bluuebunny/crossref_metadata_2025 huggingface dataset:
The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA
Authors: Liu Mingfu , Kirpekar Finn , Van Wezel Gilles P. , Douthwaite Stephen
Published: March 2003

tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic‐producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification is dependent on the presence of the methyl group donor, S‐adenosyl methionine. Analysis of the 74‐mer RNA substrate by biochemical and mass spectrometric methods shows that TlrB adds a single methyl group to the base of G748. Homologues of TlrB in other bacteria have been revealed through database searches, indicating that TlrB is the first member to be described in a new subclass of rRNA methyltransferases that are implicated in macrolide drug resistance.
The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli
Authors: Urbanowski Mark L. , Stauffer Lorraine T. , Stauffer George V.
Published: March 2003

The Escherichia coli gcvB gene encodes a small RNA transcript that is not translated in vivo. Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, the transcriptional regulators of the gcvTHP operon encoding the enzymes of the glycine cleavage system. A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity. However, this mutant has high constitutive synthesis of OppA and DppA, the periplasmic‐binding protein components of the two major peptide transport systems normally repressed in cells growing in rich medium. The altered regulation of oppA and dppA was also demonstrated using oppA–phoA and dppA–lacZ gene fusions. Although the mechanism(s) involving gcvB in the repression of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level.
Regulation of carbon metabolism in Chlamydia trachomatis
Authors: Iliffe‐Lee Emma R. , McClarty Grant
Published: March 2003

The biological significance of glycogen accumulation and how the process is regulated in Chlamydia trachomatis remains poorly defined. C. trachomatis‐infected HeLa cells were cultured in medium containing various glucose concentrations (0, 0.1, 1 or 10 mg ml−1) or in the presence of gluconeogenic carbon sources (20 mM glutamate, 20 mM malate, 20 mM α‐ketoglutarate or 20 mM oxaloacetate), and the effects of these different culture conditions on the production of infectious chlamydial elementary bodies and glycogen accumulation were monitored. When chlamydiae were cultured in glucose concentrations greater than 1 mg ml−1, optimal growth and maximal glycogen accumulation occurred. In contrast to uninfected HeLa cells, which increased their glycogen stores when grown in the presence of high glucose concentrations, chlamydial glycogen accumulation remained essentially constant. When cultured in medium supplemented with either reduced glucose concentrations or any of the gluconeogenic carbon sources, chlamydiae still grew; however, the yield of elementary bodies was substantially decreased, and there was no significant amount of glycogen accumulated by host HeLa cells or C. trachomatis. This suggests that glycogen accumulation may not be essential for chlamydial survival. Reverse transcriptase–polymerase chain reaction (RT–PCR) results indicated that, despite the fact that the source and amount of carbon available in the medium affected chlamydial glycogen accumulation, the expression of genes required for glycogen metabolism was not significantly changed. Similarly, the expression of several genes encoding key enzymes of central metabolism was not affected by alterations in carbon source or availability. Taken together, the data suggest that, unlike most free‐living bacteria, chlamydia are unable to alter the expression of genes involved in carbon metabolism in response to changes in environmental conditions.
FAP1, a homologue of human transcription factor NF‐X1, competes with rapamycin for binding to FKBP12 in yeast
Authors: Kunz Jeannette , Loeschmann Andrea , Deuter‐Reinhard Maja , Hall Michael N.
Published: March 2003

The immunosuppressive drug rapamycin binds to the peptidyl‐prolyl cis‐trans isomerase FKBP12, and this complex arrests growth of yeast cells and activated T lymphocytes in the G1 phase of the cell cycle. In yeast, loss‐of‐function mutations in FPR1, the gene encoding FKBP12, or dominant gain‐of‐function mutations in TOR1 and TOR2, the genes encoding the physical targets of the FKBP12–rapamycin complex, confer rapamycin resistance. Here, we report the cloning and characterization of a novel gene, termed FAP1, which confers resistance to rapamycin by competing with the drug for binding to FKBP12. FAP1 encodes a member of an evolutionarily conserved family of putative transcription factors that includes human NF‐X1, Drosophila melanogaster shuttle craft and previously undescribed homologues in Caenorhabditis elegans, Arabidopsis thaliana and Schizosaccharomyces pombe. We provide genetic and biochemical evidence that FAP1 interacts physically with FKBP12 in vivo and in vitro, and that it competes with rapamycin for interaction. Furthermore, mutations in the FKBP12 drug binding/active site or surface residues abolish binding to FAP1. Our results suggest that FAP1 is a physiological ligand for FKBP12 that is highly conserved from yeast to man. Furthermore, prolyl isomerases may commonly bind and regulate transcription factors.
Identification of Sat, an autotransporter toxin produced by uropathogenic Escherichia coli
Authors: Guyer Debra M. , Henderson Ian R. , Nataro James P. , Mobley Harry L. T.
Published: March 2003

Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a β‐barrel porin structure formed by the carboxy‐terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N‐terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5′ region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C‐terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK‐2 bladder and HEp‐2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.
Generation of a non‐sporulating strain of Streptomyces coelicolor A3(2) by the manipulation of a developmentally controlled ftsZ promoter
Authors: Flärdh Klas , Leibovitz Emmanuelle , Buttner Mark J. , Chater Keith F.
Published: March 2003

The differentiation of Streptomyces aerial hyphae into chains of unigenomic spores occurs through the synchronous formation of multiple FtsZ rings, leading to sporulation septa. We show here that developmental control of ftsZ transcription is required for sporulation in Streptomyces coelicolor A3(2). Three putative ftsZ promoters were detected in the ftsQ–ftsZ intergenic region. In addition, some readthrough from upstream promoter(s) contributed to ftsZ transcription. S1 nuclease protection assays and transcriptional fusions of the ftsZ promoter region to the egfp gene (for green fluorescent protein) provided evidence that ftsZ2p is a developmentally controlled promoter that is specifically upregulated in sporulating aerial hyphae. This upregulation required all the six early regulatory sporulation genes that were tested: whiA, B, G, H, I and J. The DNA sequence of the promoter indicated that it is not part of the developmental regulon that is controlled by the RNA polymerase sigma factor σWhiG. A strain in which the ftsZ2p promoter was inactivated grew normally during vegetative growth and formed aerial mycelium, but was deficient in sporulation septation. Thus, ftsZ2p was dispensable for vegetative growth, but was required for the strain to make sufficient FtsZ to support developmentally controlled multiple cell divisions in aerial hyphae.
Interplay between three global regulatory proteins mediates oxygen regulation of the Escherichia coli cytochrome d oxidase (cydAB) operon
Authors: Govantes Fernando , Orjalo Arturo V. , Gunsalus Robert P.
Published: March 2003

The Escherichia coli cydAB operon, encoding the subunits of the high‐affinity cytochrome d oxidase, is maximally transcribed in microaerobiosis as a result of the combined action of the oxygen‐responsive regulators Fnr and ArcA. Here, we report that the histone‐like protein H‐NS is an aerobic repressor of cydAB expression. ArcA is shown to antagonize H‐NS action to render cydAB expression insensitive to H‐NS repression in anaerobiosis. The targets for H‐NS‐mediated aerobic repression are the four oxygen‐regulated promoters, designated P1, P2, P3 and P4. H‐NS control is the result of H‐NS binding to an extended region within the cydAB promoter element, including sequences upstream from and overlapping the four regulated promoters. We propose a regulatory model in which oxygen control of cydAB transcription is mediated by three alternative protein–DNA complexes that are assembled sequentially on the promoter region as the cells are shifted from aerobic to microaerobic and to anaerobic conditions. According to this model, ArcA‐P plays a central role in cydAB regulation by antagonizing H‐NS repression of cydAB transcription when oxygen becomes limiting. This allows peak gene expression and subsequent repression by Fnr under fully anaerobic conditions.
The Escherichia coli histone‐like protein HU regulates rpoS translation
Authors: Balandina Anna , Claret Laurent , Hengge‐Aronis Regine , Rouviere‐Yaniv Josette
Published: March 2003

Escherichia coli HU protein is a major component of the bacterial nucleoid. HU stabilizes higher order nucleoprotein complexes and belongs to a family of DNA architectural proteins. Here, we report that HU is required for efficient expression of the sigma S subunit of RNA polymerase. This rpoS‐encoded alternative σS factor induces a number of genes implicated in cell survival in stationary phase and in multiple stress resistance. By analysis of rpoS–lacZ fusions and by pulse‐chase experiments, we show that the efficiency of rpoS translation is reduced in cells lacking HU, whereas neither rpoS transcription nor protein stability is affected by HU. Gel mobility shift assays show that HU is able to bind specifically an RNA fragment containing the translational initiation region of rpoS mRNA 1000‐fold more strongly than double‐stranded DNA. Together with the in vivo data, this finding strongly suggests that, by binding to rpoS mRNA, HU directly stimulates rpoS translation. We demonstrate here that HU, an abundant DNA‐binding, histone‐like protein, is able specifically to recognize an RNA molecule and therefore play a role in post‐transcriptional regulation.
Bacterial cell division: regulating Z‐ring formation
Authors: Harry E. J.
Published: March 2003

