bluuebunny/crossref_metadata_2025 huggingface dataset:
Emergence of recombinant strains ofHelicobacter pyloriduring human infection
Authors: Kersulyte Dangeruta , Chalkauskas Henrikas , Berg Douglas E.
Published: March 2003

Genetic recombination can be important evolutionarily in speeding the adaptation of organisms to new environments and in purging deleterious mutations. Here, we describe polymerase chain reaction (PCR), hybridization and DNA sequence‐based evidence of six such exchanges between two strains ofHelicobacter pyloriduring natural mixed infection of a patient in Lithuania. One parent strain contained the 37 kb long, virulence‐associatedcagpathogenicity island (PAI), and the other strain lacked this PAI. MostH. pylorifrom the patient had descended from thecag+parent, but had becomecag−during infection. This had resulted from transfer of DNA containing the ‘empty site’ allele from thecag−strain and homologous recombination, not from excision of thecagPAI without DNA transfer. Other cases of recombination involved genes for an outer membrane protein (omp5 andomp29; also called HP0227 and HP1342) and a putative phosphoenolpyruvate synthase (ppsA ; HP0121). Replacement of a short patch of DNA sequence (36–124 bp) was also seen. As the chance of forming any given recombinant is small, the abundance of recombinants in this patient suggests selection for particular recombinant genotypes during years of chronic infection. We suggest that genetic exchange among unrelatedH. pyloristrains, as documented here, is important because of the diversity of this gastric pathogen and its human hosts. CertainH. pylorirecombinants may grow better in a given host than either parent. The vigour of growth, in turn, could impact on the severity of disease that infection can elicit.
Identification of target promoters for the Bacillus subtilis extracytoplasmic function σ factor, σW
Authors: Huang Xuejun , Gaballa Ahmed , Cao Min , Helmann John D.
Published: March 2003

The Bacillus subtilis sigW gene encodes an extracytoplasmic function (ECF) σ factor that is expressed in early stationary phase from a sigW‐dependent autoregulatory promoter, PW. Using a consensus‐based search procedure, we have identified 15 operons preceded by promoters similar in sequence to PW. At least 14 of these promoters are dependent on σW both in vivo and in vitro as judged by lacZ reporter fusions, run‐off transcription assays and nucleotide resolution start site mapping. We conclude that σW controls a regulon of more than 30 genes, many of which encode membrane proteins of unknown function. The σW regulon includes a penicillin binding protein (PBP4*) and a co‐transcribed amino acid racemase (RacX), homologues of signal peptide peptidase (YteI), flotillin (YuaG), ABC transporters (YknXYZ), non‐haem bromoperoxidase (YdjP), epoxide hydrolase (YfhM) and three small peptides with structural similarities to bacteriocin precursor polypeptides. We suggest that σW activates a large stationary‐phase regulon that functions in detoxification, production of antimicrobial compounds or both.
Rabbit sarcoplasmic reticulum Ca2+‐ATPase replaces yeast PMC1 and PMR1 Ca2+‐ATPases for cell viability and calcineurin‐dependent regulation of calcium tolerance
Authors: Degand Ingrid , Catty Patrice , Talla Emmanuel , Thinès‐Sempoux D. , De Kerchove d'Exaerde Alban , Goffeau André , Ghislain Michel
Published: March 2003

SERCA1a, the fast‐twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca2+‐ATPase, was expressed in yeast using the promoter of the plasma membrane H+‐ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca2+‐ATPase and the vacuole PMC1 Ca2+‐ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin‐dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intracellular compartments.
Analysis of the SlyA‐controlled expression, subcellular localization and pore‐forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K‐12
Authors: Ludwig Albrecht , Bauer Susanne , Benz Roland , Bergmann Birgit , Goebel Werner
Published: March 2003

Escherichia coli K‐12 harbours a chromosomal gene, clyA (sheA, hlyE ), that encodes a haemolytic 34 kDa protein. Recombinant E. coli overexpressing the cloned clyA gene accumulated this haemolysin in the periplasm and released only very small amounts of it into the external medium. The secretion of ClyA was confined to the log phase and paralleled by the partial release of several other periplasmic proteins. Sequencing of ClyA revealed the translational start point of the clyA gene and demonstrated that the clyA gene product is not N‐terminally processed during transport. The transcription of clyA from its native promoter region was positively controlled by SlyA, a regulatory protein found in E. coli, Salmonella typhimurium and other Enterobacteriaceae. SlyA‐controlled transcription started predominantly 72 bp upstream from clyAas shown by primer extension. The corresponding putative promoter contains an unusual −10 sequence (TATGAAT) that is separated from a conventional −35 sequence by a GC‐rich spacer. Site‐directed deletion of the G in the −10 sequence abrogated the SlyA requirement for strong ClyA production, whereas a reduction in the G+C content of the spacer diminished the capability of SlyA to activate the clyA expression. Osmotic protection assays and lipid bilayer experiments suggested that ClyA forms stable, moderately cation‐selective transmembrane pores that have a diameter of about 2.5–3 nm.
Cloning and characterization of three genes (SUT1–3 ) encoding glucose transporters of the yeast Pichia stipitis
Authors: Weierstall Thomas , Hollenberg Cornelis P. , Boles Eckhard
Published: March 2003

