bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B‐band lipopolysaccharide gene cluster
Authors: Burrows L. L. , Charter D. F. , Lam J. S.
Published: November 2003

Pseudomonas aeruginosa co‐expresses A‐band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B‐band LPS, a heteropolymer with a repeating unit of 2–5 sugars which is the serotype‐specific antigen. The gene clusters for A‐ and B‐band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B‐band O‐antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz (rol), wzy (rfc), and wbpA–wbpN. A further 3 ORFs not thought to be involved with LPS synthesis were identified (hisH, hisF, and uvrB). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O‐antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup‐specific, knockout mutations confirmed it is necessary for O5 O‐antigen biosynthesis. A novel insertion sequence, IS1209, is present at the junction between the serogroup‐specific and non‐specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O‐antigen biosynthesis.
An ideR mutant of Mycobacterium smegmatis has derepressed siderophore production and an altered oxidative‐stress response
Authors: Dussurget Olivier , Rodriguez Marcela , Smith Issar
Published: November 2003

The mycobacterial IdeR protein is a homologue of the diphtheria‐toxin repressor DtxR. We have previously demonstrated that Mycobacterium tuberculosis IdeR, like DtxR, represses transcription of Corynebacterium diphtheriae iron‐regulated promoters in vivo and binds to C. diphtheriae operators in a metal‐dependent manner in vitro. We show here that ideR mutants of M. smegmatis, constructed by allelic replacement, were defective in their ability to repress siderophore biosynthesis in the presence of iron. They were also more sensitive to hydrogen peroxide and had decreased levels of catalase/peroxidase (KatG) and manganese superoxide dismutase (Mn‐SOD). This indicates that IdeR is a negative regulator of siderophore production and is required for the response to superoxide‐ and hydrogen peroxide stress. We propose that IdeR is the mycobacterial counterpart of the Escherichia coli Fur protein, i.e. it is a pleiotropic regulator that couples iron metabolism to the oxidative‐stress response.
Overexpression of the mexC–mexD–oprJ efflux operon in nfxB‐type multidrug‐resistant strains of Pseudomonas aeruginosa
Authors: Poole Keith , Gotoh Naomasa , Tsujimoto Hideto , Zhao Qixun , Wada Akihisa , Yamasaki Tetsuo , Neshat Shadi , Yamagishi Jun‐ichi , Li Xian‐Zhi , Nishino Takeshi
Published: November 2003

OprJ, overproduced in nfxB multidrug‐resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug‐resistant strain K385, were demonstrated to be immunologically cross‐reactive using an OprJ‐specific monoclonal antibody. Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N‐terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity. The N‐terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp KpnI fragment and a 10kbp BamHI fragment. Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three‐gene operon, The predicted mexC–mexD–oprJ gene products exhibit homology to the MexA–MexB–OprM components of the multidrug‐resistance efflux pump of P. aeruginosa (43–46% identity). Consistent with an implied role for mexC–mexD–oprJ in drug efflux, the mexC–mexD–oprJ‐hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug ‘exclusion’ was abrogated by energy inhibitors. The mexC and oprJ products are putative lipoproteins of a molecular mass of 40707 and 51742Da, respectively, while mexD was predicted to encode a protein of 111936Da. Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes. Overproduction of OprJ and the attendant multiple‐antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87→R change in the predicted NfxB polypeptide. OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC–mexD–oprJ expression. Consistent with this, the cloned nfxB gene repressed synthesis of a mexC–lacZ fusion in Escherichia coli. nfxB also repressed expression of a nfxB–lacZ fusion, indicating that NfxB negatively regulates its own expression. These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC–mexD–oprJ, encoding components of a second efflux pump in P. aeruginosa.
Interaction of FimB and FimE with the fim switch that controls the phase variation of type 1 fimbriae in Escherichia coli K‐12
Authors: Gally David L. , Leathart Julian , Blomfield Ian C.
Published: November 2003

The phase variation of type 1 fimbriae in Escherichia coli is associated with the site‐specific inversion of a short DNA element. Recombination at fim requires fimB and fimE, and their products are considered to be the fim recombinases. In this study, FimB and FimE were overproduced and extracts containing the proteins were shown to (i) bind to and (ii) invert the fim switch in vitro. Phenanthroline‐copper protection of DNA–protein complexes showed that both FimB and FimE bind to half‐sites that flank, and overlap with, the left and right inverted repeats (IRL and IRR, respectively) of the fim switch. Alignment of the four half‐sites identified a conserved 5′‐CA doublet; mutation of these two bases lowers the affinity of binding of both FimB and FimE to the inverted repeats, and greatly diminishes inversion of the fim switch in vivo. The specificity of the fim recombinases observed in vivo (FimB switching in both directions; FimE switching from on‐to‐off only) was maintained in vitroFurthermore, the different binding affinities of FimB and FimE for the various half‐site combinations suggests that the specificity of FimE could arise, in part, from the low affinity of FimE for IRL (off).
Two beginnings for a single purpose: the dual‐start holins in the regulation of phage lysis
Authors: Bläsi Udo , Young Ry
Published: November 2003

