bluuebunny/crossref_metadata_2025 huggingface dataset:
Signal‐sensing mechanisms of the putative osmosensor KdpD in Escherichia coli
Authors: Sugiura Akemi , Hirokawa Kozo , Nakashima Kyoko , Mizurto Takeshi
Published: October 2006

The KdpD protein is a membrane‐located sensory kinase (or signal transducer) critically involved in the regulation of the kdpABC operon that is responsible for a high‐affinity transport system in Escherichia coli. In this study, a set of KdpD mutants, each resulting in a single amino acid substitution around the membrane‐spanning regions of KdpD, was isolated. Amino acid substitutions in these KdpD mutants were located non‐randomly, particularly within the C‐terminal half of the membrane‐spanning regions. This set of KdpD mutants exhibited altered transmembrane‐signalling properties in response to external K+ and other stimuli. In particular, these mutants were found to be insensitive, if not completely, to the K+ signal. However, they were able to respond to other stimuli such as high‐salt stress, as in the wild type. Therefore, in contrast to the wild type, the cells carrying these mutations exhibited high levels of the steady‐state expression of kdp, regardless of external K+, provided that high concentrations of ionic solutes were supplemented to the cultures. More interestingly, the set of KdpD mutants could also respond to high concentrations of external non‐ionic solutes such as sucrose and D‐arabinose, thereby increasing substantially the steady‐state expression of kdp in response to the medium osmolarity. Furthermore, it was found that certain chemicals, ethanol, chlorpromazine and procaine, could function as effectors for the KdpD mutants at relatively low concentrations in the media. Based on these findings, we have examined the primary signal(s) that regulates the function of KdpD. We propose here that KdpD can be considered to be an environmental sensor that exhibits sensing mechanisms in response to both the level of K+ and the physico‐chemical state of the cytoplasmic membrane.
Helicobacter pylori hspA‐hspB heat‐shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity
Authors: Suerbaum Sebastian , Thiberge Jean‐Michel , Kansau Imad , Ferrero Richard L. , Labigne Agnès
Published: October 2006

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel‐dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat‐shock protein of the GroEL class. The gene encoding the GroEL‐like protein of H. pylori (HspB) was cloned (plLL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli. the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C‐terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel‐binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the plLL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (plLL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in ihe chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose‐binding protein (WIBP) and purified in large scale. The MBP‐HspA and MBP‐HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori‐infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic as proteins fused to the maltose‐binding protein (WIBP) and purified in large scale. The MBP‐HspA and WlBP‐HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H, py/or/‐infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.
The dipeptide permease of Escherichia coli closely resembles other bacterial transport systems and shows growth‐phase‐dependent expression
Authors: Abouhamad Walid N. , Manson Michael D.
Published: October 2006

The dipeptide permease (Dpp) of Escherichia coli transports peptides consisting of two or three L‐amino acids. The periplasmic dipeptide‐binding protein (DBP), encoded by the dppA gene, also serves as a chemoreceptor. We sequenced the dpp locus, which comprises an operon of five genes, dppABCDE. Its organization is the same as the oligopeptide permease (opp) operon of Salmonella typhimurium and the spo0K operon of Bacillus subtilis. The dpp genes are also closely related to the hbpA gene, which encodes a haem‐hinding lipoprotein, and four other genes in an unlinked operon of unknown function in Haemophilus influenzae. Each Dpp protein has an Opp, Spo0K and H. influenzae homologue. Transcription of the dpp operon initiates 165 bases upstream of the predicted dppA start codon. The start site for transcription is preceded by potential −35 and −10 regions of a σ70 promoter. During exponential growth in Luria‐Bertani (LB) broth, the level of dpp mRNA increases in two steps, one between A590 0.2 and 0.4 and one between A590 0.7 and 1.0. The 310 nucleotides between dppA and dppB include a RIP (repetitive IHF‐binding palindromic) element, whose deletion from a multi‐copy plasmid causes fivefold and 10‐fold reductions in the levels of upstream and downstream dpp mRNA, respectively.
Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue‐type plasminogen activator (t‐PA)
Authors: Sjöbring Ulf , Pohl Gunnar , Olsén Arne
Published: October 2006

Curli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue‐type plasminogen activator (t‐PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t‐PA with high affinity (1 × 108 to 2 × 108 M‐1). The binding of plasminogen and t‐PA to curli‐expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli‐expressing E. coli was readily converted to plasmin by t‐PA; both plasmin and t‐PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.
The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome‐binding site
Authors: Bibb Mervyn J. , White Janet , Ward Judith M. , Janssen Gary R.
Published: October 2006

