bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional regulation of transport and utilization systems for hexuronides, hexuronates and hexonates in gamma purple bacteria
Authors: Rodionov Dmitry A. , Mironov Andrey A. , Rakhmaninova Alexandra B. , Gelfand Mikhail S.
Published: March 2003

The comparative approach is a powerful tool for the analysis of gene regulation in bacterial genomes. It can be applied to the analysis of regulons that have been studied experimentally as well as that of regulons for which no known regulatory sites are available. It is assumed that the set of co‐regulated genes and the regulatory signal itself are conserved in related genomes. Here, we use genomic comparisons to study the regulation of transport and utilization systems for sugar acids in gamma purple bacteria Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Yersinia pestis, Erwinia chrysanthemi, Haemophilus influenzae and Vibrio cholerae. The variability of the operon structure and the location of the operator sites for the main transcription factors are demonstrated. The common metabolic map is combined with known and predicted regulatory interactions. It includes all known and predicted members of the GntR, UxuR/ExuR, KdgR, UidR and IdnR regulons. Moreover, most members of these regulons seem to be under catabolite repression mediated by CRP. The candidate UxuR/ExuR signal is proposed, the KdgR consensus is extended, and new operators for all transcription factors are identified in all studied genomes. Two new members of the KdgR regulon, a hypothetical ATP‐dependent transport system OgtABCD and YjgK protein with unknown function, are detected. The former is likely to be the transport system for the products of pectin degradation, oligogalacturonides.
A role for the Escherichia coli H‐NS‐like protein StpA in OmpF porin expression through modulation of micF RNA stability
Authors: Deighan Padraig , Free Andrew , Dorman Charles J.
Published: March 2003

When a wild‐type strain of Escherichia coli and its stpA, hns and stpA hns mutant derivatives were compared by two‐dimensional protein gel electrophoresis, the levels of expression of several proteins were found to vary. One of these was identified as the outer membrane porin protein, OmpF. In the stpA hns double mutant, the level of OmpF was downregulated dramatically, whereas in hns or stpA single mutants, it was affected only slightly. Transcription from the ompF promoter was reduced by 64% in the double mutant; however, the level of ompF mRNA was reduced by 96%. This post‐transcriptional expression was found to result from a strong reduction in the half‐life of ompF message in the double mutant. The micF antisense RNA was shown to be involved in OmpF regulation by StpA using a strain deleted for micF. Moreover, micF antisense RNA accumulated considerably in an stpA hns background. Transcriptional data from a micF–lacZ fusion and measurements of micF RNA half‐life confirmed that this was caused by transcriptional derepression of micF as a result of the hns lesion and increased micF RNA stability due to the absence of StpA (a known RNA chaperone). These data suggest a novel facet to the regulation of OmpF expression, namely destabilization of micF RNA by StpA.
About the last common ancestor, the universal life‐tree and lateral gene transfer: a reappraisal
Authors: Glansdorff N.
Published: March 2003

An organismal tree rooted in the bacterial branch and derived from a hyperthermophilic last common ancestor (LCA) is still widely assumed to represent the path followed by evolution from the most primeval cells to the three domains recognized among contemporary organisms: Bacteria, Archaea and Eucarya. In the past few years, however, more and more discrepancies between this pattern and individual protein trees have been brought to light. There has been an overall tendency to attribute these incongruities to widespread lateral gene transfer. However, recent developments, a reappraisal of earlier evidence and considerations of our own lead us to a quite different view. It would appear (i) that the role of lateral gene transfer was overemphasized in recent discussions of molecular phylogenies; (ii) that the LCA was probably a non‐thermophilic protoeukaryote from which both Archaea and Bacteria emerged by reductive evolution but not as sister groups, in keeping with a current evolutionary scheme for the biosynthesis of membrane lipids; and (iii) that thermophilic Archaea may have been the first branch to diverge from the ancestral line.
The quarternary molecular architecture of TetA, a secondary tetracycline transporter from Escherichia coli
Authors: Yin Chang‐Cheng , Aldema‐Ramos Mila L. , Borges‐Walmsley M. Ines , Taylor Robert W. , Walmsley Adrian R. , Levy Stuart B. , Bullough Per A.
Published: March 2003

TetA, a tetracycline cation/proton antiporter, was expressed in Escherichia coli with a C‐terminal tag of six histidines, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two‐dimensional crystals were obtained after reconstitution of purified protein with lipids. Electron microscopy of negatively stained crystals revealed a trigonal symmetry, from which we infer that this secondary transporter has a trimeric structure. An overall molecular envelope can be described by a triangle of side ≈ 100 Å enclosing a central stain‐filled depression. These dimensions are consistent with those obtained from projection views of single, isolated TetA particles that also display a trimeric architecture, confirming that the threefold symmetry is not simply a consequence of crystal‐packing interactions. These data represent the first direct view of the quarternary arrangement of any antibiotic efflux pump. They are fully consistent with biochemical data on TetA, which indicate that it functions as a multimer and that the monomer consists of two domains, one of which plays the major part in oligomerization interactions.
Transposition of Tn4451 and Tn4453 involves a circular intermediate that forms a promoter for the large resolvase, TnpX
Authors: Lyras Dena , Rood Julian I.
Published: March 2003

