bluuebunny/crossref_metadata_2025 huggingface dataset:
Independent acquisition and insertion into different chromosomal locations of the same pathogenicity island in Yersinia pestis and Yersinia pseudotuberculosis
Authors: Hare Janelle M. , Wagner Alexandra K. , McDonough Kathleen A.
Published: March 2003

We show that Yersinia pestis and pesticin‐sensitive isolates of Y. pseudotuberculosis possess a common 34 kbp DNA region that has all the hallmarks of a pathogenicity island and is inserted into different asparaginyl tRNA genes at different chromosomal locations in each species. This pathogenicity island (YP‐HPI) is marked by IS100, has a G + C content different from its host, is flanked by 24 bp direct repeats, encodes a putative, P4‐like integrase and contains the iron uptake virulence genes from the pgm locus of Y. pestis. These findings indicate independent horizontal acquisition of this island by Y. pestis and Y. pseudotuberculosis. The two YP‐HPI locations and their possession of an integrase gene support a model of site‐specific integration of the YP‐HPI into these bacteria.
The DNA binding protein Tfx from Methanobacterium thermoautotrophicum: structure, DNA binding properties and transcriptional regulation
Authors: Hochheimer Andreas , Hedderich Reiner , Thauer Rudolf K.
Published: March 2003

In Methanobacterium thermoautotrophicum, the fmdECB operon encoding the molybdenum formylmethanofuran dehydrogenase is directly preceded by an open reading frame tfx predicted to encode a DNA binding protein. The 16.1 kDa protein has an N‐terminal basic domain with a helix–turn–helix motif for DNA binding and a C‐terminal acidic domain possibly for transcriptional activation. We report here on the DNA binding properties of the Tfx protein heterologously overproduced in Escherichia coli. Tfx was found to bind specifically to a DNA sequence downstream of the promoter of the fmdECB operon, as shown by electrophoretic mobility shift assays and DNase I footprint analysis. Northern blot hybridizations revealed that transcription of tfx is repressed during the growth of M. thermoautotrophicum in the presence of tungstate. Based on its structure and properties, the DNA binding protein Tfx is proposed to be a transcriptional regulator composed of a basic DNA binding domain and an acidic activation domain.
Negative and positive regulation of the non‐osmoregulated ompS1 porin gene in Salmonella typhi : a novel regulatory mechanism that involves OmpR
Authors: Oropeza Ricardo , Sampieri Clara Luz , Puente José Luis , Calva Edmundo
Published: March 2003

The Salmonella typhi ompS1 gene codes for an outer membrane protein of the OmpC/OmpF porin family. It is expressed at very low levels, relative to the major porins. However, deletion analysis of the 5′ regulatory region showed that the gradual removal of nucleotides −310 to −88, upstream from the P1 major transcriptional start‐point, resulted in a stepwise increase in expression, reaching levels 10‐fold above those for the ompC major porin gene. Hence, this 222 bp segment contains cis‐acting regulatory elements involved in negative control. Primer extension analysis revealed the presence of three promoters: P1 activity was OmpR dependent; P2 was expressed at a lower level in the absence of OmpR; and P3 had a minor constitutive activity. OmpR bound preferentially to box II, an 18 bp F1/C1 canonical site, the removal (−88 to −66) of which resulted in a decrease in expression thus supporting its role in positive control. Expression of ompS1 was not induced by a set of stress conditions, including a shift in osmolarity, nor was the IHF regulator involved in negative control. An ompS1 homologue was found in E. coli K‐12, which contains a nonsense codon and a shift in the reading frame, whereas Salmonella typhimurium contains an open reading frame in this region. Thus, S. typhi ompS1 provides novel features in OmpR regulation.
Helicobacter pylori with separate β‐ and β′‐subunits of RNA polymerase is viable and can colonize conventional mice
Authors: Raudonikiene Ausra , Zakharova Natalya , Su Wan Wen , Jeong Jin‐Yong , Bryden Louis , Hoffman Paul S. , Berg Douglas E. , Severinov Konstantin
Published: March 2003

The genes encoding the β‐ and β′‐subunits of RNA polymerase (rpoB and rpoC respectively) are fused as one continuous open reading frame in Helicobacter pylori and in other members of this genus, but are separate in other bacterial taxonomic groups, including the closely related genus Campylobacter. To test whether this β–β′ tethering is essential, we used polymerase chain reaction‐based cloning to separate the rpoB and rpoC moieties of the H. pylori rpoB–rpoC fusion gene with a non‐polar chloramphenicol resistance cassette containing a new translational start, and introduced this construct into H. pylori by electrotransformation. H. pylori containing these separated rpoB and rpoC genes in place of the native fusion gene produced non‐tethered β and β′ RNAP subunits, grew well in culture and colonized and proliferated well in conventional C57BL/6 mice. Thus, the extraordinary β–β′ tethering is not essential for H. pylori viability and gastric colonization.
IS911 transposon circles give rise to linear forms that can undergo integration in vitro
Authors: Ton‐Hoang B. , Polard P. , Haren L. , Turlan C. , Chandler M.
Published: March 2003

