bluuebunny/crossref_metadata_2025 huggingface dataset:
First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non‐motile mutants deficient in endoflagella
Authors: Picardeau Mathieu , Brenot Audrey , Saint Girons Isabelle
Published: March 2003

Leptospira spp. offer many advantages as model bacteria for the study of spirochaetes. However, homologous recombination between introduced DNA and the corresponding chromosomal loci has never been demonstrated. A unique feature of spirochaetes is the presence of endoflagella between the outer membrane sheath and the cell cylinder. We chose the flaB flagellin gene, constituting the flagellar core, as a target for gene inactivation in the saprophyte Leptospira biflexa. The amino acid sequence of the FlaB protein of L. biflexa was most similar to those of spirochaetes Brachyspira hyodysenteriae (agent of swine dysentery), Leptospira interrogans (agent of leptospirosis) and Treponema pallidum (agent of syphilis). A suicide vector containing the L. biflexa flaB gene disrupted by a kanamycin marker was UV irradiated or alkali denatured before electroporation. This methodology allowed the selection of many kanamycin‐resistant colonies resulting from single and double cross‐over events at the flaB locus. The double recombinant mutants are non‐motile, as visualized in both liquid and semi‐solid media. In addition, a flaB mutant selected for further analysis was shown to be deficient in endoflagella by electron microscopy. However, most of the transformants had resulted from a single homologous recombination event, giving rise to the integration of the suicide vector. We evaluated the effect of the sacB and rpsL genes in L. biflexa as potential counterselectable markers for allelic exchange, and then used the rpsL system for the positive selection of flaB double recombinants in a streptomycin‐resistant strain. Like the flaB mutant studied above, the Strr double cross‐over mutant was non‐motile and deficient in endoflagella. Our results demonstrate that FlaB is involved in flagella assembly and motility. They also show the feasibility of performing allelic replacement in Leptospira spp. by homologous recombination.
Positive regulation of motility and flhDC expression by the RNA‐binding protein CsrA of Escherichia coli
Authors: Wei Bangdong L. , Brun‐Zinkernagel Anne‐Marie , Simecka Jerry W. , Prüß Birgit M. , Babitzke Paul , Romeo Tony
Published: March 2003

Many species of bacteria devote considerable metabolic resources and genetic information to the ability to sense the environment and move towards or away from specific stimuli using flagella. In Escherichia coli and related species, motility is regulated by several global regulatory circuits, which converge to modulate the overall expression of the master operon for flagellum biosynthesis, flhDC. We now show that the global regulator CsrA of E. coli K‐12 is necessary for motility under a variety of growth conditions, as a result of its role as an activator of flhDC expression. A chromosomally encoded flhDC′–′lacZ translational fusion was expressed at three‐ to fourfold higher levels in csrA wild‐type strains than in isogenic csrA mutants. Purified recombinant CsrA protein stimulated the coupled transcription‐translation of flhDC′–′ lacZ in S‐30 extracts and bound to the 5′ segment of flhDC mRNA in RNA mobility shift assays. The steady‐state level of flhDC mRNA was higher and its half‐life was ≈ threefold greater in a csrA wild‐type versus a csrA mutant strain. Thus, CsrA stimulates flhDC gene expression by a post‐transcriptional mechanism reminiscent of its function in the repression of glycogen biosynthesis.
Biological cost and compensatory evolution in fusidic acid‐resistant Staphylococcus aureus
Authors: Nagaev Ivan , Björkman Johanna , Andersson Dan I. , Hughes Diarmaid
Published: March 2003

Fusidic acid resistance resulting from mutations in elongation factor G (EF‐G) of Staphylococcus aureus is associated with fitness costs during growth in vivo and in vitro. In both environments, these costs can be partly or fully compensated by the acquisition of secondary intragenic mutations. Among clinical isolates of S. aureus, fusidic acid‐resistant strains have been identified that carry multiple mutations in EF‐G at positions similar to those shown experimentally to cause resistance and fitness compensation. This observation suggests that fitness‐compensatory mutations may be an important aspect of the evolution of antibiotic resistance in the clinical environment, and may contribute to a stabilization of the resistant bacteria present in a bacterial population.
Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase‐encoding gene, MAN1, and its impact on pathogenicity
Authors: Wills Elizabeth A. , Roberts Ian S. , Del Poeta Maurizio , Rivera Johanna , Casadevall Arturo , Cox Gary M. , Perfect John R.
Published: March 2003

The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP‐mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP‐mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild‐type and reconstituted strains were significantly more virulent than the knock‐out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.
Heparin‐binding outer membrane protein of chlamydiae
Authors: Stephens Richard S. , Koshiyama Kelli , Lewis Elizabeth , Kubo Aya
Published: March 2003

