bluuebunny/crossref_metadata_2025 huggingface dataset:
The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family)
Authors: Bleves Sophie , Voulhoux Romé , Michel Gérard , Lazdunski Andrée , Tommassen Jan , Filloux Alain
Published: March 2003

The xcp gene products in Pseudomonas aeruginosa are required for the secretion of proteins across the outer membrane. Four of the Xcp proteins, XcpT, U, V and W, present sequence homology to the subunits of type IV pili at their N‐termini, and they were therefore designated pseudopilins. In this study, we characterized the xcpX gene product, a bitopic cytoplasmic membrane protein. Remarkably, amino acid sequence comparisons also suggested that the XcpX protein resembles the pilins and pseudopilins at the N‐terminus. We show that XcpX could be processed by the prepilin peptidase, PilD/XcpA, and that the highly conserved glycine residue preceding the hydrophobic segment could not be mutated without loss of the XcpX function. We, therefore, classified XcpX (GspK) as the fifth pseudopilin of the system.
Evidence for pore‐forming ability by Legionella pneumophila
Authors: Kirby James E. , Vogel Joseph P. , Andrews Helene L. , Isberg Ralph R.
Published: March 2003

Legionella pneumophila is the cause of Legionnaires' pneumonia. After internalization by macrophages, it bypasses the normal endocytic pathway and occupies a replicative phagosome bound by endoplasmic reticulum. Here, we show that lysis of macrophages and red blood cells by L. pneumophila was dependent on dotA and other loci known to be required for proper targeting of the phagosome and replication within the host cell. Cytotoxicity occurred rapidly during a high‐multiplicity infection, required close association of the bacteria with the eukaryotic cell and was a form of necrotic cell death accompanied by osmotic lysis. The differential cytoprotective ability of high‐molecular‐weight polyethylene glycols suggested that osmotic lysis resulted from insertion of a pore less than 3 nm in diameter into the plasma membrane. Results concerning the uptake of membrane‐impermeant fluorescent compounds of various sizes are consistent with the osmoprotection analysis. Therefore, kinetic and genetic evidence suggested that the apparent ability of L. pneumophila to insert a pore into eukaryotic membranes on initial contact may play a role in altering endocytic trafficking events within the host cell and in the establishment of a replicative vacuole.
Use of asymmetric cell division and spoIIIE mutants to probe chromosome orientation and organization in Bacillus subtilis
Authors: Wu Ling Juan , Errington Jeffery
Published: March 2003

Soon after the onset of sporulation in Bacillus subtilis, asymmetric cell division occurs to generate the differentiating prespore and mother cell types. Formation of the septum close to the cell pole initially bisects the nucleoid destined for the prespore, trapping only about one‐third of the DNA in the small compartment. The remaining part of the chromosome is then transported through the septum. spoIIIE mutant cells fail to transfer the DNA and arrest with only partially segregated prespore chromosomes. Previous work has shown that the orientation of the chromosome at the time of septation is not random. Here, we use both physical and genetic methods to characterize the trapped DNA. The results show that the chromosome has a very specific orientation at the time of septation, consistent with the action of a centromere‐like sequence near oriC. They also demonstrate that the chromosome is folded, or otherwise organized, in a highly ordered manner.
An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli
Authors: Van Etten William J. , Janssen Gary R.
Published: March 2003

We determined the in vivo translational efficiency of ‘unleadered’lacZ compared with a conventionally leadered lacZ with and without a Shine–Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5′‐AGGA‐3′ to 5′‐UUUU‐3′ results in a 15‐fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon–anticodon complementarity or require an AUG initiation codon in a sequence‐specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon–anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.
Identification of the Chi site of Haemophilus influenzae as several sequences related to the Escherichia coli Chi site
Authors: Sourice Sophie , Biaudet Véronique , El Karoui Meriem , Ehrlich S. Dusko , Gruss Alexandra
Published: March 2003

The Escherichia coli Chi site 5′‐GCTGGTGG‐3′ modulates the activity of the powerful dsDNA exonuclease and helicase RecBCD. Genome sequence analyses revealed that Chi is frequent on the chromosome and oriented with respect to replication on the E. coli genome. Chi is also present much more frequently than predicted statistically for a random 8‐mer sequence. Although it is assumed that Chi is ubiquitous, there is virtually no proof that its features are conserved in other microorganisms. We therefore identified and analysed the Chi sequence of an organism for which the full genome sequence was available, Haemophilus influenzae. The biological test we used is based on our finding that rolling circle plasmids provide a specific substrate for RecBCD analogues in different microorganisms. Unexpectedly, several related sequences, corresponding to 5′‐GNTGGTGG‐3′ and 5′‐G(G/C)TGGAGG‐3′, showed Chi activity. As in E. coli, the H. influenzae Chi sites are frequent on the genome, which is in keeping with the need for frequent Chi sites for dsDNA break repair of chromosomal DNA. Although statistically over‐represented, this feature is less marked than that of the E. coli Chi site. In contrast to E. coli, the H. influenzae Chi motifs are only slightly oriented with respect to the replication strand. Thus, although Chi appears to have a highly conserved biological role in attenuating exonuclease activity, its sequence characteristics and statistical representation on the genome may differ according to the particular features of the host.
Transcription–repair coupling factor is involved in carbon catabolite repression of the Bacillus subtilis hut and gnt operons
Authors: Zalieckas Jill M. , Wray, Jr Lewis V. , Ferson Amy E. , Fisher Susan H.
Published: March 2003