Tn4451 is the paradigm element of a family of mobilizable chloramphenicol resistance transposons from Clostridium perfringens and Clostridium difficile. The unique feature of these 6.3 kb elements is that their excision to form a circular molecule is mediated by TnpX, a member of the large resolvase family of site‐specific recombinases. By optimizing the transposition assay system in Escherichia coli, we showed that Tn4453a from C. difficile transposed at a higher frequency than the C. perfringens element, Tn4451, and that transposition of both Tn4451 and Tn4453a was significantly enhanced by the provision of a multicopy tnpX gene in trans. The complete nucleotide sequence of Tn4453a was determined, but its comparison with Tn4451 did not reveal why it transposed at a higher frequency. Using experiments involving a chromosomal derivative of Tn4453a, we have confirmed that the circular form is the transposition intermediate. As the tnpX gene is located very close to one end of these elements, primer extension analysis was used to determine the transcription start point. The results showed that the formation of the circular intermediate creates a strong tnpX promoter, which consists of a −10 box originally located at the left end of the transposon and a −35 box originally located at the right end. The data provide strong evidence that transcription of tnpX is likely to occur from the non‐replicating circular intermediate, which would facilitate the subsequent insertion of the transient circular molecule. It is postulated that, when the transposon is in an integrated state, transcription of tnpX would depend on the presence of an appropriately spaced −35 sequence in the DNA flanking the insertion site or the presence of an alternative upstream promoter.
Differential fluorescence induction reveals Streptococcus pneumoniae loci regulated by competence stimulatory peptide
Authors: Bartilson Magdalena , Marra Andrea , Christine Jillian , Asundi Jyoti S. , Schneider William P. , Hromockyj Alexander E.
Published: March 2003

Differential fluorescence induction (DFI) in Streptococcus pneumoniae was used as a method for the discovery of genes activated in specific growth environments. Competence stimulatory peptide (CSP) was used as the model inducing system to identify differentially expressed genes. To identify CSP‐induced promoters, a plasmid library was constructed by inserting random pieces of S. pneumoniae chromosomal DNA upstream of the promoterless gfpmut2 gene in an Escherichia coli/S. pneumoniae shuttle vector. S. pneumoniae carrying the library were induced with CSP and enriched for green fluorescent protein (GFP)‐expressing bacteria using fluorescence‐activated cell sorting. A total of 886 fluorescent clones was screened, and 12 differentially activated promoter elements were identified. Sequence analysis of these clones revealed that three were associated with novel competence loci, one of which we show is essential for DNA uptake, and six are known CSP‐inducible promoters. We also explored whether competence proteins have a role in virulence and found that mutations in three CSP‐inducible genes resulted in attenuated virulence phenotypes in either of two murine infection models. These results demonstrate the utility of DFI as a method for identifying differentially expressed genes in S. pneumoniae and the potential utility of applying DFI to other Gram‐positive bacteria.
A tripeptide sequence within the nascent DaaP protein is required for mRNA processing of a fimbrial operon in Escherichia coli
Authors: Loomis Wendy P. , Koo Jovanka T. , Cheung Teresa P. , Moseley Steve L.
Published: March 2003

The biogenesis of F1845 fimbriae, a member of the Dr family of Escherichia coli adhesins, is regulated by endonucleolytic cleavage of the daaABCDPE primary transcript and differential stability of the resulting cleavage products. Processing of daa mRNA is dependent upon translation of a small open reading frame, designated daaP, which flanks the daa processing site. Here, we demonstrate that daa mRNA processing is directly coupled to daaP translation. Cleavage of the daaA–E mRNA was shown to require the tripeptide Gly–Pro–Pro (GPP), encoded by daaP codons 49–51 downstream of the processing site. Processing also required active translation through RNA located upstream of the processing site; however, processing did not depend on the amino acid sequence encoded by the region of daaP upstream of the processing site. Finally, determination of the processing site was shown to involve its location relative to the codons encoding the GPP tripeptide. These data show that translation of daaP is required in cis to promote RNA processing. These data suggest a model involving interaction of the nascent GPP tripeptide portion of the DaaP polypeptide with the ribosome, triggering cleavage of the associated mRNA at a fixed distance upstream. A model of active involvement of the ribosome in this process is proposed.
Cytochrome oxidase subunit VI of Trypanosoma brucei is imported without a cleaved presequence and is developmentally regulated at both RNA and protein levels
Authors: Tasker Maria , Timms Mark , Hendriks Ed , Matthews Keith
Published: March 2003

Mitochondrial respiration in the African trypanosome undergoes dramatic developmental stage regulation. This requires co‐ordinated control of components encoded by both the nuclear genome and the kinetoplast, the unusual mitochondrial genome of these parasites. As a model for understanding the co‐ordination of these genomes, we have examined the regulation and mitochondrial import of a nuclear‐encoded component of the cytochrome oxidase complex, cytochrome oxidase subunit VI (COXVI). By generating transgenic trypanosomes expressing intact or mutant forms of this protein, we demonstrate that COXVI is not imported using a conventional cleaved presequence and show that sequences at the N‐terminus of the protein are necessary for correct mitochondrial sorting. Analyses of endogenous and transgenic COXVI mRNA and protein expression in parasites undergoing developmental stage differentiation demonstrates a temporal order of control involving regulation in the abundance of, first, mRNA and then protein. This represents the first dissection of the regulation and import of a nuclear‐encoded protein into the cytochrome oxidase complex in these organisms, which were among the earliest eukaryotes to possess a mitochondrion.
Mutational analysis of RsrA, a zinc‐binding anti‐sigma factor with a thiol–disulphide redox switch
Authors: Paget Mark S. B. , Bae Jae‐Bum , Hahn Mi‐Young , Li Wei , Kleanthous Colin , Roe Jung‐Hye , Buttner Mark J.
Published: March 2003