High levels of expression of the transposase OrfAB of bacterial insertion sequence IS911 leads to the formation of excised transposon circles, in which the two abutted ends are separated by 3 bp. Initially, OrfAB catalyses only single‐strand cleavage at one 3′ transposon end and strand transfer of that end to the other. It is believed that this molecule, in which both transposon ends are held together in a single‐strand bridge, is then converted to the circular form by the action of host factors. The transposon circles can be integrated efficiently into an appropriate target in vivo and in vitro in the presence of OrfAB and a second IS911 protein OrfA. In the results reported here, we have identified linear transposon forms in vivo from a transposon present in a plasmid, raising the possibility that IS911 can also transpose using a cut‐and‐paste mechanism. However, the linear species appeared not to be derived directly from the plasmid‐based copy by direct double‐strand cleavages at both ends, but from preformed excised transposon circles. This was confirmed further by the observation that OrfAB can cleave a cloned circle junction both in vivo and in vitro by two single‐strand cleavages at the 3′ transposon ends to generate a linear transposon form with a 3′‐OH and a three‐nucleotide 5′ overhang at the ends. Moreover, while significantly less efficient than the transposon circle, a precleaved linear transposon underwent detectable levels of integration in vitro. The possible role of such molecules in the IS911 transposition pathway is discussed.
Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide
Authors: Stingele Francesca , Vincent Sébastien J. F. , Faber Elisabeth J. , Newell John W. , Kamerling Johannis P. , Neeser Jean‐Richard
Published: March 2003

Streptococcus thermophilus Sfi6 produces an exopolysaccharide (EPS) composed of glucose, galactose and N‐acetylgalactosamine in the molar ratio of 1:2:1. The genes responsible for the EPS biosynthesis have been isolated previously and found to be clustered in a 14.5 kb region encoding 13 genes. Transfer of this gene cluster into a non‐EPS‐producing heterologous host, Lactococcus lactis MG1363, yielded an EPS with a similar high molecular weight, but a different structure from the EPS from the native host. The structure of the recombinant EPS was determined by means of 1H homonuclear and 1H‐13C heteronuclear two‐dimensional nuclear magnetic resonance (NMR) spectra and was found to be  → 3)‐β‐d‐Glcp‐(1 → 3)‐α‐d‐Galp‐(1 → 3)‐β‐d‐Galp‐(1 →  as opposed to  → 3)[α‐d‐Galp‐(1 → 6)]‐β‐d‐Glcp‐(1 → 3)‐α‐d‐GalpNAc‐(1 → 3)‐β‐d‐Galp‐(1 →  for the wild‐type S. thermophilus Sfi6. Furthermore, L. lactis MG1363 (pFS101) was also lacking a UDP‐N‐acetylglucosamine C4‐epimerase activity, which would provide UDP‐GalNAc for a GalNAc incorporation into the EPS and probably caused the substitution of GalNAc by Gal in the recombinant EPS. This modification implies that (i) bacterial glycosyltransferases could potentially have multiple specificities for the donor and the acceptor sugar molecule; and (ii) the repeating unit polymerase can recognize and polymerize a repeating unit that differs in the backbone as well as in the side‐chain from its native substrate.
In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus
Authors: Bowman William C. , Du Shouying , Bauer Carl E. , Kranz Robert G.
Published: March 2003

It has been known for over half a century that anoxygenic photosynthetic bacteria maximally synthesize their photosystems in the absence of oxygen. During the last decade, it has become clear that this regulation is largely at the transcriptional level, with photosynthesis genes expressed only under anaerobic conditions. We describe here in vitro reconstitution of activation and repression of three photosynthesis promoters, bch (bacteriochlorophyll biosynthesis), puc (light‐harvesting II apoproteins) and puf (reaction centre and light‐harvesting I apoproteins) using purified transcription factors and RNA polymerase from Rhodobacter capsulatus. Previous genetic results have indicated that each of these three promoters is differentially regulated by three key regulators: CrtJ acting as a repressor of bch and puc and the two‐component regulators RegA/RegB, which are activators of puc and pufThese regulators are distinct from those that mediate oxygen control in enteric bacteria. Our in vitro studies show that these purified regulators directly control the expression of the housekeeping RNA polymerase at these promoters. High‐level basal expression of the bch promoter is shown to be repressed by CrtJ. The puc promoter is activated by the RegB‐phosphorylated RegA protein and additionally repressed by CrtJ. At the puc promoter, CrtJ effectively competes for promoter binding with RegA, while at the bch promoter, repression appears to be by competition for the RNA polymerase binding site. In contrast to what has been suggested previously, the RegA‐activated puf promoter is demonstrated as being recognized by the housekeeping RNA polymerase. We also discuss evidence that RegA∼P activation of the puc and puf promoters involves recruitment of RNA polymerase by different modes of protein–protein interaction.
Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica: requirement for translocators YopB and YopD, but not LcrG
Authors: Neyt Cécile , Cornelis Guy R.
Published: March 2003

