bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a partition region carried by the plasmid QpH1 of Coxiella burnetii
Authors: Lin Zhicheng , Mallavia Louis P.
Published: October 2006

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

A 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

The 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

To 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

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

The 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

The 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

A 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

A 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

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

Transposon 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

The 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

The 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

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.
Galactose‐binding site in Escherichia coli heat‐labile enterotoxin (LT) and cholera toxin (CT)
Authors: Merritt Ethan A. , Sixma Titia K. , Kalk Kor H. , van Zanten Ben A. M. , Hol Wim G. J.
Published: October 2006

The galactose‐binding site in cholera toxin and the closely related heat‐labile enterotoxin (LT) from Escherichia coli is an attractive target for the rational design of potential anti‐cholera drugs, in this paper we analyse the molecular structure of this binding site as seen in several crystal structures, including that of an LT: galactose complex which we report here at 2.2 Å resolution. The binding surface on the free toxin contains several tightly associated water molecules and a relatively flexible loop consisting of residues 51–60 of the B subunit. During receptor binding this loop becomes tightly ordered by forming hydrogen bonds jointly to the GM1 pentasaccharide and to a set of water molecules which stabilize the toxin: receptor complex.
The bacterial phosphotransferase system: new frontiers 30 years later
Authors: Saler Milton H. , Reizer Jonathan
Published: October 2006

In 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Thirty years later, we find that the PTS functions not only as a sugar‐phosphorylating system, but also as a complex protein kinase system that regulates a wide variety of metabolic processes and controls the expression of numerous genes. As a result of recent operon‐ and genome‐sequencing projects, novel PTS protein‐encoding genes have been discovered, most of which have yet to be functionally defined. Some of them appear to be involved in cellular processes distinct from those recognized previously. Fundamental aspects of past and current PTS research are briefly reviewed, and recent advances are integrated into conceptual pictures that provide guides for future research.
Signal peptides: exquisitely designed transport promoters
Authors: Izard Jennifer W. , Kendall Debra A.
Published: October 2006

Prokaryotic proteins destined for transport out of the cytoplasm typically contain an N‐terminal extension sequence, called the signal peptide, which is required for export, it is evident that many secretory proteins utilize a common export system, yet the signal sequences themselves display very little primary sequence homology. in attempting to understand how different signal peptides are able to promote protein secretion through the same pathway, the physical features of natural signal sequences have been extensively examined for similarities that might play a part in function. Experimental data have confirmed statistical analyses which highlighted dominant features of natural signal sequences in Escherichia coli: a net positive charge in the N‐terminus increases efficiency of transport; the core region must maintain a threshold level of hydrophoblcity within a range of length limitations; the central portion adopts an α‐hellcal conformation in hydrophobic environments; and the signal cleavage region is ideally six residues long, with small side‐chain amino acids in the −1 and −3 positions. This review focuses on the parallels between signal peptide physical features and their functions, which emerge when the results of a variety of experimental approaches are combined. The requirement for each property may be ascribed to a potential interaction that is critical for efficient protein export. The summation of the key physical features produces signal peptides with the flexibility to function in multiple roles in order to expedite secretion. In this way, nature has indeed evolved exquisitely tuned signal sequences.
Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coii
Authors: Eichler Knut , Bourgis Fabienne , Buchet Anne , Kleber Hans‐Peter , Mandrand‐Berthelot Marie‐Andrée
Published: October 2006

The sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified. They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H‐NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56 613 Da (CaiT), 42 564 Da (CaiA), 59311 Da (CaiC), 32 329 Da (CaiD) and 21 930 Da (CaiE). Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase. CaiD bears strong homology with enoyl hydratases/isomerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity. It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between Agrobacterium and Rhizobium.
Universal barrier to lateral spread of specific genes among microorganisms
Authors: Diaz Eduardo , Munthali Mathildah , de Lorenzo Víctor , Timmis Kenneth N.
Published: October 2006