The earliest stage of cell division in bacteria is the formation of a Z ring, composed of a polymer of the FtsZ protein, at the division site. Z rings appear to be synthesized in a bi‐directional manner from a nucleation site (NS) located on the inside of the cytoplasmic membrane. It is the utilization of a NS specifically at the site of septum formation that determines where and when division will occur. However, a Z ring can be made to form at positions other than at the division site. How does a cell regulate utilization of a NS at the correct location and at the right time? In rod‐shaped bacteria such as Escherichia coli and Bacillus subtilis, two factors involved in this regulation are the Min system and nucleoid occlusion. It is suggested that in B. subtilis, the main role of the Min proteins is to inhibit division at the nucleoid‐free cell poles. In E. coli it is currently not clear whether the Min system can direct a Z ring to the division site at mid‐cell or whether its main role is to ensure that division inhibition occurs away from mid‐cell, a role analogous to that in B. subtilis. While the nucleoid negatively influences Z‐ring formation in its vicinity in these rod‐shaped organisms, the exact relationship between nucleoid occlusion and the ability to form a mid‐cell Z ring is unresolved. Recent evidence suggests that in B. subtilis and Caulobacter crescentus, utilization of the NS at the division site is intimately linked to the progress of a round of chromosome replication and this may form the basis of achieving co‐ordination between chromosome replication and cell division.
In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae
Authors: Rice Michael C. , Bruner Michael , Czymmek Kirk , Kmiec Eric B.
Published: March 2003

Targeted gene repair directed by chimeric RNA/DNA oligonucleotides has proven successful in eukaryotic cells including animal and plant models. In many cases, however, there has been a disparity in the levels of gene correction or frequency. While the delivery of these chimera into the nucleus and the long‐term stability or purity of these molecules may contribute to this variability, understanding the molecular regulation of conversion is the key to improving or stabilizing frequency. To this end, we have identified genes that control targeted repair, using the genetically tractable organism, Saccharomyces cerevisae and a bank of yeast mutants. Results from experiments in cell‐free extracts focused our attention on RAD52, RAD1 and RAD59 as central regulatory factors. RAD1 and RAD59 appear to be required for high levels of conversion whereas RAD52 appears to act, surprisingly, in a suppressive fashion. Results from the in vitro experiments were translated into targeting experiments in vivo. Here, mutations in a fusion construct, containing a marker gene, were converted to wild type, evidenced by the expression of green fluorescence in converted cells. Because the repaired fusion gene contains a corrected neomycin sequence, cells were subsequently placed under G418 selection and conversion confirmed at the genetic level. Taken together, these results establish, for the first time, genes that participate in the regulation of targeted gene repair and provide a novel system for evaluating true frequencies of correction. Importantly, this system enables visualization of corrected (green) and uncorrected (clear) cells enabling measurements of conversion in real time.
Erythromycin inhibition of 50S ribosomal subunit formation in Escherichia coli cells
Authors: Usary Jerry , Champney W. Scott
Published: March 2003

The effects of erythromycin on the formation of ribosomal subunits were examined in wild‐type Escherichia coli cells and in an RNase E mutant strain. Pulse–chase labelling kinetics revealed a reduced rate of 50S subunit formation in both strains compared with 30S synthesis, which was unaffected by the antibiotic. Growth of cells in the presence of [14C]‐erythromycin showed drug binding to 50S particles and to a 50S subunit precursor sedimenting at about 30S in sucrose gradients. Antibiotic binding to the precursor correlated with the decline in 50S formation in both strains. Erythromycin binding to the precursor showed the same 1:1 stoichiometry as binding to the 50S particle. Gel electrophoresis of rRNA from antibiotic‐treated organisms revealed the presence of both 23S and 5S rRNAs in the 30S region of sucrose gradients. Hybridization with a 23S rRNA‐specific probe confirmed the presence of this species of rRNA in the precursor. Eighteen 50S ribosomal proteins were associated with the precursor particle. A model is presented to account for erythromycin inhibition of 50S formation.
Heterogeneity of stress gene expression and stress resistance among individual cells of Saccharomyces cerevisiae
Authors: Attfield Paul V. , Choi Hung Yoon , Veal Duncan A. , Bell Philip J. L.
Published: March 2003

