bluuebunny/crossref_metadata_2025 huggingface dataset:
A vital stain for studying membrane dynamics in bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation
Authors: Pogliano Joe , Osborne Nick , Sharp Marc D. , Abanes‐De Mello Angelica , Perez Ana , Sun Ya‐Lin , Pogliano Kit
Published: March 2003

At the onset of sporulation in Bacillus subtilis, two potential division sites are assembled at each pole, one of which will be used to synthesize the asymmetrically positioned sporulation septum. Using the vital stain FM 4‐64 to label the plasma membrane of living cells, we examined the fate of these potential division sites in wild‐type cells and found that, immediately after the formation of the sporulation septum, a partial septum was frequently synthesized within the mother cell at the second potential division site. Using time‐lapse deconvolution microscopy, we were able to watch these partial septa first appear and then disappear during sporulation. Septal dissolution was dependent on σE activity and was partially inhibited in mutants lacking the σE‐controlled proteins SpoIID, SpoIIM and SpoIIP, which may play a role in mediating the degradation of septal peptidoglycan. Our results support a model in which σE inhibits division at the second potential division site by two distinct mechanisms: inhibition of septal biogenesis and the degradation of partial septa formed before σE activation.
Specific expression of the Fusarium transposon Fot1 and effects on target gene transcription
Authors: Deschamps Francine , Langin Thierry , Maurer Patricia , Gerlinger Catherine , Felenbok Béatrice , Daboussi Marie‐Josée
Published: March 2003

The Fot1 transposon is active in some strains of the plant pathogenic fungus Fusarium oxysporum. In a high‐copy‐number strain that contains autonomous elements, we have detected a transcript of 1.7 kb hybridizing to Fot1 in very low amounts. Mapping the 3′ and 5′ termini of this transcript confirms that it corresponds to a Fot1‐specific transcript. In this strain, five independent mutants of the transgene (niaD ) encoding nitrate reductase have arisen by insertion of Fot1 into the third intron. The analysis of the effect of Fot1 insertion in these mutants shows that, depending on the orientation of Fot1 relative to niaD, different truncated chimeric niaD–Fot1 transcripts are produced. Mapping the 5′ and 3′ ends of these transcripts reveals (i) premature polyadenylation at sites present in the 5′ and 3′ untranslated regions of Fot1, and (ii) initiation of some transcripts in the 3′ part of the niaD gene at sites located immediately downstream of the Fot1 insertion. Thus, a novel promoter, associated with the end of Fot1, directs transcriptional activity outwards from the element into the coding sequence of the niaD gene. These effects demonstrate that Fot1 insertion provides an additional general mechanism controlling fungal gene expression.
The SpoIIE phosphatase, the sporulation septum and the establishment of forespore‐specific transcription in Bacillus subtilis: a reassessment
Authors: Arigoni Fabrizio , Guérout‐Fleury Anne‐Marie , Barák Imrich , Stragier Patrick
Published: March 2003

Making a spore in Bacillus subtilis requires the formation of two cells, the forespore and the mother cell, which follow dissimilar patterns of gene expression. Cell specificity is first established in the forespore under the control of the σF factor, which is itself activated through the action of the SpoIIE serine phosphatase, an enzyme targeted to the septum between the two cells. Deletion of the 10 transmembrane segments of the SpoIIE protein leads to random distribution of SpoIIE in the cytoplasm. Activation of σF is slightly delayed and less efficient than in wild type, but it remains restricted to the forespore in a large proportion of cells and the bacteria sporulate with 30% efficiency. Overexpression of the complete SpoIIE protein in a divIC mutant leads to significant σF activity, indicating that the septum requirement for activating σF can be bypassed. In contradiction to current models, we propose that genetic asymmetry is not created by unequal distribution of SpoIIE within the sporangium, but by exclusion of an inhibitor of SpoIIE from the forespore. This putative inhibitor would be a cytoplasmic molecule that interacts with SpoIIE and shuts off its phosphatase activity until it disappears specifically from the forespore.
Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa
Authors: D'Enfert Christophe , Bonini Beatriz M. , Zapella Pio D. A. , Fontaine Thierry , Da Silva Aline M. , Terenzi Héctor F.
Published: March 2003

A cAMP‐activatable Ca2+‐dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa. Using a PCR approach, A. nidulans and N. crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. Disruption of the AntreB gene encoding A. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia. Although trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A. nidulans, it is required to achieve wild‐type germination rates under carbon limitation, suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination. Furthermore, it was shown that trehalose accumulation in A. nidulans can protect germinating conidia against an otherwise lethal heat shock. Because transcription of the treB genes is not increased after a heat shock but induced upon heat shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post‐translational regulatory mechanisms, in particular cAMP‐dependent protein kinase‐mediated phosphorylation.
Recombination and clonal groupings within Helicobacter pylori from different geographical regions
Authors: Achtman Mark , Azuma Takeshi , Berg Douglas E. , Ito Yoshiyuki , Morelli Giovanna , Pan Zhi‐Jun , Suerbaum Sebastian , Thompson Stuart A. , Van Der Ende Arie , Van Doorn Leen‐Jan
Published: March 2003

