bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of the virG locus of the Agrobacterium tumefaciens plasmid pTiC58
Authors: Powell B. S. , Powell G. K. , Morris R. O. , Rogowsky P. M. , Kado C. I.
Published: October 2006

The nucleotide sequence of the virG locus of the nopaline type plasmid pTiC58 of Agrobacterium tumefaciens has been determined. It contains an open reading frame (ORF) of 759 nucleotides and has 77% homology to the virG sequences of octopine type plasmids. Differences between the sequences of the two types of Ti plasmids in the region of virG are located predominantly outside the ORF. The amino acid sequences inferred from the two virG genes show 80% homology to each other and each shows the same moderate homologies to amino acid sequences derived from genes in a family of two‐component regulatory systems. Specific differences in nucleotide and amino acid sequences as well as a structure‐function model for the gene product are discussed.
A RAS‐encoded protein in Dictyostelium discoideum is acylated and membrane‐associated
Authors: Weeks G. , Lima A. F. , Pawson T.
Published: October 2006

Dictyostelium dfscoideum synthesizes a 23000 Mr protein, p23dd‐ras, closely related to the mammalian oncogene‐encoded protein p21ras. To investigate the subcellular localization of P23dd‐ras, conditions were optimized to reduce protein degradation following cell breakage. Subcellular fractionation of D. discoideum showed that p23dd‐ras was associated predominantly with the membrane fraction during both vegetative growth and differentiation, in the absence of suitable protease inhibitors considerable amounts of a truncated form of p23dd‐ras were recovered in the cytosol fraction, suggesting that intact p23dd‐ras is attached to the membrane by a short terminal peptide sequence. Radio‐isotope labelilng of D. discoideum with myristic acid or palmitic acid in the presence of excess un‐labelled acetate resulted in radio‐isotope incorporation into a select group of proteins including p23dd‐ras. No acyl label appeared in the truncated cytoplasmic form of p23dd‐ras when ceil breakage was performed In the absence of suitable protease inhibitors, indicating that the acyl group is associated with the short terminal peptide that is cleaved. These data suggest that p23dd‐ras like its mammailan counterpart, is acylated and associated with the plasma membrane. There was no evidence during a 30‐minute pulse of methionine for a cytoplasmic precursor to the membrane‐bound p23dd‐ras suggesting that the turnover of the presumptive precursor must be much more rapid in D. discoideum than for pro‐p21ras in mammalian cells.
Penicillin‐binding proteins in β‐lactam‐resistant laboratory mutants of Streptococcus pneumoniae
Authors: Lalble G. , Hakenbeck R.
Published: October 2006

The increasing number of penicillin‐resistant clinical strains of Strepfococcus pneumoniae has raised questions about the mechanism involved. We have isolated a large number of independent, spontaneous laboratory mutants with increasing resistance against either piperacillin or cefotaxime. Both classes of mutants showed a different pathway of penicillin‐binding protein (PBP) alterations, and within each group of mutants the individual PBPs appeared to have changed at different resistance levels and in different sequences. The mutations led to decreased β‐lactam affinity and possibly to a reduction in the amount of protein present in the cell, but differences in apparent molecular weight, like those reported in low‐ and high‐level resistant pathogenic strains, were not found. Some mutants showed a high degree of cross‐resistance to a variety of pencillins and cephaiosporins independently of the acquired PBP alterations, indicating that different genotypes can be responsible for the same phenotypic expression of resistance.
DNA sequence of the gene scrA encoding the sucrose transport protein EnzymellScr of the phosphotransferase system from enteric bacteria: homology of the EnzymellScr and EnzymellBgl proteins
Authors: Ebner R. , Lengeler J. W.
Published: October 2006

The nucleotide sequence of the structural gene, scrA, which codes for sucrose‐specific EnzymellScr (EIIScr) of the phosphoenolpyruvate‐dependent carbohydrate: phosphotransferase system (PTS), was determined. EllScr requires an Enzymelll, the product of the gene crr, for full activity. The gene scrA is preceded immediately by a classical Shine‐Dalgarno sequence (AAGAGGGTA). It contains 1368 nucleotides with an increased GC‐content (58%) corresponding to a polypeptide of 455 amino acid residues (Mr 47 500). The protein has the hydropathic profile (average hydropathy +0.82) of an integral membrane protein lacking extended α‐helical structures and a signal peptide. Comparison with the sequence of the β‐glucoside‐specific Enzymell (EllBgl, 625 amino acids, Mr 66480; Bramley and Kornberg, 1987a; Schnetz et al., 1987) revealed strong homologies between EllScr and the first 458 residues of EllBgl. The 162 carboxyterminal residues of EllBgl, however, showed a high homology with the sequence of Enzymelll (Nelson et al., 1984), a homology also described recently by Bramley and Kornberg (1987b). The evolutionary and functional significance of the similarities with four other Enzymesll is discussed.
Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom‐thrB operon
Authors: Peoples O. P. , Liebl W. , Bodis M. , Maeng P. J. , Follettie M. T. , Archer J. A. , Sinskey A. J.
Published: October 2006

The complete nucleotide sequence of the Corynebacterium glutamicum hom‐thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46136 is encoded by hom and a polypeptide of Mr, 32618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1‐nuclease and deletion analysis. Located between −88, RNA start site and −219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtills promoters. Although there are no obvious attenuator‐like structures in the 5′‐untranslated region, there is a dyad‐symmetry element, which may act as an operator.
Biogenesis of F71 and F72 fimbriae of uropathogenic Escherichia coli: influence of the FsoF and FstFG proteins and localization of the Fso/FstE protein
Authors: Riegman N. , van Die I. , Leunissen J. , Hoekstra W. , Bergmans H.
Published: October 2006

The F71 and F71 P‐fimbriae of Escherichia coli are encoded by the fso (F seven one) and fst (F seven two) gene clusters, respectively (Van Die et al., 1984; 1985).