We have identified and characterized three genes, SUT1, SUT2 and SUT3, that encode glucose transporters of the yeast Pichia stipitis. When expressed in a Saccharomyces cerevisiae hxt null mutant strain that is unable to take up monosaccharides, all three proteins restored growth on glucose. Sequencing of the genes revealed open reading frames coding for 553 amino acids in the case of SUT1, and for 550 amino acids in the case of SUT2 and of SUT3. The derived protein sequences are closely related to one another, and show distinct sequence similarities to the S. cerevisiae hexose transporter family and to monosaccharide transporters of other organisms. The Sut2 and Sut3 proteins are nearly identical and differ only in one amino acid. Determination of substrate specificities and kinetic parameters of the individual Sut proteins expressed in a S. cerevisiae hxt1–7 mutant revealed Sut1, Sut2 and Sut3 as glucose transporters with Km values in the millimolar range. The proteins were also able to transport xylose and other monosaccharides, but with a considerably lower affinity. In P. stipitistranscription of SUT1 was strongly induced by glucose and was independent of the oxygen supply. In contrast, SUT2 and SUT3 were only expressed under aerobic conditions, but independent of the carbon source. Cells disrupted for the SUT1 gene did not show any obvious growth phenotype, however low‐affinity glucose uptake was lost. Further investigations suggest that the Sut proteins constitute a subfamily of glucose transporters in P. stipitis, and that other and probably unrelated proteins exist additionally mediating high‐affinity glucose and xylose uptake.
Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope
Authors: Jackson Mary , Raynaud Catherine , Lanéelle Marie‐Antoinette , Guilhot Christophe , Laurent‐Winter Christine , Ensergueix Danielle , Gicquel Brigitte , Daffé Mamadou
Published: March 2003

The antigen 85 complex of Mycobacterium tuberculosis consists of three abundantly secreted proteins. The recent characterization of a mycoloyltransferase activity associated in vitro with each of these antigens suggested that they are potentially important for the building of the unusual cell envelope of mycobacteria. To define the physiological role of these proteins, the gene coding for antigen 85C was inactivated by transposon mutagenesis. The resulting mutant was shown to transfer 40% fewer mycolates to the cell wall with no change in the types of mycolates esterifying arabinogalactan or in the composition of non‐covalently linked mycolates. As a consequence, the diffusion of the hydrophobic chenodeoxycholate and the hydrophilic glycerol, but not that of isoniazid, was found to be much faster through the cell envelope of the mutant than that of the parent strain. Taken together, these data demonstrate that: (i) antigen 85C is involved directly or indirectly in the transfer of mycolates onto the cell wall of the whole bacterium; (ii) the enzyme is not specific for a given type of mycolate; and (iii) the cell wall‐linked mycolate layer may represent a barrier for the diffusion of small hydrophobic and hydrophilic molecules.
YbtP and YbtQ: two ABC transporters required for iron uptake in Yersinia pestis
Authors: Fetherston Jacqueline D. , Bertolino Vincent J. , Perry Robert D.
Published: March 2003

Yersinia pestis, the causative agent of plague, makes a siderophore termed yersiniabactin (Ybt), which it uses to obtain iron during growth at 37°C. The genes required for the synthesis and utilization of Ybt are located within a large, unstable region of the Y. pestis chromosome called the pgm locus. Within the pgm locus, just upstream of a gene (ybtA) that regulates expression of the Ybt receptor and biosynthetic genes, is an operon consisting of 4 genes —ybtP, ybtQ, ybtX and ybtS. Transcription of the ybtPQXS operon is repressed by Fur and activated by YbtA. The product of ybtX is predicted to be an exceedingly hydrophobic cytoplasmic membrane protein that does not appear to contribute any vital function to Ybt biosynthesis or utilization in vitro. ybtP and ybtQ encode putative members of the traffic ATPase/ABC transporter family. YbtP and YbtQ are structurally unique among the subfamily of ABC transporters associated with iron transport, in that they both contain an amino‐terminal membrane‐spanning domain and a carboxy‐terminal ATPase. Cells with mutations in ybtP or ybtQ still produced Ybt but were impaired in their ability to grow at 37°C under iron‐deficient conditions, indicating that YbtP and YbtQ are needed for iron uptake. In addition, a ybtP mutant showed reduced iron accumulation and was avirulent in mice by a subcutaneous route of infection that mimics flea transmission of bubonic plague.
Bacterial genes induced within the nodule during the Rhizobium–legume symbiosis
Authors: Oke Valerie , Long Sharon R.
Published: March 2003