As an intracellular pathogen, the mechanism by which Chlamydia invade eukaryotic cells represents a cornerstone to understanding chlamydial biology. The ability of chlamydiae specifically to bind heparan sulphate or heparin and the association of this ability to bind and enter mammalian host cells was approached by searching experimentally for chlamydial outer membrane proteins that bind heparin. The 60 000 molecular weight cysteine‐rich outer membrane complex protein, OmcB, bound heparin. The ability of OmcB to bind heparin was supported by mapping the region of the protein with heparin‐binding capacity and demonstrating that an OmcB synthetic 20‐mer peptide from this region specifically bound heparin. Surface localization of OmcB was shown using monospecific antisera specific to the 20‐mer OmcB peptide that bound the surfaces of elementary bodies (EB) and by heparin‐binding peptide cross‐linking of EB surface proteins.
Repression of virulence genes by phosphorylation‐dependent oligomerization ofCsrR at target promoters in S. pyogenes
Authors: Miller Alita A. , Engleberg N. Cary , DiRita Victor J.
Published: March 2003

csrRS encodes a two‐component regulatory system that represses the transcription of a number of virulence factors in Streptococcus pyogenes, including the hyaluronic acid capsule and pyrogenic exotoxin B. CsrRS‐regulated virulence factors have diverse functions during pathogenesis and are differentially expressed throughout growth. This suggests that multiple signals induce CsrRS‐mediated gene regulation, or that regulated genes respond differently to CsrR, or both. As a first step in dissecting the csrRS signal transduction pathway, we determined the mechanism by which CsrR mediates the repression of its target promoters. We found that phosphorylated CsrR binds directly to all but one of the promoters of its regulated genes, with different affinities. Phosphorylation of CsrR enhances both oligomerization and DNA binding. We defined the binding site of CsrR at each of the regulated promoters using DNase I and hydroxyl radical footprinting. Based on these results, we propose a model for differential regulation by CsrRS.
Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity
Authors: Tseng Hsing‐Ju , Srikhanta Yogitha , McEwan Alastair G. , Jennings Michael P.
Published: March 2003

As a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain, Neisseria gonorrhoeae requires defence systems to respond to toxic oxygen species such as superoxide. It has been shown that supplementation of media with 100 µM Mn(II) considerably enhanced the resistance of this bacterium to oxidative killing by superoxide. This protection was not associated with the superoxide dismutase enzymes of N. gonorrhoeae. In contrast to previous studies, which suggested that some strains of N. gonorrhoeae might not contain a superoxide dismutase, we identified a sodB gene by genome analysis and confirmed its presence in all strains examined by Southern blotting, but found no evidence for sodA or sodC. A sodB mutant showed very similar susceptibility to superoxide killing to that of wild‐type cells, indicating that the Fe‐dependent SOD B did not have a major role in resistance to oxidative killing under the conditions tested. The absence of a sodA gene indicated that the Mn‐dependent protection against oxidative killing was independent of Mn‐dependent SOD A. As a sodB mutant also showed Mn‐dependent resistance to oxidative killing, then it is concluded that this resistance is independent of superoxide dismutase enzymes. Resistance to oxidative killing was correlated with accumulation of Mn(II) by the bacterium. We hypothesize that this bacterium uses Mn(II) as a chemical quenching agent in a similar way to the already established process in Lactobacillus plantarum. A search for putative Mn(II) uptake systems identified an ABC cassette‐type system (MntABC) with a periplasmic‐binding protein (MntC). An mntC mutant was shown to have lowered accumulation of Mn(II) and was also highly susceptible to oxidative killing, even in the presence of added Mn(II). Taken together, these data show that N. gonorrhoeae possesses a Mn(II) uptake system that is critical for resistance to oxidative stress.
Deletion of a novel protein kinase with PX and FYVE‐related domains increases the rate of differentiation of Trypanosoma brucei
Authors: Vassella Erik , Krämer Robert , Turner C. Michael R. , Wankell Miriam , Modes Christine , Van Den Bogaard Miriam , Boshart Michael
Published: March 2003

Growth control of African trypanosomes in the mammalian host is coupled to differentiation of a non‐dividing life cycle stage, the stumpy bloodstream form. We show that a protein kinase with novel domain architecture is important for growth regulation. Zinc finger kinase (ZFK) has a kinase domain related to RAC and S6 kinases flanked by a FYVE‐related zinc finger and a phox (PX) homology domain. To investigate the function of the kinase during cyclical development, a stable transformation procedure for bloodstream forms of differentiation‐competent (pleomorphic) Trypanosoma brucei strains was established. Deletion of both allelic copies of ZFK by homologous recombination resulted in reduced growth of bloodstream‐form parasites in culture, which was correlated with an increased rate of differentiation to the non‐dividing stumpy form. Growth and differentiation rates were returned to wild‐type level by ectopic ZFK expression. The phenotype is stage‐specific, as growth of procyclic (insect form) trypanosomes was unaffected, and Δzfk/Δzfk clones were able to undergo full cyclical development in the tsetse fly vector. Deletion of ZFK in a differentiation‐defective (monomorphic) strain of T. brucei did not change its growth rate in the bloodstream stage. This suggests a function of ZFK associated with the trypanosomes' decision between either cell cycle progression, as slender bloodstream form, or differentiation to the non‐dividing stumpy form.
Bacterial DNA ligases
Authors: Wilkinson Adam , Day Jonathan , Bowater Richard
Published: March 2003