With the immunocytochemical gold‐labelling technique it was demonstrated that both the FsoE and FstE proteins are non‐adhesive minor fimbrial sub‐units located at the tip of the fimbrial structure. The FsoF and FstFG proteins play an important role in the initiation of polymerization of the minor and major subunits into the fimbrial structure.
Role of the intercistronic region in post‐transcriptional control of gene expression in the histidine transport operon of Salmonella typhimurium: involvement of REP sequences
Authors: Stern M. J. , Prossnitz E. , Ames G. Ferro‐Luzzi
Published: October 2006

The high‐affinity histidine permease of Salmonella typhimurium is encoded by a four‐gene operon containing a large intercistronic region located between the first gene (hisJ) and the three distal genes (hisQ, hisM, hisP). The level of expression of hisJ is 30‐fold greater than that of hisP. In order to investigate the role of the intercistronic region in intra‐operonic control of gene expression, we have isoiated MudII‐mediated IacZ gene fusions to hisQ, hisM and hisP. We have used these fusions to isolate and analyse mutants that have altered levels of expression of the hisQ gene, the first gene downstream from the intercistronic region. The results indicate that intra‐operonic regulation is due to a combination of factors including efficiency of translational initiation, mRNA degradation, and retroregulation of hisJ expression. They also suggest that the REP (Repetitive Extragenic Palin‐dromic) sequences, which are located in the hisJ‐hisQ intercistronic region, may interfere with translation of the hisQ gene and affect upstream messenger RNA stability by protecting it from 3’to 5’nuclease degradation (in agreement with data presented by Newbury et al., 1987).
Functional analysis of different sequence elements in the Escherichia coli galactose operon P 2 promoter
Authors: Ponnambalam S. , Chan B. , Busby S.
Published: October 2006

Starting with a DNA fragment containing the galactose operon P 2 promoter, we made a series of deletions that progressively replaced DNA sequences upstream of the transcription startpoint and determined their effects on P 2 activity. The results show that specific sequences upstream of −32 are not important. Removal of the sequence 5′‐CACA‐3′ from −32 to −28 reduces P 2 activity by 50%: longer deletions to −16 further reduce activity but do not remove the information specifying the transcription startpoint. DNA sequences between −32 and −16 at galP 2 assist the isomerization of RNA polymerase from closed to open complexes rather than contributing to the initial binding of RNA polymerase. The activity of gal P 2 in the absence of −35 region sequences is dependent on the sequence TG just upstream of the − 10 hexamer, TATACT: a mutation at −14 changing the TG sequence to TT totally inactivates P 2. However, P 2 activity can be restored if the consensus −35 region sequence TTGACA is cloned 17 bp upstream of the −10 hexamer. Thus, for transcription initiation, the −10 hexamer, TATACT, must ‘cooperate’ with upstream sequences that may be located either around −35 or −14.
Characterization of the Rhizobium leguminosarum genes nodLMN involved in efficient host‐specific nodulation
Authors: Surin B. P. , Downie J. A.
Published: October 2006

Three nodulation genes, nodL, nodM and nodN, were isolated from Rhizobium leguminosarum and their DNA sequences were determined. The three genes are in the same orientation as the previously described nodFE genes and the predicted molecular weights of their products are 20105 (nodL), 65795 (nodM) and 18 031 (nodN.) Analysis of gene regulation using operon fusions showed that nodL, nodM and nodN are induced in response to flavanone molecules and that this induction is nodD‐dependent. In addition, it was shown that the nodM and nodW genes are in one operon which is preceded by a conserved ‘nod‐box’ sequence, whereas the nodL gene is in the same operon as the nodFE genes. DNA hybridizations using specific gene probes showed that strongly homologous genes are present in Rhizobium trifolii but not Rhizobium meliloti or Bradyrhizobium japonicum. A mutation within nodL strongly reduced nodulation of peas, Lens and Lathyrus but had little effect on nodutation of Vicia species. A slight reduction in nodulation of Vicia hirsuta was observed with strains carrying mutations in nodM or nodN.
Uropathogenic, Escherichia coli can express serologically identical pili of different receptor binding specificities
Authors: Lund B. , Marklund B.‐I. , Strömberg N. , Lindberg F. , Karlsson K.‐A. , Normark S.
Published: October 2006

