bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulatory functions of the three nodD genes of Rhizobium leguminosarum biovar phaseoli
Authors: Davis E. O. , Johnston A. W. B.
Published: October 2006

The three nodD genes of a strain of Rhizobium leguminosarum biovar phaseoli were cloned to study their effects on transcription of themselves and of the nodC genes of biovars phaseoli and viciae. Efficient transcription of nodD1 required nodD1 and was enhanced by exposure of the cells to bean exudate consistent with the presence of a nod‐box preceding the nolE‐nodD1 operon. Transcription of nodD2 and nodDZ was constitutive. nodC of R. leguminosarum biovar phaseoli was activated by each of the nodD genes of that biovar in the absence of inducers but expression was enhanced in cells grown with bean exudate or the flavonoids genistein or naringenin. A mutant of nodD2, tacking 60bp at its 3’end, activated nodC in the presence of inducer, but was defective in regulating certain of the nodD genes. The nodC gene of R. leguminosarum biovar viciae responded differently to the various nodD genes of R. leguminosarum biovar phaseoli than did the nodC of the latter biovar.
Developmental expression and characterization of the gene encoding spore coat protein SP60 in Dictyostelium discoideum
Authors: Widdowson D. C. C , Proffitt J. A. , Jagger P. S. , Richards A. J. , Hames B. D.
Published: October 2006

The complete coding sequence, upstream sequence and developmental expression of Dictyostelium discoideum AX2 spore coat protein gene SP60 is reported. The gene contains two exons, 154 bp and 1121 bp long, separated by a 119bp intron, and encodes a protein of 46925 molecular weight plus a 23‐amino‐acid hydrophobic leader sequence. The N‐terminus of the mature protein consists of four copies of a perfect hexapeptide repeat (GDWNNN). The central region is rich in cysteine residues, including four highly conserved cysteine‐rich repeats with homology to ‘EGF‐like’ repeats. The C‐terminus is aspartate‐rich and composed of multiple imperfect copies of a D(G/D)DYD repeat followed by several repeats of the tetrapeptide DNDW and derived sequences. A TATA box promoter motif juxtaposed to an oligo(dA) stretch lies 52 bp upstream of the main transcriptional start site of the gene. Six AC‐rich boxes occur in the region ‐327 to ‐556, all of which contain the consensus sequence CACAC. Two GC‐rich boxes and a C‐rich element (TTACCCCA) are also present upstream. Another open reading frame is positioned a short distance downstream of the SP60 gene in the opposite transcriptional orientation. Expression of the SP60 gene ceases upon disaggregation to single cells and cannot be restored by high levels of extracellular cAMP either alone or in combination with conditioned medium factors.
Integration host factor affects expression of two genes at the conjugal transfer origin of plasmid R100
Authors: Dempsey W. B. , Fee B. E.
Published: October 2006

Integration host factor (IHF) binds to two sites near the origin of transfer of the conjugative antibiotic resistance plasmid, R100. DNase I footprinting shows that one site is immediately adjacent to or/T and the gene X promoter, and another is adjacent to the traM promoter. A third site, known only from retardation gels, is near the traJ promoter. The relative promoter activities of genes X, traJ and traM are reduced in himA mutants (IHF), as measured by chloramphenicol‐resistance assays. Transcript analyses by Northern blots showed a reduction in size of the principal gene X and traJ transcripts in the absence of IHF.
Structural analysis of the pehA gene and characterization of its protein product, endopolygalacturonase, of Erwinia carotovora subspecies carotovora
Authors: Saarilahti H. T. , Heino P. , Pakkanen R. , Kalkkinen N. , Palva I. , Palva E. T.
Published: October 2006

A clone producing a potygalacturonase (EC 3.2.1.15) in Escherichia coli was isolated from a genomic library of Erwinia carotovora subspecies carotovora constructed in pUC18. The DNA segment carrying the corresponding structural gene, named pehA, contained an open reading frame (ORF) encoding a 402‐amino‐acid (aa) polypeptide with an Mr of 42849. In E. carotovora the polygalacturonase was synthesized with a 26‐aa cleavable signal peptide. The mature 376‐aa PehA had a calculated Mr of 40064 and a pi of 10.19. The pH optimum of the enzyme was about 5.5 and the temperature optimum was in the range 35–45 ° C. Analysis of the reaction products of poly‐galacturonic acid hydrolysis indicated that the PehA protein is an endopolygalacturonase. No similarity was observed between the aa sequences of PehA and other pectic enzymes of Erwinias. However, substantial similarity was detected within the C‐terminal portions of PehA and a previously described tomato polygalacturonase, suggesting that the bacterial and eukaryotic potygalacturonases may have a common origin.
Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II)
Authors: Fridén H. , Hederstedt L.
Published: October 2006