For most large phages of both Gram‐positive and Gram‐negative bacteria, there appears to be a single pathway for achieving disruption of the host envelope, requiring at least two phage‐encoded lysis functions (a holin and an endolysin). The holin is a small membrane protein which causes a non‐specific lesion in the cytoplasmic membrane, which allows the endolysin to gain access to its substrate, the peptidoglycan. The scheduling of host lysis is effected by regulatory mechanisms which govern the synthesis and activity of the holin protein accumulating in the membrane. Accordingly, aspects of expression and function of holin genes are considered here, focusing mainly on the lambdoid S genes. This group of genes, of which lambda S is the prototype, are characterized by a dual‐start motif consisting of two Met start codons separated by one or two codons, at least one of which specifies Arg or Lys. Two protein products are elaborated, differing only by two or three N‐terminal residues but apparently possessing opposing functions: the shorter polypeptide is the active holin, or lysis‐effector, whereas the longer polypeptide apparently acts as an inhibitor of holin function. Models will be considered which may account for the ability of the holin to form a ‘hole’ in the cytoplasmic membrane at a programmed time, as well as for the inhibitory properties of the longer product. Finally, we discuss recent results suggesting that the dual‐start motif can be viewed as a level of regulation superimposed on a timing function intrinsic to the canonical holin structure.
Flagellar assembly in Salmonella typhimurium
Authors: Aizawa Shin‐Ichi
Published: November 2003

The bacterial flagellum is a motility apparatus in which a long helical filament ‐ the propeller ‐ is driven by a rotary motor embedded in the cell surface. Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure. Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum. The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure. Components of the substructures do not necessarily self‐assemble, but often demand the help of other proteins. Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S. typhimurium, is reviewed.
Expression of the groESL operon is cell‐cycle controlled in Caulobacter crescentus
Authors: Avedissian Marcelo , Gomes Suely Lopes
Published: November 2003

The Caulobacter crescentus groESL operon was cloned, sequenced and found to be homologous to previously described groES and groEL genes and proteins. The size of the groESL‐specific transcript (2.3 kb) suggested that groES and groEL of C. crescentus are organized in a bicistronic operon. Heat‐shock induction of groESL mRNA is not transient, high levels of the transcript can be observed after 2 h at 40°C. Prlmer extension experiments showed that transcription initiated at two sites. Only the start site closer to the groES coding region was highly induced during heat shock. The promoter corresponding to the heat‐shock‐inducible transcript has −10 and −35 regions very similar to Escherichia coliσ32 promoters. At normal temperatures, transcription of the groESL operon is cell‐cycle controlled and both transcripts increase co‐ordinately in pre‐divisional cells. Transcription fusions with a lacZ reporter gene and deletions within the promoter region of the groESL operon have shown that no sequences upstream of the heat‐shock promoter are necessary for temporal control. An 11 bp inverted repeat, located between the heat‐shock promoter and the translation start site of groES and very similar to inverted repeats found in front of several heat‐shock genes of other bacteria, may play a role in cell‐cycle control of C. crescentus groESL expression.
The CII protein of bacteriophage 186 establishes lysogeny by activating a promoter upstream of the lysogenic promoter
Authors: Neufing Petra J. , Shearwin Keith E. , Camerotto Julianne , Egan J. Barry
Published: November 2003

We have shown previously that the cII gene product of the non‐lambdoid temperate bacteriophage 186 is required for the establishment of lysogeny. We show here that CII, a potential helix‐turn‐helix DNA‐binding protein, establishes lysogeny by activating a promoter (p E) which spans the apl/cII intergenic region, upstream of the lysogenic promoter, p L. The start site of the p E transcript (+1) has been mapped by primer extension and we have identified the CII binding determinants at p E by DNase I footprinting. CII binds to inverted repeat sequences separated by two turns of the helix, with binding half‐sites centred at the −38 and −58 positions of p E. Oligomerisation studies with purified CII protein indicate that a CII tetramer may be the species that binds to this site. We also show that p E is subject to direct negative feedback by the CI repressor.
FNR‐DNA interactions at natural and semi‐synthetic promoters
Authors: Green J. , Irvine A. S. , Meng W. , Guest J. R.
Published: November 2003

