bluuebunny/crossref_metadata_2025 huggingface dataset:
An adenosyl–cobalamin (coenzyme‐B12)‐repressed translational enhancer in the cob mRNA of Salmonella typhimurium
Authors: Ravnum Solveig , Andersson Dan I.
Published: March 2003

Expression of the cobalamin (Cbl) biosynthetic cob operon in Salmonella typhimurium is repressed by the end‐product. This regulation is conferred mainly at the translational level and involves a cobalamin‐induced folding of an RNA hairpin that sequesters the ribosomal binding site (RBS) of the cob mRNA and prevents translation initiation. A combined structural and mutational analysis shows that a cis‐acting translational enhancer (TE) element, located 83 nucleotides upstream of the Shine–Dalgarno sequence in the 5′‐untranslated region (5′‐UTR) of the cob mRNA, is required to unfold the inhibitory RBS hairpin in the absence of cobalamin. The TE element, which consists of 5 nucleotides, is proposed to confer its enhancer function in the absence of cobalamin by interacting with nucleotides in the stem of the RBS hairpin. This interaction destabilizes the RNA hairpin and allows ribosome binding. In the presence of cobalamin, the enhancer function is inhibited. As a result, the RBS hairpin forms and prevents translation initiation. Several additional RNA hairpins in the 5′‐UTR were also identified and are suggested to be important for repression. The above data suggest that normal cobalamin repression of the cob operon requires that the 5′‐UTR has a defined secondary and tertiary structure.
The puzzle of zmpB and extensive chain formation, autolysis defect and non‐translocation of choline‐binding proteins in Streptococcus pneumoniae
Authors: Bergé Mathieu , García Pedro , Iannelli Francesco , Prère Marie Françoise , Granadel Chantal , Polissi Alessandra , Claverys Jean‐Pierre
Published: March 2003

Choline‐binding proteins (CBPs) from Streptococcus pneumoniae are involved in several important processes. Inactivation of zmpB, a gene that encodes a surface‐located putative zinc metalloprotease, in a S. pneumoniae serotype 4 strain was recently reported to reveal a composite phenotype, including extensive chain formation, lysis defect and transformation deficiency. This phenotype was associated with the lack of surface expression of several CBPs, including the major autolysin LytA. LytA, normally 36 kDa in size, was reported to form an SDS‐resistant 80 kDa complex with CinA. ZmpB was therefore proposed to control translocation of CBPs to the surface, possibly through the proteolytic release of CBPs (and RecA) from CinA. Based on the use of 12 independent mariner insertions in the zmpB gene of the well‐characterized R6 laboratory strain, we could not confirm several of these observations. Our zmpB mutants: (i) did not form chains; (ii) lysed normally in the presence of deoxycholate, which indicates the presence of a functional autolysin; (iii) transformed at normal frequency; and (iv) contained bona fide CinA and LytA species. Polymorphism of ZmpB between R6 and the serotype 4 isolate could not account for the discrepancy, as inactivation of zmpB (through replacement by transposon‐inactivated zmpB R6 alleles) in the latter strain did not affect separation of daughter cells and autolysis. The conflicting observations could be explained by our finding that the reportedly serotype 4 zmpB‘mutant’ differed from its S. pneumoniae parent in lacking capsule and in exhibiting characteristic traits of the Streptococcus viridans group, including resistance to optochin.
Two‐hybrid analysis of domain interactions involving NtrB and NtrC two‐component regulators
Authors: Martínez‐Argudo Isabel , Martín‐Nieto José , Salinas Paloma , Maldonado Rafael , Drummond Martin , Contreras Asunción
Published: March 2003

Signal transduction by two‐component regulatory systems involves phosphorylation of the receiver domain of a response regulator by the transmitter domain of the cognate histidine kinase. In the NtrBC system, phosphorylation of NtrC by NtrB results in transcriptional activation of nitrogen‐regulated genes. We have used the yeast two‐hybrid system to probe interactions between domains of the NtrB and NtrC proteins fromKlebsiella pneumoniae. We constructed fusions from each of a series of proteins or protein domains to the activation and the DNA‐binding domains of GAL4 and analysed expression of GAL1:lacZandGAL1:HIS3reporters in yeast. The DNA‐binding domain of NtrC and the so‐called sensor domain of NtrB appeared to provide the major determinants for dimerization of the fusion proteins. A strong and specific interaction was also shown between NtrB and NtrC, localized to the HN region of the NtrB transmitter module and to the NtrC receiver domain, whereas other domains of these proteins do not appear to contribute to the recognition specificity. The results presented here indicate that communication between two‐component partners also involves protein–protein interactions that can be detectedin vivo, suggesting that the yeast two‐hybrid system is a powerful genetic tool for identifying functional partners of prokaryotic signal transduction pathways.
BldD is a direct regulator of key developmental genes in Streptomyces coelicolor A3(2)
Authors: Elliot Marie A. , Bibb Maureen J. , Buttner Mark J. , Leskiw Brenda K.
Published: March 2003

