bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and function of haemolysin B, P‐glycoprotein and other members of a novel family of membrane translocators
Authors: Blight M. A. , Holland I. B.
Published: October 2006

Recent studies have identified two sub‐families of highly conserved polypeptides in a wide variety of organisms concerned with the transport of many different compounds, specific for each transport protein. Both famines, represented by HisP and HlyB, respectively, have in common a highly conserved, approximately 25kD domain, containing an ATP‐binding site. The HisP sub‐family essentially consists of cytoplasmic proteins which couple energy to the import of small substrates through cytoplasmic membrane permeases in Gram‐negative bacteria. The HlyB (P‐glycoprotein) sub‐family, on the other hand, contains a second large domain which apparently acts as the transmembrane translocator itself, which in most cases drives the secretion of a variety of compounds. These membrane domains share a number of structural features which also serve to distinguish these proteins as a closely related group. Nevertheless, the compounds secreted by the HlyB sub‐family Include large polypeptides, polysaccharides and a variety of anti‐tumour drugs. We describe here the properties of each of these remarkable proteins and we speculate on their possible mechanism of action.
Homologous DNA sequences on the virulence plasmids of pathogenic Yersinia and Salmonella dublin Lane
Authors: Krause M. , Fierer J. , Guiney D.
Published: October 2006

Yersinia and Salmonella harbour plasmids that encode traits important for virulence, enabling both pathogenic genera to survive and grow in cells of the reticulo‐endothelial organs during systemic infections. We have detected DNA homology between the Salmonella dublin virulence plasmid pSDL2 and the plasmids of the pathogenic Yersinia species pestis, pseudotuberculosis, and enterocolitica. Three regions of pSDL2 were found to share homology with the virulence plasmid pIB1 of Yersinia pseudotuberculosis. Two separate hybridizing segments mapped within the previously characterized 6.4 kb vir region of pSDL2 in the Sal1 B fragment. The third homologous region involved the regions of plB1, which hybridized to the Sal1 C2 fragment of pSDL2. The virulence plasmid pCD1 from Y. pestis showed similar homology with the three regions of pSDL2. Homologies to the vir and Sal1 C2 regions of pSDL2 were also found on plasmids from Yersinia enterocolitica serotypes 0:9, 0:3 and 0:5, 27. The discovery of separate homologous regions on the virulence plasmids of Salmonella and Yersinia suggests a distant evolutionary relationship.
Tn552, a novel transposable element from Staphylococcus aureus
Authors: Rowland S.‐J. , Dyke K. G. H.
Published: October 2006

Tn552, one of several closely related β‐lactamase‐encoding transposons from Staphylococcus aureus, has a novel set of putative transposition functions. Each is homologous with a well‐characterized function from a different type of mobile genetic element. Thus, Tn552 encodes: (i) resL‐binL, a co‐integrate resolution system homologous with those of Tn3 family elements; (ii) p480, a potential transposase significantly homologous with the DNA integrases of eukaryotic retroviruses and retrotransposons; and (iii) p271, a potential ATP‐binding protein that shows homology with the B protein of phage Mu. The 3′terminal nucleotides of Tn552 (CA), adjacent to which p480 might cleave, are the same as those of retro‐viruses, retrotransposons and phage Mu. The presumptive resolvase (BinL) is very closely related to BinR, which was identified as a DNA invertase and is now shown to resolve an artificial co‐integrate in vivo. Furthermore, the structure of the derivative of Tn552 found in the staphylococcal plasmid pl258 can be explained by a BinL (or BinR)‐mediated site‐specific deletion (‘resolution’) event. Thus, pl258 contains only the right‐hand half of Tn552, which encodes the β‐lactamase and two regulatory proteins. The latter are homologous with the β‐lactamase gene repressor and co‐inducer of Bacillus licheniformis. Interestingly, the order of the regulatory genes is reversed in S. aureus compared with Bacillus licheniformis.
The Min DNA inversion enzyme of plasmid p15B of Escherichia coli 15T−: a new member of the Din family of site‐specific recombinases
Authors: Iida S. , Sandmeier H. , Hübner P. , Hiestand‐Nauer R. , Schneitz K. , Arber W.
Published: October 2006

Plasmid p15B is a bacteriophage P1‐related resident of Escherichia coli 15T−. Both genomes contain a segment in which DNA inversion occurs, although this part of their genomes is not identical. This DNA segment of p15B was cloned in a multicopy vector plasmid. Like its parent, the resulting plasmid, pAW800, undergoes complex multiple DNA inversions: this DNA inversion system is therefore called Min. The min gene, which codes for the p15B Min DNA invertase, can complement the P1 cin recombinase gene. The Min inversion system is thus a new member of the Din family of site‐specific recombinases to which Cin belongs. The DNA sequence of the min gene revealed that Min is most closely related to the Pin recombinase of the e14 defective viral element on the E. coli K12 chromosome. Like other members of the Din family, the min gene contains a recombinational enhancer element which stimulates site‐specific DNA inversion 300‐fold.
The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2‐uptake hydrogenase of Azotobacter chroococcum
Authors: Ford C. M. , Garg N. , Garg R. P. , Tibelius K. H. , Yates M. G. , Arp D. J. , Seefeldt L. C.
Published: October 2006

