bluuebunny/crossref_metadata_2025 huggingface dataset:
Export of infectious particles by Escherichia coli transfected with the RF DNA of Pf1, a virus of Pseudomonas aeruginosa strain K
Authors: Kostrikis L. G. , Reisberg S. A. , Simon M. N. , Wall J. S. , Day L. A.
Published: October 2006

Pf1 is a filamentous, single‐stranded DNA virus that has Pseudomas aeruginosa (strain K) as host. It is the longest of the filamentous bacterial viruses, and the DNA within it has the most extended conformation known. Pfl virus cannot infect Escherichia coli (strain MM294) cells, but when these cells are transfected with the double‐stranded replicative form of Pt1 DNA (RF DNA, 7.35 kb), they export low levels of infectious particles that create plaques on lawns of P. aeruginosa. Several different structural species, at least two of which are infectious, are exported. One of them, called Epf1, has virtually the same structure as Pf1, but the amount of Epf1 exported by E. coli is 104 lower than the amount of Pf1 exported by P. aeruginosa. The results imply that host factors affect not only the efficiency of virus assembly and export, but also the actual structures of the species exported.
An amplifiable and deletable chloramphenicol‐resistance determinant of Streptomyces lividans 1326 encodes a putative transmembrane protein
Authors: Dittrich W. , Betzler M. , Schrempf H.
Published: October 2006

A genetically unstable chloramphenicol resistance gene from Streptomyces lividans 1326 was cloned and characterized. This gene and adjacent DNA regions can be lost spontaneously or amplify within variants. Biochemical studies proved that chloramphenicol is not modified by an acetyltransferase or any other enzyme and that ribosomes of the resistant strain are sensitive to chloramphenicol. Sequence data revealed that the resistance gene encodes a hydrophobic protein predicted to have 12 membrane‐spanning α‐helices and a hydropathic profile similar to the membrane of proteins required for the efflux of tetracydine. Variable proportions of the amino acids (about 16‐24%) within the presumed chloramphenicol‐resistant protein are identical to various aligned tetracycline‐resistant proteins from Gram‐negative and Gram‐positive bacteria and to transporters for citrate in Klebsiella pneumonaie and for ferrichrome in Escherichia coli.
Mechanism of regulation of the formate‐hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon
Authors: Rossmann R. , Sawers G. , Böck A.
Published: October 2006

The products of a minimum of 15 genes are required for the synthesis of an active formate‐hydrogenlyase (FHL) system in Escherichia coli. All are co‐ordinately regulated in response to variations in the oxygen and nitrate concentration and the pH of the culture medium. Formate is obligately required for transcriptional activation of these genes. Analysis of the transcription of one of these genes, hycB linked to the iacZ reporter gene, revealed that oxygen and nitrate repression of transcription could be relieved completely, or partially in the case of nitrate, either by the addition of formate to the medium or by increasing the copy number of the gene encoding the transcriptional activator (fhlA) of this regulon. These studies uncovered a further level of regulation in which the transcription of hycB was reduced in cells grown on glucose. This effect was most clearly seen in aerobically grown cells when formate was added externally. Addition of cAMP overcame this glucose repression, which could be shown to be mediated by the cAMP receptor protein. These results would be consistent with the transport of formate being regulated by catabolite repression. Moreover, the repression of transcription through high pH also could be partially overcome by addition of increasing concentrations of formate to the medium, again being consistent with regulation at the level of formate import and export. Taken together, all these observations indicate that it is the intracellular level of formate that determines the transcription of the genes of the formate regulon by FhlA. This represents a novel positive feedback mechanism in which the activator of a regulon induces its own synthesis in response to increases in the concentration of the catabolic substrate, and this in turn is governed by the relative affinities of FhlA and the three formate dehydrogenase isoenzymes for formate.
Pseudomonas aeruginosa elastase and elastolysis revisited: recent developments
Authors: Galloway D. R.
Published: October 2006

With the determination of the three‐dimensional structure of elastase and the probable identification of the active site and key residues involved in proteolytic activity, our knowledge of the molecular details of this interesting protease is rapidly increasing. Pseudomonas elastase appears to be remarkably similar to the Bacillus metalloproteinase thermolysin. A further significant development has been the discovery of the lasA gene and the fact that Pseudomonas elastase and alkaline proteinase appear to act in concert with the LasA protein to display the notable elastolytic activity exhibited by isolates of this organism. Biochemical and genetic studies indicate that LasA is a second elastase which may be an important virulence factor that has been overlooked in previous studies.
Sensory transduction in the gliding bacterium Myxococcus xanthus
Authors: Zusman D. R. , McBride M. J.
Published: October 2006

