Identification and cloning of a fur homologue from Neisseria meningitidis

The iron response in a number of bacterial systems is mediated by fur (f erric u ptake r egulation)-like regulatory systems. We have cloned and characterized a gene from Neisseria meningitidis that was homologous to Escherichia coli fur. This clone was capable of modulating expression from both E. coli and neisserial iron-regulated promoters in response to iron, and it produced a protein that reacted with anti-E. coli fur serum. Although the DNA and predicted amino acid sequences were very similar to those of four other published fur homologues, meningococcal fur was the most divergent of the group. Inability to construct a meningococcal fur mutant suggested that fur may be essential in this species.     

Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity

A 2.7-kb cya A gene fragment encoding the amino-terminal end of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis has been placed under the control of the lac promoter for expression in Escherichia coli. Following induction with isopropyl beta-D-thiogalactoside, calmodulin-sensitive adenylate cyclase activity was detected in a cell extract from E. coli. The expression vector directed the synthesis of a 90-kDa polypeptide that was recognized by rabbit polyclonal antibodies raised against the catalytic subunit of B. pertussis adenylate cyclase. Inspection of the deduced amino acid sequence of the cya A gene product revealed a sequence with homology to consensus sequences for an ATP-binding domain found in many ATP-binding proteins. On the basis of the analysis of nucleotide binding proteins, a conserved lysine residue has been implicated in the binding of ATP. A putative ATP-binding domain in the B. pertussis adenylate cyclase possesses an analogous lysine residue at position 58. To test whether lysine 58 of the B. pertussis adenylate cyclase is a crucial residue for enzyme activity, it was replaced with methionine by oligonucleotide-directed mutagenesis. E. coli cells were transformed with the mutant cya A gene, and the expressed gene product was characterized. The mutant protein exhibited neither basal nor calmodulin-stimulated enzyme activity, indicating that lysine 58 plays a critical role in enzyme catalysis.     

Cloning of the repressor protein gene of iron-regulated systems in Escherichia coli K12

Summary In Escherichia coli the iron uptake systems are regulated by the fur gene product. The synthesis of the outer membrane proteins fiu, fepA, fecA, fhuA, fhuE and cir is derepressed at low iron concentrations in the medium or constitutive in a fur mutant. The fur gene region cloned into pACYC184 was analysed by restriction analysis, Tn1000 mutagenesis and complementation studies. The presence of fur ⁺ plasmids repressed synthesis of the proteins fepA, fecA, fhuE and cir in a chromosomal fur mutant. More quantitatively, the repression to wild-type levels was shown with lac fusions to the genes fiu, fepA and cir. In minicells an 18,000 dalton protein was identified as the fur gene product. Correlated with the fur protein a slightly smaller protein, possibly a degradation product, was observed. The gene fur was mapped on the E. coli chromosome near nagA at about 15.5 min.     

Cloning and Sequencing of the Vibrio parahaemolyticus fur Gene

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.     

Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA

Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo. Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180kDa. However, proteolytic fragments as small as 70kDa, which can arise during purification, also exhibit RNase E activity, in vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites. RNase E cleaves rne mRNA and autoregulates the expression of the rne gene. In addition we demonstrate RNase E-dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate-determining for degradation and reported to be cieaved by RNase K. Our data are consistent with RNase K being a proteolytic fragment of RNase E.     

Cloning of the recA gene of Bordetella pertussis and characterization of its product.

A recA gene of Bordetella pertussis was identified in a plasmid library by complementation of a recA mutation in E coli and subcloned as a 2.1-kb Sph I DNA fragment. Southern hybridization experiments showed no similarity to the E coli recA gene, but very strong similarity to other Bordetella species. E coli recA mutant cells containing the B pertussis recA gene at high gene dosage were resistant to DNA-damaging agents such as methyl methane sulfonate or 4-nitroquinoline-N-oxide, displayed induction of SOS functions, and were able to promote DNA recombination, but not induction of phage lambda. The latter phenotype distinguishes the B pertussis recA gene product from the corresponding proteins from most other Gram-negative organisms. Amino acid sequence comparisons revealed a high degree of structural conservation between prokaryotic RecA proteins.     

