bluuebunny/crossref_metadata_2025 huggingface dataset:
The purine‐cytosine permease gene of Saccharomyces cerevisiae: primary structure and deduced protein sequence of the FCY2 gene product
Authors: Weber E. , Rodriguez C. , Chevallier M. R. , Jund R.
Published: October 2006

A 2.1 kb DNA segment carrying the purine‐cytosine permease gene (FCY2) of Saccharomyces cerevisiae was sequenced, the primary structure of the protein (533 amino acids) deduced and a folding pattern in the membrane is proposed for the permease protein. Expression of the FCY2 gene product requires a functional secretory pathway and is reduced in mnn9, a mutant strain deficient in outer chain glycosylation. The FCY2 gene was mapped on the right arm of chromosome V close to the HIS1 gene.
Mechanism of polysialic acid chain elongation in Escherichia coli K1
Authors: Steenbergen S. M. , Vimr E. R.
Published: October 2006

Understanding the mechanisms of polysialic acid synthesis in Escherichia coli K1 requires a molecular description of the polymerase complex. Since the number of potential models explaining polysialic acid assembly would be constrained if only one sialyltransferase were required for this process, the phenotypes of a sialyltransferase null mutation generated by transposon mutagenesis were investigated. The chromosomal insertion mutation was mapped by Southern hybridization analysis and by complementation with plasmid subclones, demonstrating that sialyltransferase is encoded by neuS, a gene implicated previously as coding for the polymerase (Vimr et al., 1989). As expected, if only one gene encoded sialyltransferase, the null mutant had undetectable polymerase activity when assayed with endogenous or exogenous acceptors, and accumulated sugar nucleotide precursors intracellularly. Nested deletion analysis of neuS ruled out polarity effects of transposon insertion mutation and provided more precise mapping of the sialyltransferase structural gene. Maxicell analysis of the nested deletion set implicated a 34000 molecular weight polypeptide as the neuS gene product. These results, together with biochemical characterization of sialyltransferase reaction products in the wild type, indicated that CMP‐sialic acid is the probable sialosyl donor for polysialic acid elongation and that chain growth is by sequential addition of monomeric units.
LamB (maltoporin) of Salmonella typhimurium: isolation, purification and comparison of sugar binding with LamB of Escherichia coli
Authors: Schülein K. , Benz R.
Published: October 2006

LamB (maltporin) of Salmonella typhimurium was found to be more strongly associated with the murein than OmpF. It was purified in one step using a hydroxyapatite (HTP) column. Reconstitution of the pure protein with lipid bilayer membrane showed that LamB of S. typhimurium formed small ion‐permeable channels with a single channel conductance of about 90 pS in 1 M KCI and some preference for cations over anions. The conductance concentration curve was linear, which suggested that LamB of S. typhimurium does not contain any binding site for ions. Pore conductance was completely inhibited by the addition of 20mM maltotriose. Titration of the LamB‐induced membrane conductance with different sugars, including all members of the maltooligosaccharide series up to seven glucose residues, suggested that the channel contains, like LamB (maltoporin) of Escherichia coli, a binding site for sugars. The binding constant of sugars of the maltooligosaccharide series increased with increasing number of glucose residues up to five (saturated). Small sugars had a higher stability constant for sugar binding relative to LamB of E. coli. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion porin.
The ami locus of the Gram‐positive bacterium Streptococcus pneumoniae is similar to binding protein‐dependent transport operons of Gram‐negative bacteria
Authors: Alloing G. , Trombe M.C , Claverys J.P.
Published: October 2006

The complete nucleotide sequence of the ami locus of Streptococcus pneumoniae revealed the presence of six open reading frames, amiABCDEF. The predicted Ami proteins are probably involved in a transport system. The Ami A, C, D, E, and F proteins exhibit homology with components of the oligopeptide permeases (opp) of Salmonella typhimurium and Escherichia coli. Intriguingly, the AmiB protein is homologous to ArsC, a cytosolic modifier subunit of the anion pump encoded by the arsenical resistance operon of the R‐factor R773 from E. coli. Data are presented which indicate that Ami is indeed a transport system.
The cytotoxic protein YopE of Yersinia obstructs the primary host defence
Authors: Rosqvist R. , Forsberg Å. , Rimpiläinen M. , Bergman T. , Wolf‐Watz H.
Published: October 2006

It has previously been shown that the plasmid‐encoded YopE protein of Yersinia pseudotubercuiosis is a virulence determinant. In this study, HeLa cells, macrophages and mice were used as different model systems to determine the actual role of YopE in the virulence process. The YopE protein mediates a cytotoxic response on a confluent layer of HeLa cells. A prerequisite of this activity is that the pathogen binds to the cell surface. YopE also induces a cytotoxic response on mouse macrophages where it influences the ability of the pathogen to resist phagocytosis. Bacterial mutants defective in their ability to express YopE are avirulent after oral or Intra peritonea I infection but virulent following intravenous injection. On the basis of these results, we propose a role for YopE in the virulence process of Yersinia.
Ureaplasma urealyticum urease genes; use of a UGA tryptophan codon
Authors: Blanchard A.
Published: October 2006