Sensory transduction in the gliding bacterium Myxococcus xanthus is mediated by the frz genes. These genes are homologous to the chemotaxis genes of enteric bacteria and control the rate of cell reversal during gliding. Sensory transduction is hypothesized to involve the recognition of substances present in the medium at the cell surface and the subsequent stimulation of a cytoplasmic methyl‐accepting protein, FrzCD. Phosphorylation of FrzE is also involved in the sensory transduction pathway. Despite the similarities between the chemotaxis proteins of enteric bacteria and M. xanthus Frz proteins, fundamental differences exist between these different bacteria in terms of the ability of cells to recognize and respond to substances in their environment. The mechanism of directional switching and the nature of the gliding motor remain obscure. It is hoped that the study of the interaction of the Frz proteins will allow greater understanding of these problems.
K1 killer toxin, a pore‐forming protein from yeast
Authors: Bussey H.
Published: October 2006

K1 killer toxin is a secreted, pore‐forming protein that kills sensitive yeast cells. The heterodimeric toxin is processed from a precursor in the Golgi, and has allowed identification of the KEX2‐ and KEX1‐encoded proteases. The toxin binds to a glucan receptor on the cell wall of target yeast, and mutational analysis implicates both the a‐ and |5‐toxin sub‐units in receptor binding. Toxin‐resistant mutants with altered cell‐wall glucans have helped to outline a pathway of assembly of these polysaccharides. Patch‐clamp technology has demonstrated the nature of the lethal channel in toxin‐treated plasma membranes. The hydrophobic α‐subunit‐encoding region is the site of all mutations affecting channel formation. Immunity to the toxin is conferred by the toxin precursor, and immunity mutations map to the region encoding the α subunit. The precursor probably competes with the toxin to prevent channel formation in toxin‐producing cells, but the basis of this remains unknown. This toxin/immunity system has a domain structure that differs from that of other characterized toxins and has no known homologues.
H‐DNA formation by the coding repeat elements of neisserial opa genes
Authors: Belland R. J.
Published: October 2006

The coding repeat region of opa genes from Neisseria gonorrhoeae and Neisseria meningitidis determines the expression state of their respective genes through high‐frequency addition or deletion of pentanucleotide coding repeat units (CRs; CTTCT). In vitro analyses of cloned opa gene CR regions using single‐strand specific nucleases, oligonucleotide protection experiments, and modifications of non‐B‐DNA residues indicate that the regions form structures resembling H‐DNA under acidic conditions in the presence of negative supercoiling. The purine/pyrimidine strand bias and H‐palindromic nature of the repeat region are consistent with sequence requirements for H‐DNA formation. Sequences flanking the repeat elements are required to form the H‐DNA structure in vitro as judged by the pattern of exposed non‐B‐DNA residues in CR sequences synthesized as oligonucleotides to form β‐galactosidase::CR translational fusions. The fusions phase vary by addition and deletion of CR elements and the rate of phase variation increases upon induction of the fusion genes. The opa gene CR region is the first reported bacterial H‐DNA structure and is unique in that it ties within the coding sequence for the gene.
Mutational analysis supports a role for multiple structural features in the C‐terminal secretion signal of Escherichia coli haemolysin
Authors: Stanley P. , Koronakis V. , Hughes C.
Published: October 2006

We have carried out an extensive mutational analysis of the C‐terminal signal which targets the export of the 1024‐residue haemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Over 60 variants of the HlyA C‐terminal 53‐amino‐acid sequence were created by oligonucleotide‐directed mutagenesis and fused to the HlyA N‐terminal 830 residues. Transport of the HlyA derivatives by the HlyB/HlyO system was compared with the wild‐type level and the data indicate that the HlyA C‐terminal export signal lies within the last 48 amino acids and comprises three functional domains: an amphipathic, charged helix between residues 1,977 and R,996; a 13‐amino‐acid uncharged region from residue T,997 to S,1009; and an 8‐aminoacid hydroxylated tail at the extreme C‐terminus. Analogous features were found in the C‐terminal sequences of an extended family of haemolysins, leukotoxins and proteases which are secreted by HlyB/HlyD‐type translocators. In particular, all nine proteins which are secreted into the extracellular medium possess potential extended amphipathic helices. These results suggest a possible role for multiple regions of the HlyA C‐terminal export signal in which the first two domains span the membranes and the third domain remains in the cytoplasm.
Identification of gene products of the P1 operon of Mycoplasma pneumoniae
Authors: Sperker B. , Hu P.‐c. , Herrmann R.
Published: October 2006

Gene P1 of Mycoplasma pneumoniae, which codes for a major adhesin, is flanked by two sequences with open reading frames designated QRF4 and QRF6 (Inamine et al., 1988b). In order to identify proteins translated from these ORFs, gene fusions between the N‐terminus of the RNA replicase of the Escherichia coli bacteriophage MS2 and selected regions of ORF4 and ORF6 were constructed. The corresponding fusion proteins synthesized in Escherichia coli were used to immunize mice. Antisera directed against ORF4‐related sequences did not recognize M. pneumoniae antigens in Western blot analysis, but antisera directed against ORF‐6‐derived fusion proteins reacted with two M. pneumoniae proteins of 40kDa and 90kDa. In addition, some of the antisera also recognized proteins that formed in a sodium dodecyl sulphate/polyacrylamide gel a protein ladder between 115 and 145 kDa.
Identification of phosphatidylinositol‐specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor?
Authors: Mengaud J. , Braun‐Breton C. , Cossart P.
Published: October 2006

