bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperosmotic shock induces the σ32 and σE stress regulons of Escherichia coli
Authors: Bianchi Allison A. , Baneyx François
Published: March 2003

The rise in the levels of σS that accompanies hyperosmotic shock plays an important role in Escherichia coli survival by increasing the transcription of genes involved in the synthesis and transport of osmoprotectants. To determine if other stress regulons collaborate with σS in dealing with high osmolality, we used single copy fusions of lacZ to representative promoters induced by protein misfolding in the cytoplasm (dnaK and ibp ), extracytoplasmic stress [P3rpoH and htrA(degP )] and cold shock (cspA). Both the σ32‐dependent, dnaK and ibp, promoters, and the σE‐dependent, P3rpoH and htrA, promoters were rapidly but transiently induced when mid‐exponential phase cells were treated with 0.464 M sucrose. The cspA promoter, however, did not respond to the same treatment. Overproduction of the cytoplasmic domain of the σE anti‐sigma factor, RseA, reduced the magnitude of osmotic induction in λφ(P3rpoH::lacZ ) lysogens, but had no effect on the activation of the dnaK and ibp promoters. Similarly, induction of the dnaK::lacZ and ibp::lacZ fusions was not altered in either rpoS or ompR genetic backgrounds. Osmotic upshift led to a twofold increase in the enzymatic activity of the λTLF247 rpoH::lacZ translational fusion whether or not the cells were treated with rifampicin, indicating that both heat shock and exposure to high osmolality trigger a transient increase in rpoH translation. Our results suggest that the σ32, σE and σS regulons closely co‐operate in the managment of hyperosmotic stress. Induction of the σ32 and σE regulons appears to be an emergency response required to repair protein misfolding and facilitate the proper folding of proteins that are rapidly synthesized following loss of turgor, while providing a mechanism to increase the activity of σS, the primary stress factor in osmoadaptation.
Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide
Authors: Hassett Daniel J. , Ma Ju‐Fang , Elkins James G. , McDermott Timothy R. , Ochsner Urs A. , West Susan E. H. , Huang Ching‐Tsan , Fredericks Jessie , Burnett Scott , Stewart Philip S. , McFeters Gordon , Passador Luciano , Iglewski Barbara H.
Published: March 2003

Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm‐grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI‐1 (N‐(3‐oxododecanoyl)‐l‐homoserine lactone) and PAI‐2 (N‐butyryl‐l‐homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn‐SOD and Fe‐SOD, and the major catalase, KatA. The expression of sodA (encoding Mn‐SOD) was particularly dependent on PAI‐1, whereas the influence of autoinducers on Fe‐SOD and KatA levels was also apparent but not to the degree observed with Mn‐SOD. β‐Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI‐1/2‐deficient double mutant partially restored KatA activity, while the addition of PAI‐1 only was sufficient for full restoration of Mn‐SOD activity. In biofilm studies, catalase activity in wild‐type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild‐type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.
Proper ascospore maturation requires the chs1+ chitin synthase gene in Schizosaccharomyces pombe
Authors: Arellano Manuel , Cartagena‐Lirola Hugo , Nasser Hajibagheri M. A. , Durán Angel , Henar Valdivieso M.
Published: March 2003

We have cloned chs1+, a Schizosaccharomyces pombe gene with similarity to class II chitin synthases, and have shown that it is responsible for chitin synthase activity present in cell extracts from this organism. Analysis of this activity reveals that it behaves like chitin synthases from other fungi, although with specific biochemical characteristics. Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth. In contrast, chs1+ expression increases significantly during sporulation, and this is accompanied by an increase in chitin synthase activity. In addition, spore formation is severely affected when both parental strains carry a chs1 deletion, as a result of a defect in the synthesis of the ascospore cell wall. Finally, we show that wild‐type, but not chs1−/chs1 −, ascospore cell walls bind wheatgerm agglutinin. Our results clearly suggest the existence of a relationship between chs1+, chitin synthesis and ascospore maturation in S. pombe.
Characterization of the 20S proteasome from the actinomycete Frankia
Authors: Pouch Marie‐Noëlle , Cournoyer Benoit , Baumeister Wolfgang
Published: March 2003

Frankia is an actinomycete that fixes atmospheric nitrogen in symbiotic association with the root systems of a variety of non‐leguminous plants, denominated actinorhizal plants. Information on the biology of proteolysis in Frankia is almost non‐existent as it is extremely difficult to grow this organism. We have purified 20S proteasomes from Frankia strain ACN14a/ts‐r. It is composed of one α‐subunit and one β‐subunit, which assemble into the canonical structure of four rings of seven subunits each. The enzyme displayed a chymotrypsin‐like activity against synthetic substrates and was sensitive to lactacystin, a specific proteasome inhibitor. Analysis of the structural genes and the flanking regions revealed a similar organization to Rhodococcus erythropolis, Mycobacterium tuberculosis and Streptomyces coelicolor and showed that the β‐subunit is encoded with a 52‐amino‐acid propeptide that is cleaved off in the course of the assembly. We report also for the first time the in vitro assembly of chimeric proteasomes composed of Frankia and Rhodococcus erythropolis subunits, which are correctly assembled and proteolytically active.
pH‐regulated activation and release of a bacteria‐associated phospholipase C during intracellular infection by Listeria monocytogenes
Authors: Marquis Hélène , Hager Elizabeth J.
Published: March 2003

