bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of an oxytetracycline‐resistance gene, otrA, of Streptomyces rimosus
Authors: Doyle D. , McDowall K. J. , Butler M. J. , Hunter I. S. 
Published: October 2006

SummaryThe sequence of a 2657 bp DNA fragment containing the coding and regulatory regions of the oxytetracycline (OTC)‐resistance gene, otrA, from the OTC producer Streptomyces rimosus was determined. The predicted amino acid sequence of OtrA had extensive identity with tetracycline‐resistance genes from other bacteria which mediate resistance via non‐covalent ribosomal modification. The N‐terminal domain had extremely high identity with the GTP‐binding sites of elongation factors, such as EF‐G and EF‐Tu, suggesting that binding and hydrolysis of GTP is important to the function of the protein. Significant identity with EF‐G was present throughout the polypeptide. Transcriptional activity upstream of the otrA coding region was investigated. An Escherichia coli‐ type promoter, of otrA p1, was identified. Transcriptional read through of otrA from the upstream gene (otcZ) was also detected in S. rimosus cultures. A divergent promoter activity was identified with sub‐clones of the OtrA fragment in promoter probe vectors analysed in Streptomyces lividans. However, this activity was not identified in a subclone containing more than haif of the otrA coding sequence in S. lividans or at all in S. rimosus, indicating that OtrA negatively regulates the expression of the divergent transcript. The data are consistent with regulation of antibiotic production by OtrA to prevent ‘suicide’.
RNaselll activation of bacteriophage λ N synthesis
Authors: Kameyama L. , Fernandez L. , Court D. L. , Guarneros G. 
Published: October 2006

SummaryThe bacteriophage λ gene product is one of the first genes expressed during phage development. N protein allows the expression of other phage genes by altering the transcription elongation process so as to prevent transcription termination. We have found that N levels may be modulated soon after induction or infection. Using N‐lacZ fusions, we determined that cells containing RNaselll have at least a fourfold greater expression than cells defective for RNaselll. This effect is exerted at the post‐transcriptional level. RNaselll processes an RNA stem structure in the N‐ leader RNA. Removal of the stem structure by deletion increases λ expression and prevents further stimulation by RNaselll. The base of this stable stem is adjacent to the λ ribosome binding site. We present a model for control of N synthesis in which this stable stem inhibits ribosome access to the N mRNA.
A host factor absent from Lactococcus lactis subspecies lactis MG1363 is required for conjugative transposition
Authors: Bringel F. , Van Alstine G. L. , Scott J. R. 
Published: October 2006

SummaryIn matings between Lactococcus lactis strains, the conjugative transposons Tn916 and Tn919are found in the chromosome of the transconjugants in the same place as in the chromosome of the donor, indicating that no transposition has occurred. In agreement with this, the frequency of L. lactis transconjugants from intraspecies matings is the same whether the donor contains the wild‐type form of the transposon or the mutant Tn916‐int1, which has an insertion in the transposon's integrase gene. However, in intergeneric crosses with Bacillus subtilis or Enterococcus faecalis donors, Tn916 and Tn919 transpose to different locations on the chromosome of the L. lactis transconjugants. Moreover, Tn916 and Tn919 could not be transferred by conjugation from L. lactis and B. subtilis, E. faecalis or Streptococcus pyogenes. This suggests that excision of these elements does not occur in L. lactis. When cloned into E. coli with adjacent chromosomal DNA from L. lactis, the conjugative transposons were able to excise, transpose and promote conjugation. Therefore, the inability of these elements to excise in L. lactis is not caused by a permanent structural alteration in the transposon. We conclude that L. lactis lacks a factor required for excision of conjugative transposons.
Molecular cloning and transcriptional analysis of the cpeBA operon of the cyanobacterium Pseudanabaena species PCC 7409
Authors: Dubbs J. M. , Bryant D. A. 
Published: October 2006