In the Gram‐positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called σR in response to changes in the cytoplasmic thiol–disulphide status, and the activity of σR is controlled post‐translationally by an anti‐sigma factor, RsrA. In vitro, the anti‐sigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol–disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and σR‐dependent transcription, confirming that RsrA is a negative regulator of σR and a key sensor of thiol–disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti‐sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo. We also show that RsrA is a metalloprotein, containing near‐stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol–disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti‐sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free σR.
Probing the mechanism of ATP hydrolysis and substrate translocation in the AAA protease FtsH by modelling and mutagenesis
Authors: Karata Kiyonobu , Verma Chandra S. , Wilkinson Anthony J. , Ogura Teru
Published: March 2003

We have built a homology model of the AAA domain of the ATP‐dependent protease FtsH of Escherichia coli based on the crystal structure of the hexamerization domain of N‐ethylmaleimide‐sensitive fusion protein. The resulting model of the hexameric ring of the ATP‐bound form of the AAA ATPase suggests a plausible mechanism of ATP binding and hydrolysis, in which invariant residues of Walker motifs A and B and the second region of homology, characteristic of the AAA ATPases, play key roles. The importance of these invariant residues was confirmed by site‐directed mutagenesis. Further modelling suggested a mechanism by which ATP hydrolysis alters the conformation of the loop forming the central hole of the hexameric ring. It is proposed that unfolded polypeptides are translocated through the central hole into the protease chamber upon cycles of ATP hydrolysis. Degradation of polypeptides by FtsH is tightly coupled to ATP hydrolysis, whereas ATP binding alone is sufficient to support the degradation of short peptides. Furthermore, comparative structural analysis of FtsH and a related ATPase, HslU, reveals interesting similarities and differences in mechanism.
Bile‐induced ‘pili’ in Campylobacter jejuni are bacteria‐independent artifacts of the culture medium
Authors: Gaynor Erin C. , Ghori Nafisa , Falkow Stanley
Published: March 2003

In 1996, it was reported that the enteric pathogen Campylobacter jejuni produces pilus‐like appendages in response to bile salts such as deoxycholate (DOC), and that the formation of these appendages requires the putative peptidase PspA. Pili were known to be important virulence determinants in other pathogenic bacteria but had never before been observed for C. jejuni. We report here that these appendages are not pili, but are instead a bacteria‐independent morphological artifact of the growth medium. Furthermore, the pspA gene is not required for their formation. Broth cultures containing a threshold concentration of DOC inoculated with no bacteria produced identical abundant, fibrous, pilus‐like structures as those cultures that had been inoculated with C. jejuni. These fibres were also found in growth media from DOC‐containing pspA::CmR mutant cultures. These results are consistent with the absence of candidate pilin monomers in protein gel analyses as well as the dearth of pilin‐like genes and pilus formation gene clusters in the C. jejuni genome.
AraC/XylS family members, HilC and HilD, directly bind and derepress the Salmonella typhimurium hilA promoter
Authors: Schechter Lisa M. , Lee Catherine A.
Published: March 2003

During infection, Salmonella enterica serovar Typhimurium colonizes the small intestine of its hosts. This process requires a type III secretion system encoded by several genes on Salmonella pathogenicity island 1 (SPI1), a 40 kb region of DNA near centisome 63 of the Salmonella chromosome. SPI1 gene expression is controlled by a complex regulatory cascade. HilA, a member of the OmpR/ToxR family of transcriptional regulators, directly activates the expression of two SPI1 operons encoding type III apparatus components. hilA transcription is repressed by many environmental conditions and regulatory mutations. This repression requires an upstream repressing sequence (URS) located between −314 and −68 relative to the hilA transcription start site. The repressing activity of the URS is counteracted by two AraC/XylS family members named HilC and HilD. We show that HilC and HilD bind directly to the hilA promoter region in vitro. We also provide evidence that HilC and HilD bind to the same or overlapping sites within the URS. Our data are consistent with a model in which HilC and HilD derepress hilA expression by binding directly to the URS and counteracting its repressing effect in vivo.
Identification and characterization of App: an immunogenic autotransporter protein of Neisseria meningitidis
Authors: Hadi Hoda Abdel , Wooldridge Karl G. , Robinson Karen , Ala'Aldeen Dlawer A. A.
Published: March 2003