A Bacillus subtilis mutant that partially relieves carbon catabolite repression (CCR) of the hut operon was isolated by transposon mutagenesis. Characterization of this mutant revealed that the transposon had inserted into the gene, mfd, that encodes transcription–repair coupling factor. The Mfd protein is known to promote strand‐specific DNA repair by displacing RNA polymerase stalled at a nucleotide lesion and directing the (A)BC excinuclease to the DNA damage site. A set of transcriptional lacZ fusions was used to demonstrate that the mfd mutation relieves CCR of hut and gnt expression at the cis‐acting cre sequences located downstream of the transcriptional start site but does not affect CCR at sites located at the promoters. CCR of the amyE and bglPH genes, which contain cre sequences that overlap their promoters, is not altered by the mfd mutation. These results support a model in which the Mfd protein displaces RNA polymerase stalled at downstream cre sites that function as transcriptional roadblocks and reveal a new role for Mfd in cellular physiology.
BipA: a tyrosine‐phosphorylated GTPase that mediates interactions between enteropathogenic Escherichia coli (EPEC) and epithelial cells
Authors: Farris Michele , Grant Andrew , Richardson Tobias B. , O'Connor C. David
Published: March 2003

We report the functional characterization of BipA, a GTPase that undergoes tyrosine phosphorylation in an enteropathogenic Escherichia coli (EPEC) strain. BipA− mutants adhere to cultured epithelial cells but fail to trigger the characteristic cytoskeletal rearrangements found in cells infected with wild‐type EPEC. In contrast, increased expression of BipA enhances actin remodelling and results in the hyperformation of pseudopods. BipA appears to be the first example of a new class of virulence regulator, as it also controls flagella‐mediated cell motility and resistance to the antibacterial effects of a human host defence protein. Its striking sequence similarity to ribosome‐binding elongation factors suggests that it uses a novel mechanism to modulate gene expression.
The control of Azorhizobium caulinodans nifA expression by oxygen, ammonia and by the HF‐I‐like protein, NrfA
Authors: Kaminski P. Alexandre , Elmerich Claudine
Published: March 2003

The control of Azorhizobium caulinodans nifA expression in response to oxygen and ammonia involves FixLJ, FixK, NtrBC, NtrXY and the HF‐I‐like protein NrfA. The regulation is thus complex and possibly involves post‐transcriptional regulation by NrfA. The coding region of nifA was determined using a translational lacZ fusion and by site‐directed mutagenesis to identify which of four in frame AUG codons was used. The major NifA protein is translated from the second AUG codon and is predicted to consist of 613 amino acids. Primer extension analysis showed a major transcript starting 34 bp downstream from the anaerobox in wild‐type, nifA, rpoN, ntrC and nrfA strains, but not in a fixK mutant. FixK‐ and oxygen‐dependent transcription of nifA was confirmed by the analysis of four transcriptional nifA–lacZ fusions with fusion junctions at positions + 1, + 47, + 110 and + 181 with respect to the start site. Regulation by ammonia was independent of FixK and RpoN, NtrC being only partially required. Thus, there may be another type of nitrogen control that does not involve NtrC in A. caulinodans. NrfA is not required for the initiation of nifA transcription but, most probably, has an effect on nifA mRNA stability and/or translation. NrfA also restores the defect in rpoS translation to an Escherichia coli hfq mutant, indicating that HF‐I and NrfA have similar activities in both A. caulinodans and E. coli.
Stress induction of the Bacillus subtilis clpP gene encoding a homologue of the proteolytic component of the Clp protease and the involvement of ClpP and ClpX in stress tolerance
Authors: Gerth Ulf , Krüger Elke , Derré Isabelle , Msadek Tarek , Hecker Michael
Published: March 2003