SummaryThe cpeBA operon of the Group III chromatically adapting cyanobacterium Pseudanabaena species PCC 7409 was cloned, sequenced and characterized. The cpeBA genes are transcribed in green‐light‐grown cells as an abundant 1400‐nucleotide mRNA which initiates 69 nucleotides upstream from the cpeB translation start. Extensive sequence identity, extending 70 nucleotides 5′ to the transcription start, occurs among cpeBA promoters of Group II and III chromatic adapters. Cell extracts of green‐light‐grown Calothrix species PCC 7601 contain an activity which specifically binds a restriction fragment containing the Pseudanabanea species PCC 7409 cpeBA promoter. Green‐light‐dependent cpeBA transcription in Group II and III chromatically adapting cyanobacteria is suggested to be similarly controlled by a transcriptional activator.
The role of RNA polymerase in transcriptional fidelity
Authors: Libby R. T. , Gallant J. A. 
Published: October 2006

SummaryThe overall transcription of DNA has previously been demonstrated to proceed at extremely high levels of accuracy. We review the evidence that the process of transcription is subject to proof‐reading in the Hopfield sense. In addition, we speculate that the proof‐reading activity associated with transcription is subject to cyclical phase transitions. That is, during periods of low processivity associated with initiation, RNA synthesis is relatively imprecise. The transition to the elongation phase of RNA synthesis, characterized by a shift to high processivity, is accompanied by enhanced proof‐reading. A model for the damping of transcriptional errors, based on a PPi‐mediated processive pyrophosphorolysis reaction, is discussed in terms of pausing during transcription.
Molecular analysis of a complex locus from Haemophilus influenzae invoked in phase‐variable lipopolysaccharide biosynthesis
Authors: Maskell D. J. , Szabo M. J. , Butler P. D. , Williams A. E. , Moxon E. R. 
Published: October 2006

SummaryA chromosomal locus, lic3, one of several involved in lipopolysaccharide (LPS) biosynthesis by Haemophilus influenzae, was cloned and its DNA sequence determined. Iic3 comprises four closely apposed open reading frames (ORFs). ORF1 includes tandem repeats of the tetramer CAAT and two start codons out of frame with each other are found upstream of the repeats. ORF1 encodes a protein with no known homologues. 0RF2 encodes the UDP‐galactose‐4‐epimerase (galE) gene. ORF3 encodes a hydrophobic protein with no known homologues. 0RF4 encodes the adenylate kinase (adk) gene. A deletion/insertion mutation lacking the 3′ end of ORF1, all of galE, and the 5′ end of ORF3 was constructed in the parent Hib strain (RM7004). These mutants had a galE phenotype, as evidenced by galactose sensitivity, altered LPS when grown in the absence of exogenous galactose, and reduced virulence in infant rats.
Peptide transport and chemotaxis in Escherichia coli and Salmonella typhimurium: characterization of the dipeptide permease (Dpp) and the dipeptide‐binding protein
Authors: Abouhamad W. N. , Manson M. , Gibson M. M. , Higgins C. F. 
Published: October 2006

SummaryThe dipeptide permease (Dpp) is one of three genetically distinct peptide‐transport systems in enteric bacteria. Dpp also plays a role in chemotaxis towards peptides. We have devised three selections for dpp mutations based on resistance to toxic peptides (bacilysin, valine‐containing peptides, and biala‐phos). All dpp mutations mapped to a single chromosomal locus between 77 and 78 min in Salmonella typhimurium and at 79.2 min in Escherichia coli. Expression of dpp was constitutive in both species but the absolute level of expression varied widely between strains. At least in part this difference in expression levels is determined by c/s‐acting sequences. The dpp locus of E. coli was cloned. The first gene in the operon, dppA, encodes a periplasmic dipeptide‐binding protein (DBP) required for dipeptide transport and chemotaxis. Downstream of dppA are other genes required for transport but not for chemotaxis. The dipeptide‐binding protein was found to share 26.5% sequence identity with the periplasmic oligopeptide‐binding protein OppA.
DNA sequence and analysis of the bet genes encoding the osmoregulatory choline—glycine betaine pathway of Escherichia coli
Authors: Lamark T. , Kaasen I. , Eshoo M. W. , Falkenberg P. , McDougall J. , Strøm A. R. 
Published: October 2006

