bluuebunny/crossref_metadata_2025 huggingface dataset:
Population identification of western hemisphere shorebirds throughout the annual cycle
Authors: Haig S. M. , Gratto‐Trevor C. L. , Mullins T. D. , Colwell M. A.
Published: November 2003

Identification of relationships among geographically distinct populations of migratory species can provide an understanding of breeding and natal philopatry, migration pathways, and population mixing during winter. We used random amplified polymorphic DNA (RAPD) analyses to search for markers specific to difficult‐to‐differentiate shorebird species (e.g. long‐billed dowitcher Limnodromus scolopaceus and short‐billed dowitcher L. griseus) as well as geographically distinct breeding populations of Hudsonian godwits Limosa haemastica, red‐necked phalaropes Phalaropus lobatus, semipalmated plovers Charadrius semipalmatus, dunlin Calidris alpina, pectoral sandpipers C. melanotos, semipalmated sandpipers C. pusilla and western sandpipers C. mauri. Markers clearly differentiated all shorebird species. Estimates of population differentiation varied greatly among species (FST= 0.095–0.685) and correlated with interspecific variation in philopatry and geographical separation of breeding populations. We assigned individuals to putative breeding locales with greater certainty in well‐differentiated species than in poorly differentiated species. Our findings indicate specific phylogeographical structure varies among species, which has strong implications for conservation of habitats within migratory corridors. We suggest that RAPDs are useful in identifying geographical populations of migratory species and that molecular markers should be considered for tracking migratory birds throughout the annual cycle.
A hierarchical quorum‐sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary‐phase sigma factor RpoS
Authors: Latifi A. , Foglino M. , Tanaka K. , Williams P. , Lazdunski A.
Published: November 2003

In Pseudomonas aeruginosa, the production of many virulence factors and secondary metabolites is regulated in concert with cell density through quorum sensing. Two quorum‐sensing regulons have been identified in which the LuxR homologues LasR and RhIR are activated by N‐(3‐oxododecanoyl)‐l‐homo‐serine lactone (OdDHL) and N‐butanoyl‐l‐homoserine lactone (BHL) respectively. The lasR and rhIR genes are linked to the luxl homologues last and rhll, which are responsible for synthesis of OdDHL and BHL, respectively. As lasRI and rhlRI are both involved in regulating synthesis of exoenzymes such as elastase, we sought to determine the nature of their interrelationship. By using lacZ transcriptional fusions in both homologous (P. aeruginosa) and heterologous (Escherichia coli) genetic backgrounds we provide evidence that (i) lasR is expressed constitutively throughout the growth cycle, (ii) rhIR expression is regulated by LasR/OdDHL, and (iii) that RhIR/BHL regulates rhll. We also show that expression of the stationary‐phase sigma factor gene rpoS is abolished in a P. aeruginosa lasR mutant and in the pleiotropic BHL‐negative mutant PAN067. Furthermore, our data reveal that in E. coli, an rpoS‐lacZ fusion is regulated directly by RhIR/BHL. Taken together, these results indicate that P. aeruginosa employs a multilayered hierarchical quorum‐sensing cascade involving RhIR/BHL and LasR/OdDHL, interlinked via RpoS, to integrate the regulation of virulence determinants and secondary metabolites with adaptation and survival in the stationary phase.
Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei
Authors: Mach Robert L. , Strauss Joseph , Zeilinger Susanne , Schindler Martin , Kubicek Christian P.
Published: November 2003

The filamentous fungus Trichoderma reesei forms two specific, xylan‐inducible xylanases encoded by xyn1 and xyn2 to degrade the β‐1,4‐d‐xylan backbone of hemicelluloses. This enzyme system is formed in the presence of xylan, but not glucose. The molecular basis of the absence of xylanase I formation on glucose was the purpose of this study. Northern blotting of the xyn1 transcript as well as the use of the Escherichia coli hygromycin B phosphotransferase‐encoding gene (hph) as a reporter consistently showed that the basal expression of xyn1 was affected by glucose, whereas its induction by xylan remained uninfluenced. The repression of basal xyn1 transcription is mediated by the carbon catabolite repressor protein Cre1, which in vivo binds to two of four consensus sites (5‐SYG‐GRG‐3) in the xyn1 promoter, which occurred in the form of an inverted repeat. T. reesei strains, bearing a xynlv. hph reporter construct, in which four nucleotides from the middle of the inverted repeat had been removed, expressed hph on glucose at a level comparable to that observed during growth on a carbon catabolite derepressing carbon source. Northern analysis of xynl expression in a T. reesei mutant strain (RUT C‐30), which contains a truncated, non‐functional crel gene, also confirmed basal transcription of xyn1. In this strain, xyn1 transcription was still inducible by xylose or xylan to an even higher degree than in the wild‐type strain, suggesting that induction overcomes glucose repression at the level of xynl expression. Based on these data, we postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1‐mediated carbon catabolite repression.
Identification of two previously unrecognized genes (guaA and argC) important for uropathogenesis
Authors: Russo Thomas A. , Jodush Stephen T. , Brown Jennifer J. , Johnson James R.
Published: November 2003