BldD is a transcription factor required for aerial hyphae formation in the filamentous bacterium Streptomyces coelicolor. Three targets of BldD regulation were discovered by a number of means, including examination of bld gene interdependence, selective enrichment of chromosomal DNA fragments bound by BldD and searching the promoter regions of known developmental genes for matches to a previously characterized BldD binding site. The three BldD targets identified were the developmental sigma factor genes, whiG and bldN, and a previously uncharacterized gene, designated bdtA, encoding a putative transcription factor. In each target gene, the sequences bound by BldD were characterized by electrophoretic mobility shift and DNase I footprinting assays, and their alignment suggested AGTgA (n)m TCACc as a consensus BldD operator. The in vivo effect of mutation in bldD on the expression of these three target genes was assessed using S1 nuclease protection assays. In each case, target gene expression was upregulated during early colony development in the bldD background, suggesting that, in the wild type, BldD acts to repress premature expression of whiG, bldN and bdtA during vegetative growth.
Identification of a twin‐arginine leader‐binding protein
Authors: Oresnik Ivan J. , Ladner Carol L. , Turner Raymond J.
Published: March 2003

The transport and targeting of a number of periplasmic proteins is carried out by the Sec‐independent Mtt (also referred to as Tat) protein translocase. Proteins using this translocase have a distinct twin‐arginine‐containing leader. We hypothesized that specific leader‐binding proteins exist to escort proteins to the translocase complex. A fusion was constructed with the twin‐arginine leader from dimethyl sulphoxide (DMSO) reductase, subunit DmsA, to the N‐terminus of glutathione‐S‐transferase. This leader fusion was bound to a glutathione affinity column through which an Escherichia coli anaerobic cell‐free extract was passed. Proteins that bound to the leader were then separated and identified by N‐terminal sequencing, which identified DnaK and a protein originating from the uncharacterized reading frame ynfI. This gene has been designated dmsD based on the findings presented in this paper. DmsD was purified as a His6 fusion and was shown to interact with preprotein forms of DmsA and TorA (trimethyl amine N‐oxide reductase). A strain carrying a dmsD knock‐out mutation showed a loss of anaerobic growth on glycerol–DMSO medium and reduced growth on glycerol–fumarate medium. This work suggests that DmsD is a twin‐arginine leader‐binding protein.
Functional study of the Saccharomyces cerevisiae Nha1p C‐terminus
Authors: Kinclová Olga , Ramos José , Potier Serge , Sychrová Hana
Published: March 2003

Saccharomyces cerevisiae cells possess an alkali metal cation antiporter encoded by the NHA1 gene. Nha1p is unique in the family of yeast Na+/H+ antiporters on account of its broad substrate specificity (Na+, Li+, K+) and its long C‐terminus (56% of the whole protein). In order to study the role of the C‐terminus in Nha1p function, we constructed a series of 13 truncated NHA1 versions ranging from the complete one (2958 nucleotides, 985 amino acids) down to the shortest version (1416 nucleotides, 472 amino acids), with only 41 amino acid residues after the last putative transmembrane domain. Truncated NHA1 versions were expressed in an S. cerevisiae alkali metal cation‐sensitive strain (B31; ena1–4Δ nha1Δ). We found that the entire Nha1p C‐terminus domain is not necessary for either the proper localization of the antiporter in the plasma membrane or the transport of all four substrates (we identified rubidium as the fourth Nha1p substrate). Partial truncation of the C‐terminus of about 70 terminal amino acids improves the tolerance of cells to Na+, Li+ and Rb+ compared with cells expressing the complete Nha1p. The presence of the neighbouring part of the C‐terminus (amino acids 883–928), rich in aspartate and glutamate residues, is necessary for the maintenance of maximum Nha1p activity towards sodium and lithium. In the case of potassium, the participation of the long C‐terminus in the regulation of intracellular potassium content is demonstrated. We also present evidence that the Nha1p C‐terminus is involved in the cell response to sudden changes in environmental osmolarity.
Diversity in the Bordetella virulence regulon: transcriptional control of a Bvg‐intermediate phase gene
Authors: Deora Rajendar , Bootsma Hester J. , Miller Jeff F. , Cotter Peggy A.
Published: March 2003

The BvgAS signal transduction system controls the expression of at least three distinct phenotypic phases that lie along a continuum of gene expression states. The Bvg+ phase is characterized by the expression of adhesins and toxins, whereas the Bvg− phase is characterized by motility in Bordetella bronchiseptica and the expression of vrg loci in Bordetella pertussis. The Bvg‐intermediate (Bvgi) phase is characterized by the absence of Bvg‐repressed phenotypes, the expression of some, but not all, Bvg‐activated virulence factors and the presence of a recently discovered set of antigens and phenotypes that are unique to this phase. We report here the transcriptional regulation of bipA, the first‐identified Bvgi phase gene. We have mapped the bipA promoter and identified numerous BvgA binding sites in the transcriptional control region. Based on these data, we present a model in which phase‐dependent expression of bipA results from the spatial distribution and relative affinities of multiple BvgA binding sites relative to the start site of transcription.
Different localization of SeqA‐bound nascent DNA clusters and MukF–MukE–MukB complex in Escherichia coli cells
Authors: Ohsumi Katsufumi , Yamazoe Mitsuyoshi , Hiraga Sota
Published: March 2003