SummaryThe sequence was determined of 6493 nucleotides encompassing the bet genes of Escherichia coli which encode the osmoregulatory choline—glycine betaine pathway. Four open reading frames were identified: betA encoding choline dehydrogenase, a flavoprotein of 61.9kDa; betB encoding betaine aldehyde dehydrogenase (52.6kDa); betT encoding a proton‐motive‐force‐driven, high‐affinity transport system for choline (75.8kDa); and betl, capable of encoding a protein of 21.8 kDa, implicated as a repressor involved in choline regulation of the bet genes. Identification of the genes was supported by subcloning, physical mapping of λplac Mu53 insertions, amino acid sequence similarity, or N‐terminal amino acid sequencing. The bet genes are tightly spaced, with bet T located upstream of, and transcribed divergently to, the tandemly linked betlBA genes.
Binding of the immunity protein inactivates colicin M
Authors: Ölschläger T. , Turba A. , Braun V. 
Published: October 2006

SummaryColicin M (Cma) displays a unique mode of action in that it inhibits peptidoglycan and lipopolysaccharide biosynthesis through interference with bactoprenyl phosphate recycling. Protection of Cma‐producing cells by the immunity protein (Cmi) was studied. The amount of Cmi determined the degree of inhibition of in vitro peptidoglycan synthesis by Cma. In cells, immunity breakdown could be achieved by overexpression of the Cma uptake system. Full immunity was restored after raising the cmi gene copy number. In sphaeroplasts, Cmi was degraded by trypsin, but this could be prevented by the addition of Cma. The N‐terminal end includes the only hydrophobic amino acid sequence of Cmi, suggesting a function in anchoring of Cmi in the cytoplasmic membrane. It is proposed that Cmi does not act catalytically but binds Cma at the periplasmic face of the cytoplasmic membrane, thereby resulting in Cma inactivation. Two other possible modes of colicin M immunity, interference of Cmi with the uptake of Cma, and interaction of Cmi with the target of Cma, were ruled out by the data.
Common and variable domains of the flagellin gene, flaA, in Campylobacter jejuni
Authors: Fischer S. H. , Nachamkin I. 
Published: October 2006

SummaryThe organization of the flagellin gene locus in Campylobacter jejuni strain IN1 (Lior 7) was determined using the polymerase chain (PCR) reaction and a series of oligonucleotide primers. Two tandemly arranged flagellin genes of approximately 1.7 kb were found to be joined by an intervening segment of c. 0.2 kb, similar to that reported for Campylobacter coli. The 5′ flagellin gene, flaA, was generated by PCR and both strands sequenced. Comparison of the deduced amino acid sequence for C. jejuni FlaA with the published sequence for C. coli FlaA showed 77% identical amino acids between the proteins. Two common regions, C1 and C2, comprising the N‐terminal 170 amino acids and C‐terminal 100 amino acids, exhibit amino acids 94% and 96% identical to those of C. coli, respectively. The variable region, V1, comprising the middle of the protein, shows 61% identical residues with C. coli. Comparison of these regions with other bacterial flagellins reveals a similar pattern but with much less identity. Several areas within the V1 region correspond to predicted surface‐exposed regions and may represent areas in which surface epitopes are located.
Light and oxygen effects share a common regulatory DNA sequence in Rhodobacter capsulatus
Authors: Klug G. , Gad'on N. , Jock S. , Narro M. L. 
Published: October 2006

SummaryExternal factors regulate the formation of pigment protein complexes in facultatively photosynthetic bacteria. The puf operon of Rhodobacter capsulatus encodes the pigment binding proteins of the reaction centre and light‐harvesting I complex. Here we demonstrate that a single base‐pair exchange within a sequence of dyad symmetry upstream of the puf promoter affects both the oxygen regulation and the light regulation of the formation of reaction‐centre and light‐harvesting I complexes in Rhodobacter capsulatus. Our resuts imply that effects of oxygen or light ultimately act on the same regulatory DNA sequence, although it is still unknown how these environmental signals are sensed and transmitted to a transcriptional regulator.
The role of the OOP antisense RNA in coliphage λ development
Authors: Krinke L. , Mahoney M. , Wulff D. L. 
Published: October 2006

