bluuebunny/crossref_metadata_2025_split huggingface dataset:
Co‐operative autoregulation of a replication protein gene
Authors: Gammie A. E. , Crosa J. H. 
Published: October 2006

SummaryIn this work we present the localization and characterization of the repi promoter (Prepl) and show aspects of the regulation. Comparison of Prepl with other autoregulated replication protein gene promoters revealed similarities, but Prepl differs from some of these characterized promoters in not being regulated by the heat‐shock RNA polymerase. Primer extension analysis showed that Prepl is contained within five helically aligned 18 base pair repeats, or 18‐mers of the previously defined minimal origin. In addition, we find that Prepl is autoregulated by a trans‐acting product encoded in the REPI region. Purified Repl protein binds to the 18‐mer region of the origin, suggesting that the repl gene is autoregulated by the protein product. The autoregutation appears to be co‐operative since decreasing the 18‐mer binding site region results in a concomitant non‐linear loss of auto‐repression. The deletion derivatives show a decreased ability to bind the Repl protein when compared with origin DNA containing all of the binding region. The diminished capacity of the various deletion derivatives to bind Repl in vitro correlates with the loss of autorepression seen in vivo.
Chloramphenicol‐induced stabilization of cat messenger RNA in Bacillus subtilis
Authors: Dreher J. , Matzura H. 
Published: October 2006

SummaryThe expression of the chloramphenicol‐inducible chloramphenicol‐acetyltransferase gene (cat), encoded on Staphylococcus aureus plasmid pUB112, is regulated via a translational attenuation mechanism. Ribosomes, which are arrested by chloramphenicol during synthesis of a short leader peptide, activate catm RNA translation by opening a 5′‐located stem‐loop structure, thus setting free the cat ribosome‐binding site. We have determined the 5′ and 3′ ends of cat mRNA and analysed its stability in Bacillus subtilis. In the absence of the antibiotic, the half‐life of cat mRNA is shorter than 0.5 min; it is enhanced to about 8 min by sub‐inhibitory concentrations of the drug. No decay intermediates of cat mRNA could be detected, indicating a very fast degradation after an initial rate‐limiting step. ochre nonsense mutations in the 5′ region of the cat structural gene, which eliminate cat mRNA translation, did not affect its chloramphenicol‐induced stabilization. Mutations in the leader‐peptide coding region, which abolish ribosome stalling and, therefore, cat gene induction, also eliminate cat mRNA stabilization. We conclude that cat mRNA is stabilized on induction by a chloram‐phenicol‐arrested ribosome, which physically protects a nuclease‐sensitive target site in the 5′ region of cat mRNA against exo‐ or endonucleolytic initiation of degradation. This protection is analogous to ermA and ermC mRNA and seems to reflect a general mechanism for stabilization of mRNA derived from inducible antibiotic resistance genes in B. subtilis.
Purification, characterization and nucleotide sequence of the periplasmic C4‐dicarboxylate‐binding protein (DctP) from Rhodobacter capsulatus
Authors: Shaw J. G. , Hamblin M. J. , Kelly D. J. 
Published: October 2006

SummaryA periplasmic binding protein essential for high‐affinity transport of the C4‐dicarboxylates malate, succinate and fumarate across the cytoplasmic membrane of the purple photosynthetic bacterium Rhodobacter capsulatus has been purified to homogeneity and some of its ligand‐binding properties characterized. The protein was not produced in a Tn5 insertion mutant unable to transport C4‐dicarboxylates under aerobic conditions in the dark. Wild‐type DNA corresponding to the location of the transposon insertion site was subcloned and a 1.5kb section sequenced. A complete open reading frame of 999bp was identified that encoded a 333‐residue protein (DctP) with a molecular weight of 36 128 with a 26‐residue amino‐terminal signal peptide. The identity of this protein with the purified dicarboxylate‐binding protein and the position of the predicted signal peptide cleavage site was confirmed by N‐terminal sequencing. No significant homology with other proteins was detected in database searches. A GC‐rich region of dyad symmetry was located 7bp downstream of the dctP translational stop codon. This structure may be of significance in regulating the relative abundance of DctP and other dct gene products which comprise the high‐affinity dicarboxylate transport system in this bacterium.
A DNA region of 9kbp contains all genes necessary for gas vesicle synthesis in halophilic archaebacteria
Authors: Horne M. , Englert C. , Wimmer C. , Pfeifer F. 
Published: October 2006