DNA ligases join breaks in the phosphodiester backbone of DNA molecules and are used in many essential reactions within the cell. All DNA ligases follow the same reaction mechanism, but they may use either ATP or NAD+ as a cofactor. All Bacteria (eubacteria) contain NAD+‐dependent DNA ligases, and the uniqueness of these enzymes to Bacteria makes them an attractive target for novel antibiotics. In addition to their NAD+‐dependent enzymes, some Bacteria contain genes for putative ATP‐dependent DNA ligases. The requirement for these different isozymes in Bacteria is unknown, but may be related to their utilization in different aspects of DNA metabolism. The putative ATP‐dependent DNA ligases found in Bacteria are most closely related to proteins from Archaea and viruses. Phylogenetic analysis suggests that all NAD+‐dependent DNA ligases are closely related, but the ATP‐dependent enzymes have been acquired by Bacterial genomes on a number of separate occasions.
Involvement of N‐acetylmuramyl‐l‐alanine amidases in cell separation and antibiotic‐induced autolysis of Escherichia coli
Authors: Heidrich Christoph , Templin Markus F. , Ursinus Astrid , Merdanovic Melisa , Berger Jürgen , Schwarz Heinz , De Pedro Miguel A. , Höltje Joachim‐Volker
Published: March 2003

N‐acetylmuramyl‐l‐alanine amidases are widely distributed among bacteria. However, in Escherichia coli, only one periplasmic amidase has been described until now, which is suggested to play a role in murein recycling. Here, we report that three amidases, named AmiA, B and C, exist in E. coli and that they are involved in splitting of the murein septum during cell division. Moreover, the amidases were shown to act as powerful autolytic enzymes in the presence of antibiotics. Deletion mutants in amiA, B and C were growing in long chains of unseparated cells and displayed a tolerant response to the normally lytic combination of aztreonam and bulgecin. Isolated murein sacculi of these chain‐forming mutants showed rings of thickened murein at the site of blocked septation. In vitro, these murein ring structures were digested more slowly by muramidases than the surrounding murein. In contrast, when treated with the amidase AmiC or the endopeptidase MepA, the rings disappeared, and gaps developed at these sites in the murein sacculi. These results are taken as evidence that highly stressed murein cross‐bridges are concentrated at the site of blocked cell division, which, when cleaved, result in cracking of the sacculus at this site. As amidase deletion mutants accumulate trimeric and tetrameric cross‐links in their murein, it is suggested that these structures mark the division site before cleavage of the septum.
Cloning of the genes for a 4‐sulphocatechol‐oxidizing protocatechuate 3,4‐dioxygenase from Hydrogenophaga intermedia S1 and identification of the amino acid residues responsible for the ability to convert 4‐sulphocatechol
Authors: Contzen Matthias , Bürger Sibylle , Stolz Andreas
Published: March 2003

The genes for a protocatechuate 3,4‐dioxygenase (P34O‐II) with the ability to oxidize 4‐sulphocatechol were cloned from the 4‐aminobenzenesulphonate(sulphanilate)‐degrading bacterium Hydrogenophaga intermedia strain S1 (DSMZ 5680). Sequence comparisons of the deduced amino acid sequences of both subunits of the P34O‐II from H. intermedia S1 (PcaH‐II and PcaG‐II) with those of another P34O‐II, previously obtained from Agrobacterium radiobacter S2, and the corresponding sequences from the protocatechuate 3,4‐dioxygenases from other bacterial genera demonstrated that seven amino acid residues, which were conserved in all previously known P34Os (P34O‐Is), were different in both P34O‐IIs. According to previously published structural data for the P34O of Pseudomonas putidaonly two of these amino acid residues were located near the catalytical centre. The respective amino acid residues were mutated in the P34O‐I from A. radiobacter S2 by site‐specific mutagenesis, and it was found that a single amino acid exchange enabled the protocatechuate converting P34O also to oxidize 4‐sulphocatechol.
Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon
Authors: Cheng Qi , Sutanto Yuri , Shoemaker Nadja B. , Gardner Jeffrey F. , Salyers Abigail A.
Published: March 2003

Integrated self‐transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram‐positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini‐element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.
Structure, activity and evolution of the group I thiolactone peptide quorum‐sensing system ofStaphylococcus aureus
Authors: MDowell Philip , Affas Zina , Reynolds Caroline , Holden Matthew T. G. , Wood Stewart J. , Saint Sandra , Cockayne Alan , Hill Philip J. , Dodd Christine E. R. , Bycroft Barrie W. , Chan Weng C. , Williams Paul
Published: March 2003

