressed in a cellulose‐dependent manner. Each of the expressed genes contains two identically positioned introns within its coding region. The lengths and sequences of these introns are different and one is not excised from all transcripts, raising the possibility that subtly different protein products may be expressed from a common gene. Introns are also present upstream of both genes but these differ in number and position, as well as sequence and length. Endoglucanase‐like sequences could not be identified and it is suggested that variant CBHI‐like proteins may provide endoglucanase activity in this fungus.
Peptidase activity of Escherichia coli aminopeptidase A is not required for its role in Xer site‐specific recombination
Authors: McCulloch Richard , Burke Mary E. , Sherratt David J.
Published: October 2006

Xer site‐specific recombination is required for the stable inheritance of multicopy plasmids and the normal segregation of the bacterial chromosome in Escherichia coli.Two related recombinases and two accessory proteins are essential for Xer‐mediated recombination at cer, a recombination site in the plasmid ColE1 The accessory proteins, ArgR and PepA, function in ensuring that the Xer recombination reaction acts exclusively intramolecularly, converting plasmid dimers into monomers and not vice versa. PepA is an amino‐exopeptidase, but its molecular role in the Xer recombination mechanism is unclear. Here we show that a mutation directed at the presumptive active site of PepA creates a protein with no detectable peptidase activity in vitro or in vivo, but which still functions normality in Xer site‐specific recombination at cer.
Molecular characterization of intact, but cryptic, flagellin genes in the genus Shigella
Authors: Tominaga Akira , Mahmoud Mohamed Abdel‐Hakim , Mukaihara Takafumi , Enomoto Masatoshi
Published: October 2006

Flagellin genes (fliC) were detected in two species of the genus Shigella. The fliCSF gene cloned from Shigella flexneri produced normal‐type flagella in an Escherichia coli δ fliC strain while the fliCSS genes from two Shigella sonnei strains produced curly‐type flagella and their expression is repressible by Salmonella FljA repressor. The fliCSF gene (1650bp) shared high similarity with the E. coli fliCE gene not only in the 5′ and 3′ constant sequences but also in the upstream and downstream sequences. The fliCS genes (1572 bp) shared high similarity with the Salmonella typhimurium fliCs gene in the operator and 3’constant sequences and also shared high similarity with the fliCE gene in the downstream sequence, suggesting that the fliC gene has undergone horizontal transfer and recombination. Differences In nucleotide sequences of the central variable regions among the four fliC genes, including fliCE and fliCS, suggest that they started differentiation in each lineage approximately 80 million years ago. Loss of motility in Shigella seems to be evolutionarily a recent event.
Molecular characterization of PulE, a protein required for pullulanase secretion
Authors: Possot Odile , Pugsley Anthony P.
Published: October 2006

pulE, one of 14 genes specifically required for pullulanase secretion in Klebsiella oxytoca, codes for a putative nucleotide‐binding protein. Subcellular fractioNatlon indicated that the majority of PulE in Escherichia coli cells expressing all 14 secretion genes is mainly associated with the cytoplasmic membrane through both hydrophobic and non‐hydrophobic interactions. Mutational analysis revealed that one of the two regions of PulE that are conserved in many nucteotide‐binding proteins (Walker box A) is essential for pullulanase secretion. Likewise, mutations that removed aspartate residues from each of two regions immediately downstream from the Walker box A also reduced secretion. These aspartate‐rich regions are highly conserved in all 16 known PulE homologues but not in any other nucleotide‐binding proteins. Altogether, these results Indicate that PulE might belong to a new family of nucleotide‐binding proteins. The protein could not be cross‐linked to the photoactivatable ATP analogue azido‐ATP, however. Most pulE point or deletion mutations which prevented pullulanase secretion exhibited transdominance when expressed at high levels in cells producing wild‐type PulE protein. Evidence presented suggests that PulE might be a homodimer.
Arsenate reduction mediated by the plasmid‐encoded ArsC protein is coupled to glutathione
Authors: Oden Kristine L. , Gladysheva Tatiana B. , Rosen Barry P.
Published: October 2006

