bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissecting the functional role of PriA protein‐catalysed primosome assembly in Escherichia coli DNA replication
Authors: Zavitz K. H. , Marians K. J.
Published: October 2006

The multi‐functional PriA protein of Escherichia coli (formerly replication factor Y or protein n′) serves to guide the ordered assembly of the primosome, a mobile multiprotein replication priming/helicase complex. Primosome assembly is essential for bacteriophage ØX174 complementary DNA strand synthesis and ColE1‐type plasmid replication reconstituted in vitro with purified proteins. The biochemical activities of the primosome suggest that it can fulfil the primase/helicase requirement on the lagging‐strand DNA template during cellular DNA replication. However, reconstruction in vitro of DNA replication of small plasmids containing the E. coli origin of DNA replication (oriC) does not require the complete complement of primosomal proteins. Thus, the extent to which PriA‐catalysed primosome assembly participates in chromosomal replication has remained unclear. The recent isolation of the genes encoding PriA, PriB (protein n), PriC (protein n″), and DnaT (protein i) has provided the necessary tools for addressing this issue. The phenotype of mutations in these genes, and other results described in this review, suggest that assembly of the primosome catalysed by PriA does in fact contribute at some stage to normal cellular DNA replication. A model for primososme‐catalysed reactivation of a dysfunctional replication fork is discussed.
Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria
Authors: Aulkemeyer P. , Ebner R. , Heilenmann G. , Jahreis K. , Schmid K. , Wrieden S. , Lengeler J. W.
Published: October 2006

Sucrose‐positive derivatives of Escherichia coli K‐12, containing the plasmid pUR400, and of Klebsiella pneumoniae hydrolyse intracellular sucrose 6‐phosphate by means of an invertase into d‐glucose 6‐phosphate and free d‐fructose. The latter is phosphorylated by an ATP‐dependent fructokinase (gene scrK of an scr regulon) to d‐fructose 6‐phosphate. The lack of ScrK does not cause any visible phenotype in wild‐type strains of both organisms. Using genes and enzymes normally involved in d‐arabinitol metabolism from E. coli C and K. pneumoniae, derivatives of E. coli K‐12 were constructed which allowed the identification of scrK mutations on conventional indicator plates. Cloning and sequencing of scrK from sucrose plasmid pUR400 and from the chromosome of K. pneumoniae revealed an open reading frame of 924 bp in both cases — the equivalent of a peptide containing 307 amino acid residues (Mr, 39 and 34 kDa, respectively, on sodium dodecyl sulphate gels). The sequences showed overall identity among each other (69% identical residues) and to a kinase from Vibrio alginoiyticus (57%) also involved in sucrose metabolism, lower overall identity (39%) to a d‐ribose‐kinase from E. coli, and local similarity to prokaryotic, and eukaryotic phosphofructokinases at the putative ATP‐binding sites.
Molecular analysis of the Corynebacterium glutamicum gene involved in lysine uptake
Authors: Seep‐Feldhaus A. H. , Kalinowski J. , Pühler A.
Published: October 2006

Two Corynebacterium glutamicum mutants defective in lysine uptake were identified by analysing mutants resistant to S‐(2‐amtnoethyl)‐cysteine (AEC). A 5.6kb genomic DNA fragment restoring AEC sensitivity and lysine uptake was isolated. A 4.2kb subfragment was sequenced and three open reading frames were identified. Subcloning and gene disruption experiments showed that only the first open reading frame, termed lysl, is involved in lysine uptake. Lysl consists of 501 amino acids with a Mr of 53600. The hydrophobicity profile suggests that the lysl gene product is an integral membrane protein with 13 transmembrane segments. The amino acid sequence of lysl displays strong homology to that of the arcD gene product of Pseudomonas aeruginosa, which is proposed to act as an arginine–ornithine antiporter. Investigation of the influence of the lysl gene on lysine secretion suggests the existence of a separate lysine efflux system in C. glutamicum.
Co‐operative autoregulation of a replication protein gene
Authors: Gammie A. E. , Crosa J. H.
Published: October 2006

In 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

The 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

A 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

We 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

A 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

Repair 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

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

Within 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

The 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

A 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

The 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

The 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

A 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

The 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

Plasmid 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

The 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

The 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

The 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

We 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.
Molecular analysis of the virulence locus of the Salmonella dublin plasmid pSDL2
Authors: Krause M. , Roudier C. , Fierer J. , Harwood J. , Guiney D.
Published: October 2006