Our knowledge of the traits possessed by extraintestinal isolates of Escherichia coli, necessary for growth and survival in urine, is limited. To identify such determinants, transposon (TnphoA′1,4) mutant libraries of a clinical isolate (CP9) were generated and screened for derivatives exhibiting decreased growth in urine in vitro, and for mutants with active lacZ fusions that were induced in urine relative to laboratory medium. Using this approach we identified two genes, guaA (CPA24) and argC (CPI‐1), which were previously unrecognized as being important for growth in human urine. Unexpectedly, not only does CPA24 (guaA) not grow in human urine in vitro, but it is sensitive to its effects, undergoing a 2–3 log loss of viability over 6 h. By contrast, CPA24 neither grows nor is killed in M9 minimal medium and artificial urine. Therefore, we postulate that lack of guanine or its derivatives in urine, and the inability of CPA24 to synthesize these compounds de novo, prevents CPA24 from synthesizing other guanine (or derivatives)‐dependent products that are critical for growth and survival in urine. Although it seems logical that decreased growth in urine in vitro should correlate with diminished urovirulence, this concept was tested by challenging mice with CPA24 in vivo in a mouse model of urinary tract infection (UTI). Indeed, CPA24 was found to be significantly less virulent compared with its wild‐type parent CP9. CPI‐1(argC) was identified because of the significant induction of its argC::lacZ fusion in urine. Subsequent testing in urine demonstrated that its growth was significantly diminished in all urine samples tested (four females, three males). Polyamine synthesis is dependent upon, in part, the arginine biosynthetic pathway. Therefore, we tested whether the induction of argC in urine and/or the decreased growth of CPI‐1 was a result of low levels of polyamines or arginine in urine. The results suggest that low levels of arginine, but not polyamines, in human urine are responsible. When tested in vivo in the mouse model of UTI, CPI‐1 was also found to be significantly less virulent than CP9. In summary, we have established that guaA and argC are the first genes, which we are aware of, that have been shown to contribute to the growth of E. coli in urine in vitro and both have diminished urovirulence in vivo. These results support the concept that urine can be used in vitro as a screening tool to identify urovirulence traits.
FtsZ‐spirals and ‐arcs determine the shape of the invaginating septa in some mutants of Escherichia coli
Authors: Addinall Stephen G. , Lutkenhaus Joe
Published: November 2003

The essential cell division protein FtsZ forms a dynamic ring structure at the future division site. This Z‐ring contracts during cell division while maintaining a position at the leading edge of the invaginating septum. Using immunofluorescence microscopy we have characterized two situations in which non‐ring FtsZ structures are formed. In ftsZ26 (temperature sensitive, Ts) mutant cells, FtsZ‐spirals are formed and lead to formation of spirally invaginating septa, which in turn cause morphological abnormalities. In rodAsui mutant cells, which grow as spheres instead of rods, FtsZ‐arcs are formed where asymmetric septal invaginations are initiated. The FtsZ‐arcs later mature into complete FtsZ‐rings. Our data show that Z‐spirals and Z‐arcs can contract and that in doing so, they determine the shape of the invaginating septa. These results also strongly suggest that in normal cell division, FtsZ is positioned to a single nucleation site on the inner membrane, from which it polymerizes bidirectionally around the cell circumference to form the Z‐ring.
Entamoeba histolytica and Entamoeba dispar: differences in numbers and expression of cysteine proteinase genes
Authors: Bruchhaus Iris , Jacobs Thomas , Leippe Matthias , Tannich Egbert
Published: November 2003

In order to identify molecules that might be responsible for the difference in pathogenicity between the two closely related protozoan parasites Entamoeba histolytica and Entamoeba dispar, we focussed on cysteine proteinases because this class of enzymes has been considered important for pathogenic tissue destruction. By screening a genomic library derived from an E. histolytica isolate, a total of six distinct genes (ehcp1–ehcp6) encoding typical prepro‐forms of cysteine proteinases were identified which differed from each other by 40% to 85% of their nucleotide sequences. Three of these genes, ehcp1, ehcp2, and ehcp5, which exhibited high levels of expression, were found to be responsible for approximately 90% of cysteine proteinase transcripts, whereas the remaining three were either not or only marginally expressed. Expression of the different genes directly correlated with the level of activity of the respective enzymes in trophozoite lysates. Purification of the enzymes and N‐terminal sequencing revealed that virtually all cysteine proteinase activity of E. histolytica can be attributed to three enzymes namely EhCP1, EhCP2 and EhCP5. Southern blot analysis indicated that just two of these abundantly expressed genes are missing in E. dispar. On the other hand, genes analogous to four of the six genes identified in E.histolytica were found to be present in E. dispar, but only two of these are expressed within the trophozoite stage.
The Mu strong gyrase‐binding site promotes efficient synapsis of the prophage termini
Authors: Pato Martin L. , Banerjee Mary
Published: November 2003