SummaryWe determined the minimal size of the genomic region necessary for gas vesicle synthesis in halophilic archaebacteria by transformation experiments, comparative DNA sequence analysis and investigation of gas vesicle (Vac) mutants. The comparison of the three genomic regions encoding gas vesicles in Halobacterium halobium (p‐vac‐ and c‐vac‐region) and Haloferax mediterranei (mc‐vac‐region) indicates high DNA sequence similarity throughout a contiguous sequence of 9kbp. In each case, this area encompassed at least 13 open reading frames (ORFs). Ten of these ORFs (gvpD to gvpM) were located 5′ to the vac gene encoding the major gas vesicle protein, but were transcribed from the opposite strand. At least two ORFs (gvpC, and gvpN) were located 3′ to each vac gene and transcribed from the same strand as the respective vac gene. In the p‐vac‐region present on plasmid pHH1 these ORFs were transcribed as at least three units, one transcript encompassing gvpD‐gvpE, the second encompassing ORFs gvpF to gvpM, and the third unit comprising the ORFs located 3′ to the p‐vac gene. In H. halobium Vac mutants copies of the insertion elements ISH2, ISH23, ISH26 or ISH27 were found to be integrated throughout the p‐vac‐region. The de novo synthesis of gas vesicles was tested by transformation of the Vac‐negative species, Haloferax volcanii, with various subfragments of the mc‐vac‐ or p‐vac‐region cloned into vector plasmids. In contrast to a fragment containing the entire 9 kbp region, none of the subfragments tested was sufficient to promote gas vesicle synthesis. However, gas vesicle synthesis could be restored in each Vac mutant containing an ISH element when the entire transcription unit encompassing the mutated gene on pHH1 was present in the wild‐type form on the vector construct.
A gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein export
Authors: Kontinen V. P. , Saris P. , Sarvas M. 
Published: October 2006

SummaryA gene locus of Bacillus subtilis identified by mutations (prs) conferring a defect in protein secretion was cloned from a lambdaGEM‐11 expression library. The sites of three closely linked prs mutations (prs‐3, prs‐29 and prs‐40) were found to reside in a 5.3kb DNA fragment, which also complemented the secretion defect in prs‐3 and prs‐29 mutants. Partial sequencing of the fragment showed that these three mutations affect one distinct gene (prs A) encoding a putative protein of 292 amino acids (33 kDa). Sequence analysis Indicated the PrsA protein to be a lipoprotein located outside the cytoplasmic membrane. Thirty percent identity was shown to the PrtM protein of Lactococcus lactis, which is involved in the maturation of an exported proteinase. The phenotypes of prsA mutants and the structural similarity of PrsA with PrtM suggest that PrsA may have a novel function at a late phase in protein export.
The repair of double‐strand breaks and S1 nuclease‐sensitive sites can be monitored chromosome‐specifically in Saccharomyces cerevisiae using pulsed‐field gel electrophoresis
Authors: Geigl E.‐M. , Eckardt‐Schupp F. 
Published: October 2006

SummaryRepair under non‐growth conditions of DNA double‐stranded breaks (DSBs) and S1 nuclease‐sensitive sites (SSSs; e.g. DNA damage which is processed by in vitro treatment with S1 nuclease to DSBs) induced by [60Co]‐gamma‐rays (200 Gy; anoxic conditions) was monitored in a diploid repair‐competent strain of Saccharomyces cerevisiae. We used pulsed‐field gel electrophoresis (PFGE), which allows the separation of chromosome‐sized yeast DNA molecules, to determine the number of DSBs and SSSs in Individual chromosome species of yeast. Our results indicate that SSSs which have been regarded as clusters of base damage in opposite DNA strands are repaired efficiently in a repair‐proficient diploid strain of yeast. The time course of SSS repair is comparable to the one of DSB repair, indicating similarities in the molecular mechanism. Both types of repair kinetics are different for different chromosome species.
Cloning and sequencing of the structural gene for the porin protein of Bordetella pertussis
Authors: Li Z. M. , Hannah J. H. , Stibltz S. , Nguyen N. Y. , Manclark C. R. , Brennan M. J. 
Published: October 2006