A phospholipase C which cleaves phosphatidylinositol and glycosylphosphatidylinositol (GPI) anchors was identified in Listeria monocytogenes. This 36kDa protein is encoded by the gene plcA, and is homologous to the Bacillus cereus, Bacillus thuringiensis and eukaryotic phosphatidylinositol‐specific phospholipases C (PI‐PLC). Expression of the plcA gene in Escherichia coli correlates with the appearance of PI‐PLC activity in the cells. In Listeria monocytogenes, the activity is secreted to the culture medium. PI‐PLC activity was only found in the two pathogenic species of the genus Listeria, namely L. monocytogenes and L. ivanovii. PI‐PLC activity was lost and virulence decreased when the plcA gene was disrupted in the chromosome. This suggests that the PI‐PLC of L. monocytogenes might be involved in virulence.
P.70 pertactin, an outer‐membrane protein from Bordetella parapertussis: cloning, nucleotide sequence and surface expression in Escherichia coli
Authors: Li L. J. , Dougan G. , Novotny P. , Charles I. G.
Published: October 2006

The gene prn encoding the outer‐membrane protein P.70 (pertactin) from Bordetella parapertussis has been cloned in Escherichia coli and its DNA sequence determined. Analysis of the DNA sequence reveals that the gene has an open reading frame comprising 922 amino acids capable of encoding a protein with a molecular weight of 95177 (P.95). In vivo processing of this precursor yields a protein with an estimated Mr of 70kDa (P.70) which is located on the surface of B. parapertussis. Homology between the prn gene from B. parapertussis and that from Bordetella pertussis is 91.3%. The homology is 93% when the protein sequence of P.95 is aligned with that of P.93 from B. pertussis. The major differences between the P.70 pertactin from B. parapertussis and the P.69 pertactin from B. pertussis occur in the number of reiterated units within the repeat motifs found in both proteins; the sequence Gly–Gly–Xaa–Xaa–Pro is repeated four times in the P.70 pertactin, and five times in the P.69 pertactin, while the sequence Pro–Gln–Pro occurs nine times in P.70 pertactin and five times in P.69 pertactin. Cloning of the gene for P.95 in an E. coli expression vector results in the synthesis of a protein that mimics native gene expression in B. parapertussis, i.e. the P.95 protein is synthesized and subsequently processed to yield the P.70 form of the protein on the surface of the cell.
Segment IV of a Salmonella flagellin gene specifies flagellar antigen epitopes
Authors: Newton S. M. C. , Wasley R. D. , Wilson A. , Rosenberg L. T. , Miller J. F. , Stocker B. A.
Published: October 2006

Each of the two mutants isolated from a fliC (=hag, flagellin‐deficient) Escherichia coli strain made motile by a plasmid carrying the fliC gene of Salmonella muenchen by selection for motility in the presence of anti‐d (Salmonella flagellar antigen) serum had both lost and gained one or more subfactors of the wild‐type antigen. In one more mutant codon 246 was GAC (alanine) instead of GCC (asparagine); the other had a deletion of 105 base pairs, explicable by a 10bp direct repeat, starting at bases 782 and 887. The in vitro removal of a 48bp EcoRV(631)/EcoRV(679) fragment produced plasmid pLS408, which was found to lack a subfactor of wild‐type antigen d but able to confer motility on flagellin‐negative Salmonella sp.(and used for insertion of epitope‐specifying oligonucleotides at its EcoRV site). Immunoblotting with absorbed and unabsorbed sera from rabbits immunized with E. coli with wild‐type or mutated antigen d showed that the fusion proteins specified by λ gt11 with the N‐terminal part of gene lacZ joined to a restriction fragment coding for residues 145–391 of flagellin gave the same patter of parent‐specific and mutant‐specific reactions as the flagellate bacteria. Four out of five similarly selected mutants had the same 105bp deletion as the first‐isolated mutant; the fifth had a 72bp deletion made possible by a 7‐base pair direct repeat, starting at positions 649 and 721. All these changes in serological character without loss of function affected segment IV, specifying residues 182 to 308 of the total of 505, where there is little homology between different flagellar antigen alleles.
Design of camp–CRP‐activated promoters in Escherichia coli
Authors: Valentin‐Hansen P. , Hoist B. , Søgaard‐Andersen L. , Martinussen J. , Nesvera J. , Douthwaite S. R.
Published: October 2006

We have studied the doeP2 promoter of Escherichia coli to define features that are required for optimal activation by the complex of adenosine 3′, 5′ monophosphate (cAMP) and the cAMP receptor protein (CRP). Systematic mutagenesis of deoP2 shows that the distance between the CRP site and the ‐10 hexamer is the crucial factor in determining whether the promoter is activated by camp–CRP. Based on these observations, we propose that camp–CRP‐activated promoters can be created by correctly aligning a CRP target and a ‐ 10 hexamer. This idea has been successfully tested by converting both a CRP‐in‐dependent promoter and a sequence resembling the consensus ‐10 hexamer to strongly camp–CRP‐activated promoters.
Post‐transcriptional control of a sporulation regulatory gene encoding transcription factor σH in Bacillus subtilis
Authors: Healy J. , Weir J. , Smith I. , Losick R.
Published: October 2006