MukF, MukE and MukB proteins form a complex that may participate in the organization of folded sister chromosomes in Escherichia coli. We have found that a MukB–GFPuv4 fusion protein is observed as discrete fluorescent foci, which are localized within cellular spaces occupied by nucleoids, but not at the constriction site of cell division in living cells. In contrast, MukB–GFPuv4 is distributed throughout the whole cell when either MukF or MukE is absent. Statistical analysis revealed that most newborn cells have two foci of mukB–gfpUV4 at one‐quarter and three‐quarter positions in the cell length and one focus of SeqA‐bound nascent DNA at or near the middle of the cell. Subsequently, the single SeqA focus divides into two foci, and then these migrate to the one‐quarter and three‐quarter positions. Before cell division, most long cells have two SeqA foci and four MukB–GFPuv4 foci. In early stationary phase, SeqA foci disappear, but one or two foci of MukB–GFPuv4 remain. We discuss the reorganization and proper arrangement of folded sister chromosome in the cell quarter positions, which are performed after release from the long‐time cohesion of sister chromosomes.
The germ tubes of Candida albicans hyphae and pseudohyphae show different patterns of septin ring localization
Authors: Sudbery Peter E.
Published: March 2003

The location of the septin ring in the germ tubes of Candida albicans hyphae and pseudohyphae was studied using an antibody to Saccharomyces cerevisiae Cdc11p. In pseudohyphae induced by growth at 35°C in YEPD or Lee's medium, a septin ring formed at or near (mean 1.8 µm) the neck between the mother cell and the germ tube. This became double later in the cycle, and the first mitosis took place across the plane of this double ring. A septin ring also formed at the germ tube neck of developing hyphae induced by serum or growth on Lee's medium at 37°C. However, at later times, this ring became disorganized and disappeared. A second double ring then appeared 10–15 µm (mean 12.5 µm) along the length of the germ tube. The nucleus subsequently migrated out of the mother cell into the germ tube, and the first mitosis took place across the plane of this second septin ring. The relocation of the septin ring in developing hyphae provides a clear‐cut molecular distinction between hyphae and pseudohyphae. Commitment to one type of septin localization and mitosis was shown to occur early in the first mitotic cycle, well before evagination. Germ tubes of hyphae and pseudohyphae also have different widths. A point of commitment to germ tube width was also demonstrated, but occurred later in the cycle, approximately coincident with the time of evagination.
ThensdDgene encodes a putative GATA‐type transcription factor necessary for sexual development ofAspergillus nidulans
Authors: Han Kap‐Hoon , Han Kyu‐Yong , Yu Jae‐Hyuk , Chae Keon‐Sang , Jahng Kwang‐Yeop , Han Dong‐Min
Published: March 2003

The ability to reproduce both sexually and asexually is one of the characteristics of the homothalic ascomyceteAspergillus nidulans. Unlike the otherAspergillusspecies,A. nidulansundergoes sexual development that seems to be regulated by internal and external stimuli. To begin to understand the sexual reproduction ofA. nidulanswe previously isolated and characterized several NSD (never in sexual development) mutants that failed to produce any sexual reproductive organs, and identified four complementation groups,nsdA,nsdB,nsdC,andnsdD. ThensdDgene has been isolated, and it is predicted to encode a GATA‐type transcription factor with the type IVb zinc finger DNA‐binding domain. The mRNA of thensdDgene started to accumulate in the early phase of vegetative growth, and the level increased as sexual development proceeded. However, it decreased during asexual sporulation and nonsdDmRNA was detected in conidia. Deletion ofnsdDresulted in no cleistothecia (fruiting bodies) formation, even under the conditions that preferentially promoted sexual development, indicating thatnsdDis necessary for sexual development. In contrast, when thensdDgene was over‐expressed, sexual‐specific organ (Hülle cell) was formed even in submerged culture, which normally completely blocked sexual development, and the number of cleistothecia was also dramatically increased on solid medium. These results lead us to propose that thensdDgene functions in activating sexual development ofA. nidulans. Multiple copies of thensdDgene could suppressnsdB5andveA1, indicating that eithernsdDacts downstream of these genes or possibly functions in overlapping pathway(s).
Mutations in the Escherichia coli receptor FepA reveal residues involved in ligand binding and transport
Authors: Barnard Travis J. , Watson Michael E. , McIntosh Mark A.
Published: March 2003

