bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of fixLJ in the regulation of nitrogen fixation in Azorhizobium caulinodans
Authors: Kaminski P. A. , Elmerich C.
Published: October 2006

A gene bank of Azorhizobium caulinodans DNA constructed in the bacteriophage λGEM11 was screened with Rhizobium meliloti fixL and fixJ genes as probes. One positive recombinant phage, ORSλL, was isolated. The nucleotide sequence of a 3.7 kb fragment was established. Two open reading frames of 1512 bp and 613bp were identified as fixL and fixJ. Kanamycin cartridges were inserted into the cloned fixL and fixJ genes and recombined into the host genome. The resulting mutants were Nif− Fix−, suggesting that the two genes were required for symbiotic nitrogen fixation and for nitrogen fixation in the free‐living state. Using pnifH–lacZ and pnifA–lacZ fusions, it was shown that the FixLJ products controlled the expression of nifH and nifA in bacteria grown in the free‐living state.
Comparison of the class 1 outer membrane proteins of eight serological reference strains of Neisseria meningitidis
Authors: Maiden M. C. J. , Suker J. , McKenna A. J. , Bygraves J. A. , Feavers I. M.
Published: October 2006

Primers suitable for the amplification of the gene encoding the class 1 outer membrane protein of Neisseria meningitidis by the polymerase chain reaction (PCR) were designed from published DNA sequences and used to study the gene in eight meningococcal strains of different serogroup, serotype and subtype. At high annealing stringency one product, shown to correspond to the class 1 protein gene, was amplified from each strain. For three strains an additional smaller product, provisionally identified as the gene encoding the class 3 outer membrane protein, was amplified at lower annealing stringencies. Nucleotide sequence analysis of the PCR products corresponding to the class 1 proteins established the differences in the primary structure of the proteins between each of the subtypes and other outer‐membrane proteins from Neisseria spp. These differences impose constraints on possible structural models of these proteins. Most amino acid sequence variation occurred in two domains of between 8 and 17 amino acids; there was an additional region which varied mainly between classes of outer membrane protein and there were nine conserved regions. Using appropriate primers it was possible to distinguish between class 1 outer membrane protein genes from strains of different subtypes by the PCR.
Local DNA topology and gene expression: the case of the leu‐500 promoter
Authors: Lilley D. M. J. , Higgins C. F.
Published: October 2006

Many promoters are sensitive to DNA supercoiling, and it is becoming apparent that this may play an important rote in gene regulation. The twin super ‐coiled‐domain hypothesis (Liu and Wang, 1987) proposes that transcription can lead to local variation in supercoiling. The mutant leu‐500 promoter has presented a long‐standing problem to the understanding of the control of promoter function by DNA supercoiling. This promoter is activated by mutations in the gene encoding topoisomerase I, but is apparently unaffected by mutations in the genes encoding DNA gyrase. We propose a model to explain the anomalous regulation of this promoter, based on the twin super‐coiled‐domain model. This allows us to account for the unusual properties of the leu‐500 promoter, and confirms the biological importance of the twin super‐coiled‐domain model. We suggest that such topological coupling between promoters may be general, leading to co‐operativity and anti‐co‐operativity between divergent promoter pairs.
A model for asymmetric septum formation during sporulation in Bacillus subtilis
Authors: Errington J.
Published: October 2006

Many differentiation processes in both prokaryotes and eukaryotes begin with an asymmetric division, producing ‘daughter’ cells that differ in size and developmental fate. This is particularly obvious in the well‐studied prokaryotic lif cycles of Caulobacter and Bacillus. In no system, however, is the mechanism of asymmentric division understood. Here I propose a model for the mechanism o asymmetric division during sporulation in Bacillus subtilis. The model explains bth the timing and asymmetric localization of spore‐septum formation. It also explains the morphological phenotypes of various asporogenous (spo) mutants.
Characterization of the RNA polymerases of Crithidia fascicuiata
Authors: Köck J. , Cornelissen A. W. C. A.
Published: October 2006

We have characterized the crithidial RNA polymerases (RNAPs). The RNAPs from this organism were resolved by chromatography and could be classified into RNAP I, II and III because of their differential sensitivity to class‐specific RNA polymerase inhibitors, such as alpha‐amanitin (ama) and tagetitoxin. The three RNAP classes were subsequently characterized in nuclear run‐ons using class‐specific DNA templates and the inhibitory effect of ama on RNA chain elongation. These experiments showed that Crithidia fasciculata contains the normal set of eukaryotic RNAPs. However, RNAP II was found to be relatively resistant to ama, which seems to be a general feature of kinetoplastid parasites. Tagetitoxin, a potent inhibitor of purified crithidial RNAP III, does not inhibit RNAP lit in nuclear run‐on experiments, suggesting that the inhibitory effect of tagetitoxin depends on the enzyme conformation, i.e.‘naked’enzyme versus that in a transcription complex. Finally, the role of RNAP Ml in the transcription of the mini‐exon genes, which provide the 5′ end of each mRNA, is discussed.
Two distinct steps in pullulanase secretion by Escherichia coli K12
Authors: Pugsley A. P. , Poquet I. , Kornacker M. G.
Published: October 2006