Resistance to arsenate conferred on Escherichia coli by the ars operon of plasmid R773 requires both the product of the arsC gene and reduction of arsenate to arsenate. A genetic analysis was performed to identify the source of reducing potential in vivo. in addition to the ars genes, arsenate resistance required the products of the gor gene for glutathione reductase and the gshA and gshB genes for glutathione synthesis. Mutations in the trx and grx genes for thioredoxin and glutaredoxin, respectively, had no effect on arsenate resistance. Although resistance required the arsC gene, the rate of reduction of arsenate to arsenate was nearly the same in cells lacking the ars operon. In strains deficient in glutathione biosynthesis this endogenous reduction was greatly diminished, and cells exhibited increased sensitivity to arsenate. When glutathione was supplied exogenously to such mutants, resistance was restored only to cells expressing the ars operon, and only such cells had detectable arsenate reduction after addition of glutathione. Since ArsC‐catalysed reduction of arsenate provides high level resistance, physical coupling of the ArsC reaction to efflux of the resulting arsenite is hypothesised.
Lytic conversion of Escherichia coli by bacteriophage T5: blocking of the FhuA receptor protein by a lipoprotein expressed early during infection
Authors: Decker Katja , Krauel Verena , Meesmann Anke , Heller Knut J.
Published: October 2006

The nucleotide sequence of the region between the oad gene, encoding the host specificity protein, and the right‐terminal repetition of bacteriophage T5 DNA was determined. Five small open reading frames, the first of which was called IIp, were detected, which apparently formed an operon transcribed from a promoter that overlapped the oad promoter. Both promoters were confirmed by primer extension assays. Using mRNA isolated at different times after T5 infection, the IIp and oad promoters were identified as early and late promoters, respectively. The N‐terminus of the IIp gene product possess a signal sequence and a processing site characteristic of lipoproteins. After subcloning and expression of IIp, its product Lip was identified as a 7.8 kDa polypeptide. Acylation of Lip was confirmed by addition of globomycin, which resulted in the accumulation of the unprocessed precursor form. FhuA4 cells synthesizing Lip were resistant to phage T5. Resistance was caused by inhibition of adsorption of T5 to its FhuA receptor protein. Resistance could be overcome by derepression of fhuA transcription, suggesting a blocking of FhuA by direct interaction with Lip. Since Lip‐mediated T5 resistance has several aspects in common with the phenomenon of lysogenic conversion, we suggest that it should be called lytic conversion.
Why are mammalian alkaline phosphatases much more active than bacterial alkaline phosphatases?
Authors: Murphy Jennifer E. , Kantrowitz Evan R.
Published: October 2006

Mammalian alkaline phosphatases are 20‐30‐fold more active than the corresponding bacterial enzymes even though their amino acid sequences are 25–30% absolutely conserved. In the active‐site region there are two noticeable differences between the sequences of the bacterial and mammalian enzymes, in the Escherichia coli enzyme positions 153 and 328 are Asp and Lys, respectively, but in the mammalian enzymes His is observed at both of these positions. Site‐specific mutagenesis, genetic and X‐ray crystallographic data, which will be summarized here, suggest that the His substitutions at positions 153 and 328 are primarily responsible for the differences in properties between the bacterial and mammalian alkaline phosphatases.
The ins and outs of protein splicing elements
Authors: Colston M. J. , Davis E. O.
Published: October 2006

Protein splicing involves the removal of an internal protein sequence from a precursor molecule and the ligation of the two flanking sequences to produce a mature protein product, in a post‐translational event analogous to the removal of an intron from rRNA. Protein splicing introns, or‘inteins’appear to be a novel type of genetic element capable of mediating gene conversion of an‘intein‐less’allele, and hence promoting their own dissemination. The mechanism by which protein splicing is achieved is probably entirety encoded within the internal protein sequence, or intein, and does not require other accessory molecules. Although the concept of protein splicing inteins as selfish genetic elements of no immediate consequence to the host organism has emerged, this interpretation is questioned by recent evidence that in at least one example there appears to have been selection for protein splicing.
The 24 kDa N‐terminal sub‐domain of the DNA gyrase B protein binds coumarin drugs
Authors: Gilbert E. Jane , Maxwell Anthony
Published: October 2006