The structural genes (hupSL) of the membrane‐bound NiFe‐containing H2‐uptake hydrogenase (Hup) of Azotobacter chroococcum were identified by oligo‐nucleotide screening and sequenced. The small subunit gene (hupS) encodes a signal sequence of 34 amino acids followed by a 310‐amino‐acid, 34 156 D protein containing 12 cysteine residues. The large subunit gene (hupL) overlaps hupS by one base and codes for a predicted 601‐amino‐acid, 66433 D protein. There are two regions of strong homology with other Ni hydrogenases: a Cys‐Thr‐Cys‐Cys‐Ser motif near the N‐terminus of HupS and an Asp‐Pro‐Cys‐Leu‐Ala‐Cys motif near the carboxy‐terminus of HupL. Strong overall homology exists between Azotobacter, Bradyrhizobium japonicum and Rhodobacter capsulatus Hup proteins but less exists between the Azotobacter proteins and hydrogenases from Desulfovibrio strains.

Mutagenesis of either hupS or hupL genes of A. chrooooccum yielded Hup− phenotypes but some of these mutants retained a partial H2‐evolving activity. Hybridization experiments at different stages of gene segregation confirmed the multicopy nature of the Azotobacter genome.
Outer membrane proteins of Pseudomonas
Authors: Hancock R. E. W. , Siehnel R. , Martin N.
Published: October 2006

In this review, we describe the outer membrane proteins of Pseudomonas aeruginosa and related strains from the Pseudomonas fluorescens rRNA homology group of the Pseudomonadaceae, with emphasis on the physiological function and biochemical characteristics of these proteins. The use of opr (for outer membrane protein) is proposed as the genetic designation for the P. aeruginosa outer membrane proteins and letters are assigned, in conjunction with this designation, to known outer membrane proteins. Proteins whose primary functions involve pore formation, transport of specific substrates, cell structure determination and membrane stabilization are discussed. The conservation of selected proteins in the above Pseudomonas species is also examined.
Signal transduction and gene regulation through the phosphorylation of two regulatory components: the molecular basis for the osmotic regulation of the porin genes
Authors: Mizuno T. , Mizushima S.
Published: October 2006

Expression of Escherichia coli outer‐membrane porin proteins (OmpF and OmpC) is regulated by the osmolarity of the medium. EnvZ and OmpR, which are positive regulatory factors for the transcriptional osmotic regulation of the ompF and ompC genes, belong to a group of two‐component regulatory factors that respond to a variety of environmental stimuli in bacteria. EnvZ‐OmpR phosphotransfer was revealed to be involved in signal transduction in response to an osmotic stimulus, and to play a crucial physiological role in the consequent osmotic activation of the porin genes. Based on the various lines of experimental evidence, a model is proposed for the molecular mechanism underlying the osmotic regulation through phosphorylation of the activator (OmpR) by the membrane‐locate kinase (EnvZ).
Characterization of a Listeria monocytogenes‐specific protein capable of inducing delayed hypersensitivity in Listeria‐immune mice
Authors: Göhmannn S. , Wächter M. Leimeister , Schiltz E. , Goebel W. , Chakraborty T.
Published: October 2006

Recovery of the host after infection by the intracellular pathogen Listeria monocytogenes is dependent on cell‐mediated immunity. Little is known of the nature of listerial antigens that induce cell‐mediated responses in the infected host. In this study we report on the identification and cloning of an Escherichia coli recombinant encoding a listerial antigen, designated ImaA, capable of eliciting a specific delayed‐type hypersensitivity response in Listeria‐immune mice. Nucleotide sequencing of the Listeria DNA insert in plasmid pLM10 showed that the Ima A gene product consisted of 170 amino acids with a molecular weight of 17994. The predicted amino acid sequence suggests that the protein is localized to the bacterial plasma membrane or cell wall. The ImaA gene was unique to the pathogenic species L. monocytogenes and Listeria ivanovii; it was not present in any other species of the genus Listeria.
Streptococcus pneumoniae possesses canonical Escherichia coli (sigma 70) promoters
Authors: Morrison D. A. , Jaurin B.
Published: October 2006

Summary Seventeen DNA fragments from Streptococcus pneumoniae were randomly cloned in Escherichia coli with selection for promoter activity. The fragments were sequenced and the promoter locations were determined by primer extension analysis. Examination for sites similar to the E. coli major consensus promoter sequence revealed such a site in each of the seventeen fragments, located five to eight base pairs upstream of the point at which transcription was initiated in the E. coli host. Thus, the abundance of promoter activity found in pneumococcal DNA cloned in E coli hosts arises primarily from sigma‐70‐type promoter structures. Combined with the observation that such sequences are usually found just upstream of, but not within, pneumococcal genes, this implies that one class (perhaps the major class) of pneumococcal promoters closely resembles the canonical E. coli promoter consensus.
Isolation and characterization of the recA gene of Bordetella pertussis
Authors: Kuhl S. A. , McCreary R. P. , Bannan J. D. , Friedman R. L.
Published: October 2006

