bluuebunny/crossref_metadata_2025 huggingface dataset:
Fumarate reductase of Escherichia coli: an investigation of function and assembly using in vivo complementation
Authors: Condon C. , Weiner J. H.
Published: October 2006

Recombinant plasmids which carried portions of the Escherichia coli frd operon were constructed and their expression examined by in vivo complementation of E. Coli MI 1443. This strain lacked a chromosomal frd operon and was unable to grow anaerobically on glycerol and fumarate. Introduction of all four fumarate reductase subunits into E. coli MI1443 was essential for the restoration of growth. The FRD A, FRD B dimer (but neither subunit alone) was active in the benzyl viologen oxidase assay. Both FRD C and FRD D were required for membrane association of fumarate reductase and for the oxidation of reduced quinone analogues. Introduction into E. coli MI1443 of the frdABC and frdD genes on two separate plasmid vectors failed to restore anaerobic growth on glycerol and fumarate. Thus separation of the DNA coding for the FRD C and FRD D proteins affected the ability of fumarate reductase to assemble into a functional complex.
Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae
Authors: Britton P. , Cármenes R. S. , Page K. W. , Garwes D. J. , Parral F.
Published: October 2006

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced. Two non‐overlapping open reading frames (ORFs) were identified. The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43 483), was shown to be the viral nucleoprotein gene. The second ORF, found 3’to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product. The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter. Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of M, 47000, identical to the viral product, that reacted with a specific monoclonal antibody.
Isolation and molecular characterization of the gene encoding allophycocyanin B, a terminal energy acceptor in cyanobacterial phycobillsomes
Authors: Houmard J. , Capuano V. , Coursin T. , de Marsac N. Tandeau
Published: October 2006

Phycobilisomes are the major constituents of the light‐harvesting apparatus in both cyanobacteria and red algae and consist of a central core with radiating rods. From a genomic library of the cyanobacterium Calothrix 7601, a DNA fragment encoding allophycocyanin B, one of the two terminal energy acceptors of the core, was isolated and its nucleotide sequence was determined. Unlike all the other known genes encoding phycobiliproteins, the allophycocyanin B gene, apcD, is transcribed as a monocistronic unit. Mapping of the transcripts was performed and, in contrast to some of the Calothrix genes that encode rod components, transcription was shown to occur regardless of chromatic tight received during cell growth.
Identification and sequencing of the Escherichia coli cet gene which codes for an inner membrane protein, mutation of which causes tolerance to colicin E2
Authors: Drury L. S. , Buxton R. S.
Published: October 2006

Dominant mutations of the cet gene of Escherichia coli result in tolerance to colicin E2 and increased amounts of an inner membrane protein with an Mr of 42 000. We have cloned the cet+ gene and sequenced its DNA, revealing that the gene product, coded by the longest open‐reading frame, has an Mr of 49772, with five predicted transmembrane structures towards its carboxy terminus and one at its amino terminus. We have demonstrated that the cet locus does in fact code for the inner membrane protein that is present in increased amounts in cet mutants, and we have shown that this increased amount of Cet protein is the result of enhanced transcription. The cet gene is shown to be in the same operon as the phoM gene, which is required in a phoR background for expression of the structural gene for alkaline phosphatase, phoA. Although the Cet protein is not required for phoA expression, our experiments suggest that the Cet protein has an enhancing effect on the transcription of phoA. No effect of phosphate concentration on cet or phoM gene expression could be found and thus their primary function may not be connected to the phosphate regulon.
Molecular basis for the spontaneous generation of colonization‐defective mutants of Streptococcus mutans
Authors: Ueda S. , Kuramitsu H. K.
Published: October 2006

Spontaneous mutants of Streptococcus mutans GS‐5 defective in sucrose‐dependent colonization of smooth surfaces are generated at frequencies above the spontaneous mutation rate. Southern blot analysis of such mutants suggested rearrangement of the genes coding for glucosyltransferase (GTF) activity. Two strain GS‐5 homologous tandem genes, gtfB and gtfC, coding for GTF‐I and GTF‐S activities respectively, were demonstrated to undergo recombination when introduced into recombination‐proficient Escherichia coli transformants. However, the two genes were quite stable when transformed on a single DNA fragment into a recA mutant of E. coli. The DNA fragment coding for GTF activity from one S. mutans colonization‐defective mutant, SP2, was isolated and shown also to have undergone recombination between the gtfB and gtfC genes, resulting in reduced GTF activity. These results are discussed relative to the in vivo generation of colonization‐defective mutants in cultures of S. mutans.
Homology between endoglucanase Z of Erwinia chrysanthemi and endoglucanases of Bacillus subtilis and alkalophilic Bacillus
Authors: Guiseppi A. , Cami B. , Aymeric J.‐L. , Ball G. , Creuzet N.
Published: October 2006

Nucleotide sequencing of the celZ gene encoding the extracellular endoglucanase Z of Erwinia chrysanthemi indicated the presence of an open reading frame encoding 428 amino acids. The mature protein appeared to be extended by a signal peptide of 43 amino acids; this sequence is unusually long and positively charged (+5). It was shown to function as a signal peptide by fusing it to a truncated phoA gene encoding Escherichia coli alkaline phosphatase. Comparison of the encoded sequence with those of the endoglucanases of Bacillus subtilis and alkalophilic Bacillus revealed the existence of a region of extensive homology occurring in all three proteins at about the same distance from the NH2‐terminal end. These regions may be involved in substrate binding and/or catalytic sites.
Molecular cloning and nucleotide sequence of the pectin methyl esterase gene of Erwinia chrysanthemi B374
Authors: Plastow G. S.
Published: October 2006