A collection of 20 strains of Helicobacter pylori from several regions of the world was studied to better understand the population genetic structure and diversity of this species. Sequences of fragments from seven housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI, yphC ) and two virulence‐associated genes (cagA, vacA) showed high levels of synonymous sequence variation (mean percentage Ks of 10–27%) and lower levels of non‐synonymous variation (mean percentage Ka of 0.2–5.6%). Cluster analysis of pairwise differences between alleles revealed the existence of two weakly clonal groupings, which included half of the strains investigated. All six strains isolated from Japanese and coastal Chinese were assigned to the ‘Asian’ clonal grouping, probably reflecting descent from a distinct common ancestor. The clonal groupings were not totally uniform; recombination, as measured by the homoplasy test and compatibility matrices, was extremely common within all genes tested, except cagA. The fact that clonal descent could still be discerned despite such frequent recombination possibly reflects founder effects and geographical separation and/or selection for particular alleles of these genes.
Characterization of a sugar‐binding protein from Azospirillum brasilense mediating chemotaxis to and uptake of sugars
Authors: Van Bastelaere Els , Lambrecht Mark , Vermeiren Hans , Van Dommelen Anne , Keijers Veerle , Proost Paul , Vanderleyden Jos
Published: March 2003

Our approach to the isolation of plant‐inducible bacterial genes of Azospirillum brasilense, based on the analysis of protein patterns of bacteria grown in the presence and in the absence of plant root exudates, led to the identification of an acidic 40 kDa protein. Cloning and sequencing analysis of the corresponding coding DNA region revealed the presence of two open reading frames transcribed in the same orientation. The deduced ORF1 protein, which corresponds to the 40 kDa protein, is very similar to the periplasmic ChvE protein, identified in Agrobacterium tumefaciens and involved in enhanced virulence. The deduced ORF2 protein shows homology to members of the LysR family of transcriptional regulators. The function of the ChvE‐like protein in A. brasilense was investigated further. The protein, designated as SbpA (sugar binding protein A), is involved in the uptake of D‐galactose and functions in the chemotaxis of A. brasilense towards several sugars, including D‐galactose, L‐arabinose and D‐fucose. Expression of the sbpA gene requires the presence of the same sugars in the growth medium and is enhanced further in combination with carbon starvation of A. brasilense cells.
Characterization of the dual start motif of a class II holin gene
Authors: Barenboim Maxim , Chang Chung‐Yu , Dib Hajj Fadia , Young Ry
Published: March 2003

Holins are small membrane proteins that, at a genetically programmed time in a bacteriophage infective cycle, allow bacteriolytic enzymes, or endolysins, to escape to the periplasm and to attack the cell wall. Most holins fall into two sequence classes, I and II, based on the number of potential transmembrane domains (three for class I and two for class II). The prototype class I holin gene, S λ, has a dual start motif and encodes not only the effector holin, Sλ105, but also an inhibitor, Sλ107, with a Met–Lys … extension at the terminus. The prototype class II holin gene of phage 21, S 21, begins with the motif Met–Lys–Ser–Met … , and a potential RNA secondary structure overlaps the Shine–Dalgarno sequence. Here, we demonstrate that (i) two protein products are elaborated from S 21, S2171 and S2168; (ii) the shorter product is required for lysis; (iii) the longer product, S2171, inhibits S 21 function; and (iv) the Lys‐2 residue is important for the inhibitor function. Moreover, the RNA stem–loop structure is involved in the downregulation of S2171 synthesis. However, our results suggest that, in S 21, different segments of the single consensus Shine–Dalgarno sequence serve the two translational starts. These results show that the dual start motifs of class II holin genes are functionally homologous to those of class I holin genes.
The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica
Authors: Tovar Jorge , Fischer Anke , Clark C. Graham
Published: March 2003

Ultrastructural analysis of Entamoeba histolytica reveals that this intestinal human pathogen lacks recognizable mitochondria, but the presence in its genome of genes encoding proteins of mitochondrial origin suggests the existence of a mitochondrially derived compartment. We have cloned the full‐length E. histolytica gene encoding one such protein, chaperonin CPN60, and have characterized its structure and expression. Using an affinity‐purified antibody raised against recombinant protein, we have localized native E. histolytica CPN60 to a previously undescribed organelle of putative mitochondrial origin, the mitosome. Most cells contain only one mitosome, as determined by immunofluorescence studies. Entamoeba histolytica CPN60 has an amino‐terminal extension reminiscent of known mitochondrial and hydrogenosomal targeting signals. Deletion of the first 15 amino acids of CPN60 leads to an accumulation of the truncated protein in the cytoplasm. However, this mutant phenotype can be reversed by replacement of the deleted amino acids with a mitochondrial targeting signal from Trypanosoma cruzi HSP70. The observed functional conservation between mitochondrial import in trypanosomes and mitosome import in Entamoeba is strong evidence that the E. histolytica organelle housing chaperonin CPN60 represents a mitochondrial remnant.
Differential fiu–lacZ fusion regulation linked to Escherichia coli colony development
Authors: Newman Dina L. , Shapiro James A.
Published: March 2003