FepA is the Escherichia coli outer membrane receptor for ferric enterobactin, colicin D and colicin B. The transport processes through FepA are energy‐dependent, relying on the periplasmic protein TonB to interact with FepA. Through this interaction, TonB tranduces energy derived from the cytoplasmic membrane across the periplasmic space to FepA. In this study, random mutagenesis strategies were used to define residues of FepA important for its function. Both polymerase chain reaction (PCR)‐generated random mutations in the N‐terminal 180 amino acids of FepA and spontaneous chromosomal fepA mutations were selected by resistance to colicin B. The PCR mutagenesis strategy targeted the N‐terminus because it forms a plug inside the FepA barrel that is expected to be involved in ligand binding, ligand transport, and interaction with TonB. We report the characterization of 15 fepA missense mutations that were localized to three regions of the FepA receptor. The first region was a stretch of eight amino acids referred to as the TonB box. The second region included extracellular loops of both the barrel and the plug. A third region formed a cluster near the barrel wall around positions 75 and 126 of the plug. These mutations provide initial insight into the mechanisms of ligand binding and transport through the FepA receptor.
Feedback controls restrain the initiation of Escherichia coli chromosomal replication
Authors: Katayama Tsutomu
Published: March 2003

In Escherichia coli, initiation of chromosomal replication is activated by a nucleoprotein complex formed primarily between the DnaA protein and oriC (replication origin) DNA. After replicational initiation, this complex has to be inactivated in order to repress the appearance of initiation events until the next scheduled round of initiation. Studies of the mechanisms responsible for this repression have recently revealed direct coupling between these mechanisms and key elements of the replication process, suggesting that feedback‐type regulatory loops exist between the factors implicated in initiation and the elements yielded by the replication process. The loading of the ring‐shaped β‐subunit of DNA polymerase III onto DNA plays a key role in the inactivation of the DnaA protein. Duplication of oriC DNA results in hemimethylated DNA, which is inert for reinitiation. Titration of large amounts of DnaA protein to a non‐oriC locus can repress untimely initiations, and timely duplication of this locus is required for this repression in rapidly growing cells. All these systems functionally complement one another to ensure the maintenance of the interinitiation interval between two normal DNA replication cycles. The mechanisms that link the replication cycle to the progression of the cell cycle are also discussed.
Disruptions of the Ustilago maydis REC2 gene identify a protein domain important in directing recombinational repair of DNA
Authors: Kojic Milorad , Thompson Chad W. , Holloman William K.
Published: March 2003

The REC2 gene of Ustilago maydis encodes a homologue of the Escherichia coli RecA protein and was first identified in a screen for UV‐sensitive mutants. The original isolate, rec2‐1, was found to be deficient in repair of DNA damage, genetic recombination and meiosis. We report here that the rec2‐197 allele, which was constructed by gene disruption, retains some biological activity and is partially dominant with respect to REC2. The basis for the residual activity is probably as a result of expression of a diffusible product from the rec2‐197 allele that augments or interferes with REC2 functions. This product appears to be a polypeptide expressed from a remnant of the 5′ end of the open reading frame that was not removed in creating the gene disruption. The mutator activity and disturbed meiosis of rec2‐197 suggest that the Rec2 protein functions in a process that avoids spontaneous mutation and insures faithful meiotic chromosome segregation. A prediction based on the phenotype of rec2‐197 is that Rec2 protein interacts with one or more other proteins in directing these functions. To identify interacting proteins we performed a yeast two‐hybrid screen and found Rad51 as a candidate. Rec2‐197 and Rad51 appear to interact to a similar degree.
Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage φC31
Authors: Cowlishaw Deborah A. , Smith Margaret C. M.
Published: March 2003

Mutants of Streptomyces coelicolor A3(2) J1929 (ΔpglY) were isolated that were resistant to the Streptomyces temperate phage φC31. These strains could be transfected with φC31 DNA, but could not act as infective centres after exposure to phage. Thus, it was concluded that infection was blocked at the adsorption/DNA injection step. The mutants fell into three classes. Class I mutants were complemented by a gene, SCE87.05, isolated from the cosmid library of S. coelicolor A3(2). The product of SCE87.05 had good overall homology to a Mycobacterium tuberculosis hypothetical protein and regions with similarity to dolichol phosphate‐d‐mannose:protein O‐d‐mannosyltransferases. Concanavalin A (ConA) inhibited φC31 infection of S. coelicolor J1929, and this could be partially reversed by the addition of the sugar, α‐d‐methyl‐pyranoside. Moreover, glycosylated proteins from J1929, but not from the class I mutant DT1017, were detected using ConA as a probe in Western blots. Class I and II mutants were sensitive to φC31hc, a previously isolated phage exhibiting an extended host range phenotype, conferred by h. A phage with the same phenotype, φDT4002, was isolated independently, and a missense mutation was found in a putative tail gene. It is proposed that the φC31 receptor is a cell wall glycoprotein, and that the φC31h mutation compensates for the lack of glycosylation of the receptor.
Increased expression of Borrelia burgdorferi vlsE in response to human endothelial cell membranes
Authors: Hudson Charlene R. , Frye Jonathan G. , Quinn Frederick D. , Gherardini Frank C.
Published: March 2003