Cytochrome 5558 in the cytoplasmic membrane of Bacillus subtilis constitutes the anchor and electron acceptor to the flavoprotein (Fp) and iron‐sulphur protein (Ip) in succinate: quinone oxidoreductase, and seemingly contains two haem groups. EPR and MCD spectroscopic data indicate bis‐imidazole ligation of the haem. Apo‐cytochrome was found in the membrane fraction of haem‐deficient B. subtilis, suggesting that during biogenesis of the oxidoreductase the cytochrome b558 polypeptide is embedded into the membrane prior to the incorporation of haem and subsequent binding of Fp and Ip. The six His residues in cytochrome b558 were individually changed to Tyr to attempt identification of residues serving as haem axial ligands and to analyse the role of His residues for assembly and function of the oxidoreductase. From the properties of the mutants, His‐47 can be excluded as a haem ligand. The remaining His residues (at position s 13, 28, 70,113 and 155) a re located in or close to four predicted transmembrane segments. The Tyr‐28 and Tyr‐70 mutant proteins appeared to lack one of the two haems. Only the Tyr‐13 and Tyr‐47 mutant cytochromes were found to function as anchors for Fp and Ip, but the Tyr‐13 mutant cytochrome assembles into an enzymatically defective succinate: quinone oxidoreductase. It is concluded from a combination of the experimental findings, sequence comparisons and membrane topology data that His‐28, His‐70 and His‐155 are probably haem axial ligands in a dihaem cytochrome b558. His‐70 and His‐155 may be ligands to the same haem.
The influence of the Klebsiella pneumoniae regulatory gene nifL upon the transcriptional activator protein NifA
Authors: Morett E. , Kreutzer R. , Cannon W. , Buck M.
Published: October 2006

The influence of the Klebsiella pneumoniae nifL gene product upon the interaction of the transcriptional activator protein NifA with the nifH promoter has been examined using in vivo dimethylsulphate ‘footprinting’. Binding of NifA to the upstream activator sequence (UAS) of the nifH promoter in the presence of the NifL protein was observed under nitrogen‐limiting growth conditions. Growth in the presence of NH+4 or addition of NH+4 to nitrogen‐limited cells diminished the interaction of NifA with the UAS when NifL was present. Repression of nif transcription by NifL may therefore involve an interaction between NifL and NifA which reduces the affinity of NifA for the UAS.
Characterization of fourteen strains of Neisseria gonorrhoeae: structural analyses and serum reactivities
Authors: Pettit R. K. , Szuba J. C. , Judd R. C.
Published: October 2006

Resistance to normal human serum (NHS) killing in Neisseria gonorrhoeae has been associated with particular types of Protein I (PI) and lipopolysaccharide (LPS), but many exceptions exist, and the role of these structures in determining serum reactivities remains controversial. In reality, the response of the gonococcus to NHS is probably governed by several parameters involving a number of outer‐membrane (OM) components. We surveyed the serum reactivities of 14 strains of N. gonorrhoeae and characterized each of their major OM components. The strains presented a spectrum of sensitivity to pooled NHS. As assessed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping, the strains were also quite heterogeneous in terms of PI, H.8 antigen, and LPS type, and the presence of the 2‐1‐L8 epitope. Five of the strains had identical PIAs in varying LPS and H.8 backgrounds, and four had Identical PIBs in varying LPS and H.8 backgrounds. As assessed by electrophoretic migration and monoclonal antibody binding. Protein III and the 44000 Dalton protein were identical in these strains. We found no association between PI subclass and serum sensitivity, while H.8 and LPS variation appeared to be related to bactericidal responses. The diversity and close interaction of gonococcal components in the OM are undoubtedly involved in differential abilities to survive NHS killing.
Evidence for the exchange of segments between genomes during the evolution of lambdoid bacteriophages
Authors: Highton P.J. , Chang Y. , Myers R.J.
Published: October 2006

Heteroduplexes between the DNA molecules of 12 lambdoid phages were analysed by electron microscopy. The positions of the regions of base sequence homology between the DNA molecules divide them into 35 segments, most of which have a number of alternative forms (alleles), which in general must be functionally homologous but which differ in base sequence and length. The positions of the boundaries between segments in phage λ show that each segment is probably a gene or a group of genes, and that each phage genome is a different combination of the alleles of the segments.

The frequency of the occurrence of the different alleles indicates that the total number in the natural population may be small. The different combinations of alleles of separate segments, found among the phages, indicate the exchange of segments between the phages during their evolution.
Integration host factor is necessary for lysogenization of Escherichia coli by bacteriophage P2
Authors: Saha S. , Haggård‐Ljungquist E. , Nordström K.
Published: October 2006