The transcriptional regulatory gene spo0H encodes an RNA polymerase sigma factor called σH that directs gene expression at an early stage of sporulation in the Gram‐positive bacterium Bacillus subtilis. We now report that conditions that induce sporulation cause a rapid increase In the cellular concentration of σH. This increase could account for the stimulated transcription of certain σH‐controlled genes at the onset of sporulation. Experiments in which the expression of spo0H was monitored by use of a spo0H‐lacZ fusion and in which expression of spo0H was artificially manipulated by use of an isopropyl‐β‐D‐thiogalacto‐side‐inducible promoter indicate that sporulation‐induced increases in the amount of σH are not controlled at the level of the transcription of its structural gene. Rather, we infer the existence of post‐transcriptional control mechanisms that govern σH levels, and we present evidence suggesting that increases in the amount of σH at the start of sporulation are due to increased translation or stability of the spo0H mRNA and, to a lesser extent, decreased turnover of spo0H protein.
Variable antigen genes of the relapsing fever agent Borrelia hermsii are activated by promoter addition
Authors: Barbour A. G. , Burman N. , Carter C. J. , Kitten T. , Bergström S.
Published: October 2006

Borrelia hermsii, an agent of replapsing fever, avoids the host's immune response by means of multiphasic antigenic variation. Serotype specificity is determined by variable antigens called the Vmp lipoproteins. Through recombination between linear plasmids a formerly silent vmp gene replaces another vmp gene at a telomeric expression locus. We examined strain HS1 borreliae before and after a switch from serotype 7 to serotype 21. The nucleotide sequences of 5 regions of silent and expressed vmp7 and vmp 21 were determined. Silent and active vmp 7 and vmp 21 genes shared a block of homologous sequences surrounding their 5′ ends. Sequences upstream of silent vmp 7 and vmp 21 genes lacked the promoter and substantially differed from each other. In this antigenic switch a vmp gene was activated by a recombination that placed it downstream of a promoter.
The bacterial porin superfamily: sequence alignment and structure prediction
Authors: Jeanteur D. , Lakey J. H. , Pattus F.
Published: October 2006

The porins of Gram‐negative bacteria are responsible for the ‘molecular sieve’ properties of the outer membrane. They form large water‐filled channels which allow the diffusion of hydrophilic molecules into the periplasmic space. Owing to the strong hydrophilicity of their amino acid sequence and the nature of their secondary structure (beta strands), conventional hydropathy methods for predicting membrane topology are useless for this class of protein. The large number of available porin amino acid sequences was exploited to improve the accuracy of the prediction In combination with tools detecting amphipathicity of secondary structure. Using the constraints of β‐sheet structure these porins are predicted to contain 16 membrane‐spanning strands, 14 of which are common to the two (enteric and the neisserial) porin subfamilies.
Analysis of an Erwinia chrysanthemi gene cluster involved in pectin degradation
Authors: Condemine G. , Robert‐Baudouy J.
Published: October 2006

A group of four genes of Erwinia chrysanthemi involved in pectin degradation has been characterized. These four genes form independent transcription units and are regulated by the negative regulatory gene, kdgR. The functions of two of these genes are known: kduD codes for the 2‐keto‐3‐deoxygluconate oxydoreductase and kdul for the 5‐keto‐4‐deoxyuronate isomerase, two enzymes of the pectin degradation pathway. kdgC has 36% homology with pectate lyase genes of the periplasmic family but its product does not seem to have pectinolytic activity. The fourth gene, kdgF, could have a role in the pathogenicity of E. chrysanthemi. A comparison of the regulatory regions of all the genes controlled by kdgR allowed better definition of the KdgR‐binding‐site consensus.
Molecular cloning and expression in Escherichia coli K‐12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain
Authors: Bastin D. A. , Romana L. K. , Reeves P. R.
Published: October 2006

The O antigen of Escherichia coli O111 is identical in structure to that of Salmonella enterics serovar adelaide. Another O‐antigen structure, similar to that of E. coli O111 and S. enterica serovar adelaide is found in both E. coli O55 and S. enterica serovar greenside. Both O‐antigen structures contain colitose, a 3,6 dideoxyhexose found only rarely in the Enterobacteriaceae. The O‐antigen structure is determined by genes generally located in the rfb gene cluster. We cloned the rfb gene cluster from an E. coli O111 strain (M92), and the clone expressed O antigen in both E. coli K‐12 and a K‐12 strain deleted for rfb. Lipopoly‐saccharide analysis showed that the O antigen produced by strains containing the cloned DNA is polymerized. The chain length of O antigen was affected by a region outside of rfb but linked to it and present on some of the plasmids containing rfb. The rfb region of M92 was analysed and compared, by DNA hybridization, with that of strains with related O antigens. The possible evolution of the rfb genes in these O antigen groups is discussed.
The sugar‐specific outer membrane channel ScrY contains functional characteristics of general diffusion pores and substrate‐specific porins
Authors: Schülein K. , Schmid K. , Benzl R.
Published: October 2006