A strong DNA gyrase‐binding site (SGS) is located midway between the termini of the bacteriophage Mu genome and is required for efficient replicative transposition. We have proposed that the SGS promotes the efficient synapsis of the Mu prophage ends (an obligate early step in replicative transposition), and that it does so by helping to organize the prophage DNA into a supercoiled loop with the SGS at the apex of the loop and the prophage termini at the base. The positioning of the synapsing termini equidistant from the SGS is a key element in the proposed model. To test this proposal, we have constructed prophages with a second, internal right end and asked whether the natural, external right end or the internal right end is used for synapsis with the left end in the presence and absence of the SGS. In the presence of the central SGS, the natural, or outside, right end was used exclusively and very efficiently. In the absence of the central SGS, the internal right end was used preferentially and inefficiently: the efficiency of transposition decreased with increasing distance between the internal right end and the left end. Repositioning the SGS midway between the left end and an internal right end allowed highly efficient use of the internal right end. These results support a model in which gyrase can influence long‐range DNA interactions to promote efficient synapsis of Mu prophage ends.
SopE, a secreted protein of Salmonelladublin, is translocated into the target eukaryotic cell via a sip‐dependent mechanism and promotes bacterial entry
Authors: Wood Michael W. , Rosqvist Roland , Mullan Paul B. , Edwards Melissa H. , Galyov Edouard E.
Published: November 2003

The entry of Salmonella into cultured epithelial cells is dependent on genes located in several adjacent chromosomal loci. One of these loci encodes the recently identified secretory proteins, denoted Sips (Salmonella invasion proteins). SipB,C,D proteins are essential for the ability of the pathogen to invade epithelial cells. To examine if additional invasion‐associated proteins were secreted by Salmonella dublin, the genes encoding already characterized secretory proteins were inactivated to facilitate this analysis. The proteins produced and secreted by a double fliM/polar sipB mutant of S. dublin were analysed; this revealed a set of novel secreted proteins. These proteins, which we denoted Sops (Salmonella outer proteins), formed large filamentous aggregates in the medium of bacterial culture growing at 37°C. These aggregates contained five predominant proteins. Here we report the identification and characterization of one of these proteins, SopE, which is a novel invasion‐associated secretory protein of S. dublin. A specific sopE mutant of S. dublin was found to be defective for invasion into epithelial cells. Upon interaction of Salmonella with HeLa cells, SopE was found to be translocated into the cytoplasm of the target cell by extracellular bacteria. The translocation of SopE was shown to be dependent on the Sip proteins because a polar sipB mutant did not translocate SopE across the HeLa cell membrane.
Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5′ untranslated leader
Authors: Wu Chi‐Ju , Janssen Gary R.
Published: November 2003

The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine–Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Strepto‐myces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine–Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5′‐AUGC‐3′) onto the 5′ end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.
Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid‐inducible promoters by differential fluorescence induction
Authors: Valdivia Raphael H. , Falkow Stanley
Published: November 2003

The ability of Salmonella typhimurium to survive and replicate within murine macrophages is dependent on a low phagosomal pH. This requirement for an acidic vacuole suggests that low pH is an important environmental stimulus for the transcription of genes necessary for intracellular survival. To study the behaviour of acid‐inducible genes in response to the phagosomal environment, we have applied a novel enrichment strategy, termed differential fluorescence induction (DFI), to screen an S. typhimurium library for promoters that are upregulated at pH 4.5. DFI utilizes a fluorescence‐enhanced green fluorescent protein (GFP) and a fluorescence‐activated cell sorter (FACS) to perform genetic selection. In the presence of an inducing stimulus, such as low pH, a FACS is used to sort highly fluorescent bacterial clones bearing random promoters fused to the mutant GFP protein (GFPmut). This population is then amplified at neutral pH and the least fluorescent population is sorted. Sequential sorts for fluorescent and non‐fluorescent bacteria in the presence or absence of inducing conditions rapidly enriches for promoter fusions that are regulated by the inducing stimulus. We have identified eight acid‐inducible promoters and quantified their expression in response to pH 4.5 and to the phagosome milieu. These acid‐inducible promoters exhibited extensive homology to promoter regions of genes encoding for cell‐surface‐maintenance enzymes, stress proteins, and generalized efflux pumps. Only a subset of these promoters was induced in macrophages with kinetics and levels of expression that do not necessarily correlate with in vitro pH‐shock induction. This suggests that while low pH is a relevant inducer of intracellular gene expression, additional stimuli in the macrophage can modulate the expression of acid‐inducible genes.
Disruption of the gene encoding penicillin‐binding protein 2b (pbp2b) causes altered cell morphology and cease in exopolysaccharide production in Streptococcus thermophilus Sfi6
Authors: Stingele Francesca , Mollet Beat
Published: November 2003