Transcriptional analysis of the ermE gene of Saccharopolyspora erythraea, which confers resistance to erythromycin by N6‐dimethylation of 23S rRNA and which is expressed from two promoters, ermEp1 and ermEp2, revealed a complex regulatory region in which transcription is initiated in a divergent and overlapping manner. Two promoters (eryC1p1 and eryC1p2) were identified for the divergently transcribed erythromycin biosynthetic gene eryC1, which plays a role in the formation of desosamine or its attachment to the macrolide ring. Transcription from eryC1p2 starts at the same position as that of ermEp1, but on the opposite strand of the DNA helix, suggesting co‐ordinate regulation of genes for erythromycin production and resistance. ErmEp1 initiates transcription at, and one nucleotide before, the ermE translational start codon. Site‐directed and deletion mutagenesis, combined with immunochemical analysis, demonstrated that the ermEp1 transcript is translated in the absence of a conventional ribosome‐binding site to give rise to the full‐length 23S rRNA methylase. Deletion of the ‐35 region of ermEp1 reduced, but did not abolish, promoter activity, reminiscent of the‘extended ‐10’class of bacterial promoters which, like ermEp1, possess TGN motifs immediately upstream of their 10 regions and which initiate transcription seven nucleotides downstream of the ‐10 region.
Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways
Authors: Fiss Ellen H. , Yu Shengwei , Jacobs William R.
Published: October 2006

Mycobacteria produce two siderophores, mycobactin and exochelin. Mycobacterium smegmatis mutants defective in the production of exochelin were isolated using agar medium containing chrome azural S for the sensitive detection of siderophores. Cosmids of genomic libraries from M. smegmatis and Mycobacterium bovis BCG were screened for complementation of the mutation. Subcloning of the complementing M. smegmatis cosmid identified a 4.3 kb fragment required for restoring exochelin biosynthesis. Sequencing of the DNA revealed four open reading frames whose genes were named fxuA. fxuB, fxuC, and fxbA. FxuA, FxuB, and FxuC share amino acid sequence homology with the iron permeases FepG, FepC. and FepD from Escherichia coli. respectively. Deletion analysis identified fxbA as the gene required to restore exochelin biosynthesis in our mutant. Although fxbA does not share amino acid sequence homology with any of the published sequences for siderophore biosynthetic genes, it does show limited homology with the phosphoribosyl‐glycineamide formyltransferases (GAR enzymes) and methionyl‐tRNA formyltransferase over a limited region of the sequence, suggesting that fxbA may code for an enzyme which adds a formyl group in the synthesis of exochelin. A fusion of fxbA with the E. coli lacZ gene demonstrated regulation of gene expression by iron. The ability of M. smegmatis mutants to produce mycobactin in the absence of exochelin supports the hypothesis that exochelin is not a precursor of mycobactin and suggests that the siderophores have independent biosynthetic pathways. In addition, complementation of the M.
Translocation of a hybrid YopE‐adenylate cyclase from Yersinia enterocolitica into HeLa cells
Authors: Sory Marie‐Paule , Cornelis Guy R.
Published: October 2006

Pathogenic bacteria of the genus Yersinia release in vitro a set of antihost proteins called Yops. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis transfers YopE across the host cell plasma membrane. To facilitate the study of this translocation process, we constructed a recombinant Yersinia enterocolitica strain producing YopE fused to a reporter enzyme. As a reporter, we selected the calmodulin‐dependent adenylate cyclase of Borde‐tella pertussis and we monitored the accumulation of cyclic AMP (cAMP). Since bacteria do not produce calmodulin, cyclase activity marks the presence of hybrid enzyme in the cytoplasmic compartment of the eukaryotic cell. Infection of a monolayer of HeLa cells by the recombinant Y. enterocolitica strain led to a significant increase of cAMP. This phenomenon was dependent not only on the integrity of the Yop secretion pathway but also on the presence of YopB and/or YopD. It also required the presence of the adhesin YadA at the bacterial surface. In contrast, the phenomenon was not affected by cytochalasin D, indicating that internalization of the bacteria themselves was not required for the translocation process. Our results demonstrate that Y. enterocolitica is able to transfer hybrid proteins into eukaryotic cells. This system can be used not only to study the mechanism of YopE translocation but also the fate of the other Yops or even of proteins secreted by other bacterial pathogens.
Identification and characterization of new DNA replication terminators in Bacillus subtilis
Authors: Franks A.H. , Griffiths A.A. , Wake R.G.
Published: October 2006