Two rapid and convenient methods have been developed for the amplification and purification of FNR, the anaerobic transcription regulator of Escherichia coli The overproduced proteins resemble wild‐type FNR in their basic properties: oligomeric state, iron contents (up to 2.7 atoms per monomer), DNA‐binding affinities and ability to activate transcription. However, unlike previous preparations, FNR could be isolated in a form containing up to 0.25 atoms of acid‐labile sulphur per monomer. Incorporation of iron increased the Mr of FNR from 28 000 to 40 000. Under anaerobic conditions, reconstituted FNR exhibited absorption maxima at 315nm and 420 nm, which were replaced by a broad absorbance from 380 to 440 nm under aerobic conditions. These observations indicate that FNR contains one redox‐sensitive [3Fe 4S] or [4Fe 4S] centre per monomer. Footprints of FNR‐dependent promoters (ansB, fdn, fnr, narG, pflP6, pflP7 and nirB) showed protection at all of the predicted FNR sites except the pflP7 (‐57.5), ansB (‐74.5) and nirB (‐89.5) sites. An unpredicted second binding site was detected at ‐57.5 in the narG promoter. Hypersensitive sites within regions of FNR protection indicated that FNR bends DNA in a similar way to CRP. Promoters containing binding sites for FNR (FF), CRP (CC) or hybrid sites (CF or FC) were footprinted with FNR and two derivatives (FNR‐610 and FNR‐573) which activate the CCmeIR promoter in vivo. FNR preferentially protected the FNR site (FF) whereas FNR‐610 preferred CC and FNR‐573 interacted with equal affinity at all sites.
In vitro selection of optimal AbrB‐binding sites: comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three‐dimensional DNA structures
Authors: Xu Ke , Strauch Mark A.
Published: November 2003

The AbrB protein of Bacillus subtilis regulates expression of numerous genes, primarily through specific binding interactions to DNA regions containing transcriptional promoters. Although over 15 target regions for AbrB binding to chromosomally located sequences have been analysed by DNase I footprinting, no obvious consensus sequence or motif has yet emerged from their examination. Using in vitro selection techniques, we have isolated optimal AbrB‐binding sites from oligonucleotides containing 22 or 44 random base pairs. The best of these sites have an apparent in vitro Kd which is fivefold lower than a similar‐sized DNA fragment containing the sequence corresponding to the AbrB‐binding site on the spoOE gene. We tested one of the sites in vivo and found that it confers AbrB‐mediated control upon a promoter not normally regulated by AbrB. In each of four separate trials, the selected sites possess motifs that converge to a simple consensus. It is argued that the nature and spacing of these motifs produce a type of three‐dimensional DNA structure recognizable by AbrB, and that known in vivo sites, which lack these motifs, possess an approximation of the optimal structural determinant.
Purification of the Tn 10‐specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein
Authors: Aldema Mila L. , McMurry Laura M. , Walmsley Adrian R. , Levy Stuart B.
Published: November 2003

The bacterial tetracycline‐resistance determinant from Tn 10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/ac operator in a plasmid that provided fusion of TetA to a polyhis‐tidine‐carboxy terminal tail. A second plasmid provided a regulated T7 RNA polymerase. The specific activity of the TetA fusion protein was between 10–40% that of the wild‐type protein as assayed by tetracycline resistance in cells and by transport in membrane vesicles. The fusion protein, overproduced approximately 3–13‐fold, was purified by nickel chelation chromatography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an α‐helix content of 54–64%, close to the 68% predicted from the amino acid sequence by hydropathy analysis (12 membrane‐spanning helices) for the native protein in the membrane bilayer. Fluorescence studies showed binding activity of the purified protein to its substrate, the tetracycline analogue 13‐(cyclopentylthio)‐5‐hydroxy‐6‐α‐deoxyte‐tracycline. These findings suggested that the purified protein was in a native state.
The effect of the stringent response on mutation rates in Escherichia coli K‐12
Authors: Wright Barbara E.
Published: November 2003

The reversion rates of two isogenic Escherichiacoli K‐12 auxotrophs differing only in relA have been determined in the absence or presence of serine hydroxamate, which provokes the stringent response. Reversion rates of leuB−and argH−were significantly higher in the relA+ than in the relA− strain, and the reversion rates in both strains were enhanced by serine hydroxamate. A positive correlation was established between reversion rates and the synthesis of guanosine‐5′‐diphosphate‐3′‐diphosphate in the absence and presence of serine hydroxamate. It is proposed that mutation rates are dependent upon rates of transcription and upon the genes which regulate the level of the signal nucleotide, guanosine tetraphosphate.
Pneumocystis carinii telomere repeats are composed of TTAGGG and the subtelomeric sequence contains a gene encoding the major surface glycoprotein
Authors: Underwood Anthony P. , Louis Edward J. , Borts Rhona H. , Stringer James R. , Wakefield Ann E.
Published: November 2003

We have cloned a telomere and adjacent sequences from rat‐derived Pneumocystis carinii using the ability of foreign telomeres to complement a yeast artificial chromosome (YAC) deficient by one telomere in Saccharomyces cerevisiae. Characterization of the cloned DNA in the recombinant YAC demonstrated that it was a chimera of two P. carinii sequences, namely a 13.5 kb fragment of mitochondrial DNA and an 8.3 kb distal portion consisting of subtelomeric DNA. The P. carinii telomere repeat was demonstrated to be TTAGGG, the most common telomere repeat found in organisms from the animal and fungal kingdoms. Karyotype analysis confirmed that this sequence was present on all the P. carinii chromosomes. Sequence adjacent to the telomere repeats was shown by Bal31 exonuclease digestion to be located at the chromosome ends. Analysis of the subtelomeric fragment revealed homology to the gene encoding the major surface glycoprotein of P. carinii
In vitro processing activity of Bacillus subtilis polynucleotide phosphorylase
Authors: Mitra Sutapa , Hue Kim , Bechhofer David H.
Published: November 2003