During the symbiosis between the bacterium Rhizobium meliloti and plants such as alfalfa, the bacteria elicit the formation of nodules on the roots of host plants. The bacteria infect the nodule, enter the cytoplasm of plant cells and differentiate into a distinct cell type called a bacteroid, which is capable of fixing atmospheric nitrogen. To discover bacterial genes involved in the infection and differentiation stages of symbiosis, we obtained genes expressed at the appropriate time and place in the nodule by identifying promoters that are able to direct expression of the bacA gene, which is required for bacteroid differentiation. We identified 230 fusions that are expressed predominantly in the nodule. Analysis of 23 sequences indicated that only three encode proteins known to be involved in the Rhizobium–legume symbiosis, six encode proteins with homology to proteins not previously associated with symbiosis, and 14 have no significant similarity to proteins of known function. Disruption of a locus that encodes a protein with homology to a cell adhesion molecule led to a defect in the formation of nitrogen‐fixing nodules, resulting in an increased number of nitrogen‐starved plants. Our isolation of a large number of nodule‐expressed genes will help to open the intermediate stages of nodulation to molecular analysis.
ADP‐ribosylation of oncogenic Ras proteins by Pseudomonas aeruginosa exoenzyme S in vivo
Authors: Vincent Timothy S. , Fraylick Jennifer E. , McGuffie Eileen M. , Olson Joan C.
Published: March 2003

The exoenzyme S (ExoS)‐producing Pseudomonas aeruginosa strain, 388, and corresponding ExoS knock‐out strain, 388ΔexoS, were used in a bacterial and mammalian co‐culture system as a model for the contact‐dependent delivery of ExoS into host cells. Examination of DNA synthesis and Ras ADP‐ribosylation in tumour cell lines expressing normal and mutant Ras revealed a decrease in DNA synthesis concomitant with ADP‐ribosylation of Ras proteins after exposure to ExoS‐producing bacteria, but not after exposure to non‐ExoS‐producing bacteria. Examination of normal H‐Ras, K‐Ras and N‐Ras by two‐dimensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP‐ribosylation by ExoS, with H‐Ras being modified most extensively. ADP‐ribosylation of oncogenic forms of Ras was examined in vivo using cancer lines expressing mutant forms of H‐, N‐ or K‐Ras. The mutant Ras proteins were modified in a manner qualitatively similar to their normal counterparts. Using Ras/Raf‐1 co‐immunoprecipitation after co‐culture, it was found that exposure to ExoS‐producing bacteria caused a decrease in the amount of Raf‐1 associated with EGF‐activated Ras and oncogenic Ras. The results from this study indicate that ExoS ADP‐ribosylates both normal and mutant Ras proteins in vivo and inhibits signalling through Ras.
Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli
Authors: Oscarsson Jan , Mizunoe Yoshimitsu , Li Lu , Lai Xin‐He , Wieslander Åke , Uhlin Bernt Eric
Published: March 2003

Escherichia coli K‐12 carries a gene for a protein denoted ClyA or SheA that can mediate a cytolytic phenotype. The ClyA protein is not expressed at detectable levels in most strains of E. coli, but overproduction suitable for purification was accomplished by cloning the structural gene in an hns mutant strain. Highly purified ClyA protein was cytotoxic to macrophage cells in culture and caused detachment and lysis of the mammalian cells. Results from osmotic protection assays were consistent with the suggestion that the protein formed pores with a diameter of up to 3 nm. Using Acholeplasma laidlawii cells and multilamellar liposomes, we studied the effect of ClyA on membranes with varying compositions and in the presence of different ions. ClyA induced cytolytic release of the fluorescent tracer from carboxyfluorescein‐loaded liposomes, and the release was stimulated if cholesterol was present in the membranes whereas addition of calcium had no effect. Pretreatment of the ClyA protein with cholesterol inhibited the pore formation, suggesting that ClyA could bind to cholesterol. Efficient coprecipitation of ClyA with either cholesterol or 1,2,3‐trioctadecanoylglycerol in aqueous solutions showed that ClyA directly interacted with the hydrophobic molecular aggregates. We tested the possible functional importance of selected ClyA protein regions by site‐directed mutagenesis. Defined mutants of ClyA were obtained with alterations in postulated transmembrane structures in the central part and in a postulated membrane‐targeting domain in the C‐terminal part. Our results were consistent with the suggestion that particular amphiphilic segments are required for ClyA activity. We propose that these domains are necessary for ClyA to form pores.
Stress factors acting at the level of the plasma membrane induce transcription via the stress response element (STRE) of the yeast Saccharomyces cerevisiae
Authors: Moskvina Eugenia , Imre Esther‐Maria , Ruis Helmut
Published: March 2003

A variety of stress factors induces transcription via the stress response element (STRE) present in control regions of a number of genes of the yeast Saccharomyces cerevisiae. Induction of transcription involves nuclear translocation of the STRE‐binding transcription activators Msn2p and Msn4p. The primary cellular events triggering this translocation are presently not well understood. In this investigation, we have observed that a number of factors acting at the level of the yeast plasma membrane, including the antifungal agent nystatin, the steroidal alkaloid tomatine, benzyl alcohol, a number of detergents and the plasma membrane H+‐ATPase inhibitor diethylstilbestrol or mutations in the PMA1 gene encoding the plasma membrane ATPase, induce Msn2p nuclear accumulation and STRE‐dependent transcription. At least some of the stress factors acting via STREs cause an increase in plasma membrane permeability, leading to a decrease in membrane potential, which might be a primary cellular stress signal. A decrease in internal pH triggered by permeabilization of the plasma membrane or a change in cAMP levels are at least not obligatory factors in intracellular stress signal transduction. The signal transduction pathway transmitting the signal generated at the plasma membrane to Msn2p is still unknown.
Antiterminator‐dependent modulation of transcription elongation rates by NusB and NusG
Authors: Zellars Malcolm , Squires Catherine L.
Published: March 2003

