bluuebunny/crossref_metadata_2025 huggingface dataset:
ClpP participates in the degradation of misfolded protein in Lactococcus lactis
Authors: Frees Dorte , Ingmer Hanne
Published: March 2003

ClpP proteins constitute a family of homologous proteins found in both prokaryotic and eukaryotic organisms. In Escherichia coli, ClpP is the proteolytic component of a large complex also containing either the ClpA or the ClpX ATPases. We show here that the clpP gene from the Gram‐positive bacterium Lactococcus lactis encodes a 22‐kDa protein that is induced by low pH and by the t‐RNA analogue puromycin, which interferes with translation, resulting in the production of misfolded puromycyl‐containing peptides. Northern blot and primer extension analysis showed that clpP expression is also induced by heat shock and that stress induction occurs at the transcriptional level independent of the CIRCE regulatory element often implicated in stress regulation in Gram‐positive bacteria. When we disrupted the L. lactis clpP gene by insertional inactivation, the resulting mutant was more sensitive to both heat and puromycin than wild‐type cells. Furthermore, cells lacking ClpP had a reduced ability to degrade puromycyl‐containing peptides, and they synthesized heat shock proteins constitutively in the absence of stress. Thus, our data suggest that ClpP plays a major role in the degradation of misfolded proteins.
Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S‐layer proteins and the Bacillus subtilis levansucrase
Authors: Mesnage Stéphane , Tosi‐Couture Evelyne , Fouet Agnès
Published: March 2003

Many surface proteins of Gram‐positive bacteria contain motifs, about 50 amino acids long, called S‐layer homology (SLH) motifs. Bacillus anthracis, the causal agent of anthrax, synthesizes two S‐layer proteins, each with three SLH motifs towards the amino‐terminus. We used biochemical and genetic approaches to investigate the involvement of these motifs in cell surface anchoring. Proteinase K digestion produced polypeptides lacking these motifs, and stable three‐motif polypeptides were produced in Escherichia coli that were able to bind the B. anthracis cell walls in vitrodemonstrating that the three SLH motifs were organized into a cell surface anchoring domain. We also determined the function of these SLH domains by constructing chimeric genes encoding the SLH domains fused to the normally secreted levansucrase of Bacillus subtilis. Cell fractionation and electron microscopy studies showed that each three‐motif domain was sufficient for the efficient anchoring of levansucrase onto the cell surface. Proteins consisting of truncated SLH domains fused to levansucrase were unstable and associated poorly with the cell surface. Surface‐exposed levansucrase retained its enzymatic and antigenic properties.
Delivery of protein to the cytosol of macrophages using Escherichia coli K‐12
Authors: Higgins Darren E. , Shastri Nilabh , Portnoy Daniel A.
Published: March 2003

Listeriolysin O (LLO) is an essential determinant of pathogenicity whose natural biological role is to mediate lysis of Listeria monocytogenes containing phagosomes. In this study, we report that Escherichia coli expressing cytoplasmic recombinant LLO can efficiently deliver co‐expressed proteins to the cytosol of macrophages. We propose a model in which subsequent or concomitant to phagocytosis the E. coli are killed and degraded within phagosomes causing the release of LLO and target proteins from the bacteria. LLO acts by forming large pores in the phagosomal membrane, thus releasing the target protein into the cytosol. Delivery was shown to be rapid, within minutes after phagocytosis. Using this method, a large enzymatically active protein was delivered to the cytosol. Furthermore, we demonstrated that the E. coli/LLO system is very efficient for delivery of ovalbumin (OVA) to the major histocompatibility (MHC) class I pathway for antigen processing and presentation, greater than 4 logs compared with E. coli expressing OVA alone. Moreover, the time required for processing and presentation of an OVA‐derived peptide was similar to that previously reported when purified OVA was introduced directly into the cytosol by other methods. Using this system, potentially large amounts of any protein that can be expressed in E. coli can be delivered to the cytosol without protein purification. The potential use of this system for the delivery of antigenic protein in vivo and the delivery of DNA are discussed.
A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly
Authors: Taupiac Marie‐Pierre , Bébien Magali , Alami Mériem , Beaumelle Bruno
Published: March 2003

Pseudomonas exotoxin A (PE) is a cytotoxin composed of three structural domains. Domain I is responsible for cell binding, domain II for membrane translocation enabling access to the cytosol, and domain III for the catalytic inactivation of protein synthesis, which results in cell death. To investigate the role of the six α‐helices (A–F) that form the translocation domain, we deleted them successively one at a time. All mutants showed native cell‐binding and catalytic activities, indicating that deletions specifically affected translocation activity. This step of the intoxication procedure was examined directly using a cell‐free translocation assay, and indirectly by monitoring cytotoxicity. Translocation activity and log(cytotoxicity) were highly correlated, directly indicating that translocation is rate limiting for PE intoxication. Deletion of B, C and D helices resulted in non‐toxic and non‐translocating molecules, whereas mutants lacking the A or E helix displayed significant cytotoxicity albeit 500‐fold lower than native PE. We concluded that B, C and D helices, which make up the core of domain II, are essential, whereas the more peripheral A and E helices are comparatively dispensable. The last helix (F) is inhibitory for translocation because its deletion produced a mutant displaying a translocation activity 60% higher than PE, along with a three‐ to sixfold increase in cytotoxicity in all tested cell lines. This toxin is the most in vitro active PE mutant obtained until now. Finally, partial duplication of domain II did not give rise to a more actively translocated PE, but rather to a threefold less active molecule.
Two operons that encode FNR‐like proteins in Lactococcus lactis
Authors: Gostick Dominic O. , Griffin Hugh G. , Shearman Claire A. , Scott Colin , Green Jeffrey , Gasson Michael J. , Guest John R.
Published: March 2003