Colonies of strains carrying a stable λplacMu15 translational fusion displayed sharply defined intense staining at the centre on Xgal medium. The fusion was in fiu (ferric ion uptake), encoding an iron‐regulated outer membrane protein (IROMP) controlled via four overlapping ferric uptake regulator (Fur) boxes in the σ70 promoter region. Fiu–LacZ was synthesized in low amounts (< 1% of a transcriptional fiu::lacZ+ fusion), localized to membranes, and underwent processing from a large protein to one that co‐migrated with native β‐galactosidase. Intact cells synthesizing Fiu–LacZ often displayed greater enzymatic activity than permeabilized cells. The colony centre was insensitive to iron regulation observed in liquid cultures and at the colony edge. Within colonies grown on 36 μM iron citrate medium, fiu′–′lacZ protein fusion strains displayed 60‐fold higher β‐galactosidase activity in the centre, and transcriptional fiu::lacZ+ fusion strains displayed a 10‐fold centre/edge difference. On medium without added iron citrate, the centre/edge difference collapsed to < 2.2‐fold for both translational and transcriptional fusions because activity at the edge was derepressed. Iron‐insensitive fiu′–′lacZ expression in the colony centre occurred during a 6–18 h time window at the start of colony morphogenesis, corresponding to the initiation of multilayer microcolony development. A simple model for differential fiu′–′lacZ regulation is proposed whereby iron accessibility changes during colony morphogenesis.
Effect of loop deletions on the binding and transport of ferric enterobactin by FepA
Authors: Newton Salete M. C. , Igo John D. , Scott Daniel C. , Klebba Phillip E.
Published: March 2003

The siderophore ferric enterobactin enters Escherichia coli through the outer membrane (OM) porin FepA, which contains an aqueous transmembrane channel that is normally occluded by other parts of the protein. After binding the siderophore at a site within the surface loops, FepA undergoes conformational changes that promote ligand internalization. We assessed the participation of different loops in ligand recognition and uptake by creating and analysing a series of deletions. We genetically engineered 26 mutations that removed 9–75 amino acids from nine loops and two buried regions of the OM protein. The mutations had various effects on the uptake reaction, which we discerned by comparing the substrate concentrations of half‐maximal binding (Kd) and uptake (Km): every loop deletion affected siderophore transport kinetics, decreasing or eliminating binding affinity and transport efficiency. We classified the mutations in three groups on the basis of their slight, strong or complete inhibition of the rate of ferric enterobactin transport across the OM. Finally, characterization of the FepA mutants revealed that prior experiments underestimated the affinity of FepA for ferric enterobactin: the interaction between the protein and the ferric siderophore is so avid (Kd < 0.2 nM) that FepA tolerated the large reductions in affinity that some loop deletions caused without loss of uptake functionality. That is, like other porins, many of the loops of FepA are superficially dispensable: ferric enterobactin transport occurred without them, at levels that allowed bacterial growth.
Visualization of membrane domains in Escherichia coli
Authors: Fishov Itzhak , Woldringh Conrad L.
Published: March 2003

Bacterial membrane and nucleoids were stained concurrently by the lipophilic styryl dye FM 4‐64 [N‐(3‐triethylammoniumpropyl)‐4‐(6‐(4‐(diethylamino)phenyl) hexatrienyl)pyridinium dibromide] and 4′,6‐diamidino‐2‐phenylindole (DAPI), respectively, and studied using fluorescence microscopy imaging. Observation of plasmolysed cells indicated that FM 4‐64 stained the inner membrane preferentially. In live Escherichia coli pbpB cells and filaments, prepared on wet agar slabs, an FM 4‐64 staining pattern developed in the form of dark bands. In dividing cells, the bands occurred mainly at the constriction sites and, in filaments, between partitioning nucleoids. The FM 4‐64 pattern of dark bands in filaments was abolished after inhibiting protein synthesis with chloramphenicol. It is proposed that the staining patterns reflect putative membrane domains formed by DNA–membrane interactions and have functional implications in cell division.
The response to stationary‐phase stress conditions in Escherichia coli : role and regulation of the glutamic acid decarboxylase system
Authors: De Biase Daniela , Tramonti Angela , Bossa Francesco , Visca Paolo
Published: March 2003

Inducible bacterial amino acid decarboxylases are expressed at the end of active cell division to counteract acidification of the extracellular environment during fermentative growth. It has been proposed that acid resistance in some enteric bacteria strictly relies on a glutamic acid‐dependent system. The Escherichia coli chromosome contains distinct genes encoding two biochemically identical isoforms of glutamic acid decarboxylase, GadA and GadB. The gadC gene, located downstream of gadB, has been proposed to encode a putative antiporter implicated in the export of γ‐aminobutyrate, the glutamic acid decarboxylation product. In the present work, we provide in vivo evidence that gadC is co‐transcribed with gadB and that the functional glutamic acid‐dependent system requires the activities of both GadA/B and GadC. We also found that expression of gad genes is positively regulated by acidic shock, salt stress and stationary growth phase. Mutations in hns, the gene for the histone‐like protein H‐NS, cause derepressed expression of the gad genes, whereas the rpoS mutation abrogates gad transcription even in the hns background. According to our results, the master regulators H‐NS and RpoS are hierarchically involved in the transcriptional control of gad expression: H‐NS prevents gad expression during the exponential growth whereas the alternative sigma factor RpoS relieves H‐NS repression during the stationary phase, directly or indirectly accounting for transcription of gad genes.
Multiple hok genes on the chromosome of Escherichia coli
Authors: Pedersen Kim , Gerdes Kenn
Published: March 2003