Two distinct steps in the secretion of the extracellular, cell‐surface‐anchored lipoprotein pullulanase by Escherichia coli were uncoupled by allowing export of the enzyme to the cytoplasmic membrane via the signal peptide sec‐gene‐dependent general export pathway, and then inducing the pulC—0 operon of genes required for translocation to the cell surface. The secretion intermediate cofractionated mainly with intermediate‐density vesicles when cells were gently lysed and the resulting vesicles were separated by isopycnic sucrose density centrifugation. Cytoplasmic forms of pullulanase (which are not exported because they lack a functional signal peptide) are more sensitive to heat inactivation, denaturation by sodium dodecyl sulphate and carboxymethylation than the intermediate and cell‐surface forms. The latter are distinguished only by the fact that the secretion intermediate is less susceptible to proteinase K and trypsin, and is partially inaccessible to substrate or in an inactive conformation in sphaeroplasts. These and other results indicate that the secretion intermediate can acquire considerable higher‐ordered structure, including disulphide bridges, before it is transported to the cell surface; this seems to rule out the possibility that it is threaded through this membrane as a locally unfolded polypeptide.
A novel function of the cAMP‐CRP complex in Escherichia coli: cAMP‐CRP functions as an adaptor for the CytR repressor in the deo operon
Authors: Søgaard‐Andersen L. , Pedersen H. , Hoist B. , Valentin‐Hansen P.
Published: October 2006

Unlike classical bacterial repressors, the CytR repressor of Escherichia coli cannot independently regulate gene expression. Here we show that CytR binding to the deoP2 promoter relies on interaction with the master gene regulatory protein, CRP, and, furthermore, that cAMP‐CRP and CytR bind co‐operatively to deoP2. Using mutant promoters we show that tandem, properly spaced DNA‐bound cAMP‐CRP complexes are required for this co‐operative binding. These data suggest that CytR forms a bridge between tandem cAMP‐CRP complexes, and that cAMP‐CRP ftjnctions as an adaptor for CytR. The implications of this new version of negative control in E. coli on bacterial gene expression and on combinatorial gene regulation in higher organisms are discussed.
Construction and characterization of Bordetella pertussis mutants lacking the vir‐regulated P.69 outer membrane protein
Authors: Roberts M. , Fairweather N. F. , Leininger E. , Pickard D. , Hewlett E. L. , Robinson A. , Hayward C. , Dougan G. , Charles L. G.
Published: October 2006

The Bordetella pertussis P.69 protein is an immunogen with vaccine potential. The role of this protein in pathogenesis is unclear; it has been associated with the toxic adenylate cyclase and adhesion to eukaryotic cells. For further analysis of the role of P.69 in the biology of B. pertussis, we have constructed strains which specifically lack P.69.

The cloned P.69 (Prn) gene of S. pertussis was insertionally inactivated with a kanamycin‐resistance cassette. This inactivated gene was used to construct P.69− mutants of B. pertussis by allelic exchange using plasmid pRTP1.

B. pertussis P.69 strains produced normal levels of other vir‐regulated factors, including adenylate cyclase. The serotype of B. pertussis, determined by Eldering and Preston typing sera and monoclonal antibodies, was also unaffected by the presence or absence of P.69.

The ability of a prn mutant to adhere to and invade HEp2 cells was not significantly different from that of its parent strain. A strain containing a mutation in fhaB was significantly less adhesive and invasive than its parent, and a prn fhaB double mutant exhibited an even greater reduction in adhesiveness and invasiveness down to levels comparable with a Vir− strain.
Organization of the genes coding for the reaction‐centre L and M subunits and B870 antenna polypeptides α and β from the aerobic photosynthetic bacterium Erythrobacter species OCH114
Authors: Liebetanz R. , Hornberger U. , Drews G.
Published: October 2006

In the aerobic photosynthetic bacterium Erythrobacter species OCH114 the structural genes coding for the light‐harvesting (LH) complex B870 and the reaction‐centre (RC) polypeptides (the gene products of the pufB, pufA, pufL and pufM genes) are mapped on a 2.728kbp Eco RI fragment. Sequencing of this fragment revealed that the deduced amino acid sequences contain 50 (B870 β), 52 (B850 α), 283 (RCL) and 331 (RCM) residues with the corresponding molecular weights of 5592, 5814, 31364, and 37671, respectively. In the corresponding mRNA a ‘hairpin’ structure (ΔG°= ‐26.6 kcal) is predicted to be located immediately downstream of pufA. The RC and LH polypeptides are highly homologous to those of the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodo‐bacter sphaeroides and Rhodopseudomonas viridis. Directly downstream of pufM there is an open reading frame (ORF) of unknown size. Partial sequencing indicates that this ORF is highly homologous to the cytochrome subunit of the photosynthetic reaction centre from R. viridis. In the puf operon no pufQ or pufX genes could be found, but the bchA gene is located upstream of that operon. Plasmid pESS8.9 containing the 2.728kbp Eco RI fragment reconstituted a photo‐inactive mutant of Erythrobacter species OCH114. Comparative analysis of the DNA region upstream of the puf operon and of bacteriochlorophyll (Bchl) synthesis indicated that Bchl synthesis and puf gene expression are regulated differently in Erythrobacter and purple bacteria, respectively.
Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli
Authors: Zimmermann A. , Reimmann C. , Galimand M. , Haas D.
Published: October 2006