This report describes the detection and cloning of the Bordetella pertussis recA gene. Escherichia coli clones having recombinant plasmids containing the B. pertussis recA gene were isolated by complementing an E. coli RecA− mutant's inability to survive in the presence of methylmethanesulphonate (MMS). This gene was shown to complement the deficiency of E. coli RecA− strains to tolerate the DNA‐damaging effects of both a chemical agent and ultraviolet light (u.v.). Deletion mapping experiments localized the gene to a 2.5kb Stul‐EcoRI fragment, and expression of the gene in E. coli resulted in the production of a 40kD protein. These data strongly suggest that a region of the B. pertussis chromosome that encodes RecA‐like activity has been isolated and cloned.
Molecular mechanism for the antigonococcal action of lysosomal cathepsin G
Authors: Shafer W. M. , Onunka V. C. , Jannoun M. , Huthwaite L. W.
Published: October 2006

Human lysosomal cathepsin G (cat G) appears to be an important mediator of non‐oxidative killing of Neisseria gonorrhoeae ingested by human polymorpho‐nuclear leucocytes (PMNLs). Nearly isogenic strains of gonococci having variations in the structure of penicillin‐binding protein 2 (PBP2) also exhibit different levels of susceptibility to the lethal action of cat G in vitro. Accordingly, we examined the relationship between gonococcal susceptibility to cat G and PBP2 structure. The results of this study suggest that cat G has the capacity to interact with PBP2, as evidenced by its ability to inhibit binding of [3H]‐benzylpenicillin to PBP2. We also found that changes in the amino acid sequence within the transpeptidase domain of PBP2, because of certain penA mutations, modulated such interactions. We propose that PBP2 is an intracellular target for cat G and that levels of gonococcal susceptibility to cat G may be related to PBP2 structure and/or intracellular availability.
Mannose‐sensitive haemagglutination in the absence of piliation in Escherichia coli
Authors: Hultgren S. J. , Duncan J. L. , Schaeffer A. J. , Amundsen S. K.
Published: October 2006

The relationship between type 1 pilus structure and the mannose‐sensitive adhesin was investigated by analysing the properties of an 11.2 kb fragment of DNA derived from the chromosomal pil region of a type 1 piliated uropathogenic strain of Escherichia coli. The recombinant plasmids pHA9 and pSJH9, containing the cloned fragment, conferred a mannose‐sensitive haemagglutination (MSHA)‐positive but non‐piliated phenotype on recipient cells of ORN104. Most of the DNA sequences homologous to the pilA and hyp genes were not present in the 11.2kb insert, and the genetic information necessary for MSHA in the absence of piliation spanned a 6.5 kb region of the cloned fragment. The polypeptides expressed by pSJH9 were examined in minicells and Tn 1000 insertions in three genes encoding proteins of molecular weights 90 kD, 29 kD and 17kD abolished the MSHA phenotype.
Type V collagen as the target for type‐3 fimbriae, enterobacterial adherence organelles
Authors: Tarkkanen A.‐M. , Allen B. L. , Westerlund B. , Holthöfer H. , Kuusela P. , Risteli L. , Clegg S. , Korhonen T. K.
Published: October 2006

Tissue‐binding specificity of the type‐3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild‐type Kleb‐siella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type‐3 fimbriae, as well as the purified type‐3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowman's capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type‐3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type‐3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type‐3 fimbrial gene cluster in association with the pap‐encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type‐3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type‐3 fimbriae strains of Yeisinia and Salmonella, showing that the ability to use type collagen as tissue target is widespread among enteric bacteria.
Secretion of Ricinus communis glyceraldehyde‐3‐phosphate dehydrogenase by Escherichia coli
Authors: Hekman W. E. , Dennis D. T. , Miernyk J. A.
Published: October 2006

A plasmid, pWEH1, was constructed containing a fusion of the DNA encoding the signal sequence of the Escherichia coli outer‐membrane protein A to the 5′‐end of a glyceraldehyde‐3‐phosphate dehydrogenase cDNA from Ricinus communis. When expressed in E. coli, the fusion protein was secreted by the normal membrane‐potential‐dependent pathway. Processing by signal peptidase was inhibited by low concentrations of phenethyl alcohol. Quantitative cell fractionation was used to show that the mature plant protein was associated with the bacterial outer membrane. The protein could not be released from the membrane by washing with alkaline sodium carbonate. Radioactivity from [U‐14C]‐palmitate was incorporated into the heterologous protein. These results suggest that the sequence of this normally cytoplasmic enzyme contains a cryptic lipid‐modification site, and the combination of a signal sequence plus a lipid‐modification sequence results in specific targeting to the bacterial outer membrane.
Purification and characterization of RNase P from Clostridium sporogenes
Authors: Roselli D. M. , Marsh T. L.
Published: October 2006

RNase P is a multi‐subunit enzyme responsible for the accurate processing of the 5′ terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5′ terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20mM) and relatively high concentrations of NH4Cl (800mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4Cs>Rb>K>Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2 SO4 than in NH4Cl.
Pyoverdin‐facilitated iron uptake in Pseudomonas aeruginosa: immunological characterization of the ferripyoverdin receptor
Authors: Meyer J. M. , Hohnadel D. , Khan A. , Cornelis P.
Published: October 2006

A purified polyclonal antiserum directed against the isolated main 80kD IROMP (iron‐regulated outer‐membrane protein) from Pseudomonas aeruginosa PAO1 detected only the 80 kD polypeptide of outer‐membrane proteins from PAO1 cells grown in iron deficiency in Western blots. It was also shown to inhibit the uptake of 59Fe pyoverdin by PAO1 cells as well as its binding to purified outer membranes. Immunofluorescence experiments with intact PAO1 cells confirmed that the receptor is present only at the surface of cells grown under conditions of iron deficiency. All these data allow us to conclude that the 80 kD main IROMP of P. aeruginosa is indeed the receptor for the siderophore ferripyoverdin.
A rapid method for reconstitution of bacterial membrane proteins
Authors: Varadhachary A. , Maloney P. C.
Published: October 2006