Genetic and biochemical analysis of exopolysaccharide (EPS) production in lactic acid bacteria has been a growing field of interest in the food industry. We previously identified and characterized a gene cluster composed of 13 genes (epsA to epsM ) responsible for EPS production in Streptococcus thermophilus Sfi6. Here we report one further gene, pbp2b, that is connected to EPS production. Mutants with a gene disruption in pbp2b were no longer able to produce EPS, exhibited a reduced growth‐rate, and their cell morphology was altered. The predicted gene product showed significant homology to the class B penicillin‐binding proteins 2b of Streptococcus pneumoniae, Streptococcus sanguis and Streptococcus mitis involved in peptidoglycan synthesis. Upstream of pbp2b, we further identified two genes which showed significant homology to the E. colifolD and urf1′, which is an unidentified open reading frame presumed to be involved in DNA repair. Downstream of pbp2b, we identified a gene that showed homology to the Bacillus subtilis and the Escherichia colirecM or recR which, respectively, are involved in the methyl‐dependent DNA mismatch repair. In S. thermophilus, pbp2b and recM were transcribed from their own promoters as monocistronic mRNAs and are therefore organized as independent transcriptional units.
The isolation and characterization of genomic and cDNA clones coding for a cdc2‐related kinase (ThCRK2) from the bovine protozoan parasite Theileria
Authors: Kinnaird Jane H. , Logan Mary , Kirvar Erol , Tait Andrew , Carrington Mark
Published: November 2003

The tick‐transmitted protozoan parasites Theileriaannulata and Theileria parva are important intracellular pathogens of domestic cattle in tropical and sub‐tropical regions. Proliferative phases take place within both lymphocytes and erythrocytes. The lymphocyte is stimulated to enter the cell cycle by the parasite and the multinucleate parasite can establish a state in which karyokinesis and cytokinesis occur in phase with the host cell. The link between parasite nuclear division and cytokinesis is altered during the formation of merozoites (a non‐dividing, invasive, extracellular stage). These features imply a high degree of control over parasite nuclear division and cytokinesis. Two different approaches have been used to identify clones from both species which are extremely highly conserved homologues. These encode a cdc2‐related kinase which is > 60% identical to eukaryotic cyclin‐dependent kinases of the p34cdc2/p32CDK2 subfamily. There is typical conservation of kinase domains, implying an in vivo protein kinase activity for the polypeptide. The PSTAIRE region, implicated in cyclin binding, is well conserved suggesting that ThCRK2 will bind cyclin molecules closely related to the eukaryotic A/B‐type cyclins. However, there is divergence in certain key motifs potentially associated with binding of molecules that regulate the activity of the kinase. Expression patterns of RNA and protein indicate that ThCRK2 is likely to function in all dividing stages of the parasite and, taken together, the results point to a central role in the regulation of nuclear division.
A third family of allelic hsd genes in Salmonella enterica: sequence comparisons with related proteins identify conserved regions implicated in restriction of DNA
Authors: Titheradge Annette J. B. , Ternent Diane , Murray Noreen E.
Published: November 2003

Salmonella enterica serovar blegdam has a restriction and modification system encoded by genes linked to serB. We have cloned these genes, putative alleles of the hsd locus of Escherichia coli K‐12, and confirmed by the sequence similarities of flanking DNA that the hsd genes of S. enterica serovar blegdam have the same chromosomal location as those of E. coli K‐12 and Salmonella enterica serovar typhimurium LT2. There is, however, no obvious similarity in their nucleotide sequences, and while the gene order in S. enterica serovar blegdam is serB hsdM, S and R, that in E. coli K‐12 and S. enterica serovar typhimurium LT2 is serB hsdR, M and S. The hsd genes of S. enterica serovar blegdam identify a third family of serB‐linked hsd genes (type ID). The polypeptide sequence predicted from the three hsd genes show some similarities (18–50% identity) with the polypeptides of known and putative type I restriction and modification systems; the highest levels of identity are with sequences of Haemophilus influenzae Rd. The HsdM polypeptide has the motifs characteristic of adenine methyltransferases. Comparisons of the HsdR sequence with those for three other families of type I systems and three putative HsdR polypeptides identify two highly conserved regions in addition to the seven proposed DEAD‐box motifs.
The size and position of heterologous insertions in a silent locus differentially affect pilin recombination in Neisseria gonorrhoeae
Authors: Howell‐Adams Becky , Wainwright Leslie A. , Seifert H. Steven
Published: November 2003

Gonococcal pilus antigenic and phase variation result from unidirectional, RecA‐dependent recombination of DNA sequences from a silent pilin copy (pilS ) into the expressed pilin gene (pilE ). To develop a quantitative assay for pilin gene recombination that is independent of phase variation, a promoterless cat gene was inserted into pilS, and recombination of ′cat into pilE was detected by selection of chloramphenicol‐resistant (CmR) variants expressing ′cat from the pilin promoter. Although RecA‐dependent CmR variants occurred, none were generated by the simple transfer of ′cat into pilE. Instead, each CmR variant contained a new pilin locus that was a hybrid of sequences from the pilE and the pilS1::′cat loci in addition to the two starting loci. Therefore, this system could not be used to quantify antigenic variation. However, combined studies of these hybrid loci and of recombination products generated during additional pilS mutational analyses demonstrated that both the size and position of an insertion in pilS differentially affect pilin recombination. Also, the hybrid loci appear to be intermediates of antigenic variation. This enabled the creation of molecular models for the recombination reactions that result in pilin antigenic variation.
PhhR, a divergently transcribed activator of the phenylalanine hydroxylase gene cluster of Pseudomonas aeruginosa
Authors: Song Jian , Jensen Roy A.
Published: November 2003