RNA isolated from virulent Borrelia burgdorferi cells incubated with human endothelial or neurological tissue cells was subjected to subtractive hybridization using RNA from the same strain incubated in tissue culture medium alone. This RNA subtractive technique generated specific probes that hybridized to two restriction fragments (8.2 kb and 10 kb respectively) generated by EcoRI digestion of total plasmid DNA. The 10 kb EcoRI fragment localized to lp28‐1 and was subsequently identified as the variable membrane protein‐like sequence (vls) region, which includes an expression locus (vlsE) and 15 silent cassettes. vlsE encodes a 36 kDa outer surface protein that undergoes antigenic variation during animal infections. Primer extension analysis identified the 5′ end of a transcript and a putative promoter for vlsE. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) suggested that the expression of vlsE increased when virulent B. burgdorferi cells were incubated with human tissue cells or purified cell membranes isolated from those same cell lines. A 138 bp region upstream of the vlsE region that was not reported in the genome sequence was sequenced using specific 32P end‐labelled primers in a DNA cycle sequencing system at high annealing temperatures. Analysis revealed that it contained a 51 bp inverted repeat, which could form an extremely stable cruciform structure. Southern blots probed with the vlsE promoter/operator region indicated that part or all of this sequence could be found on other B. burgdorferi plasmids.
Identification of a cell wall channel of Streptomyces griseus: the channel contains a binding site for streptomycin
Authors: Kim Bong‐Hui , Andersen Christian , Benz Roland
Published: March 2003

Cells of the Gram‐positive actinomycete Streptomyces griseus were disrupted and the cell envelope was subjected to sucrose step‐gradient centrifugation. The different fractions were analysed for NADH‐oxidase activity and the formation of ion‐permeable channels in lipid bilayers. Highest channel‐forming activity and highest NADH‐oxidase activity were found in different fractions. The cell wall fraction contained an ion‐permeable channel with a single‐channel conductance of 850 pS in 1 M KCl. The channel‐forming protein, with an apparent molecular mass of 28 kDa, was purified to homogeneity using fast protein liquid chromatography after the extraction of whole cells with detergent. Single‐channel experiments suggest that the cell wall channel is wide and water‐filled. Titration experiments with streptomycin produced by S. griseus suggested that the cell wall channel binds this antibiotic with a half saturation constant of about 6 mM in 1 M KCl. The binding of streptomycin was found to be ionic strength dependent and the half saturation constant decreased to 60 µM at 0.1 M KCl. The results indicate that the 28 kDa protein represents the hydrophilic pathway through the cell wall of the Gram‐positive bacterium S. griseus.
Genetic analysis of the chromosome segregation protein Spo0J of Bacillus subtilis: evidence for separate domains involved in DNA binding and interactions with Soj protein
Authors: Autret Sabine , Nair Radha , Errington Jeffery
Published: March 2003

Spo0J and Soj belong to the ParB/ParA family of proteins involved in chromosome and plasmid segregation in bacteria. In Bacillus subtilis, Spo0J protein binds to several specific sites, parS, located on both sides of the origin of DNA replication, oriC, and apparently self‐associates to form large discrete foci visible by fluorescence microscopy. Soj protein forms large ‘patches’ probably associated with the nucleoid, which can undergo dynamic, co‐operative jumping from nucleoid to nucleoid in the presence of Spo0J. Patches of Soj protein somehow help to bring about the condensation of Spo0J foci. Soj is also a negative regulator of transcription. In the absence of Spo0J, Soj is statically distributed on each of the nucleoids in the cell and blocks the transcription of several sporulation genes. To analyse the functional interaction between Spo0J and Soj further, we have constructed and studied a collection of spo0J mutants. Most of the mutants completely prevent Spo0J from interacting with DNA. One mutation impairs the formation of compact Spo0J foci and simultaneously results in loss of Soj movement. We also isolated one spo0J mutant, in which the frequency of Soj internucleoid oscillation is highly increased. Both mutations affecting the interaction with Soj lie in the N‐terminal coding part of spo0J, whereas the substitutions affecting DNA binding lie in the mid‐ to C‐terminal coding region.
The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice
Authors: Yorgey Peter , Rahme Laurence G. , Tan Man‐Wah , Ausubel Frederick M.
Published: March 2003

We are exploiting the broad host range of the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 to elucidate the molecular basis of bacterial virulence in plants, nematodes, insects and mice. In this report, we characterize the role that two PA14 gene products, MucD and AlgD, play in virulence. MucD is orthologous to the Escherichia coli periplasmic protease and chaperone DegP. DegP homologues are known virulence factors that play a protective role in stress responses in various species. AlgD is an enzyme involved in the biosynthesis of the exopolysaccharide alginate, which is hyperinduced in mucD mutants. A PA14 mucD mutant was significantly impaired in its ability to cause disease in Arabidopsis thaliana and mice and to kill the nematode Caenorhabditis elegans. Moreover, MucD was found to be required for the production of an extracellular toxin involved in C. elegans killing. In contrast, a PA14 algD mutant was not impaired in virulence in plants, nematodes or mice. A mucDalgD double mutant had the same phenotype as the mucD single mutant in the plant and nematode pathogenesis models. However, the mucDalgD double mutant was synergistically reduced in virulence in mice, suggesting that alginate can partially compensate for the loss of MucD function in mouse pathogenesis.
The carboxy‐terminus of VirE2 from Agrobacterium tumefaciens is required for its transport to host cells by the virB‐encoded type IV transport system
Authors: Simone Mark , McCullen Colleen A. , Stahl Lisa E. , Binns Andrew N.
Published: March 2003