The hok/sok locus of plasmid R1 mediates plasmid stabilization by the killing of plasmid‐free cells. Many bacterial plasmids carry similar loci. For example, the F plasmid carries two hok homologues, flm and srnB, that mediate plasmid stabilization by this specialized type of programmed cell death. Here, we show that the chromosome of E. coli K‐12 codes for five hok homologous loci, all of which specify Hok‐like toxins. Three of the loci appear to be inactivated by the insertion elements IS150 or IS186 located close to but not in the toxin‐encoding reading frames (i.e. hokA, hokC and hokE ), one system is probably inactivated by point mutation (hokB ), whereas the fifth system is inactivated by a major genetic rearrangement (hokD ). In the ECOR collection of wild‐type E. coli strains, we identified hokA and hokC loci without IS elements. A molecular and a genetic analysis show that the hokA and hokC loci specify unstable antisense RNAs and stable toxin‐encoding mRNAs that are processed at their 3′ ends. An alignment of the mRNA sequences reveals all the regulatory elements known to be required for correct folding and refolding of the plasmid‐encoded mRNAs. The conserved elements include fbi that ensure a long‐range interaction in the full‐length mRNAs, and tac and antisense RNA target stem–loops that are required for translation and rapid antisense RNA binding of the processed mRNAs. Consistently, we find that the chromosome‐encoded mRNAs are processed at their 3′ ends, resulting in the presumed translationally active mRNAs. Despite the presence of all of the regulatory elements, the chromosome‐encoded loci do not mediate plasmid stabilization by killing of plasmid‐free cells. The chromosome‐encoded mRNAs are poorly translated in vitro, thus yielding an explanation for the lacking phenotype. These observations suggest that the chromosomal hok‐like genes may be induced by an as yet unknown signal.
Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells
Authors: Merz Alexey J. , Enns Caroline A. , So Magdalene
Published: March 2003

The pathogenic Neisseriae Neisseria meningitidis and Neisseria gonorrhoeae, initiate colonization by attaching to host cells using type IV pili. Subsequent adhesive interactions are mediated through the binding of other bacterial adhesins, in particular the Opa family of outer membrane proteins. Here, we have shown that pilus‐mediated adhesion to host cells by either meningococci or gonococci triggers the rapid, localized formation of dramatic cortical plaques in host epithelial cells. Cortical plaques are enriched in both components of the cortical cytoskeleton and a subset of integral membrane proteins. These include: CD44v3, a heparan sulphate proteoglycan that may serve as an Opa receptor; EGFR, a receptor tyrosine kinase; CD44 and ICAM‐1, adhesion molecules known to mediate inflammatory responses; f‐actin; and ezrin, a component that tethers membrane components to the actin cytoskeleton. Genetic analyses reveal that cortical plaque formation is highly adhesin specific. Both pilE and pilC null mutants fail to induce cortical plaques, indicating that neisserial type IV pili are required for cortical plaque induction. Mutations in pilT, a gene required for pilus‐mediated twitching motility, confer a partial defect in cortical plaque formation. In contrast to type IV pili, many other neisserial surface structures are not involved in cortical plaque induction, including Opa, Opc, glycolipid GgO4‐binding adhesins, polysialic acid capsule or a particular lipooligosaccharide variant. Furthermore, it is shown that type IV pili allow gonococci to overcome the inhibitory effect of heparin, a soluble receptor analogue, on gonococcal invasion of Chang and A431 epithelial cells. These and other observations strongly suggest that type IV pili play an active role in initiating neisserial infection of the mucosal surface in vivo. The functions of type IV pili and other neisserial adhesins are discussed in the specific context of the mucosal microenvironment, and a multistep model for neisserial colonization of mucosal epithelia is proposed.
TorC apocytochrome negatively autoregulates the trimethylamine N‐oxide (TMAO) reductase operon in Escherichia coli
Authors: Ansaldi Mireille , Bordi Christophe , Lepelletier Michèle , Méjean Vincent
Published: March 2003

The trimethylamine N‐oxide (TMAO) anaerobic respiratory system of Escherichia coli comprises a periplasmic terminal TMAO reductase (TorA) and a pentahaem c‐type cytochrome (TorC), which is involved in electron transfer to TorA. The structural proteins are encoded by the torCAD operon whose expression is induced in the presence of TMAO through the TorS/TorR two‐component system. By using a genomic library cloned into a multicopy plasmid, we identified TorC as a possible negative regulator of the tor operon. Interestingly, in trans overexpression of torC not only decreased the activity of a torA′–′lacZ fusion, but also dramatically reduced the amount of mature TorC cytochrome. This led us to propose that, after translocation, TorC apocytochrome downregulates the tor operon unless it is properly matured. In agreement with this hypothesis, we have shown that mini‐Tn10 insertions within genes involved in the c‐type cytochrome maturation pathway or haem biosynthesis decreased tor operon expression. Dithiothreitol (DTT), which reduces disulphide bonds and thus prevents the first step in c‐type cytochrome formation, also strongly decreases the tor promoter activity. The DTT effect is TorC dependent, as it is abolished when torC is disrupted. In contrast, overexpression of the c‐type cytochrome maturation (ccm ) genes relieved the tor operon of the negative control and allowed the bacteria to produce a higher amount of TorC holocytochrome. Therefore, the TorC negative autoregulation probably means that maturation of the c‐type cytochrome is a limiting step for Tor system biogenesis. Genetic experiments have provided evidence that TorC control is mediated by the TorS/TorR two‐component system and different from the tor anaerobic control. In our working model, TMAO and apoTorC bind to the periplasmic side of TorS, but TMAO activates TorS autophosphorylation, whereas apoTorC inhibits the TorS kinase activity.
Identification and characterization of a germination operon on the virulence plasmid pXOl of Bacillus anthracis
Authors: Guidi‐Rontani Chantal , Pereira Yannick , Ruffie Stéphanie , Sirard Jean‐Claude , Weber‐Levy Martine , Mock Michèle
Published: March 2003