Uropathogenic Escherichia coli frequently express P‐pilus adhesins that recognize Galα(1–4)Gal‐containing glycoconjugates. The P‐pilus adhesin of the, E. coli isolate J96 is encoded by the pap gene cluster and has been shown to agglutinate P1‐erythrocytes. We now describe a novel gene cluster from J96, prs, which is responsible for the agglutination of sheep erythrocytes. The structurally related gene clusters both expressed pili exhibiting the F13 antigen. Analysis of mutants of cloned prs sequences, together with trans‐complementation of pap and prs genes, identified the sheep‐specific adhesin as the 37‐kD PrsG protein. The prsG gene occupies the equivalent position in prs as occupied by papG, which specifies the Galeα(1–4)Gal‐specific adhesion of pap. PrsG was shown to be structurally distinct from PapG since PapG‐specific antiserum did not cross‐react with PrsG. Using a solid phase glycolipid receptor binding assay, PrsG was found to specify preferential binding to the Forssman antigen, a major constituent of sheep erythrocyte membranes. The binding epitope was identified as the GaINAcα(1–3)GaINAc moiety. This is the first direct evidence that serologically identical pili may present antigenically distinct adhesins, each capable of binding to a specific receptor.
Cloning and CO2‐dependent expression of the genetic region for encapsulation from Bacillus anthracis
Authors: Makino S. , Sasakawa C. , Uchida I. , Terakado N. , Yoshikawa M.
Published: October 2006

The capsule of Bacillus anthracis is an important virulence factor consisting of poly‐o‐glutamic acid. The genetic region required for the encapsulation was cloned in Escherichia coli from the capsule plasmid pTE702, using a selection procedure based on an immunodlffusion assay. The cloned region directed synthesis of the capsule both In E. coli and B. anthracis. Capsule synthesis from these clones, as in the wild type, was dependent upon the presence of CO2. However, encapsulation directed by the cloned fragment was less marked than from pTE702. Another region enhancing capsulation was shown to exist on pTE702. The minimum size of the encapsulation region was defined to within 2.7 kb DNA and shown to be essential for the encapsulation in B. anthracis.
Aerobic expression of the nar operon of Escherichia coli in a fnr mutant
Authors: Bonnefoy V. , Fons M. , Ratouchnlak J. , Pascal M.‐C. , Chippaux M.
Published: October 2006

Mutations allowing aerobic expression of the anaerobically controlled nar operon have been located in the autoregulated fnr gene. Cloning and sequencing of the mutant fnrd20 allele, and fnr mRNA quantitation by dot blot assay, revealed that the mutation was the result of an IS5 insertion into the control region of fnr that enhanced transcription of the fnr gene at least ten‐fold. Examination of the regulatory region of the negatively autoregulated fnr gene indicated that it shared homologous sequences with the positively Fnr‐controlled frd and nar operons. The increase in fnr transcription in the fnrd20 mutated allele could be partly the result of loss of autoregulation, since the IS5 separated the Fnr target site from the ‘−35’ region of the promoter.
Cloning and characterization of an albicidin resistance gene from Klebsiella oxytoca
Authors: Walker M. J. , Birch R. G. , Pemberton J. M.
Published: October 2006

A DNA fragment containing a gene for resistance to the antibiotic albicidin was isolated from Klebsiella oxytoca and shown to be expressed in Escherichia coli, where it also protected bacteriophage T7 replication from inhibition by albicidin. In vivo translation analysis demonstrated that the cloned 2.2kb DNA fragment coded for a 36 kiloDalton (kD) protein and a 25 kD protein.

The DNA sequence was determined for a 654‐base‐pair open reading frame contained within a 1.2 kb subcloned DNA fragment encoding albicidin resistance. The predicted molecular weight of the polypeptide translated from the open reading frame was 25.8 kD. A putative Shine‐Dalgarno sequence precedes the open reading frame but a potential promoter sequence was not detected. A possible rho‐independent transcription termination signal was found directly following the stop codon. The functional protein for albicidin resistance was isolated and purified. Both the molecular weight and NHg‐terminal amino acid sequence of this protein correspond with that predicted from the DNA sequence of the open reading frame. The cloned albicidin resistance gene had no effect on the tsx (nupA) nucleoside uptake gene associated with spontaneous albicidin resistance in E. coli; also, it did not complement any of a range of E. coli DNAts mutants at restrictive temperatures. The cloned resistance gene product remained intracellular in exponential cultures of K. oxytoca and E. coli. Cell‐free extracts from E. coli containing the resistance gene protected a sensitive strain of E. coli from inhibition by albicidin, as did the purified albicidin resistance protein. The mechanism of this albicidin resistance protein involved binding to albicidin to form a complex without antibiotic activity, but without catalysing further chemical modification of the antibiotic.
Determinants of translational initiation efficiency in the atp operon of Escherichia coli
Authors: McCarthy J. E. G. , Bokelmann C.
Published: October 2006