Agrobacterium tumefaciens transfers DNA from the resident ‘tumour‐inducing’ (Ti) plasmid into plant cells, where it can be stably integrated into the plant genome, ultimately resulting in crown gall tumour formation. The mobilized DNA molecule is a single‐stranded intermediate with VirD2 covalently bound to its 5′ end. Successful transport of the transferred DNA (T‐DNA) and integration of the DNA into the genome requires that additional proteins be transported to the plant as well, including the single‐stranded (ss)DNA‐binding protein, VirE2. The transport of these two different substrates occurs as a result of the activities of a type IV secretion system encoded by the virB operon. Although the substrates have been identified, the mechanism of their transport remains unknown. In the experiments described here, a region in one of these substrates, VirE2, necessary for transport is identified. The addition of a C‐terminal FLAG epitope tag to VirE2, or the deletion of its C‐terminal 18 amino acids, renders it non‐functional in A. tumefaciens. However, transgenic plants expressing either of these virE2 genes respond to virE2 mutants of A. tumefaciens by forming wild‐type tumours. These results indicate that this region of VirE2 is necessary for the protein to be transported into the plant cells, but is not necessary for its function within the plant. Additionally, these studies demonstrate that mutant forms of VirE2 lacking this region do not disrupt the activities of the VirB transporter and support the hypothesis that VirE2 and the VirD2 T‐strand are transported independently, even when they co‐exist in the same cell.
Novel mode of transcription regulation by SdiA, an Escherichia coli homologue of the quorum‐sensing regulator
Authors: Yamamoto Kaneyoshi , Yata Katsumori , Fujita Nobuyuki , Ishihama Akira
Published: March 2003

SdiA, an Escherichia coli homologue of the quorum‐sensing regulator, controls the expression of the ftsQAZ operon for cell division. Transcription of ftsQ is under the control of two promoters, upstream ftsQP2 and downstream ftsQP1, which are separated by 125 bp. SdiA activates transcription from ftsQP2 in vivo. Here, we demonstrate that SdiA facilitates the RNA polymerase binding to ftsQP2 and thereby stimulates transcription from P2. Gel shift and DNase I footprinting assays indicated that SdiA binds to the ftsQP2 promoter region between −51 and −25 with respect to the P2 promoter. Activation of ftsQP2 transcription by SdiA was observed with a mutant RNA polymerase containing a C‐terminal domain (CTD)‐deleted α‐subunit (α235) but not with RNA polymerase containing σS or a CTD‐deleted σD (σD529). In good agreement with the transcription assay, no protection of P2 was observed with the RNA polymerase holoenzymes, EσS and EσD529. These observations together indicate that: (i) SdiA supports the RNA polymerase binding to ftsQP2; and (ii) this recruitment of RNA polymerase by SdiA depends on the presence of intact σCTD. This is in contrast to the well‐known mechanism of RNA polymerase recruitment by protein–protein contact between class I factors and αCTD. In addition to the P2 activation, SdiA inhibited RNA polymerase binding to the ftsQP1 promoter and thereby repressed transcription from P1. Gel shift assays indicate weak binding of SdiA to the P1 promoter region downstream from −13 (or +112 with respect to P2). Neither αCTD nor σCTD are required for this inhibition. Thus, the transcription repression of P1 by SdiA may result from its competition with the RNA polymerase in binding to this promoter.
Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin
Authors: Pei Silvia , Doye Anne , Boquet Patrice
Published: March 2003

The Rho‐GTPases‐activating toxin CNF1 (cytotoxic necrotizing factor 1) delivers its catalytic activity into the cytosol of eukaryotic cells by a low pH membrane translocation mechanism reminiscent of that used by diphtheria toxin (DT). As DT, CNF1 exhibits a translocation domain (T) containing two predicted hydrophobic helices (H1–2) (aa 350–412) separated by a short peptidic loop (CNF1‐TL) (aa 373–386) with acidic residues. In the DT loop, the loss of charge of acidic amino acids, as a result of protonation at low pH, is a critical step in the transfer of the DT catalytic activity into the cytosol. To determine whether the CNF1 T domain operates similarly to the DT T domain, we mutated several ionizable amino acids of CNF1‐TL to lysine. Single substitutions such as D373K or D379K strongly decreased the cytotoxic effect of CNF1 on HEp‐2 cells, whereas the double substitution D373K/D379K induced a nearly complete loss of cytotoxic activity. These single or double substitutions did not modify the cell‐binding, enzymatic or endocytic activities of the mutant toxins. Unlike the wild‐type toxin, single‐ or double‐substituted CNF1 molecules bound to the HEp‐2 plasma membrane could not translocate their enzymatic activity directly into the cytosol following a low pH pulse.
Beta‐helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins
Authors: Kajava Andrey V. , Cheng Naiqian , Cleaver Ryan , Kessel Martin , Simon Martha N. , Willery Eve , Jacob‐Dubuisson Francoise , Locht Camille , Steven Alasdair C.
Published: March 2003