Ribosomal RNA is transcribed about twice as fast as messenger RNA in vivo, and this increased transcription rate requires the rrn boxA antitermination system. Because several Nus factors have been implicated in rrn antitermination, we have examined the role of NusB, NusE and NusG in controlling the rate of rrn boxA‐mediated transcript elongation. In vivo RNA polymerase transcription rates were determined by measuring the rate of appearance of lacZ transcript using a plasmid that contained an inducible T7 promoter fused to the rrn boxA sequence followed by the lacZ gene. This plasmid was introduced into Escherichia coli mutant strains that can be conditionally depleted of NusG, or that carry a deficient nusB gene or a nusE mutation. We found that, in addition to the rrn boxA antiterminator sequence, both NusG and NusB were required to maintain the high transcription rate. The nusE mutation used in this study may be specific for lambda antitermination, as it did not influence the boxA‐mediated increase in transcription rate.
The molecular characterization of the first autolytic lysozyme of Streptococcus pneumoniae reveals evolutionary mobile domains
Authors: García Pedro , González María Paz , García Ernesto , García José Luis , López Rubens
Published: March 2003

A biochemical approach to identify proteins with high affinity for choline‐containing pneumococcal cell walls has allowed the localization, cloning and sequencing of a gene (lytC ) coding for a protein that degrades the cell walls of Streptococcus pneumoniae. The lytC gene is 1506 bp long and encodes a protein (LytC) of 501 amino acid residues with a predicted Mr of 58 682. LytC has a cleavable signal peptide, as demonstrated when the mature protein (about 55 kDa) was purified from S. pneumoniae. Biochemical analyses of the pure, mature protein proved that LytC is a lysozyme. Combined cell fractionation and Western blot analysis showed that the unprocessed, primary product of the lytC gene is located in the pneumococcal cytoplasm whereas the processed, active form of LytC is tightly bound to the cell envelope. In vivo experiments demonstrated that this lysozyme behaves as a pneumococcal autolytic enzyme at 30°C. The DNA region encoding the 253 C‐terminal amino acid residues of LytC has been cloned and expressed in Escherichia coli. The truncated protein exhibits a low, but significant, choline‐independent lysozyme activity, which suggests that this polypeptide adopts an active conformation. Self‐alignment of the N‐terminal part of the deduced amino acid sequence of LytC revealed the presence of 11 repeated motifs. These results strongly suggest that the lysozyme reported here has changed the general building plan characteristic of the choline‐binding proteins of S. pneumoniae and its bacteriophages, i.e. the choline‐binding domain and the catalytic domain are located, respectively, at the N‐terminal and the C‐terminal moieties of LytC. This work illustrates the natural versatility exhibited by the pneumococcal genes coding for choline‐binding proteins to fuse separated catalytic and substrate‐binding domains and create new and functional mature proteins.
The involvement of NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high‐affinity CO2 uptake in Synechococcus sp. PCC7002 gives evidence for multiple NDH‐1 complexes with specific roles in cyanobacteria
Authors: Klughammer Barbara , Sültemeyer Dieter , Badger Murray R. , Price G. Dean
Published: March 2003

Random gene tagging was used to obtain new mutants of the marine cyanobacterium, Synechococcus sp. PCC7002, with defects in the CO2‐concentrating mechanism (CCM). Two of these mutants, K22 and A41, showed poor growth at limiting CO2. Isolation and sequencing of a 6.6 kb genomic region revealed the existence of five potential protein‐coding regions, all arranged in the same transcriptional direction. These regions code for an RbcR homologue, NdhF3 (subunit 5 of type 1 NAD(P)H dehydrogenase; NDH‐1 complex), NdhD3 (subunit 4 of NDH‐1), ORF427 and ORF133 (hypothetical proteins). Insertional mutants in ndhD3, ndhF3 and ORF427, like A41 and K22, were all incapable of inducing high‐affinity CO2 uptake and were not fully capable of inducing high‐affinity HCO3− transport. ndhD3 and ndhF3 mutants displayed P700 re‐reduction rates identical to wild‐type cells, suggesting that NdhD3 is part of a specific NDH‐1 complex that is not involved in photosynthetic cyclic electron transport. Thus, it is feasible that NdhD3, NdhF3 and ORF427 might form part of a novel NDH‐1 complex located on the cytoplasmic membrane and involved in tightly coupled energization of high‐affinity CO2 transport. The possibility of multiple, functionally distinct NDH‐1 complexes in cyanobacteria is discussed.
The staphylococcal accessory regulator (sar ) represses transcription of the Staphylococcus aureus collagen adhesin gene (cna ) in an agr‐independent manner
Authors: Blevins Jon S. , Gillaspy Allison F. , Rechtin Tammy M. , Hurlburt Barry K. , Smeltzer Mark S.
Published: March 2003