The Yersinia survival strategy is based on its ability to inject effector Yops into the cytosol of host cells. Translocation of these effectors across the eukaryotic cell membrane requires YopB, YopD and LcrG, but the mechanism is unclear. An effector polymutant of Y. pseudotuberculosis has a YopB‐dependent contact haemolytic activity, indicating that YopB participates in the formation of a pore in the cell membrane. Here, we have investigated the formation of such a pore in the plasma membrane of macrophages. Infection of PU5‐1.8 macrophages with an effector polymutant Y. enterocolitica led to complete flattening of the cells, similar to treatment with the pore‐forming streptolysin O from Streptococcus pyogenes. Upon infection, cells released the low‐molecular‐weight marker BCECF (623 Da) but not the high‐molecular‐weight lactate dehydrogenase, indicating that there was no membrane lysis but, rather, insertion of a pore of small size into the macrophage plasma membrane. Permeation to lucifer yellow CH (443 Da) but not to Texas red‐X phalloidin (1490 Da) supported this hypothesis. All these events were found to be dependent not only on translocator YopB as expected but also on YopD, which was required equally. In contrast, LcrG was not necessary. Consistently, lysis of sheep erythrocytes was also dependent on YopB and YopD, but not on LcrG.
Enhanced monomer–monomer interactions can suppress the recombination deficiency of the recA142 allele
Authors: Zaitsev Eugene N. , Kowalczykowski Stephen C.
Published: March 2003

The RecA142 protein, in which valine is substituted for isoleucine‐225, is defective for genetic recombination in vivo and for DNA strand exchange activity in vitro under conventional growth and reaction conditions respectively. However, we show that mildly acidic conditions restore both the in vitro DNA strand exchange activity and the in vivo function of RecA142 protein, suggesting that recombination function can be restored by a slight change in protein structure elicited by protonation. Indeed, we identified an intragenic suppressor of the recombination deficiency of the recA142 allele. This suppressor mutation is a substitution of leucine for glutamine at position 124. Based on the three‐dimensional structure, the Q‐124L substitution is predicted to make a new monomer–monomer contact with residue phenylalanine‐21 of the adjacent RecA monomer. The Q‐124L mutation is not allele specific, because it also suppresses the recombination deficiency of a recA deletion (Δ9), lacking nine amino acids at the amino‐terminus, presumably by reinforcing the monomer–monomer interactions that are attenuated by the Δ9 deletion. Expression of RecA(Q‐124L) protein is toxic to Escherichia coli, presumably because of enhanced affinity for DNA. We speculate as to how enhanced monomer–monomer interactions and acidic pH conditions can restore the recombination activity of some defective recA alleles.
Molecular switches — the ON and OFF of bacterial phase variation
Authors: Henderson Ian R. , Owen Peter , Nataro James P.
Published: March 2003

The expression of most bacterial genes is controlled at the level of transcription via promoter control mechanisms that permit a graded response. However, an increasing number of bacterial genes are found to exhibit an ‘all‐or‐none’ control mechanism that adapts the bacterium to more than one environment. One such mechanism is phase variation, traditionally defined as the high‐frequency ON↔OFF switching of phenotype expression. Phase variation events are usually random, but may be modulated by environmental conditions. The mechanisms of phase variation events and their significance within the microbial community are discussed here.
Specificity crosstalk among group 1 and group 2 sigma factors in the cyanobacterium Synechococcus sp. PCC7942: in vitro specificity and a phylogenetic analysis
Authors: Goto‐Seki Asako , Shirokane Masao , Masuda Susumu , Tanaka Kan , Takahashi Hideo
Published: March 2003

The chromosome of the cyanobacterium Synechococcus sp. PCC7942 contains at least one group 1 (rpoD1) and three group 2 (rpoD2, rpoD3 and rpoD4) sigma factor genes. In this study, we have analysed the structure of rpoD3 and rpoD4 and have shown that these genes are dispensable for growth at normal physiological conditions. An RNA polymerase core enzyme of the cyanobacterial strain was purified, reconstituted with the recombinant sigma factors (the rpoD1, rpoD3 and rpoD4 gene products), and the resultant holoenzymes were examined in vitro for transcription specificity. All of the holoenzymes recognized canonical promoters of Escherichia coli as well as cyanobacterial rrnA, cpcB1A1 P1a and rpoD1 promoters, although the three holoenzymes had some preference for specific promoters. These results suggest that group 1 as well as group 2 sigma factors of cyanobacteria may direct transcription initiation from the eubacterial consensus‐type promoters containing the Pribnow −10 element, and we postulate that specificity crosstalk is a common characteristic among eubacterial group 1 and group 2 sigma factors. Phylogenetic analyses revealed that most group 2 sigma factors were positioned in one of four distinct clusters. The implication of the phylogenetic tree is also discussed in this paper.
Contribution of N‐ and C‐terminal domains to the function of Hsp90 in Saccharomyces cerevisiae
Authors: Scheibel Thomas , Weikl Tina , Rimerman Ronald , Smith David , Lindquist Susan , Buchner Johannes
Published: March 2003