Global regulatory circuits of the type mediated by CRP and FNR in Escherichia coli were sought in Lactococcus lactis to provide a basis for redirecting carbon metabolism to specific fermentation products. Using a polymerase chain reaction (PCR) approach, two genes (flpA and flpB) encoding FNR‐like proteins (FlpA and FlpB) with the potential for mediating a dithiol‐disulphide‐dependent regulatory switch, were identified. Transcript analysis indicated that they are distal genes of two paralogous operons, orfX‐orfY‐flp, in which the orfX and orfY genes were predicted to encode binding domain components of cation ATPases and storage proteins respectively. The corresponding promoters were each associated with a potential FNR site (TTGAT—‐ATCAA) at positions + 4.5 (flpA operon) and −42.5 (flpB operon), suggesting that the respective operons might be negatively and positively autoregulated. The incomplete open reading frames (orfWA/B) located upstream of each operon were predicted to encode additional components of paralogous cation ATPases. No phenotypic effects were detected in flpA and flpB single mutants, but the double mutant had a lower intracellular zinc content, an increased sensitivity to hydrogen peroxide and an altered polypeptide profile (as determined by two‐dimensional gel electrophoresis): formate production was not affected. It was concluded tentatively that FlpA and FlpB regulate overlapping modulons, including systems concerned with zinc uptake, in response to metal ion or oxidative stress.
The replication checkpoint control in Bacillus subtilis: identification of a novel RTP‐binding sequence essential for the replication fork arrest after induction of the stringent response
Authors: Autret S. , Levine A. , Vannier F. , Fujita Y. , Séror S. J.
Published: March 2003

We have shown previously that induction of the stringent response in Bacillus subtilis resulted in the arrest of chromosomal replication between 100 and 200 kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively. This replication checkpoint was also shown to involve the RTP protein, normally active at the chromosomal terminus. In this study, we show that the replication block is absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP. DNA–DNA hybridization data indicated that one or more such LSTer sites mapped to gene yxcC (−128 kb from oriC ). A 7.75 kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated. This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with respect to replication, as in the chromosome. This LSTer2 site was further mapped to a 3.65 kb fragment overlapping the next40 probe. Remarkably, this fragment contains a 17 bp sequence (B′‐1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus. This B′‐1 sequence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting. Importantly, precise deletion of this sequence abolished the replication arrest. We propose that this modified B site is an essential constituent of the LSTer2 site. The differences between arrest at the normal chromosomal terminus and arrest at LSTer site are discussed.
Characterization of the BatI (Bacteroides aerotolerance) operon in Bacteroides fragilis: isolation of a B. fragilis mutant with reduced aerotolerance and impaired growth in in vivo model systems
Authors: Tang Yixin P. , Dallas Mary M. , Malamy Michael H.
Published: March 2003

YT135.2.8, a Tn4400′ insertion mutant of Bacteroides fragilis strain TM4000, grows poorly when used to infect Monika or Chinese hamster ovary (CHO) cell monolayers and is outcompeted by wild‐type strains in mixed infections. YT135.2.8 also shows defects in the rat granuloma pouch model system in monoculture and is completely outcompeted by the wild‐type strain in a mixed infection. In addition, this mutant shows defects in a new model system consisting of CHO suspension cell columns. All of these defects may be explained by the finding that YT135.2.8 shows decreased tolerance to exposure to atmospheric oxygen (less aerotolerant). The monolayer growth defect (MGD) of YT135.2.8 can be influenced significantly by the presence of sulphur‐containing reducing agents (cysteine, dithiothreitol, thiodiglycol) or the non‐sulphur reducing agent Tris‐(2‐carboxylethyl)phosphine (TCEP). The defects in YT135.2.8 can be complemented by a 6.6 kb fragment of the B. fragilis chromosome. DNA sequencing of this fragment and of the regions flanking the Tn4400′ insertion in the B. fragilis chromosome revealed the presence of five open reading frames, corresponding to genes bat (Bacteroides aerotolerance) A, B, C, D, E, which form the BatI operon; Tn4400′ inserted within batD. All of the hypothetical proteins possess one or more membrane‐spanning domains. BatA and BatB show high similarity to each other but, like BatD, they show no match to sequences of known function in the databases. BatC and BatE contain 2–4 repeated sequences similar to the tetratricopeptide repeats (TPRs) seen in many eukaryotic proteins. The function of TPR sequences in protein interactions in other systems leads to the suggestion that the Bat proteins form a complex. The BatI complex may be involved in the generation or export of reducing power equivalents to the periplasm of the B. fragilis cell.
The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli
Authors: Gort Amy Strohmeier , Ferber Daniel M. , Imlay James A.
Published: March 2003