Knowledge of gene expression and cellular responses in microorganisms is derived from analyses of populations consisting of millions of cells. Analytical techniques that provide data as population averages fail to inform of culture heterogeneity. Flow cytometry and fluorescence techniques were used to provide information on the heterogeneity of stress‐responsive gene expression and stress tolerance in individual cells within populations. A sequence of DNA encoding the heat shock and stress response elements of the Saccharomyces cerevisiae HSP104 gene was used to express enhanced green fluorescent protein (EGFP). When integrated into the genome of yeast strain W303‐1A, intrinsic expression of EGFP increased about twofold as cells progressed from growth on glucose to ethanol utilization in aerobic batch cultures. Staining of cells with orange/red fluorescent propidium iodide (PI), which only enters cells that have compromised membrane integrity, revealed that the population became more tolerant to 52°C heat stress as it progressed from growth on glucose and through the ethanol utilization phase of aerobic batch culture. Exposure of cultures growing on glucose to a mild heat shock (shift from 25°C to 37°C) resulted in significantly increased expression of EGFP in the population. However, there was heterogeneity in the intensity of fluorescence of individual cells from heat‐shocked cultures, indicating variability in the strength of stress response in the clonal population. Detailed analysis of the heterogeneity showed a clear positive trend between intensity of stress response and individual cell resistance, measured in terms of PI exclusion, to heat stress at 52°C. Further experiments indicated that, although the mean gene expression by a population is influenced by the genetic background, the heterogeneity among individual cells in clonal populations is largely physiologically based.
Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli
Authors: Sheikh Jalaluddin , Hicks Susan , Dall'Agnol Monique , Phillips Alan D. , Nataro James P.
Published: March 2003

Enteroaggregative Escherichia coli (EAEC) forms thick biofilms on the intestinal mucosa. Here, we show that most EAEC strains form a biofilm on glass or plastic surfaces when grown in cell culture medium with high sugar and osmolarity. Biofilm‐forming ability in two prototype EAEC strains required aggregative adherence fimbriae (AAF), although many other EAEC strains that do not express AAF also developed biofilms under these conditions. Ten thousand transposon mutants of EAEC strain 042 were isolated, and 100 were found to be deficient in biofilm formation. Of these, 93 were either deficient in in vitro growth or mapped to genes known to be required for AAF/II expression. Of the seven remaining insertions, five mapped to one of two unsuspected loci. Two insertions involved the E. coli chromosomal fis gene, a DNA‐binding protein that is involved in growth phase‐dependent regulation. Using reverse transcription–polymerase chain reaction (RT–PCR), we determined that the effect of fis was at the level of transcription of the AAF/II activator aggR. Biofilm formation also required the product of the yafK gene, which is predicted to encode a secreted 28 kDa protein. The yafK product is required for transcription of AAF/II‐encoding genes. Our data do not suggest a role for type 1 fimbriae or motility in biofilm formation. EAEC appears to form a novel biofilm, which may be mediated solely by AAF and may reflect its interactions with the intestinal mucosa.
Transport of cytochrome c derivatives by the bacterial Tat protein translocation system
Authors: Sanders Carsten , Wethkamp Nils , Lill Holger
Published: March 2003

An experimental system developed previously for the heterologous expression of c‐type cytochromes in Escherichia coliQ1has been adapted to monitor protein transfer across the bacteria's cytoplasmic membrane. Apocytochrome, lacking the haem cofactor and probably in an unfolded state, was readily transferred across the cytoplasmic membrane when fused to a Sec‐specific signal peptide. Furthermore, cytochrome fused to a signal peptide regarded as specific for the twin arginine transport (Tat) system was translocated in an unfolded state by the Sec apparatus. After maturation and folding in the cytoplasm, Tat‐mediated transfer of holocytochrome to the periplasm occurred. We conclude that, in addition to the nature of the specific signal peptide, the folding state of a particular protein also governs its acceptance by a given transport system.
Evidence for a rolling‐circle mechanism of phage DNA synthesis from both replicative and integrated forms of CTXφ
Authors: Moyer Kathryn E. , Kimsey Harvey H. , Waldor Matthew K.
Published: March 2003