Comparison of Staphylococcus aureus strains carrying mutations inactivating the staphylococcal accessory regulator (sar ) and/or the accessory gene regulator (agr ) suggests that sar is the primary regulatory element controlling transcription of the collagen adhesin gene (cna ) and that the regulatory effect of sar is independent of the interaction between SarA and agr. To test this hypothesis, we cloned the regions encoding each of the overlapping sar transcripts, all of which include the sarA open reading frame (ORF), and introduced each clone into cna‐positive sar and agr mutants. The introduction of each clone restored the expected sar transcripts and the temporal pattern of sar transcription. The introduction of each clone also complemented the defect in cna transcription and restored collagen binding to wild‐type levels. This was true even when the clones were introduced into a sar/agr double mutant. These results confirm the hypothesis that the sar‐mediated regulation of cna transcription occurs via an agr‐independent pathway. Direct evidence supporting this hypothesis comes from electrophoretic mobility shift assays demonstrating that SarA exhibits high‐affinity binding to cis elements upstream of the cna structural gene. We also examined the correlation between sar transcription and the production of SarA. Western blot analysis of two wild‐type strains indicated that SarA was produced in indistinguishable amounts during both the exponential and the post‐exponential growth phases.
Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNac lectin complex inhibits Entamoeba histolytica virulence
Authors: Ankri Serge , Padilla‐Vaca Felipe , Stolarsky Tamara , Koole Lucy , Katz Uriel , Mirelman David
Published: March 2003

One of the under‐represented genes identified by cDNA representational difference analysis (RDA) between avirulent Entamoeba histolytica strain Rahman and virulent strain HM‐1:IMSS was the amoebic light (35 kDa) subunit of the Gal/GalNac lectin complex. This lectin complex, which mediates the adhesion of the parasite to the target cell, also contains a heavy (170 kDa) subunit, which has the carbohydrate‐binding domain. Stable transfectants of the virulent strain in which the expression of the 35 kDa subunit was inhibited by antisense RNA were not significantly affected in their adhesion activity to mammalian or bacterial cells but were strongly inhibited in their cytopathic activity, cytotoxic activity and in their ability to induce the formation of liver lesions in hamsters. These findings suggest that the 35 kDa subunit may have a specific function in the pathogenic pathway and provides a new insight into the role of this component of the Gal/GalNac lectin complex in amoebic virulence.
Mode of action of AraR, the key regulator of l‐arabinose metabolism in Bacillus subtilis
Authors: Mota Luís Jaime , Tavares Paulo , Sá‐Nogueira Isabel
Published: March 2003

The AraR protein is a negative regulator involved in l‐arabinose‐inducible expression of the Bacillus subtilis araABDLMNPQ‐abfA metabolic operon and of the araE/araR genes that are organized as a divergent transcriptional unit. The two ara gene clusters are found at different positions in the bacterial chromosome. AraR was overproduced in Escherichia coli and purified to more than 95% homogeneity. AraR binds specifically to DNA fragments carrying the promoter region of the ara genes. DNase I protection assays showed that AraR binds to two sequences within the promoters of the araABDLMNPQ‐abfA operon and the araE gene, and to one sequence in the araR promoter. The AraR target sequences are palindromic and share high identity, defining a 16 bp AraR consensus operator sequence showing half‐symmetry, ATTTGTAC. Binding of AraR to DNA was inhibited by l‐arabinose but not by other sugars. The two operator sites within the araABDLMNPQ‐abfA operon and araE promoters are located on the same side of the DNA helix, and a pattern of enhanced and diminished DNase I cleavage was observed between them, but not in the araR promoter. Quantitative DNase I footprinting in DNA templates containing one, two or three AraR binding sites showed that the repressor binds cooperatively to the two operator sites within the metabolic operon and araE promoters but not to the site located in the araR promoter. These results are consistent with two modes for AraR transcriptional repression that might correlate with different physiological requirements: a high level of repression is achieved by DNA bending requiring two in‐phase operator sequences (metabolic operon and araE transport gene), whereas binding to a single operator, which autoregulates araR expression, is 10‐fold less effective.
Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae
Authors: Yu Jing , Peñaloza‐Vázquez Alejandro , Chakrabarty Ananda M. , Bender Carol L.
Published: March 2003