We have devised a simple method for the reconstitution of bacterial membrane proteins directly from small (1–20 ml) volumes of cell culture, thus eliminating the preparation of membrane vesicles. Cells are subjected to simultaneous lysozyme digestion and osmotic lysis, and after brief centrifugation ghosts are solubilized in 1.2% octyl‐β‐D‐glucopyranoside (octyl‐glucoside) in the presence of added carrier lipid and an osmolyte. Aliquots of the clarified supernatant are suitable for reconstitution, as documented by using extracts from three different Gram‐negative cells to recover both inorganic phosphate (Pi)‐linked antiport and oxalate: formate exchange activities in proteoliposomes. These proteoliposomes are physically stable, non‐leaky and can sustain a membrane potential and, because functional porins do not reconstitute, the artificial system has transport characteristics similar to those found when proteoliposomes are obtained using standard methods. This method should become an important tool for the screening and characterization of large numbers of strains, both wild‐type and mutant.
Analysis of the subcellular location of pullulanase produced by Escherichia coli carrying the pulA gene from Klebsiella pneumoniae strain UNF5023
Authors: Pugsley A. P. , Kornacker M. G. , Ryter A.
Published: October 2006

Three different techniques, protease accessibility, ceil fractionation and in situ immunocytochemistry, were used to study the location of the lipoprotein pullulanase produced by Escherichia coli K12 carrying the cloned pullulanase structural gene (pulA) from Klebsiella pneumoniae, with or without the K. pneumoniae genes required to transport pullulanase to the cell surface (secretion‐competent and secretion‐incompetent, respectively). Pullulanase produced by secretion‐competent strains could be slowly but quantitatively released into the medium by growing the cells in medium containing pronase. The released pullulanase lacked the N‐terminal fatty‐acylated cystelne residue (and probably also a short N‐terminal segment of the pullulanase polypeptide), confirming that the N‐terminus is the sole membrane anchor in the protein. Pullulanase produced by secretion‐incompetent strains was not affected by proteases, confirming that it is not exposed on the cell surface. Pullulanase cofractionated with both outer and inner membrane vesicles upon isopycnic sucrose gradient centrifugation, irrespective of the secretion competence of the strain. Examination by electron‐microscopy of vesicles labelled with antipullulanase serum and protein A‐gold confirmed that pullulanase was associated with both types of vesicles. When thin‐sectioned cells were examined by the same technique, pullulanase was found to be located mainly on the cell surface of the secretion‐competent cells and mainly in the proximity of the inner membrane in the secretion‐incompetent cells. Thus, while the results from three independent techniques (substrate accessibility, protease accessibility and in situ immunocytochemistry) show that pullulanase is transported to the cell surface of secretion‐competent cells, this could not be confirmed by cell‐fractionation techniques. Possible explanations for this discrepancy are discussed.
Processes of genome evolution reflected by base frequency differences among Serratia marcescens genes
Authors: Sharp P. M.
Published: October 2006

The G + C content of silent sites in codons varies greatly among Serratia marcescens genes; the value in any one gene seems to reflect a balance between mutation pressure towards high G + C content and natural selection constraining choice among synonymous codons. Interestingly, non‐coding sequences have substantially lower G + C content than silent sites thought to be under little selective constraint.
Two cyclic AMP‐regulated genes from Dictyostelium discoideum encode homologous proteins
Authors: Ramji D. P. , Richards A. J. , Jagger P. , Bleasby A. , Hames B. D.
Published: October 2006

Expression of the 7E and 2C genes late in Dictyostelium development ceases upon cell disaggregation but, in contrast to many other genes we have studied, expression is fully restored by exogenous cAMP (A. J. Richards et af., submitted). The 7E and 2C genes encode polypeptides of similar size (9220 and 10573 Daltons, respectively), each of which contains an unusually high proportion of serine plus glycine residues (41% and 59%, respectively). Each protein possesses a relatively serine‐rich N‐terminus and glycine‐rich C‐terminus and contains the conserved sequence S(X)SSS(X2)SS(X)SS(X2)SFGS. These data suggest that genes 7E and 2C may have arisen by duplication of a common ancestor. Computer analysis indicates that both gene products are probably intra‐cellular structural proteins that form extended coil structures.
Site‐directed mutagenesis of the ‘zinc finger’ DNA‐binding domain of the nitrogen‐regulatory protein NIT2 of Neurospora
Authors: Fu Y.H. , Marzluf G.A.
Published: October 2006

The major nitrogen‐regulatory gene nit‐2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen‐catabolic enzymes during conditions of nitrogen limitation. The nit‐2 gene encodes a regulatory protein of 1036 amino acid residues with a single ‘zinc finger’ and a downstream basic region, which together may constitute a DNA‐binding domain. The zinc finger domain of the NIT2 protein was synthesized nit‐3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site‐directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc‐finger motif and the downstream basic region of NIT2 are critical for its trans‐activating function in vivo and specific DNA‐binding in vitro.
Rates of assembly and degradation of bacterial ice nuclei
Authors: Watanabe N. M. , Southworth M. W. , Warren G. J. , Wolber P. K.
Published: October 2006