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500‐Å‐long secreted protein that is rich in β‐structure and contains two regions, R1 and R2, of tandem 19‐residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel β‐sheets; or (ii) a β‐helix in which the polypeptide chain is coiled to form three long parallel β‐sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional ‘covert’ copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the β‐helix model that the shaft domain – a 350 Å rod – should consist of a continuous run of these motifs, apart from loop inserts. The N‐terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin‐like structure. Drawing on crystal structures of known β‐helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins – all large secreted proteins of bacterial provenance – that have similar repeats and probably also similar structures.
Sites of positive and negative regulation in the Bacillus subtilis antiterminators LicT and SacY
Authors: Tortosa Pablo , Declerck Nathalie , Dutartre Hélène , Lindner Cordula , Deutscher Josef , Le Coq Dominique
Published: March 2003

The Bacillus subtilis homologous transcriptional antiterminators LicT and SacY control the inducible expression of genes involved in aryl β‐glucoside and sucrose utilization respectively. Their RNA‐binding activity is carried by the N‐terminal domain (CAT), and is regulated by two similar C‐terminal domains (PRD1 and PRD2), which are the targets of phosphorylation reactions catalysed by the phosphoenolpyruvate: sugar phosphotransferase system (PTS). In the absence of the corresponding inducer, LicT is inactivated by BglP, the PTS permease (EII) specific for aryl β‐glucosides, and SacY by SacX, a negative regulator homologous to the EII specific for sucrose. LicT, but not SacY, is also subject to a positive control by the general PTS components EI and HPr, which are thought to phosphorylate LicT in the absence of carbon catabolite repression. Construction of SacY/LicT hybrids and mutational analysis enabled the location of the sites of this positive regulation at the two phosphorylatable His207 and His269 within LicT‐PRD2, and suggested that the presence of negative charges at these sites is sufficient for LicT activation in vivo. The BglP‐mediated inhibition process was found to essentially involve His100 of LicT‐PRD1, with His159 of the same domain playing a minor role in this regulation. In vitro experiments indicated that His100 could be phosphorylated directly by the general PTS proteins, this phosphorylation being stimulated by P∼BglP. We confirmed that, similarly, the corresponding conserved His99 residue in SacY is the major site of the negative control exerted by SacX on SacY activity. Thus, for both antiterminators, the EII‐mediated inhibition process seems to rely primarily on the presence of a negative charge at the first conserved histidine of the PRD1.
Population density‐dependent regulation of the Bradyrhizobium japonicum nodulation genes
Authors: Loh John T. , Yuen‐Tsai Joyce P.‐Y. , Stacey Minviluz G. , Lohar Dasharath , Welborn April , Stacey Gary
Published: March 2003

The nodulation genes of Bradyrhizobium japonicum are essential for infection and establishment of a nitrogen‐fixing symbiosis. Here, we demonstrate that plant‐produced isoflavones induce nodulation gene expression in a population density‐dependent fashion. Nodulation gene induction is highest at a low population density and significantly reduced in more dense cultures. A quorum signal molecule in the conditioned medium of B. japonicum cultures mediates this repression. Repression in response to the quorum signal results from the induction of NolA which, in turn, induces NodD2 leading to inhibition of nod gene expression. Consistent with this, nolA–lacZ and nodD2–lacZ expression increased with increasing population density. Unlike the wild type, the ability to induce nodY–lacZ expression did not decline with population density in a NolA mutant. Normally, nod gene expression is repressed in planta (i.e. within nodules). However, expression of a nodY–GUS fusion was not repressed in a NolA mutant, suggesting that quorum‐sensing control may mediate in planta repression of the nod genes. Addition of conditioned medium to cultures significantly reduced nod gene expression. Treatment of inoculant cultures with conditioned medium also reduced the ability of B. japonicum to nodulate soybean plants.
Structure, organization and characterization of the gene cluster involved in the production of microcin E492, a channel‐forming bacteriocin
Authors: Lagos Rosalba , Baeza Marcelo , Corsini Gino , Hetz Claudio , Strahsburger Erwin , Castillo José Antonio , Vergara Cecilia , Monasterio Octavio
Published: March 2003

Microcin E492 is a low‐molecular‐weight, channel‐forming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post‐translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492.
GidA is an FAD‐binding protein involved in development of Myxococcus xanthus
Authors: White David J. , Merod Robin , Thomasson Bobbie , Hartzell Patricia L.
Published: March 2003