Pseudomonas aeruginosa was recently found to possess a cluster of structural genes encoding phenylalanine hydroxylase (PhhA), carbinolamine dehydratase (PhhB), and aromatic aminotransferase (PhhC). We now report the presence, in the flanking upstream region, of a divergently transcribed gene (phhR ) encoding an activator protein. Inactivation of phhR markedly reduced expression of the structural genes. PhhR belongs to the large prokaryote family of 54 enhancer‐binding proteins, and activation of the phh operon by PhhR in P. aeruginosa required rpoN. The closest homologues of PhhR are the TyrR proteins from Escherichia coli and Haemophilus influenzae. E. coli TyrR is an unusual member of the homologue family in that the transcriptional units regulated by tyrR are driven by 70 promoters. P. aeruginosaphhR was able to replace E. coli tyrR as a repressor of the aroF–tyrA operon (but not as an activator of mtr ) in the heterologous E. coli system. Two regions that resemble E. coli TyrR boxes were identified in the intervening region between phhR and phhA. We propose that one or both boxes may be the target of PhhR acting as an autogenous repressor at a 70 promoter in one direction. In the other direction, one or both boxes may be the upstream activator sequence targeted by PhhR to facilitate expression of the phh operon from a 54 promoter. The phh operon was strongly induced in fructose‐ or glucose‐based minimal medium by l‐phenylalanine. Inactivation of phhR in P. aeruginosa abolished ability to utilize either l‐phenylalanine or l‐tyrosine as a sole source of carbon for growth.
Gene disruptions indicate an essential function for the LmmCRK1 cdc2‐related kinase of Leishmania mexicana
Authors: Mottram Jeremy C. , McCready B. Philomena , Brown Kathryn G. , Grant Karen M.
Published: November 2003

The generation of homozygous null mutants for the crk1 cdc2‐related kinase of Leishmania mexicana was attempted using targeted gene disruption. Promastigote mutants heterozygous for crk1 were readily isolated with a hyg‐targeting fragment, but attempts to create null mutants by second‐round transfections with a ble‐targeting fragment yielded two classes of mutant, neither of which was null. First, the transfected fragment formed an episome; second, the cloned transfectants were found to contain wild‐type crk1 alleles as well as hyg and ble integrations. DNA‐ content analysis revealed that these mutants were triploid or tetraploid. Plasticity in chromosome number following targeting has been proposed as a means by which Leishmania avoids deletion of essential genes. These data support this theory and implicate crk1 as an essential gene, validating CRK1 as a potential drug target. L. mexicana transfected with a Trypanosoma brucei homologue, tbcrk1, was shown to be viable in an lmmcrk1 null background, thus showing complementation of function between these trypanosomatid genes. The expression of crk1 was further manipulated by engineering a six‐histidine tag at the C‐terminus of the kinase, allowing purification of the active complex by affinity selection on Ni2+–nitriloacetic acid (NTA) agarose.
A new type of transporter with a new type of cellular function: l‐lysine export from Corynebacterium glutamicum
Authors: Vrljic Marina , Sahm Hermann , Eggeling Lothar
Published: November 2003

We discovered that after deregulation of the l‐lysine biosynthesis in Corynebacterium glutamicum, l‐lysine accumulated in the cytosol and the efflux properties of this amino acid in mutants used for l‐lysine production were altered. In this study we describe the cloning and molecular identification of lysE, which encodes the translocator specifically exporting l‐lysine from the cell. The lysE gene product does not display homology to any known transporter. It is only 236 amino acids in size, with the potential to span the membrane six times. The LysE protein was oversynthesized to confirm its deduced Mr of 25 425 Da. A probable regulatory gene, lysG, is localized immediately adjacent to lysE and displays all the typical structural features of an autoregulatory transcriptional regulator of the LysR‐type family. l‐Lysine export is correlated with lysE expression. A null mutant is unable to excrete l‐lysine, whereas with overexpressed lysE, l‐lysine is exported at a rate of 3.76 nmol min−1 mg−1 dry weight, which is five times the rate that was obtained with the wild type. A deletion mutant was constructed to search for a natural function of this unique carrier. Surprisingly, growth of this mutant is abolished on a salt medium in the presence of the dipeptide Lys–Ala. The quantification of the intracellular l‐lysine concentrations revealed that, in response to peptide addition, there was an accumulation of the exceptionally high concentration of more than 1100 mM l‐lysine. These results distinguish LysE as an exporter, which: (i) structurally represents a new type of translocator; (ii) demonstrates that exporters are also present for primary metabolites such as amino acids; and (iii) serves in one physiological function to link import with export activity.
Expression and regulation of the rnc and pdxJ operons of Escherichia coli
Authors: Matsunaga James , Dyer Michael , Simons Elizabeth L. , Simons Robert W.
Published: November 2003