Summary Bordetella pertussis produces a porin protein which is a prominent outer membrane component found in both virulent and avirulent strains. N‐terminal amino acid analysis of purified B. pertussis porin was performed and this amino acid sequence was used to design an oligonucleotide that was then utilized to screen a λgt11 library containing randomly sheared fragments of DNA from B. pertussis strain 347. One clone, λBpPor, was identified and subcloned into pUC18. A portion of the DNA Insert in this subclone, pBpPor1, was sequenced and shown to contain the N‐terminal region of the structural porin gene. This truncated gene sequence was used to design an additional oligonucleotide that was used to identify a clone, pBpPor2, which overlapped with pBpPor1 and contained a termination codon. The structural gene deduced from this sequence would encode a 365‐amino‐acid polypeptide with a predicted mass of 39103 daltons. The predicted product also contains a signal sequence of 20 residues that is similar to that found in other porin genes. The predicted B. pertussis porin protein sequence contains regions that are homologous to regions found in porins expressed by Neisseria species and Escherichia coli, including the presence of phenylalanine as the carboxy‐terminal amino acid. DNA hybridization studies indicated that both virulent and avirulent strains of B. pertussis contain only one copy of this gene and that Bordetella bronchiseptica and Bordetella parapertussis contain a similar gene.
DNA sequencing and complementation/deletion analysis of the bchA‐puf operon region of Rhodobacter sphaeroides: in vivo mapping of the oxygen‐regulated puf promoter
Authors: Hunter C. N. , McGlynn P. , Ashby M. K. , Burgess J. G. , Olsen J. D. 
Published: October 2006

SummaryWithin the photosynthetic gene cluster of Rhodobacter sphaeroides the genes encoding light‐harvesting LHI and reaction‐centre complexes are transcriptionally linked in the order pufBALMX. The region stretching 1.6 kb upstream of pufB has been examined by DNA sequencing and by complementation/deletion analysis. These studies demonstrate that three open reading frames are located upstream of pufB. One open reading frame, designated bchA, terminates just Inside pufQ, which is located proximal to pufB. BchA contains a 37 bp region that functions as the oxygen‐regulated promoter for pufQ, and probably for the puf operon as a whole. We also demonstrate that the protein encoded by pufQ appears to play a role in bacteriochlorophyll biosynthesis.
The kilA operon of promiscuous plasmid RK2: the use of a transducing phage (λpklaA‐1) to determine the effects of the lethal klaA gene on Escherichia coli cells
Authors: Saltman L. H. , Kim K.‐S. , Figurski D. H. 
Published: October 2006

SummaryThe kil‐kor regulon of promiscuous plasmid RK2 includes the replication initiator gene trfA and several potentially host‐lethal kil loci (kilA, kilB, kilC, kilE), whose functions may be involved in plasmid maintenance or broad host range. The kilA locus consists of a single operon of three genes (klaA, klaB, klaC), each of which is lethal when expressed from the klaA promoter in the absence of repressors encoded by korA and korB. In this study, we examined the effects of the unregulated klaA gene on the host cell. Bacteriophage λ was used to construct a transducing phage (λpklaA‐1) that allows efficient introduction of the klaA gene into Escherichia coli. Cells lacking korA and korB (to allow uncontrolled expression of klaA) and expressing λ repressor (to prevent phage lytic growth) are killed by λpkfaA‐1. Cell death is dependent on the klaA structural gene, independent of the SOS system of the host, and is prevented by the presence of korA and korB. λpklaA‐1 was used to synchronously infect ceils lacking korA and korB to determine the effects of klaA on the cells over time. The earliest effects, visible at two hours post‐infection, are inhibition of growth of the culture, formation of elongated cells, and striking changes in the appearance of the outer membrane. After four to five hours, the viability of the culture declined sharply and macromolecular synthesis ceased. The distinct class of early events is consistent with the hypothesis that the KlaA polypeptide interacts with a specific target in the host cell.
Identification of cfxR, an activator gene of autotrophic CO2 fixation in Alcaligenes eutrophus
Authors: Windhöver U. , Bowien B. 
Published: October 2006