Whether infection by bacteriophage P2 results in lysogenization of the host or vegetative growth of the phage depends upon a race between transcription from the repressor promoter Pc and the early promoter Pe; transcription from these promoters is mutually exclusive, since the Pc repressor Cox is formed from the Pe transcript and the Pe repressor C from the Pc transcript. The involvement of integration host factor (IHF) in the lysogenization of Escherichia coli K12 by P2 was tested by comparing wild‐type and IHF‐deficient (him A and him D) mutants. No lysogenic clones were formed following infection of the mutant bacteria. A switch plasmid that contains Pc‐C‐car and Pe‐cox‐kan was used to test the choice for expression of Pc versus Pe. In the wild‐type K12 bacteria, 20% of the clones expressed Pe transcription and 80% Pc transcription, whereas all transformed IHF‐defective clones expressed transcription from Pe only. The effects of IHF on the in vivo expression of the Pe and Pc promoters were only marginal. The IHF protein was found to bind upstream of the Pe promoter, where a potential ihf sequence is located.
The function of isolated domains and chimaeric proteins constructed from the transcriptional activators NifA and NtrC of Klebsiella pneumoniae
Authors: Drummond M. H. , Contreras A. , Mitchenall L. A.
Published: October 2006

A model for the domain structure of σ54‐dependent transcriptional activators, based on sequence data, has been tested by examining the function of truncated and chimaeric proteins. Removal of the N‐terminal domain of NtrC abolishes transcriptional activation, indicating that this domain is positively required for activator function. Over‐expression of this domain as a separate peptide appears to titrate out the phosphorylating activity of NtrB. Removal of the N‐terminal domain of NifA reduces activation 3–4‐fold. The residual activity is particularly sensitive to inhibition by NifL, suggesting that the role of the N‐terminal domain is to block the action of NifL in derepressing conditions. The C‐terminal domain of NtrC showed repressor activity when expressed as a separate peptide. This domain is necessary for activator function even when NtrC binding sites are deleted from promoters. A point mutation in the ATP‐binding motif of the NtrC central domain, Ser 169 to Ala, also abolished activator function. Exchanging the N‐terminal domains of Klebsiella pneumoniae NtrC, NifA and Escherichia co/r OmpR, did not produce any hybrid activity, suggesting that N‐terminal domains in the native proteins specifically recognize the rest of the molecule.
Molecular characterization of pulA and its product, pullulanase, a secreted enzyme of Klebsielia pneumoniae UNF5023
Authors: Kornacker M. G. , Pugsley A. P.
Published: October 2006

The determined nucleotide sequence of the Klebsielia pneumoniae UNF5023 gene pulA comprises a single open reading frame coding for a 1090‐residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase of Kiebsiella aerogenes strain W70. However, the UNF5023 pullulanase lacks a collagen‐like sequence present at the N‐terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at the C‐terminus. Pullulanases with or without the collagen‐like sequence could not be separated by gel electrophoresis under denaturing or non‐denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso‐amylases. Linker mutations in the region of the UNF5023 pulA gene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino‐terminal 656 residues of pre‐pullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti‐pullulanase serum in immuno‐fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase‐specific extension of the secretion pathway are located near the N‐terminus of the pullulanase polypeptide.
Pulling the trigger: the mechanism of bacterial spore germination
Authors: Foster S. J. , Johnstone K.
Published: October 2006

In spite of displaying the most extreme dormancy and resistance properties known among living systems, bacterial endospores retain an alert environment‐sensing mechanism that can respond within seconds to the presence of specific germinants. This germination response is triggered in the absence of both germinant and germinant‐stimulated metabolism. Genes coding for components of the sensing mechanism in spores of Bacillus subtilis have been cloned and sequenced. However, the molecular mechanism whereby these receptors interact with germinants to initiate the germination response is unknown. Recent evidence has suggested that in spores of Bacillus megaterium KM, proteolytic activation of an autolytic enzyme constitutes part of the germination trigger reaction.
Transcriptional regulatory cascade of nitrogen‐fixation genes in anoxygenic photosynthetic bacteria: oxygen‐and nitrogen‐responsive factors
Authors: Kranz R. G. , Foster‐Hartnett D.
Published: October 2006

Many photosynthetic bacteria from aquatic and terrestrial habitats reduce atmospheric dinitrogen to ammonia. The synthesis of proteins required for nitrogen fixation in these microorganisms is repressed by fixed nitrogen or oxygen. Studies on the purple non‐sulphur phototroph Rhodobacter capsulatus have helped to clarify this transcriptional control and to define the factors involved in this regulation. The molecular mechanisms by which the nitrogen and oxygen status of the cell are relayed into nif gene expression or repression involve many trans‐ and cis‐ acting factors. The roles of these factors in the nif regulatory cascade of R. capsulatus are summarized. Two levels of control are present. The first level of control involves the nitrogen sensing circuitry in which at least four proteins act in a cascade. Upon nitrogen deficiency, genes involved in the second level of control are transcriptionalty activated. These genes encode regulatory proteins that subsequently activate transcription of all other nif genes under anaerobic conditions. The R. capsulatus cascade is compared to the nif regulatory cascade in Klebsiella pneumoniae, highlighting both common and unique aspects.
The par region of pSC101 affects plasmid copy number as well as stability
Authors: Manen D. , Goebel T. , Caro L.
Published: October 2006