The virulence properties of various non‐typhoid Salmonella serotypes depend on the presence of large plasmids 60–100 kb in size. We have shown previously that the virulence region on the 80 kb plasmid pSDL2 of Salmonella dublinLane maps within a 14kb SalI fragment. In this report we show that an 8.2kb region within this fragment is sufficient to express lethal disease in BALB/c mice. Sequence analysis of this segment revealed six sequential open reading frames designated vsdA–F, which encode putative proteins of 13–65kDa. Deletion analysis and location of Tn5‐oriT inserts which abolish virulence suggest that vsdA, vsdC, vsdD and vsdE are essential for virulence expression. Downstream of vsdF we discovered a locus involved in stable plasmid maintenance. Deletion of that region resulted in plasmid multimerization and instability.
The TraM protein of plasmid R1 is a DNA‐binding protein
Authors: Schwab M. , Gruber H. , Högenauer G.
Published: October 2006

The TraM protein of the resistance plasmid R1 was purified to homogeneity and used for DNA‐binding studies. Both gel retardation‐and footprint experiments showed that TraM specifically binds to DNA of plasmid R1 comprising the region between the origin of transfer and the traM gene. Several TraM molecules bind and, according to the footprint experiments, two distinct sites of specific binding exist The two sites are separated from each other by 12 nucleotides and each contains an inverted repeat. DNase I protection assays showed that the initial TraM binding occurs at these palindromic sequences. At higher protein concentrations the lengths of the DNA segments protected by TraM were increased towards the traM gene. In one region this extension leads to binding of TraM protein at its own promoters.
The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coliα‐haemolysin
Authors: Guzzo J. , Duong F. , Wandersman C. , Murgier M. , Lazdunski A.
Published: October 2006

The extracellular alkaline protease produced by Pseudomonas aeruginosa is secreted by a specific pathway, independent of the pathway used by most of the other extracellular proteins of this organism. Secretion of this protease is dependent on the presence of several genes located adjacent to the apr gene. Complementation studies have shown that PrtD, E, and F, the three secretion functions for Erwinia chrysanthemi proteases B and C (Létofféet al., 1990), can mediate the secretion of the alkaline protease by Escherichia coli. he secretion functions involved in α‐haemloysin secretion in E. coli(hlyB, hlyD, tolC) can also be used to complement alkaline protease secretion by E. coli, although less efficiently. These data indicate that protease secretion mechanisms in Pseudomonas and Erwinia are very similar and are homologous to that of E. coliα‐haemolysin.
The trpC gene of Phanerochaete chrysosporium is unique in containing an intron but nevertheless maintains the order of functional domains seen in other fungi
Authors: Schrank A. , Tempelaars C. , Sims P. F. G. , Oliver S. G. , Broda P.
Published: October 2006

The Phanerochaete chryososporium trpC gene has been isolated by complementation of an Escherichia coli trpC mutant. The full extent of the fungal gene, determined by sequence analysis, was found to be 2414bp. This includes a single intron of 50bp, the presence of which was confirmed by RNA‐primed polymerase chain reaction analysis. This feature makes the P. chrysosporium gene unique when compared to equivalent genes from other filamentous fungi. The P. chrysosporium trpC gene encodes a single protein containing three enzyme activities involved in tryptophan biosynthesis arranged in the order: NH2–GAT–IGPS–PRAI–COOH. This order is conserved in all filamentous fungi so far examined and, indeed, is the gene order within the E. coli trp operon.
The role of the ‘gearbox’ in the transcription of essential genes
Authors: Vicente M. , Kushner S. R. , Garrido T. , Aldea M.
Published: October 2006

Regulation of transcription occurs at different levels, one being in the presence of sequences specifically recognized by different forms of RNA polymerase, i.e. the promoters. Three different kinds of promoter are defined according, among other things, to their dependence on the growth rate of the cell: the house‐keeper' promoter of many metabolic genes, the stringent promoter found at several rRNA and ribosomal protein genes, and the ‘gearbox’ at genes whose products are required at higher relative amounts at lower growth rates. The identified gearbox promoters of Escherichia coli share specific homologies in the ‐10, ‐35 and upstream regions. Although there may be different types of gearbox promoters, the ‐10 sequence of one of these promoters has been found to be essential for functioning as a gearbox. This suggests the existence of specific sigma factors for its transcription. RpoS (KatF) is a likely candidate for being one of these sigma factors. Computer simulation allows us to predict that such sigma factors should, in turn, be expressed following a gearbox mode, which would then imply the existence of self‐regulated loops contributing to the expression of some genes of bacterial division. Some bacterial gene products need to be synthesized al fixed amounts per cell
Occurrence and biological function of cytochrome P450 monooxygenases in the actinomycetes
Authors: O'Keefe D. P. , Harder P. A.
Published: October 2006