The molecular chaperone Hsp90 is a regulatory component of some key signalling proteins in the cytosol of eukaryotic cells. For some of these functions, its interaction with co‐chaperones is required. Limited proteolysis defined stable folded units of Hsp90. Both an N‐terminal (N210) and a C‐terminal (262C) fragment interact with non‐native substrate proteins in vitro, but with different specificity and ATP dependence. Here, we analysed the functional properties of these Hsp90 fragments in vivo and in vitro. We determined their influence on the general viability and cell growth of Saccharomyces cerevisiae. Expression of N210 or 262C resulted in a dominant‐negative phenotype in several yeast strains tested. Their expression was not toxic, but inhibited cell growth. Further, both were unable to restore viability to Hsp90‐depleted cells. In addition, N210 and 262C influence the maturation of Hsp90 substrates, such as the glucocorticoid receptor and pp60v–Src kinase. Specifically, 262C forms partially active chaperone complexes, leading to an arrest of the chaperoned substrate at a certain stage of its maturation cycle. This demonstrates the requirement of a sophisticated and cofactor‐regulated interplay between N‐ and C‐terminal activities for Hsp90 function in vivo.
Role of flagellins from A and B loci in flagella formation of Halobacterium salinarum
Authors: Tarasov Valery Y. , Pyatibratov Michael G. , Tang Sen‐Lin , Dyall‐Smith Michael , Fedorov Oleg V.
Published: March 2003

Haloarchaeal flagella are composed of a number of distinct flagellin proteins, specified by genes in two separate operons (A and B). The roles of these flagellins were assessed by studying mutants of H. salinarum with insertions in either the A or the B operon. Cells of the flgA− mutant produced abnormally short, curved flagella that were distributed all over the cell surface. The flgA2− strain produced straight flagella, mainly found at the poles. The flgB− mutant had flagella of the same size and spiral shape as wild‐type cells, but these cells also showed unusual outgrowths, which appeared to be sacs filled with basal body‐like structures. In broth cultures of this mutant, the medium accumulated flagella with basal body‐like structures at their ends.
Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels
Authors: Ferguson Gail P. , Battista John R. , Lee Annettee T. , Booth Ian R.
Published: March 2003

The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated. Exposure of E. coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA. The activity of the KefB and KefC potassium channels protects E. coli cells against methylglyoxal and limits the amount of DNA damage. In mutants lacking KefB and KefC, methylglyoxal‐induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation. This provides evidence that acidification of the cytoplasm protects E. coli DNA against methylglyoxal. By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure. However, protection by KefB and KefC occurred independently of UvrA. Although we present evidence that exposure of E. coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal‐induced death. The implications of these findings will be discussed.
β‐1,6‐Glucan synthesis in Saccharomyces cerevisiae
Authors: Shahinian Serge , Bussey Howard
Published: March 2003

β‐1,6‐Glucan is an essential fungal‐specific component of the Saccharomyces cerevisiae cell wall that interconnects all other wall components into a lattice. Considerable biochemical and genetic effort has been directed at the identification and characterization of the steps involved in its biosynthesis. Structural studies show that the polymer plays a central role in wall structure, attaching mannoproteins via their glycosylphosphatidylinositol (GPI) glycan remnant to β‐1,3‐glucan and chitin. Genetic approaches have identified genes that upon disruption result in β‐1,6‐glucan defects of varying severity, often with reduced growth or lethality. These gene products have been localized throughout the secretory pathway and at the cell surface, suggesting a possible biosynthetic route. Current structural and genetic data have therefore allowed the development of models to predict biosynthetic events. Based on knowledge of β‐1,3‐glucan and chitin synthesis, it is likely that the bulk of β‐1,6‐glucan polymer synthesis occurs at the cell surface, but requires key prior intracellular events. However, the activity of most of the identified gene products remain unknown, making it unclear to what extent and how directly they contribute to the synthesis of this polymer. With the recent availability of new tools, reagents and methods (including genomics), the field is poised for a convergence of biochemical and genetic methods to identify and characterize the biochemical steps in the synthesis of this polymer.
A new mechanism for the control of a prokaryotic transcriptional regulator: antagonistic binding of positive and negative effectors
Authors: Schreiber Valérie , Steegborn Clemens , Clausen Tim , Boos Winfried , Richet Evelyne
Published: March 2003

MalT, the transcriptional activator of the Escherichia coli maltose regulon, self‐associates, binds promoter DNA and activates initiation of transcription only in the presence of ATP and maltotriose, the inducer. In vivo studies have revealed that MalT action is negatively controlled by the MalY protein. Using a biochemical approach, we analyse here the mechanism whereby MalY represses MalT activity. We show that MalY inhibits transcription activation by MalT in a purified transcription system. In vitro, a constitutive MalT variant (which is partially active in the absence of maltotriose) is less sensitive than wild‐type MalT to repression by MalY, as observed in vivo. We demonstrate that MalY forms a complex with MalT only in the absence of maltotriose and that, conversely, MalY inhibits maltotriose binding by MalT. Together, these results establish that MalY acts directly upon MalT without the help of any factor, and that MalY is a negative effector of MalT competing with the inducer for MalT binding.
Homing of a bacterial group II intron with an intron‐encoded protein lacking a recognizable endonuclease domain
Authors: Martínez‐Abarca Francisco , García‐Rodríguez Fernando M. , Toro Nicoláás
Published: March 2003