InStaphylococcus aureus, theagrlocus is responsible for controlling virulence gene expression via quorum sensing. As the blockade of quorum sensing offers a novel strategy for attenuating infection, we sought to gain novel insights into the structure, activity and turnover of the secreted staphylococcal autoinducing peptide (AIP) signal molecules. A series of analogues (including thel‐alanine andd‐amino acid scanned peptides) was synthesized to determine the functionally critical residues within theS. aureusgroup I AIP. As a consequence, we established that (i) the group I AIP is inactivated in culture supernatants by the formation of the corresponding methionyl sulphoxide; and (ii) the group I AIP lactam analogue retains the capacity to activateagr, suggesting that covalent modification of the AgrC receptor is not a necessary prerequisite foragractivation. Although each of thed‐amino acid scanned AIP analogues retained activity, replacement of the endocyclic amino acid residue (aspartate) located C‐terminally to the central cysteine with alanine converted the group I AIP from an activator to a potent inhibitor. The screening of clinicalS. aureusisolates for novel AIP groups revealed a variant that differed from the group I AIP by a single amino acid residue (aspartate to tyrosine) in the same position defined as critical by alanine scanning. Although this AIP inhibits group IS. aureusstrains, the producer strains possess a functionalagrlocus dependent on the endogenous peptide and, as such, constitute a fourthS. aureusAIP pheromone group (group IV). The addition of exogenous synthetic AIPs toS. aureusinhibited the production of toxic shock syndrome toxin (TSST‐1) and enterotoxin C3, confirming the potential of quorum‐sensing blockade as a therapeutic strategy.
Lre1 affects chitinase expression, trehalose accumulation and heat resistance through inhibition of the Cbk1 protein kinase in Saccharomyces cerevisiae
Authors: Versele Matthias , Thevelein Johan M.
Published: March 2003

The addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae triggers activation of the cAMP pathway with a rapid drop in stress resistance as a consequence. We have isolated the LRE1 gene as a multicopy suppressor of glucose‐induced loss of heat resistance. Overexpression of LRE1 in a wild‐type strain causes the same phenotype as observed in strains with reduced activity of the cAMP–PKA pathway: higher heat resistance and enhanced trehalose levels. Deletion of LRE1 results in the opposite phenotypes. Epistasis analysis indicates that these effects are independent of cAMP and PKA, of the protein kinases Yak1, Sch9 and Rim15 and of the transcription factors Msn2 and Msn4. Lre1 has recently been isolated in a two‐hybrid screen using the conserved protein kinase Cbk1 as a bait. Cbk1 controls the expression of CTS1 (encoding chitinase) through the transcription factor Ace2. We demonstrate here that overexpression of LRE1 represses CTS1 whereas deletion of LRE1 induces the expression of CTS1. Repression of CTS1 results in deficient cell separation as a result of inefficient degradation of the chitin ring after cytokinesis. Neither deletion nor overexpression of LRE1 has any effect on CTS1 expression in a cbk1Δ mutant, indicating that Lre1 inhibits Cbk1. In addition, we show that increased trehalose accumulation and increased heat resistance caused by overexpression of LRE1 are also the result of inhibition of Cbk1, revealing a novel control pathway for certain targets affected by PKA. The yeast genome contains a homologue of LRE1, YDR528w, which we have called HLR1 (for homologue of Lre1). Deletion and overexpression of HLR1 causes similar but less pronounced effects compared with LRE1.
The pavA gene of Streptococcus pneumoniae encodes a fibronectin‐binding protein that is essential for virulence
Authors: Holmes Ann R. , McNab Roderick , Millsap Kevin W. , Rohde Manfred , Hammerschmidt Sven , Mawdsley Jane L. , Jenkinson Howard F.
Published: March 2003

Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell‐surface anchorage signals within the primary sequence. Full‐length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid‐phase fibronectin, in a heparin‐sensitive interaction, and blocked binding of wild‐type pneumococcal cells to fibronectin. However, a C‐terminally truncated PavA′ polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2–2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2–2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C‐terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell‐surface physico‐chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104‐fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin‐binding protein plays a direct role in the pathogenesis of pneumococcal infections.
The KEX2 gene of Candida glabrata is required for cell surface integrity
Authors: Bader Oliver , Schaller Martin , Klein Sabine , Kukula Janine , Haack Katja , Mühlschlegel Fritz , Korting Hans Christian , Schäfer Wilhelm , Hube Bernhard
Published: March 2003

