bluuebunny/crossref_metadata_2025_split huggingface dataset:
An analogue of the DnaJ molecular chaperone whose expression is controlled by σS during the stationary phase and phosphate starvation in Escherichia coli
Authors: Yamashino Takafumi , Kakeda Minoru , Ueguchi Chiharu , Mizuno Takeshi 
Published: October 2006

SummaryThe Escherichia coli CbpA protein appears to be an analogue of the molecular chaperone, DnaJ, as judged from not only its structure but also its possible function. The expression of cbpA, however, was not significantly affected by up‐shift of the growth temperature. Remarkably, it was found that the expression of cbpA was induced under certain growth conditions, such as the entry of cells into stationary phase, or growth in a phosphate‐limited medium. Such conditional expression of cbpA was regulated at the transcriptional level in a σS ‐dependent manner. The structure of this σS ‐dependent cbpA promoter was clarified by determining its transcription start site. The cbpA promoter region was found to contain an unusual DNA structure (i.e. DNA curvature). From these results, it was suggested that, in contrast to DnaJ, CbpA may function as a molecular chaperone in an adaptive response to environmental stresses other than heat shock.
Analysis of membrane protein interaction: ToxR can dimerize the amino terminus of phage lambda repressor
Authors: Dziejman Michelle , Mekalanos John J. 
Published: October 2006

SummaryThe ToxR protein of Vibrio cholerae is an integral membrane protein that co‐ordinately regulates virulence determinant expression. ToxR directiy activates the cholera toxin operon, but maximal activation is achieved in the presence of ToxS, an integral membrane protein thought to interact with ToxR periplasmic sequences. Studies that substitute alkaline phosphatase sequences for the periplasmic domain of ToxR have led to a model for ToxR activation based on dimerization and ToxS interaction. We constructed λ‐ToxR chimeric proteins using the DNA‐binding domain of the phage λ repressor, which cannot effectively dimerize by itself, to assess the ability of ToxR to form dimers in Escherichia coli The results suggest that ToxR sequences can propagate dimerization, and that ToxS can influence the ability to dimerize.
Regulation of xylose utilization in Bacillus licheniformis: Xyl repressor‐xyl‐operator interaction studied by DNA modification protection and interference
Authors: Scheler Andrea , Hillen Wolfgang 
Published: October 2006

SummaryXylose utilization in Bacillus licheniformis is inducible by xylose. We establish here that the Xyl repressor recognizes and binds an xyl operator sequence located 12 nucleotides downstream from the transcription start site of the xyl operon. DNA‐retardation experiments employing xyl regulatory DNA and soluble protein extracts indicate complex formation in the presence of Xyl repressor. Two repressor–operator complexes are distinguished by different gel mobilities. They yield the same in situ copper‐phenanthroline footprint. This result suggests that a single xyl operator may be bound by different oligomers of Xyl repressor. Methylation and hydroxyl radical cleavage protection of the xyl operator by Xyl repressor binding and ethylation interference of Xyl repressor binding to the xyl operator reveals symmetrical interaction of the repressor with two half sites of the operator, which show palindromic symmetry and are located on the same side of the B‐form DNA structure.
Identification of a partition region carried by the plasmid QpH1 of Coxiella burnetii
Authors: Lin Zhicheng , Mallavia Louis P. 
Published: October 2006

Summary Coxiella burnetii is an intracellular bacterial pathogen which causes Q fever in humans and other animals. Most of the isolates found carry plasmids which share considerable homology. Unfortunately all of these plasmids remain cryptic. Initial attempts to look for secreted or membrane proteins encoded by these plasmids using TnphoA mutagenesis revealed an open reading frame on the EcoRI‐fragment C of the plasmid QpH1. Upstream DNA sequencing of the TnphoA insertions revealed a deduced peptide sequence with homology to the SopA protein which is encoded by the F plasmid in Escherichia coli. Maxi‐cell analysis showed that fragment C encoded two proteins: one was 43.5 kDa in size and designated QsopA, and a second was 38 kDa in size. These proteins are similar in molecular weight to the SopA and SopB proteins, which are essential components of the partition mechanism of the F plasmid. The region appears to be conserved in plasmids QpRS, QpDV, and QpDG, but is absent in a plasmidless isolate in which plasmid sequences have integrated into the chromosomal DNA. Complementation studies demonstrated that fragment C has a plasmid partitioning function and can restore maintenance stability of the partition‐defective mini‐F plasmid. These data suggest that fragment C carries the plasmid partition region of the plasmid QpH1.
Construction and characterization of isogenic O‐antigen variants of Salmonella typhi
Authors: Hone David M. , Harris Andrea M. , Lim Vincent , Levine Myron M. 
Published: October 2006