Listeria monocytogenes grows in the cytosol of mammalian cells and spreads from cell to cell without exiting the intracellular milieu. During cell–cell spread, bacteria become transiently entrapped in double‐membrane vacuoles. Escape from these vacuoles is mediated in part by a bacterial phospholipase C (PC‐PLC), whose activation requires cleavage of an N‐terminal peptide. PC‐PLC activation occurs in the acidified vacuolar environment. In this study, the pH‐dependent mechanism of PC‐PLC activation was investigated by manipulating the intracellular pH of the host. PC‐PLC secreted into infected cells was immunoprecipitated, and both forms of the protein were identified by SDS–PAGE fluorography. PC‐PLC activation occurred at pH 7.0 and lower, but not at pH 7.3. Total amounts of PC‐PLC secreted into infected cells increased several‐fold over controls within 5 min of a decrease in intracellular pH, and the active form of PC‐PLC was the most abundant species detected. Bacterial release of active PC‐PLC was dependent on Mpl, a bacterial metalloprotease that processes the proform (proPC‐PLC), and did not require de novo protein synthesis. The amount of proPC‐PLC released in response to a decrease in pH was the same in wild‐type and Mpl‐minus‐infected cells. Immunofluorescence detection of PC‐PLC in infected cells was performed. When fixed and permeabilized infected cells were treated with a bacterial cell wall hydrolase, over 97% of wild‐type and Mpl‐minus bacteria stained positively for PC‐PLC, in contrast to less than 5% in untreated cells. These results indicate that intracellular bacteria carry pools of proPC‐PLC. Upon cell–cell spread, a decrease in vacuolar pH triggers Mpl activation of proPC‐PLC, resulting in bacterial release of active PC‐PLC.
Differential post‐transcriptional regulation of yeast mRNAs in response to high and low glucose concentrations
Authors: Yin Zhikang , Hatton Lee , Brown Alistair J. P.
Published: March 2003

Glucose regulates yeast gene expression at both transcriptional and post‐transcriptional levels. Glucose strongly represses the transcription of the gluconeogenic genes, FBP1 and PCK1, and accelerates the degradation of their mRNAs. Together these mechanisms are responsible for the rapid decrease in gluconeogenic enzyme synthesis when yeast cells switch to glycolytic metabolism. In this study, we show that accelerated gluconeogenic mRNA degradation can be triggered by low concentrations of glucose (<0.02%). This sets the FBP1 and PCK1 mRNAs apart from other glucose‐sensitive mRNAs, such as the Ip mRNA, which only responds to high glucose concentrations (>1%). We also show that accelerated gluconeogenic mRNA degradation is co‐ordinated with transcriptional repression by common signalling components that include sugar kinases and Ras‐cAMP signalling. Furthermore, the ability of the low glucose signal to trigger accelerated gluconeogenic mRNA degradation depends upon the low glucose sensor, Snf3p, but not on the high glucose sensor, Rgt2p. Also, this response is influenced by reg1 and ume5 mutations, but not by grr1 or rgt1 mutations. Our data suggest that several signalling pathways co‐ordinate differential post‐transcriptional and transcriptional responses in yeast, depending upon the amount of glucose available in the medium.
The response regulator RssB, a recognition factor for σS proteolysis in Escherichia coli, can act like an anti‐σS factor
Authors: Becker Gisela , Klauck Eberhard , Hengge‐Aronis Regine
Published: March 2003

σS (RpoS) is the master regulator of the general stress response in Escherichia coli. Several stresses increase cellular σS levels by inhibiting proteolysis of σS, which under non‐stress conditions is a highly unstable protein. For this ClpXP‐dependent degradation, the response regulator RssB acts as a recognition factor, with RssB affinity for σS being modulated by phosphorylation. Here, we demonstrate that RssB can also act like an anti‐sigma factor for σSin vivo, i.e. RssB can inhibit the expression of σS‐dependent genes in the presence of high σS levels. This becomes apparent when (i) the cellular RssB/σS ratio is at least somewhat elevated and (ii) proteolysis is reduced (for example in stationary phase) or eliminated (for example in a clpP mutant). Two modes of inhibition of σS by RssB can be distinguished. The ‘catalytic’ mode is observed in stationary phase cells with a substoichiometric RssB/σS ratio, requires ClpP and therefore probably corresponds to sequestering of σS to Clp protease (even though σS is not degraded). The ‘stoichiometric’ mode occurs in clpP mutant cells upon overproduction of RssB to levels that are equal to those of σS, and therefore probably involves binary complex formation between RssB and σS. We also show that, under standard laboratory conditions, the cellular level of RssB is more than 20‐fold lower than that of σS and is not significantly controlled by stresses that upregulate σS. We therefore propose that antisigma factor activity of RssB may play a role under not yet identified growth conditions (which may result in RssB induction), or that RssB is a former antisigma factor that during evolution was recruited to serve as a recognition factor for proteolysis.
A chromosomally encoded regulator is required for expression of the Yersinia enterocolitica inv gene and for virulence
Authors: Revell Paula A. , Miller Virginia L.
Published: March 2003