The genes encoding cholera toxin, the principal virulence factor of Vibrio cholerae, are part of the circular single‐stranded DNA genome of CTXφ. In toxigenic V. cholerae strains, the CTXφ genome is typically found in integrated arrays of tandemly arranged CTX prophages. Infected cells that lack a chromosomal integration site harbour the CTXφ genome as a plasmid (pCTX). We studied the replication of pCTX and found several indications that this plasmid replicates via a rolling‐circle (RC) mechanism. The initiation and termination sites for pCTX plus‐strand DNA synthesis were mapped to a 22 bp sequence that contains inverted repeats and a nonanucleotide motif found in the plus‐strand origins of several RC replicons. Furthermore, similar to other RC replicons, replication of plasmids containing duplicated pCTX origins resulted in the deletion of sequences between the two origins and the formation of a single chimeric origin. Our previous work revealed that CTX prophage arrays give rise to hybrid CTX virions that contain sequences derived from two adjacent prophages. We now report that the boundaries between the sequences contributed to virions by the upstream and the downstream prophages in an array correspond to the site at which synthesis of plus‐strand pCTX DNA is initiated and terminated. These data support the model that plus‐strand CTXφ DNA is generated from chromosomal prophages via a novel process analogous to RC replication.
EmtA, a rRNA methyltransferase conferring high‐level evernimicin resistance
Authors: Mann Paul A. , Xiong Liqun , Mankin Alexander S. , Chau Andrew S. , Mendrick Cara A. , Najarian David J. , Cramer Christina A. , Loebenberg David , Coates Elizabeth , Murgolo Nicholas J. , Aarestrup Frank M. , Goering Richard V. , Black Todd A. , Hare Roberta S. , McNicholas Paul M.
Published: March 2003

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high‐level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell‐free translation assays and direct‐binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid‐borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin‐sensitive strain 30‐fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin‐binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.
FimH‐mediated autoaggregation of Escherichia coli
Authors: Schembri Mark A. , Christiansen Gunna , Klemm Per
Published: March 2003

Autoaggregation is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria. Type 1 fimbriae are surface organelles of Escherichia coli that mediate d‐mannose‐sensitive binding to various host surfaces. This binding is conferred by the minor fimbrial component FimH. In this study, we have used random mutagenesis to identify variants of the FimH adhesin that confer the ability of E. coli to autoaggregate and settle from liquid cultures. Three separate autoaggregating clones were identified, all of which contained multiple amino acid changes located within the N‐terminal receptor‐binding domain of FimH. Autoaggregation could not be inhibited by mannose, but was inhibited by growth at temperatures at or below 30°C. Using green fluorescent protein (GFP) as a reporter, we show that the autoaggregating clones do not mix with wild‐type fimbriated cells. Electron microscopy shows that autoaggregating cells produce fimbriae with a twisted and entangled appearance. We present evidence that autoaggregating versions of FimH also occur in nature. Our results stress the highly adaptive nature of the ubiquitous FimH adhesin.
Structural alterations in a type IV pilus subunit protein result in concurrent defects in multicellular behaviour and adherence to host tissue
Authors: Park Hae‐Sun Moon , Wolfgang Matthew , Van Putten Jos P. M. , Dorward David , Hayes Stanley F. , Koomey Michael
Published: March 2003

The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial–substratum and bacterial–bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial self‐aggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved amino‐terminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co‐expression of the altered alleles in the context of a wild‐type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss‐of‐function mutation in the twitching motility gene pilT. The correlations between self‐aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell–cell interactions to colonization.
Loss‐of‐function mutations in yjbD result in ClpX‐ and ClpP‐independent competence development of Bacillus subtilis
Authors: Nakano Michiko M. , Hajarizadeh Farin , Zhu Yi , Zuber Peter
Published: March 2003

Mutations in clpP and clpX have pleiotropic effects on growth and developmentally regulated gene expression in Bacillus subtilis. ClpP and ClpX are needed for expression of comK, encoding the competence transcription factor required for the expression of genes within the competence regulon. ClpP, in combination with the ATPase ClpC, degrades the inhibitor of ComK, MecA. Proteolysis of MecA is stimulated by a small protein, ComS, which interacts with MecA. Suppressor mutations (cxs) were isolated that bypass the requirement for clpX for comK expression. These were found also to overcome the defect in comK expression conferred by a clpP mutation. These mutations were identified as missense mutations (cxs‐5, ‐7 and ‐12) and a nonsense (UAG) codon substitution (cxs‐10) in the yjbD coding sequence in a locus linked to mecA. That a yjbD disruption confers the cxs phenotype, together with its complementation by an ectopically expressed copy of yjbD, indicated that the suppressor alleles bear recessive, loss‐of‐function mutations of yjbD. ClpP‐ and ClpX‐independent comK expression rendered by inactivation of yjbD was still medium‐dependent and required ComS. MecA levels in a clpP‐yjbD mutant were lower that those of clpP mutant cells and ComK protein concentration in the clpP mutant was restored to wild‐type levels by the yjbD mutation. Consequently, the yjbD mutation bypasses the defect in competence development conferred by clpP and clpX. YjbD protein is barely detectable in wild‐type cells, but is present in large amounts in the clpP mutant cells. The results suggest that the role of ClpP in competence development is to degrade YjbD protein so that ComS can productively interact with the MecA–ClpC–ComK complex. Alternatively, the result could suggest that YjbD has a negative effect on regulated proteolysis and that MecA is degraded independently of ClpP when YjbD is absent.
The gametocyte‐activating factor xanthurenic acid stimulates an increase in membrane‐associated guanylyl cyclase activity in the human malaria parasite Plasmodium falciparum
Authors: Muhia David K. , Swales Claire A. , Deng Wensheng , Kelly John M. , Baker David A.
Published: March 2003