In a search for immunogenic virulence factors in Neisseria meningitidis, we have identified a gene encoding a predicted 160 kDa protein with homology to the autotransporter family of proteins. Members of this family are secreted or surface exposed and are often associated with virulence in Gram‐negative bacterial pathogens. We named the gene adhesion and penetration protein (app), because of its extensive homology to the hap gene of Haemophilus influenzae. We reconstructed the gene with reference to genomic sequence data and cloned and expressed the protein in Escherichia coli. Rabbit antiserum raised against recombinant App reacted with proteins in all meningococcal isolates examined, which represented clonal groups responsible for the majority of meningococcal invasive disease. Antibodies to the protein were detected in the sera of patients convalescing from meningococcal infection. Purified App had strong stimulating activity for T cells isolated from a number of healthy donors and from one convalescent patient. We confirmed that App is surface localized, cleaved and secreted by N. meningitidis. Importantly, the rabbit anti‐App serum killed the organism in the presence of complement. Thus, App is conserved among meningococci, immunogenic in humans and potentially involved in virulence. It therefore merits further investigation as a component of a future multivalent vaccine.
Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP‐CRP at the Vibrio cholerae tcpPH promoter
Authors: Kovacikova Gabriela , Skorupski Karen
Published: March 2003

The expression of the Vibrio cholerae virulence factors, toxin‐co‐regulated pilus (TCP) and cholera toxin (CT), are dependent on the ability of the LysR regulator AphB to co‐operate with a second protein, AphA, to activate the expression of the membrane‐bound transcription factors TcpP and TcpH. To gain insights into the mechanism by which AphA and AphB co‐operate to activate the expression of tcpPH, we have purified these two proteins to near homogeneity and show that they are each capable of interacting with the classical tcpPH promoter at distinct binding sites. As shown by tcpP–lacZ promoter deletion experiments, gel shift and DNase I footprinting, AphA binds to and activates from a region of the promoter between −101 and −71 from the start of transcription. AphB binds to and activates from a partially overlapping downstream site between −78 and −43, and these functions are dependent upon a region of partial dyad symmetry that resembles the well‐characterized LysR‐binding motif. A single basepair difference in this region of dyad symmetry has been shown previously to play a critical role in the expression of virulence genes between the two disease‐causing biotypes of V. cholerae, classical and El Tor. We also show here that the tcpPH promoter is negatively influenced by the global regulator cAMP‐CRP. Purified CRP binds to a near‐consensus sequence in the tcpPH promoter in a cAMP‐dependent manner and protects from DNase I digestion a region that is completely within the region protected by AphA and AphB. These findings raise the possibility that the negative effect of cAMP‐CRP on virulence gene expression is the result of its ability to influence AphA‐ and AphB‐dependent transcriptional activation of tcpPH under various conditions.
Identification of the Helicobacter pylori anti‐σ28 factor
Authors: Colland Frédéric , Rain Jean‐Christophe , Gounon Pierre , Labigne Agnès , Legrain Pierre , De Reuse Hilde
Published: March 2003

Flagellar motility is essential for colonization of the human gastric mucosa by Helicobacter pylori. The flagellar filament is composed of two subunits, FlaA and FlaB. Transcription of the genes encoding these proteins is controlled by the σ28 and σ54 factors of RNA polymerase respectively. The expression of flagellar genes is regulated, but no σ28‐specific effector was identified. It was also unclear whether H. pylori possessed a checkpoint for flagellar synthesis, and no gene encoding an anti‐σ28 factor, FlgM, could be identified by sequence similarity searches. To investigate the σ28‐dependent regulation, a new approach based on genomic data was used. Two‐hybrid screening with the H. pylori proteins identified a protein of unknown function (HP1122) interacting with the σ28 factor and defined the C‐terminal part of HP1122 (residues 48–76) as the interaction domain. HP1122 interacts with region 4 of σ28 and prevents its association with the β‐region of H. pylori RNA polymerase. Thus, HP1122 presented the characteristics of an anti‐σ28 factor. This was confirmed in H. pylori by RNA dot‐blot hybridization and electron microscopy. The level of σ28‐dependent flaA transcription was higher in a HP1122‐deficient strain and was decreased by the overproduction of HP1122. The overproduction of HP1122 also resulted in H. pylori cells with highly truncated flagella. These results demonstrate that HP1122 is the H. pylori anti‐σ28 factor, FlgM, a major regulator of flagellum assembly. Potential anti‐σ28 factors were identified in Campylobacter jejuni, Pseudomonas aeruginosa and Thermotoga maritima by sequence homology with the C‐terminal region of HP1122.
Genetic analysis of the signalling pathway activated by external amino acids in Saccharomyces cerevisiae
Authors: Bernard Florent , André Bruno
Published: March 2003

The permease‐like amino acid sensor Ssy1p of Saccharomyces cerevisiae is required for transcriptional induction, in response to external amino acids, of several genes encoding peptide and amino acid permeases. Among them is AGP1 encoding a low‐affinity, broad‐specificity amino acid permease important for the utilization of amino acids as a nitrogen source. We report here data from experiments aimed at identifying components of the signalling pathway activated by Ssy1p. Overproduction of the large amino‐terminal tail of Ssy1p interferes negatively with the induction of AGP1 in wild‐type cells. Furthermore, overproduction of this domain can relieve growth defects of a ssy1 null strain, indicating that the N‐terminal tail of Ssy1p is an important functional element of the pathway. Consistent with a role for Ssy1p in the recognition of amino acids, a mutant form of the protein with a Thr to Ile substitution in the eighth predicted transmembrane domain is competent for the induction of AGP1 by leucine but not by other amino acids. In a screen for other mutants defective in the Ssy1p pathway, we confirmed that PTR3 and SSY5 encode additional factors essential for AGP1 expression in response to multiple amino acids. Data obtained by overproducing Ptr3p and Ssy5p in ssy1Δ, ptr3Δ and ssy5Δ mutants suggest that Ptr3p acts downstream from Ssy1p and Ssy5p downstream from Ptr3p in the transduction pathway. Furthermore, two‐hybrid experiments indicated that Ptr3p interacts with Ssy5p and that Ptr3p can self‐associate. Finally, the Cys‐6‐Zn2 transcription factor Uga35p/Dal81p required for the induction of AGP1 is also essential for the expression of two other genes under Ssy1p–Ptr3p–Ssy5p control, namely BAP2 and PTR2, suggesting that the protein is yet another component of the amino acid signalling pathway.
Role of vicinal cysteine pairs in metalloid sensing by the ArsD As(III)‐responsive repressor
Authors: Li Song , Chen Yanxiang , Rosen Barry P.
Published: March 2003