The primary invasion factor of Yersinia enterocolitica, invasin, is encoded by inv. inv expression is regulated in response to pH, growth phase and temperature. In vitro, inv is maximally expressed at 26°C, pH 8.0, or 37°C, pH 5.5, in early stationary phase. At 37°C, pH 8.0, inv is weakly expressed. To identify which gene(s) are required for inv regulation, we screened for transposon insertions that decreased expression of an inv–′phoA chromosomal reporter at 26°C. Of 30 000 mutants screened, two were identified that had negligible inv expression in all conditions tested. Both of these independent mutants had an insertion into the same gene, designated rovA (regulator of virulence). RovA has 77% amino acid identity to the Salmonella typhimurium transcriptional regulator SlyA. Complementation with the wild‐type rovA allele restores wild‐type inv expression as monitored by Western blot analysis, tissue culture invasion assay and alkaline phosphatase assay. There is also a significant decrease in invasin levels in bacteria recovered from mice infected with the rovA mutant; therefore, RovA regulates inv expression in vivo as well as in vitro. In the mouse infection model, an inv mutant has a wild‐type LD50, even though the kinetics of infection is changed. In contrast, the rovA mutant has altered kinetics, as well as a 70‐fold increase in the LD50 compared with wild type. Furthermore, because the rovA mutant is attenuated in the mouse model, this suggests that RovA regulates other virulence factors in addition to inv. Analysis of other proposed virulence factors such as Ail, YadA and the Yop proteins shows no regulatory role for RovA. The more severe animal phenotype combined with the lack of impact on known virulence genes aside from inv suggests RovA regulates potentially novel virulence genes of Y. enterocolitica during infection.
DNA methylation‐dependent regulation of Pef expression in Salmonella typhimurium
Authors: Nicholson Brad , Low David
Published: March 2003

Plasmid‐encoded fimbriae (Pef) expressed by Salmonella typhimurium mediate adhesion to mouse intestinal epithelium. The pef operon shares features with the Escherichia coli pyelonephritis‐associated pilus (pap) operon, which is under methylation‐dependent transcriptional regulation. These features include conserved DNA GATC box sites in the upstream regulatory region as well as homologues of the PapI and PapB regulatory proteins. Unlike Pap fimbriae, which are expressed in a variety of laboratory media, Pef fimbriae were expressed only in acidic, rich broth under standing culture conditions. Analysis of S. typhimurium grown under these conditions indicated that Pef production was regulated by a phase variation mechanism, in which the bacterial population was skewed between fimbrial expression (phase ON) and non‐expression (phase OFF) states. Leucine‐responsive regulatory protein (Lrp) and DNA adenine methylase (Dam) were required for pef transcription. In contrast, the histone‐like protein (H‐NS) and the stationary‐phase sigma factor (RpoS) repressed pef transcription. Methylation of the pef GATC II site appeared to be required for pef fimbrial expression based on analysis of a GCTC II mutant that did not express Pef fimbriae. Analysis of the DNA methylation states of pef GATC sites indicated that, under acidic growth conditions, which induced Pef production, most GATC I sites were non‐methylated, whereas GATC II and GATC X were predominantly methylated. The methylation protection at GATC I and GATC II was dependent upon Lrp and was modulated by PefI. Together, these results indicate that Pef production is regulated by DNA methylation, which is the first example of methylation‐dependent gene regulation outside of E. coli.
A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway
Authors: Canosa Inés , Sánchez‐Romero Juan Manuel , Yuste Luis , Rojo Fernando
Published: March 2003

The AlkS regulator, encoded by the alkS gene of the Pseudomonas oleovorans OCT plasmid, activates the expression of a set of enzymes that allow assimilation of alkanes. We show that the AlkS protein regulates, both negatively and positively, the expression of its own gene. In the absence of alkanes, alkS is expressed from promoter PalkS1, which is recognized by σS‐RNA polymerase, and whose activity is very low in the exponential phase of growth and considerably higher in stationary phase. AlkS was found to downregulate this promoter, limiting expression of alkS in stationary phase when alkanes were absent. In the presence of alkanes, AlkS repressed PalkS1 more strongly and simultaneously activated a second promoter for alkS, named PalkS2, located 38 bp downstream from PalkS1. Activation of PalkS2 allowed efficient transcription of alkS when alkanes were present. Transcription from PalkS2 was modulated by catabolite repression when cells were provided with a preferred carbon source. We propose that the expression of alkS is regulated by a positive feedback mechanism, which leads to a rapid increase in alkS transcription when alkanes are present. This mechanism should allow a rapid induction of the pathway, as well as a fast switch‐off when alkanes are depleted. An improved model for the regulation of the pathway is proposed.
Identification of a unique domain essential for Escherichia coli DNA topoisomerase III‐catalysed decatenation of replication intermediates
Authors: Li Zhiyu , Mondragón Alfonso , Hiasa Hiroshi , Marians Kenneth J. , DiGate Russell J.
Published: March 2003