The par locus is a segment of pSC101 that has been identified as a cis‐acting determinant of plasmid stability. We show that par also determines copy number and must, therefore, play a role in plasmid replication. The segregation defect, but not the copy‐number reduction, of par‐ replication origins is completely suppressed by a short sequence from the bacteriophage lambda gene O which is present in plasmid pKO‐4. Thus, replication and segregation functions are separable from each other.
Deletion analysis resolves cell‐binding and lytic domains of the Pasteurella leukotoxin
Authors: Cruz W. T. , Young R. , Chang Y. F. , Struck D. K.
Published: October 2006

A series of internal deletions in the IktA gene of Pasteurella haemofytica has been constructed. All of the deletions eliminated the lytic activity of the leukotoxin towards the bovine lymphoma cell line, BL‐3. Deletions removing segments of the amino‐proximal hydrophobic region, which is thought to constitute an essential membrane‐spanning domain, were found to agglutinate BL‐3 cells. Agglutination was similar to lysis by the wild‐type toxin in that it was dependent upon the presence of calcium and required expression of the lktC gene. The agglutinating deletion proteins protected BL‐3 cells from lysis by the wild‐type toxin in a competitive fashion. This suggests that these mutants bind to a surface feature of the leukocyte which interacts with the native leukotoxin. These findings demonstrate that the cell‐binding and lytic domains of the leukotoxin are separable.
Characterization of two DNA populations of Giardia lamblia
Authors: Ortega‐Pierres G. , Alonso R. , Cervantes L. , Montanez C.
Published: October 2006

Total DNA was isolated from the parasitic protozoan Giardia lamblia and separated into two distinct populations of different densities by centrifugation through CsCI gradients containing Hoechst dye 33258. The two populations obtained were characterized by restriction enzyme analysis and nucleic acid hybridization. The less‐dense population contains non‐repetitive DNA and may encode mainly structural genes, such as those for α‐ and β‐tubulin. Digestion of the DNA with several restriction endonucleases showed that the denser band was composed of a 5.5 kb unit which contains the C. lamblia ribosomal RNA cistron in tandem repeated organization.
Functional organization of the ends of IS 1: specific binding site for an IS1‐encoded protein
Authors: Zerbib D. , Prentki P. , Gamas P. , Freund E. , Galas D. J. , Chandler M.
Published: October 2006

The IS 1‐encoded protein InsA binds specifically to both ends of IS 1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel ‘foot‐printing’ and gel retardation, that the InsA binding sites are located within the 24/25bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS 1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This ‘architecture’ of the ends of IS 1 is remarkably similar to that of IS elements IS10, IS50 and IS903.
Nucleotide sequence and control of transcription of the bacteriophage T4 motA regulatory gene
Authors: Uzan M. , Brody E. , Favre R.
Published: October 2006

A 2116bp segment of the bacteriophage T4 genome encompassing the motA regulatory gene has been sequenced. In addition to motA, five open reading frames were identified in the direction of early transcription. The motA gene encodes a basic protein of 211 amino acids with a predicted molecular weight of 23559. Measurements of the rate of transcription of motA showed that the promoter of this gene is turned off after only 2 min of T4 development. This early promoter presents a structure which is richer in information than that of a classical constitutive Escherichia coli promoter. In addition to containing conserved sequences centred at ‐10 and ‐35, this promoter shares extensive homologies with other subgroups of early promoters in regions centred at +3 and at ‐55. We discuss the possible role of these different sequence determinants.
The integration‐excision system of the conjugative transposon Tn 1545 is structurally and functionally related to those of lambdoid phages
Authors: Poyart‐Salmeron C. , Trieu‐Cuot P. , Carlier C. , Courvalin P.
Published: October 2006

Excision of Tn1545 and related conjugative transposons of Gram‐positive bacteria occurs by reciprocal site‐specific recombination between non‐homologous regions of the transposon‐target junctions. Excisive recombination requires two transposon‐encoded proteins designated Xis‐Tn and Int‐Tn. We have shown that, following excision, Tn1545 is a circular structure with ends separated by either of the two hexanucleotides that were present at the transposon‐target junctions. Using a trans‐complementation assay, we have demonstrated that Int‐Tn is able to catalyse in vivo integration of a circular intermediate of Tn1545 defective for integration and excision. Comparison of integration sites suggests that limited sequence homology at the vicinity of the recombining sites is required for integration of the element. These data support the hypothesis that the integration/excision systems of conjugative transposons from Grampositive cocci and of lambdoid phages from Gramnegative bacilli have evolved from a common ancestor.
The cloning and DNA sequence of the gene for the glutathione‐regulated potassium‐efflux system KefC of Escherichia coli
Authors: Munro A. W. , Ritchie G. Y. , Lamb A. J. , Douglas R. M. , Booth I. R.
Published: October 2006