SummaryA regulatory gene, cfxR, involved in the carbon dioxide assimilation of Alcaligenes eutrophus H16 has been characterized through the analysis of mutants. The function of cfxR is required for the expression of two cfx operons that comprise structural genes encoding Calvin cycle enzymes. CfxR (34.8 kDa) corresponds with an open reading frame of 954 bp, with a translational initiation codon 167 bp upstream of the chromosomal cfx operon. The cfx operon and cfxR are transcribed divergently. The N‐terminal sequence of CfxR is very similar to those of bacterial regulatory proteins belonging to the LysR family. Heterologous expression of cfxR in Escherichia coli was achieved using the pT7‐7 system. Mobility shift experiments demonstrated that CfxR is a DNA‐binding protein with a target site upstream of both the chromosomal and the plasmid‐encoded cfx operons.
Escherichia coli rpoA mutation which impairs transcription of positively regulated systems
Authors: Thomas M. S. , Glass R.E. 
Published: October 2006

SummaryThe rpoA341 (phs) mutation of Escherichia coli results in decreased expression of several positively regulated operons and has been mapped to within or very near the rpoA gene encoding the α subunit of RNA polymerase. We have shown that plasmid‐directed synthesis of the wild‐type α subunit can complement the defective phenotypes associated with this mutation consistent with its proposed location within rpoA. This mutation was mapped by marker rescue to within a 182bp region near the 3’end of rpoA and was subsequently transferred to a plasmid by recombination in vivo. DNA sequence analysis revealed that the RpoA341 phenotype was the result of the substitution of lysine 271 by glutamate within the α polypeptide. We discuss this result in relation to our current understanding of the functional organization of the α subunit.
Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system
Authors: Gibson M. M. , Bagga D. A. , Miller C. G. , Maguire M. E. 
Published: October 2006

SummaryThe CorA Mg2+ transport system of Salmonella typhimurium mediates both influx and efflux of Mg2+. Mutations at the corA locus (83.5min) confer resistance to Co2+. Using transposon mutagenesis, three additional Co2+ resistance loci (corB, corC, and corD) were found and mapped to 55, 15, and 3min, respectively, on the S. typhimurium chromosome. No mutations corresponding to the reported corB locus at 95min in Escherichia coli were obtained. The corB, corC, and corD mutations confer levels of Co2+ resistance intermediate between those of the wild‐type and corA mutations.Isogenic strains were constructed containing combinations of transposon insertion mutations in each of the three Co2+ resistance loci to assess their influence on the CorA Mg2+ transport system. The Vmax and Km values for 28Mg2+ or for 57Co2+ and 63Ni2+ influx, analogues of Mg2+ transported by the CorA system, were changed less than twofold compared with the wild‐type values, regardless of the mutation(s) present. However, while efflux of 28Mg2+ through the CorA system was decreased threefold in strains carrying one or two mutant alleles among corB, corC, or corD, efflux was completely abolished in either a corA or a corBCD strain. Thus, although the corA gene product is necessary and sufficent to mediate Mg2+ influx, Mg2+ efflux requires the presence of a wild‐type allele of at least one of the corB, corC or corD loci.
Regulation of toxA and regA by the Escherichia coli fur gene and identification of a Fur homologue in Pseudomonas aeruginosa PA103 and PA01
Authors: Prince R. W. , Storey D. G. , Vasil A. I. , Vasil M. L. 
Published: October 2006

SummaryA multicopy plasmid containing the Escherichia coli fur gene was introduced into Pseudomonas aeruginosa strain PA103C. This strain contains a toxA‐lacZ fusion integrated into its chromosome at the toxA locus. Beta‐galactosidase synthesis in this strain is regulated by iron, as is seen for exotoxin A production. Beta‐galactosidase synthesis and exotoxin A production in PA103C containing multiple copies of E. coli fur was still repressed in low iron conditions. The transcription of regA, a positive regulator of toxA, was also found to be inhibited by multiple copies of the E. coli fur gene. In addition, the ability of PA103C containing multiple copies of E. coli fur to produce protease was greatly reduced relative to PA103C containing a vector control.A polyclonal rabbit serum containing antibodies that recognize E. coli Fur was used to screen whole‐cell extracts from Vibrio cholerae, Shigella flexneri, Salmonella typhimurium and Pseudomonas aeruginosa. All strains tested expressed a protein that was specifically recognized by the anti‐Fur serum. These results and those described above suggest that Fur structure and function are conserved in a variety of distinct bacterial genera and that at least some of these different genera use this regulatory protein to control genes encoding virulence factors.
Radiation resistance in Schizosaccharomyces pombe
Authors: Subramani S. 
Published: October 2006