A 17‐amino‐acid residue domain has been identified in Escherichia coli DNA topoisomerase III (Topo III) that is essential for Topo III‐mediated resolution of DNA replication intermediates in vitro. Deletion of this domain reduced Topo III‐catalysed resolution of DNA replication intermediates and decatenation of multiply linked plasmid DNA dimers by four orders of magnitude, whereas reducing Topo III‐catalysed relaxation of negatively supercoiled DNA substrates only 20‐fold. The presence of this domain has been detected in multiple plasmid‐encoded topoisomerases, raising the possibility that these enzymes may also be decatenases.
Non‐hydrolysable GTP‐γ‐S stabilizes the FtsZ polymer in a GDP‐bound state
Authors: Scheffers Dirk‐Jan , Den Blaauwen Tanneke , Driessen Arnold J. M.
Published: March 2003

FtsZ, a tubulin homologue, forms a cytokinetic ring at the site of cell division in prokaryotes. The ring is thought to consist of polymers that assemble in a strictly GTP‐dependent way. GTP, but not guanosine‐5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S), has been shown to induce polymerization of FtsZ, whereas in vitro Ca2+ is known to inhibit the GTP hydrolysis activity of FtsZ. We have studied FtsZ dynamics at limiting GTP concentrations in the presence of 10 mM Ca2+. GTP and its non‐hydrolysable analogue GTP‐γ‐S bind FtsZ with similar affinity, whereas the non‐hydrolysable analogue guanylyl‐imidodiphosphate (GMP‐PNP) is a poor substrate. Preformed FtsZ polymers can be stabilized by GTP‐γ‐S and are destabilized by GDP. As more than 95% of the nucleotide associated with the FtsZ polymer is in the GDP form, it is concluded that GTP hydrolysis by itself does not trigger FtsZ polymer disassembly. Strikingly, GTP‐γ‐S exchanges only a small portion of the FtsZ polymer‐bound GDP. These data suggest that FtsZ polymers are stabilized by a small fraction of GTP‐containing FtsZ subunits. These subunits may be located either throughout the polymer or at the polymer ends, forming a GTP cap similar to tubulin.
Saccharomyces cerevisiae PAU genes are induced by anaerobiosis
Authors: Rachidi Najma , Martinez Marie‐Joséée , Barre Pierre , Blondin Bruno
Published: March 2003

Saccharomyces cerevisiae PAU genes constitute the largest multigene family in yeast, with 23 members located mainly in subtelomeric regions. The role and regulation of these genes were previously unknown. We detected PAU gene expression during alcoholic fermentation. An analysis of PAU gene regulation using PAU–lacZ fusions and Northern analyses revealed that they were regulated by anaerobiosis. PAU genes display, however, different abilities to be induced by anaerobiosis and this appears to be related to their chromosomal localization; two subtelomeric copies are more weakly inducible than an interstitial one. We show that PAU genes are negatively regulated by oxygen and repressed by haem. Examination of PAU gene expression in rox1Δ and tup1Δ strains indicates that PAU repression by oxygen is mediated by an unknown, haem‐dependent pathway, which does not involve the Rox1p anaerobic repressor but requires Tup1p. Given the size of the gene family, PAU genes could be expected to be important during yeast life and some of them probably help the yeast to cope with anaerobiosis.
Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae
Authors: Morona Judy K. , Paton James C. , Miller David C. , Morona Renato
Published: March 2003