Sex is an obligate step in the life cycle of the malaria parasite and occurs in the midgut of the mosquito vector. With both Plasmodium falciparum and Plasmodium berghei, the tryptophan metabolite xanthurenic acid induces the release of motile male gametes from red blood cells (exflagellation), a prerequisite for fertilization. The addition of cGMP or phosphodiesterase inhibitors to cultures of mature gametocytes has also been shown to stimulate exflagellation. Here, we demonstrate that there is a guanylyl cyclase activity associated with mature P. falciparum gametocyte membrane preparations, which is dependent on the presence of Mg2+/Mn2+ but is inhibited by Ca2+. Significantly, this activity is increased on addition of xanthurenic acid. In contrast, a xanthurenic acid precursor (3‐hydroxykynurenine), which is not an inducer of exflagellation, does not induce this guanylyl cyclase activity. These results therefore suggest that xanthurenic acid‐induced exflagellation may be mediated by activation of the parasite cGMP signalling pathway.
Phosphoserine modification of the enteropathogenic Escherichia coli Tir molecule is required to trigger conformational changes in Tir and efficient pedestal elongation
Authors: Warawa Jonathan , Kenny Brendan
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) virulence is correlated with intimate adherence to gut epithelial cells, loss of absorptive microvilli and reorganization of host cytoskeletal proteins into pedestal‐like structures beneath the adherent bacteria. These processes depend on Tir (i) being inserted into the plasma membrane; (ii) being tyrosine phosphorylated; and (iii) interacting with the bacterial outer membrane protein, intimin. However, phosphorylation on other undefined residues leads to ≈ 5 kDa and ≈ 2 kDa increases in Tir apparent molecular mass within host cells. In this study, we show that equivalent shifts can be induced in vitro by phosphorylation of Tir on two serine (S434 and S463) residues by protein kinase A (PKA). Our data suggest that the sequential addition of two phosphate groups triggers conformational changes in Tir structure that may supply the energy to insert Tir into the plasma membrane. PKA was also shown to modify Tir within host cells on S434 to induce the ≈ 5 kDa shift. Whereas modification of S434 was not essential to generate an actin‐nucleating molecule, it was required for Tir to induce pedestal elongation efficiently. This study not only increases our understanding of the mechanism by which phosphorylation induces shifts in Tir apparent molecular mass and suggests a mechanism by which Tir may be inserted into the plasma membrane, but also reveals a role for non‐tyrosine phosphorylation in Tir function and identifies the first kinase that can modify Tir in vitro or in vivo.
Synergy between the N‐ and C‐terminal domains of InlB for efficient invasion of non‐phagocytic cells by Listeria monocytogenes
Authors: Jonquières Renaud , Pizarro‐Cerdá Javier , Cossart Pascale
Published: March 2003

InlB is a Listeria monocytogenes protein promoting entry in non‐phagocytic cells, and has been shown recently to activate the hepatocyte growth factor receptor (HGFR or Met). The N‐terminal domain of InlB (LRRs) binds and activates Met, whereas the C‐terminal domain of InlB (GW modules) mediates loose attachment of InlB to the listerial surface. As HGF activation of Met is tightly controlled by glycosaminoglycans (GAGs), we tested if GAGs also modulate the Met–InlB interactions. We show that InlB‐dependent invasion of non‐phagocytic cells decreases up to 10 times in the absence of GAGs, and that soluble heparin releases InlB from the bacterial surface and promotes its clustering. Furthermore, we demonstrate that InlB binds cellular GAGs by its GW modules, and that this interaction is required for efficient InlB‐mediated invasion. Therefore, GW modules have an unsuspected dual function: they attach InlB to the bacterial surface and enhance entry triggered by the LRRs domain. Our results thus provide the first evidence for a synergy between two host factor‐binding domains of a bacterial invasion protein, and reinforce similarities between InlB and mammalian growth factors.
Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator
Authors: Jablonowski Daniel , Frohloff Frank , Fichtner Lars , Stark Michael J. R. , Schaffrath Raffael
Published: March 2003