The kinetics of ice‐nucleus assembly from newly synthesized nucleation protein were observed following induction of nucleation gene expression in the heterologous host Escherichia coli. Assembly was significantly slower for the small proportion of ice nuclei active above ‐4.4°C; this was consistent with the belief that these nuclei comprise the largest aggregates of nucleation protein. The kinetics of nucleus degradation were followed after inhibiting protein synthesis. Nucleation activity and protein showed a concerted decay, indicating that most of the functional ice nuclei are in equilibrium with a single cellular pool of nucleation protein. A minority of the ice nuclei decayed much more slowly than the majority; presumably their nucleation protein was distinct either by virtue of different structure or different subcellular compartmentalization, or because of its presence in a metabolically distinct subpopulation of cells.
The importance of the 5′‐region in regulating the stability of sdh mRNA in Bacillus subtilis
Authors: Melin L. , Friden H. , Dehlin E. , Rutberg L. , von Gabain A.
Published: October 2006

The decay of the polycistronic Bacillus subtilis sdh mRNA was analysed using probes specific for each of the component cistrons, sdhC, sdhA and sdhB. In exponentially growing cells, the entire sdh mRNA seems to decay with an ‘all or nothing’ mechanism and with a uniform half‐life of 2–3 min for all cistrons. In stationary‐phase cells, the half‐life of the 5′‐part had dropped to about 0.6 min whereas that of the 3′‐part was about 1.2 min. Decay of sdh mRNA was also measured in exponentially growing cells containing a ‘down‐mutation’ in the ribosomal binding site preceding sdhC which decreases the expression of sdhC by about 90%. The mutation has a moderate effect on expression of the downstream cistron sdhA. In this mutant, the half‐life of the 5′‐part of sdh mRNA was about 0.5 min (i.e. the same as in stationary phase wild‐type cells) and the half‐life of the 3′‐part about 1.3 min. Also, analysis of the decay of an sdh‐cat fusion transcript revealed that the sdh (5′) part decayed more rapidly than the cat part and this difference was more pronounced in stationary‐phase cells compared to exponentially growing cells. The results of these experiments demonstrate the importance of the 5‐ segment of sdh mRNA in controlling the stability of the transcript under different growth conditions.
Cryptic α‐toxin gene in toxic shock syndrome and septicaemia strains of Staphylococcus aureus
Authors: O'Reilly M. , Kreiswirth B. , Foster T. J.
Published: October 2006

The majority of clinical isolates of Staphylococcus aureus that produce toxic shock syndrome toxin‐1 (TSST‐1) fail to express α‐toxin, despite having a copy of the hla gene in the chromosome. The hla gene was cloned from an Hla‐ TSST‐1+ strain, Todd 555, which had been isolated from a case of toxic shock syndrome in the USA. Of the 630 bases of the Todd 555 gene sequenced, 46 differed from the hla gene sequence of strain Wood 46. The defect in α‐toxin expression was shown to be due to a nonsense mutation which converted a CAG glutamine codon in the equivalent position in the functional Wood 46 sequence to a TAG stop codon. The same mutation was present in the hla gene cloned from a human septicaemia strain (V37) isolated in Dublin. The nonsense mutation of Todd 555 was suppressed by the supE 44 mutation in Escherichia coli resulting in haemolytic activity in cell lysates. Hybrid hla genes were formed by splicing fragments of hla from Todd 555 and Wood 46. Expression of one such chimaeric hla gene in S. aureus demonstrated that the Todd 555 hla gene has a functional agr‐regulated promoter. The silent hla gene may be a cryptic gene in S. aureus.
Analysis of site‐directed mutations in the α‐and β‐subunits of Klebsiella pneumoniae nitrogenase
Authors: Kent H. M. , Baines M. , Gormal C. , Smith B. E. , Buck M.
Published: October 2006

Using directed mutagenesis, amino acid substitutions have been made in the α‐ and β‐subunits of the Klebsiella pneumoniae nitrogenase component 1 at positions normally occupied by conserves cysteine or tyrosine residues. Nif', Nif and intermediate pheno‐types have been obtained. To extend our earlier biochemical characterization (Kent et al, 1989) the electrophoretic mobiliy of component 1 of the mutant and wild‐type nitrogenases has been analysed by non‐denaturing gel electrophoresis. The major and minor forms of component 1 separated by this methodology have been probed for by using both polyclonal and monoclonal antibodies. All Nif mutants exhibited a distribution of electrophoretic forms of component 1 comparable to the wild type, and the abundance of the major from found in purified nitrogennase correlated approximately with the specific activity of the extract. In contrast, after electrophoresis, component 1 from Nif mutants exhibited either a major low‐mobility from or a fast‐moving from. Analysis of co‐factor (FeMoco) allowed us to conclude that changing cysteine 275 to alanine in the α‐submit produces component 1 defective in its interaction with FeMoco. Substitution of other con‐served cysteine residues by alanine appears to prevent early steps in nitrogebnase assembly or to promote degradation. Two single mutations (cysteine89 to alanine in the β‐subunit) which are tightly Nif can be combined to produce a weakly active nitrogenase, indicating regions involved in the interaction between subunits.
Mutagenesis, cloning and complementation analysis of C4‐dicarboxylate transport genes from Rhodobacter capsulatus
Authors: Hamblin M. J. , Shaw J. G. , Curson J. P. , Kelly D. J.
Published: October 2006

