bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic analysis of the Erwinia chrysanthemi 3937 chrysobactin iron‐transport system: characterization of a gene cluster involved in uptake and biosynthetic pathways
Authors: Franza T. , Enard C. , van Gijsegem F. , Expert D. 
Published: October 2006

SummaryTwenty of the twenty‐two MudII1734 insertions impairing the chrysobactin iron‐assimilation system of Erwinia chrysanthemi 3937 were localized to a 50kbp genomic insert contained in the R‐prime plasmid, R′4 (Enard et al., 1988). Using the conjugative plasmid pULB1 10 (RP4::mini‐Mu) and the generalized transducing phage ΦEC2, we located this iron‐transport region and the two unlinked mutations on the chromosome linkage map. Chrysobactin is a catechol‐type siderophore and, as we have previously observed with the entA locus of Escherichia coli, the E. chrysanthemi‐derived R′4 was found to complement E. coli entB and entE mutations. A 2.9 kb Eco Ri and a 4.8kb BamHI fragment in the R′4 sharing homology with the E. coli entCEBAP15 operon DNA were subcloned. These fragments were used as DNA/DNA hybridization probes to screen a wild‐type gene library, yielding a recombinant cosmid (pEC7) able to complement mutations disrupting the 2,3‐drhydroxy‐benzoic acid biosynthetic pathway in both Erwinia and Escherichia spp. as well as the E. coli entE mutation. Physical mapping of the genomic MudII1734 insertions corresponding to these mutations led to the identification of a cluster of genes confined to a DNA sequence of about 10 kb required for both biosynthetic and receptor functions.
Characterization of the opa (class 5) gene family of Neisseria meningitidis
Authors: Aho E. L. , Dempsey J. A. , Hobbs M. M. , Klapper D. G. , Cannon J. G. 
Published: October 2006

SummaryClass 5 outer membrane proteins of Neisseria meningitidis show both phase‐ and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used λgt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.
Type 1 fimbriae mutants of Escherichia coli K12: characterization of recognized afimbriate strains and construction of new fim deletion mutants
Authors: Blomfjeld I. C. , McClain M. S. , Eisenstein B. I. 
Published: October 2006

SummaryWe have used Southern hybridization analysis to characterize the extent of fim homology in recognized type 1 fimbriae mutants of Escherichia coli K12, including strains HB101, P678‐54, and VL584. We have found extensive homology in strain HB101, and confirm that P678‐54 lacks the majority of fim DNA. Strain VL584 contains a deletion of the entire fim region. We have used a new allelic exchange procedure to generate novel fim deletion derivatives of strains MG1655, MM294, and YMC9. To increase the utility of the new deletion strains we also isolated recA derivatives of each mutant. These strains facilitate the isolation, characterization, and manipulation of cloned fimbriae genes from diverse sources.
Enhancement of the invasive ability of Neisseria gonorrhoeae by contact with HeclB, an adeno‐carcinoma endometrial cell line
Authors: Chen J. C.‐R. , Bavoil P. , Clark V. L. 
Published: October 2006

SummarySince Neisseria gonorrhoeae is an obligate pathogen, there is no animal model for identification of virulence factors for this bacterium. An alternative model for assessment of gonococcal virulence is invasion of the adenocarcinoma endometrial cell line, HeclB. Pre‐incubation of gonococci with glutaraldehyde‐fixed HeclB cells eliminated the six‐ to eight‐hour lag in entry of bacteria into a fresh HeIIB monolayer seen with unpreincubated gonococci or gonococci pre‐incubated in tissue‐culture medium alone. Gonococci tightly bound to fixed HecIB cells were more Invasive than cells free in the tissue‐culture medium, suggesting that actual contact with HecIB cells was required for the enhancement of invasive ability. Chloramphenicol addition during the preincubation prevented the enhanced invasion. Preincubated gonococci were not more adherent to HecIB cells, suggesting that a stage in invasion after binding of gonococci to HecIB cells was enhanced. The enhanced invasion occurred only when gonococci were preincubated with HecIB ceils and not with HEp‐2, HeLa, Chang or CHO cells. This eukaryotic cell specificity for induction of enhanced invasion may indicate a role for invasion in gonococcal infection of the endometrium.
Regulation of retrotransposition in Saccharomyces cerevisiae
Authors: Curcio M. J. , Garfinkel D. J. 
Published: October 2006

SummaryRetrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element Ty (Transposon yeast), found in the yeast Saccharomyces cerevisiae, is a model system for the study of retrotransposons because of the experimental tools that exist to manipulate and detect transposition. Ty transposition can be elevated to levels exceeding one transposition event per cell when an element is expressed from an inducible yeast promoter. In addition, individual genomic Ty elements can be tagged with a retrotransposition indicator gene that allows transposition events occurring at at a rate of 10−5 to 10−7 per element per cell division to be detected phenotypically. These systems are being used to elucidate the mechanism of Ty transposition and clarify how Ty transposition is controlled.
A Bacillus subtilis dipeptide transport system expressed early during sporulation
Authors: Mathiopoulos C. , Mueller J. P. , Slack F. J. , Murphy C. G. , Patankar S. , Bukusoglu G. , Sonenshein A. L. 
Published: October 2006

SummaryTwo previously identified Bacillus subtilis DNA segments, dciA and dciB, whose transcripts accumulate very rapidly after induction of sporulation, were found in the same 6.2 kb transcription unit, now known as the dciA operon. Analysis of the sequence of the dciA operon showed that its putative products are homologous to bacterial peptide transport systems. The product of the fifth gene, DciAE, is similar to peptide‐binding proteins from Escherichia coli and Salmonella typhimurium (DppA and OppA) and B. subtilis (OppA). A null mutation in dciAE abolished the ability of a proline auxotroph to grow in a medium containing the dipeptide Pro–Gly as sole proline source, suggesting that the dciA operon encodes a dipeptide transport system.
Transcriptional regulation of a Bacillus subtilis dipeptide transport operon
Authors: Slack F. J. , Mueller J. R , Strauch M. A. , Mathiopoulos C. , Sonenshein A. L. 
Published: October 2006

SummaryThe Bacillus subtilis dciA operon, which encodes a dipeptide transport system, was induced rapidly by several conditions that caused the cells to enter stationary phase and initiate sporulation. The in vivo start point of transcription was mapped precisely and shown to correspond to a site of transcription initiation in vitro by the major vegetative form of RNA polymerase. Post‐exponential expression was prevented by a mutation in the spo0A gene (whose product is a known regulator of early sporulation genes) but was restored in a spo0A abrB double mutant. This implicated AbrB, another known regulator, as a repressor of dciA. In fact, purified AbrB protein bound to a portion of the dciA promoter region, protecting it against DNase I digestion. Expression of dciA in growing cells was also repressed independently by glucose and by a mixture of amino acids; neither of these effects was mediated by AbrB.
Regulation of nitrogen fixation in Azorhizobium caulinodans: identification of a fixK‐like gene, a positive regulator of nifA
Authors: Kaminski P. A. , Mandon K. , Arigoni F. , Desnoues N. , Elmerich C. 
Published: October 2006

SummaryThe nucleotide sequence of a 1 kb fragment upstream of Azorhizobium caulinodans fixL was established. An open reading frame of 744 bp was identified as a fixK homologue. A kanamycin cartridge was inserted into the cloned fixK‐1ike gene and recombined Into the host genome. The resulting mutant was Nif− Fix− suggesting that FixK was required for nitrogen fixation both in symbiotic conditions and in the free‐living state. Using a pfixK–lacZ fusion, the FixLJ products were shown to control the expression of fixK. Using a pnifA–lacZ fusion, the FixK product was shown to regulate positively the transcription of nifA in bacteria grown in the free‐living state. In addition, a double ntrC–fixL mutant was constructed and was shown to be completely devoid of nitrogenase activity. A model of regulation, based on these data, is presented and might explain the unusual ability of A. caulinodans to fix nitrogen both under symbiotic conditions and in the free‐living state.
Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin‐resistant clinical isolates of Streptococcus pneumoniae
Authors: Laible G. , Spratt B. G. , Hakenbeck R. 
Published: October 2006

SummaryPenicillin resistance in pneumococci is due to the appearance of high molecular‐weight penicillin‐binding proteins (PBPs) that have reduced affinity for the antibiotic. We have compared the PBX2x genes (pbpX) of one penicillin‐susceptible and five penicillin‐resistant clinical isolates of Streptococcus pneumoniae isolated from various parts of the world. All of the resistant isolates contained a low‐affinity form of PBP 2x. The 2kb region of the two penicillin‐susceptible isolates differed at only eight nucleotide sites (0.4%) and resulted in one single amino acid difference in PBP 2x. In contrast, the sequences of the PBP 2x genes from the resistant isolates differed overall from those of the susceptible isolates at between 7 and 18% of nucleotide sites and resulted in between 27 and 86 amino acid substitutions in PBP 2x. The altered PBP 2x genes consisted of regions that were similar to those of susceptible strains (<3% diverged), alternating with regions that were very different (18–23% diverged). The presence of highly diverged regions within the PBP 2x genes of the resistant isolates contrasts with the uniformity of the sequences of the amylomaltase genes from the same isolates, and with the uniformity of the PBP 2x genes in the two susceptible isolates. It suggests that the altered PBP 2x genes have arisen by localized interspecies recombinational events involving the PBP 2x genes of closely related streptococci, as has been suggested to occur for altered PBP 2b genes (Dowson etal., 1989b). The PBP 2x genes from the resistant isolates could transform the susceptible strain R6 to increased levels of resistance to β‐lactam antibiotics. Indicating that the altered forms of PBP 2x in the resistant isolates contribute to their resistance to penicillin.
Bacillus subtilis chytochrome oxidase mutants: biochemical analysis and genetic evidence for two aa3‐type oxidases
Authors: van der Oost J. , von Wachenfeld C. , Hederstedt L. , Saraste M. 
Published: October 2006