ArsD is a 120‐residue repressor that regulates expression of the arsRDABC arsenical resistance operon of plasmid R773 in Escherichia coli. ArsD is released from arsRDABC promoter DNA by binding of the compounds with the metalloids As(III) or Sb(III). ArsD has three vicinal cysteine pairs, Cys‐12 and Cys‐13, Cys‐112 and Cys‐113 and Cys‐119 and Cys‐120. In this study, the role of these three cysteine pairs was investigated. Mutation or deletion of Cys‐119–Cys‐120 had no effect on repression or metalloid responsiveness in vivo or in vitro. Mutagenesis of either the Cys‐12–Cys‐13 pair or the Cys‐112–Cys‐113 pair had no effect on repression but produced loss of inducibility, suggesting that both Cys‐12–Cys‐13 and Cys‐112–Cys‐113 may be required for As(III) or Sb(III) responsiveness. Assays of binding of wild‐type and mutant ArsDs by As(III) affinity chromatography showed that each of the three vicinal cysteine pairs is capable of binding As(III) independently. The effect of As(III) or Sb(III) on intrinsic protein fluorescence was used to examine the properties of individual cysteine pairs. The fluorescence of Trp‐97 was shown to be quenched by the addition of Sb(III) or As(III). The vicinal Cys‐112–Cys‐113 pair was required for the majority of the metalloid‐dependent quenching of Trp‐97 fluorescence. The data are consistent with a model in which Cys‐12–Cys‐13 and Cys‐112–Cys‐113 form independent As(III) binding sites, both of which are required for in vivo ArsD function.
A point mutation in an unusual Sec7 domain is linked to brefeldin A resistance in a Plasmodium falciparum line generated by drug selection
Authors: Baumgartner Frank , Wiek Sabine , Paprotka Kerstin , Zauner Stefan , Lingelbach Klaus
Published: March 2003

The malaria parasite Plasmodium falciparum has an unusual organization of its secretory compartments. As an approach to a functional identification of auxiliary proteins involved in secretion, a parasite line was generated by drug selection that is resistant to brefeldin A, an inhibitor of the secretory pathway. In the resistant line, neither protein secretion nor parasite viability were affected by the drug. The analysis of a sec7 domain, a conserved structure of guanine nucleotide exchange factors (ARF‐GEF) required for the activation of ADP‐ribosylation factors, revealed a single methionine–isoleucine substitution in the resistant parasite line. ARF‐GEFs are key molecules in the formation of transport vesicles and the main targets of brefeldin A. The methionine residue in this position of sec7 domains is highly conserved and confers brefeldin A sensitivity. Unlike other eukaryotes that have multiple ARF‐GEFs, the plasmodial genome encodes a single sec7 domain. This domain shows a distinct structural difference to all sec7 domains analysed so far; two conserved subdomains that are essential for protein function are separated in the plasmodial protein by an insertion of 146 amino acids.
Co‐evolution of the agrocinopine opines and the agrocinopine‐mediated control of TraR, the quorum‐sensing activator of the Ti plasmid conjugation system
Authors: Oger Philippe , Farrand Stephen K.
Published: March 2003

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is controlled by a hierarchical system in which opines, substrates produced by crown gall tumours, induce a quorum‐sensing system. The cascade results from the control of expression of traR, the quorum‐sensing activator, by a regulator responsive to the opine. In the two cases studied to date, the gene arrangements responsible for the cascade differ remarkably, suggesting that considerable diversity exists among the many Ti‐like plasmids in the agrobacteria. In this study, we demonstrated that the novel Ti plasmid pTiChry5 is induced to transfer at high frequency by extracts from tumours initiated by strain Chry5. The purified inducer had the chemical and biological properties of agrocinopines C and D, a set of sugar phosphodiester opines known to induce transfer of another Ti plasmid, pTiBo542. The T‐region of pTiChry5 contained a gene whose product, called AcsChry5, is virtually identical to the agrocinopine C+D synthase from the T‐region of pTiBo542. The two genes are less closely related to acs of pTiC58, which is responsible for the production of agrocinopines A+B, a similar but not identical set of phosphodiester opines by tumours induced by strain C58. Agrocinopines A+B induce transfer of pTiC58 but did not induce transfer of pTiChry5. A single copy of traR was identified at the 11 o'clock region of pTiChry5, where it is part of a two‐gene operon called arcChry5. Although altered by deletions, arcChry5 is related to the five‐gene arc operon that controls the expression of traR on pTiC58. Expression of traRChry5 was induced by agrocinopines C+D and the opines isolated from Chry5 tumours but not by agrocinopines A+B. A mutation in traRChry5 abolished transfer, and transfer was restored by complementation in trans. We conclude that the agrocinopine opines and the corresponding opine‐meditated conjugal regulatory regions of pTiChry5 and pTiC58 share a common origin, but that the opine signals for the two Ti plasmids have evolved divergently through changes in the opine synthase enzymes. The alterations in the opines, in turn, necessitated a co‐evolutionary change in the opine recognition systems responsible for controlling expression of the traR genes on these two types of Ti plasmids.
New erythromycin derivatives from Saccharopolyspora erythraea using sugar O‐methyltransferases from the spinosyn biosynthetic gene cluster
Authors: Gaisser Sabine , Lill Rachel , Wirtz Gabriele , Grolle Friederike , Staunton James , Leadlay Peter F.
Published: March 2003