Candida glabrata has emerged as one of the most common causes of candidosis. In order to identify factors that are necessary for viability and pathogenicity of this fungal pathogen, we analysed the role of the KEX2 gene, which codes for a regulatory endoproteinase that is known to process certain virulence factors in Candida albicans. The KEX2 gene from C. glabrata was cloned and found to have 51% and 62% identity and high structural similarities to the homologous counterparts in C. albicans and Saccharomyces cerevisiae. KEX2 was expressed at all time points investigated during growth in complex medium. In order to investigate the role of this putative regulatory proteinase, Kex2‐deficient mutants were produced. In addition to known kex2 phenotypes, such as pH and calcium hypersensitivity, the mutants grew in cellular aggregates and were found to be hypersensitive to several antifungal drugs that target the cell membrane, including azoles, amorolfine and amphotericin B. Ultrastructural investigation after exposure to low doses of itraconazole showed azole‐specific alterations such as enlarged vacuoles and proliferation of the cytoplasmatic membrane in the kex2 mutants, but not in the control strains. In contrast, antifungals such as 5‐flucytosine and hydroxypyridones inhibited growth of the kex2 mutants and the control strains to the same extent. In an in vitro model of oral candidosis, kex2 mutants showed reduced tissue damage in the presence of itraconazole compared with the control infections. These data suggest that Kex2 is involved in the processing of proteins that are essential for cell surface integrity of C. glabrata.
Accumulation of metal‐binding peptides in fission yeast requires hmt2+
Authors: Vande Weghe Jennifer G. , Ow David W.
Published: March 2003

The fission yeast Schizosaccharomyces pombe detoxifies cadmium by synthesizing phytochelatins, peptides of the structure (γ‐GluCys)nGly, which bind cadmium and mediate its sequestration into the vacuole. The fission yeast protein HMT2, a mitochondrial enzyme that can oxidize sulphide, appears to be essential for tolerance to multiple forms of stress, including exposure to cadmium. We found that the hmt2– mutant is unable to accumulate normal levels of phytochelatins in response to cadmium, although the cells possess a phytochelatin synthase that is active in vitro. Radioactive pulse–chase experiments demonstrated that the defect lies in two steps: the synthesis of phytochelations and the upregulation of glutathione production. Phytochelatins, once formed, are stable. hmt2– cells accumulate high levels of sulphide and, when exposed to cadmium, display bright fluorescent bodies consistent with cadmium sulphide. We propose that the precipitation of free cadmium blocks phytochelatin synthesis in vivo, by preventing upregulation of glutathione production and formation of the cadmium–glutathione thiolate required as a substrate by phytochelatin synthase. Thus, although sulphide is required for phytochelatin‐mediated metal tolerance, aberrantly high sulphide levels can inhibit this pathway. Precise regulation of sulphur metabolism, mediated in part by HMT2, is essential for metal tolerance in fission yeast.
The role of the yeast plasma membrane SPS nutrient sensor in the metabolic response to extracellular amino acids
Authors: Forsberg Hanna , Gilstring C. Fredrik , Zargari Arezou , Martínez Paula , Ljungdahl Per O.
Published: March 2003

In response to discrete environmental cues, Saccharomyces cerevisiae cells adjust patterns of gene expression and protein activity to optimize metabolism. Nutrient‐sensing systems situated in the plasma membrane (PM) of yeast have only recently been discovered. Ssy1p is one of three identified components of the Ssy1p–Ptr3p–Ssy5 (SPS) sensor of extracellular amino acids. SPS sensor‐initiated signals are known to modulate the expression of a number of amino acid and peptide transporter genes (i.e. AGP1, BAP2, BAP3, DIP5, GAP1, GNP1, TAT1, TAT2 and PTR2) and arginase (CAR1). To obtain a better understanding of how cells adjust metabolism in response to extracellular amino acids in the environment and to assess the consequences of loss of amino acid sensor function, we investigated the effects of leucine addition to wild‐type and ssy1 null mutant cells using genome‐wide transcription profile analysis. Our results indicate that the previously identified genes represent only a subset of the full spectrum of Ssy1p‐dependent genes. The expression of several genes encoding enzymes in amino acid biosynthetic pathways, including the branched‐chain, lysine and arginine, and the sulphur amino acid biosynthetic pathways, are modulated by Ssy1p. Additionally, the proper transcription of several nitrogen‐regulated genes, including NIL1 and DAL80, encoding well‐studied GATA transcription factors, is dependent upon Ssy1p. Finally, several genes were identified that require Ssy1p for wild‐type expression independently of amino acid addition. These findings demonstrate that yeast cells require the SPS amino acid sensor component, Ssy1p, to adjust diverse cellular metabolic processes properly.
FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co‐localization with FtsZ during Escherichia coli cell division
Authors: Chen Joseph C. , Beckwith Jon
Published: March 2003