A phosphate‐dependent exonuclease activity was identified in purified protein fractions from Bacillus subtilis that were selected for binding to poly(I)‐poly(C) agarose. Based on the characteristics of the degradation products and the absence of this activity in a pnpA strain, which contains a transposon insertion in the B. subtilis PNPase gene (Luttinger et al., 1996 — accompanying paper), this exonuclease activity was shown to be due to polynucleotide phosphorylase (PNPase). Processive 3′‐to‐5′ exonucleolytic degradation of an SP82 phage RNA substrate was stalled at a particular site. Structure probing of the RNA showed that the stall site was downstream of a particular stem‐loop structure. A similar stall site was observed for an RNA that comprised the intergenic region between the B. subtilis rpsO and pnpA genes. The ability to initiate degradation of a substrate that had a stem structure at its 3′ end differed for the B. subtilis and Escherichia coli PNPase enzymes.
The TraB protein, which mediates the intermycelial transfer of the Streptomyces plasmid pSN22, has functional NTP‐binding motifs and is localized to the cytoplasmic membrane
Authors: Kosono Saori , Kataoka Masakazu , Seki Tatsuji , Yoshida Toshiomi
Published: November 2003

The traB gene on the Streptomyces conjugative plasmid pSN22 is required for intermycelial plasmid transfer and the mobilization of chromosomal markers (Cma). The predicted amino acid sequence of TraB contains one Walker type‐A and two type‐B NTP‐binding motifs. Site‐directed mutagenesis revealed that the type‐A motif and one of the type‐B motifs, 109 amino acid residues downstream of the type‐A motif, were essential for both plasmid transfer and Cma. The second type‐B sequence could be changed without any phenotypic effect. A modified traB gene was constructed, resulting in the production of a functional protein with an amino‐terminal c‐Myc epitope tag for immunological analysis. This protein was associated with the cytoplasmic membrane, suggesting that TraB is a membrane protein that uses energy from ATP hydrolysis to transport DNA between mycelia. The c‐Myc tagging of TraB decreased the efficiency of intramycelial plasmid spread, suggesting that TraB is involved in both inter‐ and intramycelial transfer processes.
Escherichia coli K‐12 genes essential for the synthesis of c‐type cytochromes and a third nitrate reductase located in the periplasm
Authors: Grove J. , Tanapongpipat S. , Thomas G. , Griffiths L. , Crooke H. , Cole J.
Published: November 2003

The ‘aeg46.5 ’ operon was originally detected as an ‘anaerobically expressed gene’ located at minute 46.5 on the Escherichia coli linkage map. Subsequent results from the E. coli Genome Sequencing Project revealed that the ‘aeg46.5 ’ promoter was located in the centisome 49 (minute 47) region. Downstream from this promoter are 15 genes, seven of which are predicted to encode a periplasmic nitrate reductase and eight encode proteins homologous to proteins essential for cytochrome c assembly in other bacteria. All of these genes, together with the ‘aeg46.5 ’ promoter, have been subcloned on a 20 kb EcoRI fragment from Kohara phage 19D1. Evidence is presented that, as predicted, the region includes structural genes for two c‐type cytochromes of mass 16 kDa and 24 kDa, which are transcribed from the previously described ‘aeg46.5 ’ promoter, and that the first seven genes encode a functional nitrate reductase. We, therefore, propose that they should be designated nap (nitrate reductase in the periplasm) genes. Plasmids encoding the entire 20 kb region, or only the downstream eight genes, complemented five mutations resulting in total absence of all five known c‐type cytochromes in E. coli, providing biochemical evidence that these are ccm (for cytochrome c maturation) genes. The ccm region was transcribed both from the FNR‐dependent, NarL‐ and NarP‐regulated nap promoter (formerly the ‘aeg46.5 ’ promoter) and from constitutive or weakly regulated promoters apparently located within the downstream nap and ccm genes.
Heterologous expression and self‐assembly of the S‐layer protein SbsA of Bacillus stearothermophilus in Escherichia coli
Authors: Kuen Beatrix , Sára Margit , Lubitz Werner
Published: November 2003

The cell surface of Bacillus stearothermophilus PV72 is covered by a regular surface layer (S‐layer) composed of a single species of protein, SbsA, with a molecular weight of 130 000. Recently, the sequence of the corresponding gene (sbsA) has been determined. The SbsA coding region including the signal sequence was cloned as a polymerase chain reaction (PCR) product into a low‐copy‐number vector under the transcriptional control of the λpL promoter. Expression of sbsA was shown to be thermally inducible from the resulting vector pBK4 in a strain of Escherichia coli expressing the λcI857 from the chromosome. As shown by ultrathin sectioning of whole cells and immunogold labelling using SbsA‐specific antibodies, expression of sbsA in E. coli led to accumulation of sheet‐like self‐assembling products of the protein in the cytoplasm. No SbsA protein was detected either in the periplasm or in the supernatant fractions. Long‐term expression of sbsA from pBK4, including in the late stationary phase, did not lead to degradation of SbsA.
Multicopy single‐stranded DNA of Escherichia coli enhances mutation and recombination frequencies by titrating MutS protein
Authors: Maas Werner K. , Wang Chi , Lima Tania , Hach Angela , Lim Dongbin
Published: November 2003