Using a previously developed expression system based on the erythromycin‐producing strain of Saccharopolyspora erythraea, O‐methyltransferases from the spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14‐membered polyketide macrolactone. The spnI, spnK and spnH methyltransferase genes were expressed individually in the S. erythraea mutant SGT2, which is blocked both in endogenous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryCIII. Exogenous 3‐O‐rhamnosyl‐erythronolide B was efficiently converted into 3‐O‐(2′‐O‐methylrhamnosyl)‐erythronolide B by the S. erythraea SGT2 (spnI) strain only. When 3‐O‐(2′‐O‐methylrhamnosyl)‐erythronolide B was, in turn, fed to a culture of S. erythraea SGT2 (spnK), 3‐O‐(2′,3′‐bis‐O‐methylrhamnosyl)‐erythronolide B was identified in the culture supernatant, whereas S. erythraea SGT2 (spnH) was without effect. These results confirm the identity of the 2′‐ and 3′‐O‐methyltransferases, and the specific sequence in which they act, and they demonstrate that these methyltransferases may be used to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway. In contrast, 3‐O‐(2′,3′‐bis‐O‐methylrhamnosyl)‐erythronolide B was found not to be a substrate for the 4′‐O‐methyltransferase SpnH. Although rhamnosylerythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosyl‐erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl‐erythronolide precursors using the S. erythraea strain SGT2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway. 3‐O‐(2′‐O‐methylrhamnosyl)‐erythromycin D was tested and found to be significantly active against a strain of erythromycin‐sensitive Bacillus subtilis.
Variation in 16S‐23S rRNA intergenic spacer regions among Bacillus subtilis 168 isolates
Authors: Shaver Yevette J. , Nagpal Madan L. , Fox Karen F. , Rudner Rivka , Fox Alvin
Published: March 2003

The genome of the Bacillus subtilis 168‐type strain contains 10 ribosomal RNA (rRNA) operons. In the intergenic spacer region (ISR) between the 16S and 23S rRNA genes, five rRNA operons, rrnI‐H‐G and rrnJ‐W, lack a trinucleotide signature region. Precise determination of molecular weight (MW), using electrospray mass spectrometry (MS), of the polymerase chain reaction (PCR) products from a segment of the ISR from the 168‐type strain and B. subtilis 168‐like strain 23071 demonstrated 114 and 111 basepair (bp) PCR products (due to the presence or absence of the insert in the operons) as predicted from sequence. However, PCR of the ISR segment for five other B. subtilis 168 isolates generated only a 114 bp PCR product, suggesting the presence of the trinucleotide signature region in all rRNA operons for these strains. Additional genetic variability between the seven B. subtilis 168 isolates was demonstrated by restriction fragment length polymorphism (RFLP) of the rRNA operons, with three distinct patterns found upon Southern blot analysis. The 168‐type strain and three others (23066, 23067, and 23071) exhibited the same Southern pattern. Thus, operon deletion is not responsible for the absence of a 111 bp product on MS analysis for strains 23066 and 23067. Restriction analysis confirmed the presence of the trinucleotide signature region in the ISR of all rRNA operons for five B. subtilis 168 isolates; sequencing of rrnW/H from a representative strain also upheld this finding. These results help provide a better understanding of variations in sequence, operon number and chromosomal organization, both within a genome and among isolates of B. subtilis subgroup 168. It is also hypothesized that the presence of the trinucleotide insert in certain rRNA operons may play a role in rRNA maturation and protein synthesis.
The TolQ–TolR proteins energize TolA and share homologies with the flagellar motor proteins  MotA–MotB
Authors: Cascales Eric , Lloubès Roland , Sturgis James N
Published: March 2003