SummaryA 7.5 kb Kpnl‐generated fragment, from within the rfb cluster of Salmonella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) which is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, these rfb sequences of S. typhimurium were integrated into the rfb clusters of wild‐type Salmonella typhi Vi‐positive strain ISP 1820 (i.e. serotype 09,12; Vi+ H‐d), S. typhi Vi‐negative strain H400 (i.e. serotype 09,12; Vi−; H‐d), and a double aro mutant of S. typhi ISP 1820, strain CVD 906, resulting in the isolation of strains H325, H404 and CVD 906‐O4, respectively. Immunoblot analysis of lipopolysaccharide (LPS) purified from H325, H404 and CVD 906‐O4 demonstrated that these 8trains express the 04 antigen (an abequose residue) in place of the O9 antigen (a tyvelose residue) in the LPS molecule. Hence, the serotype of H325 is O4,12; Vi+; H‐d and the serotype of H404 is O4,12; Vi−; H‐d. DNA hybridization analysis of chromosomal DNA from H325, H404 and CVD 906‐O4 confirmed that a precise recombination event within sequences flanking rfbSE of S. typhi (which encodes the enzymes necessary for cytidine diphosphate‐tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary for O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906‐O4. The resistance of each strain to the bactericidal effects of guinea‐pig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit similar resistance patterns in GPC, strain H400 is sensitive to the bactericidal effects of GPC. The results suggest that the development of the O‐antigen serotype diversity of Salmonella is probably the result of both sequence divergence and recombination
Location of a lepidopteran specificity region in insecticidal crystal protein CryllA from Bacillus thuringiensis
Authors: Liang Y. , Dean D. H. 
Published: October 2006

SummaryThe Bacilius thuringiensis insecticidal crystal protein CryllA has both high mosquito activity and gypsy moth activity; in contrast CryllB, which is 87% homologous, displays no mosquito activity and has a threefold lower gypsy moth activity. The regions responsible for specificity against gypsy moth (Lymantria dispar) and mosquito (Aedes aegypti) larvae were located by introducing Mlul and Xhol sites into homologous positions within the putative domain ii of both cryllA and cryllB genes, which divided almost equally the respective second domains into three regions. Taking advantage of naturally occurring Nhel and Narl sites that border the putative domain II, a set of seven chimeric proteins were produced by exchanging all combinations of those regions Isetween CryllA and CryllB. Analysis of the toxicity of these chimeric proteins demonstrated that the lepidopteran and dipteran specificity regions of CryllA were not collnear. While the specificity region of CryllA against mosquito larvae involved region 1 and probably also region 2, the specificity region of CryllA against gypsy moth larvae was located within region 2.
Nucleoid partitioning in Escherichia coli during steady‐state growth and upon recovery from chloramphenicol treatment
Authors: van Helvoort Joop M. L. M. , Woldringh Conrad L. 
Published: October 2006

SummaryTo distinguish between a gradual or an abrupt movement of the Escherichia coli nucleoid during partitioning we determined the distances between nucleoid borders and cell poles. Measurements were performed on fixed but hydrated cells and on living cells growing in steady state. The distance between nucleoid outer border and cell pole remained constant in cells with either one or two nucleoids. Thus the nucleoid outer borders moved gradually during the partition process. To study partitioning during recovery from protein‐synthesis inhibition cells were treated with chloramphenicol. After growth resumption, cells and nucleoids first elongated before partitioning occurred. Again, no indication of a rapid displacement of the nucleoid to one‐quarter and three‐quarter positions in the cell was observed.
Five Listeria monocytogenes genes preferentially expressed in infected mammalian cells: plcA, purH, purD, pyrE and an arginine ABC transporter gene, arpJ
Authors: Klarsfeld André D. , Goossens Pierre L. , Cossart Pascale 
Published: October 2006