The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, was cloned and sequenced. The amount of clpP‐specific mRNA increased after heat shock, salt and ethanol stress, as well as after treatment with puromycin. Two transcriptional start sites upstream of the clpP structural gene were identified, preceded by sequences resembling the consensus sequences of promoters recognized by σA andσB transcriptional factors of the B. subtilis RNA polymerase respectively. Transcription initiation occurred predominantly at the putative σA‐dependent promoter in exponentially growing cells and was induced under stress conditions. After exposure to stress, initiation of transcription also increased at the σB‐dependent promoter, but to a lesser extent, indicating that clpP belongs to a double promoter‐controlled subgroup of class III general stress genes in B. subtilis. In a sigB mutant strain, clpP remained heat and stress inducible at the σA‐dependent promoter. BgaB–reporter gene fusions, carrying either the σA‐ or the σB‐dependent promoter, showed a higher bgaB induction at the σA‐dependent promoter, whereas a significantly lower level of induction was measured at the σB‐dependent promoter. The σA‐dependent promoter appeared to be crucial for the heat‐inducible transcription of clpP. A CIRCE (controlling inverted repeat of chaperone expression) element, the characteristic regulation target of class I heat shock genes such as dnaK and groESL, was not found between the transcriptional and translational start sites. Mutants lacking either the proteolytic component ClpP or the regulatory ATPase component ClpX were phenotypically distinct from the wild type. Both mutants produced chains of elongated cells and exhibited severely impaired growth under stress conditions and starvation. Comparison of two‐dimensional protein gels from wild‐type cells with those from clpP and clpX mutant cells revealed several changes in the protein pattern. Several proteins, such as GroEL, PpiB, PykA, SucD, YhfP, YqkF, YugJ and YvyD, which were found preferentially in higher amounts in both clpP and clpX mutants, might be potential substrates for the ClpXP protease.
A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis
Authors: Cervin Marguerite A. , Spiegelman George B. , Raether Brian , Ohlsen Kari , Perego Marta , Hoch James A.
Published: March 2003

The Spo0JA and Spo0JB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid‐partitioning proteins, respectively, and mutation of spo0JB prevents the expression of stage II genes of sporulation. This phenotype is a consequence of Spo0JA activity in the absence of Spo0JB, and its basis was unknown. In the studies reported here, Spo0JA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor Spo0A and RNA polymerase with the spoIIG promoter. This repressor‐like activity is likely to be the basis for preventing the onset of differentiation in vivo. Spo0JB is known to neutralize Spo0JA activity in vivo and also to interact with a mitotic‐like apparatus responsible for chromosome partitioning. These data suggest that Spo0JA and Spo0JB form a regulatory link between chromosome partition and development gene expression.
DNA sequence determinant for Flp‐induced DNA bending
Authors: Luetke Karen H. , Sadowski Paul D.
Published: March 2003

The Flp site‐specific recombinase from Saccharomyces cerevisiae induces DNA bending upon interaction with the Flp recognition target (FRT) site. The minimal FRT site comprises the inverted a and b binding elements, which flank a central 8 bp core region. The DNA bend in a complex of two Flp monomers bound to the FRT site is located in the middle of the core region. When the central AT basepair was replaced with a CG, the DNA bend was positioned at the outside end of the core region adjacent to the a binding element. The other basepairs surrounding the central AT basepair were not important to the position of Flp‐induced bends. The change also decreased Flp‐mediated cleavage of the top strand of the FRT site and increased Flp‐mediated cleavage of the bottom strand. The overall recombination proficiency of the site was impaired. We conclude that the central AT basepair provides a point of flexure in the FRT site, which Flp uses to position the bend in dimeric Flp–DNA complexes, and that the structure of the core DNA influences the functionality of the site.
A developmentally regulated gene encoding a repressor‐like protein is essential for sporulation in Streptomyces coelicolor A3(2)
Authors: Ryding N. Jamie , Kelemen Gabriella H. , Whatling Carl A. , Flärdh Klas , Buttner Mark J. , Chater Keith F.
Published: March 2003

whiH is one of several known loci specifically needed for the orderly multiple sporulation septation of aerial hyphae of Streptomyces coelicolor A3(2) and for the expression of at least some late sporulation genes. DNA complementing whiH mutants was located immediately upstream of hrdB, which encodes the principal σ factor of S. coelicolor. Sequencing revealed a gene whose disruption gave rise to a typical whiH mutant phenotype. Four whiH mutants contained base changes or a frameshift in this gene. The deduced product of whiH is related to a large family of bacterial regulatory proteins, the most similar being several repressors (such as GntR of Bacillus subtilis) responsive to carboxylate‐containing intermediates in carbon metabolism. Transcription of whiH was initiated at a single promoter, PwhiH. Levels of whiH mRNA were developmentally regulated, increasing sharply when aerial mycelium was present, and reaching a maximum approximately when spores were first detectable. Transcript levels were markedly increased in a whiH mutant, indicating the possible involvement of WhiH in negative regulation of its own production. PwhiH was directly dependent on the σ factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.
A lethal role for lipid A in Salmonella infections
Authors: Khan Shahid A. , Everest Paul , Servos Spiros , Foxwell Neale , Zähringer Ulrich , Brade Helmut , Rietschel Ernst Th. , Dougan Gordon , Charles Ian G. , Maskell Duncan J.
Published: March 2003