A functional DNA replication terminator of Bacillus subtilis contains two overlapping binding sites, A and B, for the replication terminator protein (RTP). A degenerate 17‐mer oligonucleotide corresponding to the consensus B site has been used to detect four new terminators in the B. subtilis chromosome, in addition to the previously identified and closely spaced IRI and IRII. All the new terminators lie in the terminus region of the chromosome, on both sides of IRI and IRII, with their positions spanning <1O% of its length. Their DNA sequences are characterized by clearly identifiable A‐ and B‐binding sites. They bind RTP in a manner indistinguishable from IRI, although precise affinities have not been compared. Each new terminator is functional in causing fork arrest when present in a plasmid replicating in B. subtilis. Three of the four were tested for polarity in fork‐arrest activity and exhibited the polarity expected. The total of six terminators now identified in B. subtilis have been named TerI‐TerVI. TerI and TerII correspond to the previously identified IRI and IRII, respectively. The chromosomal orientations of all but one of the terminators (TerIV) have been established and they conform to an arrangement similar to that in Escherichia coli in which two opposed groups of polar terminators provide a replication‐fork trap ensuring that the approaching forks meet within a restricted region of the chromosome. The development of a strikingly similar arrangement of terminators in the two organisms, despite the lack of any detectable similarity in their respective DNA terminators and terminator proteins, emphasizes the importance of the replication‐fork trap in each case.
A new RNA polymerase sigma factor, σF is required for the late stages of morphological differentiation in Streptomyces spp.
Authors: Potúčková Laura , Kelemen Gabriella H. , Findlay Kim C. , Lonetto Michael A. , Buttner Mark J. , Kormanec Ján
Published: October 2006

A gene (sigF) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well‐conserved region 2.2 of the eubacterial σ70 family. Homologues were present in other Streptomyces spp., and that of the genetically well‐studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 (S. coelicolor) and 295 (S. aureofaciens) amino acid residues, both showing greatest similarity to σB of Bacillus subtilis. However, while σB is involved in stationary‐phase gene expression and in the general stress response in B. subtilis, σF affects morphological differentiation in Streptomyces, Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. σF is proposed to control the late stages of spore development in Streptomyces.
Co‐ordinate, temperature‐sensitive regulation of the three Yersinia enterocolitica flagellin genes
Authors: Kapatral Vinayak , Minnich Scott A.
Published: October 2006

Yersinia enterocolitica cells, when cultured at 30°C or below, are flagellated and motile. Cells cultured at 37°C or above lack flagella and are non‐motile. To identify flagellin genes that are a target of this temperature‐dependent regulation, a library of Y. enterocolitica genomic inserts in a phage λ vector was probed with the Salmonella typhimuriumfliC (flagellin) gene. A DNA fragment subcloned from a recombinant phage which hybridizes with the probe complements a non‐motile S. typhimuriumfliC−fljB− (flagellin‐minus) mutant. DNA sequence analysis shows that Y. enterocolitica contains three tandem flagellin genes, designated fleA, fleB and fleC. All three genes are co‐ordinately transcribed at low, but not high, temperature from fliA‐dependent (σF) promoters. Flagellin transcription arrests rapidly after upshift to 37°C (host temperature). In contrast, flagellin transcription resumes only after several generations when cells cultured at 37°C are downshifted to 28°C.
A physical genome map of the Burkholderia cepacia type strain
Authors: Rodley Philip D. , Römling Ute , Tümmler Burkhard
Published: October 2006

Burkholderia cepacia (basonym Pseudomonas cepacia), the type speciesof the new genus Burkholderia, is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil‐borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type‐strain ATCC 25416 was constructed by applying two‐dimensional pulsed‐field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 Spel, 11 Swal, 11 Pacl, 11 Pmel and six l‐Ceul sites, resulting in an average resolution of 1O5 kbp. Random single‐hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase‐size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia. The phylogenetically related species Burkholderia glumae, Burkholderia pickettii, Burkholderia solanacearum, Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase‐size replicon.
Cloning and molecular characterization of two genes encoding adhesion proteins involved in Trichomonas vaginalis cytoadherence
Authors: Alderete J.F. , O'Brien Jennifer L. , Arroyo Rossana , Engbring Jean A. , Musatovova Oxana , Lopez Omero , Lauriano Crystal , Nguyen John
Published: October 2006

Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellate Trichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno‐crossreactive recombinant proteins that possessed functional properties equal to the T. vaginalis AP65 adhesin. Analysis of full‐length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701‐base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 63087 daltons, respectively. Comparison of the full‐length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction‐enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now called ap65−1 and ap65−2) are present in the T. vaginalis genome in at least two copies each. Northern analysis detected high levels of transcript of ∼1.8 kb for both ap65−1 and ap65‐2 genes in trichomonads grown only in high‐iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence of T. vaginalis to host cells nor prevented binding of the trichomonad AP65 to HeLa cells in a ligand assay.
GvpCs with reduced numbers of repeating sequence elements bind to and strengthen cyanobacterial gas vesicles
Authors: Kinsman R. , Walsby A.E. , Hayes P.K.
Published: October 2006