The kefC gene of Escherichia coli encodes a potassium‐efflux system that is regulated by glutathione metabolites. The close proximity of the E. coli kefC gene to the folA gene, encoding dihydrofolate reductase, has been utilized to clone the structural gene for the system from a Clarke‐Carbon plasmid. The cloned gene has been refined to a region of DNA approximately 2.1 kb in length using exonuclease III‐generated deletions and random Muc/M1734 (IacZ) insertions. The direction of transcription has been deduced from the orientation of the Mu insertions in the cloned DNA. A hybrid protein consisting of approximately two thirds of the KefC protein fused to β‐galactosidase has been shown to be membrane‐located. The DNA sequence of the gene has been determined and an open reading frame of 1.86kb has been located which could encode a protein of 620 amino acids (7901 0Da). Using the T7 expression system a membrane protein, of apparent molecular mass 55–60 kDa, has been shown to be encoded by the kefC gene. The predicted protein sequence shows a highly hydrophobic amino‐terminus and a strongly hydrophilic carboxy‐terminus, Comparison of the amino acid sequence of the kefC gene product with those of two glutathione‐utilizing enzymes, glyoxalase and dehalogenase, has revealed some similarities.
Gene 32 transcription and mRNA processing in T4‐related bacteriophages
Authors: Loayza D. , Carpousis A. J. , Krisch H. M.
Published: October 2006

We have analysed transcription and mRNA processing for the gene 32 region of five phages related to T4. Two different organizations of gene 32 proximal promoters were found. In T4 and M1, middle‐ and late‐mode promoters are separated by 50 nucleotides and located within an upstream open reading frame. In T2, K3, Ac3, and Ox2, the 626 bp T4 sequence that includes these promoters is replaced by a 59 bp sequence containing overlapping middle and late promoters. The RNase E‐dependent processing of the g32 mRNAs is conserved in all of the phages. The processing site immediately upstream of g32 in T4 and M1 has been replaced in the other phages by a different sequence that is also cleaved by RNase E. The remarkable conservation of these regulatory features, despite the sequence divergences, suggests that they play an important role in the control of gene expression.
The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene
Authors: Taraseviciene L. , Miczak A. , Apirion D.
Published: October 2006

A DNA clone complementing the rne‐3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the me‐3071 mutation has been mapped. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by this DNA fragment (110000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne‐3071 and the ams mutations cause similar patterns of RNA synthesis, show that the me gene — a gene specifying the processing endonuclease RNase E — and the ams gene — a gene that affects mRNA stability — are identical.
Genetic studies of cleavage‐initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same
Authors: Melefors Ö. , von Gabain A.
Published: October 2006

We show in the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli amsts strain (originally defined by a prolonged bulk mRNA half‐life) and in the rnets strain (originally defined by aberrant 9S RNA processing). The temperature‐sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus. A 5.8 kb fragment from this genomic DNA segment was cloned into a low‐copy plasmid and used to transform the amsts and rnets strains. This resulted in growth at the non‐permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA. Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Amsts and the Rnets phenotype with regard to the ompA cleavages. In addition we showed that the amsts strain suppresses 9S RNA processing to 5S RNA to the same extent as the rnets strain, and that the rnets strain has a prolonged bulk mRNA half‐life, as was reported for the amsts strain. Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing. We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K.
Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature‐sensitive pSC101 replicon
Authors: Blomfield I. C. , Vaughn V. , Rest R. F. , Eisenstein B. I.
Published: October 2006

To facilitate efficient allelic exchange of genetic information into a wild‐type strain background, we improved upon and merged approaches using a temperature‐sensitive plasmid and a counter‐selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild‐type chromosomal sequences, at either the fimB–A or lacZ–A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn5, but, unlike another similar cassette, it lacks IS1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature‐sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non‐selective conditions at 42°C, and then in the presence of sucrose at 30°C, allowing positive selection for both plasmid excision and curing. Despite marked locus‐specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non‐essential parts of the E. coli chromosome.
Identification and molecular characterization of a transcriptional regulator from Pseudomonas aeruginosa PAO1 exhibiting structural and functional similarity to the FNR protein of Escherichia coli
Authors: Sawers R. G.
Published: October 2006