The gene encoding pectin methyl esterase (pme) has been cloned from Erwinia chrysanthemi B374. Expression of pme in Escherichia coli allowed the enzyme to be characterized. Pectin methyl esterase (PME) was found to have an apparent molecular weight of 36000 Daltons and an isoelectric point of approximately 9.9. The structural gene was sequenced and consists of a 1098‐bp open reading frame encoding a polypeptide of 39318 Daltons, which includes an amino‐terminal signal peptide. The isolation of the Erwinia gene provides a simple method for the production of PME free from depolymerizing pectinases thereby extending its potential uses.
Mercury and tetracycline resistance genes and flanking repeats associated with methicillin resistance on the chromosome of Staphylococcus aureus
Authors: Skinner S. , Inglis B. , Matthews P. R. , Stewart P. R.
Published: October 2006

Sections of a cloned 27 kb segment of chromosomal DNA, associated with resistance to four antimicrobial agents in a clinical isolate of methicillin‐resistant Staphylococcus aureus (MRSA), were tested for their ability to determine resistance when transformed into a sensitive laboratory strain of S. aureus. This was achieved by inserting the sections into a newly constructed shuttle vector, amplifying the recombinant DNA In E. coli, and transforming protoplasts of the sensitive S. aureus strain. Two sections of the cloned DNA were found to determine resistance separately to mercuric ion and to tetracycline, in both S. aureus and Escherichia coli.
Identification and Characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum
Authors: Santero E. , Toukdarian A. , Humphrey R. , Kennedy C.
Published: October 2006

Five Tn5‐induced Nif− mutants of Azotobacter vinelandii were characterized as regulatory mutants because they were restored to Nif+ by the introduction of constitutively expressed nifA from Klebsiella pneumoniae. The mutants fell into two different classes on the basis of hybridization to a Rhizobium leguminosarum nifA gene probe and by complementation with cosmids isolated from pLAFRI gene banks of A.vineiandii and Azotobacter chroococcum. One mutant, MV3, was located in or near a nifA gene. The others, MV12, MV16, MV18 and MV26, defined a new regulatory gene, which has been called nfrX. The lack of expression of different nif‐lacZ fusions confirmed the regulatory phenotype of all five mutant strains. The ability of both nifA and nfrX mutants to grow on nitrogen‐free medium with vanadium, but not on medium with molybdenum, suggests that neither gene is required for expression of the alternative V‐containing nitrogenase of A. vinelandii. A fragment carrying Tn5 and flanking DNA from MV3 was used as a probe to isolate the nifA region of A. chroococcum. Ligation of two adjacent EcoRI fragments of A. chroococcum yielded an intact nifA gene that activated expression of nifH‐lac fusions and also restored MV3 to Nif+. The four nfrX mutants were complemented by pLAFRI cosmids pLV163 and pLCI 21. The nfrX gene was subcloned from pLV163 and located within a 3.2 kb fragment. To determine whether nfrX might be found in other nitrogen‐fixing organisms, DNA from 13 different species was hybridized to an nfrX probe. The failure to observe hybridization suggests that nfrX may be specific to nif regulation in Azotobacter.
Multiplicity of §‐endotoxin genes with different insecticidal specificities in Bacillus thuringiensis aizawai 7.29
Authors: Sanchis V. , Lereclus D. , Menou G. , Chaufaux J. , Lecadet M‐M.
Published: October 2006

The hypothesis according to which multiple and different 5‐endotoxin genes could determine the host‐range specificity of the lepidopteran strains of Bacillus thuringiensis is being checked in the case of strains aizawai 7.29 (serotype 7) and entomocidus 601 (serotype 6). From these strains, several crystal protein genes, belonging to different structural types, have been isolated. One of the cloned genes that is not present in strain entomocidus 601 is duplicated in strain aizawai 7.29. This gene belongs to a previously characterized type of crystal protein gene and encodes a protein preferentially active against Pieris brassicae. Two other genes, of presumed chromosomal location, are present In both strains and each displays a unique physical map. In both strains the two genes are in close proximity and in the same orientation. The first, which belongs to a new type of crystal protein gene, encodes a 130–140 kD protein that is not significantly active against the two insect species tested. The other new type of crystal protein gene directs the synthesis of a polypeptide preferentially active against Spodoptera littoralis.
The integral membrane protein from a virulent isolate of transmissible gastroenteritis virus: molecular characterization, sequence and expression in Escherichia coli
Authors: Britton P. , Cármenes R. S. , Page K. W. , Garwes D. J.
Published: October 2006

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones. Part of a new clone and a previously reported clone were sequenced and used to construct the viral gene for integral membrane protein. A single open reading frame (ORF) encoding a polypeptide of 262 amino acids, relative molecular mass (Mr) 29459, was identified. The positive identification of the polypeptide as the integral membrane protein was demonstrated by the production in E. coli of a chimaeric protein comprising most of the ORF encoding the Mr 29459 polypeptide and β‐galactosidase. The chimaeric protein reacted with a specific monoclonal antibody to viral integral membrane protein and antibodies raised against the chimaeric protein immune precipitated the viral protein. Comparison with the sequence of an avirulent isolate indicates amino acid residues that may be important in pathogenicity.
The structure of a plasmid of Chlamydia trachomatis believed to be required for growth within mammalian cells
Authors: Comanducci M. , Ricci S. , Ratti G.
Published: October 2006