Escherichia coli K–12 strain PS1–28–37 carries the multicopy plasmid pPSO28–37 containing a DNA fragment coding for two of the proteins that enable bacteria to utilize sucrose as sole carbon source. One of the different gene products of the plasmid is the outer membrane protein, ScrY. This protein was isolated and purified by chromatography across a gel filtration column. Reconstitution experiments with lipid by layer membrane demonstrated that ScrY formed ion‐permeable channels with properties very similar to those of general diffusion pores of enteric bacteria. The presence of sugars in the aqueous phase led to a dose‐dependent block of ion transport through the channel, like the situation found with LamB (maltoporin) of Escherichia coli and Salmonella typhimurium. The binding constants of a variety of different sugars were determined. The stability constant for malto‐oligosaccharide binding increased with increasing numbers of glucose residues. Disaccharides generally had a larger binding constant than monosaccharides. The binding of different sugars to ScrY and LamB of E. coli discussed with respect to the kinetics of sugar movement through the channel.
IS901, a new member of a widespread class of atypical insertion sequences, is associated with pathogenicity in Mycobacterium avium
Authors: Kunze Z. M. , Wall S. , Appelberg R. , Silva M. T. , Portaels F. , McFadden J. J.
Published: October 2006

An insertion sequence (IS901), found in pathogenic strains of Mycobacterium avium, but absent in M. avium complex isolates from patients with acquired immune deficiency syndrome (AIDS), has been isolated and sequenced. This insertion element has a nucleotide sequence of 1472 bp, with one open reading frame (0RF1), which codes for a protein of 401 amino acids. The amino acid sequence, terminal ends and target site of IS901 are similar to those of IS900, present in Mycobacterium paratuberculosis. However, the DNA sequences of these two IS elements exhibit only 60% homology, compared to a DNA homology of 98% between their respective hosts. IS901, like IS900, appears to belong to a family of related insertion elements present in actinomycetes and other bacteria. M. avium strains containing IS900 were found to be more virulent in mice than closely related strains lacking IS901. IS901 may be a useful tool for the study of the genetics of virulence in the M. avium complex and for obtaining stable integration of foreign genes into mycobacteria.
Escherichia coli DNA helicases: mechanisms of DNA unwinding
Authors: Lohman T. M.
Published: October 2006

DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair. These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown. Although it is clear that not all helicases unwind duplex DNA in an identical way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action. For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric. The oligomeric nature of helicases provides them with multiple DNA‐binding sites, allowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either single‐stranded and duplex DNA simultaneously or two strands of single‐stranded DNA. Modulation of the relative affinities of these binding sites for single‐stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases. The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oligomeric structures, with emphasis on the E. coli Rep, RecBCD and phage T7 gene 4 helicases.
Bacillus subtilis chemotaxis: a deviation from the Escherichia coli paradigm
Authors: Bischoff D. S. , Ordal G. W.
Published: October 2006

In Escherichia coli, chemotactic sensory transduction is believed to involve phosphoryl transfer for excitation, and changes in receptor methylation for adaptation. In Bacillus subtilis, changes in degree of receptor methylation do not bring about adaptation. Novel methylation reactions are believed to be involved in excitation in B. subtillis. The main chemotaxis proteins of E. coli—CheA, CheB, CheR, CheW and CheY—are present in B. subtilis but play somewhat different roles in the two organisms. Several unique chemotaxis proteins are also present in B. subtilis. Some of the properties of B. subtilis chemotaxis are also seen in Halobacterium halobium, suggesting that there may be a similar underlying mechanism that predates the evolutionary separation of the bacteria from the archaea and eucarya.
Pseudomonas aeruginosa AlgB, which modulates the expression of alginate, is a member of the NtrC subclass of prokaryotic regulators
Authors: Goldberg J. B. , Dahnke T.
Published: October 2006

The Pseudomonas aeruginosa exopolysaccharide alginate is an important virulence factor in chronic pulmonary infections of cystic fibrosis patients. We determined the nucleotide sequence of the gene, algB, which regulates the level of exopolysaccharide produced by mucoid P. aeruginosa. The predicted amino acid sequence of AlgB revealed a high degree of similarity to the regulatory proteins in the NtrC subclass of ‘two‐component regulatory systems’. AlgB expression in Escherichia coli minicells showed a molecular weight of ˜ 50 000 Da, comparable to that of the inferred amino acid sequence (49 318 Da). We show that algB is transcriptionally active in mucoid strains of P. aeruginosa and regulates the expression of the alginate biosynthetic gene, algD, thereby resulting in increased expression of alginate in mucoid P. aeruginosa.
Molecular characterization of mutations of nlt‐4, the pathway‐specific regulatory gene which controls nitrate assimilation in Neurospora crassa
Authors: Yuan G.‐F. , Marzluf G. A.
Published: October 2006