Salmonella infections in naturally susceptible mice grow rapidly, with death occurring only after bacterial numbers in vivo have reached a high threshold level, commonly called the lethal load. Despite much speculation, no direct evidence has been available to substantiate a role for any candidate bacterial components in causing death. One of the most likely candidates for the lethal toxin in salmonellosis is endotoxin, specifically the lipid A domain of the lipopolysaccharide (LPS) molecule. Consequently, we have constructed a Salmonella mutant with a deletion–insertion in its waaN gene, which encodes the enzyme that catalyses one of the two secondary acylation reactions that complete lipid A biosynthesis. The mutant biosynthesizes a lipid A molecule lacking a single fatty acyl chain and is consequently less able to induce cytokine and inducible nitric oxide synthase (iNOS) responses both in vivo and in vitro. The mutant bacteria appear healthy, are not sensitive to increased growth temperature and synthesize a full‐length O‐antigen‐containing LPS molecule lacking only the expected secondary acyl chain. On intravenous inoculation into susceptible BALB/c mice, wild‐type salmonellae grew at the expected rate of approximately 10‐fold per day in livers and spleens and caused the death of the infected mice when lethal loads of approximately 108 were attained in these organs. Somewhat unexpectedly, waaN mutant bacteria grew at exactly the same rate as wild‐type bacteria in BALB/c mice but, when counts reached 108 per organ, mice infected with mutant bacteria survived. Bacterial growth continued until unprecedentedly high counts of 109 per organ were attained, when approximately 10% of the mice died. Most of the animals carrying these high bacterial loads survived, and the bacteria were slowly cleared from the organs. These experiments provide the first direct evidence that death in a mouse typhoid infection is directly dependent on the toxicity of lipid A and suggest that this may be mediated via pro‐inflammatory cytokine and/or iNOS responses.
Genetic and biochemical characterization of mutations affecting the carboxy‐terminal domain of the Escherichia coli molecular chaperone DnaJ
Authors: Goffin Laurence , Georgopoulos Costa
Published: March 2003

DnaJ is a universally conserved heat shock protein involved in protein folding. DnaJ contains four conserved domains. The N‐terminal ‘J‐domain’ has been shown to be responsible for the recruitment of its specific DnaK partner protein. The ‘Gly/Phe’‐ and ‘Cys‐rich’ domains have been implicated in stabilizing interactions with DnaK. DnaJ is also able to interact independently with unfolded or native polypeptides. Very little is known regarding such binding/chaperone abilities, but it has been suggested that the least conserved carboxy‐terminal domain could contribute to these properties. To gain insight into the biological activity of this fourth domain, we deleted two relatively conserved patches of amino acid residues, a ‘G‐rich’ cluster and a ‘G–D–L–Y–V’ motif, resulting in the DnaJΔ[230–238] and DnaJΔ[242–246] mutant proteins respectively. Both mutant proteins are partially defective in stimulating the ATPase activity of DnaK and in preventing aggregation of firefly luciferase in vitro. Both mutants have lost the ability to regulate the σ32‐dependent heat shock response, as shown in vivo using a heat shock transcriptional fusion. Furthermore, and unlike wild‐type DnaJ, DnaJΔ[242–246] is unable to assist the DnaK‐dependent refolding of denatured luciferase. In agreement with these results, we found that DnaJΔ[242–246] is unable to restore either the temperature‐sensitive phenotype or the motility defect of a dnaJ null mutation. Substitution of amino acids [242–246] by five alanines leads to similar phenotypic defects, suggesting that altering the ‘G–D–L–Y–V’ motif leads to partial loss of DnaJ activity. Our data clearly support a role in the intrinsic chaperone/substrate binding ability of the carboxy‐terminal domain of DnaJ.
Identification of histidine residues in Wolinella succinogenes hydrogenase that are essential for menaquinone reduction by H2
Authors: Gross Roland , Simon Jörg , Lancaster C. Roy D. , Kröger Achim
Published: March 2003

The cytochrome b subunit (HydC) of Wolinella succinogenes hydrogenase binds two haem B groups. This is concluded from the haem B content of the isolated hydrogenase and is confirmed by the response of its cytochrome b to redox titration. In addition, three of the four haem B ligands were identified by characterizing mutants with the corresponding histidine residues replaced by alanine or methionine. Substitution in HydC of His‐25, His‐67 or His‐186, which are, in addition to His‐200, predicted to be haem B ligands, caused the loss of quinone reactivity of the hydrogenase, while the activity of benzylviologen reduction was retained. The corresponding mutants did not grow with H2 as electron donor and either fumarate or polysulphide as terminal electron acceptor. The mutants grown with formate and fumarate did not catalyse electron transport from H2 to fumarate or to polysulphide, or quinone reduction by H2, in contrast to the wild‐type strain. Cytochrome b was not reduced by H2 in the Triton X‐100 extract of the mutant membranes, which contained wild‐type amounts of the mutated HydC protein. Substitution in HydC of His‐122, His‐158 or His‐187, which are predicted not to be haem B ligands, yielded mutants with wild‐type properties. Substitution in HydA of His‐188 or of His‐305 resulted in mutants with the same properties as those lacking one of the haem B ligands of HydC. His‐305 is located in the membrane‐integrated C‐terminal helix of HydA. His‐188 of HydA is predicted to be a ligand of the distal iron–sulphur centre that may serve as the direct electron donor to the haem B groups of HydC. The results suggest that each of the three predicted haem B ligands of HydC tested (out of four) is required for electron transport from H2 to either fumarate or polysulphide, and for quinone reactivity. This also holds true for the two conserved histidine residues of HydA.
The high‐pathogenicity island of Yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn tRNA genes
Authors: Buchrieser Carmen , Brosch Roland , Bach Sandrine , Guiyoule Annie , Carniel Elisabeth
Published: March 2003