During cell division in Gram‐negative bacteria, the cell envelope invaginates and constricts at the septum, eventually severing the cell into two compartments, and separating the replicated genetic materials. In Escherichia coli, at least nine essential gene products participate directly in septum formation: FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ and ZipA. All nine proteins have been localized to the septal ring, an equatorial ring structure at the division site. We used translational fusions to green fluorescent protein (GFP) to demonstrate that FtsQ, FtsL and FtsI localize to potential division sites in filamentous cells depleted of FtsN, but not in those depleted of FtsK. We also constructed translational fusions of FtsZ, FtsA, FtsQ, FtsL and FtsI to enhanced cyan or yellow fluorescent protein (ECFP or EYFP respectively), GFP variants with different fluorescence spectra. Examination of cells expressing different combinations of the fusions indicated that FtsA, FtsQ, FtsL and FtsI co‐localize with FtsZ in filaments depleted of FtsN. These localization results support the model that E. coli cell division proteins assemble sequentially as a multimeric complex at the division site: first FtsZ, then FtsA and ZipA independently of each other, followed successively by FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN.
A three‐protein inhibitor of polar septation during sporulation in Bacillus subtilis
Authors: Eichenberger Patrick , Fawcett Paul , Losick Richard
Published: March 2003

We present evidence for a three‐protein inhibitor of polar division that locks in asymmetry after the formation of a polar septum during sporulation in Bacillus subtilis. Asymmetric division involves the formation of cytokinetic Z‐rings near both poles of the developing cell. Next, a septum is formed at one of the two polar Z‐rings, thereby generating a small, forespore cell and a mother cell. Gene expression under the control of the mother‐cell transcription factor σE is needed to block cytokinesis at the pole distal to the newly formed septum. We report that this block in polar cytokinesis is mediated partly by σE‐directed transcription of spoIID, spoIIM and spoIIP, sporulation genes that were known to be involved in the subsequent process of forespore engulfment. We find that a spoIID, spoIIM and spoIIP triple mutant substantially mimicked the bipolar division phenotype of a σE mutant and that cells engineered to produce SpoIID, SpoIIM and SpoIIP prematurely were inhibited in septum formation at both poles. Consistent with the hypothesis that SpoIID, SpoIIM and SpoIIP function at both poles of the sporangium, a GFP–SpoIIM fusion localized to the membrane that surrounds the engulfed forespore and to the potential division site at the distal pole.
Haem utilization in Vibrio cholerae involves multiple TonB‐dependent haem receptors
Authors: Mey Alexandra R. , Payne Shelley M.
Published: March 2003

Vibrio cholerae has multiple iron transport systems, one of which involves haem uptake through the outer membrane receptor HutA. A hutA mutant had only a slight defect in growth using haemin as the iron source, and we show here that V. cholerae encodes two additional TonB‐dependent haem receptors, HutR and HasR. HutR has significant homology to HutA as well as to other outer membrane haem receptors. Membrane fractionation confirmed that HutR is present in the outer membrane. The hutR gene was co‐transcribed with the upstream gene ptrB, and expression from the ptrB promoter was negatively regulated by iron. A hutA, hutR mutant was significantly impaired, but not completely defective, in the ability to use haemin as the sole iron source. HasR is most similar to the haemophore‐utilizing haem receptors from Pseudomonas aeruginosa and Serratia marcescens. A mutant defective in all three haem receptors was unable to use haemin as an iron source. HutA and HutR functioned with either V. cholerae TonB1 or TonB2, but haemin transport through either receptor was more efficient in strains carrying the tonB1 system genes. In contrast, haemin uptake through HasR was TonB2 dependent. Efficient utilization of haemoglobin as an iron source required HutA and TonB1. The triple haem receptor mutant exhibited no defect in its ability to compete with its Vib– parental strain in an infant mouse model of infection, indicating that additional iron sources are present in vivo. V. cholerae used haem derived from marine invertebrate haemoglobins, suggesting that haem may be available to V. cholerae growing in the marine environment.
Fate of germinated Bacillus anthracis spores in primary murine macrophages
Authors: Guidi‐Rontani Chantal , Levy Martine , Ohayon Helene , Mock Michele
Published: March 2003

We investigated the fate of germinated Bacillus anthracis spores after their germination in Swiss murine peritoneal macrophages and in the cell line RAW264.7. We found that the lethal toxin and the oedema toxin are germ‐associated factors that are essential for the survival of the vegetative form in host cells. We also found that pX02 is not involved in this complex pathogenic process. By transmission electron microscopy, we showed the tight interaction between the exosporium of the spore and the phagosomal membrane of the macrophage. Our data strongly suggest that the B. anthracis toxinogenic, unencapsulated Sterne strain (7702) does not multiply within macrophages. These results contributed to reveal the strategies used by B. anthracis to survive within the host and to reach the external medium where they proliferate.
A molecular mechanism for the repression of transcription by the H‐NS protein
Authors: Rimsky Sylvie , Zuber Florent , Buckle Malcolm , Buc Henri
Published: March 2003

The H‐NS protein is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Although H‐NS does not exhibit a high DNA sequence specificity, a number of H‐NS‐responsive promoters have been shown to contain regions of intrinsic DNA curvature located either upstream or downstream of the transcription start point. We have studied H‐NS binding to DNA and in vitro transcriptional regulation by H‐NS at several synthetic promoters with or without curved sequences inserted upstream of the Pribnow box. We show how such inserts determine the final organization of H‐NS‐containing nucleoprotein complexes and how this affects transcription. We refine a two‐step mechanism for the constitution of H‐NS assemblies that are efficient in regulation.
A two‐component signal transduction system involved in nickel sensing in the cyanobacterium Synechocystis sp. PCC 6803
Authors: López‐Maury Luis , García‐Domínguez Mario , Florencio Francisco J. , Reyes José C.
Published: March 2003