Many species within the order Actinomycetales contain one or more soluble cytochrome P450 monooxygenases, often substrate‐inducible and responsible for a variety of xenobiotic transformations. The individual cytochromes exhibit a relatively broad substrate specificity, and some strains have the capacity to synthesize large amounts of the protein(s) to compensate for low catalytic turnover with some substrates. All three of the Streptomyces cytochromes sequenced to date are exclusive members of one P450 family, CYP105. In several instances, monooxygenase activity arises from induction of a P450 and associated ferredoxin, or of a P450 only, suggesting that some essential electron donor proteins (reductase and ferredoxin) are not coordinately regulated with the cytochrome. The overall properties of these systems suggest an adaptive strategy whose twofold purpose is to maintain a competitive advantage via the production of secondary metabolites, and, whenever possible, to utilize unusual growth substrates by introducing metabolites from these reactions into the more substrate‐specific primary metabolic pathways.
IIIegitimate integration of non‐replicative vectors in the genome of Rhodococcus fascians upon electro‐transformation as an insertional mutagenesis system
Authors: Desomer J. , Crespi M. , Van Montagu M.
Published: October 2006

Electrotransformation of Rhodococcus fascians by non‐replicating plasmids containing a suitable resistance marker resulted in stable transformants by integration of these constructs at various sites in the genome, thereby generating different mutations. Tagged genes could be isolated in Escherichia coli owing to the presence of a CoIE1 replicon and an ampicillin resistance gene in the inserted sequences.

Southern analysis and nucleotide sequencing revealed that recombination can occur at defined locations in the plasmid, while no site preference for target sequences could be detected. Low homology between the recombining sequences indicates illegitimate recombination. The specificity of the plasmid sites could be explained by assuming a linear recombination intermediate, generated by cleavage of the transformed plasmid.
Characterization of two insertion sequences, IS701 and IS702, from the cyanobacterium Calothrix species PCC 7601
Authors: Mazel D. , Bernard C. , Schwarz R. , Castets A. M. , Houmard J. , de Marsac N. Tandeau
Published: October 2006

We describe the characterization of two insertion elements, IS701 and IS702, isolated from Calothrix species PCC 7601. These insertion elements were cloned from spontaneous pigmentation mutants. Both show the characteristics of typical bacterial insertion sequences, i.e. they present long terminal inverted repeats and they duplicate target DNA upon insertion. These elements share no homology with the only other cyanobacterial insertion sequence described so far, IS891. At least 15 copies of IS701 and 9 copies of IS702 were detected by hybridization experiments in the Calothrix 7601 genome. Their occurrence in several cyanobacterial strains is also reported.
In vivo analysis of integration of membrane proteins in Escherichia coli
Authors: Ito K. , Akiyama Y.
Published: October 2006

The in vivo process of membrane protein integration was studied by pulse‐labelling Escherichia coli cells, and assessing integral anchoring of labelled proteins to the lipid bilayer based on their resistance to alkali extraction. To conduct this experiment, conditions for extracting E. coli proteins with alkali were refined, and the immunoprecipitation procedures were improved to allow effective detection of integral membrane proteins. Examination of pulse‐labelled, integral membrane proteins, including lactose permease (LacY), SecY, cytochrome o subunit II and leader peptidase revealed that all were in the alkali‐insoluble fraction, indicating that membrane integration of these proteins takes place rapidly in wild‐type cells. However, when LacY was synthesized in excess from a multicopy plasmid, significant proportions were found in the alkali‐soluble fraction, indicating that the solubility in alkali is not an intrinsic property of the protein, and suggesting that LacY depends on some limited cellular factor for membrane integration. The unintegrated species of LacY sedimented slowly through an alkaline sucrose gradient. The secY24 mutant cells accumulated higher proportions of unintegrated LacY molecules at lower levels of overproduction than the sec+ cells. LacY overproduction in wild‐type cells was found to inhibit processing (export) of β‐lactamase but not of OmpA and OmpF. These results are interpreted to mean that integration of LacY depends on multiple cellular components, one of which is also involved in export of β‐lactamase.
Membrane topology of the integral membrane components, OppB and OppC, of the oligopeptide permease of Salmonella typhimurium
Authors: Pearce S. R. , Mimmack M. L. , Gallagher M. P. , Gileadi U. , Hyde S. C. , Higgins C. F.
Published: October 2006