A gene library of chromosomal DNA from Pseudomonas aeruginosa contained a DNA fragment which was able to restore anaerobic growth to an Escherichia coli fnr deletion mutant on glycerol/nitrate medium. The cloned gene (termed anr) was sequenced and shown to encode a protein of 244 amino acids with a calculated molecular weight of 27 129. The deduced amino acid sequence of the anr gene product showed considerable similarity to the FNR protein from E. coli. Expression of the anr gene in a T7 promoter/polymerase system identified ANR as a 31 kDa protein. Transcriptional analysis of the anr gene showed that it is monocistronic but apparently lacks the equivalent sites for negative autoregulation which have been shown to be present in the promoter region of the E. coli fnr gene. The ANR protein was shown to activate transcription of the pfl gene in E. coli in response to anaerobiosis, as well as being able to restore the activity of three anaerobicaliy inducible enzymes. A P. aeruginosa mutant incapable of growing anaerobically with nitrate or on arginine was fully complemented by the anr gene, indicating that it probably has a function in controlling anaerobic gene expression in Pseudomonas. Further corroboration for this assumption was provided by S1 nuclease analysis of transcription of the multiple promoters of the E. coli pfl operon in P. aeruginosa. Transcription was induced by oxygen limitation and was completely ANR‐dependent in both aerobic and anaerobic cells. Removal of the upstream regulatory sequence of the pfl operon, which includes the sequences required for FNR‐dependent regulation in E. coli, removed ANR‐dependent transcriptional control of the remaining pfl promoters, irrespective of the cellular oxygen status. These results imply that the mechanisms by which ANR and FNR regulate transcription are fundamentally similar.
Mutations in the aphA‐2 gene of transposon Tn5 mapping within the regions highly conserved in aminoglycoside‐phosphotransferases strongly reduce aminoglycoside resistance
Authors: Blázquez J. , Davies J. , Moreno F.
Published: October 2006

Aminoglycoside‐phosphotransferases contain several conserved amino acid sequence motifs. Using hydroxylamine we have obtained five independent missense mutations within the aphA‐2 gene of transposon Tn5. Four of the mutations dramatically reduced antibiotic resistance. Two were identical and included the replacement of His‐188 with Tyr. One other resulted from the replacement of Gly‐189 with Asp. These three mutations map within the first of the conserved motifs. The replacement of Asp‐261 with Asn maps to the third of these structural motifs. A mutation diminishing but not eliminating aminoglycoside resistance resulted from replacement of the conserved Val‐36 with Met. By site‐directed mutagenesis three additional mutants were obtained: His‐188 was replaced with Leu and Ser, and Arg‐211 within the second conserved motif was substituted by Gly. All three showed reduced levels of resistance to kanamycin. Our results show that these conserved motifs are essential for the biological activity of aminoglycoside phosphotransferases.
The Haemophilus influenzae capsulation gene cluster: a compound transposon
Authors: Kroll J. S. , Loynds B. M. , Moxon E. R.
Published: October 2006

The population of capsulate Haemophilus influenzae is divided into two phylogenetic divisions. Here we show that in division I strains the capsulation (cap) gene cluster lies between direct repeats of a novel insertion sequence (IS)‐like element, IS 1016. cap has apparently been mobilized in the chromosome as a compound transposon by IS 1016, and the repeats have provided a molecular substrate for reversible cap gene amplification, with augmentation of capsule production, through unequal homologous recombination. Such amplification has occurred in serotype b strains, but in these a large direct repeat of cap genes has become fixed in the population. We have found a 1.2kb deletion at one end of this duplicated cap b locus, removing most of one copy of the polysaccharide export gene bexA. We have shown that this makes capsulation dependent on preservation of the direct repeat structure in order to avoid recombination‐mediated loss of the other copy of bexA. Type b strains with this cap configuration are disseminated worldwide and currently cause nearly all invasive Haemophilus infections, leading us to speculate that the 1.2 kb deletion occurred in an ancestral type b strain and conferred significant biological advantage.
Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host‐range Gram‐negative cosmid vector pJRD215
Authors: Hermans J. , Martin C. , Huijberts G. N. M. , Goosen T. , de Bont J. A. M.
Published: October 2006

The transformation of Mycobacterium aurum and Mycobacterium smegmatis with the Gram‐negative RSF1010‐derived cosmid pJRD215 is described. The plasmid is stably maintained in both species and the antibiotic resistance determinants for kanamycin and streptomycin are expressed. Southern blot analysis shows that rearrangements take place both in M. aurum and in M. smegmatis. The use of pJRD215 in mycobacterial cloning systems is discussed.
Macronuclear and micronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1α in Stylonychia lemnae
Authors: Bierbaum P. , Dönhoff T. , Klein A.
Published: October 2006

The micronuclear and macronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1α in the hypotrich ciliate Stylonychia lemnae were compared. The two sequences are generally colinear. The coding sequence of the micronuclear gene is, however, interrupted by a 64 bp insert flanked by a 2 bp direct repeat in a gene region which is moderately conserved among EF 1α genes of different organisms. The insertion site is distinct from known intron positions in eukaryotic EF 1α genes. The insert sequence shows inverted repeats at its ends and thus exhibits typical features of an internal eliminated sequence (IES). Comparison with other such sequences in the related organism Oyxtricha nova shows that the IES falls into a new group of such elements. The macronuclear gene exhibits a strikingly limited codon usage, which cannot be simply explained by the overall base composition of the DNA but probably also relates to the very high copy number of the macronuclear gene and the putative high amount of the gene product.
The Entamoeba histolytica rDNA episome: nuclear localization, DNAase I sensitivity map, and specific DNA‐protein interactions
Authors: Zurita M. , Alagón A. , Vargas‐Villarreal J. , Lizardi P. M.
Published: October 2006