The nit‐4 genes of three conventional Neurospora crassa mutations and of the closely related species, Neurospora intermedia, have been isolated by amplifying the genomic DNA with the polymerase chain reaction. Nucleotide sequencing has revealed that the three nit‐4 mutants, alleles 15,1214, and 2994, are the result of a missense mutation, a nonsense mutation and a frameshift mutation, respectively. The nucleotide sequence of the NIT4 protein coding region of a nit‐4 mutant (allele 2994) and of N. intermedia have been determined and compared with that of wild‐type N. crassa. The molecular characteristics confirm that the mutated gene of 2994 originated from N. intermedia and was introgressed into N. crassa. The polyglutamine domains of the N. crassa wild type, the 2994 mutant, or N. intermedia cannot replace an upstream glutamine‐rich domain which is essential for nit‐4 function.
Iron regulates growth of Trichomonas vaginalis and the expression of immunogenic trichomonad proteins
Authors: Lehker M. W. , Alderete J. F.
Published: October 2006

Iron is an essential nutrient for Trichomonas vaginalis and is acquired via highly specific receptor‐mediated mechanisms from the host. Responses of T. vaginalis to conditions of iron limitation or iron excess were analysed in order to determine whether iron levels in the growth medium regulate certain properties of the parasite. When compared with organisms grown in excess iron, iron limitation resulted in ≥80% lower rates of protein synthesis and ≥3‐fold decreases in cell densities. These parasites also exhibited generation times of 10 hours, 2.5‐fold longer than organisms grown in the usual complex medium. Iron‐restricted growth also resulted in increased binding of lactoferrin by trichomonads, which paralleled elevated expression of the lactoferrin‐binding receptor protein having a relative molecular mass of 136000 daltons (136 kDa). A Mr 126kDa protein was concomitantly repressed in low‐iron‐grown parasites. The greater amounts of lactoferrin bound by iron‐depleted T. vaginalis organisms corresponded with both the expression of additional receptors onto trichomonal surfaces and increased affinity of the receptor for the lactoferrin molecule. Finally, immunoblot analysis of parasites grown under high‐ and low‐iron conditions using sera from patients with trichomoniasis further revealed the synthesis by T. vaginalis of at least 19 iron‐regulated immunogens, and patients' sera also detected the lactoferrin receptor. These data not only show the overall importance of iron to the biology of this protozoan, but illustrate the in vivo iron modulation of gene expression of the biofunctional lactoferrin receptor and other immunogens.
Ferrioxamine uptake in Yersinia enterocolitica: characterization of the receptor protein FoxA
Authors: Báumler Andreas J. , Hantke Klaus
Published: October 2006

The gene for the high‐affinity outer membrane ferrioxamine receptor FoxA of Yersinia enterocolitica was cloned in Escherichia coli K‐12. A foxA mutant of Yersinia could be complemented by the cloned DNA fragment. The FoxA encoding region was sequenced and an open reading frame encoding 710 amino acids, including a signal sequence of 26 amino acids, was deduced. The mature FoxA protein consisted of 684 amino acids and had a molecular mass of 75768 Da. FoxA shared 33% amino acid sequence homology with FhuA, the ferrichrome receptor of Escherichia coli. Based on the homologies with FhuA and other TonB‐dependent receptors a topological model of FoxA is presented.
IRE1 encodes a putative protein kinase containing a membrane‐spanning domain and is required for inositol phototrophy in Saccharomyces cerevisiae
Authors: Nikawa Jun‐Ichi , Yamashita Satoshi
Published: October 2006

A novel gene, IRE1, of Saccharomyces cerevisiae was cloned through genetic complementation of a myo‐ inositol auxotrophic mutant. The predicted amino acid sequence indicated that IRE1 encodes a protein of 126983 Da with two highly hydrophobic regions, probably a signal sequence and a membrane‐spanning region. The carboxy‐terminal region of IRE1 showed close sequence similarity to the catalytic domains of protein kinases. Disruption of the IRE1 locus caused myo‐inositol auxotrophy. The IRE1 product is very likely a protein kinase required for myo‐inositol synthesis.
Molecular genetic analysis of the Rhizobium meliloti fixK promoter: identification of sequences involved in positive and negative regulation
Authors: Waelkens F. , Foglia A. , Morel J.‐B. , Fourment J. , Batut J. , Boistard P.
Published: October 2006

Transcription of the Rhizobium meliloti fixK gene is induced in symbiotic and microaerobic growth conditions by the FixL/FixJ modulator/effector pair. Transcription of fixK is also negatively autoregulated. By 5’deletion analysis, the involvement in negative regulation of a DNA region between ‐514 and ‐450 with respect to the transcription start was demonstrated. Site‐directed mutagenesis allowed us to show that a sequence homologous to the binding site of the Escherichia coli Fnr protein, centred at position ‐487, participates in this effect. However, deletion or mutagenesis of this Fnr‐like sequence does not completely eliminate FixK‐dependent repression, which suggests that either an additional DNA region is involved in negative regulation or that it is mediated at the level of fixLJ transcription. Deletion analysis also allowed the definition of a DNA region involved in FixJ‐mediated activation of the fixK promoter, between ‐79 and ‐42. Different point mutations in the ‐60, ‐45 and ‐35 regions were shown to affect promoter activity. In some cases, the activity of mutant promoters could be partly or fully restored by increasing the expression of the fixLJ regulatory genes. In an E. coli strain harbouring a plasmid with fixLJ under the control of an inducible (p‐tac) promoter.
The spo0A gene is implicated in the maintenance of non‐complementing diploids in Bacillus subtilis
Authors: Le Dérout J. , Thaler David S. , Guillén Nancy , Hirschbein Luisa
Published: October 2006