Pathogenicity islands (PAIs) have been identified in several bacterial species. A PAI called high‐pathogenicity island (HPI) and carrying genes involved in iron acquisition (yersiniabactin system) has been previously identified in Yersinia enterocolitica and Yersinia pestis. In this study, the HPI of the third species of Yersinia pathogenic for humans, Y. pseudotuberculosis, has been characterized. We demonstrate that the HPI of strain IP32637 has a physical and genetic map identical to that of Y. pestis. A gene homologous to the bacteriophage P4 integrase gene is located downstream of the asn tRNA locus that borders the HPI of strain IP32637. This int gene is at the same position on the HPI of all three pathogenic Yersinia species. However, in contrast to Y. pestis 6/69, the HPI of Y. pseudotuberculosis IP32637 is not invariably adjacent to the pigmentation segment and can be inserted at a distance ≥ 190 kb from this segment. Also, in contrast to Y. pestis and Y. enterocolitica, the HPI of Y. pseudotuberculosis IP32637 can precisely excise from the chromosome, and, strikingly, it can be found inserted in any of the three asn tRNA loci present on the chromosome of this species, one of which is adjacent to the pigmentation segment. The pigmentation segment, which is present in Y. pestis but not in Y. enterocolitica, is also present and well conserved in all strains of Y. pseudotuberculosis studied. In contrast, the presence and size of the HPIs vary depending on the serotype of the strain: an entire HPI is found in strains of serotypes I only, a HPI with a 9 kb truncation in its left‐hand part that carries the IS100 sequence and the psn and ybtE genes characterizes the strains of serotype III, and no HPI is found in strains of serotypes II, IV and V.
The structure of an ECF‐σ‐dependent, light‐inducible promoter from the bacterium Myxococcus xanthus
Authors: Martínez‐Argudo Isabel , Ruiz‐Vázquez Rosa M. , Murillo Francisco J.
Published: March 2003

Expression of the Myxococcus xanthus gene crtI is controlled by a light‐inducible promoter. The activity of this promoter depends on CarQ, a σ factor of the extracytoplasmic function (ECF) subfamily. Here, we show that the minimum DNA stretch reproducing normal expression of crtI extends from a few bases upstream of the −35 position to a site well downstream of the transcriptional start. The downstream DNA contains an enhancer‐like element that remains active when displaced upstream of the promoter. Experimental evidence is provided for the activity of the crtI promoter being critically dependent on a pentanucleotide sequence centred at the −31 position. The similarity of this sequence with the consensus for ECF‐σ‐dependent promoters from other bacteria is discussed. The activity of the crtI promoter also depends on certain basepairs at the −10 region. Hence, the operation of ECF‐σ‐factors seems to require binding to two different DNA sites, although the −10 sequences of different ECF‐σ‐dependent promoters are unrelated to one another, and the ECF‐σ‐factors themselves lack the conserved domain known to mediate binding of other σ‐factors to the −10 DNA site.
The type III secretion determinants of the flagellar anti‐transcription factor, FlgM, extend from the amino‐terminus into the anti‐σ28 domain
Authors: Chilcott Gavin S. , Hughes Kelly T.
Published: March 2003

The flagellar‐specific anti‐sigma factor, FlgM, inhibits the expression of late flagellar genes until the hook–basal body structure is assembled and competent for export of the flagellins and hook‐associated proteins (flagellar late proteins). FlgM monitors this assembly checkpoint by being a substrate for export via the hook–basal body structure, which includes a type III protein secretion complex. Amino acid sequence alignment of late‐secreted flagellar proteins identified a region of homology present in the amino‐terminus of FlgM and the other late flagellar proteins, but not in flagellar proteins secreted earlier during flagellar biosynthesis. Single amino acid substitutions at specific positions within this motif decreased the export of FlgM. Deletion of this region (S3‐P11) resulted in lower intracellular FlgM levels, but did not prevent recognition and export by the flagellar‐specific secretion system. Mutations were isolated in a second region of FlgM spanning residues K27 to A65 that exhibited increased anti‐σ28 activity. These FlgM ‘hyperinhibitor’ mutants were secreted less than wild‐type FlgM. Mutations that interfere with the secretion of FlgM without abolishing anti‐σ28 activity have a negative effect upon the secretion of a His‐tagged FlgM mutant that lacks anti‐σ28 activity. Models are proposed to explain the dominant negative phenotype of the FlgM secretion mutants reported in this study.
Type III secretion by Salmonella typhimurium does not require contact with a eukaryotic host
Authors: Daefler Simon
Published: March 2003