The spores of Bacillus anthracis, the agent of anthrax disease, germinate within professional phagocytes, such as murine macrophage‐like RAW264.7 cells and alveolar macrophages. We identified a cluster of germination genes extending for 3608 nucleotides between the pag and atxA genes on the B. anthracis virulence plasmid pXOl. The three predicted proteins (40, 55 and 37 kDa in size) have significant sequence similarities to B. subtilis, B. cereus and B. megaterium germination proteins. Northern blot analysis of total RNA from sporulating cells indicated that the gerX locus was organized as a tricistronic operon (gerXB, gerXA and gerXC). Primer extension analysis identified a major potential transcriptional start site 31 bp upstream from the translation initiation codon of gerXB. Expression of the gerX operon was studied using a gerXB–lacZ transcriptional fusion. Expression began 2.5–3 h after the initiation of sporulation and was detected exclusively in the forespore compartment. A gerX null mutant was constructed. It was less virulent than the parental strain and did not germinate efficiently in vivo or in vitro within phagocytic cells. These data strongly suggest that gerX‐encoded proteins are involved in the virulence of B. anthracis.
Displacement of cellular proteins by functional analogues from plasmids or viruses could explain puzzling phylogenies of many DNA informational proteins
Authors: Forterre Patrick
Published: March 2003

Comparative genomics has revealed many examples in which the same function is performed by unrelated or distantly related proteins in different cellular lineages. In some cases, this has been explained by the replacement of the original gene by a paralogue or non‐homologue, a phenomenon known as non‐orthologous gene displacement. Such gene displacement probably occurred early on in the history of proteins involved in DNA replication, repair, recombination and transcription (DNA informational proteins), i.e. just after the divergence of archaea, bacteria and eukarya from the last universal cellular ancestor (LUCA). This would explain why many DNA informational proteins are not orthologues between the three domains of life. However, in many cases, the origin of the displacing genes is obscure, as they do not even have detectable homologues in another domain. I suggest here that the original cellular DNA informational proteins have often been replaced by proteins of viral or plasmid origin. As viral and plasmid‐encoded proteins are usually very divergent from their cellular counterparts, this would explain the puzzling phylogenies and distribution of many DNA informational proteins between the three domains of life.
Multiple insertions of fimbrial operons correlate with the evolution of Salmonella serovars responsible for human disease
Authors: Folkesson Anders , Advani Abdolreza , Sukupolvi Soila , Pfeifer John D. , Normark Staffan , Löfdahl Sven
Published: March 2003

On centisome 7, Salmonella spp. contain a large region not present in the corresponding region of Escherichia coli. This region is flanked by sequences with significant homology to the E. coli tRNA gene aspV and the hypothetical E. coli open reading frame yafV. The locus consists of a mosaic of differentially acquired inserts forming a dynamic cs7 region of horizontally transferred inserts. Salmonella enterica subspecies I, responsible for most Salmonella infections in warm‐blooded animals, carries a fimbrial gene cluster (saf) in this region as well as a regulatory gene (sinR). These genes are flanked by inverted repeats and are inserted in another laterally transferred region present in most members of Salmonella spp. encoding a putative invasin (pagN ). S. enterica subspecies I serovar Typhi, the Salmonella serovar that causes the most severe form of human salmonellosis, contains an additional insert of at least 8 kb in the sinR–pagN intergenic region harbouring a novel fimbrial operon (tcf ) similar to the coo operon encoding the CS1 fimbrial adhesin expressed by human‐specific enterotoxigenic E. coli. It is suggested that the multiple insertions of fimbrial genes that have occurred in the cs7 region have contributed to phylogenetic diversity and host adaptation of Salmonella spp.
A gene required for the regulation of photosynthetic light harvesting in the cyanobacterium Synechocystis 6803
Authors: Emlyn‐Jones Daniel , Ashby Mark K. , Mullineaux Conrad W.
Published: March 2003