Sequence analysis of a 7.5 kb DNA plasmid isolated from Chlamydia trachomatis shows 8 open reading frames (ORFs) regularly spaced along most of the sequence. One of these ORFs encodes a 451‐amino‐acid polypeptide highly homologous to the DnaB protein of Escherichia coli. A region between ORFs 6 and 7 contains a cluster of alternating ATs and a 22 bp sequence tandemly repeated 4 times, suggesting a replication control region. Several ORFs correspond to plasmid‐specific polypeptides that have been described. Codons ending with A or T are more frequent, as might be expected from the high A/T content (64%) of the plasmid, and codon usage is similar to that of the C. trachomatis chromosomal gene, omp1L2.
Reiterated DNA sequences defining genomic diversity within the species Mycoplasma hyorhinis
Authors: Taylor M. A. , Ferrell R. V. , Wise K. S. , McIntosh M. A.
Published: October 2006

Genomic restriction fragments isolated from Mycoplasma hyorhinis and Mycoplasma hyopneumoniae were shown by DNA hybridization and nucleotide sequence analyses to contain sequences common to these two species, as well as another porcine‐derived mycoplasma, Mycoplasma flocculare. Intraspecies hybridization experiments using these fragments as probes indicated that the sequence is highly redundant in several strains of M. hyorhinis, but that there is diversity in the sizes of restriction fragments detected among these strains. In contrast, repetition of the sequence was limited in M. hyopneumoniae and M. flocculare, and no homologies to this repeated element were apparent in mycoplasma species isolated from animal hosts other than the swine. The reiterated sequence may reflect intraspecies genomic diversification in M. hyorhinis and its selective presence in otherwise unrelated species raises the possibility that it has been horizontally transmitted between these organisms.
Mutations of putP that alter the lithium sensitivity of Salmonella typhimurium
Authors: Myers R. S. , Maloy S. R.
Published: October 2006

The putP gene encodes the major proline permease in Salmonella typhimurium that couples transport of proline to the sodium electrochemical gradient. To identify residues involved in the cation binding site, we have isolated putP mutants that confer resistance to lithium during growth on proline. Wild‐type S. typhimurium can grow well on proline as the sole carbon source in media supplemented with NaCl, but grows poorly when LiCl is substituted for NaCl. In contrast to the growth phenotype, proline permease is capable of transporting proline via Na+/proline or Li+/proline symport. Therefore, we selected mutants that grow well on media containing proline as the sole carbon source in the presence of lithium ions. All of the mutants assayed exhibit decreased rates of Li+pro‐line and Na+/proline cotransport relative to wild type. The location of each mutation was determined by deletion mapping: the mutations cluster in two small deletion intervals at the 5′ and 3′ termini of the putP gene. The map positions of these lithium resistance mutations are different from the locations of the previously isolated substrate specificity mutations. These results suggest that Lir mutations may define domains of the protein that fold to form the cation binding site of proline permease.
Molecular cloning of a Salmonella typhi LT‐like enterotoxin gene
Authors: Fernández M. , Sierra‐Madero J. , de la Vega H. , Vázquez M. , López‐Vidal Y. , Ruíz‐Palacios G. M. , Calva E.
Published: October 2006

Diarrhoea is a common event during typhoid fever; nevertheless, the possible participation of a diarrhoea‐inducing enterotoxin has not been described (Roy et al., 1985). Recombinant bacteriophage λ FDC1 was isolated from a genomic library of Salmonella typhi, the causal agent of typhoid fever, by screening with a probe for the B subunit gene of the heat‐labile, cholera‐like, Escherichia coli enterotoxin (LT). λ FDC1 codes for an enterotoxin that causes secretion in rat ileal loops, that elongates Chinese hamster ovary (CHO) cells, that is recognized by antibodies against LT, and does not bind in vitroto ganglioside GM1. These results should allow further studies towards elucidating a possible role for the S. typhi enterotoxin in the pathogenesis of typhoid fever.
Sequences required for regulation of arginine biosynthesis promoters are conserved between Bacillus subtilis and Escherichia coli
Authors: Smith M. C. M. , Czaplewski L. , North A. K. , Baumberg S. , Stockley P. G.
Published: October 2006

The region required for regulation of a previously characterized arginine‐regulatable promoter upstream from the argC gene in the argCAEBD‐cpa‐argF cluster of Bacillus subtilis was defined by integration of argC‐lacZ translational fusions into the chromosome at a site distant from the arginine loci. Some sequence similarity was detected between the argC regulatory region and the well‐characterized Escherichia coli arginine operators (ARG boxes). This similarity was shown to be functional in vivo in that the B. subtilis repressor regulated the E. coli arginine genes, but the E. coli repressor, even when encoded by a multicopy plasmid, could not repress the B. subtilis argC promoter. In vitro binding studies using purified repressors on DNA fragments encoding operators from both E. coli and B. subtilis demonstrated interactions by both proteins.
Molecular characterization of a gene from Saccharopolyspora erythraea (Streptomyces erythraeus) which is involved in erythromycin biosynthesis
Authors: Dhillon N. , Hale R. S. , Cortes J. , Leadlay P. F.
Published: October 2006