The discovery of superoxide dismutase (CuZnSOD) within the periplasms of several Gram‐negative pathogens suggested that this enzyme evolved to protect cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. However, its presence in some non‐pathogenic bacteria implies that there may be a role for this SOD during normal growth conditions. We found that sodC, the gene that encodes the periplasmic SOD of Escherichia coli, is repressed anaerobically by Fnr and is among the many antioxidant genes that are induced in stationary phase by RpoS. Surprisingly, the entry of wild‐type E. coli into stationary phase is accompanied by a several‐hour‐long period of acute sensitivity to hydrogen peroxide. Induction of the RpoS regulon helps to diminish that sensitivity. While mutants of E. coli and Salmonella typhimurium that lacked CuZnSOD were not detectably sensitive to exogenous superoxide, both were killed more rapidly than their parent strains by exogenous hydrogen peroxide in early stationary phase. This sensitivity required prior growth in air. Evidently, periplasmic superoxide is generated during stationary phase by endogenous metabolism and, if it is not scavenged by CuZnSOD, it causes an unknown lesion that augments or accelerates the damage done by peroxide. The molecular details await elucidation.
The interdomain linker of Escherichia coli initiation factor IF3: a possible trigger of translation initiation specificity
Authors: De Cock Eve , Springer Mathias , Dardel Frédéric
Published: March 2003

Initiation factor IF3 is responsible for the accuracy of translation initiation in bacteria, by destabilizing complexes involving non‐initiator tRNA and/or non‐start codons. This proofreading is performed on the 30S subunit to which IF3 binds selectively. IF3 has an unusual architecture, with two globular domains connected by a mobile, positively charged linker. Here, we have investigated the function of this flexible tether by probing its conformation when IF3 is bound to the ribosomal RNA. Using site‐directed mutagenesis of the linker region, we have also selectively modified its length, its flexibility and its chemical composition. The function of the mutant genes was assayed in vivo, and the structural and biochemical properties of some of the corresponding variant proteins were characterized in vitro. The two isolated domains of IF3 were also co‐expressed in order to test the requirement for their covalent attachment. The results indicate that the physical link between the two domains of IF3 is essential for the function of this protein, but that the exact length and chemical composition of the linker can be varied to a large extent. A model is presented in which the extended linker would act as a ‘strap’, triggering a conformational change in the 30S subunit, which would then ensure initiator tRNA selection.
The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface
Authors: Steinhauer Josefa , Agha Rabia , Pham Thao , Varga Andrew W. , Goldberg Marcia B.
Published: March 2003

Shigella flexneri is an intracellular pathogen that is able to move within the cytoplasm of infected cells by the continual assembly of actin onto one pole of the bacterium. IcsA, an outer membrane protein, is localized to the old pole of the bacterium and is both necessary and sufficient for actin assembly. IcsA is slowly cleaved from the bacterial surface by the protease IcsP (SopA). Absence of IcsP leads to an alteration in the distribution of surface IcsA, such that the polar cap is maintained and some IcsA is distributed along the lateral walls of the bacillus. The mechanism of unipolar localization of IcsA and the role of IcsP in its unipolar localization are incompletely understood. Here, we demonstrate that cleavage of IcsA occurs exclusively in the outer membrane and that IcsP is localized to the outer membrane. In addition, we show that IcsA at the old pole is susceptible to cleavage by IcsP and that native IcsP is active at the pole. Taken together, these data indicate that IcsP cleaves IcsA over the entire bacterial surface. Finally, we show that, immediately after induction from a tightly regulated promoter, IcsA is expressed exclusively at the old pole in both the icsP−icsA− and the icsA− background. These data demonstrate that unipolar localization of IcsA results from its direct targeting to the pole, followed by its diffusion laterally in the outer membrane.
The amino‐terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small‐molecular‐weight GTP‐binding proteins
Authors: Pederson Kristin J. , Vallis Amy J. , Aktories Klaus , Frank Dara W. , Barbieri Joseph T.
Published: March 2003