A gene required for the short‐term regulation of photosynthetic light harvesting (the state transition) has been identified in the cyanobacterium Synechocystis sp. PCC6803. The open reading frame is designated sll1926 in the complete Synechocystis gene sequence. The deduced amino acid sequence has no homologues in current sequence databases and no recognizable sequence motifs. It encodes a putative integral membrane protein of 16 kDa, which we have designated RpaC (regulator of phycobilisome association C). Fluorescence measurements of an insertional inactivation mutant of rpaC (Δsll1926) show that it is specifically unable to perform state transitions. Δsll1926 has approximately wild‐type levels of PS1, PS2 and phycobilisomes. Measurements of oxygen evolution and uptake show Δsll1926 to have no deficiency in electron transport rates. In vitro [γ‐32P]‐ATP labelling experiments suggest that RpaC is not the 15 kDa membrane phosphoprotein previously implicated in state transitions. Δsll1926 grows more slowly than the wild type only at very low light intensities.
Expression and heat‐responsive regulation of a TFIIB homologue from the archaeon Haloferax volcanii
Authors: Thompson Dorothea K. , Palmer John R. , Daniels Charles J.
Published: March 2003

Multiple divergent genes encoding the eukaryal‐like TFIIB (TFB) transcription initiation factor have been identified in the archaeon Haloferax volcanii. Expression of one of these TFB‐encoding genes, referred to here as tfb2, was induced specifically in response to heat shock at the transcription level. A time course for tfb2 induction demonstrated that mRNA levels increased as much as eightfold after 15 min at 60°C. A transcription fusion of the tfb2 promoter region with a stable RNA reporter gene confirmed the heat responsiveness of the tfb2 core promoter, and immunoblot analysis using antibodies generated against a recombinant His‐tagged TFB2 showed that the protein levels of one TFB increased slightly in response to elevated temperatures. An archaeal consensus TATA element (5′‐TTTATA‐3′) was located 110 bp upstream of the translation start site and appeared to be used for both basal and heat shock‐induced expression. The long DNA leader region (79 bp) preceding the predicted AUG translation start codon for TFB2 contained a T‐rich sequence element located 22 bp downstream of the transcription start site. Using an in vivo transcription termination assay, we demonstrated that this T‐rich element can function as a sequence‐dependent transcription terminator, which may serve to downregulate expression of the tfb2 gene under both non‐heat shock and heat shock conditions.
Towards deciphering the Helicobacter pylori cytotoxin
Authors: Reyrat Jean‐Marc , Pelicic Vladimir , Papini Emanuele , Montecucco Cesare , Rappuoli Rino , Telford John L
Published: March 2003

VacA, the major exotoxin produced by Helicobacter pylori, is composed of identical 87 kDa monomers that assemble into flower‐shaped oligomers. The monomers can be proteolytically cleaved into two moieties, one of 37 and the other of 58 kDa, named P37 and P58 respectively. The most studied property of VacA is the alteration of intracellular vesicular trafficking in eukaryotic cells leading to the formation of large vacuoles containing markers of late endosomes and lysosomes. However, VacA also causes a reduction in transepithelial electrical resistance in polarized monolayers and forms ion channels in lipid bilayers. The ability to induce vacuoles is localized mostly but not entirely in P37, whereas P58 is mostly involved in cell targeting. Until recently, H. pylori isolates were classified as tox+ or tox−, depending on whether they induced vacuoles in HeLa cells or not. Today, we know that almost all strains are cytotoxic. The major difference between tox+ and tox− resides in the cell binding domain, which exists in two allelic forms, only one of which is toxic for HeLa cells. The two forms, named m1 and m2, are found predominantly in Western and Chinese isolates respectively.
Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature‐tagged transposon mutagenesis
Authors: Camacho Luis Reinaldo , Ensergueix Danielle , Perez Esther , Gicquel Brigitte , Guilhot Christophe
Published: March 2003

Tuberculosis remains the greatest cause of death worldwide due to a single pathogen. In order to identify the genes required for the pathogenicity of Mycobacterium tuberculosis, a functional genomic approach was developed. A library of signature‐tagged transposon mutants of this bacterium was constructed and screened for those affected in their multiplication within the lungs of mice. From 1927 mutants tested, 16 were attenuated for their virulence. The insertions harboured by the selected mutants were mapped on the M. tuberculosis genome and most of the mutated loci appeared to be involved in lipid metabolism or transport across the membrane. Four independent mutations identified a cluster of virulence genes located on a 50 kb chromosomal region. These genes might be involved in the production of phthiocerol and phenolphthiocerol derivatives, a group of molecules restricted to eight mycobacterial species, seven of them being either strict or opportunistic pathogens. The interaction of five mutant strains with mouse bone marrow macrophages was investigated. These five mutants were still able to multiply in this cell type. However, in three cases, there was a growth defect in comparison with the wild‐type strain. The other two strains exhibited no clear difference from the virulent strain, MT103, in this model. This study, which is the first global research of virulence factors of M. tuberculosis, opens the way to a better understanding of the molecules that are key players in the interaction of this pathogen with its host.
Analysis of three clustered polygalacturonase genes in Erwinia chrysanthemi 3937 revealed an anti‐repressor function for the PecS regulator
Authors: Nasser William , Shevchik Vladimir E. , Hugouvieux‐Cotte‐Pattat Nicole
Published: March 2003