Anaerobic growth of Pseudomonas aeruginosa on nitrate or arginine requires the anr gene, which codes for a positive control element (ANR) capable of functionally complementing an fnr mutation in Escherichia coli. The anr gene was sequenced; it showed 51% identity with the fnr gene at the amino acid sequence level. Four cysteine residues known to be essential in the FNR protein are conserved in ANR. The anr gene product (deduced Mr 27129) was visualized by the maxicell method and migrated like a 32kDa protein in gel electrophoresis under denaturing conditions. An anr mutant of P. aeruginosa constructed by gene replacement was defective in nitrate respiration, arginine deiminase activity, and hydrogen cyanide biosynthesis, underscoring the diverse metabolic functions of ANR during oxygen limitation. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all had a functional analogue of ANR, indicating that similar anaerobic control mechanisms exist in these bacteria.
The Rhizobium meliloti exoZl exoB fragment of megaplasmid 2: ExoB functions as a UDP‐glucose 4‐epimerase and ExoZ shows homology to NodX of Rhizobium leguminosarum biovar viciae strain TOM
Authors: Buendia A. M. , Enenkel B. , Köplin R. , Niehaus K. , Arnold W. , Pünier A.
Published: October 2006

A 2.6kb Clal‐Bam HI DNA fragment of megaplasmid 2 of Rhizobium meliloti 2011 was found to carry genes involved in exopolysaccharide synthesis and infection of alfalfa nodules. The analysis of the nucleotide sequence of this DNA fragment revealed the existence of two open reading frames (ORFs) running in opposite directions. Plasmid integration mutagenesis showed that these ORFs are organized as two monocistronic transcription units. One of the ORFs represents a new exo gene designated exoZ, which is involved in, but not essential for, the production of acidic exopolysaccharide. However, exoZ is not necessary for nodule formation with alfalfa. The ExoZ protein was found to show homology (23.3%) to the NodX protein of the R. leguminosarum biovar viciae strain TOM, known to be essential for nodulating the primitive Afghanistan pea. The second identified ORF corresponds to the exoB locus. The deduced amino acid sequence of the ExoB protein is homologous (39.6%) to that of the Escherichia coli GalE protein. In R meliloti, exoB codes for a UDP‐glucose 4‐epimerase. A deficiency in the activity of this enzyme fully accounts for all the multiple carbohydrate defects that have been observed in exoB mutants.
Structure of two retrons of Escherichia coli and their common chromosomal insertion site
Authors: Lim D.
Published: October 2006

It has been shown that certain strains of myxobacteria and of Escherichia coli have a genetic element encoding a reverse transcriptase (RT). This element, called a ‘retron’, produces a covalently linked RNA–DNA compound (msDNA–RNA). Here, I report the complete nucleotide sequence of retron EC‐86, the retron in E. coli B, together with its flanking regions. Retron EC‐86 contains genes for msDNA–RNA (msd, and msr), a gene for RT (ret) and a gene for an open reading frame whose function is unknown. The upstream junction is composed of the sequence GCGCGCGC, but there are no direct or inverted repeats at the retron–host junctions. It is also shown that another retron of E. coli, EC‐67, which was isolated originally from the clinical strain CL1 and was later found to be present also in a clinical E. coli isolate from Brazil, is inserted at the same chromosomal site as retron EC‐66. Retron EC‐67 contains only msd, msr, and ret. I suggest that these two retrons were independently inserted into the same site of their host strains via a novel mechanism of integration.
Complement activation and antibody binding by pneumolysin via a region of the toxin homologous to a human acute‐phase protein
Authors: Mitchell T. J. , Andrew P. W. , Saunders F. K. , Smith A. N. , Boulnois G. J.
Published: October 2006

Pneumolysin, a membrane‐damaging toxin, is known to activate the classical complement pathway. We have shown that 1 μg ml−1 of pneumolysin can activate complement, which is a much lower level than observed previously. We have identified two distinct regions of pneumolysin which show homology with a contiguous sequence within acute‐phase proteins, including human C‐reactive protein (CRP). Site‐directed mutagenesis of the pneumolysin gene was used to change residues common to pneumolysin and CRP. Some of the modified toxins had a reduced ability both to activate complement and bind antibody. We suggest that the ability of pneumolysin to activate complement is related to its ability to bind the Fc portion of immunoglobulin G.
Characterization of a Legionella pneumophila gene encoding a lipoprotein antigen
Authors: Engleberg N. C. , Howe D. C. , Rogers J. E. , Arroyo J. , Eisenstein B. I.
Published: October 2006

A prominent 19kDa surface antigen of Legionella pneumophila, cloned in Escherichia coli, was found to be intimately associated with peptidoglycan. The DNA region encoding this antigen was mapped on an 11.9kb plasmid by means of deletion analysis and transposon mutagenesis. PhoA+ gene fusions, generated by TnphoA insertions into this region, confirmed the presence of a gene encoding a secreted protein. PhoA+ transposon insertions were also associated with loss of the 19 kDa antigen in immunoassay s using a monoclonal antibody (mAb1E9) and the replacement of the 19kDa antigen with larger fusion proteins in immunoblots using Legionella immune serum. A 1540bp PstI fragment carrying the gene was sequenced, and the open reading frame encoding the antigen was identified. The gene encodes a polypeptide 176 amino acid residues long and 18913Da in size. The presence of a signal sequence of 22 amino acids with a consensus sequence for cleavage by signal peptidase II indicates that the antigen is a lipoprotein, and striking similarity with peptidoglycan‐associated lipoproteins (PALs) from E. coli (51% amino acid homology) and Haemophilus influenzae (55% homology) is noted. We conclude that the 19kDa antigen of L. pneumophila is the structural equivalent of the PAL found in other Gram‐negative species and suggest that its post‐translational acylation may explain its potency as an immunogen.
Evolved neomycin phosphotransferase from an isolate of Klebsiella pneumoniae
Authors: Lee K.‐Y. , Hopkins J. D. , Syvanen M.
Published: October 2006