A 7.3 kbp DNA fragment, encompassing the erythromycin (Em) resistance gene (ermE) and a portion of the gene cluster encoding the biosynthetic genes for erythromycin biosynthesis in Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has been cloned in Streptomyces lividans using the plasmid vector pIJ702, and its nucleotide sequence has been determined using a modified dideoxy chain‐termination procedure. In particular, we have examined the region immediately 5′ of the resistance determinant, where the tandem promoters for ermE overlap the promoters for a divergently transcribed coding sequence (ORF). Disruption of this ORF using an integrational pIJ702‐based plasmid vector gave mutants which were specifically blocked in erythromycin biosynthesis, and which accumulated 3‐O‐α‐L‐mycarosylerythronolide B: this behaviour is identical to that of previously described eryC1 mutants. The eryC1‐gene product, a protein of subunit Mr 39200, is therefore involved either as a structural or as a regulatory gene in the formation of the deoxyamino‐sugar desosamine or in its attachment to the macro‐lide ring.
Transcriptionai induction of Streptomyces cacaoiβ‐lactamase by a β‐lactam compound
Authors: Forsman M. , Lindgren L. , Haggström B. , Jaurin B.
Published: October 2006

The soil bacterium Streptomyces cacaoi produces an extracellular β‐lactamase. The β‐lactamase expression could be induced by the β‐lactam compound 6‐amino penicillinoic acid (6‐APA). In liquid cultures, a 50‐fold increase in β‐lactamase expression was observed within the first three hours after addition of 6‐APA. Using the cloned β‐lactamase gene as a probe, it was shown that this increase was mediated at the level of transcriptional initiation. The start point of the induced β‐lactamase transcript was determined, and the nucleotide sequence of the promoter region was analysed. No noticeable homology was found to control regions of inducible β‐lactamase genes of other bacteria. A striking feature was the presence of six direct repeats (ten base pairs each) upstream of the promoter region. Thus, an example of an inducible regulatory gene system in this Gram‐positive microorganism is presented. Also, the primary structure of the β‐lactamase was deduced, showing a high degree of homology with class A β‐lactamases.
Isolation of Erwinia chrysanthemi mutants altered in pectinolytic enzyme production
Authors: Hugouvieux‐Cotte‐Pattat N. , Robert‐Baudouy J.
Published: October 2006

Various mutations in the pectin catabolic pathway of Erwinia chrysanthemi were isolated by selection of Mu‐lac insertions, resulting in expression of the lac genes inducible by pectin degradation products. This approach allowed us to isolate lacZ fusions with the genes pelC, pelD, ogl and pem, encoding pectate lyases PLc and PLd, oligogalacturonate lyase and pectin methytesterase, respectively. Moreover, we obtained mutations affecting the regulation of pectinolytic enzymes; a locus called peel appeared to be involved in induction of pectate tyases and pectin methylesterase. A second locus, called pect, may encode an activator protein acting on pectate lyase production. Both peel and pecL expression are induced in the presence of pectic polymers. The expression of the pem gene was studied in more detail by analysis of the pem‐lacZ fusions. The expression of pem appears to be controlled by the negative regulatory gene kdgR, which controls alt the genes involved in pectin degradation (pem, pel, ogl, kduD, kduf, kdgK, kdgA). This study confirmed that 2‐keto‐3‐deoxy‐gluconate is a key intermediate for the induction of the pectin catabolic pathway. The three genes pem, pelD and pecl were localized in the same region, near the ade‐377 marker on the genetic map of the E. chrysanthemi strain 3937. The pem gene was located more precisely on an 18kb DNA fragment containing the pelADE cluster. However, this 18 kb DNA fragment did not complement the pecl mutation. The pecL mutations were located near the ile‐2 marker on the genetic map of E. chrysanthemi strain 3937.
Mitochondrial cytochrome b genes with a six‐nucleotide deletion or single‐nucleotide substitutions confer resistance to antimycin A in the yeast Kluyveromyces lactis
Authors: Coria R. , Garcfa M. , Brunner A.
Published: October 2006

Extrachromosomal mutants resistant to antimycin, from the yeast Kluyveromyces lactis, have been isolated, genetically characterized, and assigned to two specific genetic loci (Brunner et al., 1987). In the present work the cytochrome b nucleotide sequence from six of these mutants was determined. Five mutants had single point mutations, corresponding to transverslons. In one mutant, a six‐base‐pair deletion, beginning at nucleotide 689, was observed. The amino acid sequence derived from the coding strand showed that, in three independent antimycin‐resistant mutants, a change of asparagine 31 into lysine took place (two of these mutants are also resistant to diuron). Two other mutants showed a change from lysine 228 into isoleucine (or methionine). Leucine 230, isoleucine 231, and threonine 232, were lost in the deletion mutant and were replaced by serine.
pH‐regulated gene expression in Salmonella: genetic analysis of aniG and cloning of the earA regulator
Authors: Foster J. W. , Aliabadi Z.
Published: October 2006

The recently described aniG locus exhibits a series of unique regulatory features. The gene is exogenously coinduced by acid and D‐mannose, its expression is maximal under anaerobiosis, and the system is regulated in an unusual manner by cyclic AMP. The external acid regulatory locus, earA, is a repressor protein that mediates the acid and mannose control of aniG. The earA locus was cloned and found to code for a 33 K protein associated with membrane‐ and soluble fractions. A second locus, earB, was located immediately upstream from earA. The earB locus or its product interferes with the repression of aniG by EarA. Mutations in crp and cya were found to prevent transcription of aniG but only in an earA+ background. Analysis of an earA‐cat fusion established that crp does not affect earA expression.