Summary Listeria monocytogenes is a bacterial pathogen that multiplies within the cytosol of eukaryotic cells. To identify Listeria genes with preferentially intracellular expression (pic genes), a library of Tn917‐lac Insertion mutants was screened for transcriptional fusions to lacZ with higher expression inside a macrophage‐like cell line than in a rich broth medium. Five pic genes with up to 100‐fold induction inside cells were identified. Three of them (purH, purD and pyrE) were involved in nucleotide biosynthesis. One was part of an operon encoding an ABC (ATP‐binding cassette) transporter for arginine. The corresponding mutants were not affected in intracellular growth, cell‐to‐cell spread or virulence, except for the transporter mutant, whose LD50 after intravenous infection of mice was twofold higher than the wild‐type. The fifth gene was plcA, a previously identified virulence gene that encodes a phosphatidylinositol‐phospholipase C., and is cotranscribed with prfA, a gene encoding a pleiotropic transcriptional activator of known virulence genes. Although plcA expression is known to depend on PrfA, a prfA promoter–lacZ fusion was highly expressed both Inside and outside cells. Furthermore, in the presence of cellobiose, a disac‐charide recently shown to repress plcA and hly expression, plcA and hly mRNA levels were dramatically reduced without any decrease in the monocistronic prfA mRNA levels. These results demonstrate that virulence gene activation does not depend only on prfA transcript accumulation.
Mutant Escherichia coli arginine repressor proteins that fail to bind l‐arginine, yet retain the ability to bind their normal DNA‐binding sites
Authors: Burke Mary , Merican Amir F. , Sherratt David J. 
Published: October 2006

SummaryThe Escherichia coli arginine repressor (ArgR) is an l‐arginine‐dependent DNA‐binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site‐specific recombination at cer and related recombination sites in plasmids. Site‐directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine‐independent manner. Both mutants support Xer site‐specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild‐type ArgR.
Redundancy of the conserved His residue in Azotobacter vinelandii NifL, a histidine autokinase homologue which regulates transcription of nitrogen fixation genes
Authors: Woodley Paul , Drummond Martin 
Published: October 2006

SummaryThe NifL protein of Azotobacter vinelandii inhibits NifA, the activator of nif (nitrogen fixation) transcription, in response to oxygen and fixed nitrogen. NifL shows strong homology in its C‐terminal domain to the histidine autokinase domains of the canonical two‐component sensor proteins, including the region around His‐304, which corresponds to the residue known to be phosphorylated in other systems. To examine the mechanism of sensory transduction by NifL, mutations encoding 10 substitutions for His‐304 were introduced into the A. vinelandii chromosome. Regulation of nif transcription was measured using acetylene reduction and RNA blots. The substitutions His‐304 → Arg and His‐304 → Pro impaired regulation by both fixed nitrogen and oxygen, but substitution of Ala, Phe, Ile, Lys, Asn, Ser, Thr, Val had no effect. None of the mutants, including His‐304 → Arg and His‐304 → Pro, excreted ammonium during diazotrophy, a phenotype of nifL deletion mutants, suggesting that the molecular basis of this effect differs from that responsible for the inhibition of nif transcription. The data show conclusively that phosphorylation of His‐304 is not essential for any of the known functions of A. vinelandii NifL. Homology to the family of histidine autokinases is therefore inadequate evidence for a mechanism of sensory transduction involving phosphorylation of the conserved histidine residue.
Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones
Authors: Ferrero L. , Cameron B. , Manse B. , Lagneaux D. , Crouzet J. , Famechon A. , Blanche F. 
Published: October 2006