A new aminoglycoside resistance gene (aphA 1‐IA6) confers high‐level resistance to neomycin. The sequence of apA 1‐IAB is closely related to aphA 1 found in the transposons Tn4352, 7n903 and Tn602. For example, aphA 1‐IAB differs from aphA 1–903 at five nucleotides that result in four amino acid replacements. The enzyme encoded by aphA 1‐IAB has a significantly higher turnover number with neomycin, kanamycin and G418 as substrates than does the aphA 1–903 enzyme. A parsimonious phylogenetic tree suggests that aphA 1‐IAB evolved from an ancestral form that is closely related or identical to the aphA 1 found in Tn903. The excess of replacement substitutions over silent substitutions in aphA I‐IAB, as well as its convergence toward aphA 3 from Staphylococcus aureus, is indicative of selective evolution. Our hypothesis to explain these results is that aphA 1‐IAB evolved under the selective pressure of neomycin use in relatively recent times.
Repression and induction of the nag regulon of Escherichia coll K‐12: the roles of nagC and nagA in maintenance of the uninduced state
Authors: Plumbridge J. A.
Published: October 2006

The nag regulon located at 15.5 min on the Escherichia coli chromosome consists of two divergent operons, nagE and nagBACD, encoding genes involved in the uptake and metabolism of N‐acetylglucosamine. Null mutations have been created in each of the genes by insertion of antibiotic resistance cartridges. The phenotypes of the strains carrying the insertions in nagE, B and A were consistent with the previous identification of gene products: nagE, EIINag, the N‐acetylglucosamine specific transporter of the phosphotransferase system and nagB and nagA, the two enzymes necessary for the degradation of N‐acetylglucosamine. Insertions in the napC result in derepression of the nag genes, which is consistent with earlier observations that the nagC gene encodes the repressor of the regulon. Insertions in nagA also provoke a derepression, implying that nagA has a role in the regulation of the expression of the nag regulon as well as in the degradation of the amino‐sugars. N‐acetylglucosamine‐6‐phosphate, the intra‐cellular product of N‐acetylglucosamine transport and the substrate of the nagA gene product, is shown to be an inducer of the regulon and this suggests how nagA mutations result in derepression: the absence of N‐acetylglucosamine‐6‐phosphate deacetylase allows N‐acetylglucosamine‐6‐phosphate to accumulate and induce the regulon.
Partial deletion of the Rhizobium phaseoli CFN23 symbiotic plasmid implies a concomitant amplification of plasmid DNA sequences
Authors: Soberón‐Chávez G. , Nájera R. , Calva E. , Fernández M.
Published: October 2006

Rhizobium leguminosarum biovar phaseol CFN23 loses its ability to nodulate beans at a high frequency because of a deletion of part of its symbiotic (pSym) plasmid (Soberón‐Chávez et al., 1986). We report here that at least 80kb of pSym are deleted upon loss of the symbiotic phenotype; the deletion removes the nitrogenase structural nifHDK and the common nod ABC genes. The size of the deleted pSym is not reduced, since it is accompanied by an amplification of other pSym plasmid sequences. This genetic rearrangement is similar to the deletion and amplification of yeast mitochondrial DNA leading to‘petite’mutations.
Complementation of DNA repair‐deficient Escherichia coli by the yeast Apn1 apurinic/apyrimidinic endonuclease gene
Authors: Ramotar D. , Popoff S. C. , Demple B.
Published: October 2006

The Saccharomyces cerevisiae APN1 gene encoding an AP endonuclease/3′ ‐diesterase was engineered in vitro for expression in Escherichia coli. The expression vector directs the synthesis in E. coli of a Mr 40500 protein that reacts with anti‐Apn1 antibodies and has the DNA‐repair activities characteristic of Apn1 isolated from yeast. A band corresponding to Apn1 was observed in DNA repair activity gels only with extracts of E. coli harbouring the APN1 expression plasmid. Expression of Apn1 conferred resistance to oxidants and alkylating agents in E. coli lacking exonuclease III and endonuclease IV. For H2O2 damage, this rescue effect was correlated with the repair of oxidative lesions in the bacterial chromosome by the Apn1 protein. Thus, Apn1 can function in bacteria in a manner similar to its proposed multiple functions in yeast.
Evidence that the entire length of a kinetoplast DNA minicircle is transcribed in Trypanosoma cruzi
Authors: Thertulien R. , Harth G. , Haidaris C. G.
Published: October 2006