RmInt1 is a functional group II intron found in Sinorhizobium meliloti where it interrupts a group of IS elements of the IS630‐Tc1 family. In contrast to many other group II introns, the intron‐encoded protein (IEP) of RmInt1 lacks the characteristic conserved part of the Zn domain associated with the IEP endonuclease activity. Nevertheless, in this study, we show that RmInt1 is capable of inserting into a vector containing the DNA spanning the RmInt1 target site from the genome of S. meliloti. Efficient homing was also observed in the absence of homologous recombination (RecA− strains). In addition, it is shown that RmInt1 is able to move to its target in a heterologous host (S. medicae). Homing of RmInt1 occurs very efficiently upon DNA target uptake (conjugation/electroporation) by the host cell resulting in a proportion of invaded target of 11–30%. Afterwards, the remaining intronless target DNA is protected from intron invasion.
Mutational analysis of the functional motifs of RuvB, an AAA+ class helicase and motor protein for Holliday junction branch migration
Authors: Iwasaki Hiroshi , Han Yong‐Woon , Okamoto Takashi , Ohnishi Takayuki , Yoshikawa Manabu , Yamada Kazuhiro , Toh Hiroyuki , Daiyasu Hiromi , Ogura Teru , Shinagawa Hideo
Published: March 2003

Escherichia coli RuvB protein, together with RuvA, promotes branch migration of Holliday junctions during homologous recombination and recombination repair. The RuvB molecular motor is an intrinsic ATP‐dependent DNA helicase with a hexameric ring structure and its architecture has been suggested to be related to those of the members of the AAA+ protein class. In this study, we isolated a large number of plasmids carrying ruvB mutant genes and identified amino acid residues important for the RuvB functions by examining the in vivo DNA repair activities of the mutant proteins. Based on these mutational studies and amino acid conservation among various RuvBs, we identified 10 RuvB motifs that agreed well with the features of the AAA+ protein class and that distinguished the primary structure of RuvB from that of typical DNA/RNA helicases with seven conserved helicase motifs.
Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity
Authors: Baliga Nitin S. , DasSarma Shiladitya
Published: March 2003

Transcription from the bop promoter in the haloarchaeon Halobacterium NRC‐1, is highly induced under oxygen‐limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non‐B structure was found 3′ to the TATA box within an 11 bp alternating purine–pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single‐base substitutions of A(r)G either 23 or 19 bp 5′ to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5′ to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3′ end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.
Redox regulation of AMP synthesis in yeast: a role of the Bas1p and Bas2p transcription factors
Authors: Pinson Benoît , Gabrielsen Odd S. , Daignan‐Fornier Bertrand
Published: March 2003

Expression of yeast AMP synthesis genes (ADE genes) was severely affected when cells were grown under oxidative stress conditions. To get an insight into the molecular mechanisms of this new transcriptional regulation, the role of the Bas1p and Bas2p transcription factors, known to activate expression of the ADE genes, was investigated. In vitro, DNA‐binding of Bas1p was sensitive to oxidation. However, this sensitivity could not account for the regulation of the ADE genes because we showed, using a BAS1‐VP16 chimera, that Bas1p DNA‐binding activity was not sensitive to oxidation in vivo. Consistently, a triple cysteine mutant of Bas1p (fully resistant to oxidation in vitro) was unable to restore transcription of the ADE genes under oxidative conditions. We then investigated the possibility that Bas2p could be the oxidative stress responsive factor. Interestingly, transcription of the PHO5 gene, which is dependent on Bas2p but not on Bas1p, was found to be severely impaired by oxidative stress. Nevertheless, a Bas2p cysteine‐free mutant was not sufficient to confer resistance to oxidative stress. Finally, we found that a Bas1p–Bas2p fusion protein restored ADE gene expression under oxidative conditions, thus suggesting that redox sensitivity of ADE gene expression could be due to an impairment of Bas1p/Bas2p interaction. This hypothesis was further substantiated in a two hybrid experiment showing that Bas1p/Bas2p interaction is affected by oxidative stress.
Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high‐affinity ferrous iron transporter
Authors: Velayudhan Jyoti , Hughes Nicky J. , McColm Andrew A. , Bagshaw Julie , Clayton Chris L. , Andrews Simon C. , Kelly David J.
Published: March 2003