While the physiological role of aniG/earA is unclear, this system serves as a model for external pH‐regulated gene expression. The present data indicate that it is used to sense the presence of mannose in an acidic extracellular environment. This is particularly intriguing in that the system is not involved in the utilization of mannose as a carbon source.
Stability of ColE1‐like and pBR322‐like plasmids in Escherichia coli
Authors: Ayala‐Sanmartin J. , Gómez‐Eichelmann M. C.
Published: October 2006

The average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1‐like and pBR322‐like plasmids. From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated. Experimentally determined plasmid stability was compared with that calculated using the variance of the units of partition and the ratio between the generation times of plasmid‐free and of plasmid‐carrying cells, assuming that the units of partition are distributed randomly between daughter cells.

Stability of the pBR322‐like plasmids present mainly as monomers in the bacterial host was consistent with random partitioning, whereas pBR322‐like plasmids, present mainly as dimers, and the ColE1‐like plasmid showed greater stability than that predicted with random partitioning at cell division.
Mutations in genes downstream of the rpoN gene (encoding σ54) of Klebsiella pneumoniae affect expression from σ54‐dependent promoters
Authors: Merrick M. J. , Coppard J. R.
Published: October 2006

Two open reading frames (ORFs), designated ORF95 and ORF162, downstream of the Klebsiella pneumoniaeσ54 structural gene (rpoN) have been sequenced and shown to encode polypeptides of 12kD and 16kD, respectively. ORFs homologous to ORF95 are present downstream of four out of five rpoN genes sequenced to date from a range of Gram‐negative bacteria, and ORF162 is also conserved, at least in Pseudomonas putida. Chromosomal mutations have been created in each gene using a kan cassette and both have the same phenotype, i.e. they cause an increase in the level of expression from σ54‐dependent promoters. We propose that the products of both genes function to modulate the activity of Eσ54, although a physiological role for these proteins has not yet been identified.
Analysis of the yopA gene encoding the Yop1 virulence determinants of Yersinia spp.
Authors: Skurnik M. , Wolf‐Watz H.
Published: October 2006

The Yop proteins of Yersinia are important virulence determinants. The Yop1 protein sequences of Yersinia pestis, Yersinia pseudotuberculosis, and two Yersinia enterocolitica serotypes, O:3 and O:8, deduced from the nucleotide sequences of the corresponding yopA genes, were compared. Most differences were found in the hydrophilic domains of the proteins, whereas the hydrophobic domains were conserved. The amino acid sequences revealed a signal sequence 25 amino acids long. No cysteine residues were present, even though Yop1 forms a polymeric structure.

The transcription startpoint of yop4 was determined by primer extension. The coding region and transcription startpoint were separated by a leader sequence 270 nucleotides long. The yopA promoter sequence of Y. pestis is identical to the corresponding sequence of Y. pseudotuberculosis and transcription studies revealed that this promoter is active in Y. pestis. Thus, the inability of Y. pestis to express the Yop1 protein is due to a single base pair deletion In the coding region of the yopA gene of Y. pestis (Rosqvist et al., 1988).
Sense and antisense transcripts of traM, a conjugal transfer gene of the antibiotic resistance plasmid R100
Authors: Dempsey W. B.
Published: October 2006

The region of the antibiotic resistance plasmid R100 that encodes the plasmid‐specific transfer gene traM has two tandemly aligned promoters separated by 145 nucleotides. The principal transcripts are 705 and 562 nucleotides long. Minor transcripts are 1550 and 1700 nucleotides long. The 705‐base transcript appears to encode an 11 kD traM protein. The 562‐base transcript does not encode a detectable protein. When sub‐cloned on short fragments, the promoter for the 562‐base transcript initiates efficiently but that for the 705 site does not. The 3′ ends of the 705 and 562 base transcripts end inside the traJ ORF. Thus they provide additional sense RNA to compete with traJ for finP, the antisense translational regulator of traJ. A model is proposed for the participation of these sense and antisense transcripts in the control of expression of the traJ gene.
Expression of gonococcal protein II in Escherichia coli by translational fusion
Authors: Palmer L. , Brooks G. F. , Falkow S.
Published: October 2006

Aprotein II (P.II) gene from Neisseria gonorrhoeae was cloned in Escherichia coli and characterized by DNA sequence analysis. As with other reported P.II sequences, this gene contains an ATG initiation codon which is out of frame with respect to the remainder of the P.II amino acid sequence. A translational fusion was constructed in E. coli which linked the P.II sequence to the signal peptide of β‐lactamase. This P.II fusion differs from the gonococcal protein only in the first seven residues at the N terminus. In E. coli, the P.II fusion product exhibits properties analogous to those of P.II in N. gonorrhoeae. The P.II fusion product is a major component of the E. coli outer membrane and it is exposed on the cell surface. The P.II fusion protein also exhibits the heat‐modifiable phenotype of gonococcal P.II.
Transcription of the sulA‐ompA region of Escherichia Coli during the SOS response and the role of an antisense RNA molecule
Authors: Cole S. T. , Honoré N.
Published: October 2006