Alginate, a co‐polymer of O‐acetylated β‐1,4‐linked d‐mannuronic acid and l‐guluronic acid, has been reported to function in the virulence of Pseudomonas syringae, although genetic studies to test this hypothesis have not been undertaken previously. In the present study, we used a genetic approach to evaluate the role of alginate in the pathogenicity of P. syringae pv. syringae 3525, which causes bacterial brown spot on beans. Alginate biosynthesis in strain 3525 was disrupted by recombining Tn5 into algL, which encodes alginate lyase, resulting in 3525.L. Alginate production in 3525.L was restored by the introduction of pSK2 or pAD4033, which contain the alginate biosynthetic gene cluster from P. syringae pv. syringae FF5 or the algA gene from P. aeruginosa respectively. The role of alginate in the epiphytic fitness of strain 3525 was assessed by monitoring the populations of 3525 and 3525.L on tomato, which is not a host for this pathogen. The mutant 3525.L was significantly impaired in its ability to colonize tomato leaves compared with 3525, indicating that alginate functions in the survival of strain 3525 on leaf surfaces. The contribution of alginate to the virulence of strain 3525 was evaluated by comparing the population dynamics and symptom development of 3525 and 3525.L in bean leaves. Although 3525.L retained the ability to form lesions on bean leaves, symptoms were less severe, and the population was significantly reduced in comparison with 3525. These results indicate that alginate contributes to the virulence of P. syringae pv. syringae 3525, perhaps by facilitating colonization or dissemination of the bacterium in planta.
β‐Lactam resistance in Streptococcus pneumoniae: penicillin‐binding proteins and non‐penicillin‐binding proteins
Authors: Hakenbeck Regine , Grebe Thorsten , Zähner Dorothea , Stock Jeffry B.
Published: March 2003

The β‐lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of β‐lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no β‐lactamase‐producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the β‐lactams, the penicillin‐binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non‐PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non‐PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non‐PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during β‐lactam treatment counteract the effects caused by the inhibition of penicillin‐binding proteins.
Delayed nucleoid segregation in Escherichia coli
Authors: Huls P. G. , Vischer N. O. E. , Woldringh C. L.
Published: March 2003

To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry. In pbpB(Ts) and ftsZ strains growing as filaments at 42°C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild‐type cells. Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers. From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D′ period (time between termination and nucleoid separation). Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28°C) showed a long D′ period (42 min versus 18 min in the parent) indicative of an extended segregation time. The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation. We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.
Novel sensing mechanisms and targets for the cAMP–protein kinase A pathway in the yeast Saccharomyces cerevisiae
Authors: Thevelein Johan M. , De Winde Johannes H.
Published: March 2003

The cAMP–protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae plays a major role in the control of metabolism, stress resistance and proliferation, in particular in connection with the available nutrient conditions. Extensive information has been obtained on the core section of the pathway, i.e. Cdc25, Ras, adenylate cyclase, PKA, and on components interacting directly with this core section, such as the Ira proteins, Cap/Srv2 and the two cAMP phosphodiesterases. Recent work has now started to reveal upstream regulatory components and downstream targets of the pathway. A G‐protein‐coupled receptor system (Gpr1–Gpa2) acts upstream of adenylate cyclase and is required for glucose activation of cAMP synthesis in concert with a glucose phosphorylation‐dependent mechanism. Although a genuine signalling role for the Ras proteins remains unclear, they appear to mediate at least part of the potent stimulation of cAMP synthesis by intracellular acidification. Recently, several new targets of the PKA pathway have been discovered. These include the Msn2 and Msn4 transcription factors mediating part of the induction of STRE‐controlled genes by a variety of stress conditions, the Rim15 protein kinase involved in stationary phase induction of a similar set of genes and the Pde1 low‐affinity cAMP phosphodiesterase, which specifically controls agonist‐induced cAMP signalling. A major issue that remains to be resolved is the precise connection between the cAMP–PKA pathway and other nutrient‐regulated components involved in the control of growth and of phenotypic characteristics correlated with growth, such as the Sch9 and Yak1 protein kinases. Cln3 appears to play a crucial role in the connection between the availability of certain nutrients and Cdc28 kinase activity, but it remains to be clarified which nutrient‐controlled pathways control Cln3 levels.
The Tir‐binding region of enterohaemorrhagic Escherichia coli intimin is sufficient to trigger actin condensation after bacterial‐induced host cell signalling
Authors: Liu Hui , Magoun Loranne , Luperchio Steve , Schauer David B. , Leong John M.
Published: March 2003

Enterohaemorrhagic Escherichia coli (EHEC) has emerged as an important agent of diarrhoeal disease. Attachment to host cells, an essential step during intestinal colonization by EHEC, is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (A/E) lesion, directly beneath bound bacteria. The outer membrane protein intimin is required for the formation of this structure, as is Tir, a bacterial protein that is translocated into the host cell and is thought to function as a receptor for intimin. To understand intimin function better, we fused EHEC intimin to a homologous protein, Yersinia pseudotuberculosis invasin, or to maltose‐binding protein. The N‐terminal 539 amino acids of intimin were sufficient to promote outer membrane localization of the C‐terminus of invasin and, conversely, the N‐terminal 489 amino acids of invasin were sufficient to promote the localization of the C‐terminus of intimin. The C‐terminal 181 residues of intimin were sufficient to bind mammalian cells that had been preinfected with an enteropathogenic E. coli strain that expresses Tir but not intimin. Binding of intimin derivatives to preinfected cells correlated with binding to recombinant Tir protein. Finally, the 181‐residue minimal Tir‐binding region of intimin, when purified and immobilized on latex beads, was sufficient to trigger A/E lesions on preinfected mammalian cells.
The Helicobacter pylori neutrophil‐activating protein is an iron‐binding protein with dodecameric structure
Authors: Tonello Fiorella , Dundon William G. , Satin Barbara , Molinari Maurizio , Tognon Giuseppe , Grandi Guido , Del Giudice Giuseppe , Rappuoli Rino , Montecucco Cesare
Published: March 2003