Nucleotide sequence analysis of a Ureaplasma urealyticum DNA fragment, homologous to cloned urease genes of other prokaryotes, revealed three consecutive open reading frames. The molecular weights of the three deduced polypeptides are 11.2kD, 13.6kD and 66.6 kD. These values are consistent with the size of the three subunits previously reported for purified native urease. A significant sequence homology was found between the three polypeptides of the ureaplasmal urease and the single polypeptide of jack bean (Canavalia ensiformis) urease. Codon usage indicates that UGA is a tryptophan codon in this mollicute. Use of polymerase chain reactions has disclosed the existence of genetic polymorphism among the urease genes of different serotypes of U. urealyticum.
Genetic functions and cell–cell interactions in the pheromone‐inducible plasmid transfer system of Enterococcus faecalis
Authors: Dunny G. M.
Published: October 2006

Pheromone‐inducible plasmid transfer is a novel form of bacterial conjugation which has, to date, been observed only in Enterococcus (Streptococcus) faecalis. This process includes several important stages of interaction between the donor and recipient cell. The initial interaction is the transmission of a chemical signal from the recipient to the donor cell. Recent evidence has shown that the signal is in the form of a small hydrophobic peptide, which is capable of inducing a complex mating response in the donor cell at concentrations as low as 1–5 molecules per responder cell. Most E. faecalis strains produce multiple pheromones, each of which induces a response only in cells carrying a particular plasmid (or member of a family of related plasmids). Genetic functions ascribed to the pheromone response include: (i) cell‐cell aggregation, which promotes initial close contact between mating cells; (ii) surface exclusion, which prevents plasmid transfer between aggregated donor cells; and (iii) highly efficient DNA transfer, which requires other unidentified functions in addition to aggregation. The first two processes appear to be mediated by proteinaceous surface antigens.
The ‘Bayer bridges’ confronted with results from improved electron microscopy methods
Authors: Kellenberger E.
Published: October 2006

In electron micrographs of conventionally prepared thin sections of Escherichia coli one observes (i) a wavy appearance of the two membranes showing frequent appositions (named adhesion sites) and (ii) intermembrane bridges after plasmolysis which, it is claimed, occur at the adhesion sites and are related to intermembrane protein transport (transmigration). When chemical fixation is replaced by cryofixation, the observations are very different, (a) The two membranes are equally spaced and no contacts, adhesions or other sorts of connections are visible, (b) After plasmolysis the protoplast is shrunken, but the typical bridges are no longer produced. (c) In addition, when peptidoglycan is stained on conventionally prepared sections, it is revealed as a 7‐nm‐thick sacculus which is not interrupted at the sites of apposition. In view of the new observations, the structural concepts derived from conventionally prepared material must be revised. It is proposed that the intermembrane space is entirely filled by a gel, the outer part of which is the 7 nm thick, very stable, chemically resistant peptidoglycan (or murein). The inner part is much less stable and is proposed to undergo rapid autolytic changes upon cell death. The large ‘Bayer bridges’ might then tentatively be explained as an artificial post‐mortem enhancement of either a stream of proteins transmigrating across the periplasm or of a pre‐existing, but not yet resolved, structure. This enhancement probably occurs during the 7‐10 min between plasmolysis and fixation that are prescribed for the procedure necessary for revealing‘Bayer bridges'.
Integrity of Escherichia coli P pili during biogenesis: properties and role of PapJ
Authors: Tennent J. M. , Lindberg F. , Normark S.
Published: October 2006

The papJ gene of uropathogenic Escherichia coli is required to maintain the integrity of Galα(1‐4)Gal‐binding P pili. Electron microscopy and ELISA have established that strains carrying the papJ1 mutant allele have a large amount of pilus antigen free of the cells. In contrast to the whole pili released by strains unable to produce the PapH pilus anchor, the free papJ1 pili consist of variably sized segments that appear to result from Internal breakages to the pilus. The DNA sequence of papJ is presented and its gene product identified as an 18kD periplasmic protein that possesses homology with nucleotide‐binding proteins. PapJ may function as a ‘molecular chaperone’ directly or indirectly establishing the correct assembly of PapA subunits in the P pilus.
Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence
Authors: Barny M. A. , Guinebretière M. H. , Marçais B. , Coissac E. , Paulin J. P. , Laurent J.
Published: October 2006

Phage MudIIPR13 insertional mutagenesis of Erwinia amylovora CFBP1430 allowed us to isolate 6900 independent CmR mutants. The frequencies of different auxotrophs in this population indicated that MudIIPR13 had inserted randomly in E. amylovora. Screening of 3500 CmR mutants on (i) apple calli and (ii) pear and apple seedlings led to the isolation of 19 non‐pathogenic prototrophic single mutants, four of which expressed a LacZ+ hybrid protein. Expression of the fusion proteins was temperature sensitive. The 19 mutants could be separated into two classes according to their behaviour on tobacco: 13 were unable to elicit the hypersensitive response on tobacco (Hrp−) while six still could (Dsp−). The 19 MudIIPR13 insertions all mapped in the same virulence region. The MudIIPR13 insertions of Hrp− mutants were all clustered on the left part of this region, white the MudIIPR13 insertions of Dsp− mutants were located on the right part. All of the mutants except one, which proved to have a large deletion of the entire virulence region, could be complemented functionally by cosmids from an E amylovora CFBP1430 genomic library. No hybridization was observed between the cosmid pPV130, which complemented 12 hrp::MudIIPR13 mutations, and the hrp genes from Pseudomonas syringae pv. phaseolicola (Lindgren et al., 1986), P. syringae pv. tomato (N. J. Panopoulos, unpublished data) or P. solanacearum (Boucher et al., 1987). Further analysis of the large virulence region will allow mapping of the border of the virulence region and facilitate the study of the function and regulation of the hrp and dsp genes.
Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor
Authors: Domenighini M. , Relman D. , Capiau C. , Falkow S. , Prugnola A. , Scarlato V. , Rappuoli R.
Published: October 2006