In Streptococcus pneumoniae, the first four genes of the capsule locus (cpsA to cpsD) are common to most serotypes. By analysis of various in‐frame deletion and site‐directed mutants, the function of their gene products in capsular polysaccharide (CPS) biosynthesis was investigated. We found that while CpsB, C and D are essential for encapsulation, CpsA is not. CpsC and CpsD have similarity to the amino‐terminal and carboxy‐terminal regions, respectively, of the autophosphorylating protein‐tyrosine kinase Wzc from Escherichia coli. Alignment of CpsD with Wzc and other related proteins identified conserved Walker A and B sequence motifs and a tyrosine rich domain close to the carboxy‐terminus. We have shown that CpsD is also an autophosphorylating protein‐tyrosine kinase and that point mutations in cpsD affecting either the ATP‐binding domain (Walker A motif) or the carboxy‐terminal [YGX]4 repeat domain eliminated tyrosine phosphorylation of CpsD. We describe, for the first time, the phenotypic impact of these two mutations on polysaccharide production and show that they affect CPS production differently. Whereas a mutation in the Walker A motif resulted in loss of encapsulation, mutation of the tyrosines in the [YGX]4 repeat domain resulted in an apparent increase in encapsulation and a mucoid phenotype. These data suggest that autophosphorylation of CpsD at tyrosine attenuates its activity and reduces the level of encapsulation. Additionally, we demonstrated that CpsC is required for CpsD tyrosine phosphorylation and that CpsB influences dephosphorylation of CpsD. These results are consistent with CpsD tyrosine phosphorylation acting to negatively regulate CPS production. This has implications for the function of CpsC/CpsD homologues in both Gram‐positive and Gram‐negative bacteria and provides a mechanism to explain regulation of CPS production during pathogenesis.
The gene for a halophilic β‐galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum
Authors: Patenge Nadja , Haase Andrea , Bolhuis Henk , Oesterhelt Dieter
Published: March 2003

Investigations of transcriptional regulation and the characterization of promoters in homologous expression systems are most easily performed using suitable reporter genes. Presumably because of the high internal salt concentration in halophilic Archaea, the successful application of the commonly used reporter genes has not been reported so far. Recently, the gene for an extremely halophilic β‐galactosidase (bgaH) from Haloferax alicantei has become available. After transformation of Halobacterium salinarum with a vector‐carrying bgaH, the enzyme activity in cell lysates could be readily determined by a simple colorimetric assay and colonies could be screened for activity on plates containing Xgal substrate. Expression of bgaH under the control of various halobacterial promoters of known strength led to different specific β‐galactosidase activities in the lysates. Using Northern blot hybridization and semiquantitative RT‐PCR, it was shown that the bgaH transcript level corresponded to the specific enzyme activity. Therefore, the bgaH gene of Haloferax alicantei appears to be a useful tool for in vivo studies of gene expression in Halobacterium salinarum and possibly other halophilic Archaea.
The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates d‐xylose reductase gene expression
Authors: Hasper Alinda A. , Visser Jaap , De Graaff Leo H.
Published: March 2003

Screening of an Aspergillus niger differential cDNA library, constructed by subtracting cDNA fragments of a xlnR loss‐of‐function mutant from wild‐type cDNA fragments, resulted in the cloning of the gene encoding d‐xylose reductase (xyrA). Northern blot analysis using an A. niger wild‐type strain, a xlnR multiple‐copy strain and a xlnR loss‐of‐function mutant confirmed that the xyrA gene is regulated by XlnR, the transcriptional activator of the xylanolytic enzyme system in A. niger. d‐xylose reductase catalyses the NADPH‐dependent reduction of d‐xylose to xylitol, which is the first step in d‐xylose catabolism in fungi. Until now, XlnR was shown to control the transcription of genes encoding extracellular hydrolytic enzymes involved in cellulose and xylan degradation. In the present study, we show that A. niger is able to harmonize its sugar metabolism and extracellular xylan degradation via XlnR by regulating the expression of XyrA.
A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea
Authors: Gaisser Sabine , Reather James , Wirtz Gabriele , Kellenberger Laurenz , Staunton James , Leadlay Peter F.
Published: March 2003

The biological activity of polyketide antibiotics is often strongly dependent on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin‐producing strain of Saccharopolyspora erythraea, for detection of hybrid glycoside formation, and this system has been used to demonstrate that an amino sugar characteristic of 14‐membered macrolides (d‐desosamine) can be efficiently attached to a 16‐membered aglycone substrate. First, the S. erythraea mutant strain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 from Streptomyces antibioticus, which transfers l‐oleandrose, has recently been shown to transfer rhamnose to the oxygen at C‐3 of erythronolide B and 6‐deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3‐O‐rhamnosyl‐erythronolide B and 3‐O‐rhamnosyl‐6‐deoxyerythronolide B were produced. Having thus validated the expression system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 from Streptomyces fradiae to utilise a different amino sugar, tylM2 was integrated into S. erythraea SGT2, and the resulting strain was fed with the 16‐membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glycoside was isolated in good yield and characterized as 5‐O‐desosaminyl‐tylactone, indicating that TylM2 may be a useful glycosyltransferase for combinatorial biosynthesis. 5‐O‐glucosyl‐tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone in these cells.
Enzyme I and HPr from Lactobacillus casei: their role in sugar transport, carbon catabolite repression and inducer exclusion
Authors: Viana Rosa , Monedero Vicente , Dossonnet Valérie , Vadeboncoeur Christian , Pérez‐Martínez Gaspar , Deutscher Josef
Published: March 2003