A 1.3kb cDNA (cDNA52) was derived from Trypanosoma cruzi trypomastigote mRNA. Using single stranded probes in Northern blots, we identified the putative coding strand of cDNA52. In addition, a minor band was detected in RNA from epimastigotes that was absent in RNA from trypomastigotes. Nucleotide sequence analysis revealed that cDNA52 was highly homologous to T. cruzi kinetoplast DNA minicircle sequences. All four conserved regions of T. cruzi minicircles were identified in cDNA52. Using several criteria, we demonstrated that the hybridization signals were not caused by contaminating minicircle DNA in the RNA preparations. The data provide direct evidence for the unprecedented finding that the entire length of a kDNA minicircle is transcribed in T. cruzi.
Expression of the gene encoding glycerol‐3‐phosphate dehydrogenase (glpD) in Bacillus subtilis is controlled by antitermination
Authors: Holmberg C. , Rutberg B.
Published: October 2006

The Bacillus subtilis glpD gene encodes glycerol‐3‐phosphate (G3P) dehydrogenase. A sigma A type promoter and the transcriptional startpoint for glpD were identified. Between the transcriptional start‐point and glpD there is an inverted repeat followed by a run of T residues. The inverted repeat prevents expression of a reporter gene, xylE, when positioned between this gene and a constitutive promoter. Expression of xylE, like expression of glpD, is induced by G3P and repressed by glucose. Induction also requires the product of the glpP gene. Our results suggest that glpD expression is controlled by antitermination of transcription. The inverted repeat appears to be a target for induction by G3P and GlpP. We speculate that glucose repression is mediated via an inhibitory effect on synthesis or activity of GlpP.
Prevalence of gene sequences coding for hypervariable regions of Opa (protein II) in Neisseria gonorrhoeae
Authors: Brooks G. F. , Olinger L. , Lammel C. J. , Bhat K. S. , Calvello C. A. , Palmer M. L. , Knapp J. S. , Stephens R. S.
Published: October 2006

Opas (protein IIs) are a family of surface‐exposed proteins of Neisseria gonorrhoeae. Each strain of N. gonorrhoeae has multiple (10–11) genes encoding for Opas. Identifiable elements in opa genes include the coding repeat within the signal sequence, conserved 5′ and 3′ regions, and hypervariable regions (HV1 and HV2) located within the structural gene. N. gonorrhoeae strains appear to have many biological properties in common that are either HV‐region‐mediated or associated with the presence of specific HV regions, suggesting that HV regions could be found in many clinical isolates. Oligonucleotides from three source strains representing three conserved regions of opa, 12 HV1 regions, and 14 HV2 regions were used by dot blot analysis to probe 120 clinicial isolates of N. gonorrhoeae. The probe for the coding repeat hybridized to all 120 strains, the 3′ conserved‐region probe reacted with 98% of the strains, and the 5′ conserved‐region probe with 90% of the strains. Nine HV1 probes hybridized to 3.3–39.2% of the strains, and 13 of the HV2 probes hybridized to 1.7–25% of the isolates. Analysis of the number of probes that hybridized to each of the isolates showed that 19% did not hybridize with any of the HV1 probes and 25% did not hybridize with any of the HV2 probes. Approximately three‐quarters of the isolates hybridized with one, two or three of the HV1 probes or one, two or three of the HV2 probes; 89% of the isolates hybridized to least one HV1 or oe HV2 probe. The data indicate that some genes encoding HV regions of N. gonorhoeae Opa proteins are widely distributed in nature.
A class of gyrB mutants, substantially unaffected in DNA topology, suppresses the Escherichia coli K12 fts Z84 mutation
Authors: Ruberti I. , Crescenzi F. , Paolozzi L. , Ghelardini P.
Published: October 2006

Previous work in our laboratory suggested that DNA topology could be implicated in the regulation of the division gene ftsZ. To settle this question, we have selected and characterized mutants in the gyrB gene able to phenotypically suppress the defects of the ftsZ84 mutation. No strict correlation was found between the degree of plasmid DNA relaxation and the level of suppression of the thermosensitivity of the ftsZ84 strain. Interestingly, the class of mutants that shows maximal suppression is substantially unaffected in DNA topology. In addition, the amount of ffsZ‐specific mRNA in this class of mutants is comparable to that present in the ftsZ84 strain. These results hint that the ability of these gyrB mutants to correct the effects of the ftsZ84 mutation is largely unrelated to the function of the GyrB (as a part of DNA gyrase) in the control of DNA superhelicity and suggest hitherto unsuspected interaction between the ftsZ and gyrB gene products.
Analysis of the gluconate (gnt) operon of Bacillus subtilis
Authors: Reizer A. , Deutscher J. , Saier M. H. , Reizer J.
Published: October 2006