The nucleotide sequence of the structural gene for filamentous haemagglutinin (FHA), fhaB, a crucial adherence factor for Bordetella perfussis, has been determined. Its 10774 nucleotides are far more than necessary to encode the 220kD biologically active, mature polypeptide product, suggesting a role for co‐or post‐translational processing. Fusion proteins derived from various portions of the fhaB open reading frame (ORF) were used to generate polyclonal antisera. Western immunoblot analysis of purified FHA and Bordetella sp. whole cell extracts with these antisera indicated that the 220kD product is encoded by the 5 portion of the ORF and that the smaller polypeptide species are breakdown products of this polypeptide. These data, as well as N‐terminal amino acid sequencing of the major polypeptide species, suggest a scheme for the proteolytic processing of an FHA precursor polypeptide.
Recent advances in peptide chain termination
Authors: Craigen W. J. , Lee C. C. , Caskey C. T.
Published: October 2006

Peptide chain termination occurs when a stop codon is decoded by a release factor. In Escherichia coli two codon‐specific release factors (RF1 and RF2) direct the termination of protein synthesis, while in eukaryotes a single factor is required. The E. coli factors have been purified and their genes isolated. A combination of protein and DNA sequence data reveal that the RFs are structurally similar and that RF2 is encoded in two reading frames. Frame‐shifting from one reading frame to the next occurs at a rate of 50%, is regulated by the RF2‐specific stop codon UGA, and involves the direct interaction of the RF2 mRNA with the 3’end of the 16S rRNA. The RF genes are located in two separate operons, with the RF1 gene located at 26.7 min and the RF2 gene at 62.3 min on the chromosome map. Ribosomal binding studies place the RF‐binding region at the interface between the ribosomal subunits. A possible mechanism of stop‐codon recognition is reviewed.
DNA from diverse sources manifests cryptic low‐level transcription in Escherichia coli
Authors: Miller W. G. , Simons R. W.
Published: October 2006

We present evidence that DNA from diverse prokaryotic and eukaryotic sources gives rise to low‐level fusion expression in Escherichia coli promoter‐probe vectors. This expression may be as high as 10% of the E coli lac UV5 promoter. Although expression does not correlate with the presence of obvious E. coli promoter‐like sequences, it is blocked by transcriptional terminators. Furthermore, transcription across the fusion junction is detected at levels that correlate with fusion expression. We suggest that this Mow‐level transcription'(LLT) results from infrequent initiation by RNA polymerase at random sites and/or weak promoters. We propose that LLT has biological significance. In some instances, it may provide an advantageous basal level of gene expression, and we suggest that this may be true for the E. coli lac Y gene. In other instances, LLT may be detrimental, in which case it may be blocked by mechanisms such as RNA secondary structure or transcriptional polarity. We present evidence to show that activation of the IS 10 transposase gene by LLT is blocked at the translational level.
Sequence analysis of Enterococcus faecalis aggregation substance encoded by the sex pheromone plasmid pAD1
Authors: Galli D. , Lottspeich F. , Wirth R.
Published: October 2006

The location of the structural gene for aggregation substance on the sex pheromone plasmid pAD1 of Enterococcus faecalis was determined using an oligonucleotide deduced from the N‐terminal amino acid sequence of the purified protein. The nucleotide sequence was determined for the corresponding region and two open reading frames (ORFs) could be identified. ORF1 codes for a small (Mr 13160) acidic protein of unknown function. The gene for aggregation substance (named asa1 was found to code for a protein of 1296 amino acids (Mr 142248). The protein has a signal peptide of 43 amino acids (the resulting (Mr for mature aggregation substance is 137429) and contains in its C‐terminal region a proline‐rich sequence, previously characterized as being involved in cell wall association, which is followed by a membrane anchor. The membrane anchor showed significant similarity to that of other Gram‐positive organisms, but no other similarities to surface proteins from Gram‐positive bacteria were found. In particular, no repeats on the DNA or protein level could be detected for pAD1‐specific aggregation substance. The protein contains the amino acid motifs Arg‐Gly‐Asp‐Ser and Arg‐Gly‐Asp‐Val (once each), which, it is proposed, play a crucial role in adherence to eukaryotic cells.
Insertion of an extra amino acid is the main cause of the low affinity of penicillin‐binding protein 2 in penicillin‐resistant strains of Neisseria gonorrhoeae
Authors: Brannigan J. A. , Tirodimos I. A. , Zhang Q.‐Y. , Dowson C. G. , Spratt B. G.
Published: October 2006