The type III secretion system encoded by pathogenicity island I in Salmonella typhimurium delivers proteins to the external milieu and into the eukaryotic host cell. The principal factor in induction of the secretion system was found to be a change in the pH of the culture medium from acidic to mildly alkaline. The synthesis of components of the secretion machinery and the production and secretion of substrates occur simultaneously and do not require contact with a eukaryotic host cell. This argues against the concept that type III secretion in S. typhimurium is a process in which the delivery of a presynthesized pool of substrates is triggered by contact with a eukaryotic host cell.
The structural and functional organization of H‐NS‐like proteins is evolutionarily conserved in Gram‐negative bacteria
Authors: Bertin Philippe , Benhabiles Nora , Krin Evelyne , Laurent‐Winter Christine , Tendeng Christian , Turlin Evelyne , Thomas Annick , Danchin Antoine , Brasseur Robert
Published: March 2003

The structural gene of the H‐NS protein, a global regulator of bacterial metabolism, has been identified in the group of enterobacteria as well as in closely related bacteria, such as Erwinia chrysanthemi and Haemophilus influenzae. Isolated outside these groups, the BpH3 protein of Bordetella pertussis exhibits a low amino acid conservation with H‐NS, particularly in the N‐terminal domain. To obtain information on the structure, function and/or evolution of H‐NS, we searched for other H‐NS‐related proteins in the latest databases. We found that HvrA, a trans‐activator protein in Rhodobacter capsulatus, has a low but significant similarity with H‐NS and H‐NS‐like proteins. This Gram‐negative bacterium is phylogenetically distant from Escherichia coli. Using theoretical analysis (e.g. secondary structure prediction and DNA binding domain modelling) of the amino acid sequence of H‐NS, StpA (an H‐NS‐like protein in E. coli ), BpH3 and HvrA and by in vivo and in vitro experiments (e.g. complementation of various H‐NS‐related phenotypes and competitive gel shift assay), we present evidence that these proteins belong to the same class of DNA binding proteins. In silico analysis suggests that this family also includes SPB in R. sphaeroides, XrvA in Xanthomonas oryzae and VicH in Vibrio cholerae. These results demonstrate that proteins structurally and functionally related to H‐NS are widespread in Gram‐negative bacteria.
First molecular characterization of a uridine diphosphate galacturonate 4‐epimerase: an enzyme required for capsular biosynthesis in Streptococcus pneumoniae type 1
Authors: Muñoz Rosario , López Rubens , De Frutos Mercedes , García Ernesto
Published: March 2003

Uridine diphosphate galacturonate 4‐epimerases (UDPGLEs) are enzymes that convert UDP‐glucuronate into UDP‐galacturonate. Although the presence of UDPGLEs has been reported in prokaryotic and eukaryotic organisms, the genes coding for these enzymes are completely unknown. The galacturonic acid‐containing capsular polysaccharide of Streptococcus pneumoniae type 1 is synthesized through the action of a specific UDPGLE. We have constructed a defined deletion mutant in the cap1J gene (one of the 15 cap1 genes responsible for the synthesis of the type 1 capsule) that exhibited an unencapsulated phenotype. This mutant was unable to synthesize UDPGLE, suggesting that Cap1J was the type 1‐specific UDPGLE of S. pneumoniae. Escherichia coli cells harbouring the recombinant plasmid pRMM38 (cap1J ) overproduced a 40 kDa protein, characterized as Cap1J on the basis of the N‐terminal amino acid sequence analysis, and expressed high levels of enzymatically active Cap1J epimerase. Cap1J was partially purified, although purification to electrophoretic homogeneity inactivated the enzyme irreversibly. The enzyme has the following characteristics: Kmfor UDP‐glucuronate, 0.24 mM; pH optimum, 7.5; equilibrium constant (in the direction of UDP‐galacturonate formation), 1.3; and an approximate Mr of 80 000 for the active form. The Cap1J protein exhibited a fluorescence emission spectrum similar to that of NADH. Upon inactivation with p‐hydroxymercuribenzoate, the addition of NAD+ and 2‐mercaptoethanol were sufficient to reactivate the enzyme. Among several compounds tested, UDP‐galactose and UDP‐xylose exhibited the highest inhibition of the UDPGLE activity. Inactivation of UDPGLE activity was also observed in the presence of UMP and several reducing sugars. To our knowledge, this is the first example of a thoroughly molecular characterization of a UDPGLE.
Selective advantages created by codon ambiguity allowed for the evolution of an alternative genetic code inCandidaspp.
Authors: Santos Manuel A. S. , Cheesman Caroline , Costa Vitor , Moradas‐Ferreira Pedro , Tuite Mick F.
Published: March 2003