SummaryThe ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127° on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene‐replacement using a phleomycin‐resistance marker. The mutant was unable to oxidize N,N,N′,N′‐tetramethyl‐p‐phenylene‐diamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild‐type membranes confirmed the presence of two haem A‐containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from CtaC, was found to express neither of these enzymes. Radioactive haem‐labelling was used to identify sub‐unit ii, which contains a haem C, and cytochrome c‐550 among the membrane‐bound c‐type cytochromes of B. subtilis.
Molecular chaperones and protein translocation across the Escherichia coli inner membrane
Authors: Kumamoto C.A. 
Published: October 2006

SummaryProteins that are able to translocate across biological membranes assume of loosely folded structure. In this review it is suggested that the loosely folded structure, referred to here as‘per‐folded conformation', is a particular structure that interacts favourably with components of the export apparatus. Two soluble factors, SecB and GroEL, have been implicated in maintenance of the pre‐folded conformation and have been termed‘molecular chaperones'. Results suggest that SecB may be a chaperone that is specialized for binding to exported protein precursors, while GroEL may be a general folding modulator that binds to many intracellular proteins.
Identification of a central regulator of stationary‐phase gene expression in Escherichia coli
Authors: Lange R. , Hengge‐Aronis R. 
Published: October 2006

SummaryDuring carbon‐starvation‐induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30–50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C‐starvation‐regulated genes we isolated and characterized chromosomal C‐starvation‐ induced csi::lacZ fusions using the λplac Mu system. One operon fusion (csi 2::lacZ) has been studied in detail. csi 2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two‐dimensional gel electrophoresis, cells carrying csi 2::lacZ did not synthesize at least 16 proteins present In an isogenic csi 2+ strain. Cells containing csi 2::lacZ or csi 2::Tn10 did not produce glycogen, did not develop thermo‐tolerance and H2O2 resistance, and did not induce a stationary‐phase‐specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild‐type cells during prolonged starvation. We conclude that csi 2::lacZ defines a regulatory gene of central importance for stationary phase E. coli cells. These results and the cloning of the wild‐type gene corresponding to csi 2 demonstrated that the csi 2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi 2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose ‘rpoS’ (‘σs’) as appropriate designations.
Repression and derepression of conjugation of plasmid R1 by wild‐type and mutated finP antisense RNA
Authors: Koraimann G. , Koraimann C. , Koronakis V. , Schlager S. , Högenauer G. 
Published: October 2006

SummaryThe finP gene of plasmid R1 is located between the genestraM and traJ, partially overlapping the first few nucleotides of the latter. It codes for a repressor of the conjugation system. The product of this gene is a small RNA of 72 nucleotides and, because it is transcribed from the opposite DNA strand, it is complementary to the 5′ non‐translated sequences, the ribosome‐binding site, and the first two codons of traJ mRNA. The finP transcript is present in much higher concentrations in R1 than in R1‐19 containing cells, the latter being a derepressed mutant of the former. A synthetic finP gene expressed from a synthetic lambda PL promoter markedly reduced the conjugation frequency of pDB12, a multicopy derivative of R l‐19. Mutagenesis of finP showed that only finP loop II mutants have lost the ability to repress conjugation of R1‐19 intrans. They are also the only ones which derepress conjugal DNA transfer of R1, probably by competing for the finO product, a molecule needed as corepressor for maximal activity. Mutations interrupting potential open reading frames of finP do not abolish finP repressor activity. Hence finP acts as an antisense RNA blocking the expression of the traJ gene by interacting with traJ mRNA through loop II.
Virulence‐associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis
Authors: Okada N. , Sasakawa C. , Tobe T. , Yamada M. , Nagai S. , Talukder K. A. , Komatsu K. , Kanegasaki S. , Yoshikawa M. 
Published: October 2006