Bacillus subtilis can exist in a diploid state in which two genetically distinct chromosomes co‐exist in the same cell and yet only one of them is expressed, thereby determining the phenotype. Such cells are called non‐complementing diploids (Ncds). In this study, two types of experiments are reported which indicate that a previously known pleiotropic gene, spo0A, plays a role in the maintaining the diploid state, as follows, (i) When protoplasts of two Spo0A mutant strains were fused, the resulting products continued to segregate cells of both parental phenotypes for many more divisions than had been reported previously, (ii) When a stable Ned (an Ncd in which the unexpressed markers are not spontaneously activated at a detectable level) harbouring a chloramphenicol acetyltransferase gene on the silent chromosome was transformed with spo0A null alleles the transformants often expressed chloramphenicol acetyltransferase activity. Together these results indicate that the spo0A gene is involved in maintenance of the diploid state in both unstable and stable Ncds.
Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli
Authors: Sauter M. , Böhm R. , Böck A.
Published: October 2006

In‐frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system. The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity. It was found that hycA, the promoter‐proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA. Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both. None of the mutations affected hydrogenase 1 or 2 activity. A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems. HycH, the product of the most promoter‐distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.
Involvement of a possible chaperonin in the efficient expression of a cloned CryllA δ‐endotoxin gene in Bacillus thuringiensis
Authors: Crickmore Neil , Ellar David J.
Published: October 2006

The Bacillus thuringiensis cryllAδ‐endotoxin gene is found as the third‐gene in a three‐gene operon, with a sporulation‐dependent promoter lying upstream of the first gene, orf1. We show here that the polypeptide product of the middle gene (orf2) is required for efficient expression of the toxin gene. In the absence of a functional ORF2 polypeptide the toxin does not form the crystalline inclusions characteristic of other known Bacillus thuringiensis toxins. We discuss the importance of this finding with respect to the possible role of chaperonins in the crystallization of these proteins.
icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread
Authors: Allaoui Abdelmounaaïm , Mounier Joëlle , Prévost Marie‐Christine , Sansonetti Philippe J. , Parsot Claude
Published: October 2006

Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. We have characterized the icsB gene which is located on the virulence plasmid pWR100. After inactivation of icsB, the mutant strain remained invasive, but formed abnormally small plaques on HeLa cell monolayers, colonized only the peripheral cells of Caco‐2 islets, and was unable to provoke a keratoconjunctivitis in guinea‐pigs. Examination of infected HeLa cells showed that the icsB mutant was able to lyse the phagocytic vacuole and to form protrusions at the surface of infected cells, but, unlike the wild type, remained trapped in protrusions surrounded by two membranes. These results indicate that IcsB is involved in the lysis of the protrusions, a step necessary for intercellular spread.
In vivo characterization of tus gene expression in Escherichia coli
Authors: Roecklein Bryan A. , Kuempel Peter L.
Published: October 2006

The tus gene encodes a DNA‐binding protein (Tus) that inhibits replication forks at specific block‐sites within the terminus region of the Escherichia coli chromosome. One of these block‐sites, TerB, is adjacent to the tus gene. Using primer extension and a promoter fusion to characterize in vivo expression, we have demonstrated that the tus transcription start site is within TerB, and that Tus protein autoregulates expression at this weak promoter We have also demonstrated that a minority of tus transcripts are initiated from an upstream region that contains two additional open reading frames. This readthrough transcription into tus is reduced in the presence of Tus protein.
An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity
Authors: Tang C. M. , Cohen J. , Holden D. W.
Published: October 2006

Invasive pulmonary aspergillosis, usually caused by Aspergillus fumigatus, is a life‐threatening condition of immunosuppressed patients. We have created a mutant strain of this fungus that lacks an extracellular alkaline protease (AFAlp). This was accomplished by transformation of A. fumigatus with a plasmid containing a selectable marker for hygromycin B resistance, and a 504 bp segment of the AFAlp gene, obtained by polymerase‐chain‐reaction‐based amplification of A. fumigatus genomic DNA. Approximately 25% of transformants resulted from disruption of the AFAlp gene. SDS‐polyacrylamide gel etectrophoresis of proteins from the culture filtrate of a strain carrying the AFAlp gene disruption showed that it lacked a major protein of 33 kDa. Furthermore, in contrast to the culture filtrate from wild‐type cells, the mutant had undetectable activity on azocollagen and elastin‐Congo red, over a broad pH range. This shows that AFAlp accounts for most, if not all, of the extracellular elastinolytic activity of A. fumigatus, and that the mutant strain will be useful in assessing the role of AFAlp in pathogenicity.
Phylogeny of LCR‐1 and OXA‐5 with class A and class D β‐lactamases
Authors: Couture France , Lachapelle Jean , Levesque Roger C.
Published: October 2006