We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate–carbohydrate phosphotransferase system (PTS). Northern blot analysis revealed that these two genes are organized in a single‐transcriptional unit whose expression is partially induced. The PTS plays an important role in sugar transport in L. casei, as was confirmed by constructing enzyme I‐deficient L. casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose). Phosphorylation of HPr at Ser‐46 is assumed to be important for the regulation of sugar metabolism in Gram‐positive bacteria. L. casei ptsH mutants were constructed in which phosphorylation of HPr at Ser‐46 was either prevented or diminished (replacement of Ser‐46 of HPr with Ala or Thr respectively). In a third mutant, Ile‐47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P–Ser–HPr for its target protein CcpA. The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non‐PTS sugars ribose or maltose. The ptsH mutants synthesizing Ser‐46–Ala or Ile‐47–Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P–Ser–HPr/CcpA‐mediated mechanism of CCR is common to most low G+C Gram‐positive bacteria. In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P–Ser–HPr participates also in inducer exclusion.
Inhibition of pyruvate‐ferredoxin oxidoreductase gene expression in Giardia lamblia by a virus‐mediated hammerhead ribozyme
Authors: Dan Meixia , Wang Alice L. , Wang Ching C.
Published: March 2003

Giardia lamblia is a primitive eukaryotic microorganism that derives its metabolic energy primarily from anaerobic glycolysis. In trophozoites, pyruvate‐ferredoxin oxidoreductase (PFOR) converts pyruvate to acetyl‐CoA with the transfer of a pair of electrons to ferredoxin, which can then reduce metronidazole and activate it into a potent antigiardiasis agent. It is unclear, however, whether this anaerobic disposal of electrons is essential for the energy metabolism in Giardia. In the present study, cDNAs encoding hammerhead ribozyme flanked with various lengths of antisense PFOR RNA were cloned into a viral vector pC631pac derived from the genome of giardiavirus (GLV). RNA transcripts of the plasmids showed high cleavage activities on PFOR mRNA in vitro. They were introduced into GLV‐infected G. lamblia trophozoites by electroporation and stablized in the transfected cells via serial passages under puromycin selection. PFOR mRNA and enzyme activity in the transfected cells were decreased by 46–60% with the ribozyme PRzS flanked with 20 nt PFOR antisense RNA on each arm and by 69–80% with the ribozyme PRzL flanked with 600 and 1500 nt PFOR antisense RNA. PRzS without the inserted ribozyme or ribozyme flanked with alcohol dehydrogenase E antisense RNA showed no effect on PFOR mRNA and activity. The ribozyme‐transfected cells demonstrated significantly enhanced resistance to metronidazole and grew equally well under anaerobic and aerobic conditions. In contrast, the wild‐type cells grew slightly better anaerobically than the transfectants but did not grow at all in aerobic conditions. Thus, the reduced PFOR expression enables Giardia to grow under molecular oxygen and the presence of PFOR enhances the anaerobic growth of Giardia with an increased susceptibility towards metronidazole. In addition, this study demonstrated for the first time the feasibility of using a viral RNA vector to express a ribozyme targeted at a specific mRNA in G. lamblia to reduce the expression of a specific gene.
Staphylococcal α‐toxin: repair of a calcium‐impermeable pore in the target cell membrane
Authors: Valeva Angela , Walev Iwan , Gerber Angela , Klein Jochen , Palmer Michael , Bhakdi Sucharit
Published: March 2003

Staphylococcal α‐toxin forms heptameric pores that render membranes permeable for monovalent cations. The pore is formed by an amphipathic β‐barrel encompassing amino acid residues 118–140 of each subunit of the oligomer. Human fibroblasts are susceptible to α‐toxin but are able to repair the membrane lesions. Thereby, toxin oligomers remain embedded in the plasma membrane and exposed to the extracellular medium. In this study, we sought to detect structural changes occurring in the pore‐forming sequence during lesion repair. Single cysteine substitution mutants were labelled with the environmentally sensitive fluorochrome acrylodan and, after mixing with wild‐type toxin, incorporated into hybrid heptamers on fibroblast membranes. Formation of the lipid‐inserted β‐barrel was accompanied by characteristic fluorescence emission shifts. After lesion repair, the environment of the residues at the outer surface of the β‐barrel remained unchanged, indicating continued contact with lipids. However, the labelled residues oriented towards the channel lumen underwent a green to blue shift in fluorescence, indicating reduced exposure to water. Pore closure proceeded in the presence of calmodulin inhibitors and of microtubule disruptors; however, it was prevented by cytochalasin D and by inhibitors of lipid metabolism. Our findings reveal the existence of a novel mechanism of membrane repair that may consist in constriction of the inserted proteinaceous pore within the lipid bilayer.
Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p
Authors: Zhang Xiaoting , De Micheli Michelle , Coleman Sean T. , Sanglard Dominique , Moye‐Rowley W. Scott
Published: March 2003