Pseudomonas aeruginosa delivers exoenzyme S (ExoS) into the intracellular compartment of eukaryotic cells via a type III secretion pathway. Intracellular delivery of ExoS is cytotoxic for eukaryotic cells and has been shown to ADP‐ribosylate Ras in vivo and uncouple a Ras‐mediated signal transduction pathway. Functional mapping has localized the FAS‐dependent ADP‐ribosyltransferase domain to the carboxyl‐terminus of ExoS. A transient transfection system was used to examine cellular responses to the amino‐terminal 234 amino acids of ExoS (ΔC234). Intracellular expression of ΔC234 elicited the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose‐dependent manner. Expression of ΔC234 did not inhibit the expression of two independent reporter proteins, GFP and luciferase, or induce trypan blue uptake, which indicated that expression of ΔC234 was not cytotoxic to CHO cells. Carboxyl‐terminal deletion proteins of ΔC234 were less efficient in the elicitation of CHO cell rounding than ΔC234. Cytoskeleton rearrangement elicited by ΔC234 was blocked and reversed by the addition of cytotoxic necrotizing factor 1 (CNF‐1). CNF‐1 catalyses the deamidation of Gln‐63 of members of the Rho subfamily of small‐molecular‐weight GTP‐binding proteins, resulting in protein activation. This implies a role for small‐molecular‐weight GTP‐binding proteins in the disruption of actin by ΔC234. Together, these data identify ExoS as a cytotoxin that possesses two functional domains. Intracellular expression of the amino‐terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl‐terminal domain of ExoS possesses FAS‐dependent ADP‐ribosyltransferase activity and is cytotoxic to eukaryotic cells.
P‐type ATPase spf1 mutants show a novel resistance mechanism for the killer toxin SMKT
Authors: Suzuki Chise , Shimma Yoh‐ichi
Published: March 2003

SMKT, a killer toxin produced by the halotolerant yeast Pichia farinosa KK1, consists of α and β subunits with folding remarkably similar to that of the fungal killer toxin KP4, a Ca2+ channel inhibitor. The budding yeast Saccharomyces cerevisiae is sensitive to SMKT. To understand the killing mechanism of SMKT, we isolated SMKT‐resistant mutants of S. cerevisiae and characterized them. Five spf mutants (sensitivity to the P. farinosa killer toxin) fell into a single genetic complementation group, designated spf1. The SPF1 gene was cloned by complementation of the mutant phenotype. The SPF1 gene encodes a putative P‐type ATPase of 1215 amino acid residues that contains 12 membrane‐spanning regions. Gene disruption revealed that the SPF1 gene is not essential for viability but is required for the sensitivity to SMKT. The spf1 disruptant showed some phenotypes characteristic of glycosylation‐defective mutants and secreted underglycosylated invertase. Fluorescence‐activated cell‐sorting analysis and indirect immunofluorescence microscopy showed that SMKT interacts with the cell surface of the resistant cells but not with that of sensitive cells, suggesting a novel resistance mechanism for this toxin. The glycosylation‐defective phenotype and possible killer‐resistant mechanisms are discussed in comparison with the Golgi Ca2+ pump Pmr1p.
In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K‐12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding
Authors: Andersen Christian , Bachmeyer Christoph , Täuber Harald , Benz Roland , Wang Jiang , Michel Valérie , Newton Salete M. C. , Hofnung Maurice , Charbit Alain
Published: March 2003

The trimeric protein LamB of Escherichia coli K‐12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18‐stranded antiparallel β‐barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single‐, ΔL4, ΔL5, ΔL6, and double‐loop deletions, ΔL4 + ΔL5 and ΔL5 + ΔL6, abolished maltoporin functions, but not the double deletion ΔL4 + ΔL6 and the triple deletion ΔL4 + ΔL5 + ΔL6. While deletion of the central variable portion of loop L9 (ΔL9v) affected maltoporin function only moderately, the combination of ΔL9v with the double deletion of loops L4 and L6 (triple deletion ΔL4 + ΔL6 + ΔL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate‐binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBΔL9v), of two loops L4 and L6 (LamBΔL4 +ΔL6), of three loops L4, L5 and L6 (LamBΔL4 +ΔL5 + ΔL6) or of L4, L6 and L9v (LamBΔL4 + ΔL6 +ΔL9v) had relatively little influence on the carbohydrate‐binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBΔL4) or L6 (LamBΔL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column, suggest that maltooligosaccharides enter the LamB channel from the cell surface side with the non‐reducing end in advance. The absence of some of the loops leads to obstruction of the channel from the outside, which results in a considerable difference in the on‐rate of carbohydrate binding from the extracellular side compared with that from the periplasmic side.
Mechanisms of neisserial serum resistance
Authors: Vogel Ulrich , Frosch Matthias
Published: March 2003

Pathogenic Neisseria use a variety of mechanisms to survive the bactericidal action of the complement system. Serum resistance is a crucial virulence factor for the development of severe meningococcal disease, meningococcal meningitis and disseminated gonococcal infection. Furthermore, local inflammation at the site of gonococcal infection exposes the bacteria to moderate concentrations of complement factors. We review current concepts of neisserial serum resistance with emphasis on porins and polysaccharides exposed on the neisserial surface and their interaction with components of normal human serum.
Control of DNA topology during thermal stress in hyperthermophilic archaea: DNA topoisomerase levels, activities and induced thermotolerance during heat and cold shock in Sulfolobus
Authors: López‐García Purificación , Forterre Patrick
Published: March 2003