A genetic circuit to suppress the lateral spread of cloned genes from recombinant to indigenous microorganisms in the environment has been developed. It is based on the endonucleolytic activity of the bacterial toxin colicin E3, which has a distinct target at the 3 end of the 16S ribosomal RNA; this sequence is conserved in virtually all prokaryotic and many eukaryotic genera. Cleavage at this sequence separates the mRNA binding sites from the remainder of the 16S rRNA, thereby inhibiting protein synthesis. While host bacteria carrying the genes for both colicin production and colicin immunity are perfectly viable, lateral transfer of the E3 gene to non‐immune reciptents results in killing of such recipients. This genetic circuit decreases operational transfer frequencies of cloned genes linked to the E3 gene among a variety of bacterial genera by four to five orders of magnitude, in combination with transposon cloning vectors, the circuit is predicted to reduce the rate of lateral spread of specific genes to ecologically insignificant levels. This system therefore represents a useful tool both to explore the evolutionary and ecological consequences of experimentally reducing lateral gene spread among microorganisms, and to increase the ecological predictability of novel recombinant microorganisms.
A phosphorylated DNA‐binding protein is specific for the red‐light signal during complementary chromatic adaptation in cyanobacteria
Authors: Sobczyk André , Bely Alexandra , de Marsac Nicole Tandeau , Houmard Jean
Published: October 2006

Complementary chromatic adaptation is a mechanism by which some cyanobacteria that are able to synthesize phycoerythrin can adapt their pigment (phycobili‐protein) content to the incident wavelengths of the light. In Calothrix sp. PCC 7601 it concerns phycoerythrin (cpe operon), synthesized under green light, and phycocyanin‐2 (cpc2 operon), expressed under red light, and involves transcriptional controls. With cell‐free extracts from Calothrix sp. PCC 7601 grown under various light regimes, a protein designated RcaD was found by gel retardation experiments to specifically bind to the cpc2 promoter region and to be present only in red‐light‐grown cells. This protein was partially purified and its binding activity was shown to be sensitive to an alkaline phosphatase treatment. RcaO can protect two regions of the cpc2 promoter sequence against degradation by DNase I. Because its activity is detected only under the conditions required for cpc2 expression, we propose that RcaD is a positive effector of transcription.
Specific initiation of transcription at a cyanobacterial promoter with RNA polymerase purified from Calothrix sp. PCC 7601
Authors: Schyns Ghislain , Sobczyk André , de Marsac Nicole Tandeau , Houmard Jean
Published: October 2006

Although in cyanobacteria many genes have been shown to be transcriptionally controlled by specific stimuli, little is known about promoter structure and the form of RNA polymerase that recognizes individual promoters. RNA polymerase holoenzyme has been purified from Calothrix sp. PCC 7601. its polypeptide composition resembles that of the plant chloroplast enzymes. To study transcription in cyanobacteria further, we have analysed the promoter‐recognition properties of the purified enzyme. In vitro transcription was assayed with the promoter of the phycocyanin gene (cpc1) that is expressed whatever the incident light conditions. Transcription initiation at the same start point as in vivo was obtained with the Calothrix sp. PCC 7601 purified enzyme and the Escherichia coli core enzyme supplemented with a Calothrix sp. PCC 7601 sigma factor, but not with the E. coli holoenzyme.
ppGpp concentration, growth without PBP2 activity, and growth‐rate control in Escherichia coli
Authors: Joseleau‐Petit Danièle , Thévenet Danielle , D'Arl Richard
Published: October 2006

Escherichia coli strains partially induced for the stringent response are resistant to mecillinam, a β‐lactam antibiotic which specifically inactivates penicillin‐binding protein 2, the key enzyme determining cell shape. We present evidence that mecillinam resistance occurs whenever the intracellular concentration of the nucleotide ppGpp (guanosine 3’‐diphosphate 5’‐diphosphate), the effector of the stringent response, exceeds a threshold level. First, the ppGpp concentration was higher in a mecillinam‐resistant mutant than in closely related sensitive strains. Second, the ppGpp pool was controlled by means of a plasmid carrying a ptac‐relA′ gene coding for a hyperactive (p)ppGpp synthetase, ReiA′; increasing the ppGpp pool by varying the concentration of lac operon inducer IPTG resulted in a sharp threshold ppGpp concentration, above which cells were mecillinam resistant. Third, the ppGpp pool was increased by using poor media; again, at the lowest growth rate studied, the cells were mecillinam resistant, in all experiments, cells with a ppGpp concentration above 140pmoles/A600 were mecillinam resistant whereas those with lower concentrations were sensitive. We discuss a possible role for ppGpp as transcriptional activator of cell division genes whose products seem to become limiting in the presence of mecillinam, when cells form large spheres. We confirmed the well‐known inverse correlation between growth rate and ppGpp concentration but, surprisingly, for a given growth rate, the ppGpp concentration was lower in poor medium than in richer medium in which RelA is induced. We conclude that, for E. coli growing in poor media, the concentration of the nucleotide ppGpp is not the major growth rate determinant.
The opaH locus of Neisseria gonorrhoeae MS11A is involved in epithelial cell invasion
Authors: Waldbeser Lillian S. , Ajioka Richard S. , Merz Alexey J. , Puaoi Deidre , Lin Lan , Thomas Mary , So Magdalene
Published: October 2006