In the cyanobacterium Synechocystis sp. PCC 6803, genes for Ni2+, Co2+, and Zn2+ resistance are grouped in a 12 kb gene cluster. The nrsBACD operon is composed of four genes, which encode proteins involved in Ni2+ resistance. Upstream from nrsBACD, and in opposite orientation, a transcription unit formed by the two genes rppA and rppB has been reported previously to encode a two‐component signal transduction system involved in redox sensing. In this report, we demonstrate that rppA and rppB (here redesigned nrsR and nrsS respectively) control the Ni2+‐dependent induction of the nrsBACD operon and are involved in Ni2+ sensing. Thus, expression of the nrsBACD operon was not induced by Ni2+ in a nrsRS mutant strain. Furthermore, nrsRS mutant cells showed reduced tolerance to Ni2+. Whereas the nrsBACD operon is transcribed from two different promoters, one constitutive and the other dependent on the presence of Ni2+ in the medium, the nrsRS operon is transcribed from a single Ni2+‐inducible promoter. The nrsRS promoter is silent in a nrsRS mutant background suggesting that the system is autoregulated. Purified full length NrsR protein is unable to bind to the nrsBACD‐nrsRS intergenic region; however, an amino‐terminal truncated protein that contains the DNA binding domain of NrsR binds specifically to this region. Our nrsRS mutant, which carries a deletion of most of the nrsR gene and part of the nrsS gene, does not show redox imbalance or photosynthetic gene mis‐expression, contrasting with the previously reported nrsR mutant.
Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase
Authors: Makinoshima Hideki , Nishimura Akiko , Ishihama Akira
Published: March 2003

Cultures of Escherichia coli could be separated into more than 15 cell populations, each forming a discrete band after Percoll gradient centrifugation. The cell separation was found to result from the difference in buoyant density but not the size difference. The cell density increases upon transition from exponential growth to stationary phase. Exponential phase cultures formed at least five discrete bands with lower densities, whereas stationary phase cultures formed more than 10 bands with higher densities. Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative immunoblotting. These findings together suggest that the growth phase‐coupled transition of E. coli phenotype is discontinuous.
A cytoplasmic coiled‐coil domain is required for histidine kinase activity of the yeast osmosensor, SLN1
Authors: Tao Wei , Malone Cheryl L. , Ault Addison D. , Deschenes Robert J. , Fassler Jan S.
Published: March 2003

The yeast histidine kinase, Sln1p, is a plasma membrane‐associated osmosensor that regulates the activity of the osmotic stress MAP kinase pathway. Changes in the osmotic environment of the cell influence the autokinase activity of the cytoplasmic kinase domain of Sln1p. Neither the nature of the stimulus, the mechanism by which the osmotic signal is transduced nor the manner in which the kinase is regulated is currently clear. We have identified several mutations located in the linker region of the Sln1 kinase (just upstream of the kinase domain) that cause hyperactivity of the Sln1 kinase. This region of histidine kinases is largely uncharacterized, but its location between the transmembrane domains and the cytoplasmic kinase domain suggests that it may have a potential role in signal transduction. In this study, we have investigated the Sln1 linker region in order to understand its function in signal transduction and regulation of Sln1 kinase activity. Our results indicate that the linker region forms a coiled‐coil structure and suggest a mechanism by which alterations induced by osmotic stress influence kinase activity by altering the alignment of the phospho‐accepting histidine with respect to the catalytic domain of the kinase.
Identification of N‐acetylgalactosamine‐containing glycoproteins PEB3 and CgpA in Campylobacter jejuni
Authors: Linton Dennis , Allan Elaine , Karlyshev Andrey V. , Cronshaw Andrew D. , Wren Brendan W.
Published: March 2003