Plasmid topology varies transiently in hyperthermophilic archaea during thermal stress. As in mesophilic bacteria, DNA linking number (Lk) increases during heat shock and decreases during cold shock. Despite this correspondence, plasmid DNA topology and proteins presumably involved in DNA topological control in each case are different. Plasmid DNA in hyperthermophilic archaea is found in a topological form from relaxed to positively supercoiled in contrast to the negatively supercoiled state typical of bacteria, eukaryotes and mesophilic archaea. We have analysed the regulation of DNA topological changes during thermal stress in Sulfolobus islandicus (kingdom Crenarchaeota), which harbours two plasmids, pRN1 and pRN2. In parallel with plasmid topological variations, we analysed levels of reverse gyrase, topoisomerase VI (Topo VI) and the small DNA‐binding protein Sis7, as well as topoisomerase activities in crude extracts during heat shock from 80°C to 85–87°C, and cold shock from 80°C to 65°C. Quantitative changes in reverse gyrase, Topo VI and Sis7 were not significant. In support of this, inhibition of protein synthesis in S. islandicus during shocks did not alter plasmid topological dynamics, suggesting that an increase in topoisomerase levels is not needed for control of DNA topology during thermal stress. A reverse gyrase activity was detected in crude extracts, which was strongly dependent on the assay temperature. It was inhibited at 65°C, but was greatly enhanced at 85°C. However, the intrinsic reverse gyrase activity did not vary with heat or cold shock. These results suggest that the control of DNA topology during stress in Sulfolobus relies primarily on the physical effect of temperature on topoisomerase activities and on the geometry of DNA itself. Additionally, we have detected an enhanced thermoresistance of reverse gyrase activities in cultures subject to prolonged heat shock (but not cold shock). This acquired thermotolerance at the enzymatic level is abolished when cultures are treated with puromycin, suggesting a requirement for protein synthesis.
Stimulation of transposition of the Mycobacterium tuberculosis insertion sequence IS6110 by exposure to a microaerobic environment
Authors: Ghanekar Kiran , McBride Alan , Dellagostin Odir , Thorne Stephen , Mooney Rachel , McFadden Johnjoe
Published: March 2003

The Mycobacterium tuberculosis‐specific insertion sequence IS6110/986 has been widely used as a probe because of the multiple polymorphism observed among different strains. To investigate transposition of IS6110, a series of artificially constructed composite transposons containing IS6110 and a kanamycin resistance marker were constructed. The composite transposons were inserted into a conditionally replicating, thermosensitive, Escherichia coli–mycobacterial shuttle vector and introduced into M. smegmatis mc2155. Lawns of transformants were grown at the permissive temperature on kanamycin‐supplemented agar and subsequently prevented from further growth by shifting to the non‐permissive temperature. Under normal atmospheric conditions, kanamycin‐resistant papillae appeared after only about 5–6 weeks of incubation. However, these events were not associated with transposon mobilization. In contrast, lawns that were exposed to a 48 h microaerobic shock generated kanamycin‐resistant papillae after only 6–14 days. These events were generated by conservative transposition of the IS6110 composite transposon into the M. smegmatis chromosome, with loss of the shuttle vector. In common with other IS3 family elements, transposition of IS6110 is thought to be controlled by translational frameshifting. However, we were unable to detect any significant frameshifting within the putative frameshifting site of IS6110, and the level of frameshifting was not affected by microaerobic incubation. The finding that transposition of IS6110 is stimulated by incubation at reduced oxygen tensions may be relevant to transposition of IS6110 in M. tuberculosis harboured within TB lesions.
A constitutive role for GPI anchors in Saccharomyces cerevisiae : cell wall targeting
Authors: De Sampaïo Guillaume , Bourdineaud Jean‐Paul , Lauquin Guy J.‐M.
Published: March 2003

GPI anchors are widely represented among organisms and have several cellular functions. It has been proposed that in yeast there are two groups of GPI proteins: plasma membrane‐resident proteins, such as Gas1p or Yap3p, and cell wall‐targeted proteins, such as Tir1p or α‐agglutinin. A model has been proposed for the plasma membrane retention of proteins from the first group because of a dibasic motif located just upstream of the GPI‐anchoring signal. The results we report here are not in agreement with such a model as we show that constructs containing the C‐terminal parts of Gas1p and Yap3p are also targeted to the cell wall. We also detect the genuine Gas1p after cell wall treatment with Quantazyme or Glucanex glycanases. In addition, we show that the GPI‐anchoring signal from the human placental alkaline phosphatase (PLAP) is not compatible with the yeast machinery unless the human transamidase hGpi8p is co‐expressed. In this condition, this human signal is able to target a protein to the cell wall. Moreover, TIR1 proved to be a multicopy suppressor of Δgas1 mutation. The present findings suggest a constitutive role for GPI anchors in yeast: the cell wall targeting of proteins.
A role for the leucine‐responsive regulatory protein and integration host factor in the regulation of the Salmonella plasmid virulence (spv ) locus in Salmonella typhimurium
Authors: Marshall David G. , Sheehan Brian J. , Dorman Charles J.
Published: March 2003