SummaryShigellae are the causative agents of bacillary dysentery and are capable of invading epithelial cells, multiplying therein and spreading into adjacent cells. To identify genes on the chromosome associated with the virulence phenotype, 9114 independent Tn5 insertion mutants were isolated in a virulent strain of Shigella flexneri. By using an in vitro assay for intercellular spread or an animal infection model, the Serény test, 50 chromosomal Tn5 mutants with reduced virulence were identified. The 50 mutants were characterized with respect to their virulence phenotypes, including three different mutations that affect invasion of epithelial cells, bacterial metabolism and structure of lipopolysaccharide. Mutants with reduced invasive ability were further characterized and it was found that two of them had decreased levels of IpaB, C and D antigens as well as the mRNA for the ipaBCD operon encoded by the large virulence plasmid, suggesting that positive regulatory elements for the IpaBCD operon are encoded by the chromosome. Assignment of the 50 Tn5 insertions of the mutants to the 19 Notl restriction fragments of the chromosomal DNA has permitted the identification of at least nine virulence‐associated chromosomal loci.
Leader peptidase
Authors: Dalbey R. E. 
Published: October 2006

SummaryThe Escherichia coli leader peptidase has been vital for unravelling problems in membrane assembly and protein export. The role of this essential peptidase is to remove amino‐terminal leader peptides from exported proteins after they have crossed the plasma membrane. Strikingly, almost ail periplasmic proteins, many outer membrane proteins, and a few inner membrane proteins are made with cleavable leader peptides that are removed by this peptidase. This enzyme of 323 amino acid residues spans the membrane twice, with its large carboxyl‐terminal domain protruding into the periplasm. Recent discoveries show that its membrane orientation is controlled by positively charged residues that border (on the cytosolic side) the transmembrane segments. Cleavable pre‐proteins must have small residues at –1 and a small or aliphatic residue at –3 (with respect to the cleavage site). Leader peptidase does not require a histidine or cysteine amino acid for catalysis. Interestingly, serine 90 and aspartic acid 153 are essential for catalysis and are also conserved in a mitochondrial leader peptidase, which is 30.7% homologous with the bacterial enzyme over a 101‐residue stretch.
Alternative sigma factors and the regulation of flagellar gene expression
Authors: Helmann J. D. 
Published: October 2006

SummarySynthesis of bacterial flagella and the accompanying array of chemotaxis receptors and transducers represents a major commitment of energy and resources for a growing bacterial cell and is subject to numerous levels of regulation. Genes for flagellar and chemotaxis proteins are expressed in a complex transcriptional cascade. This regulatory hierarchy acts to ensure that the highly expressed filament structural protein, flagellin, is synthesized only after a prerequisite set of structural proteins has been expressed and properly assembled. Recent evidence suggests that many bacteria utilize an alternative sigma (σ) subunit, similar in specificity to the Bacillus subtilisσ28 protein, to direct transcription of flagellin, chemotaxis and motility genes. In Caulobacter crescentus and Campylobacter spp., both a σ54‐like factor and a σ28‐like factor participate in the transcription of flagellar and chemotaxis genes. Conversely, a σ28‐like factor controls non‐motility functions in at least one non‐flagellated organism.
Evidence for a TonB‐dependent energy transduction complex in Escherichia coli
Authors: Skare J. T. , Postle K. 
Published: October 2006

Summary Escherichia coli TonB protein is required for the active transport of vitamin B12 and Fe(III)‐siderophore complexes across the outer membrane, infection by bacteriophages T1 and φ80, and sensitivity to B‐group colicins. TonB appears to function as an energy transducer in these processes, coupling cytoplasmic membrane electrochemical potential to receptors in the outer membrane. Previous reports have demonstrated that chromosomally encoded TonB is functionally unstable in the absence of protein synthesis (half‐life ∼15–30 minutes) and have shown that plasmid‐encoded, overexpressed TonB is chemically unstable (half‐life ∼5 minutes). In contrast, this study has shown that chromosomally encoded TonB was chemically stable for greater than 90 minutes while maintaining its functional instability. These data suggest that proteolytic degradation of TonB protein is not the basis of its functional instability. Auxiliary proteins such as ExbB also play a role in TonB‐dependent energy transduction. In this study, we have shown that the chemical half‐life of chromosomally encoded TonB in an exbB::Tn10 mutant was reduced at least 18‐fold, suggesting that TonB is a part of a cytoplasmic membrane complex that includes, at the minimum, ExbB. These results also suggest that the chemical instability of plasmid‐encoded TonB resulted when the TonB ExbB ratio was too high and are consistent with previous observations that plasmid‐encoded ExbB can stabilize plasmid‐encoded TonB. The exbB mutation also resulted in a significant decrease in TonB function as measured by the ability of cells to adsorb bacterio‐phage Φ80. In a previous study, strains carrying a tolQ nonsense mutation in combination with an exbB mutation were observed to mimic a tonB phenotype, suggesting that either ExbB or TolQ can alternatively activate TonB. In contrast, we have shown that neither the chemical half‐life nor the function of TonB are affected by the tolQ mutation, and thus, unlike ExbB, TolQ plays a minimal role in TonB‐dependent processes.