We have previously shown that the gas‐vesicle protein GvpC is present on the outer surface of the gas vesicle, can be reversibly removed and rebound to the surface, and increases the critical collapse pressure of the gas vesicle. The GvpC molecule, which contains five partially conserved repeats of 33 amino acids (33‐RR) sandwiched between 18 N‐terminal and 10 C‐terminal amino acids, is present in a ratio of 1:25 with the GvpA molecule, which forms the ribs of the gas vesicle. By using recombinant techniques we have now made modified versions of GvpC that contain only the first two, three or four of the 33‐amino‐acid repeats. All of these proteins bind to and strengthen gas vesicles that have been stripped of their native GvpC. Recombinant proteins containing three or four repeats bind in amounts that give the same ratio of 33‐RR:GvpA (i.e. 1:5) as the native protein, and they restore much of the strength of the gas vesicle; the protein containing only two repeats binds at a lower ratio (1:7.7), however, and restores less of the strength. Ancestral proteins with only two, three or four of the 33‐amino‐acid repeats would have been functional in strengthening the gas vesicle but the progressive increase in number of repeats would have provided strength with increased efficiency.
The stationary‐phase sigma factor σS (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium
Authors: Soo Lee In , Lin Jyhshiun , Hall Holly K. , Bearson Bradley , Foster John W.
Published: October 2006

The acid tolerance response (ATR) of log‐phase Salmonella typhimurium is induced by acid exposures below pH 4.5 and will protect cells against more extreme acid. Two systems are evident: a transiently induced system dependent on the iron regulator Fur that provides a moderate degree of acid tolerance and a more effective sustained ATR that requires the alternate sigma factor σS encoded by rpoS. Differences between the acid responses of virulent S. typhimurium and the attenuated laboratory strain LT2 were attributed to disparate levels of RpoS caused by different translational starts. The sustained ATR includes seven newly identified acid shock proteins (ASPs) that are dependent upon σS for their synthesis. It is predicted that one or more of these ASPs is essential for the sustained system. The sustained ATR also provided cross‐protection to a variety of other environmental stresses (heat, H2O2 and osmolarity); however, adaptation to the other stresses did not provide significant acid tolerance. Therefore, in addition to starvation, acid shock serves as an important signal for inducing general stress resistance. Consistent with this model, σS proved to be induced by acid shock. Our results also revealed a connection between the transient and sustained ATR systems. Mutations in the regulator atbR are known to cause the overproduction of ten proteins, of which one or more can suppress the acid tolerance defect of an rpoS mutant. One member of the AtbR regulon, designated atrB, was found to be co‐regulated by σS and AtbR. Both regulators had a negative effect on atrB expression. The results suggest AtrB serves as a link between the sustained and transient ATR systems. When σS concentration are low, a compensatory increase in AtrB is required to engage the transiently induced, RpoS‐independent system of acid tolerance. Results also suggest different acid‐sensitive targets occur in log‐phase versus stationary‐phase cells.
Characterization of a new rho mutation that relieves polarity of Mu insertions
Authors: Peters Joseph E. , Benson Spencer A.
Published: October 2006

We report the identification and characterization of a new rho mutation, rho614, that relieves polarity of Mu insertions in Escherichia coli The mutation was identified by its ability to suppress the polarity of the Mu‐mediated Φ(lamB′—′lacZ)hyb 61‐4 fusion that is located at codon four of the lamB signal sequence. The rho614 mutation alters residue 80 in the proposed RNA‐binding domain of Rho and is recessive to wild‐type rho. We suggest that in the presence of the rho614 allele transcripts initiated at the Mu promoter PcM fail to terminate at presumptive Rho‐dependent termination sites, namely rut1 and rut2, and continue into the 3′′lamB gene allowing a LamB+ phenotype. This contention is supported by deletion analysis of the region and the observation that insertional inactivation of genes that reduce transcription from PcM, clpP (ATP protease), himA (IHF‐α), and himD (IHF‐β), block the LamB+ phenotype. rho614, rho4 and rho201 alleles suppress the polarity of a malK::Mu insertion on the downstream ′lamB gene. However, the polarity of the ΦlamB′‐′lacZ)hyb 61–4 insertion is only suppressed by the rho614 mutation. We propose that the rho614 mutation allows suppression of transcriptional polarity without interfering with translation initiation signals of the truncated ′lamB gene. In addition to identifying a new rho mutation and Rho‐dependent terminator sequence, this system provides a means of studying Rho protein/terminator relationships through the identification of new classes of rho mutations.
An Agrobacterium virulence factor encoded by a Ti plasmid gene or a chromosomal gene is required for T‐DNA transfer into plants
Authors: Pan Shen Q. , Jin Shouguang , Boulton Margaret I. , Hawes Martha , Gordon Milton P. , Nester Eugene W.
Published: October 2006