A number of lines of evidence suggest that the N‐terminal sub‐domain of the DNA gyrase B protein contains the binding site for the coumarin antibiotics. We have engineered a clone which encodes a 24 kDa protein which represents this domain. Bacteria which overproduce this protein show an elevated level of resistance to coumarins, suggestive of binding of the 24 kDa protein to the drugs In vivo. In vitro we find that the 24 kDa protein does not interact with the gyrase A or B proteins or with DNA, and fails to hydrolyse ATP or show significant binding to ATP, ADP or ADPNP. However, we show that the 24 kDa protein binds coumarin drugs as tightly as the Intact B protein. A number of experiments suggest that the Interaction of the coumarins with the protein is predominantly hydrophobic in nature.
Synthesis of ribosomal proteins during growth of Streptomyces coelicolor
Authors: Blanco Gloria , Rodicio M. Rosario , Puglia Anna Maria , Méndez Carmen , Thompson Charles J. , Salas José A.
Published: October 2006

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse‐labelled with [35 S]‐methionine, separated by two‐dimensional poly‐acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ‐galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10‐L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.
Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi
Authors: Bassler Bonnie L. , Wright Miriam , Silverman Michael R.
Published: October 2006

Density‐dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) In the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density‐dependent response. Transposon Tn5 Insertions in the recombinant clone were isolated, and the mutations were transferred to the genome of V. harveyi for examination of mutant phenotypes. Expression of luminescence in V. harveyi strains with transposon insertions in one locus, luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild‐type bacteria. Sequence analysis of luxO revealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two‐component, signal transduction proteins. The constitutive phenotype of LuxO∼ mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalling system.
Post‐transcriptional regulation of the groEL1 gene of Streptomyces albus
Authors: Servant Pascale , Thompson Charles J. , Mazodier Philippe
Published: October 2006

Thermally induced expression of the heat‐shock gene groEL is subject to post‐transcriptional regulation in Streptomyces albus. When S. albus cells were shifted from 30°C to 41°C, synthesis of three GroEL‐like proteins was induced from two genes transcribed from associated promoters P1 and P2. Surprisingly, analyses of transcriptional fusions of these promoters with various reporter genes Indicated constitutive expression independent of heat shock. In contrast, neo expression was thermally inducible as a GroELI‐APH translational fusion protein. Furthermore, expression of the groEL1‐neo gene was heat Inducible even after the groEL1 promoter region was replaced by a heteroiogous non‐heat‐inducible promoter such as the Escherichia coli lac promoter. Finally, synthesis of GroE proteins, as well as the GroEL‐APH fusion protein, was heat inducible when their transcription was inhibited by rifampicin. Post‐transcriptional regulatory signals needed for heat‐induced GroEL1 synthesis were mapped within of the groEL 1 structural gene.
Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors
Authors: Green J. , Guest J. R.
Published: October 2006

FNR Is a transcriptional regulator that controls gene expression in response to oxygen limitation in Escherichia coli. The NADH dehydrogenase II gene (ndh) is repressed by FNR under anaerobic conditions. Repression is not simply due to occlusion of the promoter (‐35 and ‐10) region by FNR because adjacent pairs of FNR monomers were found to bind at two sites centred at ‐50.5 and ‐94.S in the ndh promoter region without preventing RNA polymerase binding. However, contact between RNA polymerase and the ‐132 to ‐62 region of the non‐coding strand of ndh DNA, and RNA polymerase‐mediated open complex formation, were prevented by bound FNR. The upstream FNR‐binding site (‐94.5) was needed for efficient FNR‐dependent repression of ndh transcription if. vitro, and also for repression of an ndh‐lacZ fusion in vivo. Anaerobic ndh repression may thus involve the binding of two pairs of FNR monomers upstream of the ‐35 region, which prevents essential RNA polymerase‐DNA contacts in the upstream region as well as inhibiting RNA polymerase function by direct FNR interaction. Expression of the ndh‐lacZ fusion in an fnr deletion strain was enhanced by anaerobic growth in rich medium or minimal medium supplemented with amino acids. Furthermore, two proteins (Mr 12000 and 35000) which interact with and may activate transcription from the ndh promoter under these conditions were detected by gel retardation analysis. These putative amino acid‐responsive activators may thus offset FNR‐mediated repression and maintain a low level of anaerobic ndh expression for regulating the NAD+NADH ratio during growth in rich media.
Beta‐lactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus
Authors: Reeves Philip J. , Douglas Paul , Salmond George P. C.
Published: October 2006