The transcriptional pattern of the 22 min region of the Escherichia coli chromosome containing the linked sulA and ompA genes, which encode an SOS‐inducible inhibitor of cell division and a constitutively expressed, major outer membrane protein, respectively, has been re‐examined. During normal growth, the sulA gene was repressed whereas the ompA gene produced a stable 1250 nucleotide transcript. Counter‐transcription of sulA occurred from a promoter situated in the sulA‐ompA intergenic region and the product of this transcriptional circuit, named isf, is a 353 nucleotide untranslated RNA. Since the isf RNA is complementary to the 3‐end of the sulA transcript, it could modulate sulA function by serving as an anti‐messenger.

On induction of the SOS‐response, massive transcription of sulA took place, resulting in the ‘silencing’ of the isf gene, production of an abundant ∼615 nucleotide sulA mRNA and a novel hybrid transcript of ∼2100 nucleotides encoding both the SulA and OmpA proteins. Production of the latter RNA species, caused by transcription reading through the sulA terminator, the intergenic region and the coding sequences, was accompanied by a decrease in the abundance of the ompA mRNA as a result of promoter occlusion. However, the amount of OmpA protein produced was only slightly reduced.
Transcriptional regulation of F plasmid gene srnB: rifampicin‐promoted in vitro readthrough of a terminator in the leader region
Authors: Akimoto S. , Sakikawa T. , Ono K. , Ono T. , Ohnishi Y.
Published: October 2006

srnB is an F‐plasmid encoded gene, otherwise silent, whose expression is induced by added rifampicin, leading to the release of cellular Mg2+ and degradation of stable RNA. In the absence of rifampicin, transcripts from the srnB gene were relatively short. S1 nuclease mapping revealed that the short mRNA species terminated within the leader, at the 3’end of a potential stem‐and‐loop structure. A deletion in the stem‐loop resulted in constitutive synthesis of the mRNA that extended beyond the termination site into the structural gene. Even with the wild‐type gene, transcription continued beyond the terminator sequence in the presence of added rifampicin. Most of the transcripts synthesized in the presence of rifampicin were long enough to encode the srnB protein. We hypothesize from these results that RNA polymerase associated with rifampicin can read through the terminator to induce srnB expression.
Characterization of Rhizobium phaseoli Sym plasmid regions involved in nodule morphogenesis and host‐range specificity
Authors: Cevallos M. A. , Vázquez M. , Dávalos A. , Espín G. , Sepúlveda J. , Quinto C.
Published: October 2006

Two nodulation regions from the symbiotic plasmid (pSym) of Rhizobium phaseoil CE‐3 were identified. The two regions were contained in overlapping cosmids pSM927 and pSM991. These cosmids, in a R phaseoli pSym‐cured strain background, induced ineffective nodules on Phaseolus vulgaris roots.

Transconjugants of Rhizobium meliloti harbouring pSM991 induced nodule‐like structures on bean roots, suggesting that this cosmid contains host‐range determinants.

Analysis of deletions and insertional mutations in the sequences of pSM991 indicated that the genes responsible for the induction and development of nodules in P. vulgaris are organized in two regions 20kb apart. One region, located in a 6.8kb Eco RI fragment, includes the common nodABC genes. The other region, located in a 3.5kb EcoRI fragment, contains information required for host‐range determination.
An overlap between osmotic and anaerobic stress responses: a potential role for DNA supercoiling in the coordinate regulation of gene expression
Authors: Bhriain N. Ni , Dorman C. J. , Higgins C. F.
Published: October 2006

The regulation of several genes in response to osmotic and anaerobic stress has been examined. We have demonstrated a clear overlap between these two regulatory signals. Thus, the osmotically induced proU and ompC genes require anaerobic growth for optimum induction while the anaerobically induced tppB gene is also regulated by osmolarity. Furthermore, normal expression of tppB and ompC requires the positive regulatory protein OmpR, yet this requirement can be partially, or even fully, overcome by altering the growth conditions. Finally, the pleiotropic, anaerobic regulatory locus, oxrC, is also shown to affect expression of the osmoticalty regulated proU gene. The oxrC mutation is shown to affect the level of negative supercoiling of plasmid DNA and its effects on gene expression can be explained as secondary consequences of altered DNA topology. We suggest that there is a class of ‘stress‐regulated’ genes that are regulated by a common mechanism in response to different environmental signals. Furthermore, our data are consistent with the notion that this regulatory overlap is mediated by changes in DNA supercoiling in response to these environmental stresses.
A novel, non‐invasive promoter probe vector: cloning of the osmoregulated proU promoter of Escherichia coli K12
Authors: Park S. F. , Stirling D. A. , Hulton C. S. J. , Booth I. R. , Higgins C. F. , Stewart G. S. A. B.
Published: October 2006