In order to produce a successful infection, Neisseria gonorrhoeae (GC) must attach to and invade mucosal epithelial cells. To identify GC gene products involved in this early interaction with host cells we constructed a gene bank derived from a clinical isolate of GC, and isolated a clone which had the capacity to adhere to the human endometrial adenocarcinoma tissue‐culture line HEC‐1‐B. The cloned sequence was identified as a member of the opa gene family whose protein products have been associated with virulence. The GC chromosome contains numerous variant opa genes which, in MS11, are designated opaA‐K. Previous work showed that expression of opaC confers a highly invasive phenotype upon strain MS11. When our cloned opa gene was mutated and returned to the GC MS11A chromosome by transformation and homologous recombination, we isolated one transformation that was significantly reduced in its invasive capacity. The locus mutated in this transformant was identified as opaH. Our resuits indicate that invasive‐ness of GC for human epithelail cells can be determined by more than one opa gene in strain MS11 A.
The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F‐like plasmids
Authors: Anthony Karen G. , Sherburne Craig , Sherburne Richard , Frost Laura S.
Published: October 2006

The effects of defined mutations In the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating‐pair formation in liquid media by the transfer systems of the F‐Iike plasmids pOX38 (F), ColB2 and R100‐1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responslbie for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. CoIB2 transfer was more strongly affected by mutations in the heptose II‐heptose III region of the LPS (rfaF) whereas R100‐1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating‐pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F‐like plasmids (F, ColB2, R100‐1) were used to complement this mutation. Unexpectediy, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.
Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis‐acting function dispensable for chromosomal gene transfer
Authors: Pettis Gregg S. , Cohen Stanley N.
Published: October 2006

The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101‐induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid‐borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer‐associated pocking phenomenon, and interfered with the ability of tra+ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis‐acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt‐like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.
Synergism of mosquitocidal toxicity between CytA and CrylVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis
Authors: Wu Dong , Johnson Jeffrey J. , Federici Brian A.
Published: October 2006

The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG‐14 isolate of B. thuringiensis subsp. morrisoni is at least 20‐fold greater than any of the four mosquitocidal proteins of which It is composed (CytA, CrylVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CrylVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bio‐assays against first instars of Aedes aegypti. The LC50 for the CytA inclusion was 60 ng ml−1, whereas the LC50 for the CryIVD was 85ng ml−1 In comparison, the LC50s for different combinations of CytA and CrylVD inclusions ranged from 12–15 ng ml−1, 4–5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild‐type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni Is due to synergism among three or four of their major proteins.
Regulation of R100 conjugation requires traM in cis to traJ
Authors: Dempsey Walter B.
Published: October 2006

Deletion mutants of R100‐1 were constructed by classical methods to remove various segments of the traM open reading frame, pTraM‐binding sites and the traM promoters. Complementation tests showed that traM was efficiently complemented only when the frans‐acting fragment contained both the complete traM gene and the adjacent traJ promoter and leader sequences. The conclusion is that traM and traJ constitute a complex operon. A deletion mutant lacking all of the fraJ gene, and one containing a frameshifting traM deletion, retained the ability to transfer at a low level, thereby showing that neither pTraM nor pTraJ is absolutely essential for transfer.
Mutants of Escherichia coli Trp repressor with changes of conserved, helix‐turn‐helix residue threonine 81 have altered DNA‐binding specificities
Authors: Pfau James , Arvidson Dennis N. , Youderian Philip
Published: October 2006

Threonine is found at the third position of the second α‐helix in the helix‐turn‐helix motifs of most bacterial DNA‐binding proteins. To investigate the role of this conserved residue in Escherichia coli Trp repressor function, plasmids encoding mutant Trp repressers with each of the 19 amino acid changes of Thr‐81 were made by site‐directed mutagenesis. All 19 changes decrease the activity of Trp holorepressor, indicating that the Thr‐81 side‐chain is critical for TrpR function. Three mutant repressors, Ser‐81, Lys‐81 and Arg‐81, retain partial DNA‐binding activity and inhibit transcription from the wild‐type trp promoter/operator complex; challenge‐phage assays show that Ser‐81 and Lys‐81 holorepressors have altered DNA‐binding specificities. The side‐chain of Thr‐81 may make direct contacts with base pairs 4 and 3 of the trp operator, consistent with the nuclear magnetic resonance solution structures of the holorepressoroperator complex.
σS‐dependent growth‐phase induction of the csgBA promoter in Escherichia coli can be achieved in vivo by σ70 in the absence of the nucleoid‐associated protein H‐NS
Authors: Arnqvist Anna , Olsén Arne , Normark Staffan
Published: October 2006