The Tol–Pal system of Escherichia coli is required for the maintenance of outer membrane stability. Recently, proton motive force (pmf) has been found to be necessary for the co‐precipitation of the outer membrane lipoprotein Pal with the inner membrane TolA protein, indicating that the Tol–Pal system forms a transmembrane link in which TolA is energized. In this study, we show that both TolQ and TolR proteins are essential for the TolA–Pal interaction. A point mutation within the third transmembrane (TM) segment of TolQ was found to affect the TolA–Pal interaction strongly, whereas suppressor mutations within the TM segment of TolR restored this interaction. Modifying the Asp residue within the TM region of TolR indicated that an acidic residue was important for the pmf‐dependent interaction of TolA with Pal and outer membrane stabilization. Analysis of sequence alignments of TolQ and TolR homo‐logues from numerous Gram‐negative bacterial genomes, together with analyses of the different tolQ–tolR mutants, revealed that the TM domains of TolQ and TolR present structural and functional homologies not only to ExbB and ExbD of the TonB system but also with MotA and MotB of the flagellar motor. The function of these three systems, as ion potential‐driven molecular motors, is discussed
Cellular location and temperature‐dependent assembly of IncHI1 plasmid R27‐encoded TrhC‐associated conjugative transfer protein complexes
Authors: Gilmour Matthew W. , Lawley Trevor D. , Rooker Michelle M. , Newnham Peter J. , Taylor Diane E.
Published: March 2003

Conjugal transfer of IncHI plasmid DNA between Gram‐negative bacteria is temperature sensitive, as mating is optimal between 22°C and 30°C but is inhibited at 37°C. R27, isolated from Salmonella enterica serovar Typhi, is an IncHI1 plasmid of 180 kbp that has been sequenced completely. The gene encoding green fluorescent protein (GFP) was inserted into R27 in frame with trhC. TrhC is a mating pair formation (Mpf) protein that is essential for plasmid transfer and H‐pilus production. Fluorescence microscopy allowed visualization of the TrhC–GFP fusion protein, and Escherichia coli cells were examined for the subcellular localization and temperature‐dependent production of TrhC–GFP. At 27°C, TrhC–GFP was found at the periphery of cells as discrete foci, indicating an association of TrhC within protein complexes in the bacterial cell membrane, whereas at 37°C, little fluorescence was detected. These foci probably represent the intracellular position of protein complexes involved in conjugative transfer, as the formation of foci was dependent upon the presence of other Mpf proteins. During temperature shift experiments from 37°C to 27°C, a long lag period was required for generation of GFP foci. Conversely, during short shifts from 27°C to 37°C, the GFP foci remained stable. These results suggest that the expression of transfer genes in the Tra2 region of R27 is temperature dependent. Subcellular localization of TrhC was verified by cellular fractionation. Expression patterns of TrhC–GFP were confirmed with immunoblot analysis and reverse transcriptase–polymerase chain reaction (RT–PCR). These results allow us to propose mechanisms to explain the temperature‐sensitive transfer of R27.
The ZbYME2 gene from the food spoilage yeast Zygosaccharomyces bailii confers not only YME2 functions in Saccharomyces cerevisiae, but also the capacity for catabolism of sorbate and benzoate, two major weak organic acid preservatives
Authors: Mollapour Mehdi , Piper Peter W.
Published: March 2003

A factor influencing resistances of food spoilage microbes to sorbate and benzoate is whether these organisms are able to catalyse the degradation of these preservative compounds. Several fungi metabolize benzoic acid by the β‐ketoadipate pathway, involving the hydroxylation of benzoate to 4‐hydroxybenzoate. Saccharomyces cerevisiae is unable to use benzoate as a sole carbon source, apparently through the lack of benzoate‐4‐hydroxylase activity. However a single gene from the food spoilage yeast Zygosaccharomyces bailii, heterologously expressed in S. cerevisiae cells, can enable growth of the latter on benzoate, sorbate and phenylalanine. Although this ZbYME2 gene is essential for benzoate utilization by Z. bailii, its ZbYme2p product has little homology to other fungal benzoate‐4‐hydroxylases studied to date, all of which appear to be microsomal cytochrome P450s. Instead, ZbYme2p has strong similarity to the matrix domain of the S. cerevisiae mitochondrial protein Yme2p/Rna12p/Prp12p and, when expressed as a functional fusion to green fluorescent protein in S. cerevisiae growing on benzoate, is largely localized to mitochondria. The phenotypes associated with loss of the native Yme2p from S. cerevisiae, mostly apparent in yme1,yme2 cells, may relate to increased detrimental effects of endogenous oxidative stress. Heterologous expression of ZbYME2 complements these phenotypes, yet it also confers a potential for weak acid preservative catabolism that the native S. cerevisiae Yme2p is unable to provide. Benzoate utilization by S. cerevisiae expressing ZbYME2 requires a functional mitochondrial respiratory chain, but not the native Yme1p and Yme2p of the mitochondrion.
In vivo aggregation of the HET‐s prion protein of the fungus Podospora anserina
Authors: Coustou‐Linares Virginie , Maddelein Marie‐Lise , Bégueret Joël , Saupe Sven J.
Published: March 2003

We have proposed that the [Het‐s] infectious cytoplasmic element of the filamentous fungus Podospora anserina is the prion form of the HET‐s protein. The HET‐s protein is involved in a cellular recognition phenomenon characteristic of filamentous fungi and known as heterokaryon incompatibility. Under the prion form, the HET‐s protein causes a cell death reaction when co‐expressed with the HET‐S protein, from which it differs by only 13 amino acid residues. We show here that the HET‐s protein can exist as two alternative states, a soluble and an aggregated form in vivo. As shown for the yeast prions, transition to the infectious prion form leads to aggregation of a HET‐s–green fluorescent protein (GFP) fusion protein. The HET‐s protein is aggregated in vivo when highly expressed. However, we could not demonstrate HET‐s aggregation at wild‐type expression levels, which could indicate that only a small fraction of the HET‐s protein is in its aggregated form in vivo in wild‐type [Het‐s] strains. The antagonistic HET‐S form is soluble even at high expression level. A double amino acid substitution in HET‐s (D23A P33H), which abolishes prion infectivity, suppresses in vivo aggregation of the GFP fusion. Together, these results further support the model that the [Het‐s] element corresponds to an abnormal self‐perpetuating aggregated form of the HET‐s protein.
Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation
Authors: Yu Rosa R. , DiRita Victor J.
Published: March 2003