Non‐β‐lactamase‐ producing, penicillin‐resistant strains of Neisseria gonorrhoeae (CMRNG strains) produce altered forms of penicillin‐binding protein 2 (PBP2) that have decreased affinity for penicillin. A feature of PBP2 from all CMRNG strains is the presence of an additional residue (Asp‐345A) that is absent from PBP2 of penicillin‐sensitive strains. The role of the additional aspartic acid residue in the decreased affinity of PBP2 is unclear as PBP2 of all previously examined CMRNG strains possess several other amino acid sequence alterations, in addition to the insertion of Asp‐345A, compared to PBP2 of penicillin‐sensitive strains. Site‐directed mutagenesis has been used to insert the Asp‐345A codon into the penA gene from a penicillin‐sensitive gonococcus. The resulting penA gene expressed an altered form of PBP2 that had a decreased affinity for benzylpenicillin and was able to transform a pencillin‐sensitive strain of N. gonorrhoeae to an increased level of resistance to benzylpenicillin. Insertion of amino acids other than aspartic acid did not produce forms of PBP2 that provided increased resistance to penicillin. Removal of the Asp‐345A codon from the penA gene of a CMRNG strain reduced its ability to transform a penicillin‐sensitive strain to an increased level of penicillin resistance. The reduction in the affinity of PBP2 in CMRNG strains is therefore largely, although not exclusively, due to the insertion of Asp‐345A. Clinical isolates that produce altered forms of PBP2 that differ from that of penicillin‐sensitive strains only in the insertion of Asp‐345A have been identified.
Octanoylation of the lipoyl domains of the pyruvate dehydrogenase complex in a lipoyl‐deficient strain of Escherichia coli
Authors: Ali S. T. , Moir A. J. G. , Ashton P. R. , Engel P. C. , Guest J. R.
Published: October 2006

The overexpression of a subgene encoding a hybrid lipoyl domain of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli has previously bee shown to result in the formation of lipoylated an unlipoylated products. Overexpression of the same subgene in a lipoic acid biosynthesis mutant growing under lipoate‐deficient conditions has now bee shown to produce domains modified by octanoylation as well as unmodified domains. It was concluded from the mass of a lipoyl‐binding‐site peptide that the modification involves N6‐octanoylation of the lysin residue (Lys244) that is normally lipoylated, and this was confirmed by the trypsin‐insensitivity of the corresponding Lys244‐Ala245 bond, and the absence c modification in a mutant domain in which Lys244 is replaced by Gin. This novel protein modification raise interesting questions concerning the pathway of lipoic acid biosynthesis and the mechanism of enzyme lipoylation.
Molecular aspects of phosphate transport in Escherichia coli
Authors: Rao N. N. , Torriani A.
Published: October 2006

Escherichia coli transports inorganic phosphate (Pi) by the low‐affinity transport system, Pit. When the level of the external Pi is lower than 20μM, another transport system, Pst, is induced with a Kt of 0.25μM. An outer‐membrane porin, PhoE, with a Km of about 1μM is also induced. The outer membrane allows the intake of organic phosphates which are degraded to Pi by phosphatases in the periplasm. The Pi‐binding protein will capture the free Pi produced in the periplasm and direct it to the transmembrane channel of the cytoplasmic membrane. The channel consists of two proteins, PstA and PstC, which have six and five transmembrane helices, respectively. On the cytoplasmic side of the membrane the channel is linked to the PstB protein, which carries a nucleotide (probably ATP)‐binding site. PstB probably provides the energy required by the channel to free Pi. The Pst system has two functions in E. coli: (i) the transport of Pi, and (ii) the negative regulation of the phosphate regulon (a complex of 20 proteins mostly related to organic phosphate transport). It is remarkable that these two functions are not related, since the repressibility of the regulon depends on the integral structure of Pst (PiBP + PstA + PstC + PstB) and not on the Pi transported. Another gene of the pst operon, phoU, produces a protein involved in the negative regulation of the Pho regulon, but the mechanism of this function has not been explained. Thus the regulatory function of the Pst system remains obscure. Its basal level, present when Pi is abundant, is sufficient to repress the Pho regulon but the negative regulatory function is lost upon Pi starvation.
Construction and characterization of a protective antigen‐deficient Bacillus anthracis strain
Authors: Cataldi A. , Labruyère E. , Mock M.
Published: October 2006

The pag gene coding for protective antigen (PA), one of the three toxin components of Bacillus anthracis, has been cloned into the mobilizable shuttle vector pAT187 and transferred by conjugation from Escherichia coli to B. anthracis. Using this strategy, an insertionally mutated pag gene constructed and characterized in E. coli, was introduced into B. anthracis Sterne strain. This transconjugant was used to select a recombinant clone (RP8) carrying the inactivated pag gene on the toxin‐encoding piasmid, pXO1. Strain RP8 was deficient for PA while still producing the two other toxin components, i.e. lethal factor (LF) and edema factor (EF). In contrast to spores from the wild‐type Sterne strain, spores prepared from RP8 were totally non‐lethal in mice. These results clearly establish the central role played by PA in B. anthracis pathogenicity.
Sequence, localization and function of the invasin protein of Yersinia enterocoiitica
Authors: Young V. B. , Miller V. L. , Falkow S. , Schoolnik G. K.
Published: October 2006