The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe2+‐ and Fe3+‐citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was ≈ 10‐fold lower than in the wild type (with 0.5 μM Fe), irrespective of whether Fe was supplied in the Fe2+ or Fe3+ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe2+ and Fe3+ transport. The growth inhibition exhibited by the feoB mutant in Fe‐deficient media was relieved by human holo‐transferrin, holo‐lactoferrin and Fe3+‐dicitrate, but not by FeSO4. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe2+ transport in the wild type suggested the presence of high‐ and low‐affinity uptake systems. The high‐affinity system (apparent Ks = 0.54 μM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe2+ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe2+ and Fe3+ uptake implied the concerted involvement of both an Fe3+ reductase and FeoB in the uptake of Fe supplied as Fe3+. Taken together, the results are consistent with FeoB‐mediated Fe2+ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low‐pH, low‐O2 environment of the stomach.
Response of Saccharomyces cerevisiae to severe osmotic stress: evidence for a novel activation mechanism of the HOG MAP kinase pathway
Authors: Van Wuytswinkel O. , Reiser V. , Siderius M. , Kelders M. C. , Ammerer G. , Ruis H. , Mager W. H.
Published: March 2003

The HOG/p38 MAP kinase route is an important stress‐activated signal transduction pathway that is well conserved among eukaryotes. Here, we describe a novel mechanism of activation of the HOG pathway in budding yeast. This mechanism operates upon severe osmostress conditions (1.4 M NaCl) and is independent of the Sln1p and Sho1p osmosensors. The alternative input feeds into the HOG pathway MAPKK Pbs2p and requires activation of Pbs2p by phosphorylation. We show that, upon severe osmotic shock, Hog1p nuclear accumulation and phosphorylation is delayed compared with mild stress. Moreover, both events lost their transient pattern, presumably because of the absence of negative feedback mediated by Ptp2p tyrosine phosphatase, which we found to be localized in the nucleus. Under severe osmotic stress conditions, the delayed nuclear accumulation correlates with a delay in stress‐responsive gene expression. Severe osmoshock leads to a situation in which active and nuclear‐localized Hog1p is transiently unable to induce transcription of osmotic stress‐responsive genes. It also appeared from our studies that the Sho1p osmosensor is less active under severe osmotic stress conditions, whereas the Sln1p/Ypd1p/Ssk1p sensor and signal transducer functions normally under these circumstances.
YscP, a Yersinia protein required for Yop secretion that is surface exposed, and released in low Ca2+
Authors: Stainier I. , Bleves S. , Josenhans C. , Karmani L. , Kerbourch C. , Lambermont I. , Tötemeyer S. , Boyd A. , Cornelis G. R.
Published: March 2003

The Yersinia Ysc apparatus is made of more than 20 proteins, 11 of which have homologues in many type III systems. Here, we characterize YscP from Yersinia enterocolitica. This 515‐residue protein has a high proline content, a large tandem repetition and a slow migration in SDS–PAGE. Unlike the products of neighbouring genes, it has a counterpart only in Pseudomonas aeruginosa and it varies even between Yersinia Ysc machineries. An yscPΔ97−465 mutant was unable to secrete any Yop, even under conditions overcoming feedback inhibition of Yop synthesis. Interestingly, a cloned yscPΔ57−324 from Yersinia pestis introduced in the yscPΔ97−465 mutant can sustain a significant Yop secretion and thus partially complemented the mutation. This explains the leaky phenotype observed with the yscP mutant of Y. pestis. In accordance with this secretion deficiency, YscP is required for the delivery of Yop effectors into macrophages. Mechanical shearing, immunolabelling and electron microscopy experiments showed that YscP is exposed at the bacterial surface when bacteria are incubated at 37°C in the presence of Ca2+ and thus do not secrete Yops. At 37°C, when Ca2+ ions are chelated, YscP is released like a Yop protein. We conclude that YscP is a part of the Ysc injectisome which is localized at the bacterial surface and is destabilized by Ca2+ chelation.
Inactivation of the gene for phospholipid N‐methyltransferase in Sinorhizobium meliloti: phosphatidylcholine is required for normal growth
Authors: De Rudder Karel E. E. , López‐Lara Isabel M. , Geiger Otto
Published: March 2003

In phosphatidylcholine (PC)‐containing prokaryotes, only the methylation pathway of PC biosynthesis was thought to occur. However, a second choline‐dependent pathway for PC formation, the PC synthase (Pcs) pathway, exists in Sinorhizobium (Rhizobium) meliloti in which choline is condensed with CDP‐diacylglyceride. Here, we characterize the methylation pathway of PC biosynthesis in S. meliloti. A mutant deficient in phospholipid N‐methyltransferase (Pmt) was complemented with a S. meliloti gene bank and the complementing DNA was sequenced. A gene coding for a S‐adenosylmethionine‐dependent N‐methyltransferase was identified as the sinorhizobial Pmt, which showed little similarity to the corresponding enzyme from Rhodobacter sphaeroides. Upon expression of the sinorhizobial Pmt, besides phosphatidylcholine, the methylated intermediates of the methylation pathway, monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine, are also formed. When Pmt‐deficient mutants of S. meliloti are grown on minimal medium, they cannot form PC, and they grow significantly more slowly than the wild type. Growth of the Pmt‐deficient mutant in the presence of choline allows for PC formation via the Pcs pathway and restores wild‐type‐like growth. Double knock‐out mutants, deficient in Pmt and in Pcs, are unable to form PC and show reduced growth even in the presence of choline. These results suggest that PC is required for normal growth of S. meliloti.
IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formation
Authors: Niebuhr Kirsten , Jouihri Noureddine , Allaoui Abdelmounaaim , Gounon Pierre , Sansonetti Philippe J. , Parsot Claude
Published: March 2003