The putative Kluyveromyces lactis zymocin target complex, TOT, from Saccharomyces cerevisiae comprises five Tot proteins, four of which are RNA polymerase II (RNAP II) Elongator subunits. Recently, two more Elongator subunit genes, ELP6 (TOT6) and ELP4 (TOT7), have been identified. Deletions of both TOT6 and TOT7 result in the complex tot phenotype, including resistance to zymocin, thermosensitivity, slow growth and hypersensitivity towards drugs, thus reinforcing the notion that TOT/Elongator may be crucial in signalling zymocicity. Mutagenesis of ELP3/TOT3, the Elongator histone acetyltransferase (HAT) gene, revealed that zymocin sensitivity could be uncoupled from Elongator wild‐type function, indicating that TOT interacts genetically with zymocin. To test the possibility that zymocin functions by affecting RNAP II activity in a TOT/Elongator‐dependent manner, global poly(A)+ mRNA levels were found to decline drastically on zymocin treatment. Moreover, cells overexpressing Fcp1p, the RNAP II carboxy‐terminal domain phosphatase, acquired partial zymocin resistance, whereas cells underproducing RNAP II became zymocin hypersensitive. This suggests that zymocin may convert TOT/Elongator into a cellular poison toxic for RNAP II function and eventually leading to the observed G1 cell cycle arrest.
Characterization of the interaction partners of secreted proteins and chaperones of Shigella flexneri
Authors: Page Anne‐Laure , Fromont‐Racine Micheline , Sansonetti Philippe , Legrain Pierre , Parsot Claude
Published: March 2003

The type III secretion (TTS) system of Gram‐negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that are secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two‐hybrid system to investigate the interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His‐tagged IpgC protein to co‐purify truncated IpaB and IpaC proteins, we showed that the chaperone‐binding domain was unique and located in the N‐terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process.
A maize‐specifically expressed gene cluster in Ustilago maydis
Authors: Basse Christoph W. , Kolb Sebastian , Kahmann Regine
Published: March 2003

The corn pathogen Ustilago maydis requires its host plant maize for development
and completion of its sexual cycle. We have identified the fungal mig2–1
gene as being specifically expressed during this biotrophic stage. Intriguingly,
mig2–1 is part of a gene cluster comprising five highly homologous
and similarly regulated genes designated mig2–1 to mig2–5.
Deletion analysis of the mig2–1 promoter provides evidence for negative
and positive regulation. The predicted polypeptides of all five genes lack significant
homologies to known genes but have characteristic N‐terminal secretion sequences.
The secretion signals of mig2–1 and mig2–5 were shown
to be functional, and secretion of a full length Mig2–1‐eGFP fusion protein
to the extracellular space was demonstrated. The central domains of the Mig2 proteins
are highly variable whereas the C‐termini are strongly conserved and share a characteristic
pattern of eight cysteine residues. The mig2 gene cluster was conserved in
a wide collection of U. maydis strains. Interestingly, some U. maydis
isolates from South America had lost the mig2–4 gene as a result of
a homologous recombination event. Furthermore, the related Ustilago scitaminea
strain, which is pathogenic on sugar cane, appears to lack the mig2 cluster.
We describe a model of how the mig2 cluster might have evolved and discuss its possible role in governing host interaction.
An ABC‐type, high‐affinity urea permease identified in cyanobacteria
Authors: Valladares Ana , Montesinos María Luz , Herrero Antonia , Flores Enrique
Published: March 2003

Urea is an important nitrogen source for many microorganisms, but urea active transporters have not been characterized at a molecular level in any bacterium. Cells of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 exhibited the capacity to take up [14C]‐urea from low‐concentration (<1 μM) urea solutions. The Ks of Anabaena cells for urea was about 0.11 μM, and the observed uptake activity involved the transport and metabolism of urea. In contrast to urease, which was constitutively ex‐pressed, expression of the high‐affinity urea uptake activity was subjected to nitrogen control. In an Anabaena ureG (urease–) mutant, a concentrative, active transport of urea could be demonstrated. We found that a mutant of open reading frame (ORF) sll0374 from the Synechocystis genomic sequence lacked urea transport activity. This ORF encoded a conserved component of an ABC‐type transporter, but it is not clustered together with any other possible transporter‐encoding gene. An Anabaena homologue of sll0374, urtE, was isolated and found to be part of a cluster of genes, urtABCDE, putatively encoding all the elements of an ABC‐type permease. Although the longest transcript that we could detect only covered urtABC, the impairment of urea transport by inactivation of urtA, urtB or urtE suggested that the whole gene cluster is expressed producing the urea permease. Expression was induced under nitrogen‐limiting conditions, and a complex promoter regulated by the cyanobacterial global nitrogen control transcription factor NtcA was found upstream from urtA. Our work adds urea to the known substrates of the versatile class of ABC‐type transporters and suggests the involvement of a transporter of this superfamily in urea scavenging by some bacteria in natural environments.
Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling
Authors: Betts Joanna C. , Lukey Pauline T. , Robb Linda C. , McAdam Ruth A. , Duncan Ken
Published: March 2003