Escherichia coli rnc–era–recO operon (rnc operon) expression is negatively autoregulated at the level of message stability by ribonuclease III (RNase III), which is encoded by the rnc gene. RNase III, a double‐stranded RNA‐specific endoribonuclease involved in rRNA and mRNA processing and degradation, cleaves a stem‐loop structure in the 5′ untranslated leader, initiating rapid decay of the rnc operon mRNA. Here, we examine rnc operon expression and regulation in greater detail. Northern, primer extension, and lacZ fusion analyses show that a single promoter (rncP ) specifies two principal mRNAs: the 1.9 kb rnc–era transcript and the less‐abundant 3.7 kb RNA encoding rnc–era–recO and the downstream pdxJ and acpS genes. A 1.3 kb pdxJ–acpS RNA is transcribed from a promoter (pdxP ) located within recO. About 70% of pdxJ transcription depends on transcription from rncP. Both promoters were characterized genetically. RNase III reduces 1.9 kb and 3.7 kb transcript levels and stability, and corresponding effects are seen with genetic fusions. These detailed studies enabled us to show that the first 378 nucleotides of the rnc transcript comprise a portable RNA stability element (rncO) that contains all of the cis‐acting elements required for RNase III‐initiated decay of the rnc mRNA as well as the heterologous lacZ transcript. Moreover, mutations in rncO that block RNase III cleavage also block control, showing that RNase III initiates mRNA decay by cleaving at a single site.
Nickel binding and immunological properties of the C‐terminal domain of the Helicobacter pylori GroES homologue (HspA)
Authors: Kansau Imad , Guillain Florent , Thiberge Jean‐Michel , Labigne Agnès
Published: November 2003

Helicobacter pylori synthesizes a heat‐shock protein of the GroES class. The gene encoding this protein (heat‐shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N‐terminal domain (domain A) homologous with other GroES proteins, and a C‐terminal domain (domain B) corresponding to 27 additional residues resembling a metal‐binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose‐binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP‐fused proteins allowed us to conclude that HspA binds nickel ions by means of its C‐terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP–HspA binds nickel ions with an apparent dissociation constant (Kd) of 1.8 μM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99–100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co‐expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant‐encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.
The N‐domain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae
Authors: Virji Mumtaz , Watt Suzanne M. , Barker Stephanie , Makepeace Katherine , Doyonnas Regis
Published: November 2003

Using COS (African green monkey kidney) cells transfected with cDNAs encoding human cell surface molecules, we have identified human cellular receptors for meningococcal virulence‐associated Opa proteins, which are expressed by the majority of disease and carrier isolates. These receptors belong to the immunoglobulin superfamily of adhesion molecules and are expressed on epithelial, endothelial and phagocytic cells. Using soluble chimeric receptor molecules, we have demonstrated that meningococcal Opa proteins bind to the N‐terminal domain of biliary glycoproteins (classified as BGP or CD66a) that belong to the CEA (CD66) family. Moreover, the Opa proteins of the related pathogen Neisseriagonorrhoeae, responsible for urogenital infections, also interact with this receptor, making CD66a a common target for pathogenic neisseriae. Over 95% of Opa‐expressing clinical and mucosal isolates of meningococci and gonococci were shown to bind to the CD66 N‐domain, demonstrating the presence of a conserved receptor‐binding function in the majority of neisserial Opa proteins.
Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic neisseriae
Authors: Virji Mumtaz , Makepeace Katherine , Ferguson David J. P. , Watt Suzanne M.
Published: November 2003

Opa protein‐expressing pathogenic neisseriae interact with CD66a‐transfected COS (African green monkey kidney) and CHO (Chinese hamster ovary) cells. CD66a (BGP) is a member of carcinoembryonic antigen (CEA, CD66) family. The interactions occur at the N‐terminal domain of CD66a, a region that is highly conserved between members of the CEA subgroup of the CD66 family. In this study, we have investigated the roles of CD66 expressed on human epithelial cells and polymorphonuclear phagocytes (PMNs) in adhesion mediated via Opa proteins. Using human colonic (HT29) and lung (A549) epithelial cell lines known to express CD66 molecules, we show that these receptors are used by meningococci. A monoclonal antibody, YTH71.3, against the N‐terminal domain of CD66, but not 3B10 directed against domains, A1/B1, inhibited meningococcal adhesion to host cells. When acapsulate bacteria expressing Opa proteins were used, large numbers of bacteria adhered to HT29 and A549 cells. In addition, both CD66a‐transfected CHO cells and human epithelial cells were invaded by Opa‐expressing meningococci, suggesting that epithelial cell invasion may occur via Opa–CD66 interactions. In previous studies we have shown that serogroup A strain C751 expresses three Opa proteins, all of which mediate non‐opsonic interactions with neutrophils. We have examined the mechanisms of these interactions using antibodies and soluble chimeric receptors. The results indicate that the nature of their interactions with purified CD66a molecules and with CD66 on neutrophils is alike and that these interactions occur at the N‐terminal domain of CD66. Thus, the Opa family of neisserial ligands may interact with several members of the CD66 family via their largely conserved N‐terminal domains.
Transcriptional regulation of zwf, encoding glucose‐6‐phosphate dehydrogenase, from the cyanobacterium Nostoc punctiforme strain ATCC 29133
Authors: Summers Michael L. , Meeks John C.
Published: November 2003