Multicopy single‐stranded DNA (msDNA) molecules consist of single‐stranded DNA covalently linked to RNA. In Escherichia coli, such molecules are encoded by genetic elements called retrons. The DNA moieties of msDNAs have characteristic stem‐loop structures, and most of these structures contain mismatched base pairs. Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids. In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E. coli. To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs. We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination. We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.
Characterization of the CheAS/CheZ complex: a specific interaction resulting in enhanced dephosphorylating activity on CheY‐phosphate
Authors: Wang Hui , Matsumura Philip
Published: November 2003

The cheA gene encodes two overlapping polypeptides with a common carboxyl terminus: CheAL and CheAS. CheAL plays a central role in the Escherichiacoli chemotaxis signalling pathway by autophosphorylation and transferring the phosphate to both CheY and CheB. On the other hand, the physiological functions of CheAS remain unknown.

We have observed that overproduction of CheAS in wild‐type cells increased counterclockwise‐biased flagellar rotation, and this effect is dependent on the presence of CheZ. CheZ specifically facilitates CheY‐phosphate (CheY‐P) dephosphorylation and generates a smooth swimming signal. A physical interaction was detected between CheZ and CheAS in wild‐type cell lysates by immunoprecipitation. The CheAS/CheZ interaction does not require other chemotaxis components, as we could form the complex using purified CheAS and CheZ proteins. The ability of CheAS to bind to CheZ depends on its being in the reduced state. We found that under non‐reducing conditions, CheAS appears to form intermolecular disulphide bonds and loses the ability to bind to CheZ. Finally, the CheAS/CheZ complex formed in vitro shows a greater dephosphorylating activity on CheY‐P than does free CheZ.
Cation and sugar selectivity determinants in a novel family of transport proteins
Authors: Poolman Bert , Knol Jan , Van Der Does Chris , Henderson Peter J. F. , Liang Wei‐Jun , Leblanc Gérard , Pourcher Thierry , Mus‐Veteau Isabelle
Published: November 2003

A new family of homologous membrane proteins that transport galactosides–pentoses–hexuronides (GPH) is described. By analysing the aligned amino acid sequences of the GPH family, and by exploiting their different specificities for cations and sugars, we have designed mutations that yield novel insights into the nature of ligand binding sites in membrane proteins. Mutants have been isolated/constructed in the melibiose transport proteins of Escherichia coliKlebsiella pneumoniae and Salmonella typhimurium, and the lactose transport protein of Streptococcus thermophilus which facilitate uncoupled transport or have an altered cation and/or substrate specificity. Most of the mutations map in the amino‐terminal region, in or near amphipathic α‐helices II and IV, or in interhelix‐loop 10–11 of the transport proteins. On the basis of the kinetic properties of these mutants, and the primary and secondary structure analyses presented here, we speculate on the cation binding pocket of this family of transporters. The regulation of the transporters through interaction with, or phosphorylation by, components of the phosphoenolpyruvate:sugar phosphotransferase system is also discussed.
The plasmid replicator AMA1 in Aspergillus nidulans is an inverted duplication of a low‐copy‐number dispersed genomic repeat
Authors: Aleksenko Alexei , Clutterbuck A. John
Published: November 2003

The AMA1 sequence was isolated from a genomic library of Aspergillus nidulans on the basis of its ability to enhance transformation frequency and generate phenotypically unstable transformants in this fungus. These properties were previously shown to be the result of extrachromosomal replication of AMA1‐bearing plasmids. Here we demonstrate that AMA1 is an inverted duplication of a sequence which has other isolated genomic copies. These sequences (mobile Aspergillus transformation enhancers, or MATEs) share a high degree of sequence similarity and exhibit some features characteristic of mobile elements, including a potential Met‐tRNA priming site, similar to that found in retrotransposons of the Ty‐copia group. The nucleotide sequence does not encode any extended polypeptides but contains ARS‐consensus matches and a multiply repeated ‘Spe’ motif, which may be described as a symmetrically duplicated topoisomerase I recognition site. This motif was shown to be a target for illegitimate recombination events. The mobility of members of the MATE family is inferred from the observation that their chromosomal locations are highly variable between wild Aspergillus isolates. The inverted duplication AMA1 is present in laboratory strains derived from the Glasgow isolate but not in other wild isolates tested. This indicates that the inverted duplication AMA1 is of recent evolutionary origin and probably does not exert any conserved function in the chromosome. We discuss possible connections between structural features of AMA1 and its ability to promote extrachromosomal plasmid replication.
The molecular defences against reactive oxygen species in yeast
Authors: Moradas‐Ferreira P. , Costa V. , Piper P. , Mager W.
Published: November 2003