The neutrophil‐activating protein (HP‐NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP‐NAP and both bacterial DNA‐protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP‐NAP and Dps. Electron microscopy revealed that HP‐NAP forms hexagonal rings of 9–10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP‐NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP‐NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP‐NAP is an unusual, small, ferritin that folds into a four‐helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.
LipC, a second lipase of Pseudomonas aeruginosa, is LipB and Xcp dependent and is transcriptionally regulated by pilus biogenesis components
Authors: Martínez Alejandro , Ostrovsky Paula , Nunn David N.
Published: March 2003

We have isolated cosmids that complement a Pseudomonas aeruginosa export‐impaired mutant by increasing growth on lipid agar, a medium that requires lipase expression and export. These cosmids encode a previously unidentified lipase, LipC, which has high homology to the P. aeruginosa lipA gene product. Like LipA, LipC activity requires the chaperone activity of the lipB gene product and a functional xcp gene cluster for export. However, expression of LipC is barely detectable in a wild‐type background. Transposon insertions that increase lipC promoter activity have been obtained that inactivate two pilus biogenesis genes, pilX and pilY1. This suggests that these proteins either directly or indirectly repress the expression of LipC and may be involved in transducing an extracellular signal that regulates this lipase.
Critical determinants of host receptor targeting by Neisseria meningitidis and Neisseria gonorrhoeae : identification of Opa adhesiotopes on the N‐domain of CD66 molecules
Authors: Virji Mumtaz , Evans Debbie , Hadfield Andrea , Grunert Fritz , Teixeira Ana M. , Watt Suzanne M.
Published: March 2003

The human pathogens Neisseria meningitidis and Neisseria gonorrhoeae express a family of variable outer membrane opacity‐associated (Opa) proteins that recognize multiple human cell surface receptors. Most Opa proteins target the highly conserved N‐terminal domain of the CD66 family of adhesion molecules, although a few also interact with heparan sulphate proteoglycans. In this study, we observed that at least two Opa proteins of a N. meningitidis strain C751 have the dual capacity to interact with both receptors. In addition, all three Opa proteins of C751 bind equally well to HeLa cells transfected with cDNA encoding the carcinoembryonic antigen [CEA (CD66e)] subgroup of the CD66 family, but show distinct tropism for CGM1‐ (CD66d) and NCA (CD66c)‐expressing cells. Because the C751 Opa proteins make up distinct structures via the surface‐exposed hypervariable domains (HV‐1 and HV‐2), these combinations appear to be involved in tropism for the distinct CD66 subgroups. To define the determinants of receptor recognition, we used mutant proteins of biliary glycoprotein [BGP (CD66a)] carrying substitutions at several predicted exposed sites in the N‐domain and compared their interactions with several Opa proteins of both N. meningitidis and N. gonorrhoeae. The observations applied to the molecular model of the BGP N‐domain that we constructed show that the binding of all Opa proteins tested occurs at the non‐glycosylated (CFG) face of the molecule and, in general, appears to require Tyr‐34 and Ile‐91. Further, efficient interaction of distinct Opa proteins depends on different non‐adjacent amino acids. In the three‐dimensional model, these residues lie in close proximity to Tyr‐34 and Ile‐91 at the CFG face, making continuous binding domains (adhesiotopes). The epitope of the monoclonal antibody YTH71.3 that inhibits Opa/CD66 interactions was also identified within the Opa adhesiotopes on the N‐domain. These studies define the molecular basis that directs the Opa specificity for the CD66 family and the rationale for tropism of the Opa proteins for the CD66 subgroups.
Salmonella typhimurium leucine‐rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems
Authors: Miao Edward A. , Scherer Christina A. , Tsolis Renée M. , Kingsley Robert A. , Adams L. Garry , Bäumler Andreas J. , Miller Samuel I.
Published: March 2003

Salmonellae encode two virulence‐associated type III secretion systems (TTSS) within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Two Salmonella typhimurium genes, sspH1 and sspH2, that encode proteins similar to the Shigella flexneri and Yersinia species TTSS substrates, IpaH and YopM, were identified. SspH1 and SspH2 are proteins containing leucine‐rich repeats that are differentially targeted to the SPI1 and SPI2 TTSS. sspH2 transcription was induced within RAW264.7 macrophages, and was dependent upon the SPI2‐encoded regulator ssrA/ssrB. In contrast, sspH1 transcription is independent of SPI2, and is not induced after bacterial phagocytosis by eukaryotic cells. Infection of eukaryotic cells with strains expressing a SspH2–CyaA fusion protein resulted in SPI2 TTSS‐dependent cAMP increases. In contrast, SspH1–CyaA‐mediated cAMP increases were both SPI1 and SPI2 TTSS dependent. sspH2‐like sequences were found in most Salmonella serotypes examined, whereas sspH1 was detected in only one S. typhimurium isolate, indicating that the copy number of sspH genes can be variable within Salmonella serotypes. S. typhimurium deleted for both sspH1 and sspH2 was not able to cause a lethal infection in calves, indicating that these genes participate in S. typhimurium virulence for animals.
The synthesis of Rhodobacter capsulatus HupSL hydrogenase is regulated by the two‐component HupT/HupR system
Authors: Dischert Wanda , Vignais Paulette M. , Colbeau Annette
Published: March 2003