The gluconate (gnt) operon of Bacillus subtilis includes the gntR, gntK, gntP, and gntZ genes, respectively encoding the transcriptional repressor of the operon, gluconate kinase, the gluconate permease, and an unidentified open reading frame (Fujita and Fujita, 1987). We have compared the proteins encoded by the gnt operon of B. subtilis with published sequences and showed that (i) the gluconate repressor is homologous to several putative regulatory proteins in Escherichia coli, (ii) the gluconate kinase of B. subtilis is homologous to xylulose kinase, glycerol kinase and fucose kinase in E. coli (20‐26% identity; 12‐59 S.D.), (iii) the gluconate permease exhibits a C‐terminal domain which is homologous to a hydrophobic protein encoded by an unidentified open reading frame (dsdAp) which precedes the dsdA gene of E. coli (39% identity; 19 S.D.), and (iv) the gntZ gene product is homologous to 6‐phosphogluconate dehydrogenases of other bacteria and of animals (48‐56%; 82‐178 S.D.), thereby suggesting that the B. subtilis gntZ encodes 6‐phosphogluconate dehydro‐genase. Several conserved regions of the sequenced 6‐phosphogluconate dehydrogenases can serve as signature patterns of this protein. Computer analyses have indicated that the previously reported sequences of the porcine and ovine 6‐phosphogluconate dehydrogenases, as well as the hypothetical DsdAp protein, are probably erroneous. The probable reasons for the errors are reported along with the proposed revised sequences.
Identification and characterization of a conserved outer‐membrane protein of Neisseria gonorrhoeae
Authors: Judd R. C. , Strange J. C. , Pettit R. K. , Shafer W. M.
Published: October 2006

A previous study in our laboratory identified a surface‐exposed peptidoglycan‐associated protein of Neisseria gonorrhoeae which had an apparent molecular mass of 44000 daltons (44kDa) (Hill and Judd, 1989). This paper reports results which confirm that the 44 kDa protein is surface‐exposed, and that the protein is expressed in, and is structurally invariant among, 14 strains of N. gonorrhoeae. The fact that the 44kDa outer‐membrane protein is found in a conserved form in all gonococci examined strongly suggests that ft is crucial to the bacterium's survival. Moreover, it appears that this protein is a penicillin‐binding protein (PBP3) (Shafer and Judd, 1991). This invariant, surface‐exposed, peptidoglycan‐associated outer‐membrane protein deserves further investigation to elucidate its role in the immunobiology of N. gonorrhoeae, and its possible use as an immunoprophylactic reagent.
The cephamycin biosynthetic genes pcbAB, encoding a large multidomain peptide synthetase, and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in Acremonium chrysogenum and Penicillium chrysogenum
Authors: Coque J. J. R. , Martin J. F. , Calzada J. G. , Liras P.
Published: October 2006

A 34 kb fragment of the Nocardia lactamdurans DNA carrying the cluster of early cephamycin biosynthetic genes was cloned in λ EMBL3 by hybridization with probes internal to the pcbAB and pcbC genes of Penicillium chrysogenum and Streptomyces griseus. The pcbAB and pcbC genes were found to be closely linked together in the genome of N. lactamdurans. The pcbAB gene of N. lactamdurans showed the same orientation as the pcbC gene, in contrast to the divergent expression of the genes in the pcbAB‐pcbC cluster of P. chrysogenum and Acremonium chrysogenum. The pcbAB gene encodes a large (3649 amino acids) multidomain δ‐(L‐α‐aminoadipyl)‐L‐cysteinyl‐D‐valine synthetase with a deduced Mr, of 404134. This enzyme contains three repeated domains and a consensus thioesterase active‐site sequence. The pcbC gene encodes a protein of 328 amino acids with a deduced Mr of 37469, which is similar to other isopenicillin N synthases except that it lacks one of two cysteine residues conserved in all other isopenicillin N synthases. The different organization of the pcbAB‐pcbC gene cluster in N. lactamadurans and Streptomyces clavuligerus relative to P. chrysogenum and A. chrysogenum is intriguing in relation to the hypothesis of horizontal transference of these genes from actinomycetes to filamentous fungi by a single transfer event.
Nucleotide sequence of the endoglucanase C gene (cenC) of Cellulomonas fimi, its high‐level expression in Escherichia coli, and characterization of its products
Authors: Coutinho J. B. , Moser B. , Kilburn D. G. , Warren R. A. J. , Miller R. C.
Published: October 2006

The cenC gene of Cellulomonas fimi, encoding endoglucanase CenC, has an open reading frame of 1101 codons closely followed by a 9bp inverted repeat. The predicted amino acid sequence of mature CenC, which is 1069 amino acids long, is very unusual in that it has a 150‐amino‐acid tandem repeat at the N‐terminus and an unrelated 100‐amino‐acid tandem repeat at the C‐terminus. CenC belongs to subfamily E1 of the β‐1,4‐glycanases. High‐level expression in Escherichia coli of cenC from a 3.6kbp fragment of C. fimi DNA leads to levels of CenC which exceed 10% of total cell protein. Most of the CenC is in the cytoplasm in an inactive form. About 60% of the active fraction of CenC is in the periplasm. The catalytic properties of the active CenC are indistinguishable from those of native CenC from C. fimi. The Mr of CenC from E. coli and C. fimi is approximately 130 kDa. E. coli and C. fimi also produce an endoglucanase, CenC', of approximate Mr, 120kDa and with the same N‐terminal amino acid sequence and catalytic properties as CenC. CenC’appears to be a proteolytic product of CenC. CenC and CenC’can bind to cellulose and to Sephadex. CenC is the most active component of the C. fimi cellulase system isolated to date.
Topographic analysis of the toxic Gef protein from Escherichia coli
Authors: Poulsen L. K. , Refn A. , Molin S. , Andersson P.
Published: October 2006