Mutagenesis of the vir region on the Ti plasmid of Agrobacterium tumefaciens revealed a new locus, virJ, that is induced by the plant‐wound signal molecule, acetosyringone (AS). virJ lies between virA and virB, and is transcribed in the same direction. The amino acid sequence of virJ is similar to a region of a previously characterized chromosomal gene, acvB, required for virulence. virJ can complement the avirulent phenotype of an acvB mutant, indicating that virJ and acvB encode the same factor required for tumorigenesis. Southern analysis revealed that virJ is present on the Ti plasmid of an octopine but not a nopaline strain whereas acvB is present on the chromosomes of both octopine and nopaline strains. While virJ is regulated by AS under the control of the virA/virG two‐component regulatory system, acvB is not induced by AS. VirJ possesses a putative signal peptide and was found predominantly in the periplasmic fraction. The strain lacking both acvB and virJ had an impaired ability to transfer T‐DNA into plant cells, suggesting that the factor encoded by virJ or acvB is required for T‐DNA transfer from A. tumefaciens to plant cells. acvB is the first chromosomal gene implicated in T‐DNA transfer, but whether it functions specifically for this process is not clear. We hypothesize that virJ evolved from acvB, presumably for a more specialized role in tumorigenesis.
The — 16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli
Authors: Voskuil Martin I. , Voepel Kai , Chambliss Glenn H.
Published: October 2006

The promoter (amyP) of the Bacillus subtilisα‐amylase gene, which is recognized by EτA, has a three out of six match to the consensus promoter in both the −35 and −10 hexamers. Oligonucleotide‐directed mutagenesis was used to identify important bases for promoter utilization in the spacer sequence between the hexamers. Mutations in the sequence TGTG extending from positions −18 to −15 (the −16 region) caused a 5–94‐fold decrease in α‐amylase production. A G‐C transversion at position −15 was the most detrimental mutation: it essentially eliminated amyP utilization in B. subtilis and in Escherichia coli. Mutating the −35 and −10 hexamers to the EτA consensus promoter increased α amylase production 56‐fold in B. subtilis and fivefold in E. coli Introducing the −15 G to C transversion into the consensus promoter reduced α‐amylase production threefold, in contrast to the 94‐fold reduction for the wild‐type promoter in B. subtilis. The −15 G to C transversion did not reduce α‐amylase synthesis directed by the consensus promoter in E. coli. The α amylase gene is subject to two forms of transcriptional regulation: catabolite repression and temporal regulation. None of the mutants constructed in this study had any effect on either type of regulation. The −16 region, especially the G at position −15, appears to be moderately conserved in B. subtilis and in other Gram‐postitive organisms and weakly conserved in E. coli. The evidence suggests that the −16 region is an additional region of EτA promoters in B. subtilis and Eτ70 promoters in E. coli, essential in some weak promoters such as the α‐amylase promoter but, of little benefit to very strong promoters.
Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B
Authors: Von Eichel‐Streiber Christoph , Zu Heringdorf Dagmar Meyer , Habermann Ernst , Sartingen Sabine
Published: October 2006