SummaryThe fission yeast Schizosaccharomyces pombe serves as an excellent alternative and complementary model system for the analysis of genes and gene products involved in DNA repair. This brief review outlines the advantages of S. pombe and describes the radiation‐sensitive mutants available for the analysis of DNA repair and recombination mechanisms in this organism. The progress in the cloning and characterization of representative genes is also described.
deoP1 promoter and operator mutants in Escherichia coli: isolation and characterization
Authors: Dandanel G. , Hammer K. 
Published: October 2006

SummaryPlasmid DNA containing deoP1, one of the two major promoters of the deo operon, has been mutagenized using hydroxylamine, and promoter down‐mutations and operator mutations were selected. The isolated mutants are all located within a 16bp palindromic sequence containing the –10 region of deoP1. The results show that RNA polymerase and DeoR repressor compete for the same DNA target. The deoP1 promoter activity is dependent on a TG motif one base pair upstream of the –10 consensus sequence. The sequence of the deo operator site was further verified by use of a synthetic linker.
Structural and genetic analysis of the bvg locus in Bordetella species
Authors: Arico B. , Scarlato V. , Monack D. M. , Falkow S. , Rappuoli R. 
Published: October 2006

SummaryThe bvg locus contains two genes, bvgA and bvgS, which control the expression of the virulence‐associated genes in Bordetella species by a system similar to the two‐component systems used by a variety of bacterial species to respond to environmental stimuli. We determined the nucleotide sequence of the bvg loci of Bordetella parapertussis and Bordetella bronchiseptica and compared them with the previously determined sequence of Bordetella pertussis. The nucleotide and amino acid sequences of the bvg loci of these species are well conserved in those regions coding for the protein domains which have putative kinase and DNA‐binding activities. In marked contrast, the region of BvgS that codes for the protein domain with putative sensor activity shows a high degree of variability. In total, we find 198 base‐pair changes in the bvg loci of B. parapertussis and B. bronchiseptica relative to the bvg locus of B. pertussis. One hundred and seventy‐three of these base‐pair changes are identical in S. parapertussis and B. bronchiseptica. Tills confirms our previous observation that B. parapertussis and B. bronchiseptica are more related to each other than to B. pertussis. We have mapped the mutations that cause phase changes in B. bronchiseptica and we have found that in three cases these are due to spontaneous deletions in the bvgS gene.The wild‐type bvg locus present on a multicopy plasmid cannot complement avirulent derivatives of B. bronchiseptica to wild‐type levels, but it can do so when the bvgA gene on the plasmid is inactivated. This suggests that hyperexpression of bvgA down‐regulates the bvg system.
Analysis of the topology of the cytochrome d terminal oxidase complex of Escherichia coli by alkaline phosphatase fusions
Authors: Newton G. , Yun C.‐H. , Gennis R. B. 
Published: October 2006

SummaryThe cytochrome d complex of Escherichia coli is a heterodimer located in the bacterial cytoplasmic membrane, where it functions as a terminal oxidase of the aerobic respiratory chain. The topology of each of the two subunits of the cytochrome d complex was analysed by the genetic method involving alkaline phosphatase gene fusions. These fusions were generated by both an in vivo method using the transposon TnphoA and an in vitro method of construction. A total of 48 unique fusions were isolated and the whole‐cell alkaline phosphatase‐specific activities were determined. Data from these fusions, in combination with information from other studies, provide the basis for two‐dimensional models for each of the two subunits, defining the way in which the subunits fold in the inner membrane of E. coli.
The replication of plasmid pSC101
Authors: Manen D. , Caro L. 
Published: October 2006

SummaryThe origin of replication of plasmid pSC101 presents features reminiscent of those found in a number of plasmids. As for those plasmids, many details about the way it initiates its replication are beginning to be known, but the regulation of this process will not be easily understood.
3′ processing in Dictyostelium: unusual sequence requirements and interaction with a downstream promoter
Authors: Maniak M. , Nellen W. 
Published: October 2006

SummaryWe have determined the sequence requirements for 3′‐processing in Dictyostelium and find that a single AATAAA site, embedded in an A/T‐rich environment is sufficient. A synthetic oligonucleotide containing the additional GT/T‐rich element, which is necessary for 3′‐processing in higher eukaryotes, is not used in Dictyostelium. On the basis of reports suggesting termination signals (upstream terminators) in Dictyostelium promoters, we investigated possible interactions between processing signals and regulatory elements. Our data suggest that upstream termination enhances transcription from a downstream promoter.