SummaryWe have made a derivative of bacteriophage λ that makes no OOP antisense RNA. The mutant phage carries a point mutation that inactivates the OOP promoter, po. The phages λ+ and λp o‐ have identical plaque morphologies, one‐step growth curves, and frequencies of lysogenization of a sensitive host. OOP RNA synthesis is weakly repressed by the Escherichia coli LexA protein. Consonant with this inducibility of OOP RNA synthesis by ultraviolet light, we find a two‐fold greater phage burst following ultraviolet induction of a λ+ than of a λp o− prophage. In λ+ infections, OOP RNA causes two cleavage events in dl mRNA: one is in the 3′ ‐end of the coding region, and the second is in the intercistronic region between the dl and O genes. The dl gene fragments are subject to additional hydrolytic events, and dl mRNA levels are several‐fold lower in λ+ than in λp o‐ infections late in the infection cycle. However, O mRNA levels are almost unaffected by the P o‐ mutation.
Mucoid Pseudomonas aeruginosa in cystic fibrosis: signal transduction and histone‐like elements in the regulation of bacterial virulence
Authors: Deretic V. , Mohr C. D. , Martin D. W. 
Published: October 2006

SummaryThe profuse production of the exopolysaccharide alginate results in mucoidy, a critical virulence factor expressed by Pseudomonas aeruginosa during chronic respiratory tract infections in cystic fibrosis. Studies of the regulation of this pathogenic determinant have unravelled at least two levels of control, including bacterial signal transduction systems and histone‐like elements. Although only in its initial phase, an understanding of the dual control of mucoidy may help to illuminate adaptive processes that depend on the combination of these regulatory factors. Integration of specific signals transduced by the two‐component systems with inputs generated by the general state of bacterial nucleoids may govern the expression of certain virulence determinants and provide a framework facilitating selection of pheno‐types successful under particular environmental conditions and selective pressures.
The gef gene from Escherichia coli is regulated at the level of translation
Authors: Poulsen L. K. , Refn A. , Molin S. , Andersson P. 
Published: October 2006

SummaryWe describe post‐transcriptional regulation of the chromosomal gene, gef, from Escherichia coli. The gef gene is a member of a gene family consisting of the chromosomal gef and relF genes from Escherichia coli and the hok, flmA, smB, and pndA genes, which are situated on conjugative plasmids. All the genes encode small, toxic proteins of approximately 50 amino acids which are functionally and structurally homologous. Furthermore, the gene family shares post‐transcriptional regulation of expression, albeit by different mechanisms. We demonstrate here that translation of gef is coupled to an upstream open reading frame which, in turn, is regulated by a transacting factor, probably an antisense RNA.
In vitro activation of Escherichia coli prohaemolysin to the mature membrane‐targeted toxin requires HlyC and a low molecular‐weight cytosolic polypeptide
Authors: Hardie K. R. , Issartel J.‐P. , Koronakis E. , Hughes C. , Koronakis V. 
Published: October 2006

SummaryThe c. 110kDa haemolysin toxin secreted by Escherichia coli and other pathogenic Gram‐negative bacteria is synthesized as the non‐toxic precursor, prohaemolysin (proHlyA), which is unable to target mammalian cell membranes until activated intracellularly by an unknown mechanism dependent upon the coexpressed c. 20kDa protein, HlyC. We have established in vitro post‐translational activation of proHlyA in membrane‐depleted cell extract fractions from E. coli recombinant strains containing (separately) the proHlyA and HlyC proteins. In vitro activation was calcium‐independent and effective over a pH range of 6 to 9 and at temperatures from 42°C to 4°C. HlyC cell extract was also able to activate proHlyA which had been secreted out of cells containing the export proteins HlyB and HlyD. Fractionation of HlyC cell extracts by sucrose gradient centrifugation and molecular weight chromatography revealed activating fractions as having a molecular mass of 40kDa, suggesting that the HlyC activator is present physiologically in a multimeric form. Cell extracts containing activation‐competent HlyC and proHlyA were inactive following dialysis, but activity was restored by complementation with a cell extract lacking both proteins. HlyC and proHlyA proteins which were overproduced separately from recombinant expression plasmids were inactive following purification, but activity could again be restored with a Hly‐negative cell extract. These experiments demonstrated that HlyC is not sufficient for activation; an additional cellular factor is required. The cellular factor was found in enterobacteria but not other bacteria or eukaryotic cells. It was cytosolic, protease‐sensitive, and behaved as a c. 10kDa polypeptide in a number of assays including dialysis, sucrose gradient centrifugation, and gel filtration chromatography. Thus activation was possible in a defined in vitro reaction containing only purified proHlyA, HlyC, and the cellular factor. Ki stic studies in which the relative concentrations of the three components of proHlyA activation were varied suggested that neither HlyC nor the cellular factor acts as a conventional enzyme, with each participating in a finite number of activation events.
Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation
Authors: Pla J. , Sanchez M. , Patacios P. , Vicente M. , Aldea M. 
Published: October 2006