Strain 1470 is the standard typing strain for serogroup F of Clostridium difficile containing both toxin genes, toxA‐1470 and toxB‐1470. A polymerase chain reaction (PCR)‐based approach to the sequencing of the total toxB‐1470 gene identified an open reading frame (ORF) of 7104 nucleotides. In comparison with the previously sequenced toxB of C. difficile VPI10463, the toxB‐1470 gene has 16 additional nucleotides, 13 within the 5′‐untranslated region and three within the coding region. The Mr of ToxB‐1470 is 269 262, with an isoelectric point (IP) of 4.16. The equivalent values for ToxB are Mr 269 709 and IP 4.13. In comparison with ToxB, ToxB‐1470 differs primarily in the N‐terminal region between positions 1 and 868 where 148 amino acids residues are changed. The C‐terminal region between residues 869–2367 is highly conserved with only six amino acid alterations. Dot matrix comparison of ToxB‐1470 with ToxA and ToxB reveals the highest homology between ToxB‐1470 and ToxB. Thus ToxB‐1470 did not originate from recombination between ToxA and ToxB. On cultured endothelial cells, from porcine pulmonary artery, purified ToxB‐1470 is less potent than ToxB. The cytopathic effects of ToxB‐1470 are indistinguishable from those caused by the lethal toxin (LT) of Clostridium sordellii, but are clearly different from the patterns observed after exposure of endothelial cells to ToxA and ToxB of C. difficile (VPI10463) or α‐toxin (Tcnα) of Clostridium novyi. The LT‐like action of ToxB‐1470 was not due to altered internalization processes, as microinjection and addition to the medium induced identical effects on the cells. Since the differences between ToxB and ToxB‐1470 are clustered within the N‐terminal third of the respective proteins, we conclude that these domains carry the toxic determinants. A three‐domain structure is proposed for the family of large clostridal cytotoxins.
Gradient of genomic diversity in the Pseudomonas aeruginosa chromosome
Authors: Römling Ute , Greipel Joachim , Tümmler Burkhard
Published: October 2006

In 545 Pseudomonas aeruginosa strains, mainly collected from patients with cystic fibrosis, Spel‐Dral macrorestriction fragment lenght diversity was scanned for using probes of known map position on th P.earuginosa PAO chromosome. Southern analysis of the 60 unrelated clones uncovered a gradient of macrorestriction fragment lenght polymorphisms (RFLPs) from the origin of replication towards the auxotroh‐poor region of the P. aeruginosa population in the region encompassed by the rrn operons. The oriC‐reactive Spel fragment was conserved in nearly all isolates examined. Few fragment lenght classes were seen for the alga60‐, algR‐ and toxA‐encoding Spel fragments. Fragment siz varied within one class by up to 20 kb. Two probes from the auxotroph‐poor region detected a broad size range for the Spel fragment, suggestiong extensive genomic deversity in these reions. Subclonalvariation of fragment size was detected at all investigated loci in at least one of the analysed clones, but within one particlular clone, Spel‐RFLPs were found at only few loci.
RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases
Authors: Shuman Stewart , Schwer Beate
Published: October 2006

mRNA capping entails GMP transfer from GTP to a 5′ diphosphate RNA end to form the structure G(5′)ppp(5′)N. A similar reaction involving AMP transfer to the 5′ monophosphate end of DNA or RNA occurs during strand joining by polynucleotide ligases. In both cases, nucleotidyl transfer occurs through a covalent lysyl‐NMP intermediate. Sequence conservation among capping enzymes and ATP‐dependent ligases in the vicinity of the active site lysine (KxDG) and at five other co‐linear motifs suggests a common structural basis for covalent catalysis. Mutational studies support this view. We propose that the cellular and DNA virus capping enzymes and ATP‐dependent ligases constitute a protein superfamily evolved from a common ancestral enzyme. Within this superfamily, the cellular capping enzymes display more extensive similarity to the ligases than they do to the poxvirus capping enzymes. Recent studies suggest that eukaryotic RNA viruses have evolved alternative pathways of cap metabolism catalysed by structurally unrelated enzymes that nonetheless employ a phosphoramidate intermediate. Comparative analysis of these enzymes, particularly at the structural level, should illuminate the shared reaction mechanism while clarifying the basis for nucleotide specificity and end recognition. The capping enzymes merit close attention as potential targets for antiviral therapy.
Evidence for two aromatic amino acid‐binding sites, one ATP‐dependent and the other ATP‐independent, in the Escherichia coli regulatory protein TyrR
Authors: Wilson Timothy J. , Argaet Victor P. , Howlett Geoffrey J. , Davidson Barrie E.
Published: October 2006

In Escherichia coli, genetic regulation of aromatic amino acid biosynthesis and uptake is effected by the protein TyrR, which acts via ligand‐mediated repression and activation. Characterization of the interactions of tyrosine, phenylalanine and tryptophan with TyrR revealed the presence of two separate aromatic amino acid‐binding sites, one ATP‐dependent, the other ATP‐independent. Binding to the ATP‐dependent site induces the self‐association of TyrR. Using sedimentation equilibrium analyses, dissociation constants for this site in the dimeric and hexameric forms of TyrR were determined to be 330 μM and 24 μM, respectively, for tyrosine, and 55 mM and 3.7 mM, respectively, for phenylalanine. Tryptophan bound with a strength similar to that of phenylalanine, and both phenylalanine and tryptophan competed with the binding of tyrosine. The ATP‐independent site, which has not been observed previously, was characterized by ultraviolet (u.v.) difference spectroscopy and a sedimentation‐velocity meniscus‐depletion method. Phenylalanine bound co‐operatively to this site, exhibiting half‐saturation at 260 µM. Tryptophan competed weakly with phenylalanine, half‐saturation occurring at 1.2 mM. No binding of tyrosine to this site could be detected. We propose that the binding of phenylalanine or tryptophan to this ATP‐independent site is responsible for phenylalanine‐ and tryptophan‐mediated regulation by TyrR.
Transcription‐induced deletions in Escherichia coli plasmids
Authors: Vilette Didier , Ehrlich S. Dusko , Michel Bénédicte
Published: October 2006