A gene encoding a homologue of the Escherichia coli GidA protein (glucose‐inhibited division protein A) lies immediately upstream of aglU, a gene encoding a WD‐repeat protein required for motility and development in Myxococcus xanthus. The GidA protein of M. xanthus shares about 48% identity overall with the small (≈ 450 amino acid) form of GidA from eubacteria and about 24% identity overall with the large (≈ 620 amino acid) form of GidA from eubacteria and eukaryotes. Each of these proteins has a conserved dinucleotide‐binding motif at the N‐terminus. To determine if GidA binds dinucleotide, the M. xanthus gene was expressed with a His6 tag in E. coli cells. Purified rGidA is a yellow protein that absorbs maximally at 374 and 450 nm, consistent with FAD or FMN. Thin‐layer chromatography (TLC) showed that rGidA contains an FAD cofactor. Fractionation and immunocytochemical localization show that full length GidA protein is present in the cytoplasm and transported to the periplasm of vegetative‐grown M. xanthus cells. In cells that have been starved for nutrients, GidA is found in the cytoplasm. Although GidA lacks an obvious signal sequence, it contains a twin arginine transport (Tat) motif, which is conserved among proteins that bind cofactors in the cytoplasm and are transported to the periplasm as folded proteins. To determine if GidA, like AglU, is involved in motility and development, the gidA gene was disrupted. The gidA– mutant has wild‐type gliding motility and initially is able to form fruiting bodies like the wild type when starved for nutrients. However, after several generations, a stable derivative arises, gidA*, which is indistinguishable from the gidA– parent on vegetative medium, but is no longer able to form fruiting bodies. The gidA* mutant releases a heat‐stable, protease‐resistant, small molecular weight molecule that acts in trans to inhibit aggregation and gene expression of wild‐type cells during development.
Constitutive competence for genetic transformation in Streptococcus pneumoniae caused by mutation of a transmembrane histidine kinase
Authors: Lacks Sanford A. , Greenberg Bill
Published: March 2003

Competence for DNA uptake and genetic transformation in Streptococcus pneumoniae is regulated by a quorum‐sensing system. A competence‐stimulating polypeptide (CSP) is secreted by the bacteria and acts back on the cells via a transmembrane histidine kinase. This enzyme phosphorylates a response regulator that activates synthesis of a SigH‐like protein. The new sigma factor enables expression of a set of proteins transcribed from a novel promoter. A mutation called trt had been found that circumvented this regulation. The mutant cells are constitutively competent; that is, they can be transformed at low cell densities, in the presence of proteases that attack CSP, or during growth at low pH. In this work, cells containing trt were shown to be competent even in the presence of a comAB mutation that blocks secretion of CSP. The trt mutation was localized to comD, the gene encoding the transmembrane histidine kinase. A DNA segment of the trt mutant corresponding to comCDE was cloned, and it was shown to contain the trt mutation by its ability to confer constitutive competence. A two‐step assay, which was based on transfer of trt to a wild strain and screening for transformability in the presence of trypsin, served to locate the trt mutation precisely. It corresponds to a GC→AT transition, which changes Asp299 in the histidine kinase to Asn. This alteration in the carboxyl terminal half of the protein, which is cytoplasmically located and contains the phosphorylase activity, presumably alters the enzyme conformation so that it is permanently activated, independent of signals from the transmembrane domain. These results may help illuminate the mechanism by which external signals affect kinase action in two‐component regulatory systems, and they may be of practical value in facilitating genetic studies by rendering pneumococcal strains permanently competent.
InlA‐ but not InlB‐mediated internalization of Listeria monocytogenes by non‐phagocytic mammalian cells needs the support of other internalins
Authors: Bergmann Birgit , Raffelsbauer Diana , Kuhn Michael , Goetz Monika , Hom Silke , Goebel Werner
Published: March 2003

To determine the contribution of the previously identified internalins, InlA, InlB, InlC, InlE, InlG, and InlH, to internalization of Listeria monocytogenes by
non‐professional phagocytic mammalian cells, we constructed mutants with various
combinations of deletions in the respective inl genes. Internalization of
these mutants into the epithelial‐like Caco‐2 and the microvascular endothelial HBMEC
cell lines were studied. Deletion of the inlGHE gene cluster, or of the single
genes, led to a two to fourfold increased internalization by HBMEC and other non‐phagocytic
mammalian cells. Invasion into HBMEC was totally blocked in the absence of InlB,
and InlB‐dependent internalization did not require the presence of any of the other
internalins. Internalization by Caco‐2 cells was reduced to a level of about 1%
in the absence of InlA and InlB, and was most efficient in the presence of InlA,
InlB and InlC and in the absence of InlG, InlH and InlE. InlB and InlA, in each case
in the absence of the other internalins, led (compared with the wild‐type strain)
to reduced internalization of about 20% and less than 10% respectively.
InlA‐dependent internalization (in the absence of InlB) required the additional function
of InlC and InlGHE. The deletion of inlGHE enhanced the expression of InlA
and InlB. The increased amount of InlA led to an increase in early association of
L. monocytogenes with Caco‐2 cells without enhancing its uptake in the absence
of the other internalins, whereas the larger amount of InlB did not enhance early
association of L. monocytogenes with HBMEC but led to an increase in internalization
of L. monocytogenes. The results suggest that InlB is able to induce phagocytosis
in HBMEC and (at a lower efficiency) in Caco‐2 cells by itself, but InlA needs the
supportive functions of the other internalins to trigger phagocytosis. None of these
internalins seems to be required for cell‐to‐cell spread by L. monocytogenes,
as shown by microinjection of Caco‐2 cells with appropriate inl mutants.