Invasion of epithelial cells by Shigella flexneri involves entry and intercellular dissemination. Entry of bacteria into non‐phagocytic cells requires the IpaA–D proteins that are secreted by the Mxi–Spa type III secretion machinery. Type III secretion systems are found in several Gram‐negative pathogens and serve to inject bacterial effector proteins directly into the cytoplasm of host cells. In this study, we have analysed the IpgD protein of S. flexneri, the gene of which is located on the virulence plasmid at the 5′ end of the mxi–spa locus. We have shown that IpgD (i) is stored in the bacterial cytoplasm in association with a specific chaperone, IpgE; (ii) is secreted by the Mxi–Spa type III secretion system in amounts similar to those of the IpaA–D proteins; (iii) is associated with IpaA in the extracellular medium; and (iv) is involved in the modulation of the host cell response after contact of the bacterium with epithelial cells. This suggests that IpgD is an effector that might be injected into host cells to manipulate cellular processes during infection.
Salmonella induces macrophage death by caspase‐1‐dependent necrosis
Authors: Brennan Molly A. , Cookson Brad T.
Published: March 2003

We provide evidence that Salmonella typhimurium kills phagocytes by an unusual proinflammatory mechanism of necrosis that is distinguishable from apoptosis. Infection stimulated a distinctly diffuse pattern of DNA fragmentation in macrophages, which contrasted with the marked nuclear condensation displayed by control cells undergoing chemically induced apoptosis. In apoptotic cells, DNA fragmentation and nuclear condensation result from caspase‐3‐mediated proteolysis; caspases also subvert necrotic cell death by cleaving and inactivating poly ADP‐ribose polymerase (PARP). Caspase‐3 was not activated during Salmonella infection, and PARP remained in its active, uncleaved state. Another hallmark of apoptosis is sustained membrane integrity during cell death; yet, infected macrophages rapidly lost membrane integrity, as indicated by simultaneous exposure of phosphatidylserine with the uptake of vital dye and the release of the cytoplasmic enzyme lactate dehydrogenase. During experimentally induced necrosis, lethal ion fluxes through the plasma membrane can be prevented by exogenous glycine; similarly, glycine completely blocked Salmonella‐induced cytotoxicity. Finally, inhibition of the interleukin (IL)‐1‐converting enzyme caspase‐1 blocked the death of infected macrophages, but not control cells induced to undergo apoptosis or necrosis. Thus, Salmonella‐infected macrophages are killed by an unusual caspase‐1‐dependent mechanism of necrosis.
Activation of the Anabaena nir operon promoter requires both NtcA (CAP family) and NtcB (LysR family) transcription factors
Authors: Frías José E. , Flores Enrique , Herrero Antonia
Published: March 2003

A region of the genome of the heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120 containing the ntcB gene was identified. This region is located upstream from the nir operon involved in nitrate assimilation in this cyanobacterium. An Anabaena ntcB mutant was able to use ammonium and dinitrogen as sources of nitrogen for growth but was unable to assimilate nitrate. Enzymes of the nitrate reduction system were not synthesized in the ntcB mutant under derepression conditions. The transcription start‐point of the Anabaena nir operon, which has been shown to be subjected to ammonium‐stimulated repression and whose expression requires the global nitrogen regulator NtcA, was only weakly used in the ntcB mutant. The expression of the ntcB gene in strain PCC 7120 was also subjected to repression by ammonium and was found to take place from an NtcA‐activated promoter located 31 bp upstream from the start of the ntcB gene. NtcB binds to the nir promoter region in vitro and protects a region localized just upstream from the NtcA‐binding site in footprinting assays. These results showed that NtcB, a LysR‐family protein, is required in addition to NtcA, a CAP‐family protein, for the expression of genes encoding proteins specifically involved in nitrate assimilation in Anabaena sp. PCC 7120.
Identification of proteases with shared functions to the proprotein processing protease Krp1 in the fission yeast Schizosaccharomyces pombe
Authors: Ladds Graham , Davey John
Published: March 2003

Many secretory proteins are synthesized as inactive proproteins that undergo proteolytic activation as they travel through the eukaryotic secretory pathway. The best characterized family of processing enzymes are the prohormone convertases or kexins, and these are responsible for the processing of a wide variety of prohormones and other precursors. Recent work has identified other proteases that appear to be involved in proprotein processing, but characterization of these enzymes is at an early stage. Krp1 is the only kexin identified in the fission yeast Schizosaccharomyces pombe, in which it is essential for cell viability. We have used a genetic screen to identify four proteases with specificities that overlap Krp1. Two are serine proteases, one is a zinc metalloprotease (glycoprotease) and one is an aspartyl protease that belongs to the recently described yapsin family of processing enzymes. All four proteases support the growth of a yeast strain lacking Krp1, and each is able to process the P‐factor precursor, the only substrate currently known to be processed by Krp1.
Export of active green fluorescent protein to the periplasm by the twin‐arginine translocase (Tat) pathway in Escherichia coli
Authors: Thomas Joanna D. , Daniel Richard A. , Errington Jeff , Robinson Colin
Published: March 2003