Several species of the genusCandidadecode the standard leucine CUG codon as serine. This and other deviations from the standard genetic code in both nuclear and mitochondrial genomes invalidate the notion that the genetic code is frozen and universal and prompt the questions ‘why alternative genetic codes evolved and, more importantly, how can an organism survive a genetic code change?’ To address these two questions, we have attempted to reconstruct the early stages ofCandida albicansCUG reassignment in the closely related yeastSaccharomyces cerevisiae. These studies suggest that this genetic code change was driven by selection using a molecular mechanism that requires CUG ambiguity. Such codon ambiguity induced a significant decrease in fitness, indicating that CUG reassignment can only be selected if it introduces an evolutionary edge to counteract the negative impact of ambiguity. We have shown that CUG ambiguity induces the expression of a novel set of stress proteins and triggers the general stress response, which, in turn, creates a competitive edge under stress conditions. In addition, CUG ambiguity inS. cerevisiaeinduces the expression of a number of novel phenotypes that mimic the natural resistance to stress characteristic ofC. albicans. The identification of an evolutionary advantage created by CUG ambiguity is the first experimental evidence for a genetic code change driven by selection and suggests a novel role for codon reassignment in the adaptation to new ecological niches.
A single amino acid, outside the AlcR zinc binuclear cluster, is involved in DNA binding and in transcriptional regulation of the alc genes in Aspergillus nidulans
Authors: Nikolaev Igor , Cochet Marie‐Françoise , Lenouvel François , Felenbok Béatrice
Published: March 2003

In Aspergillus nidulans, the transcriptional activator AlcR mediates specific induction of a number of alc genes. The AlcR DNA‐binding domain is a zinc binuclear cluster that differs from the other members of the Zn2Cys6 family in several respects. Of these, the most remarkable is its ability to bind in vitro as a monomer to single sites, whereas only repeated sites (direct or inverted) are necessary and functional in vivo. Deletion of the first five amino acids (following the N‐terminal methionine) upstream of the AlcR zinc cluster or mutation of a single residue, Arg‐6, impairs the AlcR in vitro binding mainly to symmetrical sites. In vivo, the same mutations result in the inability of A. nidulans to grow on ethanol. The alc− phenotype results from a drastic decrease in activation of its own transcription and, in addition, that of the two structural genes, alcA and aldA, required for ethanol oxidation. This defect seems to be correlated to the inability of the Arg‐6 AlcR mutant protein to bind to AlcR palindrome targets, which are essential in the three alc promoters. AlcR shows a unique pattern of binding and of transactivation among the Zn2Cys6 family.
HAP4, the glucose‐repressed regulated subunit of the HAP transcriptional complex involved in the fermentation–respiration shift, has a functional homologue in the respiratory yeast Kluyveromyces lactis
Authors: Bourgarel D. , Nguyen C.‐C. , Bolotin‐Fukuhara M.
Published: March 2003

In Saccharomyces cerevisiae, the heteromeric HAP transcription factor is necessary for optimal growth on respiratory carbon sources. One of its components, the Hap4p protein, is necessary for transcriptional activation. The same protein is also the regulatory part of the complex in response to carbon sources, as HAP4 is strongly induced during the shift from fermentative to respiratory metabolism in S. cerevisiaeWe report here the characterization of a new gene from the respiratory yeast Kluyveromyces lactis, obtained by heterologous complementation of a Δhap4 S. cerevisiae mutant strain. The deduced sequence of the protein (643 amino acids) exhibits two small domains (11 and 16 amino acids respectively) highly homologous to corresponding domains of ScHap4p, while the overall similarity is rather weak. Additional experiments were performed to confirm the functional homology of this new gene with ScHAP4, which we named KlHAP4. The importance of the small highly conserved N‐terminal sequence was confirmed by in vitro mutagenesis. All the mutations that interfere with the Hap4p–Hap2/3/5 interaction were localized in it. The discovery of the same regulatory protein in two metabolically distinct yeast species raises the question of its functional significance during evolution.
The identification of a new family of sugar efflux pumps in Escherichia coli
Authors: Liu Jia Yeu , Miller Paul F. , Gosink Mark , Olson Eric R.
Published: March 2003

Using a functional cloning strategy with an Escherichia coli genomic plasmid library, we have identified a new family of sugar efflux proteins with three highly homologous members in the E. coli genome. In addition, two open reading frames, one present in Yersinia pestis and the other in Deinococcus radioduransappear to encode closely related proteins. An in vitro transport assay using inside‐out membrane vesicles prepared from overproducing strains was used to demonstrate that members of this new family can efflux [14C]‐lactose. As sugar efflux phenomena have been reported previously in several bacterial species including E. coli, the identification of a new family of sugar efflux proteins may help to reveal the physiological role of sugar efflux in metabolism. It is proposed that the E. coli members of this family, whose functions were previously unknown, be given the gene family designation SET for sugar efflux transporter.
Bacterial secreted proteins are required for the internalization of Campylobacter jejuni into cultured mammalian cells
Authors: Konkel Michael E. , Kim Bong J. , Rivera‐Amill Vanessa , Garvis Steven G.
Published: March 2003