Characterization of functions that render DNA susceptible to rearrangement is important for a better understanding of genome instability. In a previous work, we showed that sequences located downstream of a strong promoter are particularly prone to deletion. In this paper, the parameters that influence transcription‐induced deletions were studied. pBR322 derivatives carrying the M13 (+) replication origin and a PTac‐dependent transcription region were used. Deletion formation was analysed in the presence of the replication protein of M13, which introduces a nick at the phage replication origin, and in a rep− strain to avoid M13‐driven replication. Our study showed that: (i) 4 h after induction of transcription, a few per cent of the plasmids have experienced a deletion; (ii) these deletions result in joining of the M13 replication origin to a nucleotide located in or downstream of the transcribed region; (iii) deletion formation strongly depends on the orientation of transcription, on promoter strength and transcript length, but is independent of translation; (iv) formation of transcription‐induced supercoiling domains does not induce deletion formation. We propose that deletions in the transcribed region result from collisions between converging replication and transcription machineries.
Salmonella typhimurium responses to a bactericidal protein from human neutrophils
Authors: Qi Shu‐Yun , Li Yan , Szyroki Alexander , Giles Ian G. , Moir Arthur , David O'Connor C.
Published: October 2006

Bactericidal/permeability‐increasing protein [BPI] is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram‐negative bacteria via the lipid A component of lipopolysaccharide. To obtain information about the responses of Salmonella typhimurium to cell‐surface damage by BPI, two‐dimensional gel electrophoresis and N‐terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment. The majority of the affected proteins are involved in central metabolic processes. Upon addition of BPI, the β‐subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11‐fold. Three of the latter were identified as lipoamide dehydrogenase, enoyl‐acyl carrier protein reductase, and the heat‐shock protein HtpG. Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes. A conserved direct‐repeat motif is present in the regulatory regions of several BPI‐inducible genes, including the bipA gene. Only one of the BPI‐responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A. We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S. typhimurium even though they bind with high affinity to the same cell‐surface component.
Detection of induced β‐galactosidase activity in individual non‐culturable cells of pathogenic bacteria by quantitative cytological assay
Authors: Nwoguh C.E. , Harwood C.R. , Barer M.R.
Published: October 2006

One Escherichia coli and two Flac+Salmonella strains were carbon and nitrogen stressed at 37°C over 35 days in the presence or absence of chloramphenicol; the number, activity and culturability of cells in the resultant populations were studied. Active cells were enumerated by fluorescence microscopy after treatment with the lac inducer IPTG and cytological assay for β‐galactosidase. In all experiments, active and total cell counts remained within a three‐fold range of each other and their initial values, while culturability fell by >108‐fold and 103‐fold in chloramphenicol‐treated and untreated preparations, respectively. Quantitative image analysis revealed different distributions of cell‐specific fluorescence and indicated a progressive decline in the levels of induced enzyme activity in both E. coli and Salmonella enteritidis. It was concluded that the non‐culturable cells studied retained inducible enzyme activity and that this activity did not result from a starvation‐induced programme of gene expression. Whether or not such active but non‐culturable cells are viable, they are clearly responsive and have the potential to influence their environment. The assay described can be applied to heterogeneous populations and environments and shows considerable potential for the study of gene expression at the single cell level.
Identification and characterization of a cell envelope protein of Haemophilus influenzae contributing to phase variation in colony opacity and nasopharyngeal colonization
Authors: Weiser Jeffrey N. , Chong Suzanne T.H. , Greenberg Danielle , Fong Winifred
Published: October 2006