Transcription and translation of the atp genes encoding the subunits δ, α, γ, and ɛ of the Escherichia coli H+‐ATPase were studied. The nature and quantities of the respective transcripts initiated from different promoters were compared with overall expression rates thus yielding accurate information about relative translational efficiency and its coupling to mRNA levels. Part of the highly efficient subunit c gene translational initiation region (TIR) was used as a tool in manipulating the TIRs of the other genes. Rate control of atp cistron translation occurs at the initiation level and is determined locally by each gene's TIR. In this way, individual subunit synthesis rates are set to match the requirements for H+‐ATPase assembly. There is no (or very restricted) translational coupling between the cistrons. Translational initiation rates of the normally weakly expressed atp genes could be increased by up to a factor of 27 by manipulating the sequences upstream of the start codons, despite biased codon usages. In the presence of an improved upstream sequence, the N‐terminal sequence of the subunit γ gene exerted a limiting effect. This could be relieved by altering the sequence of the first seven codons. The levels of subunit γ mRNA were more sensitive to changes in translational efficiency than the concentrations of the other atp mRNAs. The relationships between initiation efficiency and primary and secondary structure in the natural and manipulated atp TIRs are discussed in detail.
Molecular characterization of malQ, the structural gene for the Escherichia coli enzyme amylomaltase
Authors: Pugsley A. P. , Dubreuil C.
Published: October 2006

The structural gene for the Escherichia coli enzyme amylomaltase, malQ, is the second gene in the malPQ operon. The nucleotide sequence of malQ shows that the gene encodes an Mr 78360 protein close to the experimentally determined Mr of purified amylomaltase (72000–74000). The malQ initiation codon was identified by sequence analysis of clustered deletions around the 5′ end of the gene. One of these deletions removed the first 5 bases from the malQ coding sequence. Strains carrying a plasmid with this truncated malQ gene under lacZ promoter control and out‐of‐frame with the first four codons of lacZ were Mal−. The Mal+ phenotype could be restored by inserting small, random fragments of E. coli chromosomal DNA into the unique EcoRI site. Nucleotide sequencing showed that the inserts either joined the lacZ and malQ sequences in frame, or contained a new translation start signal and coding sequence in frame with malQ. These results indicate that amylomaltase could be useful as a reporter protein in gene fusion studies.
Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non‐haemolytic classical strain 569B
Authors: Aim R. A. , Stroeher U. H. , Manning P. A.
Published: October 2006

The El Tor haemolysin, product of hlyA, is exported from Vibrio cholerae as a Mr 80000 protein which can be subsequently cleaved to give two proteins of Mr 65000 and 15000. Nucleotide sequence analysis has demonstrated that hlyA encodes a protein of Mr 82250 with a potential 18‐amino‐acid signal sequence.

The non‐haemolytic classical strain 569B has been shown to have a structural gene defect rather than a defect in secretion. By non‐reciprocal recombination it was possible to transfer this defect onto a plasmid and show that a truncated hlyA product of Mr 27000 is made in Escherichia coli K‐12 minicells. Nucleotide sequence analysis demonstrates an 11‐base‐pair deletion which would result in a Mr 26940 protein probably loosely associated with the membrane.
Expression of four virulence antigens of Shigella flexneri is positively regulated at the transcriptional level by the 30 kilo Dalton virF protein
Authors: Sakai T. , Sasakawa C. , Yoshikawa M.
Published: October 2006

On the virulence plasmid of Shigella flexneri the virG region required for cell‐to‐cell spread of the bacteriumencodes a 130 kiloDalton (kD) antigen and Region‐2essential for the bacterial invasion of epithelial cells encodes 57, 43 and 39 kD antigens. The expression of these four antigens is positively regulated by the 30 kD protein encoded by virF, whose nucleotide sequence had been determined and which was previously found to be essential for virulence. An approximately 3.8kilobase (kb) RNA transcript is found to be transcribed by the virG region and is positively regulated by the virF protein resulting in increased production of the 130 kD antigen. The virF sequence is conserved among all shigellae and enteroinvasive Escherichia coli.
Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium
Authors: Luchansky J. B. , Muriana P. M. , Klaenhammer T. R.
Published: October 2006

Plasmid DNA was introduced by electroporation into Bacillus, Enterococcus, LactobacHlus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Propionibacterium and Staphylococcus as an alternative to competent‐cell or protoplast transformation. Plasmid‐containing transformants were recovered in these recipients at frequencies ranging from 101 105 transformants μg−1 of pGK12. Several parameters of the protocol, including DNA concentration, voltage, plating regimen and electroporation buffers were evaluated to determine conditions that improved transformation frequencies for Lactobacillus acidophilus. Using optimized conditions, the following plasmids were introduced into L. acidophilus: pAMB1, pC194, pGB354, pGKV1, pSA3, pTRK13, pTV1 andpVA797. The ability to transfer plasmid DNA via electroporation will greatly facilitate the application of recombinant DNA methodology and transposon technology to Gram‐positive bacteria for cloning and analysis of significant genes.
Functional domains of colicin A
Authors: Baty D. , Frenette M. , Lioubès R. , Geli V. , Howard S. P. , Pattus F. , Lazdunski C.
Published: October 2006

A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed. The receptor‐binding properties of each protein were also analysed. From these results, we suggest that the NH2‐terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane. The central region of colicin A (residues 173 to 336) contains the receptor‐binding domain. The COOH‐terminal domain (residues 389 to 592) carries the pore‐forming activity.
Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae
Authors: Lee B. C. , Schryvers A. B.
Published: October 2006