Presented here is the first evidence that Campylobacter jejuni secrete proteins upon co‐cultivation with host cells and in INT 407 cell‐conditioned medium. A C. jejuni gene designated ciaB for Campylobacter invasion antigen B was identified, using a differential screening technique, which is required for this secretion process and the efficient entry of this bacterium into a host cell. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73 154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins associated with the invasion of host cells from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Confocal microscopic examination of C. jejuni‐infected cells revealed that CiaB was translocated into the cytoplasm of the host cells. Culturing C. jejuni with INT 407 cells or in INT 407‐conditioned medium resulted in the secretion of at least eight proteins, ranging in size from 12.8 to 108 kDa, into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all eight proteins, indicating that CiaB is required for the secretion process. The identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization and the pathogenesis of C. jejuni‐mediated enteritis.
The R28 protein of Streptococcus pyogenes is related to several group B streptococcal surface proteins, confers protective immunity and promotes binding to human epithelial cells
Authors: Stålhammar‐Carlemalm Margaretha , Areschoug Thomas , Larsson Charlotte , Lindahl Gunnar
Published: March 2003

The R28 protein is a surface molecule expressed by some strains of Streptococcus pyogenes (group A streptococcus). Here, we present evidence that R28 may play an important role in virulence. Sequence analysis demonstrated that R28 has an extremely repetitive sequence and can be viewed as a chimera derived from the three surface proteins Rib, α and β of the group B streptococcus (GBS). Thus, the gene encoding R28 may have originated in GBS. The R28 protein promotes adhesion to human epithelial cells, as shown by experiments with an R28‐negative mutant and by the demonstration that antibodies to highly purified R28 inhibited adhesion. In a mouse model of lethal intraperitoneal S. pyogenes infection, antibodies to R28 conferred protective immunity. However, the virulence of an R28‐negative mutant was similar to that of the parental strain in the intraperitoneal infection model. Together, these data indicate that R28 represents a novel type of adhesin expressed by S. pyogenes and that R28 may also act as a target for protective antibodies at later stages of an infection. We consider the hypothesis that R28 played a pathogenetic role in the well‐known epidemics of childbed fever (puerperal fever), which were caused by S. pyogenes. A role for R28 in these epidemics is suggested by epidemiological data.
Inactivation of the gene (cpe ) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe‐positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops
Authors: Sarker Mahfuzur R. , Carman Robert J. , McClane Bruce A.
Published: March 2003

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non‐food‐borne GI illnesses, such as antibiotic‐associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock‐out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non‐food‐borne GI disease isolate carrying a plasmid‐borne cpe gene). Western blot analyses confirmed that neither cpe knock‐out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild‐type cpe gene. When the virulence of the wild‐type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild‐type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock‐out mutants induced these intestinal effects. However, full sporulation‐associated virulence could be restored by complementing these cpe knock‐out mutants with a recombinant plasmid carrying the wild‐type cpe gene, which confirms that the observed loss of virulence for the cpe knock‐out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe‐positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe‐positive C. perfringens type A isolates.
The widely conserved Era G‐protein contains an RNA‐binding domain required for Era function in vivo
Authors: Johnstone Brian H. , Handler Aaron A. , Chao Diana K. , Nguyen Vi , Smith Michael , Ryu Seung Y. , Simons Elizabeth L. , Anderson Paul E. , Simons Robert W.
Published: March 2003

Era is a small G‐protein widely conserved in eubacteria and eukaryotes. Although essential for bacterial growth and implicated in diverse cellular processes, its actual function remains unclear. Several lines of evidence suggest that Era may be involved in some aspect of RNA biology. The GTPase domain contains features in common with all G‐proteins and is required for Era function in vivo. The C‐terminal domain (EraCTD) bears scant similarity to proteins outside the Era subfamily. On the basis of sequence comparisons, we argue that the EraCTD is similar to, but distinct from, the KH RNA‐binding domain. Although both contain the consensus VIGxxGxxI RNA‐binding motif, the protein folds are probably different. We show that bacterial Era binds RNA in vitro and can form higher‐order RNA–protein complexes. Mutations in the VIGxxGxxI motif and other conserved residues of the Escherichia coli EraCTD decrease RNA binding in vitro and have corresponding effects on Era function in vivo, including previously described effects on cell division and chromosome partitioning. Importantly, mutations in L‐66, located in the predicted switch II region of the E. coli Era GTPase domain, also perturb binding, leading us to propose that the GTPase domain regulates RNA binding in response to unknown cellular cues. The possible biological significance of Era RNA binding is discussed.