The Salmonella plasmid virulence (spv ) genes of Salmonella typhimurium are activated at the level of transcription as the bacteria enter stationary phase in vitro or in response to signals received during intracellular growth. Activation requires the LysR‐like transcription factor SpvR and the alternative sigma factor RpoS. In this report, we show by biochemical and genetic analyses that two chromosomally encoded DNA‐binding proteins contribute to the control of spv expression. These are the integration host factor (IHF), which binds to DNA sequences upstream of the spvR regulatory gene, and the leucine‐responsive regulatory protein (Lrp), which binds to sequences upstream of the spvABCD operon. Under all conditions tested, inactivation of IHF expression reduces the level of spvR transcription by twofold. It also alters the response of the spv regulon to loss of DNA gyrase activity, consistent with a role for IHF in organizing DNA structure in the vicinity of the spvR promoter. Lrp represses spvA gene expression by up to fivefold and Lrp‐mediated repression is antagonized by leucine. The Lrp binding site upstream of the spvA gene overlaps one of the binding sites for the positive regulator SpvR, suggesting a mechanism by which Lrp repression is exerted. This is a first demonstration of a role for Lrp in controlling genes that are also subject to intracellular regulation. These data show that the spv virulence genes belong simultaneously to several regulons in the cell, raising the possibility that spv expression can be fine‐tuned in response to multiple environmental inputs.
Analysis of Shigella flexneri Wzz (Rol) function by mutagenesis and cross‐linking: Wzz is able to oligomerize
Authors: Daniels Craig , Morona Renato
Published: March 2003

The modal length or degree of polymerization (dp) of the Shigella flexneri O‐antigen is determined in an unknown manner by the Wzz/Rol protein. The Wzz protein is anchored into the cytoplasmic membrane by two transmembrane domains (TM1 amino acids 32–52; TM2 amino acids 295–315) with the central loop of the protein located in the periplasm. Plasmids were constructed encoding hybrid Wzz proteins consisting of regions of S. flexneri Wzz (WzzSF) and Salmonella typhimurium Wzz (WzzST). These imparted O‐antigen modal chain lengths that implied that the carboxy‐terminal region of Wzz was involved in chain length determination. Site‐directed mutagenesis was undertaken to investigate the functional significance of highly conserved residues in amino‐/carboxy‐terminal domains of WzzSF. Some of the WzzSF variants resulted in O‐antigen modal chain lengths much shorter than those of wild‐type WzzSF, whereas other mutants inactivated WzzSF function entirely and a third class had a longer O‐antigen chain length distribution. The data indicate that amino acids throughout the length of the WzzSF protein are important in determination of O‐antigen modal chain length. In vivo cross‐linking experiments were performed to investigate the interactions between Wzz proteins. The experiments indicated that the WzzSF protein is able to form dimers and oligomers of at least six WzzSF proteins. A carboxy‐terminal‐truncated WzzSF protein having the amino terminal 194 amino acids was able to oligomerize, indicating that the amino‐terminal region is sufficient for the Wzz–Wzz interaction observed. Shortened WzzSF proteins having internal deletions in the amino‐terminal region were also able to oligomerize, suggesting that residues 59–194 are not essential for oligomerization. Cross‐linking of WzzSF proteins with mutationally altered residues showed that loss of WzzSF function may be correlated to a reduced/altered ability to form oligomers, and that mutational alteration of glycine residues in the TM2 segment affects WzzSF–WzzSF dimer mobility in SDS polyacrylamide gels. These results provide the first evidence of protein–protein interactions for proteins involved in O‐antigen polysaccharide biosynthesis.
Positive co‐regulation of the Escherichia coli carnitine pathway cai and fix operons by CRP and the CaiF activator
Authors: Buchet Anne , Nasser William , Eichler Knut , Mandrand‐Berthelot Marie‐Andrée
Published: March 2003

Activation of the two divergent Escherichia coli cai and fix operons involved in anaerobic carnitine metabolism is co‐dependent on the cyclic AMP receptor protein (CRP) and on CaiF, the specific carnitine‐sensitive transcriptional regulator. CaiF was overproduced using a phage T7 system, purified on a heparin column and ran as a 15 kDa protein on SDS–PAGE. DNase I footprinting and interference experiments identified two sites, F1 and F2, with apparently comparable affinities for the binding of CaiF in the cai–fix regulatory region. These sites share a common perfect inverted repeat comprising two 11 bp half‐sites separated by 13 bp, and centred at −70 and −127 from the fix transcription start site. They were found to overlap the two low‐affinity binding sites, CRP2 and CRP3, determined previously for CRP. Gel shift assays and footprinting experiments suggest that CaiF and CRP bind co‐operatively to the F1/CRP2 and F2/CRP3 sites of the intergenic cai–fix region. Moreover, they appeared to serve the simultaneous binding of each other, giving rise to an original multiprotein CRP–CaiF complex enabling RNA polymerase recruitment and local DNA untwisting, at least at the fix promoter. Using random mutagenesis, two CaiF mutants impaired in transcription activation were isolated. The N‐terminal A27V mutation affected the structural organization of the activator, whereas the central I62N mutation was suggested to interfere with DNA binding.
Cloning and functional analysis of Asa373, a novel adhesin unrelated to the other sex pheromone plasmid‐encoded aggregation substances of Enterococcus faecalis
Authors: Muscholl‐Silberhorn Albrecht
Published: March 2003