SummaryA 4.6 kb Staphylococcus aureus DNA fragment containing DNA gyrase‐like genes (grlA and grlB) was cloned and sequenced. The proteins GrlA and GrlB exhibit more than 30% identity with E. coli DNA topoisomerase IV sub units and with the gyrase subunits from S. aureus and Escherichia coli. The combined E. coli cell extracts of GrlA and GrlB overproducing strains catalysed ATP‐dependent relaxation and decatenation specific to DNA topoisomerase IV. The temperature‐sensitive phenotype of Salmonella typhimurium parC and parE mutants was complemented by the S. aureus grlA and grlB genes, when the two genes were co‐expressed. These results show that GrlA and GrlB are the subunits of S. aureus DNA topoisomerase IV. The GyrA subunit of DNA gyrase has been previously defined as a primary target of quinolones based on genetic and biochemical experiments essentially carried out in E. coli. Single‐point mutations occurring in the‘quinolone resistance‐determining region’(QRDR) of GyrA were found in bacteria exhibiting quinolone resistance, the most common mutation being a substitution of Ser‐83 on the E. coli GyrA sequence. We analysed eight S. aureus fluoroquinolone‐resistant clinical isolates and observed that mutations in the QRDR of GyrA are not present in the low‐quinolone‐resistant isolates. In contrast, Ser‐80 of GrlA, which corresponds to Ser‐83 of E. coli GyrA, is substituted to Phe or Tyr in both high‐ and low‐quinolone‐resistant isolates. We propose that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.
Use of digitized video microscopy with a fluorogenic enzyme substrate to demonstrate cell‐ and compartment‐specific gene expression in Salmonella enteritidis and Bacillus subtilis
Authors: Lewis P. J. , Nwoguh C. E. , Barer M. R. , Harwood C. R. , Errington J. 
Published: October 2006

SummaryA rapid and sensitive method for detection of cell‐ and compartment‐specific gene expression in individual cells of both Gram‐negative and Gram‐positive microorganisms is described. The method combines the use of gene fusions to lacZ, and a fluorogenic β‐galactosidase substrate, fluorescein‐di‐(β‐d‐galactopyranoside), with digitized video microscopy. All of the reporter constructs tested were successfully detected. Secondary staining of the cells with a nucleic acid‐specific dye, propidium iodide, allowed cells devoid of nucleic acid to be identified, while cell nucleoid shape and the morphological stage of development could be correlated with the location of β‐galactosidase activity. The double‐staining procedure was used to show that gene expression can be induced in non‐culturable cells of Salmonella enteritidis produced by carbon/nitrogen starvation. The resolution was sufficient to distinguish between cells at different morphological stages of sporulation in Bacillus subtilis. This highly sensitive and rapid method may have many other applications in basic and applied microbiology.
The fimbrial gene cluster of Haemophilus influenzae type b
Authors: van Ham S. Marieke , van Alphen Loek , Mool Frits R. , van Putten Jos P. M. 
Published: October 2006

Summary Haemophilus infuenzae infections are preceded by airway colonization, a process facilitated by fimbriae. Here, we identified the complete fimbrial gene cluster of H. influenzae type b. HifA forms the major subunit. HifB, a periplasmic chaperone, and HifC, an outer membrane usher, are typical assembly genes; their inactivation abolished fimbriae formation. HifD and HifE are putative minor subunits, both participating in fimbriae biogenesis. Inactivation of either one drastically reduced fimbriae expression. HifD represents a novel type of fimbrial subunit with lipoprotein characteristics, pointing to a membrane‐associated function of HifD. Transcription of all fimbrial genes is coregulated through two clustered promoters. The flanking of the fimbrial gene cluster by repetitive extragenic palindromic sequences together with a partial duplication of an adjacent unrelated operon indicated that the cluster was once inserted in the H. influenzae genome as a mobile virulence unit.
Identification of an HP1 phage protein required for site‐specific excision
Authors: Esposito Dominic , Scocca John J. 
Published: October 2006

SummaryTransposon insertion mutagenesis and transformation were used to locate genes responsible for excision in the temperate phage HP1 of Haemophilus influenzae. A 6.5 kb segment of DNA near the left end of the phage genome was sequenced, and 11 new open reading frames were identified. Two face‐to‐face overlapping promoter sequences organized these open reading frames into two operons transcribed in opposite directions. Interruption of the first open reading frame in the rightward operon created lysogens unable to produce phages. Provision of the uninterrupted open reading frame In trans restored phage production. The gene identified by this procedure, cox, was cloned and the protein product was expressed at high levels in Escherichia coli. The Cox protein is a 79‐residue basic protein with a predicted strong helix‐turn‐helix DNA‐binding motif. Extracts induced to express high levels of Cox contained a 9 kDa protein. These extracts inhibited integrative recombination and were required for excisive recombination mediated by HP1 integrase. The HP1 cox gene location is similar to that of the homologous excisive and regulatory genes from coli‐phages P2 and 186. These phages appear to share a distinctive organization of recombination proteins and transcriptional domains differing markedly from phage lambda and its relatives.
A novel illegitimate recombination event: precise excision and reintegration with the Mu gem mutant prophage
Authors: Ghelardini P. , Liébart J. C. , Zenzo G. Di , Michell G. , D'Ari R. , Paolozzi L. 
Published: October 2006