We have constructed a novel promoter probe plasmid pSB40, containing a unique lac‐α‐tetracycline marker gene tandem, which allows for both positive and negative selection of active promoters. Promoters cloned in pSB40 can be readily mobilized as EcoR1 cassettes. Using this vector we have performed a non‐invasive analysis of the E. coli chromosome for promoters regulated by osmotic upshift. Only one such promoter, subsequently identified as part of the proU operon, was isolated. A sequence of 253bp, sufficient to mediate osmotic regulation of the proU promoter, was defined. This E coli promoter was normally regulated in Salmonella typhimurium, Klebsiella and Citrobacter but not in Shigella. A proU‐luxAB fusion plasmid was constructed and used to monitor in vivo real‐time kinetics of proU induction following osmotic upshock.
Differential gene expression during sporulation in Bacillus subtilis: regulation of the spoVJ gene
Authors: Errington J. , Wootten L. , Dunkerley J.C. , Foulger D.
Published: October 2006

The process of spore formation in the Gram‐positive bacterium Bacillus subtilis is a simple developmental system controlled by 50 or more genes. The complex pattern of regulatory interactions between these genes is beginning to be elucidated. spoVJ is a poorly characterized locus in which mutations affect spore development at a relatively late stage (Stage V). We have now cloned and physically characterized the SpoVJ locus, and analysed its expression by lacZ fusion. Expression of spoVJ is temporally delayed until about two hours after the initiation of sporulation. Its expression is also spatially restricted to the mother cell compartment; as such, it represents the earliest known mother‐cell‐specific event. Control of spoVJ transcription is complex: expression is dependent upon the products of all of the spo0 genes and on some of the spoII genes butitis independent of all later genes except spoIIID. As spoIIID mutations do not affect prespore development, this gene must be an important early determinant of mother‐cell‐specific gene expression.
Reverse transcriptase in bacteria
Authors: Lim D. , Maas W. K.
Published: October 2006

Reverse transcriptase, discovered in 1970 in retroviruses, has until recently been found only in eukaryotic organisms. Recently it was shown to occur in two groups of bacteria: myxobacteria and Escherichia coli. The gene for reverse transcriptase is part of a chromosomal genetic element that codes for the production of a branched DNA‐RNA compound. In this compound a single‐stranded DNA is connected to RNA at a specific G residue by a 2′‐5’phosphodiester linkage. The precursor for the DNA‐RNA compound is a folded messenger RNA, in which the specific G residue is the initiation point for reverse transcription. In the final DNA‐RNA compound, the portion of the RNA transcribed by reverse transcriptase is eliminated by RNase H. The DNA‐RNA compound is present in several hundred copies per cell. Its biological function is unknown at present.
Molecular cloning and expression of a locus (mdoA) implicated in the biosynthesis of membrane‐derived oligosaccharides in Escherichia coli
Authors: Lacroix J.‐M. , Tempête M. , Menichi B. , Bohin J.‐P.
Published: October 2006

Mutants of Escherichia coli defective in the mdoA locus are blocked at an early stage in the biosynthesis of membrane‐derived oligosaccharides. The mdoA locus has now been cloned into multicopy plasmids. A 5kb DNA fragment is necessary to complement mdoA mutations. Cells harbouring the mdoA+ plasmid produced three to four times more MDO than wild‐type cells. MDO overproduction did not affect the degree of MDO substitution with sn‐1‐phosphoglycerol residues. The biosynthesis of MDO remained under osmotic control in overproducing strains.
Molecular and genetical analysis of the fructose‐glucose transport system in the cyanobacterium Synechocystis PCC6803
Authors: Zhang C.‐C. , Durand M.‐C. , Jeanjean R. , Joset F.
Published: October 2006

Complementation for glucose transport capacity of deficient mutants from Synechocystis PCC6803 allowed the cloning of the corresponding gene, glcP. The protein predicted from one open reading frame (ORF) in the DNA sequence was 468 residues long. It showed 46–60% amino acid sequence homology and similarity in size and predicted structure (including twelve probable membrane‐spanning regions) with a group of non‐phosphorylating sugar transporters from mammals, yeasts and Escherichia coli. A second ORF, 64 base pairs downstream from glcP, was detected. Its function, dispensable under auto‐ and heterotrophic conditions, could not be determined. Genetic analysis of mutants confirmed that the resistance to fructose, acquired simultaneously with the deficiency in glucose transport, resulted from mutations in the glcP gene, whose approximate location could be determined.
Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257
Authors: Rouch D. A. , Messerotti L. J. , Loo L. S. L. , Jackson C. A. , Skurray R. A.
Published: October 2006

Trimethoprim resistance mediated by the Staphylococcus aureus multi‐resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789‐790 bp), the outside two of which are flanked by directly repeated 8‐bp target sequences. IS257 has imperfect terminal inverted repeats of 27‐28 bp and encodes for a putative transposase with two potential α‐helix‐turn‐α‐helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram‐negative bacteria and with ISS 1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim‐resistant DHFRs from Gram‐negative bacteria and with chromosomally encoded DHFRs from Gram‐positive and Gram‐negative bacteria. 5’to dfrA is a thymidylate synthetase gene, designated thyE.
Distribution of msDNAs among serotypes of enteropathogenic Escherichia coli strains
Authors: Lim D. , Gomes T. A. T. , Maas W. K.
Published: October 2006