The inv locus of Yersinia enterocolitica is sufficient to convert a non‐invasive Escherichia coli K12 strain into a microorganism that is able to penetrate cultured mammalian cells. The nucleotide sequence of inv reveals an open reading frame corresponding to an 835‐amino‐acid protein that is homologous to the invasin protein from Yersinia pseudotuberculosis. A polyclonal antiserum elicited by a synthetic peptide corresponding to the C‐terminal 88 amino acids of this open reading frame detected a unique 100kD protein in cell lysates of Y. enterocolitica strain 8081c and in an E. coli strain harbouring the cloned inv gene. This protein localized to the outer membranes of both microorganisms and was cleaved by low concentrations of extracellular trypsin. HEp‐2 cells were shown to attach to surfaces coated with bacterial outer membranes containing invasin and this attachment was destroyed by treatment of the membranes with trypsin. Thus it appears that the invasin protein from Y. enterocolitica is able to mediate both attachment to and entry of cultured epithelial cells.
Protein transfer into the recipient cell during bacterial conjugation: studies with F and RP4
Authors: Rees C. E. D. , Wilkins B. M.
Published: October 2006

Transfer of donor cell proteins to the recipient bacterium was examined in F‐ and RP4‐mediated conjugation. Transfer of a 120kD polypeptide, identified as the larger product of the plasmid DNA primase gene, was readily detected during RP4‐promoted conjugation. The protein was transmitted to the cytoplasm of the recipient, presumably complexed to the transferred ssDNA. F DNA was transferred without detectable association with any cytoplasmic tra protein or with the ssDNA‐binding protein encoded by the plasmid. However, a 92kD protein, possibly F TraD product, was transmitted to the membrane fraction of the recipient cell.
On the evolutionary origins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system
Authors: Wu L.‐F. , Saier M. H.
Published: October 2006

The genes encoding the proteins of the fructose‐specific phosphotransferase system (PTS) of Rhodobacter capsulatus were sequenced, and the deduced amino acyl sequences of the energy‐coupling protein, Enzyme I, and the transport protein, Enzyme IItru, were compared with published sequences. Enzyme I was found to be homologous to pyruvate: phosphate dikinase of plants, while Enzyme IIfru was found to be homologous to the insulin‐responsive glucose facilitator of mammals. The evolutionary and functional implications of these findings are discussed.
What are antibiotics? Archaic functions for modern activities
Authors: Davies J.
Published: October 2006

Secondary metabolites are proposed to have played important roles in the evolution of the reactions of living forms on earth, in effecting and modulating reactions during biochemical evolution by chemical and structural interaction with ‘receptor’ sites in primitive macromolecular templates. For example, in the evolution of the translation system, as the polymerizing reactions became more complex and proteins became involved, the low molecular‐weight effectors were functionally replaced by polypeptides, but retained their ability to interact with receptor sites in nucleic acids and proteins. Many of these low molecular‐weight effectors now play a different role, that of antagonists, by interacting with the original receptor sites in contemporary activity as antibiotics.
Characterization of divergent NtrA‐dependent promoters in the anaerobically expressed gene cluster coding for hydrogenase 3 components of Escherichia coli
Authors: Lutz S. , Böhm R. , Beier A. , Böck A.
Published: October 2006

The regulatory region of two divergently oriented transcriptional units involved in the formation of the gas‐evolving hydrogenase (isoenzyme 3) of Escherichia coli was investigated. DNA sequence analysis revealed the existence of a 210bp non‐coding region containing two sequences showing homology to ‐24/‐12 NtrA‐dependent promoters. These sequences were arranged in a divergent orientation entirety consistent with their being involved in transcribing the divergent operons. Through S1 protection experiments it could be shown that transcription of both promoters was NtrA‐dependent and that it was regulated in an identical manner: oxygen repressed expression, as did anaerobic growth in the presence of nitrate; transcription was induced in cells grown anaerobically in the absence of exogenous electron acceptors and formate was found to be obligately required for this anaerobic induction. Lying at an approximately equal distance between both promoters was a short stretch of DNA which showed similarity to the sequence previously identified (Birkmann and Böck, 1989a) as being necessary for formate induction of the fdhF gene.
Characterization of the traT gene and mutants that increase outer membrane permeability from the Salmonella typhimurium virulence plasmid
Authors: Sukupolvi S. , Vuorio R. , Qi S.‐Y. , O'Connor D. , Rhen M.
Published: October 2006

The nucieotide sequence of the traT gene present in the virulence‐associated pfasmid of Salmonella typhimurium was determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino‐terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previousfy focalrzed to the traT gene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6‐5: the introduction, by site‐directed mutagenesis, of either positively or negatively charged amino acids or the helix‐disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer membrane.
Duplication and variation of the thermostable direct haemolysin (tdh) gene in Vibrio parahaemolyticus
Authors: Nishibuchi M. , Kaper J. B.
Published: October 2006