The nucleotide sequences of blaLCR‐1 and blaOXA‐5β‐lactamase genes have been determined. Polypeptide products of 260 and 267 amino acids with estimated molecular masses of 27 120 Da and 27387 Da were obtained for the mature form of LCR‐1 and OXA‐5 proteins. A progressive alignment was used to evaluate the extent of identity between LCR‐1 and OXA‐5 with 29 other β‐lactamase amino acid sequences. The data showed that both belong to class D. We identified amino acids conserved in 24 positions for class A β‐lactamases and in 28 positions for five class D enzymes. The structural similarities between class A and class D β‐lactamases are more extensive than indicated by earlier biochemical studies with overall 16% identity between both classes. From the alignment, dendograms were constructed with a distance‐matrix and parsimony methods which defined three major groups of proteins subdivided into clusters giving insight on β‐lactamase phylogeny and evolution.
Occurrence of a Photosystem II polypeptide in non‐photosynthetic membranes of cyanobacteria
Authors: Smith Dawn , Bendall Derek S. , Howe Christopher J.
Published: October 2006

Cytoplasmic and thylakoid membranes have been purified from the cyanobacteria Anacystis nidulans R2 and Phormidium laminosum by sucrose density gradient centrifugation. Probing of Western blots of proteins from these purified membrane fractions with antibodies directed against the 33 kDa polypeptide of Photosystem II from pea indicates that this protein is present in both the thylakoid and cytoplasmic membranes, rather than just the thylakoid membranes. This has been confirmed by immunogold labelling of cells. Oxygen evolution assays have been used to show that the 33 kDa polypeptide is not assembled into a functional Photosystem II complex in the cytoplasmic membranes. This may be due to the absence of other Photosystem II components.
A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae
Authors: Yu Jun , Webb Helen , Hirst Timothy R.
Published: October 2006

A strain of Vibrio cholerae, which had been engineered to express high levels of the non‐toxic B subunit (EtxB) of Escherichia coli heat‐labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200‐amino‐acid exported protein, with a Cys–X–X–Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation In E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active‐site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.
Isolation of phosphorylation‐deficient mutants of the Rhizobium Meliloti two‐component regulatory protein, FixJ
Authors: Weinstein Michael , Lois Augusto F. , Monson Ellen K. , Ditta Gary S. , Helinski Donald R.
Published: October 2006

Rhizobium meliloti FixL and FixJ are members of a symbiotically essential two‐component system that regulates nitrogen‐fixation genes in response to environmental oxygen concentrations. FixL is a membrane protein that is thought to relay information about oxygen availability to FixJ via a phosphotransfer mechanism. FixJ increases expression of the nifA and fixK genes by activating transcription of the nifA and fixK promoters (p‐nifA and p‐fixK, respectively). In this study, we examined the relationship between the in vivo activity of FixJ as a transcriptional regulator and its ability to be phosphorylated in vitro by the sensor FixL. FixJ mutants were isolated that showed decreased activity on p‐nifA in Escherichia coli. Most of the FixJ mutant proteins also showed decreased activity on the fixK promoter. These mutants were analysed in R. meliloti for activity on p‐nifA during vegetative growth, where similarities and differences were observed when compared with their phenotypes in E. coli. Three mutants showing significantly less activity in R. meliloti were examined for symbiotic activity in planta and were found to be ineffective. When these three mutant FixJ proteins were examined in vitro for their ability to be phosphorylated by FixL, two mutants were found to have a significantly decreased ability to accept phosphate from FixL. These findings are discussed in relation to signal transduction in the FixLJ system.
Molecular characterization of pcp, the structural gene encoding the pyrrolidone carboxylyl peptidase from Streptococcus pyogenes
Authors: Cleuziat P. , Awadé A. , Robert‐Baudouy J.
Published: October 2006

This paper describes the cloning of a gene (pep) coding for pyrrolidone carboxylyl peptidase (PYRase), an enzyme which selectively removes N‐terminal pyroglutamic acid residues from polypeptldes. This gene was isolated from Streptococcus pyogenes by construction of a gene library with a bacteriophage λ‐derived cosmid‐Escherichia coli host system. Nucleotide sequence determination of a 1.3 kb restriction fragment revealed a 645 bp open reading frame encoding a 215‐amino‐acid product of Mr, 23 135 consistent with the 26 kDa polypeptide obtained from in vivo overexpression in E. coll. Southern hybridization confirmed that pep is a single‐copy gene on the S. pyogenes chromosome. 5’and 3’endpoint mapping of the 0.7 kb specific transcript observed by Northern analysis permitted the identification of transcriptional initiation and termination signals. Structural features of the pep gene product from S. pyogenes are discussed and compared with that from Bacillus subtilis. The lack of sequence identity with any other known protein or nucleotide sequence suggests that this enzyme belongs to a new class of peptidase.