The synthesis of the membrane‐bound [NiFe]hydrogenase of Rhodobacter capsulatus (HupSL) is regulated negatively by the protein histidine kinase, HupT, and positively by the response regulator, HupR. It is demonstrated in this work that HupT and HupR are partners in a two‐component signal transduction system. The binding of HupR protein to the hupS promoter regulatory region (phupS ) was studied using gel retardation and footprinting assays. HupR protected a 50 bp region localized upstream from the binding site of the histone‐like integration host factor (IHF) regulator. HupR, which belongs to the NtrC subfamily, binds to an enhancer site (TTG‐N5‐CAA) localized at −162/−152 nt. However, the enhancer‐binding HupR protein does not require the RpoN sigma factor for transcriptional activation, as is the case for NtrC from enteric bacteria, but functions with σ70‐RNA polymerase, as is the case for R. capsulatus NtrC. Besides, unlike NtrC from Escherichia coli, HupR activates transcription in the unphosphorylated form and becomes inactive by phosphorylation. This was demonstrated by replacing the putative phosphorylation site (D54) of the HupR protein with various amino acids or by deleting it using site‐directed mutagenesis. Strains expressing mutated hupR genes showed high hydrogenase activities even in the absence of H2, indicating that hupSL transcription is activated by the binding of unphosphorylated HupR protein. Strains producing mutated HupRD54 proteins were derepressed for hupSL expression as were HupT− mutants. It is shown that the phosphorylated form of HupT was able to transfer phosphate to wild‐type HupR protein but not to mutated D54 HupR proteins. Thus, it is concluded that HupT and HupR are the partners of a two‐component regulatory system that regulates hupSL gene transcription.
Negative constrained DNA supercoiling in archaeal nucleosomes
Authors: Musgrave David , Forterre Patrick , Slesarev Alexei
Published: March 2003

Archaeal histones have significant sequence and structural similarity to their eukaryal counterparts. However, whereas DNA is wrapped in negatively constrained supercoils in eukaryal nucleosomes, it has been reported that DNA is positively supercoiled by archaeal nucleosomes. This was inferred from experiments performed at low temperature and low salt concentrations, conditions markedly different from those expected for many archaea in vivo. Here, we report that the archaeal histones HMf and HTz wrap DNA in negatively constrained supercoils in buffers containing potassium glutamate (K‐Glu) above 300 mM, either at 37°C or at 70°C. This suggests that high salt concentrations allow an alternate archaeal nucleosome topology: a left‐handed tetramer rather than the right‐handed tetramer seen in low salt conditions. In contrast, the archaeal histone MkaH produces DNA negative supercoiling at all salt concentrations, suggesting that this duality of structure is not possible for this atypical protein, which is formed by the association of two histone folds in a single polypeptide. These results extend the already remarkable similarity between archaeal and eukaryal nucleosomes, as it has been recently shown that DNA can be wrapped into either positive or negative supercoils around the H3/H4 tetramer. Negative supercoiling could correspond to the predominant physiological mode of DNA supercoiling in archaeal nucleosomes.
The thermostable α‐l‐rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial α‐l‐rhamnoside hydrolase, a new type of inverting glycoside hydrolase
Authors: Zverlov Vladimir V. , Hertel Christian , Bronnenmeier Karin , Hroch Angelika , Kellermann J. , Schwarz Wolfgang H.
Published: March 2003

An α‐l‐rhamnosidase clone was isolated from a genomic library of the thermophilic anaerobic bacterium Clostridium stercorarium and its primary structure was determined. The recombinant gene product, RamA, was expressed in Escherichia coli, purified to homogeneity and characterized. It is a dimer of two identical subunits with a monomeric molecular mass of 95 kDa in SDS polyacrylamide gel electrophoresis. At pH 7.5 it is optimally active at 60°C and insensitive to moderate concentrations of Triton X100, ethanol and EDTA. It hydrolysed p‐nitrophenyl‐α‐l‐rhamnopyranoside, naringin and hesperidin with a specific activity of 82, 1.5 and 0.46 U mg−1 respectively. Hydrolysis occurs by inversion of the anomeric configuration as detected using 1H‐NMR, indicating a single displacement mechanism. Naringin was hydrolysed to rhamnose and prunin, which could further be degraded by incubation with a thermostable β‐glucosidase. The secondary structure of RamA consists of 27% α‐helices and 50% β‐sheets, as detected by circular dichroism. The primary structure of the ramA gene has no similarity to other glycoside hydrolase sequences and possibly is the first member of a new enzyme family.