Co‐ordinate expression of many virulence genes in the human pathogen Vibrio
cholerae is under the direct control of the ToxT protein, including genes whose
products are required for the biogenesis of the toxin‐co‐regulated pilus (TCP) and
cholera toxin (CTX). This work examined interactions between ToxT and the promoters
of ctx and tcpA genes. We found that a minimum of three direct repeats
of the sequence TTTTGAT is required for ToxT‐dependent activation of the ctx
promoter, and that the region from –85 to –41 of the tcpA promoter
contains elements that are responsive to ToxT‐dependent activation. The role of H‐NS
in transcription of ctx and tcpA was also analysed. The level of activation
of ctx–lacZ in an E. coli hns– strain was
greatly increased even in the absence of ToxT, and was further enhanced in the presence
of ToxT. In contrast, H‐NS plays a lesser role in the regulation of the tcpA
promoter. Electrophoretic mobility shift assays demonstrated that 6×
His‐tagged ToxT directly, and specifically, interacts with both the ctx and
tcpA promoters. DNase I footprinting analysis suggests that there may be two
ToxT binding sites with different affinity in the ctx promoter and that ToxT
binds to –84 to –41 of the tcpA promoter. In vitro transcription experiments demonstrated that ToxT alone is able to activate transcription from both promoters. We hypothesize that under conditions appropriate for ToxT‐dependent gene expression, ToxT binds to AT‐rich promoters that may have a specific secondary conformation, displaces H‐NS and stimulates RNA polymerase resulting in transcription activation.
A fadD mutant of Sinorhizobium meliloti shows multicellular swarming migration and is impaired in nodulation efficiency on alfalfa roots
Authors: Soto María José , Fernández‐Pascual Mercedes , Sanjuan Juan , Olivares José
Published: March 2003

Swarming is a form of bacterial translocation that involves cell differentiation and is characterized by a rapid and co‐ordinated population migration across solid surfaces. We have isolated a Tn5 mutant of Sinorhizobium meliloti GR4 showing conditional swarming. Swarm cells from the mutant strain QS77 induced on semi‐solid minimal medium in response to different signals are hyperflagellated and about twice as long as wild‐type cells. Genetic and physiological characterization of the mutant strain indicates that QS77 is altered in a gene encoding a homologue of the FadD protein (long‐chain fatty acyl‐CoA ligase) of several microorganisms. Interestingly and similar to a less virulent Xanthomonas campestris fadD(rpfB) mutant, QS77 is impaired in establishing an association with its host plant. In trans expression of multicopy fadD restored growth on oleate, control of motility and the symbiotic phenotype of QS77, as well as acyl‐CoA synthetase activity of an Escherichia coli fadD mutant. The S. meliloti QS77 strain shows a reduction in nod gene expression as well as a differential regulation of motility genes in response to environmental conditions. These data suggest that, in S. meliloti, fatty acid derivatives may act as intracellular signals controlling motility and symbiotic performance through gene expression.
Independent regulation of the divergent Escherichia coli nrfA and acsP1 promoters by a nucleoprotein assembly at a shared regulatory region
Authors: Browning Douglas F. , Beatty Christine M. , Wolfe Alan J. , Cole Jeffrey A. , Busby Stephen J. W.
Published: March 2003

Expression from the Escherichia coli nrfA promoter (pnrfA) is activated by both the FNR protein (an anaerobically triggered transcription activator) and the NarL or NarP proteins (transcription activators triggered by nitrite and nitrate). Under anaerobic conditions, FNR binds to a site centred at position –41.5 at pnrfA and activates transcription. Further activation, induced by the presence of nitrite, results from the binding of NarL and NarP to a site centred at position –74.5. A second promoter (pacsP1), which directs transcription into the adjacent gene encoding acetyl coenzyme A synthetase (acs), is overlapping and divergent to pnrfA. Despite extensive overlap of regulatory elements, pnrfA and pacsP1 are regulated independently. We demonstrate that at least two nucleoid‐associated factors bind to the nrfA–acs intergenic region. The Fis protein binds to a site centred at position –15 (in relation to pnrfA transcription), whereas the IHF protein binds to a site centred at position –54. Both Fis and IHF repress in vivo expression from pacsP1, but have smaller repressive effects on expression from pnrfA. Gel retardation assays were used to investigate the pairwise binding of FNR, NarL, Fis and IHF proteins to the nrfA–acs intergenic region. The binding of NarL and IHF is mutually exclusive, whereas all other combinations can bind simultaneously. Experiments in which deletions and point mutations were introduced into the upstream region of pnrfA demonstrated that an additional factor must bind upstream to inhibit FNR‐dependent transcription. We conclude that the nrfA–acs intergenic region is folded into an ordered nucleoprotein structure that permits the two divergent promoters to be regulated independently in response to different physiological signals.