SummaryAn ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild‐type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ+ allele is unable to be replicated at the same pace as the chromosome, the cells become non‐viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.
The three‐dimensional structure of the regular surface protein of Comamonas acidovorans derived from native outer membranes and reconstituted two‐dimensional crystals
Authors: Engelhardt H. , Gerbt‐Rleger S. , Santarius U. , Baumelster W. 
Published: October 2006

SummaryThe three‐dimensional structure of the regular surface protein (p4 symmetry, lattice constant a=b=10.5 nm) of Comamonas acidovorans has been determined to a resolution of about 1.5 nm by means of electron microscopy and image processing. Three‐dimensional reconstructions were performed using native outer membranes and artificial two‐dimensional crystals of the surface protein, which was selectively solubilized by deoxycholate and recrystallized on carbon films. The two‐fold symmetric morphological complex is composed of two identical monomers which are in tight contact with the outer membrane and presumably anchored to it by a small hydrophobic domain.
Purification and mutant analysis of Citrobacter freundii AmpR, the regulator for chromosomal AmpC β‐lactamase
Authors: Bartowsky E. , Normark S. 
Published: October 2006

SummaryAmpR, the transcriptional regulator for the Citrobacter freundii ampC β‐lactamase gene, was purified. The purified AmpR had DNA‐binding activity, the same molecular mass (32 kDa) on sodium dodecyl sulphate polyacrylamide gel electrophoresis as previously described, and N‐terminal sequencing of the first 15 amino acids was in agreement with that predicted from the nucleotide sequence. Two mutants were isolated that abolish DNA‐binding and β‐lactamase induction and which map in the amino‐ and carboxyl‐terminal ends of AmpR, respectively. The mutation in the amino terminus (S35F) was located in a helix‐turn‐helix region showing high homology to other members of the LysR regulator family. Therefore this mutation may directly abolish the contact between AmpR and its operator sequence. It is suggested that the C‐terminal mutation (Y264N) affects subunit interactions in AmpR. One constitutive mutant was isolated which mapped in the centre of the ampR gene. This G102E mutant leads to constitutive β‐lactamase expression in the absence of both β‐lactam inducer and ampG, a gene essential for induction in wild‐type enterobacteria. Another mutant protein, D135Y, showed wild‐type properties In an ampG* and an ampG::kan background, but could, unlike wild‐type AmpR, activate the ampC gene in an ampG1 mutant background. It is thought that ampG1 is a missense mutant. These two types of ampR mutants suggest that activation of ampC transcription is dependent on the conversion of AmpR into a transcriptional activator and that this activation may normally involve interactions with AmpG.
Marker exchange mutagenesis of the hydN genes in Desulfovibrio fructosovorans
Authors: Rousset M. , Dermoun Z. , Chippaux M. , Bélaich J. P. 
Published: October 2006

SummaryA strain of Desulfovibrio fructosovorans deleted from the hydN [NiFe]hydrogenase structural gene was constructed. A plasmid carrying a 7 kb DNA fragment on which the hydN gene had been replaced by the npt reporter gene (kanamycin‐resistant, KnR) was introduced into D. fructosovorans by electroporation. Southern analysis of one KnR clone demonstrated that the hydN gene had been eliminated by marker exchange. This mutant, which was devoid of the [NiFe]hydrogenase gene, still showed a 10% residual hydrogenase activity. Its ability to grow efficiently with H2 as sole energy source is discussed. This is the first report, in a member of the sulphate‐reducing bacteria, of a successful transformation and concomitant homologous recombination leading to a fully controlled genotype.