Transposon mutagenesis was used to isolate insertion mutants of the photosynthetic bacterium Rhodobacter capsulatus which were unable to grow under aerobic conditions in the dark on malate, succinate or fumarate as sole carbon sources. Of five mutants isolated, all were deficient in C4‐dicarboxylate transport. However, these mutants were still capable of photoheterotrophic growth, although at a slower rate than the wild type, on malate and succinate (but not fumarate). The mutated locus (designated dct) was complemented in trans using a cosmid gene bank. Subcloning and complementation analysis indicated that at least three closely linked genes essential for aerobic dicarboxylate transport were contained within an 8.3kb region of the Rhodobacter capsulatus chromosome.
Gene sequences and comparison of the fimbrial subunits representative of Bacteroides nodosus serotypes A to I: class I and class II strains
Authors: Mattick J. S. , Anderson B. J. , Cox P. T. , Dalrymple B. P. , Bills M. M. , Hobbs M. , Egerton J. R.
Published: October 2006

We have determined the nucleotide sequences of the genes encoding the fimbrial subunits representative of the known Bacteroides nodosus serogroups. All of the genes are preceded by a highly conserved region which includes the likely promoter and transcriptional regulator sites as well as the ribosome‐biding site, and are followed within a short but variable distance by a sequence with the characteristics of a transcription termination or attenuation signal. Based on sequence and organization, the subunits can be divided into two major classes called I (serogroups A, B, C., E, F, G, and I) and II (serogroups D and H). All contain the same seven‐amino‐acid positively charged leader sequence and conserved hydrophobic amino‐terminal sequence typical of type 4 fimbriae. Beyond this point the class II subunits are quite different from class I and share features more in common with those from other type 4 fimbriate bacteria, such as Moraxella bovis and Pseudomonas aeruginosa. The larger class I may be further subdivided into two subsets: (i) ((A, E, F)(B, I)) and (ii) (C, G). These proteins exhibit three major clusters of variation, at either end of the presumptive disulphide loop which spans the central third of the protein, and near the carboxy‐terimus, with dispersed changes in between. The length of the mature subunits varies from 152–156 amino acids, and the variation Includes small insertions or deletions in the variable clusters between more conserved domains. The class II subunits are 149 amino acids in length and contain two pairs of cysteine residues: one is at the end of the amino‐terminal conserved region, and the other is at the end of the protein. The major variation occurs in the central region of the molecule, and again small insertions or deletions are required to align adjacent conserved domains. There is also a striking absence of silent codon changes in the 5′ coding region of all of these genes, indicating that these sequences have a secondary genetic function, probably in recombi‐national exchange.
Catabolite repression of α amylase gene expression in Bacillus subtilis involves a trans‐acting gene product homologous to the Escherichia coli lacl and galR repressors
Authors: Henkin T. M. , Grundy F. J. , Nicholson W. L. , Chambliss G. H.
Published: October 2006

Expression of the α‐amylase gene of Bacillus subtilis is controlled at the transcriptional level, and responds to the growth state of the cell as well as the availability of rapidly metabolizable carbon sources. Glucose‐mediated repression has previously been shown to involve a site near the transcriptional start‐point of the amyE gene. In this study, a transposon insertion mutation was characterized which resulted in loss of glucose repression of amyE gene expression. The gene affected by this mutation, which was localized near 263° on the B. subtilis chromosomal map, was isolated and its DNA sequence was determined. This gene, designated ccpA, exhibited striking homology to repressor genes of the lac and gal repressor family. The ccpA gene was found to be allelic to alsA, previously identified as a regulator of acetoin biosynthesis, and may be involved in catabolite regulation of other systems as well.
Intervening sequences (IVSs) in the 23S ribosomal RNA genes of pathogenic Yersinia enterocolitica strains. The IVSs in Y enterocolitica and Salmonella typhimurium have a common origin
Authors: Skurnik M. , Toivanen P.
Published: October 2006

The 23S ribosomal RNA (rRNA) was shown to be in two fragments in pathogenic Yersinia enterocolitica. The cleavage site in the structural gene of the 23S rRNA was occupied by an intervening sequence (IVS) of about 100 nucleotides, analogous to IVSs found in salmonellae (Burgin et al., 1990). Nucleotide sequences of IVSs of several Y. enterocolitica strains revealed that the IVSs of the highly virulent Y. enterocolitica serotypes strains, and the IVS of Salmonella typhimurium were about 90% similar. On the other hand, the IVSs of the highly and the poorly virulent Y. enterocolitica serotypes were only about 60% similar. These results give the impression that at some point during the IVS evolution, the highly virulent Y. enterocolitica and S. typhimurium both received their IVSs at about the same time from the same source, and that the poorly virulent serotypes received their IVSs earlier. We also found that strain LB5010, derived by extended mutagenization of S. typhimurium LT2, had lost the IVSs originally present in LT2, and that this loss had created a new ‘hairpin loop’ which substituted for the original ‘hairpin loop’.
The ferric‐pseudobactin receptor PupA of Pseudomonas putida WCS358: homology to TonB‐dependent Escherichia coli receptors and specificity of the protein
Authors: Bitter W. , Marugg J. D. , de Weger L. A. , Tommassen J. , Weisbeek P. J.
Published: October 2006