SummaryThe bacteriophage Mu is known to insert its DNA more or less randomly within the Escherichia coli chromosome, as do transposable elements, but unlike the latter, precise excision of the prophage, thereby restoring the original sequence, is not observed with wild‐type Mu, although it has been reported with certain defective mutants. We show here that the mutant prophage Mu gem2ts can excise precisely from at least three separate loci —malT, Iac and thyA (selected as Mal+, Lac+ and Thy+, respectively). This excision occurs under permissive conditions for phage development, is observed in fully immune (c+) lysogens, and is independent of RecA and of Mu transposase. Mu gemts2 excision is invariably accompanied by reintegration of a Mu gem2ts prophage elsewhere in the chromosome, in the case of Mal+ revertants, this prophage is systematically located at 94min on the E. coli chromosome. Mu gem2ts excision therefore sheds some light on the long‐standing paradox of the lack of precise Mu excisio.
Transport of haemin across the cytoplasmic membrane through a haemin‐specific periplasmic binding‐protein‐dependent transport system in Yersinia enterocolitica
Authors: Stojiljkovic Igor , Hantke Klaus 
Published: October 2006

SummaryThe Yersinia enterocolitica O:8 periplasmic binding‐protein‐dependent transport (PBT) system for haemin was cloned and characterized. It consisted of four proteins: the periplasmic haemin‐binding protein HemT, the haemin permease protein HemU, the ATP‐binding hydrophilic protein HemV and the putative haemin‐degrading protein HemS. Y. enterocolitica strains mutated in hemU or hemV genes were unable to use haemin as an iron source whereas those mutated in the hemT gene were able to use haemin as an iron source. As Escherichia coli strains expressing only the haemin outer membrane receptor protein HemR from Y. enterocolitica were capable of using haemin as an iron source the existence of an E. coli K‐12 haemin‐specific PBT system is postulated. The first gene in the Y. enterocolitica haemin‐specific PBT system encoded a protein, HemS, which is probably involved in the degradation of haemin in the cytoplasm. The presence of the hemS gene was necessary to prevent haemin toxicity in E. coli strains that accumulate large amounts of haemin in the cytoplasm. We propose a model of haemin utilization in Y. enterocolitica in which HemT, HemU and HemV proteins transport haemin into the cytoplasm where it is degraded by HemS thereby liberating the iron.
The nIpD gene is located in an operon with rpoS on the Escherichia coli chromosome and encodes a novel lipoprotein with a potential function in ceil wall formation
Authors: Lange Roland , Hengge‐Aronis Regine 
Published: October 2006

Summary rpoS is the structural gene for σs, which is a second vegetative sigma subunit of RNA polymerase in Escherichia coli and is involved in the expression of many stationary phase‐Induced genes. Upstream of rpoS is an open reading frame (ORF) whose function and regulation have not been studied. Strong overproduction of its gene product using the IPTG‐inducible tec promoter leads to the formation of bulges at the cell septum and the cell poles, and in rapidly growing cells brings about cell lysis, indicating that the gene product has a hydrolytic function in cell wall formation or maintenance. This is corroborated by sequence homology to lysostaphin, a cell wait lytic exoenzyme synthesized by two Staphylococcus strains. Using globomycin, a specific Inhibitor of signal peptidase II, we demonstrate that the product of the ORF is a novel lipoprotein (NIpD). Two transcriptional start sites for nIpD have been localized. In contrast to rpoS, nIpD is not induced during entry into stationary phase. Growth‐phase‐regulated transcription of rpoS is initiated at additional sites within the nIpD ORF, but the nIpD promoters contribute substantially to the basal level of rpoS expression in exponentially growing cells, indicating that nIpD and rpoS form an operon.