The relationship between phenotypic variation and nucleotide sequence variation of the gene encoding Vibrio parahaemolyticus thermostable direct haemolysin (tdh gene) was examined. Strains showing a typical haemolysin‐positive phenotype carried two chromosomal gene copies (designated tdh1 and tdh2) while fdh‐gene‐positive strains showing a weakly positive or negative haemolysin phenotype possessed only a single chromosomal gene copy. Both gene copies from a typical haemolysin‐positive strain were cloned and sequenced and possessed 97.2% homo‐logy. Comparison of the amino acid sequence predicted from the nucleotide sequence with the protein sequence determined by Edman degradation as well as construction of a tdh1‐deficient yet haemolytic strain of V. parahaemolyticus suggest that the tdh2 locus is primarily responsible for the haemolytic phenotype. Two other tdh gene copies were cloned from a phenotypically negative strain which was unusual in that it contained one gene copy on a plasmid (designated tdh4) in addition to a single copy on the chromosome (tdhS). Both tdh3 and tdh4 were expressed in Escherichia coli and TDHs with haemolytic activity were produced. These gene copies were sequenced and shared 96.7% homology with the tdh1 gene. The V. parahaemolyticus strain carrying tdh3 and tdh4 gene copies did not produce detectable amounts of tdh‐specific RNA transcript. It seems, therefore, that differences in the transcriptional control are primarily responsible for the differences seen in haemolytic phenotype.
Functional analysis of the fsoC gene product of the F71 (fso) fimbrial gene cluster
Authors: Riegman N. , Acton D. , Sakkers R. , van Die I. , Hoekstra W. , Bergmans H.
Published: October 2006

Contrary to what would be expected from data in the literature, mutations in the fsoC gene of the F71 (fso) P‐fimbrial gene cluster do not completely block fimbrial biogenesis. fsoC mutants still express small amounts of fimbriae of normal length, which carry the non‐adhesive minor subunit protein, FsoE, but lack the adhesin, FsoG. The FsoC protein operates at the same stage in fimbrial biogenesis as the FsoF and FsoG proteins. The data suggest that FsoC, FsoF and FsoG interact to form an initiation complex for fimbrial biogenesis.
Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli
Authors: Gygi D. , Nicolet J. , Frey J. , Cross M. , Koronakis V. , Hughes C.
Published: October 2006

The gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild‐type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemotytic activity were attained similarly by the E. coli post‐translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram‐negative human and animal pathogens.
Mechanism of post‐segregational killing by the hoklsok system of plasmid R1: sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid‐free cells
Authors: Gerdes K. , Thisted T. , Martinussen J.
Published: October 2006

The hok/sok system of plasmid R1, which mediates plasmid stabilization via killing of plasmid‐free segregants, encodes two genes: hok and sok. The hok gene product is a potent cell‐killing protein. The expression of hok is regulated post‐transcriptionally by the sok gene‐encoded repressor, an antisense RNA complementary to the hok mRNA leader region. We show here that the hok mRNA is very stable, while the sok RNA decays rapidly. We also observe a new hok mRNA species which is 70 nucleotides shorter in the 3′‐end than the full‐length hok transcript. The appearance of the truncated hok mRNA was found to be regulated by the sok antisense RNA. Furthermore, the presence of the truncated hok mRNA was found to be correlated with efficient expression of the Hok protein. On the basis of these findings, we propose an extended model in order to explain the killing of plasmid‐free segregants by the hok/sok system.
Interconversion of the DNA‐binding specificities of two related transcription regulators, CRP and FNR
Authors: Spiro S. , Gaston K. L. , Bell A. I. , Roberts R. E. , Busby S. J. W. , Guest J. R.
Published: October 2006

In Escherichia coli, FNR and CRP are homologous transcriptional regulators which recognize similar nucleotide sequences via DNA‐binding domains containing analogous helix‐turn‐helix motifs. The molecular basis for recognition and discrimination of their target sites has been investigated by directed amino acid substitutions in the corresponding DNA‐recognition helices. In FNR, Glu‐209 and Ser‐212 are essential residues for the recognition of FNR sites. A V208R substitution confers CRP‐site specificity without loss of FNR specificity, but this has adverse effects on anaerobic growth. In contrast, changes at two (V206R and E209D) or three (V208R, S212G and G216K) positions in FNR endow a single CRP‐site binding specificity. In reciprocal experiments, two substitutions (R180V and G184S) were required to convert the binding specificity of CRP to that of FNR. Altering Asp‐199 in FNR failed to produce a positive control phenotype, unlike substitutions at the comparable site in CRP. Implications for the mechanism of sequence discrimination by FNR and CRP are discussed.
The bex locus in encapsulated Haemophilus influenzae: a chromosomal region involved in capsule polysaccharide export
Authors: Kroll J. S. , Loynds B. , Brophy L. N. , Moxon E. R.
Published: October 2006

The nucleotide sequence of a 5.1 kb region in the Haemophiius influenzae type b capsulation locus has been determined and found to contain four open reading frames: bexD, bexC, bexB, and bexA. Comparison of the deduced products of bexC, bexB, and bexA to known proteins, and TnphoA mutagenesis, suggests that they form components of an ATP‐driven polysaccharide export apparatus. Furthermore, close sequence similarity between BexA and BexB and products of the kpsT and kpsM genes at the Escherichia coli K5 capsulation locus (Smith et al., 1990 — accompanying paper) suggests that capsulation genes in these organisms may have a common ancestry.
Molecular analysis of the Escherichia coli K5 kps locus: identification and characterization of an inner‐membrane capsular polysaccharide transport system
Authors: Smith A. N. , Boulnois G. J. , Roberts I. S.
Published: October 2006