A genetic element, called a retron, is present in certain Escherichia coli strains. It consists of genes for the production of a covalently linked DNA‐RNA compound and a reverse transcriptase. The presence of a retron can be detected by testing for a satellite DNA band by polyacrylamide gel electrophoresis. This DNA band consists of the DNA portion of the DNA–RNA compound and is called msDNA (multicopy single‐stranded DNA). In a survey of intestinal E. coli isolates we detected msDNAs in classical enteropathogenic (EPEC) strains and in strains with aggregative adherence to tissue‐culture cells (AA), but not in enteroinvasive (EIEC) and enterotoxigenic (ETEC) strains. Among 76 EPEC strains belonging to 14 different serotypes, msDNA was found to be present in 7 serotypes. In total, five different types of msDNA were found, although within each serotype, the msDNAs were the same. These results suggest that different retrons are clonally inherited.
Involvement of OmpF during reception and translocation steps of colicin N entry
Authors: Bourdineaud J.‐P. , Frerobe H.‐P. , Lazdunski C. , Pagès J.‐M.
Published: October 2006

[125I]‐colicin N binds to OmpF receptor sites (70000 per cell) with an average Kassoc of 3.2 × 106 M−1 at 23°C. Monoclonal antibody directed against a cell‐surface‐exposed epitope of OmpF is able to compete with the binding of the colicin in vitro and also to protect against colicin N in vivo. OmpF is an absolute requirement for colicin N uptake. OmpC cannot serve as a substitute for OmpF during translocation across the outer membrane under receptor bypass conditions, which is in contrast to colicin A. Colicin N does not cross‐react with various monoclonal antibodies directed against colicin A.
Effects of N‐methyl‐D‐aspartate receptor ligands on yeast sporulation
Authors: Piñón R.
Published: October 2006

Earlier studies have suggested that glutamate may play an important role in the transition between the mitotic (vegetative) and meiotic (sporulative) stages of the life cycle in the yeast Saccharomyces cerevisiae. Glutamate is also a major excitatory neurotransmitter in the vertebrate brain, and its actions are mediated by the excitatory amino acid (EAA) family of receptors, the three best‐characterized of which are the N‐methyl‐d‐aspartate (NMDA), quisqualate (Q), and kainate (K) receptors. As an initial test of the possibility that glutamate action in S. cerevisiae might be mediated by an EAA‐like receptor mechanism, the effects of ligands that define the functional domains of the vertebrate NMDA receptor have been examined. The responses of S. cerevisiae cells to ligands that act at four distinct sites on the NMDA receptor provide the first evidence for an NMDA‐like receptor‐mediated system involved in the control of yeast sporulation.
Uncharged tRNA, protein synthesis, and the bacterial stringent response
Authors: Goldman E. , Jakubowski H.
Published: October 2006

Uncharged tRNA has been shown in vivo to have an active role both in the stringent response, and in modulating the rate of translational elongation. Both of these effects appear to be mediated by codon–anticodon interactions on the ribosome. Although the involvement of uncharged tRNA in the stringent response was expected from in vitro experiments, it has only recently been confirmed in vivo. Inhibition of translation by cognate uncharged tRNA was not expected, and a model is proposed in which excess uncharged tRNA competes with charged tRNA (in ternary complex) for the 30S component of the ribosomal A site. When uncharged tRNA is in sufficient excess over charged tRNA, interaction of uncharged tRNA with the 50S component of the A site occurs as well, leading to a stringent response. The cell has a continuum of responses to decreasing aminoacyl‐tRNA levels: in moderately limited conditions, the proportion of uncharged tRNA increases, and the translation rate is slowed; under more severe limitations, uncharged tRNA provokes a stringent response, with pleiotropic consequences for the cell.
Structure, localization and function of FanF, a minor component of K99 fibrillae of enterotoxigenic Escherichia coii
Authors: Simons B. L. , Willemsen P. T. J. , Bakker D. , Roosendaal B. , De Graaf F. K. , Oudega B.
Published: October 2006

The DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG.

A specific mouse antiserum against FanF was prepared by constructing and purifying a hybrid CroLacZ‐FanF protein. Minicell analysis, immunoblotting and immunoelectronmicroscopy revealed a pool of FanF in the periplasm of K99‐producing cells and showed, furthermore, that FanF Is a minor component of K99 fibrillae, present at the top and in or along the shaft of the K99 fibrillar structures.

A fanF mutant plasmid was constructed. Cells harbouring this plasmid produced all K99‐specific proteins, except FanF, but produced 0.1% of the K99 fibrillae relative to ‘normal’ K99‐producing cells. Electron microscopic observations showed that cells defective in fanF produce only a few (apparently short) K99 fibrillae. FanF, therefore, was supposed to play a role in initiation and elongation of K99 fibrillae formation.

Thin‐layer chromatography experiments involving purified receptor material showed that FanF is not required for binding of K99 fibrillae to the ganglioside receptor. Fibrillae produced by an adhesion‐negative strain carrying a mutation in the K99 major fibrillar subunit were shown to contain a normal amount of FanF.
Mapping of the msDNA operon in the chromosome of Escherichia coli B
Authors: Lim D. , Maas W. K.
Published: October 2006

An msDNA operon, consisting of genes for msDNA and a reverse transcriptase, is present in Escherichia coli B and absent from E. coli K12. We have found that the msDNA operon is located on a DNA fragment, longer than 15 kb, that is absent from E. coli K12. Using conjugation, P1 transduction, and nucleic acid hybridization between E. coli B and E. coli K12 strains, we have located the position of the msDNA operon on the E. coli B chromosome at a site that corresponds to minute 19 on the genetic map and to position 900 on the physical map of the E. coli K12 chromosome.