The chromosomal gef gene of Escherichia coli is a member of the gef gene family which encodes strongly toxic proteins of about 50 amino acids. We demonstrate here that the Gef protein is detectable by anti‐peptide antibodies. Furthermore, we show that Gef is anchored in the cytoplasmic membrane by the N‐terminal part of the protein, and that the C‐terminal part is localized in the periplasm in a dimeric form with at least one disulphide bond. By mutagenesis of gef it is shown that the periplasmic portion of Gef encodes the toxic domain and that the dimerization of Gef is not essential for the toxic effect.
Possible role of calmodulin in the secretion of Entamoeba histolytica electron‐dense granules containing collagenase
Authors: Muñoz M. de L. , Moreno M. A. , Pérez‐Garcia J. N. , Tovar G. R. , Hernandez V. I.
Published: October 2006

Entamoeba histolytica cells secrete electron‐dense granules (EDGs) that have collagenase activity. To study the possible involvement of calmodulin (CaM) on EDG secretion, the effect of several CaM antagonists (TFP, R24571, W‐7, W‐5, dibucaine and DL‐propranolol) was tested on this cellular function. Except for W‐5 and dibucaine, the rest of these compounds inhibited EDG secretion. Transmission electron microscopy of collagen‐activated trophozoites showed numerous EDGs located in or near the surface membrane. In contrast, trophozoites incubated with TFP showed no EDGs. Protein kinase C inhibitors (H‐7, ML‐9) had no effect on EDG secretion, suggesting that CaM antagonists acted by selectively inhibiting CaM. These results suggest that a CaM‐dependent process is involved in EDG secretion.
Expression of the fibronectin‐binding components of Streptococcus pyogenes in Escherichia coli demonstrates that they are proteins
Authors: Talay S. R. , Ehrenfeld E. , Chhatwal G. S. , Timmis K. N.
Published: October 2006

The fibronectin‐binding components (fbcs) of two clinical isolates and a culture collection strain of Streptococcus pyogenes have been analysed. Western immunoblotting of bacterial lysates which had been fractionated on polyacrylamide gels revealed trypsin‐sensitive fibronectin‐binding species. The genes specifying the fbcs were cloned from all three strains and expressed in Escherichia coli using a λ EMBL3 vector. An fbc gene from the culture collection strain was subcloned and expressed in the E. coli expression vector pJLA601, and subjected to deletion analysis. The fibronectin‐binding domain was thereby localized within a 40 kDa truncated peptide encoded by the 1000 bp C‐terminal region of the gene. Southern hybridization experiments demonstrated that the analysed gene was present in the parental S. pyogenes chromosome, but not in the DNA of fbc expressing λ clones obtained from the two clinical isolates. Further evidence for the existence of at least two different types of fbcs in group A streptococci was provided by Western blot analysis of recombinant phage tysates which revealed a complex series of fibronectin‐binding species ranging from 120 to 200 kDa in size and showing strain‐dependent variation in their patterns. As was the case with parental streptococcal strains all of the recombinant fbcs were protease‐sensitive, and treatment with trypsin or pronase resulted in a total loss of fibronectin‐binding activity. Competitive inhibition experiments indicated that lipoteichoic acid was not a significant fbc in the tested streptococcal strains. The biological separation of the principal fbcs of S. pyogenes from lipoteichoic acid, through the cloning and expression of their determinants in the non‐lipoteichoic acid‐producing organism E. coli K‐12, provides unequivocal proof of the protein nature of these components.
Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits
Authors: Calder K. M. , McEwen J. E.
Published: October 2006

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while sub‐units IV, V, VI, VII, Vila and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase sub‐units in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mito‐chondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse‐chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear‐encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial‐encoded subunits I, II and HI with the nuclear‐encoded subunit IV was investigated. Relative to the parental COX7+ strain, the cox7 mutant strain showed no association of sub‐unit IV with subunit I, and decreased association of subunits IV and II. These results provide direct evidence for a role for subunit VII in assembly between mitochondrial‐encoded and nuclear‐encoded cytochrome c oxidase subunits.
Secretion of a potential virulence factor, a fungal ribonucleotoxin, during human aspergillosis infections
Authors: Lamy B. , Moutaouakil M. , Latge J.‐P. , Davies J.
Published: October 2006

We show by cloning and nucleotide sequence analysis that the 18kDa antigen found in the urine of patients suffering from aspergillosis is related to the fungal protein toxins restrictocin and mitogillin. These are inhibitors of translation which act by catalytic inactivation of eukaryotic ribosomes; they may be implicated in the pathogenesis of the disease.
Tn7: a target site‐specific transposon
Authors: Craig N. L.
Published: October 2006

The bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low‐frequency and display little target site‐selectivity. By contrast, Tn7 inserts at high‐frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coil where Tn7 has been most extensively studied, Tn7 transposes at low‐frequency and inserts into many different sites.

Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7‐encoded proteins, probably host‐encoded proteins and also rather large cis‐acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.
Structure and function of pneumolysin, the multifunctional, thiol‐activated toxin of Streptococcus pneumoniae
Authors: Boulnois G. J. , Paton J. C. , Mitchell T. J. , Andrew P. W.
Published: October 2006

Pneumolysin is a thiol‐activated membrane‐damaging, multifunctional toxin and a known virulence factor of Streptococcus penumoniae. The toxin can interfere with the functioning of both cellular and soluble components of the human immune system which protects against penumococcal infection. Different amino acids within the toxin which are important in promoting oligomerization of the toxin in membranes and for the generation of functional lesions have been identified by site‐directed mutagenesis. Pneumolysin can also activate the classical pathway of complement, and this appears to involve antibody binding (via Fc) by a region of the toxin homologous to C‐reactive protein, a human acute‐phase protein also capable of classical pathway activation and implicated in host defence against pneumococcal infection.
Microbial pathogenesis and tyrosine dephosphorylation: surprising‘bedfellows'
Authors: Clemens J. C. , Guan K. , Bliska J. B. , Falkow S. , Dixon J. E.
Published: October 2006

The importance of parasite‐directed phosphatases in such diseases as smallpox and the bubonic plague emphasizes the need to understand the molecular events associated with normal function of protein tyrosine phosphatase in eukaryotic cells.
The kis and kid genes of the parD maintenance system of plasmid R1 form an operon that is autoregulated at the level of transcription by the co‐ordinated action of the Kis and Kid proteins
Authors: Ruiz‐Echevarria. M. J. , Berzal‐Herranz A. , Gerdes K. , Diaz‐Orejas R.
Published: October 2006

Stability mediated by the parD system of plasmid R1 is modulated by a killer protein, Kid, and by an antagonist of this function, Kis. Determination of the 5′ ends of ParD transcripts, revealed that the genes coding for these proteins are transcribed from a single promoter. Analysis of the 3′ end of the ParD RNAs indicated the existence of two transcripts: one of them coding for the Kis and Kid proteins, and the other coding only for Kis. Analysis of the effects of parD+ and kis‐ recombinants on the β‐galactosidase levels expressed by different transcriptional and translational parD‐lacZfusions, and on the ParD RNA levels determined by a derepressed parD mutant, indicated that the Kis and Kid proteins repress co‐ordinately the parD system at the transcriptional level. We discuss the relevance of these results in terms of the activities of the Kis and Kid proteins and in the context of the stabilization mediated by parD.
An Agrobacterium two‐component regulatory system for the detection of chemicals released from plant wounds
Authors: Winans S. C.
Published: October 2006

Crown gall tumorigenesis by Agrobacterium tumefaciens requires the co‐ordinate transcriptional induction of a set of pathogenesis genes. At least three classes of environmental stimuli act synergistically to induce these genes, (i) monocyclic aromatic hydrocarbons such as acetosyringone, coniferyl alcohol, and vanillin, (ii) neutral or acidic monosaccharides such as glucose and glucuronic acid, and (iii) acidic pH. Three proteins are required to sense and respond to these stimuli: (i) VirA, a transmembrane sensory protein and histidine protein kinase. (ii) VirG, a transcriptional activator which is phosphorylated by phosphoryl VirA, and (iii) ChvE, a periplasmic sugar‐binding protein. VirA and VirG are members of the so‐called two‐component family of regulatory proteins. This regulatory system continues to offer new discoveries in the areas of signal transduction. host‐microbe interactions, and host range.
Characterization of a plasmid from Helicobacter pylori encoding a replication protein common to plasmids in Gram‐positive bacteria
Authors: Kleanthous H. , Clayton C. L. , Tabaqchali S.
Published: October 2006

A 1.5kb cryptic plasmid was isolated from Helicobacter pylori. Low‐stringency hybridization analysis using this plasmid as a DNA probe revealed base sequence homology with other plasmids in this species. Nucleotide sequence analysis identified an open reading frame encoding a putative polypeptide of 25 kDa. This protein showed marked amino acid sequence similarity to replication‐initiation proteins commonly found in small plasmids endogenous to Gram‐positive bacteria which replicate by the ‘rolling‐circle’ mechanism. Sequence motifs corresponding to the origins‐of‐replication consensus sequences were found on this cryptic plasmid. DNA and oligonucleotide probes to these plasmid replication sequences were used in hybridization analysis to identify similar sequences in other H. pylori plasmids. We believe this is the first plasmid isolated from a Gram‐negative bacterium to show replication determinants characteristic of the ‘rolling‐circle’ group of plasmids from Gram‐positive bacteria. The cloned plasmid will be used to develop a shuttle‐vector for H. pylori.
Site‐specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis
Authors: Martin C. , Mazodier P. , Mediola M. V. , Gicquel B. , Smokvina T. , Thompson C. J. , Davies J.
Published: October 2006

A method which allowed the stable integration of DNA fragments at a single site (attB) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens, pSAM2. Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site (attP) were constructed and introduced to M. smegmatis by electroporation. Transformants showed stable integration of the plasmid into a single site (attB) of the mycobacterial genome. This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2.
An O antigen can interfere with the function of the Yersinia pseudotuberculosis invasin protein
Authors: Voorhis D. L. , Dillon S. , Formal S. B. , Isberg R. R.
Published: October 2006

Escherichia coli strains harbouring the Yersinia pseudotuberculosis inv gene are able to enter cultured mammalial cells. We show here that this property is not shared by all enteric bacteria, since Shigella flexneri 2a cured of its virulence‐associated plasmid and harbouring the inv gene is unable to enter mammalian cells efficiently. Mapping studies showed that the region of the chromosome responsible for this phenotype includes rfaB, a locus involved in the production of O antigen. S. flexneri 2a strains that express O antigen were unable to enter mammalian cells, even though invasin was efficiently expressed and localized, showing that this structure interferes with invasin activity. The O antigen either masks invasin or sterically hinders the ability of the mammalian cell receptor to bind this protein.