The complete nucleotide sequence has been determined of a region of the Escherichia coli K5 antigen gene cluster postulated to encode functions for the translocation of capsular polysaccharide across the inner membrane. This revealed two genes, designated kpsM and kpsT, organized in a single transcriptional unit. Analysis of the predicted amino acid sequence of the KpsM and KpsT proteins indicates that they may function as dual components in a polysaccharide export system analogous to the periplasmic binding protein‐dependent transport systems of Gram‐negative bacteria. We propose that the KpsT protein acts as an energizer, coupling ATP hydrolysis to the transport process mediated by the KpsM protein. Extensive sequence homology between the KpsM and KpsT proteins and the products of the bexB and bexA genes present in the capsulation (cap) locus of Haemophilus influenzae, indicates that a common mechanism for the export of polysaccharide across the inner membrane may exist in these two microorganisms.
Exopolysaccharide production in Rhizobium and its role in invasion
Authors: Gray J. X. , Rolfe B. G.
Published: October 2006

A complex interaction between rhizobia and specific legume plants results in the formation of nitrogen‐fixing root nodules. The necessity for signal exchange and a chemically based recognition system between the symbiotic partners has been appreciated for some time, but the details are only gradually being elucidated. The two basic nodule ontogenies exhibit different requirements for Rhizobium exopolysaccharides. These surface exopolysaccharide molecules of Rhizobium are synthesized at a membrane complex, which is regulated by both transcriptional and post‐trans‐lational controls. The acidic exopolysaccharide probably plays both a passive and an active role during the invasion process.
Proteinase inhibition, immunoglobulin‐binding proteins and a novel antimicrobial principle
Authors: Björck L.
Published: October 2006

Recent work has demonstrated that a tripeptide derivative mimicking the active proteinase‐binding site of cystatin C, a human cysteine proteinase inhibitor, can block growth of group A streptococci and replication of herpes simplex virus (HSV). In the case of HSV, intact cystatin C was also found to inhibit replication of the virus. Many streptococcal strains and HSV‐infected cells produce immunoglobulin (Ig)‐binding proteins, and a possible connection between such proteins and proteolytic activity was indicated by the finding that bacterial Ig‐binding proteins also show affinity for proteinase inhibitors. The significance of these various observations is not clear, but available data suggest that proteinases play a role in vital microbial functions (e.g. viral replication) and may be utilized as targets for antimicrobial agents. The results discussed here also indicate that peptide derivatives based on the structure of proteinase inhibitors occurring in nature could be used as such agents.
A family of ATPases involved in active partitioning of diverse bacterial plasmids
Authors: Motallebi‐Veshareh M. , Rouch D. A. , Thomas C. M.
Published: October 2006

Low copy‐number bacterial plasmids F (the classical Escherichia coli sex factor) and prophage P1 encode partitioning functions which may provide fundamental insights into the active processes which ensure that bacterial genomes are segregated to both daughter cells prior to cell division. These partitioning systems involve two proteins: ParA and ParB. We report that IncC from the broad host‐range plasmid RK2 is a member of the family of ParA partitioning proteins and that these proteins (as well as related proteins encoded by plasmids from Agrobacterium tume‐faciens and Chlamydia trachomatis) contain type I nucleotide‐binding motifs. Also, we show that the cell division inhibitor MinD is homologous to members of the ParA family. Sequence comparisons of ParB proteins suggest that they may contain sites for phosphorylation. We propose that ATP hydrolysis by the ParA protein may result in phosphorylation of the ParB protein, thereby causing a conformational shift necessary to separate paired plasmid molecules at the cell division plane.
Cloning and physical mapping of the sor genes for L‐sorbose transport and metabolism from Klebsiella pneumoniae
Authors: Wöhrl B. M. , Lengeler J. W.
Published: October 2006

The sor genes of Klebsiella pneumoniae KAY2026, which enable the bacterium to metabolize the ketose L‐sorbose, have been cloned on an 8.3kb DNA fragment into the multicopy plasmid, pACYC184. The genes were mapped by restriction analysis, by deletion mapping and by insertion mutagenesis with Tn1725. The corresponding gene products were identified by the maxicell technique. The structural genes sorD, sorA and sorE code for a D‐glucitol‐6‐P dehydrogenase (27 kilodalton (kD)), an Enzymell (EIISor) activity specific for L‐sorbose and an L‐sorbose‐1‐P reductase (45kD). Besides these genes for known functions, three additional genes were discovered: sorC, coding for a transcriptional 40kD regulatory protein, and sorF and sorB, coding for two proteins of 14kD and 19kD, respectively, involved in transport. The genes form an operon (gene order sorCpCDFBAE) and are inducible by L‐sorbose.
Studies on Clostridium acetobutylicum ginA promoters and antisense RNA
Authors: Janssen P. J. , Jones D. T. , Woods D. R.
Published: October 2006

