bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryThe 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

SummaryResistance 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

SummaryHeteroduplexes 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

SummaryWhether 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

SummaryA 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

SummaryThe 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

SummaryIn 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

SummaryMany 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

SummaryThe 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

SummaryA 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

SummaryTotal 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

SummaryThe 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

SummaryA 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

SummaryExcision 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

SummaryThe 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

SummaryWe 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

SummaryA 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

SummaryWe 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

SummaryTo 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.