Lactoferrin (LF) and transferrin (TF) are postulated to be important physiological sources of iron for Neisseria gonorrhoeae. A dot binding assay involving the use of gonococcal total membranes derived from cells grown in iron‐limited conditions demonstrated the presence of separate receptors for LF and TF. The ligand and functional specificities of these receptors were examined in competition‐binding and growth experiments. The results indicate that the LF and TF receptors are highly specific for the human protein, suggesting that this property may be partially responsible for conferring the human host specificity of N. gonorrhoeae.
Identification of the membrane component of the anion pump encoded by the arsenical resistance operon of R‐factor R773
Authors: San Francisco M. J. D. , Tisa L. S. , Rosen B. P.
Published: October 2006

The arsenical resistance (ars) operon of the conjugative R‐factor R773 encodes an ATP‐driven anion extrusion pump, producing bacterial resistance to arsenicals. There are three structural genes, of which the product of the middle gene, arsB, has not previously been identified. From nucleotide sequence data, the ArsB protein is predicted to be a 45577 Dalton hydrophobic protein. A mini‐Mu transposition procedure was used to construct an arsB‐lacZ gene fusion, producing a hybrid ArsB‐β‐galactosidase protein which was localized in the inner membrane. The operon was cloned into a T7 RNA polymerase expression vector. In addition to the previously identified ArsA and ArsC proteins, the cells synthesized an inner membrane protein with an apparent mass of 36 kD identified as the ArsB protein.
Penicillin‐binding protein 2 genes of non‐β‐lactamase‐producing, penicillin‐resistant strains of Neisseria gonorrhoeae
Authors: Dowson C. G. , Jephcott A. E. , Gough K. R. , Spratt B. G.
Published: October 2006

Oligonucleotides that correspond to regions of the penicillin‐binding protein 2 gene (penA) that differ between penicillin‐sensitive and penicillin‐resistant strains have been used as probes to classify the penA genes in a collection of penicillin‐resistant gonococci isolated in Britain. 44/47 of those gonococcal strains that had minimal inhibitory concentrations of ≥0.25 μg benzylpenicillin per ml contained extensively altered penA genes which appeared to be very similar (or identical) to one or other of the two classes of altered penA genes that have been described previously. Since these two classes of altered penA genes are related, it appears that the great majority of the altered penA genes of non‐β‐lactamase‐producing penicillin‐resistant gonococci have a clonal origin. The other three penicillin‐resistant strains had altered penA genes that were different to those described previously. A crucial step in the development of the altered forms of PBP2 with decreased affinity for penicillin appears to have been the insertion of an extra codon within the transpeptidase domain of the penA gene. This insertion was found in the penA gene of all gonococci with minimal inhibitory concentrations of >0.016μg benzylpenicillin per ml but was not found in any strains with minimal inhibitory concentrations of ≤0.016μg per ml.
Conserved lipoprotein H.8 of pathogenic Neisseria consists entirely of pentapeptide repeats
Authors: Woods J. P. , Spinola S. M. , Strobel S. M. , Cannon J. G.
Published: October 2006

The pathogenic Neisseria, N. gonorrhoeae and N. meningitidis, possess an outer membrane protein (OMP), designated H.8, with a conserved monoclonal antibody (MAb)‐binding epitope. We determined the DNA sequence of a gonococcal H.8 gene, and confirmed the relationship between the cloned gene and the H.8 OMP by constructing a gonococcal mutant lacking H.8. The predicted H.8 OMP is a lipoprotein 71 amino acids in length, composed of 13 repeats of a consensus sequence AAEAP with perfect 5‐residue periodicity. The AAEAP units form a repeating epitope that comprises the entire predicted sequence of the protein.
Extensive re‐modelling of the transpeptidase domain of penicillin‐binding protein 2B of a penicillin‐resistant South African isolate of Streptococcus pneumoniae
Authors: Dowson C. G. , Hutchison A. , Spratt B. G.
Published: October 2006

Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (>1000‐fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin‐binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin‐binding protein 2B from three penicillin‐sensitive strains of S. pneumoniae and from a penicillin‐resistant South African strain. The amino acid sequences of the transpeptidase domains of PBP2B of the three penicillin‐sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin‐resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the‘penicillin‐resistant’form of PBP2B is the substitution of seven consecutive residues in a region that is predicted to form a loop at the bottom of the penicillin‐binding site.
Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: nucleotide sequence of ptsX, ptsH and the 5’‐end of ptsl and evidence for a ptsHI operon
Authors: Gonzy‐Tréboul G. , Zagorec M. , Rain‐Guion M.‐C. , Steinmetz M.
Published: October 2006

The nucleotide sequence of a 1689bp fragment of the Bacillus subtiiis locus containing ptsX (a crr‐like gene), ptsH (coding for HPr), and the 5’‐end of ptsl (coding for Enzyme I) was determined. The deduced amino acid sequences of ptsH and the N‐terminal part of ptsl were compared to those of Streptococcus faecalis and Escherichia coli. Transcription fusion demonstrated that ptsHl constitutes an operon. An open reading frame overlapping the main part of ptsH in the opposite sense was shown to be expressed in vivo, using protein fusions with β‐galactosidase. The deduced amino acid sequence of ptsX showed significant homology with that of Salmonella typhimurium glucose‐specific Enzyme III. ptsX was preceded by an open reading frame whose amino acid sequence showed strong homology with the C‐terminal part of E.coli Enzyme IIGlc.
Nucleotide sequences of the pbpX genes encoding the penicillin‐binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime‐resistant mutant, C506
Authors: Laible G. , Hakenbeck R. , Sicard M. A. , Joris B. , Ghuysen J.‐M.
Published: October 2006