It was demonstrated recently that there is a system of general protein glycosylation in the human enteropathogen Campylobacter jejuni. To char‐ acterize such glycoproteins, we identified a lectin, Soybean agglutinin (SBA), which binds to multiple C. jejuni proteins on Western blots. Binding of lectin SBA was disrupted by mutagenesis of genes within the previously identified protein glycosylation locus. This lectin was used to purify putative glycoproteins selectively and, after sodium dodecyl sulphate‐ polyacrylamide gel electrophoresis (SDS–PAGE), Coomassie‐stained bands were cut from the gels. The bands were digested with trypsin, and peptides were identified by mass spectrometry and database searching. A 28 kDa band was identified as PEB3, a previously characterized immunogenic cell surface protein. Bands of 32 and 34 kDa were both identified as a putative periplasmic protein encoded by the C. jejuni NCTC 11168 coding sequence Cj1670c. We have named this putative glycoprotein CgpA. We constructed insertional knockout mutants of both the peb3 and cgpA genes, and surface protein extracts from mutant and wild‐type strains were analysed by one‐ and two‐dimensional polyacrylamide gel electrophoresis (PAGE). In this way, we were able to identify the PEB3 protein as a 28 kDa SBA‐reactive and immunoreactive glycoprotein. The cgpA gene encoded SBA‐reactive and immunoreactive proteins of 32 and 34 kDa. By using specific exoglycosidases, we demonstrated that the SBA binding property of acid‐glycine extractable C. jejuni glycoproteins, including PEB3 and CgpA, is a result of the presence of α‐linked N‐acetylgalactosamine residues. These data confirm the existence, and extend the boundaries, of the previously identified protein glycosylation locus of C. jejuni. Furthermore, we have identified two such glycoproteins, the first non‐flagellin campylobacter glycoproteins to be identified, and demonstrated that their glycan components contain α‐linked N‐acetylgalactosamine residues.
ActP controls copper homeostasis in Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti preventing low pH‐induced copper toxicity
Authors: Reeve Wayne G. , Tiwari Ravi P. , Kale Neetin B. , Dilworth Michael J. , Glenn Andrew R.
Published: March 2003

Two ‘calcium‐irreparable’ acid‐sensitive mutants were identified after mutagenizing Rhizobium leguminosarum bv. viciae and Sinorhizobium
meliloti with Tn5. Each mutant contains a single copy of the transposon
which, inserted within the actP gene, prevents expression of a P‐type ATPase
that belongs to the CPx heavy metal‐transporting subfamily. Here, we show that both
actP‐knockout mutants show sensitivity to copper; omission of this heavy metal
from low pH‐buffered media restores acid tolerance to these strains. Furthermore,
complementation of the mutant phenotype requires only the actP gene. An actP–gusA
fusion in R. leguminosarum was transcriptionally regulated by copper in a
pH‐dependent manner. Downstream to actP in both organisms is the hmrR
gene that encodes a heavy metal‐responsive regulator
(HmrR) that belongs to the merR class of regulatory genes. Insertional inactivation
of hmrR abolished transcriptional activation of actP by copper ions
and increased the basal level of its expression in their absence. These observations
suggest that HmrR can regulate actP transcription positively and negatively. We show that copper homeostasis is an essential mechanism for the acid tolerance of these root nodule bacteria since it prevents this heavy metal from becoming overtly toxic in acidic conditions.
Spa15 of Shigella flexneri, a third type of chaperone in the type III secretion pathway
Authors: Page Anne‐Laure , Sansonetti Philippe , Parsot Claude
Published: March 2003

The type III secretion (TTS) pathway is used by numerous Gram‐negative pathogens to inject virulence factors into eukaryotic cells. In addition to a functional TTS apparatus, secretion of effector proteins depends upon specific chaperones. Using a two‐hybrid screen in yeast and a co‐purification assay in Shigella flexneri, we demonstrated that Spa15, which is encoded by an operon for components of the TTS apparatus, is associated in the cytoplasm with three proteins that are secreted by the TTS pathway, IpaA, IpgB1 and OspC3. Spa15 was found to be necessary for stability of IpgB1 but not IpaA, and for secretion of IpaA molecules that were stored in the cytoplasm but not those that were synthesized while the secretion apparatus was active. The ability of Spa15 to associate with several non‐homologous secreted proteins, the presence of Spa15 homologues in other TTS systems and the location of the corresponding genes within operons for components of the TTS apparatus suggest that Spa15 belongs to a new class of TTS chaperones.
Enhanced bacterial virulence through exploitation of host glycosaminoglycans
Authors: Menozzi Franco D. , Pethe Kevin , Bifani Pablo , Soncin Fabrice , Brennan Michael J. , Locht Camille
Published: March 2003

Present in the extracellular matrix and membranes of virtually all animal cells, proteoglycans (PGs) are among the first host macromolecules encountered by infectious agents. Because of their wide distribution and direct accessibility, it is not surprising that pathogenic bacteria have evolved mechanisms to exploit PGs for their own purposes, including mediating attachment to target cells. This is achieved through the expression of adhesins that recognize glycosa‐minoglycans (GAGs) linked to the core protein of PGs. Some pathogens, such as Bordetella pertussis and Chlamydia trachomatis,
may express more than one GAG‐binding adhesin. Bacterial interactions with PGs may
also facilitate cell invasion or systemic dissemination, as observed for Neisseria
gonorrhoeae and Mycobacterium tuberculosis respectively. Moreover, pathogenic bacteria can use PGs to enhance their virulence via a shedding of PGs that leads to the release of effectors that weaken the host defences. The exploitation of PGs by pathogenic bacteria is thus a multifaceted mechanistic process directly related to the potential virulence of a number of microorganisms.