The oligopeptide permease of Salmonella typhimurium is a periplasmic binding protein‐dependent transport system. Five gene products, OppABCDF, are required for the functioning of this transporter, two of which (OppB and OppC) are highly hydrophobic, integral membrane proteins and are responsible for mediating passage of peptides across the cytoplasmic membrane. OppB and OppC are each predicted, from their sequences, to span the membrane many times. In this paper we describe experimental evidence confirming these predictions using a combination of biochemical, immunological and genetic procedures. Each of these two proteins is shown to span the membrane six times, with the N‐ and C‐termini both being located at the cytoplasmic face of the membrane. Opp is apparently a typical member of the ABC (ATP‐binding cassette) superfamily of transporters. These findings, therefore, have general implications for the organization and function of other ABC transporters, including the human multidrug resistance protein and the product of the cystic fibrosis gene.
The copy number of plasmid pLS1 is regulated by two trans‐acting plasmid products: the antisense RNA II and the repressor protein, RepA
Authors: del Solar G. , Espinosa M.
Published: October 2006

The promiscuous plasmid pLS1 encodes two transacting elements that regulate its copy number: protein RepA and antisense RNA II. In vitro transcription showed that RNAs for both repressors are synthesized from two promoters, PAB and PII From PAB, genes encoding RepA (transcriptional repressor) and RepB (initiator of replication) are cotranscribed, the target of RepA being located within PAB Mutants in repA or in PAB are still sensitive to RepA. However, cloning of the repA gene in a compatible replicon did not result in incompatibility towards pLS1. From PII, the 50‐nucleotide RNA II is synthesized. The main incompatibility determinant towards pLS1 corresponds to the coding sequence for RNA II. The RNA II target could be reduced to 21 nucleotides, including the RepB initiation of translation signals. We propose that plasmids of the pLS1 family (pE194, pADB201, and pLB4) share functional and structural characteristics for the regulation of their copy numbers.
Inducible DNA repair and differentiation in Bacillus subtilis: interactions between global regulons
Authors: Yasbin Ronald E. , Cheo David L. , Bayles Ken W.
Published: October 2006

The SOS response of Escherichia coli has become a paradigm for the study of inducible DNA repair and recombination processes in many different organisms. While these studies have demonstrated that the components of the SOS response appear to be highly conserved among bacterial species, as with most models, there are some significant variations. Perhaps the best example of this comes from an analysis of the SOS‐like system of the developmental organism. Bacillus subtilis. Accordingly, the most striking difference is the complex developmental regulation of the SOS system as this organism differentiates into its competent state. In this review we have given an overview of the elements that comprise the SOS system of B. subtilis. Additionally, we have summarized our most recent findings regarding the regulation of this regulon. Using these results along with new findings from other laboratories we have provided provocative molecular models for the regulation of the B. subtilis SOS system in response to DNA damage and during competent cell formation.
Molecular analysis of the rfb gene cluster of Salmonella serovar muenchen (strain M67): the genetic basis of the polymorphism between groups C2 and B
Authors: Brown P. K. , Romana L. K. , Reeves P. R.
Published: October 2006

The rfb (O antigen) gene cluster of group C2 Salmonella differs from that of group B in a central region of 12.4kb: we report the sequencing of this region of strain M67 (group C2) and a subsequent comparison with the central region of strain LT2 (group B). We find a block of seven open reading frames unique to group C2 which encode the O antigen polymerase (rfc) and the transferases responsible for assembly of the group C2 O antigen. The remaining rfb genes are common to strains M67 and LT2, but rfbJ (GDP‐abequose synthase) and rfbM and rfbK (GDP‐mannose synthesis), which are immediately adjacent to the central region, are highly divergent. All these genes have a low G+C content and appear to have been recent additions to Salmonella enterica. We discuss the evolutionary significance of the arrangement and divergence of the genes in the polymorphism of the rfb cluster.
Involvement of the Arc system in redox regulation of the Cob operon in Salmonella typhimurium
Authors: Andersson Dan I.
Published: October 2006

Transcription of the cobalamin biosynthetic (cob) genes in Salmonella typhimurium is induced during anaerobic growth. Two mutants defective in induction of the Cob operon were isolated. The mutants showed decreased anaerobic induction, both during conditions of fermentation and anaerobic respiration, relative to the wild type. The mutant genes were localized by Hfr and transduction mapping to 100 minutes and 69 minutes on the chromosome. The mutants were sensitive to The dye toluidine blue. The mapping and the dye sensitivity suggest that these mutants are defective in The arcA and arcB genes, not previously identified in Salmonella typhimurium. Thus The regulatory function of the Arc system is expanded to include anaerobic activation of The Cob operon.
AIDA‐I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a precursor molecule
Authors: Benz Inga , Schmidt M. Alexander
Published: October 2006