There is rapidly expanding interest into the protective systems against reactive oxygen species (ROS) in the eukaryotic cell, now that the links between oxidative damage, various disease states, and ageing, are firmly established in higher organisms. Yeast molecular genetics should be able to provide powerful insight into these mechanisms; this potential is now starting to be exploited. A number of primary antioxidant activities and systems of metal‐ion homeostasis or detoxification have now been demonstrated to contribute to oxidative‐stress protection in yeast. Also, evidence is emerging that the oxidative‐stress response of this organism is complex, involving separate transcription‐factor responses to peroxide, superoxide anion and metal ions.
C‐terminal domains of general regulatory factors Abf1p and Rap1p in Saccharomyces cerevisiae display functional similarity
Authors: Gonçalves Paula M. , Maurer Kick , Van Nieuw Amerongen Geerten , Bergkamp‐Steffens Karin , Mager Willem H. , Planta Rudi J.
Published: November 2003

Abf1p and Rap1p are global regulatory factors which play an essential role in the transcription activation of yeast ribosomal protein genes. This functional link prompted us to investigate whether these factors may be functionally interchangeable. We focused on the indispensable C‐terminal portions of both factors and performed mutual domain swaps. The functional capacity of the resulting hybrid proteins was subsequently examined using yeast strains conditionally expressing either the ABF1 or the RAP1 gene. Both the Abf1p–Rap1p and the Rap1p–Abf1p fusion proteins were found to be able to complement the growth defect of the respective strains. Furthermore, Abf1p and Rap1p are both able to promote transcription of a reporter gene through a combination of the respective binding site and a T‐rich promoter element. These data strongly suggest that the C‐terminal domains of Abf1p and Rap1p have, at least partially, identical functions. Finally, a deletion analysis of the so far largely uncharacterized C‐terminal domain of Abf1p was performed, which revealed that two regions of 50 amino acids can perform all essential Abf1p functions.
Cloning and expression of the Haemophilus influenzae transferrin receptor genes
Authors: Loosmore Sheena M. , Yang Yan‐ping , Coleman Debbie C. , Shortreed Jean M. , England Diane M. , Harkness Robin E. , Chong Pele S.‐C. , Klein Michel H.
Published: November 2003

The genomic transferrin receptor genes (tbpA and tbpB ) from two strains of Haemophilus influenzae type b (Hib) and two strains of non‐typable H. influenzae (NTHi) have been cloned and sequenced. The deduced protein sequences of the H. influenzae tbpA genes were 95–100% conserved and those of the tbpB genes were 66–100% conserved. The tbpB gene from one strain of NTHi was found to encode a truncated Tbp2 protein. The tbpB genes from four additional NTHi strains were amplified by the polymerase chain reaction (PCR) utilizing primers derived from the conserved N‐terminal sequences of Tbp1 and Tbp2 and were found to encode full‐length proteins. Although several bacterial species express transferrin receptors, when the Tbp1 and Tbp2 sequences from different organisms were compared, there was only limited homology. Recombinant Tbp1 and Tbp2 proteins were expressed from Escherichia coli and antisera were raised to the purified proteins. There was significant antigenic conservation of both Tbp1 and Tbp2 amongst H. influenzae strains, as determined by Western blot analysis. In a passive model of bacteraemia, infant rats were protected from challenge with Hib after transfer of anti‐rTbp2 antiserum, but not after anti‐rTbp1 antiserum.
Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1
Authors: Comstock Laurie E. , Johnson Judith A. , Michalski Jane M. , Morris, Jr J. Glenn , Kaper James B.
Published: November 2003

Vibrio cholerae serogroup O139 Bengal is the first documented serogroup other than O1 to cause epidemic cholera. The O139 Bengal strains are very similar to V. cholerae serogroup O1 biotype El Tor strains. The major differences between the two serogroups are that O139 Bengal contains a distinct O antigen and produces a polysaccharide capsule. We previously described three TnphoA mutants of O139 strain AI1837 which abolish both O antigen and capsule production. These TnphoA insertions were mapped to a 21.5 kb EcoRI fragment of the O139 chromosome. We describe here the cloning and mapping of this 21.5 kb EcoRI fragment and it was shown to complement each of the mutants in trans to produce O antigen and capsule. The EcoRI fragment contains 13 kb of DNA that is specific to O139 and 8.5 kb of DNA that is common to O1 and O139. Sequence analysis of the 13 kb of O139‐specific DNA revealed that it contains 11 open reading frames all of which are transcribed in the same direction. Eight of the 11 open reading frames are homologous to sugar biosynthesis genes from other organisms. Using extended polymerase chain reactions, we show that the extent of the DNA region in O139 that is not present in O1 is approximately 35kb. The site of insertion of this O139‐specific DNA in the O1 chromosome was mapped to the rfbO1 region. We also demonstrate that O139 Bengal strain AI1837 contains a deletion of 22 kb that in serogroup O1 strains contains the rfb region. Therefore, O139 Bengal probably arose from an O1 strain that had undergone genetic rearrangements including deletion of the O1 rfb region and acquisition of a 35 kb region of DNA which encodes O139 surface polysaccharide.
Specific induction by glycine of the gene for the P‐subunit of glycine decarboxylase from Saccharomyces cerevisiae
Authors: Sinclair David A. , Hong Seung‐Pyo , Dawes Ian W.
Published: November 2003