Haemophilus influenzae undergoes spontaneous phase variation in colony morphology. Organisms from transparent colonies efficiently colonize the nasopharynx in an infant rat model of H. influenzae carriage, whereas organisms from more opaque colonies are deficient at colonization. A genetic approach relying on the transformability of H. influenzae was used to identify a locus contributing to opacity variation. By screening a library of chomosomal DNA from an opaque variant of strain Rd, it was possible to isolate a single clone capable of transforming a transparent Rd host to a more opaque phenotype. A region containing two genes, designated oapA and oapB, was identified. The deduced amino acid sequence of oapB has similarity to a consensus sequence for bacterial lipoproteins. Genetically defined mutations in oapA were transformed into the transparent Rd to confirm that this gene is required for expression of the transparent colony phenotype. Although oapA lacks a signal sequence, gene fusions to phoA show that OapA is secreted in H. influenzae and undergoes phase variation in expression. Mutagenesis of oapA in strain Rd, and type b strain Eagan, resulted in loss of the ability to colonize the nasopharynx of infant rats. The type b mutant, however, was as virulent as its parent strain when inoculated intraperitoneally. This suggests that the contribution of OapA to pathogenesis is limited to events associated with colonization of the mucosal surface.
Identification of base pairs important for OmpR‐DNA interaction
Authors: Pratt Leslie A. , Silhavy Thomas J.
Published: October 2006

OmpR, the transcriptional regulator of the ompF and ompC porin genes, is a member of a novel class of DNA‐binding proteins. The mechanism(s) by which this class of proteins interacts with target DNA sites is not understood. To address this issue, we investigated the nature of the DNA sequences recognized by OmpR. A 36 bp DNA fragment was identified that is capable of supporting OmpR‐DNA interaction in vivo. The base pairs within this region of DNA that are critical to this interaction were identified by isolating mutations within the fragment that hinder normal OmpR‐DNA binding. The results obtained provide insights concerning the nature of the sequences recognized by OmpR and also support a model in which co‐operative binding is involved in OmpR‐DNA interaction.
Evolution of the chaperonin families (HSP60, HSP 10 and TCP‐1) of proteins and the origin of eukaryotic cells
Authors: Gupta Radhey S.
Published: October 2006

The members of the 10 kDa and 60 kDa heat‐shock chaperonin proteins (Hsp10 and Hsp60 or Cpn10 and Cpn60), which form an operon in bacteria, are present in all eubacteria and eukaryotic ceil organelles such as mitochondria and chloroplasts. In archaebacteria and eukaryotic cell cytosol, no close homologues of Hsp10 or Hsp60 have been identified. However, these species (or ceil compartments) contain the Tcp‐1 family of proteins (distant homologues of Hsp60). Phylogenetic analysis based on global alignments of Hsp60 and Hsp10 sequences presented here provide some evidence regarding the evolution of mitochondria from a member of the α‐subdivision of Gram‐negative bacteria and chloroplasts from cyanobacterial species, respectively. This inference is strengthened by the presence of sequence signatures that are uniquely shared between Hsp60 homologues from α‐purple bacteria and mitochondria on one hand, and the chloroplasts and cyanobacterial hsp60s on the other. Within the α‐purple subdivision, species such as Rickettsia and Ehrlichia, which live intracellularly within eukaryotic cells, are indicated to be the closest relatives of mitochondrial Homologues, In the Hsp60 evolutionary tree, rooted using the Tcp‐1 homologue, the order of branching of the major groups was as follows: Gram‐positive bacteria — cyanobacteria and chloroplasts — chlamydiae and spirochaetes —β and γ‐Gram‐negative purple bacteria —α‐purple bacteria — mitochondria. A similar branching order was observed independently in the Hsp10 tree. Multiple Hsp60 homologues, when present in a group of species, were found to be clustered together in the trees, indicating that they evolved by independent gene‐duplication events. This review also considers in detail the evolutionary relationship between Hsp50 and Tcp‐1 families of proteins based on two different models (viz. archaebacterial and chimeric) for the origin of eukaryotic cell nucleus. Some predictions of the chimeric model are also discussed.
Bacterial transposases and retroviral integrases
Authors: Polard P. , Chandler M.
Published: October 2006

Transposable genetic elements have adopted two major strategies for their displacement from one site to another within and between genomes. One involves passage through an RNA intermediate prior to synthesis of a DNA copy while the other is limited uniquely to DNA intermediates. For both types of element, recombination reactions involved in integration are carried out by element‐specific enzymes. These are called transposases in the case of DNA elements and integrases in the case of the best‐characterized RNA elements, the retroviruses and retrotransposons. In spite of major differences between these two transposition strategies, one step in the process, that of insertion, appears to be chemically identical. Current evidence suggests that the similarities in integration mechanism are reflected in amino acid sequence similarities between the integrases and many transposases. These similarities are particularly marked in a region which is thought to form part of the active site, namely the DDE motif. In the light of these relationships, we attempt here to compare mechanistic aspects of retroviral integration with transposition of DNA elements and to summarize current understanding of the functional organization of integrases and transposases.