The out gene cluster of Erwinia spp. encodes the proteins of the general secretory pathway (GSP) apparatus that is required for pectinase and cellulase secretion. We have used fusions between Erwinia carotovora subsp. carotovora (Ecc) out genes and the topology probe blaM to assess the ability of Out protein regions to export BlaM across the cytoplasmic membrane in Escherichia coli and Ecc. For the outO gene product (an NMePhe peptidase), seven transmembrane regions have been identified and one more is predicted. The region of OutO with the highest level of hydrophilicity is likely to exist as a large cytoplasmic loop, located between two hydrophobic domains, and is positioned towards the N‐terminus of the protein. When BlaM was fused on the C‐terminal side of the last hydrophobic stretch of OutO, the resulting hybrid protein transferred the BlaM moiety to the periplasm whilst retaining OutO activity. Removal of a portion of this hydrophobic stretch resulted in the loss of OutO activity, suggesting that there are tight constraints on the topological integrity of OutO for maintaining catalytic function. When outG, ‐H, ‐I, ‐J, ‐K and ‐N were fused to blaM, the resulting phenotype suggested that the majority of each protein was targeted to the periplasm. Our results indicate that these six Out proteins, when produced by E. coli or Ecc, each adopt, at least temporarily, a type II bitopic conformation in the cytoplasmic membrane. For OutG, ‐H, ‐I and ‐J this probably represents the membrane topology prior to processing by OutO in Ecc. When produced in vivo from a T7 gene 10 promoter construct, the outG product was processed in Ecc whereas the outO mutant RJP249 failed to process pre‐OutG. BlaM fusions positioned on the C‐termtnal side of the hydrophobic stretches of pre‐OutG, ‐H, ‐I, and ‐J were processed by wild‐type Ecc but not RJP249 or E. coli DHL Thus the periplasmic domains of these proteins play no role in the peptidase cleavage reaction. An OutG‐BlaM fusion construct was used to demonstrate NMePhe peptidase activity in other bacterial strains including E. carotovora subsp. carotovora (ATCC39048), E. carotovora subsp. atroseptica (SCR11043) and Erwinia chrysanthemi (3937).
Replication of a Bacillus subtilis oriC plasmid in vitro
Authors: Moriya S. , Firshein W. , Yoshikawa H. , Ogasawara N.
Published: October 2006

We constructed an in vitro replication system specific for a Bacillus subtilis oriC plasmid using a soluble fraction derived from cell extracts of B. subtilis. DNA polymerase III and two initiation proteins, DnaA and DnaB, were required for in vitro replication as observed in vivo. Both upstream and downstream DnaA box regions of the dnaA gene were required as cis‐elements for in vitro synthesis of the B. subtilis oriC plasmid as well as for in vivo activity. The replication was semi‐conservative and only one round of replication occurred within 15min. These results indicate that in vitro replication faithfully reproduced in vivo replication. To elucidate the site of initiation and the direction of replication, we analysed replicative intermediates generated in vitro in the presence of various concentrations of ddGTP by two methods. First, analysis of restriction fragments around the dnaA gene showed a high level of incorporation of the radioactive substrate, indicating that replication began within the vicinity of the dnaA gene. Second, using 2‐dimensional gel electrophoresis, bubble arcs were detected only on fragments containing the DnaA box region downstream of the dnaA gene, indicating that the initiation site resided within this region. The distribution of the bubble arcs suggested that both bidirectional and unidirectional replication occurred in vitro.
MycDB: an integrated mycobacterial database
Authors: Bergh Staffan , Cole Stewart T.
Published: October 2006

As part of ongoing efforts to Investigate the molecular biology of the human pathogens in the genus Mycobacterium, a customized database was developed specifically for these organisms and implemented in ACEDB database manager software. The data loaded include the IMMYC Antigen List, details of reagents available from the CDC/WHO Antibody Bank, more than 1 Mb of sequences of mycobacterial genes and proteins from public databases, the physical maps of Mycobacterium leprae and Mycobacterium tuberculosis developed at the institut Pasteur, as well as a subset of the references found in MedLine. The ACEDB software allows both quick and intuitive access to the data and to connections between facts by a simple mouse‐driven interface, as well as by more powerful query mechanisms.