Structural and functional features of the extrachromosomal DNA element that contains the ribosomal RNA genes of Entamoeba histolytica were studied using a variety of techniques. Using in situ hybridization, the element was found to be distributed along the inner phase of the nuclear membrane in the trophozoite stage; it appears to be part of the so‐called peripheral chromatin. DNAase I‐sensitive regions on the episome were mapped and found to correspond to the borders of the ribosomal RNA coding region. Other DNAase I‐sensitive regions were found to correspond to DNA containing a 145bp sequence that exists in the episome as tandem repeats. Electrophoretic shift assays and footprinting experiments demonstrate the existence of specific nuclear factors that bind specifically to the 145bp repeat. Preliminary analysis of the binding factors showed that a 28 kDa polypeptide is a likely candidate for a specific DNA:protein interaction involving the repetitive element. These results suggest that a protein‐binding domain within the 145bp repeat may have a specific function in the episome.
The spoIIIA operon of Bacillus subtilis defines a new temporal class of mother‐cell‐specific sporulation genes under the control of the σE form of RNA polymerase
Authors: Illing N. , Errington J.
Published: October 2006

We have cloned and characterized a 5 kbp region of the Bacillus subtilis chromosome and show that it contains the promoter‐proximal part of the spoIIIA locus. The locus consists of a polycistronic operon containing at least three genes. We show that the operon is regulated at the transcrlptional level, from a promoter that is first activated about 80 minutes after the induction of sporulation, immediately after septation. Expression of spoIIIA in different spo mutant backgrounds correlates with the ability of each strain to synthesize the sporulation‐specific sigma factor, σE. Moreover, synthesis of σE in vegetative cells by use of an inducible promoter causes expression of mother‐cell‐specific genes spoIID, spoIIIA, and spoIIID, but not the prespore‐specific genes, spoIIIG and spoVA. We suggest that σE may be the primary determinant of mother‐cell‐specific gene expression and that the SpoIIID protein exerts an additional level of regulation on spoIIIA, apparently by acting as a transcriptional repressor. Since the onset of spoIIID expression occurs about 10 minutes after that of spoIIIA, spoIIIA expression is transient. Thus spoIIIA defines a third temporal class of gene controlled by the σE form of RNA polymerase.
IS900‐promoted stable integration of a foreign gene into mycobacteria
Authors: England P. M. , Wall S. , McFadden J.
Published: October 2006

An artificial mycobacterial transposon was constructed by placing two copies of the insertion sequence IS900 flanking a kanamycin resistance gene into a non‐(mycobacterial) replicating vector. Constructs were introduced into mycobacteria by electroporation and transposition events conferring kanamycin resistance were selected. Integration of IS900 into several genomic sites was analysed by Southern blotting and shown to involve both simple insertions and cointegrate formation, suggesting that IS900 can transpose by a replicative mechanism. Kanamycin resistance of IS900‐integrated transformants was shown to be stable in the absence of selection.
Computer modelling of the NAD binding site of ADP‐ribosylating toxins: active‐site structure and mechanism of NAD binding
Authors: Domenighini M. , Montecucco C. , Ripka W. C. , Rappuoli R.
Published: October 2006

Summary Five ADP‐ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site‐directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three‐dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.
987P fimbrial gene identification and protein characterization by T7 RNA polymerase‐induced transcription and TnphoA mutagenesis
Authors: Schifferli D. M. , Beachey E. H. , Taylor R. K.
Published: October 2006

The 987P fimbrial gene cluster has been previously cloned as a 12 kb fragment from prototype strain 987. Gene products encoded by the whole clone were analysed by utilizing an in vivo system based on the induction of transcription by T7 RNA polymerase. The sensitivity of this technique permitted us to identify new proteins involved in 987P fimbriation. In total, eight proteins were detected, their genes (fasA to fasH) were mapped and their orientation of transcription determined. Several of the gene products demonstrated typical properties of exported proteins. Precursor and processed forms could be correlated after inhibiting protein transport with ethanol. The detection of enzymatically active fusion proteins after TnphoA (Tn5IS50L::phoA) mutagenesis supported and complemented these results. One protein encoded by the 12kb fragment was found not to be related to fimbriation but rather the product of the STla gene, identified as a component of a Tn1681‐like transposon.
Chlamydia trachomatis contains a protein similar to the Legionella pneumophila mip gene product
Authors: Lundemose A. G. , Birkelund S. , Fey S. J. , Larsen P. Mose , Christiansen G.
Published: October 2006