CAP1 encodes a basic region‐leucine zipper (bZip) transcriptional regulatory protein that is required for oxidative stress tolerance in Candida albicans. Cap1p is a homologue of a Saccharomyces cerevisiae bZip transcription factor designated Yap1p that is both required for oxidative stress tolerance and localized to the nucleus in response to the presence of oxidants. Oxidant‐regulated localization of Yap1p to the nucleus requires the presence of a carboxy‐terminal cysteine residue (C629) that is conserved in Cap1p as C477. To examine the role of this conserved cysteine residue, C477 was replaced with an alanine residue. This mutant protein, C477A Cap1p, was analysed for its behaviour both in S. cerevisiae and C. albicans. Wild type and C477A Cap1p were able to complement the oxidant hypersensitivity of a Δyap1 S. cerevisiae strain. Whereas a Yap1p‐responsive lacZ fusion gene was oxidant inducible in the presence of YAP1, the C. albicans Cap1p derivatives were not oxidant responsive in S. cerevisiae. Introduction of wild type and C477A Cap1p‐expressing plasmids into C. albicans produced differential resistance to oxidants. Glutathione reductase activity was found to be inducible by oxidants in the presence of Cap1p but was constitutively elevated in the presence of C477A Cap1p. Western blot assays indicate Cap1p is post‐translationally regulated by oxidants. Green fluorescent protein fusions to CAP1 showed that this protein is localized to the nucleus only in the presence of oxidants while C477A Cap1p is constitutively nuclear localized. Directly analogous to S. cerevisiae Yap1p, regulated nuclear localization of C. albicans Cap1p is crucial for its normal function.
Molecular basis of Salmonella‐induced enteritis
Authors: Wallis Timothy S. , Galyov Edouard E.
Published: March 2003

Salmonella pathogenesis is a complex and multifactorial phenomenon. Many genes required for full virulence in mice have been identified, but only a few of these have been shown to be necessary for the induction of enteritis. Likewise, at least some of the Salmonella virulence factors affecting enteritis do not appear to be required for infection of systemic sites in mice. This suggests that subsets of virulence genes influence distinct aspects of Salmonella pathogenesis. Recently, considerable progress has been made in characterizing the virulence mechanisms influencing enteritis caused by non‐typhoid Salmonella spp. The Salmonella pathogenicity island‐1‐encoded type III secretion system mediates the translocation of secreted effector proteins into target epithelial cells. These effector proteins are key virulence factors required for Salmonella intestinal invasion and the induction of fluid secretion and inflammatory responses.
Comparative proteome analysis of Helicobacter pylori
Authors: Jungblut P. R. , Bumann D. , Haas G. , Zimny‐Arndt U. , Holland P. , Lamer S. , Siejak F. , Aebischer A. , Meyer T. F.
Published: March 2003

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high‐resolution two‐dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two‐dimensional electrophoresis (2‐DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2‐DE patterns with their identified protein species were added to the dynamic 2D‐PAGE database (http://www.mpiib‐berlin.mpg.de/2D‐PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.
GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: a mechanism for disruption of actin microfilament structure
Authors: Von Pawel‐Rammingen Ulrich , Telepnev Maxim V. , Schmidt Gudula , Aktories Klaus , Wolf‐Watz Hans , Rosqvist Roland
Published: March 2003

The YopE cytotoxin of Yersinia pseudotuberculosis is an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid‐encoded type III secretion system. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. Here, we show that YopE exhibits a GTPase‐activating protein (GAP) activity and that the presence of YopE stimulates downregulation of Rho, Rac and Cdc42 activity. YopE has an arginine finger motif showing homology with those found in other GAP proteins. Exchange of arginine 144 with alanine, located in this arginine finger motif, results in an inactive form of YopE that can no longer stimulate GTP hydrolysis by the GTPase. Furthermore, a yopE(R144A) mutant is unable to induce cytotoxicity on cultured HeLa cells in contrast to the corresponding wild‐type strain. Expression of wild‐type YopE in cells of Saccharomyces cerevisiae inhibits growth, while in contrast, expression of the inactive form of YopE, YopE(R144A), does not affect the yeast cells. Co‐expression of proteins belonging to the Rho1 pathway of yeast, Rho1, Rom2p, Bck1 and Ste20, suppressed the growth phenotype of YopE in yeast cells. These results provide evidence that YopE exhibits a GAP activity to inactivate RhoGTPases, leading to depolymerization of the actin stress fibres in eukaryotic cells and growth inhibition in yeast.
Targeted gene disruption in Candida albicans wild‐type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates
Authors: Wirsching Stephanie , Michel Sonja , Morschhäuser Joachim
Published: March 2003

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole‐susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild‐type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild‐type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP‐mediated, site‐specific recombination (MPAR‐flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole‐resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock‐out mutations in any C. albicans wild‐type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.
Suppression of a DnaX temperature‐sensitive polymerization defect by mutation in the initiation gene, dnaA, requires functional oriC
Authors: Blinkova Alexandra , Ginés‐Candelaria Edwin , Ross Julie D. , Walker James R.
Published: March 2003