The initial step in the uptake of iron via ferric pseudobactin by the plant‐growth‐promoting Pseudomonas putida strain WCS358 is binding to a specific outer‐membrane protein. The nucleotide sequence of the pupA structural gene, which codes for a ferric pseudobactin receptor, was determined. It contains a single open reading frame which potentially encodes a polypeptide of 819 amino acids, including a putative N‐terminal signal sequence of 47 amino acids. Significant homology, concentrated in four boxes, was found with the TonB‐dependent receptor proteins of Escherichia coli. The pupA mutant MH100 showed a residual efficiency of 30% in the uptake of 55HFe3+ complexed to pseudobactin 358, whereas the iron uptake of four other pseudobactins was not reduced at all. Cells of strain WCS374 supplemented with the pupA gene of strain WCS358 could transport ferric pseudobactin 358 but showed no affinity for three other pseudobactins. It is concluded that PupA is a specific receptor for ferric pseudobactin 358, and that strain WCS358 produces at least one other receptor for other pseudobactins.
Transcriptional analysis of the restriction and modification genes of bacteriophage P1
Authors: Sharrocks A. D. , Hornby D. P.
Published: October 2006

Bacteriophage P1 res and mod genes encode the restriction and modification polypeptides of the Type III restriction enzyme EcoP1. Northern blot analysis using res‐ and mod‐specific probes revealed the presence of two separate transcripts in strains harbouring the Eco P1 restriction and modification genes. Furthermore, by constructing a series of fusions with a promoterless lacZ gene, we show that both the res and mod genes are transcribed from separate promoters. A more detailed investigation of the mod promoter region revealed two promoters located some 70 and 140 bp upstream from the translational start codon. In addition, another pair of promoters and a further separate promoter are located more than 500 bp upstream from this start codon. Two short open reading frames are located between these distal and proximal promoter clusters.

Transcription of the res gene is initiated from within the mod open reading frame from two adjacent promoters. In addition a functional promoter is located on the antisense strand close to the res promoter region. The relationship between the transcription units of the res and mod genes is discussed.
Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2)
Authors: Jiang X.‐M. , Neal B. , Santiago F. , Lee S. J. , Romana L. K. , Reeves P. R.
Published: October 2006

The rfb gene cluster of Salmonella LT2 has been cloned and sequenced. The genes rfbA, rfbB, rfbD, rfbF, rfbG, rfbK, rfbM and rfbP were located individually and the gene rfbL was located outside the cluster. Approximately 16 open reading frames were found in the region which is essential for the expression of O antigen. The gene products of rfbB and rfbG were found to have homology with the group of dehydrogenase and related enzymes described previously. Analysis of the G+C ratio of the rfb cluster extended the area of low‐G+C composition previously found in the sequence of rfbJ to the whole rfb gene cluster. Three to five segments with discrete G+C contents and codon adaptation indices are present in the rfb region, indicating a heterogeneous origin of these segments. Potential promoters were found near the start of the rfb region, supporting the possibility that the rfb gene cluster is an operon.
The role of σF in prespore‐specific transcription in Bacillus subtilis
Authors: Partridge S. R. , Foulger D. , Errington J.
Published: October 2006

Sporulation in Bacillus subtilis is a simple developmental system in which a single cell undergoes differentiation to two ‘sister’ cells, namely the prespore and the sporangium. Prespore‐specific gene expression is largely dependent on the synthesis of a transcription factor, σG. Transcription of spolllG, the gene encoding σG, is under precise temporal and spatial control, requiring the products of at least eight genes that are expressed in the pre‐divisional cell. Here we show that the product of one of these genes, another sigma factor, σF, is by itself sufficient to direct transcription of spolllG in non‐sporulating cells. The results indicate that the cell‐specificity of prespore gene expression is determined by a mechanism that exerts temporal and spatial control over the activity of σF.
Calcium in bacteria: a solution to which problem?
Authors: Norris V. , Chen M. , Goldberg M. , Voskuil J. , McGurk G , Holland B.
Published: October 2006

Calcium and calcium‐binding proteins including those resembling calmodulin are implicated in numerous diverse processes in bacteria. These processes include chemotaxis, sporulation, virulence, the transport of sugars and proteins, phosphorylation, heat shock, the initiation of DNAS replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipids synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism metabolism which have yet to be discovered.
ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria
Authors: Hulton C. S. J. , Higgins C. F. , Sharp P. M.
Published: October 2006

We describe a family of highly conserved, Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences, 14 of which have been identified in Escherichia coli and Salmonella typhimurium and a further three in other enterobacterial species (Yersinia pseudotubercuiosis, Kiebsiella pneumoniae and Vibrio cholerae). ERIC sequences are 126 bp long and appear to be restricted to transcribed regions of the genome, either in intergenic regions of polycistronic operons or in untranslated regions upstream or downstream of open reading frames. ERIC sequences are highly conserved at the nucleotide sequence level but their chromosomal locations differ between species. Several features of ERIC sequences resemble those of REP sequences (Stern et al., 1984) although the nucleotide sequence is entirely different. The question of whether ERICs have a specific function, or represent a form of 'selfish’DNA, is discussed.
Formation of Rhizobium phaseoli symbiotic plasmids by genetic recombination
Authors: Soberón‐Chavez G. , Nájera R. , Espín G. , Moreno S.
Published: October 2006

We report here the formation of symbiotic plasmids (pSyms), by genetic recombination between rearranged pSyms, which lack symbiotic information, and resistance plasmids carrying parts of different symbiotic plasmids (R's). This recombination was found to occur both between plasmids derived from different Rhizobium phaseoli isolates, and between plasmids derived from strains obtained from the same original isolate.