A 27kDa Chlamydia trachomatis L2 protein was characterized by the use of monoclonal antibodies and by two‐dimensional gel electrophoresis. The protein was shown to be located in the membrane of reticulate bodies as well as elementary bodies. Its synthesis could be detected from 10 hours post‐infection. Cloning and sequence analysis of the distal part of the gene revealed an open reading frame of 175 amino acids. Comparison of the deduced amino acid sequence with the NBRF data base revealed significant homology between the 27kDa chlamydial membrane protein and the product of the macrophage infectivity potentiator (mip) gene of Legionella pneumophila.
Folding in vitro and transport in vivo of pre‐β‐lactamase are SecB independent
Authors: Laminet A. A. , Kumamoto C. A. , Plückthun A.
Published: October 2006

The rate of folding of the precursor of β‐lactamase is not influenced by the presence of SecB under conditions in which GroEL/ES retards the folding. Wild‐type β‐lactamase and several mutants in the signal or the mature protein, affecting either transport or enzyme kinetics and probably folding, were examined for total expression, total enzymatic activity, and transported β‐lactamase (in vivo resistance) in secB‐ and secB+ strains. We conclude that there is no indication of any relevant interaction between SecB and pre‐β‐lactamase in vitro, nor did the secB‐ mutation affect the transport of wild–type β‐lactamase or any of the mutants in vivo. Thus, putative Escherichia coli‘folding modulators’must be of limited specificity.
Control of pilus expression in Neisseria gonorrhoeae as an original system in the family of two‐component regulators
Authors: Taha M. K. , Dupuy B. , Saurin W. , So M. , Marchal C.
Published: October 2006

We have previously reported the identification of two genes, pilA and pilB, which act in trans to regulate pilus expression in Neisseria gonorrhoeae. Here we show that PilA and PilB have amino acid sequence similarities with members of the two component 'sensor‐regulator’family of proteins. PilB has homology with histidine kinase sensors. Alkaline phosphatase fusions to the predicted sensor and transmitter domains are described. Their PhoA activity and cellular location suggest that PilB is inserted in the cytoplasmic membrane and predict periplasmic and cytoplasmic locations for the sensor and the transmitter domains, respectively. PilA has homology with response regulators in its N‐terminal part, and with components of the eukaryotic protein secretory apparatus (SRP 54 and SRP receptor) as well as two Escherichia coli gene products in its C‐terminal part. In particular, it contains a putative GTP‐binding site. Mini‐transposon insertions into different regions of pilA were obtained. The phenotypes and genotypes of these mutants and preliminary biochemical studies of the gene products of two of these mutants lend further support to the hypothesis that PitA is a DNA‐binding response regulator and confirm that it participates in an essential function in the bacterium.
Sequences downstream from the transcriptional start site are essential for microaerobic, but not symbiotic, expression of the Rhizobium meliloti nifHDK promoter
Authors: Wang Y.‐P. , Birkenhead K. , Dobson A. , Boesten B. , O'Gara F.
Published: October 2006

Deletion analysis studies have been carried out on the nifHDK promoter (p1) of R. meliloti in the expression of this promoter under both free‐living microaerobic and symbiotic conditions. Deletion of a region down‐stream (+17 to +61) from the promoter element resulted in low levels of expression under free‐living microaerobic conditions However, wild‐type levels of expression were obtained during symbiosis with Alfalfa plants. The sequences in this region were designated the‘downstream sequences'. The pattern of expression observed when the downstream sequences were deleted was similar to that observed when a previously identified upstream activator sequence (UAS) was deleted. Only when both the downstream sequences and the UAS were deleted, did activity form the P1 promoter become significantly decreased during symbiosis. Expression studies of the P1 promoter in a nifA mutant background indicate that nifA is required for symbiotic expression of P1 which is enhanced by the presence of the downstream sequences.
The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation
Authors: Perego M. , Higgins C. F. , Pearce S. R. , Gallagher M. P. , Hoch J. A.
Published: October 2006

Bacillus subtilis spo0K mutants are blocked at the first step in sporulation. The spo0K strain was found to contain two mutations: one was linked to the trpS locus, and the other was elsewhere on the chromosome. The mutation linked to trpS was responsible for the sporulation defect (spo‐). The unlinked mutation enhanced this sporulation deficiency but had no phenotype on its own. The spo‐ mutation was located in an operon of five genes highly homologous to the oligopeptide transport (Opp) system of Gram‐negative species. Studies with toxic peptide analogues showed that this operon does indeed encode a peptide‐transport system. However, unlike the Opp system of Salmonella typhimurium, one of the two ATP‐binding proteins, OppF, was not required for peptide transport or for sporulation. The OppA peptide‐binding protein, which is periplasmically located in Gram‐negative species, has a signal sequence characteristic of lipo‐proteins with an amino‐terminal lipo‐amino acid anchor. Cellular location studies revealed that OppA was associated with the cell during exponential growth, but was released into the medium in stationary phase. A major role of the Opp system in Gram‐negative bacteria is the recycling of cell‐wall peptides as they are released from the growing peptidoglycan. We postulate that the accumulation of such peptides may play a signaling role in the initiation of sporulation, and that the sporulation defect in opp mutants results from an inability to transport these peptides.