The gene encoding glucose‐6‐phosphate dehydrogenase (G6PD), zwf, in Nostocpunctiforme strain ATCC 29133 is part of a four‐gene operon that also encodes fructose bisphosphatase (fbp), transaldolase (tal ) and a gene product termed OpcA, which is cotranscribed with zwf and essential for G6PD activity. The effect of exogenous nitrogen and carbon sources on transcription of these genes was investigated. Growth in the presence of ammonium yielded low levels of transcripts encoding all genes of the operon, while growth under nitrogen‐fixing conditions resulted in a large increase of transcripts encoding for fbp and zwf–opcA. When cells are grown in the presence of fructose, levels of transcripts encoding tal and zwf–opcA were increased, relative to levels in ammonium‐grown cells. These results indicate that this facultatively heterotrophic cyanobacterium can respond to changes in its environment by altering transcription of genes involved in carbon catabolism. Primer extension identified five 5′ ends corresponding to the major regulated transcripts which we conclude arise from independent transcriptional start points.
Effect of the deletion of the σ32‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance
Authors: Qi Haiyan , Menzel Rolf , Tse‐Dinh Yuk‐Ching
Published: November 2003

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli. The P1 promoter of topA has previously been shown to be a σ32‐dependent heat‐shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is >10‐fold more sensitive to heat treatment (52°C) than the wild type. After brief treatment at 42°C, wild‐type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52°C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100‐fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat‐survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat‐inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse‐labelling pattern of proteins at 42°C (displayed on SDS–polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.
Genetically probing the regions of ribose‐binding protein involved in permease interaction
Authors: Eym Yongbin , Park Yongkyu , Park Chankyu
Published: November 2003

The ribose‐binding protein (RBP) of Escherichia coli, located in the periplasm, binds to ribose and mediates transport and chemotaxis. The regions on the tertiary structure of RBP that interact with the membrane permease, an ABC transporter, were genetically probed by screening a mutation using the chimeric receptor Trz. Trz is a hybrid protein between the periplasmic domain of chemoreceptor Trg and the cytoplasmic portion of osmosensor EnvZ, which provides a system for monitoring the chemotactic interaction of RBP on MacConkey agar plates when coupled with a reporter lacZ fused to an ompC gene. The expression of ompC can be increased by an interaction of ribose‐bound RBP with Trz. A transport defect, either in the binding protein or in the membrane permease, causes a signalling‐constitutive Lac+ phenotype of Trz even in the absence of ribose. This appears to be due to the presence of a small amount of ribose, which is normally taken up by the high‐affinity transport system. By taking advantage of this, we have designed a system for genetic screening that permits a selection for mutations in the binding protein, causing specific defects in permease interaction but not in tactic interaction. Mutant RBPs that were isolated were unable to perform normal ribose uptake and to utilize ribose as a carbon source, while other functions such as taxis and sugar‐binding properties were not substantially affected. The mutational changes were repeatedly found in several residues of RBP, concentrating on three surface regions and comprising two domains of the tertiary structure. We suggest that the two regions, including residues 52 and 166, are specifically involved in the permease interaction while the third region, including residues 72, 134, and others, recognizes both the permease and the chemosensory receptor.
Expression of the tolQRA genes of Escherichia coli K‐12 is controlled by the RcsC sensor protein involved in capsule synthesis
Authors: Clavel Thierry , Lazzaroni Jean Claude , Vianney Anne , Portalier Raymond
Published: November 2003

The tolQRABpal cluster of Escherichia coli K‐12 encodes proteins involved in the maintenance of cell‐envelope integrity. In addition, toi/pal mutations result in a mucoid colony phenotype at low temperature. The synthesis of capsular polysaccharides by the cps genes is controlled by the positive regulator RcsA and the two‐component RcsC/RcsB system. It was shown that the mucoid phenotype of the tol/pal mutants was due to an rcsCB‐dependent activation of the cps genes. Furthermore, we have identified a mutation in the rcsC gene that decreased the activity of a tolA‐lac operon fusion independently of RcsA and partially independently of RcsB activators. The corresponding rcsC338 mutation resulted in a Glu to Lys substitution at residue 338 of RcsC. This mutation induced mucoidy even at high temperature. We propose that RcsC modulates the phosphorylated forms of RcsB and an uncharacterized regulatory protein involved in the control of the tolQRA genes in an opposite manner. Moreover, our findings strengthen the previous suggestion that RcsC senses some alterations in the cell surface such as those induced by tol, pal or rfa mutations, and activates capsule synthesis to protect the cell against deleterious agents.
Involvement of σ54 in exponential silencing of the Pseudomonas putida TOL plasmid Pu promoter
Authors: Cases IIdefonso , de Lorenzo Victor , Pérez‐Martín José
Published: November 2003