We also present evidence on the formation of a functional symbiotic plasmid by recombination of an R, carrying nif and nod genes from strain CFN42, and an indigenous plasmid present in this strain (pCFN42e), which was thought to be unrelated to its symbiotic plasmid (pCFN42d). These data are discussed with respect to the stability and transfer of Rhizobium symbiotic information.
Replication mutants of Staphylococcus aureus macrolide‐lincosamide‐streptogramin B resistance plasmid pT48
Authors: Catchpole I. , Dyke K. G. H.
Published: October 2006

Copy‐number mutants of Staphylococcus aureus macrolide‐lincosamide‐streptogramin B (MLS) resistance plasmid pT48 were isolated by their resistance to the non‐inducing macrolide, tylosin. One mutant plasmid, pcopD3, showed a three‐ to five‐fold cis‐dominant increase in copy number, and nucleotide sequence analysis revealed that the mutant had a single base change within the replication region. All other pT48 mutants examined had the unusual pheno‐type of increased plasmid multimerization and elevated copy number. These mutants were effective in trans and DNA sequencing showed that plasmids with this phenotype were deleted in one of two ways. The deletions caused similar alterations to the C‐ter‐minus of the wild‐type pT48 Rep protein. The two types of mutant Rep proteins terminate with the same pentapeptide sequence: Ala‐Asn‐Glu‐lle‐Asp. The multimerization phenotype of these mutants can be explained by defective termination of rolling‐circle type replication.
The ArsR protein is a trans‐acting regulatory protein
Authors: Wu J. , Rosen B. P.
Published: October 2006

The arsR gene encodes the regulatory protein of the plasmid‐encoded arsenical resistance operon. A series of in‐frame fusions was constructed between the C‐terminally truncated arsR gene and the coding region for the mature form of β‐lactamase (blaM). Fusions containing most of the arsR gene were still inducible by arsenicals. Fusions containing less than 102 residues of the 117‐residue ArsR protein were constitutive. When a wild‐type arsR gene was placed in trans, the constitutive constructs were again inducible. The results demonstrate that the ArsR protein is a trans‐acting regulatory protein which controls its own expression.
Mitochondrial gene sequences show fungal homology for Pneumocystis carinii
Authors: Pixley F. J. , Wakefield A. E. , Banerji S. , Hopkin J. M.
Published: October 2006

A 6.8 kjlobase fragment of mJtochondrial DNA from Pneumocystis carinii encodes for apocytochrome b, NADH dehydrogenase subunits 1, 2, 3, and 6, cytochrome oxidase subunit II, and the small subunit of ribosomal RNA. Comparative sequence analysis with a series of organisms representative of the fungal and protozoan groups shows that R carinii has, consistently, an average similarity of 60% with the fungi but only 20% with the protozoa. The data indicate homology with the fungi for this opportunistic pathogen.
Characterization of a polygalacturonase gene of Aspergillus niger RH5344
Authors: Ruttkowski E. , Khanh N. Q. , Wientjes F.‐J. , Gottschalk M.
Published: October 2006

We have cloned a gene encoding a polygalacturonase (PG) in the filamentous fungus Aspergillus niger RH5344. The structural gene comprises 1141 bp codIng for 362 amino acids and the open reading frame is disrupted by one intron of 52 bp. Eukaryotic consensus sequences for transcription regulation are found only in deviated forms. The biological functionality of the isolated PG gene was established by retransformation in A. niger and Aspergillus awamori. In addition, we have found that the PG protein of A. niger shares significant similarities with PG proteins from tomato and Erwinia carotovora. Comparison of the three enzymes revealed a highly conserved region in their C‐terminal region probably comprising the elements of substrate binding and the catalytic centre.
A chimeric nucleotide‐binding protein, encoded by a hisP–malK hybrid gene, is functional in maltose transport in Salmonella typhimurium
Authors: Schneider E. , Walter C.
Published: October 2006

We have isolated a hybrid gene, composed of the first 455 nucleotides of hisP and nucieotides 275–1107 of malK, the genes coding for the nucleotide‐binding components of the high‐affinity transport systems for histidine and maltose in Salmonella typhimurium, respectively. The fusion had occurred by recombination within 11 homologous base pairs located between the two DNA fragments. In the chimeric protein peptidic motifs A and B, proposed to be part of the nucleotide‐binding fold, originate from HisP and MalK, respectively. Plasmid pES42‐39, harbouring the hybrid gene, was shown to complement only a malK mutation but failed to complement a hisP deletion mutation. The chimeric protein was identified by immunoblotting as a protein with an apparent molecular mass of 49kDa. Removal of the C‐terminal 77 amino acid residues from the chimeric protein resulted in the loss of function in transport. In contrast, 51 amino acid residues could be removed from the C‐terminus of wild‐type MalK without any effect. Upon overproduction the chimeric protein, as wild‐type MalK, inhibited expression of the malB regulon. However, both truncated proteins, when overproduced, did not exhibit this activity. Based on these results, a tentative model of the functional domains of MalK is presented.