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin‐binding proteins (PBPs) which reduce their affinity for β‐lactam antibiotics. PBP2x is one of the high‐Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime‐resistant laboratory mutants. In this study, we have sequenced a 2564 base‐pair chromosomal fragment from the penicillin‐sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base‐pair open reading frame was found which coded for a protein having an Mr of 82.35 kD, a value which is in good agreement with the Mr of 80–85kD measured by SDS‐gel electrophoresis of the PBP2x protein itself. The N‐terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime‐resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the β‐lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from β‐lactam‐resistant strains are known. The penicillin‐binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N‐terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin‐resistant Staphylococcus aureus PBP2′. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low‐Mr PBPs.
Positive control of colanic acid synthesis in Escherichia coli by rmpA and rmpB, two virulence‐plasmid genes of Kiebsiella pneumoniae
Authors: Nassrf X. , Honoré N. , Vasselon T. , Cole S. T. , Sansonetti P. J.
Published: October 2006

In Kiebsiella pneumoniae, the mucoid phenotype, which is a virulence factor, is distinct from capsule production. It is positively controlled by a plasmid gene, designated rmpA. When introduced into certain Escherichia coli strains, rmpA induces expression of a mucoid phenotype, which results from overproduction of colanic acid at 30° C but not at 37°C. In E. coli, production of colanic acid is regulated by three genes: rcsA and rcsB which act as positive regulators, and rcsC which is a negative effector. In this work we present evidence that the rmpA gene complemented an rcsA, Ion double mutant of E. coli, but not an rcsA, ion+ isolate. This leads to the suggestion that rmpA expressed an rcsA‐like activity and like rcsA, was negatively controlled at post‐transcriptional level by the Lon protease. The nucleotide sequence of rmpA is reported. No homology could be found between the 27 kiloDalton RcsA protein and the deduced amino acid sequence of the 15.5 kiloDalton RmpA protein. Another gene, rmpB, which was required in E. coli recA isolates for full expression of rmpA at 30° C, has been identified on the K. pneumoniae virulence plasmid and shown to encode a 37 kiloDalton protein. Although rmpB was closely linked to rmpA, it was not present on the same transcriptional unit. These results suggested that induction of colanic acid synthesis by the K. pneumoniae virulence gene rmpA, was, at least in E. coli, under the control of the RecA network via rmpB, which may act as a positive regulator of rmpA. We conclude that these plasmid genes may function in K. pneumoniae as regulatory genes controlling the mucoid phenotype, which is itself encoded by the chromosome.
CRP/cAMP‐ and CytR‐regulated promoters in Escherichia coli K12: the cdd promoter
Authors: Valentin‐Hansen P. , Holst B. , Josephsen J. , Hammer K. , Albrechtsen B.
Published: October 2006

Transcriptional regulation of the deoP2 promoter by the cyclic AMP/cyclic AMP receptor protein complex (cAMP/CRP) and the CytR repressor requires two high‐affinity CRP targets located around ‐41 and ‐93 bp preceding the start site for transcription. Here we report the structure of cddP, another CRP/CytR‐regulated promoter. In common with what was found in deo, the cdd promoter also contains multiple CRP targets. Thus, using the DNasel footprinting procedure, tandem CRP binding sites were identified around ‐41 and ‐93. These findings support a general model for CytR binding and CytR regulation, in which (i) CytR and the CRP/cAMP complex bind to similar or Identical targets, (ii) two or more targets are necessary for proper binding of CytR to a promoter region, and (iii) CytR represses transcription by antagonizing cAMP/CRP activation.
The Yersinia yop regulon
Authors: Cornelis G. R. , Biot T. , de Rouvroit C. Lambert , Michiels T. , Mulder B. , Sluiters C. , Sory M.‐P. , Van Bouchaute M. , Vanooteghem J.‐C.
Published: October 2006

Growth of yersiniae is restricted at 37°C in the absence of calcium ions. This phenomenon correlates with the massive release of a set of proteins called Yops. Growth restriction and Yops production are governed by a 70 kb plasmid called pYV. yop genes are distributed throughout pYV and constitute a thermoactivated regulon controlled by the gene virF. The transcription activator VirF is a member of a new family of regulators including those of the arabinose and rhamnose operons as well as a regulator of enteric colonization pili. The role of calcium ions on the release of Yops remains largely unknown.
A family of genes encoding a cell‐killing function may be conserved in all Gram‐negative bacteria
Authors: Poulsen L. K. , Larsen N. W. , Molin S. , Andersson P.
Published: October 2006