The stationary‐phase‐specific sigma factor σS (RpoS/KatF) is required for Escherichia coli to induce expression of fibronectin‐binding curli organelles upon reaching stationary phase. We show that the csgA gene which encodes the curlin subunit protein belongs to a dicistronic operon, csgBA. The transcriptional start site of csgBA was determined and an AT‐rich upstream activating sequence (UAS) required for transcriptional activation was identified. The pcsgBA promoter is not specific for σS since the same promoter sequence can be used by Eσ70in vivo in a strain lacking nucleoid‐associated protein H‐NS and σS. Transcription remained growth‐phase induced and dependent upon the UAS in such a double mutant. Furthermore, we demonstrate that an additional operon, hdeAB, which is also dependent upon σS for transcription, can be transcribed by Eσ70in vivo in the absence of H‐NS by utilizing the phdeAB promoter. Two other genes known to be under the control of σS for expression, bolA and katE, remained transcriptionally silent in the absence of H‐NS. It is suggested that a subset of E. coli promoters can be recognized by both EσS and Eσ70in vivo but H‐NS interacting with these sequences prevents formation of succesful transcription‐initiation complexes with Eσ70.
Molecular characterization and transcription of the histone H2B gene from the protozoan parasite Trypanosoma cruzi
Authors: García‐Salcedo José A. , Oliver José L. , Stock Roberto P. , González Antonio
Published: October 2006

The structure, genomic organization and transcription of the gene encoding histone H2B in the protozoan parasite Trypanosoma cruzi have been studied. This gene consists of a 746‐nucleotlde unit, tandemly repeated at least 18 times in each of two clusters. DNA probes corresponding to histones H2B and H3 hybridized to different chromosomes revealing that the genes coding for these two histones are not physically linked in the genome of T. cruzi. The primary transcription product of the H2B gene is processed by trans‐splicing and polyadenylation. Inhibition of DNA synthesis with aphidicolin resulted in the reduction of histone H2B mRNA to undetectable levels in about two hours, suggesting that its abundance is regulated throughout the cell cycle as it occurs in other eukaryotes. in addition, a concomitant inhibition of translation by cycloheximide reverted this effect indicating that de novo protein synthesis is required for RNA instability. Histone mRNA abundance was dependent on the life‐cycle stage of T. cruzi: abundant in amastigotes and epimastigotes, the dividing forms in the host cell and the insect vector, respectively, white undetected in trypomastigotes, the parasite's non‐dividing life stage.
Gene expression in mycobacteria: transcriptional fusions based on xylE and analysis of the promoter region of the response regulator mtrA from Mycobacterium tuberculosis
Authors: Curcic R. , Dhandayuthapani S. , Oeretic V.
Published: October 2006

Understanding promoter regulation and signal‐transduction systems in pathogenic mycobacteria is critical for uncovering the processes that govern interactions of these bacteria with the human host. In order to develop additional genetic tools for analysis of mycobacterial promoters, the xylE gene from Pseudomonas was tested as a transcriptional fusion reporter in fast‐ and slow‐growing mycobacteria. initially, its utility was demonstrated by expression behind the hsp60 promoter in Mycobacterium smegmatis and Mycobacterium bovis BCG. The presence of an active promoter in front of the promoterless xylE cassette on a plasmid was scored by development of a bright yellow colour upon spraying of mycobacterial colonies on plates with a solution of catechol. The gene product of xylE, catechol 2,3 dioxygenase, was measurable in sonic extracts and whole cells, permitting quantitative determination of promoter activity in both fast‐ and slow‐growing mycobacteria. The xylE‐based mycobacterial transcriptional fusion plasmid pRCX3 was constructed and used to assess promoter activity within the sequences located upstream of the newly characterized Mycobacterium tuberculosis H37Rv response regulator mtrA, a member of the superfamily of bacterial signal‐transduction systems.
Glycopeptide resistance mediated by enterococcal transposon Tn 1546 requires production of VanX for hydrolysis of D‐alanyl‐D‐alanine
Authors: Reynolds Peter E. , Depardieu Florence , Dutka‐Malen Sylvie , Arthur Michel , Courvalin Patrice
Published: October 2006

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

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