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes including several pectate lyases encoded by the pel genes. We characterized a novel cluster of pectinolytic genes consisting of the three adjacent genes pehV, pehW and pehX, whose products have polygalacturonase activity. The high similarity between the three genes suggests that they result from duplication of an ancestral gene. The transcription of pehV, pehW and pehX is dependent on several environmental conditions. They are induced by pectin catabolic products and this induction results from inactivation of the KdgR repressor which controls almost all the steps of pectin catabolism. The presence of calcium ions strongly reduced the transcription of the three peh genes. Their expression was also affected by growth phase, osmolarity, oxygen limitation and nitrogen starvation. In addition, the pehX transcription is affected by catabolite repression and controlled by the activator protein CRP. PecS, which was initially isolated as a repressor of virulence factors, acts as an activator of the peh transcription. We showed that the three regulators KdgR, PecS and CRP act by direct interaction with the promoter regions of the peh genes. Analysis of simultaneous binding of KdgR, PecS, CRP and RNA polymerase indicated that the activator effect of PecS results from a competition between PecS and KdgR for the occupation of overlapping binding sites. Thus, to activate peh transcription, PecS behaves as an anti‐repressor against KdgR.
Steps in the development of a Vibrio cholerae El Tor biofilm
Authors: Watnick Paula I. , Kolter Roberto
Published: March 2003

We report that, in a simple, static culture system, wild‐type Vibrio cholerae El Tor forms a three‐dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose‐sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three‐dimensional biofilm architecture.
Flagellar proteins and type III‐exported virulence factors are the predominant proteins secreted into the culture media of Salmonella typhimurium
Authors: Komoriya Kaoru , Shibano Naoko , Higano Tomomi , Azuma Norihiro , Yamaguchi Shigeru , Aizawa Shin‐Ichi
Published: March 2003

We analysed all major proteins secreted into culture media from Salmonella typhimurium. Proteins in culture supernatants were collected by trichloroacetic acid precipitation, separated in SDS–polyacrylamide gels and analysed by amino acid sequencing. Wild‐type strain SJW1103 cells typically gave rise to nine bands in SDS gels: 89, 67, 58, 52, 50, 42, 40, 35 and (sometimes) 28 kDa. A search of the sequences in the available databases revealed that they were either flagellar proteins or virulence factors. Six of them were flagella specific: FlgK or HAP1 (58 kDa), FliC or flagellin (52 kDa), FliD or HAP2 (50 kDa), FlgE or hook protein (42 kDa), FlgL or HAP3 (35 kDa) and FlgD or hook‐cap protein (28 kDa). The other four bands were specific for virulence factors: SipA (89 kDa), SipB (67 kDa), SipC (42 kDa) and InvJ (40 kDa). The 42 kDa band was a mixture of FlgE and SipC. We also analysed secreted proteins from more than 30 flagellar mutants, and they were categorized into four groups according to their band patterns: wild type, mot type, polyhook type and master gene type. Virulence factors were constantly secreted at a higher level in all flagellar mutants except a Δmot (motAB deletion) mutant, in which the amounts were greatly reduced. A new morphological pathway of flagellar biogenesis including protein secretion is presented.
The MET3 promoter: a new tool for Candida albicans molecular genetics
Authors: Care R. S. , Trevethick J. , Binley K. M. , Sudbery P. E.
Published: March 2003

A central technique used to investigate the role of a Candida albicans gene is to study the phenotype of a cell in which both copies of the gene have been deleted. To date, such investigations can only be undertaken if the gene is not essential. We describe the use of the Candida albicans MET3 promoter to express conditionally an essential gene, so that the consequences of depletion of the gene product may be investigated. The effects of environmental conditions on its expression were investigated, using GFP as a reporter gene. The promoter showed an ≈85‐fold range of expression, according to the presence or absence of either methionine or cysteine in concentrations in excess of 1 mM. In the presence of either amino acid, expression was reduced to levels that were close to background. We used URA3 as a model to demonstrate that the MET3 promoter could control the expression of an essential gene, provided that a mixture of both methionine and cysteine was used to repress the promoter. We describe an expression vector that may be used to express any gene under the control of the MET3 promoter and a vector that may be used to disrupt a gene and simultaneously place an intact copy under the control of the MET3 promoter. During the course of these experiments, we discovered that directed integration into the RP10 locus gives a high frequency of transformation, providing a means to solve a long‐standing problem in this field.
The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance
Authors: Merrell D. Scott , Camilli Andrew
Published: March 2003

Vibrio cholerae is a facultative pathogen of humans that must survive exposure to inorganic and organic acids in the stomach and small intestine. To learn more about the mechanisms by which this pathogen colonizes the intestinal tract, we used a recombinase gene fusion reporter to identify transcripts induced during infection in an adult rabbit model of cholera. One of the genes identified was cadA, which encodes an inducible lysine decarboxylase. CadA was also induced during infections of the suckling and adult mouse intestines, and in vitro under conditions of low pH and high lysine concentration. We show that V. cholerae is capable of mounting an acid tolerance response (ATR) to both inorganic and organic acid challenges. Mutational analyses revealed a significant role for cadA, but not for speF, which encodes an ornithine decarboxylase, in both inorganic and organic ATR. Potential roles for toxR, toxT and rpoS in ATR were examined, and it was found that toxR plays a ToxT‐independent role in mediating organic ATR, whereas rpoS played no detectable role in either ATR. Transcriptional analysis showed that the toxR defect in ATR is not caused by decreased cadA transcription. Despite induction of cadA in these animal models, competition assays revealed that neither cadA nor speF alone or together were required for colonization of suckling or adult mice. However, acid‐adapted wild‐type V. cholerae exhibited a major competitive advantage over unadapted cells during colonization of suckling mice.
The putative DNA translocase SpoIIIE is required for sporulation of the symmetrically dividing coccal species Sporosarcina ureae
Authors: Chary Vasant K. , Hilbert David W. , Higgins Michael L. , Piggot Patrick J.
Published: March 2003