The relF gene in Escherichia coli is related to the hok gene on plasmid R1. Both genes encode small proteins which, when overexpressed In E. coli lead to collapse of the membrane potential and cell death. A third gene, designated gef, which encodes a homologous cell‐toxic protein, has been isolated from E. coli DNA. Both gef and relF are transcribed in E. coli and subject to post‐transcriptional regulation which, in the case of gef, is coupled to translation of a leader sequence. The finding of homologous sequences in such distantly related bacteria as Agrobacterium and Rhizobium species suggests an important physiological role.
The Escherichia coli regulatory protein MetJ binds to a tandemly repeated 8bp palindrome
Authors: Davidson B. E. , Girons I. Saint
Published: October 2006

Site‐directed oligonucleotide mutagenesis has been used to isolate thirty four new mutants in the regulatory region of the Escherichia coliK12 gene, metF. The mutants include single base pair (bp) substitutions and insertions, double bp substitutions and one 7bp deletion. The effects of these and another five previously described mutants on the transcriptional regulation of metF have been analysed by using a metF′‐lac'Z fusion in a low copy‐number plasmid. These data, and those obtained from DNAse protection studies using pure MetJ with wild‐type and mutant metF operator DNA, show that the metF operator is comprised of five tandem 8bp repeat units that overlap the ‐10 region of the metF promoter. In the presence of the co‐repressor S‐adenosylmethionine, the DNAse protection studies yielded dissociation constants of 150nM and 300 nM for the interaction of MetJ with repeat units 1 to 4 and repeat unit 5, respectively. In the absence of co‐repressor, the dissociation constants obtained for these interactions are four to five times greater. It is proposed that regulation at the metF operator requires four molecules of MetJ dimer to bind to the five 8bp repeat units to form a tandem, overlapping array. Interactions between MetJ molecules make an essential contribution to the stability of this protein‐DNA complex.
RNA polymerase heterogeneity in Streptomyces coelicolor A3(2)
Authors: Buttner M. J.
Published: October 2006

Recent genetic and biochemical experiments have revealed the existence of at least seven different Sigma factors in Streptomyces coelicolor and demonstrated a role for alternative sigma factors in the control of differentiation and also in the transcription of primary metabolic genes. S. coelicolor has four genes predicted to encode sigma factors that are very closely related to the σ70 polypeptide of Escherichia coli.
Genes for biosynthesis and assembly of CS3 pili of CFA/II enterotoxigenic Escherichia coli: novel regulation of pilus production by bypassing an amber codon
Authors: Jalajakumari M. B. , Thomas C. J. , Halter R. , Manning P. A.
Published: October 2006

The complete nucleotide sequence of the 4746bp HindIII fragment encoding the genes for the biosynthesis and assembly of CS3 pili has been determined. By site‐directed mutagenesis in conjunction with analysis of the plasmid‐encoded proteins in minicells, the actual reading frames for the various products have been determined. This demonstrated that the genes for four of the proteins (63 kD, 48 kD, 33 kD, and 20kD in size) are encoded entirely within the same open reading frame as a fifth protein (104kD). However, for synthesis of this latter protein, suppression or readthrough of an internal amber codon is required. Termination at this codon is also necessary for synthesis of the former proteins. Two further proteins are also encoded within the HindIII fragment: a 27 kD precursor of a periplasmic protein and the 17.5kD precursor of the major CS3 fimbrial subunit.
Regulation of the RAD2 gene of Saccharomyces cervisiae
Authors: Siede W. , Robinson G. W. , Kalainov D. , Malley T. , Friedberg E. C.
Published: October 2006

Regulation of the DNA damage‐inducible RAD2 gene was investigated in yeast cells transformed with centromeric plasmids containing RAD2‐lacZ fusion constructs. Deletion analysis defined several regions in the 350 bp region upstream of the translational start codon which are required for induction of β‐galactosidase activity. No deletions resulted in constitutivety enhanced expression. We therefore conclude that induction of RAD2 by DNA‐damaging agents is positively regulated. Two domains required for induction have a similar sequence and are located ∼70 and ∼140bp upstream of the major transcriptional start site. Four other sequence domains required for induction contain uninterrupted poly(dA) poly(dT) stretches 9‐13 bp long. Deletion of some of these AT‐rich domains also affects constitutive expression of RAD2. Expression of RAD2 is not cell‐cycle‐regulated in mitotic cells. However, meiosis is accompanied by increased steady‐state levels of RAD2 mRNA in the absence of DNA damage. This enhanced transcription is not dependent on the presence of upstream sequences required for regulation of induction by DNA damage. Increased steady‐state levels of RAD2 mRNA are induced by cycloheximide in asynchronously dividing populations of cells, but not in non‐replicating cells arrested in G1 phase of the cell cycle. Following exposure to u.v. irradiation induction is also dramatically reduced in non‐replicating cells.
Nickel deficiency gives rise to the defective hydrogenase phenotype of hydc and fnr mutants in Escherichia coli
Authors: Wu L.‐F. , Mandrand‐Berthelot M.‐A. , Waugh R. , Edmonds C. J. , Holt S. E. , Boxer D. H.
Published: October 2006