The glycine decarboxylase complex (GDC) is composed of four subunits referred to as H‐, L‐, P‐, and T‐proteins. The Saccharomyces cerevisiaeGCV2 gene, encoding the P‐protein has been cloned by complementation of the gsd2 mutation which prevents cells converting glycine to serine or using glycine as the sole nitrogen source. The gene, located on the right arm of chromosome XIII adjacent to TPS1, encodes a product with a Mr of 114 385. Expression of GCV2 was induced by the addition of more than 200 μM glycine in the medium, and a maximal sixfold induction occurred above 1 mM. This response was specific to glycine and was not observed for any other amino acid. Under the same conditions, the intracellular level of glycine increased up to 30‐fold. The levels of P‐ and L‐protein transcripts and GDC activity were also elevated in cells grown in the presence of glycine. Deletion analysis of the GCV2 promoter delimited the control region which contains putative regulatory sites for GCN4 and GLN3 transcription factors.
Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae
Authors: Spellerberg Barbara , Cundell Diana R. , Sandros Jens , Pearce Barbara J. , Idänpään‐Heikkilä Ilona , Rosenow Carsten , Masure H. Robert
Published: November 2003

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcβ1‐3Gal, GalNAcβ1‐4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H2O2 and CO2. This mutant produced decreased concentrations of H2O2 and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down‐regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo
DsbA is required for stability of the type IV pilin of enteropathogenic Escherichia coli
Authors: Zhang Hong‐Zhong , Donnenberg Michael S.
Published: November 2003

The periplasmic Escherichia coli enzyme DsbA catalyses the efficient formation of disulphide linkages in numerous extracytoplasmic proteins. Enteropathogenic E. coli, a major cause of infantile diarrhoea worldwide, expresses a type IV fimbria known as the bundle‐forming pilus that promotes adherence to tissue‐culture cells. In this study, we report that transposon insertions in the dsbA locus abolish adherence and dramatically reduce the level of bundlin, the major structural subunit of the pilus encoded by the bfpA locus. Adherence and bundlin levels are restored by complementation with the cloned dsbA gene. DsbA has no effect on bfpA transcription as measured with bfpA–lacZ fusions. Replacement of either cysteine codon 129 or 179 of bfpA with a serine codon results in reduced levels of bundlin, similar to the effect of the dsbA mutation. As is the case with dsbA mutants, this decreased level of bundlin is not due to decreased transcription. The half‐life of bundlin as detected by pulse‐chase experiments is dramatically reduced in a dsbA mutant in comparison to the wild type. The effect of DsbA on bundlin oxidation is independent of signal‐peptide processing. Thus, we demonstrate that the DsbA enzyme is critical for the biogenesis of a type IV fimbria because of the essential role of a disulphide bond in the stability of the major structural subunit. These data illuminate the early steps in the biogenesis of type IV fimbriae by demonstrating that newly synthesized prepilin is a transmembrane protein accessible to periplasmic and cytoplasmic processing enzymes.
Establishing differential gene expression in sporulating Bacillus subtilis: phosphorylation of SpoIIAA (anti‐anti‐σF) alters its conformation and prevents formation of a SpoIIAA/SpoIIAB/ADP complex
Authors: Magnin Thierry , Lord Matthew , Errington Jeffrey , Yudkin Michael D.
Published: November 2003

Sigma‐factor F (σF) is a key transcription factor that initiates prespore development in Bacillus subtilis. Its activity is controlled by an anti‐sigma factor, SpoIIAB, which is also a protein kinase that phosphorylates the anti‐anti‐sigma factor SpoIIAA. We have examined our earlier prediction that SpoIIAA must undergo a major change in its properties when phosphorylated. Upon gel filtration in the presence of ADP, SpoIIAA‐P was eluted from a Superdex column much later than SpoIIAB, whereas SpoIIAA was co‐eluted with SpoIIAB, indicating the formation of a protein/protein complex. The complex contained ADP, and had two monomers of SpoIIAA to each SpoIIAB dimer. Its dissociation constant was 13 μM. Gel permeation on high‐performance liquid chromatography (HPLC) suggested an apparent molecular mass for SpoIIAA‐P which was much higher (23.5 kDa) than that of SpoIIAA (15.8 kDa), but Ferguson plots showed that SpoIIAA‐P was not a phosphorylated dimer of SpoIIAA. Our tentative conclusion, that SpoIIAA and SpoIIAA‐P differ markedly in conformation, was confirmed by the results of partial digestion with chymotrypsin.
Antagonistic involvement of FIS and H‐NS proteins in the transcriptional control of hns expression
Authors: Falconi Maurizio , Brandi Anna , La Teana Anna , Gualerzi Claudio O. , Pon Cynthia L.
Published: November 2003

Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns. These sites extend from −282 to +25 with two sites, closely flanking the DNA bend located at −150 from the transcriptional startpoint, partly overlapping the H‐NS binding sites involved in the transcriptional autorepression of hns. The interplay between FIS, H‐NS and the hns promoter region were studied by examining the effects of FIS and H‐NS on in vitro transcription of hns–cat fusions, as well as looking at the effect of FIS on preformed complexes containing H‐NS and a DNA fragment derived from the hns promoter region. Taken together, our data suggest that in the cell, FIS and H‐NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns
Allelic polymorphism and site‐specific recombination in the opc locus of Neisseria meningitidis
Authors: Seiler Andrea , Reinhardt Richard , Sarkari Jasmine , Caugant Dominique A. , Achtman Mark
Published: November 2003

The opc gene is widespread in epidemic and endemic Neisseria meningitidis, but most strains of certain epidemic clones (ET‐37 complex, Cluster A4) and a few random endemic isolates lack an opc gene. Four percent of the 1148 bp that contain opc plus the surrounding intergenic region was polymorphic (18 alleles), and many of the alleles contained a 230 bp insertion at a fixed location in the intergenic region. The presence or absence of the insertion reflects site‐specific recombination. The alleles are stably inherited within clonal groupings for up to at least 50 years, with rare cases of horizontal genetic exchange. Most statistical methods indicated significant intragenic recombination events within this dataset.
Erythromycin production in Saccharopolyspora erythraea does not require a functional propionyl‐CoA carboxylase
Authors: Donadio Stefano , Staver Michael J. , Katz Leonard
Published: November 2003

Using an oligonucleotide corresponding to the consensus sequence for the biotin‐binding motif, two unlinked genetic loci, bpl1 and bpl2, were cloned from the erythromycin producer Saccharopolyspora erythraea and the nucleotide sequences of a c. 4 kb segment from each determined. The two loci share a virtually identical segment of 1746 nucleotides, coinciding with most of the genes designated bcpA1 and bcpA2 present in bpl1 and bpl2, respectively. The deduced sequences of these genes are highly similar to that of the α‐chain of mammalian propionyl‐CoA carboxylase. Upstream of bcpA2 lies pccB, the gene encoding the β‐chain of this enzyme. Mutant strains carrying frameshift mutations in bcpA1 and pccB were constructed, but we failed to isolate insertional mutants in bcpA2. Propionyl‐CoA carboxylase activity was undetectable in the pccB mutant, but was unaffected in the bcpA1‐defective strain. These results indicate that pccB encodes the β‐chain of propionyl‐CoA carboxylases, and suggest that the α‐chain of this enzyme, which is likely to be encoded by bcpA2, is shared with some other essential biotin‐dependent enzyme. The pccB mutation had no impact on erythromycin production in complex medium.
Polynucleotide phosphorylase is required for the rapid degradation of the RNase E‐processed rpsO mRNA of Escherichia coli devoid of its 3′ hairpin
Authors: Braun Frédérique , Hajnsdorf Eliane , Régnier Philippe
Published: November 2003

The monocistronic transcript of rpsO undergoes an endonucleolytic cleavage downstream of the coding sequence, which removes the hairpin of the transcription terminator and initiates the rapid degradation of the message. We demonstrate here that the two rne‐dependent cleavages, on both sides of the transcription terminator, are catalysed by RNase E in vitro and that the RNase E‐processed rpsO message is rapidly degraded by polynucleotide phosphorylase, while RNase II produces stable decay intermediates. Moreover, we show that RNase E cuts in vitro the coding sequence of the rpsO mRNA at several sites which are not detected in vivo
Regulation of competence development and sugar utilization in Haemophilus influenzae Rd by a phosphoenolpyruvate:fructose phosphotransferase system
Authors: Macfadyen Leah P. , Dorocicz Irene R. , Reizer Jonathan , Saier Jr Milton H. , Redfield Rosemary J.
Published: November 2003

Changes in intracellular cAMP concentration play important roles in Haemophilus influenzae, regulating both sugar utilization and competence for natural transformation. In enteric bacteria, cAMP levels are controlled by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in response to changes in availability of the preferred sugars it transports. We have demonstrated the existence of a simple PTS in H. influenzae by several methods. We have cloned the H. influenzaeptsI gene, encoding PTS Enzyme I; genome analysis locates it in a pts operon structurally homologous to those of enteric bacteria. In vitro phosphorylation assays confirmed the presence of functional PTS components. A ptsI null mutation reduced fructose uptake to 1% of the wild‐type rate, and abolished fructose fermentation even when exogenous cAMP was provided. The ptsI mutation also prevented fermentation of ribose and galactose, but utilization of these cAMP‐dependent sugars was restored by addition of cAMP. In wild‐type cells the non‐metabolizable fructose analogue xylitol prevented fermentation of these sugars, confirming that the fructose PTS regulates cAMP levels. Development of competence under standard inducing conditions was reduced 250‐fold by the ptsI mutation, unless cells were provided with exogenous cAMP. Competence is thus shown to be under direct nutritional control by a fructose‐specific PTS.