The adherence mechanisms of enteropathogenic Escherichia coli (EPEC) to epithelial cells are still not understood. To study the molecular basis of the diffuse adherence (DA) phenotype exhibited by diarrhoeagenic E. coli expressing classical EPEC serotypes we investigated strain 2787 (O126:H27) isolated from a case of infantile diarrhoea. A 6.0 kb plasmid‐derrved DNA fragment mediates the DA phenotype and encodes the 100 kDa adhesin protein AIDA‐I (adhesin involved in diffuse adherence). Sequencing of the entire fragment revealed two open reading frames which encoded proteins of 45 kDa and 132 kDa, respectively. The 132 kDa protein has been identified as an AIDA‐I precursor protein. After cleavage of the signal sequence further processing at the C‐terminus of the 132 kDa precursor leads to the mature ∼100 kDa AIDA‐I. While the exact function of the cytopiasmic 45 kDa protein is not known, preliminary evidence indicates that it is necessary for the correct maturation of AIDA‐I. The AIDA‐l precursor exhibits significant homology with the virG(icsA) protein of Shigella flexneri which seems to be involved in the intercellular spread of invasive Shigella organisms.
PCR‐amplified length polymorphisms in tRNA intergenic spacers for categorizing staphylococci
Authors: Welsh John , McClelland Michael
Published: October 2006

The intergenic spacers between some adjacent tRNA genes were shown to be polymorphic in length when closely related Staphylococcus species were compared. A simple procedure was developed to detect and sequence these tRNA intergenic length polymorphisms (tRNA‐ILPs). A comparison of homologous tRNA gene sequences flanking these ILPs in three Staphylococcus species was used to derive primers for high‐stringency amplification of the ILPs by the polymerase chain reaction (PCR). The detection of tRNA‐ILPs by PCR allowed the classification of virtuatly all strains from the five species of Staphylococcus that were examined. The procedure used to identify, sequence and derive primers for PCR detection of tRNA‐ILPs in Staphylococcus should be applicable to many other genera of eubacteria. These primers could be used on uncultured material such as clinical samples.
Early events in yeast mitochondrial protein targeting
Authors: Verner Keith
Published: October 2006

Protein import into mitochondria involves a number of complex steps occurring in the cytosol, on the mitochondrial surface, and inside the organelle. Once an initial interaction between mitochondrial proteins and their specific receptors occurs, the proteins are transported into the organelle in a series of reactions involving (in the case of a protein to be translocated into the mitochondrial matrix) the mitochondrial membrane potential, ATP hydrolysis and an undetermined number of membrane components. Inside the organelle, mitochondrial proteins are processed and sorted to their final intramitochondrial destinations. The earliest steps in the import process take place in the cytosol and include the synthesis of the mitochondrial proteins themselves, their interaction with cytosolic factors, and perhaps the establishment of cotranslational import complexes on the mitochondrial surface. These early events are important because it is during this phase that the system as a whole is most sensitive to cytosolic conditions that may exert control over the entire import process.
Differential regulation of two genes encoding lysyl‐tRNA synthetases in Escherichia coli: lysU‐constitutive mutations compensate for a lysS null mutation
Authors: Kawakami K. , Ito K. , Nakamura Y.
Published: October 2006

Lysyl‐tRNA synthetases are synthesized in Escherichia coli from two distinct genes, lysS and lysU, which are regulated differentially. A strain which is null for lysS, the constitutive gene, was created by gene disruption (lysS1) and exhibited cold‐sensitive lethality. Hence, lysS is dispensable at high temperatures. This cold sensitivity was suppressed by a multi‐copy plasmid carrying lysU, the inducible gene. These data are interpreted as indicating that lysS is functionally replaceable by lysU for cell growth, and that the cold sensitivity of lysS1 is caused by insufficient expression of lysU at low temperatures. To investigate the mechanism of lysU expression, cold‐resistant bypass mutations were isolated from lysS1, and named als (for abandonment of lysS.). Two als mutations which were linked to lysU contain IS2 insertions upstream of the lysU promoter. They caused a 16–19‐fold increase in the lysU‐mRNA level. Furthermore, deletion mutations created immediately upstream of the lysU promoter restored growth of lysS1. These results suggest that transcription of lysU is negatively controlled by a cis‐element located upstream of the promoter.