The Clostridium acetobutylicum glnA gene has two transcript start sites under the control of promoters P1 and p2. Initiation of transcription was regulated by nitrogen and a downstream region was implicated in the regulation of transcript initiation by nitrogen in Escherichia coli. Putative antisense RNA was produced from a single downstream transcript start site under the control of P3. An up‐promoter mutation in P3 resulted in lower levels of glutamine synthetase (GS) activity. Putative antisense RNA had a role in down‐regulating GS expression but was not involved in regulation by nitrogen. Deletion of downstream inverted repeat sequences resulted in very low levels of GS activity.
The pYV plasmid of Yersinia encodes a lipoprotein, YlpA, related to TraT
Authors: China B. , Michiels T. , Cornelis G. R.
Published: October 2006

A series of lipoproteins was detected in the membrane fraction of Yersinia enterocolitica W227, a typical strain from serotype O:9. At least two of them, YlpA and YlpB, are encoded by the pYV plasmid. The sequence of ylpA reveals the presence of a typical lipoprotein signal peptide. The mature YlpA protein would be 223 residues long with a calculated molecular weight of 23798 for the proteic moiety of the molecule. YlpA shares 88% identical residues with the TraT protein encoded by plasmid pED208, 80% identity with TraT proteins encoded by plasmids R100 and F, and 77% identity with the TraT protein encoded by the virulence plasmid of Salmonella typhimurium. The ylpA gene hybridized with the pYV plasmid of Yersinia pseudotuberculosis, suggesting that this gene is conserved among Yersinia spp. The production of YlpA is controlled by virF and only occurs at 37°C in the absence of Ca2+ ions. This co‐regulation with the yop genes suggests that ylpA is a virulence determinant. However, mutations in ylpA clearly affect neither the resistance to human serum nor the virulence for intravenously inoculated mice.
Tandem DNA‐bound cAMP‐CRP complexes are required for transcriptional repression of the deoP2 promoter by the CytR repressor in Escherichia coli
Authors: Sogaard‐Andersen L. , Mellegaard N. E. , Douthwaite S. R. , Valentin‐Hansen P.
Published: October 2006

We have studied the deoP2 promoter in Escherichia coli to define features important for its interaction with the CytR repressor. As is characteristic for CytR‐regulated promoters, deoP2 encodes tandem binding sites for the activating complex cAMP‐CRP. One of these sites, CRP‐1, overlaps the ‐ 35 region, and is sufficient for activation; the second site, CRP‐2, centred around‐93, is indispensable for repression. Here we demonstrate, by means of in vivo titration, that CytR interaction with deoP2 depends not only on CRP‐2, but also on CRP‐1 and the length and possibly the sequence separating these two sites. Also, point mutations in either CRP site reduce or abolish CytR titration; however, no co‐operativity is observed in the interaction of CytR with the two CRP binding sites. Furthermore, the reduction in CytR titration parallels the reduction in binding of cAMP‐CRP to the mutated CRP sites in vitro. These observations are not easily explained by current models for the action of prokaryotic repressors; instead we favour a model in which the interaction of CytR with deoP2 depends on the presence of tandem DNA‐bound cAMP–CRP complexes.
Conservation of a hexapeptide sequence in the anchor region of surface proteins from Gram‐positive cocci
Authors: Fischetti V.A , Pancholi V. , Schneewind O.
Published: October 2006

summary

A highly conserved hexapeptide sequence (both at the protein‐and DNA level) has been identified within the C‐terminal end of all 11 known surface proteins from Gram‐positive cocci. The hexapeptide, with the consensus sequence LPXTGE, is located about 9 amino acids N‐terminal from the C‐terminal hydrophobic domain which is found in all these surface molecules. The conservation of the hexapeptide, despite sequence variation within the regions flanking it, suggests that it is important for the attachment of these proteins within the cell.
Selenocysteine: the 21st amino acid
Authors: Böck A. , Forchhammer K. , Heider J. , Leinfelder W. , Sawers G. , Veprek B. , Zinoni F.
Published: October 2006

Great excitement was elicited in the field of selenium biochemistry in 1986 by the parallel discoveries that the genes encoding the selenoproteins glutathione peroxidase and bacterial formate dehydrogenase each contain an in‐frame TGA codon within their coding sequence. We now know that this codon directs the incorporation of selenium, in the form of selenocysteine, into these proteins. Working with the bacterial system has led to a rapid increase in our knowledge of selenocysteine biosynthesis and to the exciting discovery that this system can now be regarded as an expansion of the genetic code. The prerequisites for such a definition are co‐translational insertion into the polypeptide chain and the occurrence of a tRNA molecule which carries selenocysteine. Both of these criteria are fulfilled and, moreover, tRNASec even has its own special translation factor which delivers it to the translating ribosome. It is the aim of this article to review the events leading to the elucidation of selenocysteine as being the 21st amino acid.
Physiological roles of the DnaK and GroE stress proteins: catalysts of protein folding or macromolecular sponges?
Authors: LaRossa R. A. , Van Dyk T. K.
Published: October 2006

When organisms ranging from microbes to man are subjected to certain environmental stresses a characteristic heat shock’ response is observed. In Escherichia coli this response is characterized by the induction of several proteins, three of which are the 70 kilodalton product of the dnaK gene, the 60 kilodalton product of the groEL (mopA) gene and the 15 kilodalton product of the groES (mopB) gene. In this review, utilizing enteric bacteria as model organisms, we focus on the role of these proteins within the context provided by well‐established functions of other heat shock products. These facts serve as a starting point from which to speculate upon the in vivo role of these proteins during steady‐state growth.