The spoIIIE gene of Sporosarcina ureae encodes a 780‐residue protein, showing 58% identity to the SpoIIIE protein of Bacillus subtilis, which is thought to be a DNA translocase. Expression of the S. ureae spoIIIE gene is able to restore sporulation in a B. subtilis spoIIIE mutant. Inactivation of the S. ureae spoIIIE gene blocks sporulation of S. ureae at stage III. Within the limits of detection, the sporulation division in S. ureae shows the same symmetry, or near symmetry, as the vegetative division (in contrast to the highly asymmetric location of the sporulation division for B. subtilis), and so it is inferred that SpoIIIE facilitates chromosome partitioning during sporulation, even when the division is not grossly asymmetric. It is suggested that chromosome partitioning lags behind division during sporulation but not during vegetative growth.
Two roles for integration host factor at an enhancer‐dependent nifA promoter
Authors: Wassem Roseli , De Souza Emanuel Maltempi , Yates Marshall Geoffrey , Pedrosa Fábio de Oliveira , Buck Martin
Published: March 2003

Control of transcription in prokaryotes often involves direct contact of regulatory proteins with RNA polymerase. For the σ54 RNA polymerase, regulatory proteins bound to distally located enhancers engage the polymerase via DNA looping. The σ54‐dependent nifA promoter of Herbaspirillum seropedicae (Hs) is activated under nitrogen‐limiting growth conditions. Potential enhancers for the nitrogen control activators NTRC and NIFA and binding sites for integration host factor (IHF) and σ54‐holoenzyme were identified. DNA footprinting experiments showed that these sites functioned for protein binding. Their involvement in the promoter regulation was explored. In vitro, activation of the Hs nifA promoter by NTRC is stimulated by the DNA bending protein IHF. In marked contrast, activation by NIFA is greatly reduced by IHF, thus diminishing potentially destabilizing autoactivation of the nifA promoter by NIFA. Additionally, high levels of NIFA appear to limit NTRC‐dependent activation. This inhibition is IHF dependent. Therefore, IHF acts positively and negatively at the nifA promoter to restrict transcription activation to NTRC and one signal transduction pathway.
Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae
Authors: Kirn Thomas J. , Lafferty Margaret J. , Sandoe Carola M. P. , Taylor Ronald K.
Published: March 2003

The toxin‐co‐regulated pilus (TCP), a type 4 pilus that is expressed by epidemic strains of Vibrio cholerae O1 and O139, is required for colonization of the human intestine. The TCP structure is assembled as a polymer of repeating subunits of TcpA pilin that form long fibres, which laterally associate into bundles. Previous passive immunization studies have suggested that the C‐terminal region of TcpA is exposed on the surface of the pilus fibre and has a critical role in mediating the colonization functions of TCP. In the present study, we have used site‐directed mutagenesis to delineate two domains within the C‐terminal region that contribute to TCP structure and function. Alterations in the first domain, termed the structural domain, result in altered pilus stability or morphology. Alterations in the second domain, termed the interaction domain, affect colonization and/or infection by CTX‐bacteriophage without affecting pilus morphology. In vitro and in vivo analyses of the tcpA mutants revealed that a major function of TCP is to mediate bacterial interaction through direct pilus–pilus contact required for microcolony formation and productive intestinal colonization. The importance of this function is supported by the finding that intragenic suppressor mutations that restore colonization ability to colonization‐deficient mutants simultaneously restore pilus‐mediated bacterial interactions. The alterations resulting from the suppressor mutations also provide insight into the molecular interactions between pilin subunits within and between pilus fibres.
The mitochondrial protein targeting suppressor (mts1) mutation maps to the mRNA‐binding domain of Npl3p and affects translation on cytoplasmic polysomes
Authors: Gratzer Sabine , Beilharz Traude , Beddoe Travis , Henry Michael F. , Lithgow Trevor
Published: March 2003

In all eukaryotic organisms, messenger RNA (mRNA) is synthesized in the nucleus and then exported to the cytoplasm for translation. The export reaction requires the concerted action of a large number of protein components, including a set of shuttle proteins that can exit and re‐enter the nucleus through the nuclear pore complex. Here, we show that, in Saccharomyces cerevisiae, the shuttle protein Npl3p leaves the nuclear pore complex entirely and continues to function in the cytoplasm. A mutation at position 219 in its RNA‐binding domain leaves Npl3p lingering in the cytoplasm associated with polysomes. Yeast cells expressing the mutant Npl3(L‐219S) protein show alterations in mRNA stability that can affect protein synthesis. As a result, defects in nascent polypeptide targeting to subcellular compartments such as the mitochondria are also suppressed.