pAM373 of Enterococcus faecalis deviates from the various other representatives of sex pheromone plasmids in that it encodes a clumping‐mediating adhesin, Asa373, unrelated to the highly conserved aggregation substances typical of this plasmid class. The use of a new general cloning strategy and sequencing of the corresponding gene has confirmed that Asa373 represents a novel type of adhesin embedded in a DNA sequence very similar to sex pheromone plasmid pPD1. To prove the specific function of the relatively small protein (75.6 kDa vs 137 kDa for pAD1‐encoded Asa1) in cell aggregation, an expression vector, pERM‐ex1, was constructed, allowing reliable and stable expression of proteins in E. faecalis. The expression of Asa373 in E. faecalis indeed resulted in constitutive clumping, whereas non‐polar disruption of the gene in the original pAM373 abolished clumping capacity. Expression in a strain (INY3000) defective in binding substance — which for the other aggregation substances constitutes the attachment site on the mating partner — did not alter Asa373‐dependent clumping; this implies a separate mechanism in cell–cell interaction for this adhesin. Some amino acid motifs of Asa373 link the protein to adhesins of oral streptococci and other cell surface proteins. Comparison of the leader sequence of asa373 with those of several other aggregation substances revealed a highly conserved translational unit possibly involved in the regulation of asa373 expression.
Interaction between the F plasmid TraA (F‐pilin) and TraQ proteins
Authors: Harris Robin L. , Sholl K. April , Conrad Michael N. , Dresser Michael E. , Silverman Philip M.
Published: March 2003

Elaboration of conjugative (F) pili by F+ strains of Escherichia coli requires the activities of over a dozen F‐encoded DNA transfer (Tra) proteins. The organization and functions of these proteins are largely unknown. Using the yeast two‐hybrid assay, we have begun to analyse binary interactions among the Tra proteins required for F‐pilus formation. We focus here on interactions involving F‐pilin, the only known F‐pilus subunit. Using a library of F tra DNA fragments that contained all the F genes required for F pilus formation in a yeast GAL4 activation domain vector (pACTII), we transformed yeast containing a plasmid (pAS1CYH2traA) encoding a GAL4 DNA‐binding domain–F‐pilin fusion. Doubly transformed cells were screened for GAL4‐dependent gene expression. This screen repeatedly identified only a single Tra protein, TraQ, previously identified as a likely F‐pilin chaperone. The F‐pilin–TraQ interaction appeared to be specific, as no transcriptional activation was detected in yeast transformants containing pACTIItraQ plasmids and the Salmonella typhi pED208 traA gene cloned in pAS1CYH2. Two traQ segments isolated in the screen against F‐pilin were tested for complementation of a traQ null allele in E. coli. One, lacking the first 11 (of 94) TraQ amino acids, restored DNA donor activity, donor‐specific bacteriophage sensitivity and membrane F‐pilin accumulation to wild‐type levels. The second, lacking the first 21 amino acids, was much less effective in these assays. Both TraQ polypeptides accumulated in E. coli as transmembrane proteins. The longer, biologically active segment was fused to the GAL4 DNA‐binding domain gene of pAS1CYH2 and used to screen the tra fragment library. The only positives from this screen identified traA segments. The fusion sites between the traA and GAL4 segments identified the hydrophobic, C‐terminal domain IV of F‐pilin as sufficient for the interaction. As TraQ is the only Tra protein required for the accumulation of inner membrane F‐pilin, the interaction probably reflects a specific, chaperone‐like function for TraQ in E. coli. Attempts to isolate an F‐pilin–TraQ complex from E. coli were unsuccessful, suggesting that the interaction between the two is normally transient, as expected from previous studies of the kinetics of TraA membrane insertion and processing to F‐pilin.
Genome‐wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway
Authors: Jung Un Sung , Levin David E.
Published: March 2003

The cell integrity pathway of Saccharomyces cerevisiae monitors cell wall remodelling during growth and differentiation. Additionally, this pathway responds to environmental stresses that challenge the integrity of the cell wall. We conducted a genome‐wide survey of genes whose expression was altered in response to activation of Mpk1/Slt2, the MAP kinase, under the control of cell integrity signalling. We identified 25 genes whose regulation was altered by Mpk1 activity. Among these, 20 were positively regulated by Mpk1, and five were negatively regulated. Most of the genes identified encode either known or suspected cell wall proteins or enzymes involved in cell wall biogenesis. These include glycosyl‐phosphatidylinositol (GPI) proteins, members of the Pir family of cell wall proteins, Mpk1 itself and others. All of the regulation detected was mediated by the Rlm1 transcription factor, a MADS‐box protein that is phosphorylated and activated by Mpk1, but for which no transcriptional targets had been identified. A similar pattern of regulation was observed when cell integrity signalling was induced by environmental stress (i.e. temperature upshift).
Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes
Authors: Hodgson David A.
Published: March 2003