Hydrogenase activity and other hydrogenase‐related functions can be restored to hydC mutants by the specific addition of nickel salts to the growth medium. These mutants are defective in all three hydrogenase isoenzymes and the restoration is dependent upon protein synthesis. The cellular nickel content of the mutant when grown in LB medium is less than 1% of that of the parental strain. Partial suppression of the hydrogenase phenotype of hydC mutants occurs when growth takes place in a different medium. This correlates with an increased cellular nickel content. The phenotype of the mutant is also fully suppressed by growth in media of very low magnesium content. Such media facilitate nickel uptake via the magnesium transport system, which leads to the acquisition of a normal cellular nickel content. Mutations in the fnr gene, which encodes a transcriptional regulator for several anaerobically expressed enzymes, abolishes hydC expression and gives rise to a defective hydrogenase phenotype. The hydrogenase phenotype of fnr is closely similar to that of hydC in all respects examined. The hydrogenase activity of fnr strains can be restored by the presence of a functional hydC gene on a multicopy plasmid. The hydrogenase phenotype of fnr strains therefore arises indirectly via suppression of hydC, which leads to a low cellular nickel content. Nickel has no influence on fumarate reductase or nitrate reductase activities in fnr strains. The hydrogen‐metabolism phenotype of fnr strains is, therefore, dependent upon their ability to acquire nickel from growth media. It is likely that hydC encodes a specific transport system for nickel.
Multi‐gene amplification: simultaneous detection of three virulence genes in diarrhoeal stool
Authors: Frankel G. , Giron J. A. , Valmassoi J. , Schoolnik G. K.
Published: October 2006

Enterotoxigenic Escherichia coli (ETEC) and Shigella account for a substantial proportion of acute diarrhoeal illnesses among Third‐World children. Rapid detection of these infectious agents in faeces followed by the prompt implementation of public health measures could help reduce their spread during the early phase of epidemics. Towards this end, three pairs of synthetic oligonucleotide primers were prepared and shown to hybridize specifically to the genes encoding the heat‐stable (ST) and the heat‐labile (LT) enterotoxins of ETEC and to invasion‐associated loci (ial) of the large Shigella virulence plasmid. When the three primer pairs were used together in the polymerase chain reaction (PCR), the three corresponding genetic loci could be simultaneously amplified using DNA extracted directly from stool; the amplified products were readily detected by ST‐, LT‐ and ial‐specific, alkaline phosphatase‐labelled oligonucleotide probes (AP probes). The performance of this system was evaluated in a Mayan community in southeastern Mexico, where diarrhoeal illnesses are a common cause of childhood morbidity and mortality. Using only simple and inexpensive laboratory equipment, multigene amplification with these primers and probes led to the identification of ETEC and/or Shigella in the stools of 20 out of 71 children with diarrhoea; the procedure could be completed in seven hours and was more sensitive than conventional diagnostic tests or DNA probes used without amplification.
Diversity of the outer membrane protein P2 gene from major clones of Haemophilus influenzae type b
Authors: Munson R. , Bailey C. , Grass S.
Published: October 2006

In previous studies, it has been demonstrated that outer membrane protein P2 from Haemophilus influenzae type b has porin activity and that antibody directed against P2 is protective in an infant rat bacteraemic model.

Outer membrane protein subtyping has been employed to subclassify type b Haemophilus isolates. Strain MinnA has the outer membrane protein subtype 1H and is representative of the dominant clonal group of disease‐producing isolates in the United States. In the present study, the P2 gene from strain MinnA was employed to probe EcoRI‐ and PvuII‐digested chromosomal DNA from 24 Haemophilus influenzae type b isolates representative of the common outer membrane protein subtype groups observed throughout the world. Restriction fragment length polymorphisms were identified for the members of the outer membrane protein subtype 3L group, but not for the other subtypes examined.

The P2 gene from each of four prototype isolates was then cloned, sequenced and compared to the previously reported sequence of the strain MinnA gene. The P2 gene from each of two isolates with the outer membrane protein subtype 3L was identical to the MinnA P2 sequence. The P2 gene from a subtype 2L isolate differed by a single nucleotide and the gene from a subtype 6U isolate differed by 13 nucleotides. Thus, the P2 protein is highly conserved among type b isolates.
Salmonella as an intracellular parasite
Authors: Finiay B. B. , Falkow S.
Published: October 2006

Salmonella species are facultative intracellular parasites, capable of penetrating (invading), surviving, and often multiplying within diverse eukaryotic cell types, including epithelial and phagocytic cells. These processes are essential for virulence, and involve both bacterial and host cell products. The use of cultured eukaryotic cells and other model systems has facilitated the study of bacterial‐host cell interactions, and has led to a better understanding of the genetic and molecular basis of Salmonella pathogenicity.
The expression of the DNA ligase gene of Escherichia coli is stimulated by relaxation of chromosomal supercoiling
Authors: Liebart J. C. , Paolozzi L. , Camera M. G. , Pedrini A. M. , Ghelardini P.
Published: October 2006

Many genes of Escherichia coli have been shown to be sensitive to DNA superhelicity. The superhelicity of the chromosome is itself also supercoiling‐dependent. We have developed a general strategy for investigating how a particular gene responds to changes in DNA topology. This approach is used to study the E. coli ligase gene. The thermosensitivity of the E. coli ligts251 mutation can be phenotypically suppressed by mutations which map close to, or in, the gyrB gene and which affect the degree of DNA supercoillng. The level of suppression correlates with the degree of DNA relaxation observed, suggesting that the gene encoding the E. coli DNA ligase is activated by relaxation of the chromosomal DNA.