Temperature sensitivity of DNA polymerization and growth, resulting from mutation of the τ and γ subunits of Escherichia coli DNA polymerase III, are suppressed by Cs,Sx mutations of the initiator gene, dnaA. These mutations simultaneously cause defective initiation at 20°C. Efficient suppression, defined as restoration of normal growth rate at 39°C to essentially all the cells, depends on functional oriC. Increasing DnaA activity in a strain capable of suppression, by introducing a copy of the wild‐type allele, increasing the suppressor gene dosage or introducing a seqA mutation, reversed the suppression. This suggests that the suppression mechanism depends on reduced activity of DnaACs,Sx. Models that assume that suppression results from an initiation defect or from DnaACs,Sx interaction with polymerization proteins during nascent strand synthesis are proposed.
Subcellular localization of Dna‐initiation proteins of Bacillus subtilis: evidence that chromosome replication begins at either edge of the nucleoids
Authors: Imai Yukiho , Ogasawara Naotake , Ishigo‐oka Daisuke , Kadoya Ryosuke , Daito Tsutomu , Moriya Shigeki
Published: March 2003

We examined the intracellular distribution of Bacillus subtilis Dna‐initiation proteins by immunofluorescence microscopy to visualize the initiation complex of replication in vivo. DnaA was distributed throughout the cytoplasm, but both DnaB and DnaI were always detected as foci during the cell‐division cycle. Interaction of DnaI with the DnaC helicase by the yeast two‐hybrid assay suggests that DnaI acts as a helicase loader. The number of DnaB and DnaI foci within the cell exceeded that of oriC. Although the foci were not always co‐localized with oriC, they seemed to be localized near the outer or inner edges of the nucleoids at initiation of replication. When the replication cycle was synchronized in cells using a temperature‐sensitive dnaA mutant, duplication of the oriC region was observed predominantly near an edge of the nucleoid. Before initiation occurred, each one of the DnaB and DnaI foci was frequently observed near there. Furthermore, DnaX–GFP (DnaX is a component of DNA polymerase III) foci were detected near either of the edges of the nucleoids at the onset of replication. These results suggest that the replisome is recruited into oriC near either edge of the nucleoids to initiate chromosome replication in B. subtilis.
Glycosylation deficiency phenotypes resulting from depletion of GDP‐mannose pyrophosphorylase in two yeast species
Authors: Warit Saradee , Zhang Nianshu , Short Andrea , Walmsley Richard M. , Oliver Stephen G. , Stateva Lubomira I.
Published: March 2003

The genes encoding GDP‐mannose pyrophosphorylase from Saccharomyces cerevisiae (SRB1/PSA1) and Candida albicans (CaSRB1) were expressed under the control of the tightly regulated promoters of MET3 and CaMET3 respectively. Northern analysis showed that the addition of methionine effectively blocks the transcription of pMET3‐SRB1/PSA1 and pCaMET3CaSRB1 expression cassettes, which had been integrated into the genomes of appropriate mutants. Methionine‐mediated repression of CaSRB1 caused loss of viability in C. albicans, demonstrating that, as in S. cerevisiae, the gene is essential for growth. Depletion of GDP‐mannose pyrophosphorylase had a highly pleiotropic effect in the two yeasts. The major phenotypes observed were lysis, failure of cell separation and/or cytokinesis, impaired bud growth and bud's site selection, clumping and flocculation, as well as increased sensitivity to a wide range of antifungal drugs and cell wall inhibitors, and impaired hyphal switching ability. These phenotypes resulted from defects in glycosylation, as demonstrated by reduced affinity for Alcian blue and sensitivity to hygromycin B. Our results provide new information about the roles of protein glycosylation in yeast and, in particular, the steps that require GDP‐mannose in the fungal pathogen C. albicans.
Signature‐tagged and directed mutagenesis identify PABA synthetase as essential for Aspergillus fumigatus pathogenicity
Authors: Brown Jeremy S. , Aufauvre‐Brown Agnès , Brown Juliane , Jennings Joanne M. , Arst Herbert , Holden David W.
Published: March 2003

Signature‐tagged mutagenesis (STM) is a method that has been used to screen for genes required for in vivo survival of pathogenic bacteria, but has not been used to investigate a eukaryotic pathogen in an animal model of disease. We have adapted STM to identify genes required for in vivo growth of the opportunistic fungal pathogen Aspergillus fumigatus. Using a mouse model of invasive pulmonary aspergillosis, we have isolated several mutant strains with defects in their ability to replicate in vivo. One strain unable to cause lethal infection was further characterized and found to have an insertion into the promoter of a gene (pabaA) encoding para‐aminobenzoic acid synthetase, an enzyme catalyzing a late step in the biosynthesis of folate. The complete inability of this strain, and other pabaA− strains constructed in this study by targeted gene deletion, to cause lethal infection in mice confirms the importance of the folate synthesis pathway for in vivo survival of this pathogen. The successful application of STM to A. fumigatus demonstrates that in vivo genetic analysis of eukaryotic pathogens is feasible and could result in the identification of potential targets, such as para‐aminobenzoic acid synthetase, for novel antifungal therapies.