The search for new TB drugs that rapidly and effectively sterilize the tissues and are thus able to shorten the duration of chemotherapy from the current 6 months has been hampered by a lack of understanding of the metabolism of the bacterium when in a ‘persistent’ or latent form. Little is known about the condition in which the bacilli survive, although laboratory models have shown that Mycobacterium tuberculosis can exist in a non‐growing, drug‐resistant state that may mimic persistence in vivo. Using nutrient starvation, we have established a model in which M. tuberculosis arrests growth, decreases its respiration rate and is resistant to isoniazid, rifampicin and metronidazole. We have used microarray and proteome analysis to investigate the response of M. tuberculosis to nutrient starvation. Proteome analysis of 6‐week‐starved cultures revealed the induction of several proteins. Microarray analysis enabled us to monitor gene expression during adaptation to nutrient starvation and confirmed the changes seen at the protein level. This has provided evidence for slowdown of the transcription apparatus, energy metabolism, lipid biosynthesis and cell division in addition to induction of the stringent response and several other genes that may play a role in maintaining long‐term survival within the host. Thus, we have generated a model with which we can search for agents active against persistent M. tuberculosis and revealed a number of potential targets expressed under these conditions.
c‐Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs
Authors: Stein Markus , Bagnoli Fabio , Halenbeck Robert , Rappuoli Rino , Fantl Wendy J. , Covacci Antonello
Published: March 2003

The human pathogen Helicobacter pylori colonizes the mucous layer of the

stomach. During parasitic infection, freely swimming bacteria adhere to the gastric

epithelial cells and trigger intracellular signalling pathways. This process requires

the translocation of the effector protein CagA into the host cell through a specialized

type IV secretion system encoded in the cag pathogenicity island. Following transfer, CagA is phosphorylated on tyrosine re‐sidues by a host cell kinase. Here, we describe how the tyrosine phosphorylation of CagA is restricted to a previously identified repeated sequence called D1. This sequence is located in the C‐terminal half of the protein and contains the five‐amino‐acid motif EPIYA, which is amplified by duplications in a large fraction of clinical isolates. Tyrosine phosphorylation of CagA is essential for the activation process that leads to dramatic changes in the morphology of cells growing in culture. In addition, we observed that two members of the src kinases family, c‐Src and Lyn, account for most of the CagA‐specific kinase activity in host cell lysates. Thus, CagA translocation followed by tyrosine phosphorylation at the EPIYA motifs promotes a growth factor‐like response with intense cytoskeletal rearrangements, cell elongation effects and increased cellular motility.
afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2)
Authors: Lee Ping‐Chin , Umeyama Takashi , Horinouchi Sueharu
Published: March 2003

AfsR is a pleiotropic, global regulator that controls the production of actinorhodin, undecylprodigiosin and calcium‐dependent antibiotic in Streptomyces coelicolor
A3(2). AfsR, with 993 amino acids, is phosphorylated on serine and threonine residues
by a protein serine/threonine kinase AfsK and contains an OmpR‐like DNA‐binding fold
at its N‐terminal portion and A‐ and B‐type nucleotide‐binding motifs in the middle
of the protein. The DNA‐binding domain, in‐dependently of the nucleotide‐binding
domain, contributed the binding of AfsR to the upstream region of afsS that
locates immediately 3′ to afsR and encodes a 63‐amino‐acid protein.
No transcription of afsS in the ΔafsR background and restoration
of afsS transcription by afsR on a plasmid in the same genetic background
indicated that afsR served as a transcriptional activator for afsS.
Interestingly, the AfsR binding site overlapped the promoter of afsS, as determined
by DNase I protection assay and high‐resolution S1 nuclease mapping. The nucleotide‐binding
domain contributed distinct ATPase and GTPase activity. The phosphorylation of AfsR
by AfsK greatly enhanced the DNA‐binding activity and modulated the ATPase activity.
The DNA‐binding ability of AfsR was independent of the ATPase activity. However,
the ATPase activity was essential for transcriptional activation of afsS, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. Thus, AfsR turns out to be a unique transcriptional factor, in that it is modular, in which DNA‐binding and ATPase activities are physically separable, and the two functions are modulated by phosphorylation on serine and threonine residues.