This is the first report of generalized transduction in the Gram‐positive, food‐borne pathogen Listeria monocytogenes. Bacteriophages were isolated from the environment and from lysogens, or were obtained from other laboratories. Of the 59 bacteriophages tested, 34 proved to be capable of transduction. We exploited the ability of L. monocytogenes to grow at room temperature and isolated bacteriophages that were incapable of growth at 37°C. Transductions at this temperature therefore eliminated transductant killing and lysogeny, as did inclusion of citrate and the use of a low multiplicity of infection. Transducing bacteriophages were found for each of the well‐characterized L. monocytogenes strains: EGD, 10403, Mack (serotype1/2a), L028 (serotype 1/2c), Scott A (serotype 4b) and strains from the Jalisco and Halifax, Nova Scotia outbreaks (serotype 4b). P35 (φLMUP35) is a particularly useful generalized transducing bacteriophage with a wide host range (75% of all serotype 1/2 strains tested). Its disadvantages are that it is small and transduction is relatively infrequent. U153(φCU‐SI153/95) is larger than P35 and transduction frequency increased 100‐fold, but it has a very narrow host range. We demonstrated interstrain transduction and used transduction to test linkage between transposon insertions and mutant phenotypes in a variety of strains.
Two opposing effects of mismatch repair on CTG repeat instability in Escherichia coli
Authors: Schmidt Kristina H. , Abbott Catherine M. , Leach David R. F.
Published: March 2003

The expansion of normally polymorphic CTG microsatellites in certain human genes has been identified as the causative mutation of a number of hereditary neurological disorders, including Huntington's disease and myotonic dystrophy. Here, we have investigated the effect of methyl‐directed mismatch repair (MMR) on the stability of a (CTG)43 repeat in Escherichia coli over 140 generations and find two opposing effects. In contrast to orientation‐dependent repeat instability in wild‐type E. coli and yeast, we observed no orientation dependence in MMR−E. coli cells and suggest that, for the repeat that we have studied, orientation dependence in wild‐type cells is mainly caused by functional mismatch repair genes. Our results imply that slipped structures are generated during replication, causing single triplet expansions and contractions in MMR− cells, because they are left unrepaired. On the other hand, we find that the repair of such slipped structures by the MMR system can go awry, resulting in large contractions. We show that these mutS‐dependent contractions arise preferentially when the CTG sequence is encoded by the lagging strand. The nature of this orientation dependence argues that the small slipped structures that are recognized by the MMR system are formed primarily on the lagging strand of the replication fork. It also suggests that, in the presence of functional MMR, removal of 3 bp slipped structures causes the formation of larger contractions that are probably the result of secondary structure formation by the CTG sequence. We rationalize the opposing effects of MMR on repeat tract stability with a model that accounts for CTG repeat instability and loss of orientation dependence in MMR− cells. Our work resolves a contradiction between opposing claims in the literature of both stabilizing and destabilizing effects of MMR on CTG repeat instability in E. coli.
Methylcitrate synthase from Aspergillus nidulans: implications for propionate as an antifungal agent
Authors: Brock Matthias , Fischer Reinhard , Linder Dietmar , Buckel Wolfgang
Published: March 2003

Aspergillus nidulans was used as a model organism to investigate the fungal propionate metabolism and the mechanism of growth inhibition by propionate. The fungus is able to grow slowly on propionate as sole carbon and energy source. Propionate is oxidized to pyruvate via the methylcitrate cycle. The key enzyme methylcitrate synthase was purified and the corresponding gene mcsA, which contains two introns, was cloned, sequenced and overexpressed in A. nidulans. The derived amino acid sequence of the enzyme shows more than 50% identity to those of most eukaryotic citrate synthases, but only 14% identity to the sequence of the recently detected bacterial methylcitrate synthase from Escherichia coli. A mcsA deletion strain was unable to grow on propionate. The inhibitory growth effect of propionate on glucose medium was enhanced in this strain, which led to the assumption that trapping of the available CoA as propionyl‐CoA and/or the accumulating propionyl‐CoA itself interferes with other biosynthetic pathways such as fatty acid and polyketide syntheses. In the wild‐type strain, however, the predominant inhibitor may be methylcitrate. Propionate (100 mM) not only impaired hyphal growth of A. nidulans but also synthesis of the green polyketide‐derived pigment of the conidia, whereas in the mutant pigmentation was abolished with 20 mM propionate.
A NapC/NirT‐type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes
Authors: Simon Jörg , Gross Roland , Einsle Oliver , Kroneck Peter M. H. , Kröger Achim , Klimmek Oliver
Published: March 2003

Wolinella succinogenes can grow by anaerobic respiration with nitrate or nitrite using formate as electron donor. Two forms of nitrite reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic nitrite reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric nitrite reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes nitrite reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N‐terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane‐bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C‐terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram‐positive bacteria, cyanobacteria and chloroplasts. The residual N‐terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin‐like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (ΔnrfJ,ΔnrfIJ and ΔnrfAIJ) were studied. Mutants ΔnrfAIJ and ΔnrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant ΔnrfJ showed wild‐type properties. The NrfA protein formed by mutant ΔnrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.