The twin‐arginine translocation (Tat) system targets cofactor‐containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin‐arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin‐arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild‐type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild‐type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.
Two atypical mobilization proteins are involved in plasmid CloDF13 relaxation
Authors: Núñez Belén , De La Cruz Fernando
Published: March 2003

The mobilization region of plasmid CloDF13 was localized to a 3.6 kb DNA segment that was analysed by transposon mutagenesis and DNA sequencing. Analysis of the DNA sequence allowed us to identify two mobilization genes and the CloDF13 origin of conjugative transfer (oriT), which was localized to a 661 bp segment at one end of the mobilization (Mob) region. Thus, the overall organization was oriT–mobB–mobC. Plasmid CloDF13 DNA was isolated mainly as a relaxed form that contained a unique strand and site‐specific cleavage site (nic). The position of nic was mapped to the sequence 5′‐GGGTG/GTCGGG‐3′ by primer extension and sequencing reactions. Analysis of Mob− insertion mutants showed that mobC was essential for CloDF13 relaxation in vivo. The sequence of mobC predicts a protein (MobC) of 243 amino acids without significant similarity to previously reported relaxases. In addition to MobC, the product of mobB was also required for CloDF13 mobilization and for oriT relaxation in vivo. mobB codes for a protein (MobB) of 653 amino acids with three predicted transmembrane segments at the N‐terminus and the NTP‐binding motifs characteristic of the TraG family of conjugative coupling proteins. Membership of the TraG family was confirmed by the fact that CloDF13 mobilization by plasmid R388 was independent of TrwB and only required PILW. However, contrary to the activities found for other coupling proteins, MobB was required for efficient oriT cleavage in vivo, suggesting an additional role for this particular protein during oriT processing for mobilization. Additionally, the cleavage site produced by the joint activities of MobB and MobC was shown to contain unblocked ends, suggesting that no stable covalent intermediates between relaxase and DNA were formed during the nic cleavage reaction. This is the first report of a conjugative transfer system in which niccleavage results in a free nicked‐DNA intermediate.
The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix
Authors: Zogaj Xhavit , Nimtz Manfred , Rohde Manfred , Bokranz Werner , Römling Ute
Published: March 2003

Production of cellulose has been thought to be restricted to a few bacterial species such as the model organism Acetobacter xylinus. We show by enzymatic analysis and mass spectrometry that, besides thin aggregative fimbriae, the second component of the extracellular matrix of the multicellular morphotype (rdar) of Salmonella typhimurium and Escherichia coli is cellulose. The bcsA, bcsB, bcsZ and bcsC genes responsible for cellulose biosynthesis are not regulated by AgfD, the positive transcriptional regulator of the rdar morphotype. Transcription of the bcs genes was not co‐expressed with the rdar morphotype under any of the environmental conditions examined. However, cellulose biosynthesis was turned on by the sole expression of adrA, a gene encoding a putative transmembrane protein regulated by agfD, indicating a novel pathway for the activation of cellulose synthesis. The co‐expression of cellulose and thin aggregative fimbriae leads to the formation of a highly hydrophobic network with tightly packed cells aligned in parallel in a rigid matrix. As the production of cellulose would now appear to be a property widely distributed among bacteria, the function of the cellulose polymer in bacteria will have to be considered in a new light.
The Caulobacter crescentus flagellar gene, fliX, encodes a novel trans‐acting factor that couples flagellar assembly to transcription
Authors: Muir Rachel E. , O'Brien Tina M. , Gober James W.
Published: March 2003

The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early class II components of the basal body complex to the expression of class III and IV genes, which encode extracytoplasmic structures of the flagellum. The transcription of class III/IV flagellar genes is activated by the response regulator factor, FlbD. Gain of function mutations in flbD, termed bfa, can bypass the transcriptional requirement for the assembly of class II flagellar structures. Here we show that the class II flagellar gene fliX encodes a trans‐acting factor that couples flagellar assembly to FlbD‐dependent transcription. We show that the overexpression of fliX can suppress class III/IV gene expression in both wild‐type and flbD‐bfa cells. Introduction of a bfa allele of flbD into cells possessing a deletion in fliX restores motility indicating that FliX is not a structural component of the flagellum, but rather a trans‐acting factor. Furthermore, extragenic motile suppressors which arise in ΔfliX cells map to the flbD locus. These results indicate that FlbD functions downstream of FliX in activating class III/IV transcription. β‐Lactamase fusions to FliX and analysis of cellular fractions demonstrate that FliX is a cytosolic protein that demonstrates some peripheral association with the cytoplasmic membrane. In addition, we have isolated a mutant allele of fliX that exhibits a bfa‐like phenotype, restoring flbD‐dependent class III/IV transcription in strains that contain mutations in class II flagellar structural genes. Taken together, these results indicated both a positive and negative regulatory function for FliX in coupling the assembly of class II basal body components to gene expression.