The σ54‐dependent Pu promoter of the TOL plasmid pWWO of Pseudomonas putida becomes activated by the prokaryotic enhancer‐binding XyIR protein when cells encounter m‐xylene in the medium. However, even in the presence of the aromatic inducer, Pu activity is silenced in vivo during rapid exponential growth of the cells in rich medium. Various elements known to be involved in the control of the transcriptional activity of the promoter were examined to ascertain the mechanism by which expression of Pu is limited during the exponential phase of growth. A truncated and fully constitutive XyIR derivative deleted of its signal reception N‐terminal domain was found to be subjected to the same exponential silencing as the wild‐type XyIR when exposed to m‐xylene. This indicated that the phenomenon is not due to a late activation of XyIR by the aromatic effector. A Pu variant in which the integration host factor (IHF)‐binding site had been functionally replaced by a statically curved DNA segment showed the same induction pattern, thus ruling out variations in the intracellular levels of IHF changes during growth as the element responsible for the inactivity of Pu in rapidly growing cells. On the contrary, overproduction of the σ54 factor allowed Pu expression during exponential phase. As σ54 protein levels remained approximately constant during growth, the exponential silencing of Pu could be caused ultimately by changes in the activity of the factor itself. This effect may not be exclusive to Pu, but could be a general co‐regulation mechanism in σ54‐dependent promoters that connects transcription of a specific set of genes with the general physiological status of the cells.
Use of chimeric recombinant polypeptides to analyse conformational, surface epitopes on trypanosome variant surface glycoproteins
Authors: Hsia Ru‐ching , Beals Thomas , Boothroyd John C.
Published: November 2003

Identification of surface‐exposed epitopes on the variant surface glycoproteins (VSGs) of African trypanosomes has been complicated by the observation that most such epitopes are highly conformational. As a result, whenever the molecule is broken down for analysis, the epitope is generally lost. We have exploited the existence of closely related gene families to create chimeric molecules in which particular segments of one VSG are placed in the analogous position of a related but antigenically distinct VSG. The process is used in both a positive and negative manner, so that the epitope can be specifically added or destroyed in a given chimera. As an example, we have used this approach to identify the regions involved in reactivity to a monoclonal antibody specific for VSG117 on the surface of live trypanosomes. We show that while deletion of almost any region of VSG117 results in loss of reactivity to this monoclonal antibody, substituting particular regions with the corresponding segment of the structurally related but antigenically distinct VSG FM8.5 restores reactivity in most but not all cases, thereby delimiting the antigenically key regions. Likewise, substituting key regions from VSG117 into FM8.5 confers reactivity on the resulting chimeras. This approach circumvents some of the problems that result from the highly conformational nature of VSG and should allow further elucidation of the biologically relevant antigenic topology of VSGs.
The katE gene, which encodes the catalase HPII of Mycobacterium avium
Authors: Milano A. , De Rossi E. , Gusberti L. , Heym B. , Marone P. , Riccardi G.
Published: November 2003

Disseminated Mycobacterium avium‐Mycobacterium intracellular disease is a prevalent opportunistic infection in patients with acquired immune deficiency syndrome (AIDS). These pathogens are generally resistant to isoniazid (INH), a powerful antituberculosis drug. It is now generally accepted that the INH susceptibility of Mycobacterium tuberculosis results from the transformation of the drug into a toxic derivative, as a result of the action of the enzyme catalase‐peroxidase (HPI), encoded by the katG gene. It has been speculated that the presence of a second catalase (HPII) in some mycobacterial species, but lacking in M. tuberculosis, may impair the action of INH. In this report, the nucleotide sequence of the M. avium katE gene, encoding catalase HPII, is described. This enzyme shows strong similarity to Escherichia coli catalase HPII and eukaryotic catalases. All amino acids previously postulated as participating directly in catalysis by liver catalase and most of the amino acids binding the prosthetic group are conserved in M. avium catalase HPII. The enzyme is expressed in E. coli and is inhibited by 3‐amino ‐l,2,4 triazole (AT). Furthermore, Southern blot hybridizations and polymerase chain reaction experiments demonstrate the distribution of katE gene in several mycobacterial species.

To evaluate the potentially antagonistic effect of HPII catalase on INH susceptibility, the katE gene was transformed into M. tuberculosis H37Rv and the minimum inhibitory concentration (MIC) for INH was determined. Despite strong expression of the katEgene, no change in MIC was observed, thus ruling out a possible contribution of this enzyme to the natural resistance of M. avium to the drug.

The availability of the gene probe, encoding the second mycobacterial catalase HPII, should open the way for the development of new drugs and diagnostic tests to combat drug‐resistant pathogen strains.