Candidate cis-elements for human renin gene expression in the promoter region

The regulation of renin gene expression, the rate‐limiting enzyme of the system, is thought to be fundamental to the total system. Previously, we mapped six putative cis‐elements in the promoter region of the human renin gene with nuclear proteins from human chorionic cells and human renal cortex by DNase I protection assay (footprint A–F). Each footprint contains Ets motif like site (A), HOXñPBX recognition sequence (B), unknown sequence as DNA binding consensus (C), CRE (D), COUP‐TFII (ARP‐1) motif like site (E), and AGE3 like site (F). Footprint D has been characterized by means of functional studies as the genuine human renin gene CRE interacting with CREB in cooperation with the site of footprint B. To obtain further clues to the specific expression in the promoter region, these putative cis‐elements were conducted to a consensus‐specific binding assay to compare renin‐producing and non‐renin‐producing cells by EMSA and electromobility super‐shift assay. Different sequence‐specific DNA/protein binding was obtained among the different cell lines with footprint B site, with COUP‐TFII (ARP‐1) motif like site and possibly with footprint F site. The results implicate these putative cis‐elements and each corresponding trans‐factor in the specific expression of the human renin gene in the promoter region. Further functional characterization of these elements would provide important data for a better understanding of human renin gene expression. © 2004 Wiley‐Liss, Inc.     

Isolation and characterization of the recA gene of Bordetella pertussis

This report describes the detection and cloning of the Bordetella pertussis recA gene. Escherichia coli clones having recombinant plasmids containing the B. pertussis recA gene were isolated by complementing an E. coli RecA- mutant's inability to survive in the presence of methylmethanesulphonate (MMS). This gene was shown to complement the deficiency of E. coli RecA- strains to tolerate the DNA-damaging effects of both a chemical agent and ultraviolet light (u.v.). Deletion mapping experiments localized the gene to a 2.5 kb StuI-EcoRI fragment, and expression of the gene in E. coli resulted in the production of a 40 kD protein. These data strongly suggest that a region of the B. pertussis chromosome that encodes RecA-like activity has been isolated and cloned.     

Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures

Escherichia coli containing a cloned gene encoding the Bordetella pertussis serotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole-cell extracts. To engineer plasmids capable of directing the expression in E. coli of high levels of this product, both as a pre-protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda P(L) and P(R) promoters and the E. coli atpE translational initiation region. These constructs did not result in the expression of fimbrial subunit at detectable levels in several E. coli strains including DH5. However, they did in E. coli CAG629, which is lon protease and heat shock protein deficient. Both pre-protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane. Electron microscopic investigation of the cell surface of E. coli cells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures. The methionylated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity. After dialysis in appropriate conditions it was seen to autoassemble into protein polymers. Antibodies raised against polymerized recombinant subunit reacted weakly with whole B. pertussis serotype 2 fimbriae in immunodot blot assays. However, such antibodies reacted in Western blots equally well with the recombinant and wild-type form of the fimbrial subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Osmotic Shock Induction of the Expression of Vibrio fischeri lux Genes in Escherichia coli Cells

The effect of osmotic shock on the expression of genes in the lux regulon of marine bacteria Vibrio fischeri was studied in cells ofEscherichia coli. Bioluminescence of cells was shown to drastically increase, when cells were exposed to osmotic shock at the early logarithmic growth phase, at far lower optic densities as compared to the critical optic density characteristic. The expression of lux genes induced by osmotic shock is determined by the two-component regulatory system RcsC–RcsB. A nucleotide sequence in the regulatory region of the luxR gene homologous to the RcsB-box consensus of E. coli is assumed to be a primary site for this system.     

Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co-localization with the Spo0J partitioning protein

We have cloned the rpoD gene coding for the major sigma factor of Bordetella pertussis . The deduced amino acid sequence reveals a protein of 733 residues which has extensive amino acid homology with the principal σ factors of a number of divergent prokaryotes. It is larger than most σ factors identified to date, having a molecular mass of 81.3 kDa. We have designated this factor sigma 80. In a heterologous complementation assay, B. pertussis rpoD was able to complement the Escherichia coli rpoD temperature-sensitive mutant UQ285. Furthermore, B. pertussis rpoD conferred better specificity to the E. coli RNA polymerase, allowing increased expression of the B. pertussis virulence-associated fha promoter, but could not activate the ptx and cya promoters in the E. coli UQ285 strains carrying the B. pertussis bvg locus. We discuss the implications of these results on the mechanisms involved in the activation of virulence-associated promoters.     

Fusion expression and immunogenicity of <Emphasis Type="Italic">Bordetella pertussis</Emphasis> PTS1-FHA protein: implications for the vaccine development

Mutants of pertussis toxin (PT) S1 subunit and filamentous hemagglutinin (FHA) type I immunodominant domain from Bordetella pertussis (B. pertussis) are considered to be effective candidate antigens for acellular pertussis vaccines; however, the substantial progress is hampered in part for the lack of a suitable in vitro expression system. In this paper, the gene sequences of a S1 mutant C180-R9K/E129G (mS1) and a truncated peptide named Fs from FHA type I immunodominant domain were linked together and constructed to pET22b expression vector as a fusion gene; after inducing with IPTG, it was highly expressed in E. coli BL21 (DE3) as inclusion body. The fusion protein FsmS1 was purified from cell lysates and refolded successfully. The result of Western blotting indicate that it was able to react with both anti-S1 and anti-FHA McAbs; antiserum produced from New Zealand white rabbits immunized with this protein was able to recognize both native PT and FHA antigens as determined by western blotting. These data have provided a novel feasible method to produce PT S1 subunit and FHA type I immunodominant domain in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against B. pertussis infection.     

Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis.

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis, a 45 kd secreted protein, is synthesized as a 1706 amino acid precursor. We have shown that this precursor is a bifunctional protein, carrying both adenylate cyclase and haemolytic activities. The 1250 carboxy-terminal amino acids of the precursor showed 25% similarity with Escherichia coli alpha-haemolysin (HlyA) and 22% similarity with Pasteurella haemolytica leucotoxin. Three open reading frames were identified downstream from the cyaA gene: cyaB, cyaD and cyaE, coding for polypeptides of 712, 440 and 474 amino acid residues, respectively. As for E. coli alpha-haemolysin, secretion of B.pertussis adenylate cyclase and haemolysin requires the expression of additional genes. The gene products of cyaB and cyaD are highly similar to HlyB and HlyD, known to be necessary for the transport of HlyA across the cell envelope and for its release into the external medium. Complementation and functional studies indicate that the B.pertussis adenylate cyclase-haemolysin bifunctional protein is secreted by a mechanism similar to that described for E.coli alpha-haemolysin, requiring, in addition to the cyaB and cyaD gene products, the presence of a third gene product specified by the cyaE gene.     

Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes

The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endo-proteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the wild type precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of prov WF. In contrast, cotransfection of fur cDNA with a recombinat derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.     

The fur gene from Klebsiella pneumoniae: characterization,genomic organization and phylogenetic analysis

The Fur (ferric uptake regulator) protein controls the expression of a number of bacterial virulence determinants including those involved in iron uptake. The fur gene was cloned and characterized from Klebsiella pneumoniae. The gene is preceded by a single autoregulated promoter whose −10 region overlaps the putative Fur binding site. The autoregulated nature of the K. pneumoniae fur gene and functionality of the encoded Fur repressor were tested in Fur titration and complementation assays. A partial open reading frame upstream from the fur gene was identified as a flavodoxin (fldA) gene. An open reading frame located 50 bases downstream from the fur stop codon appears to be a truncated citA gene that, if functional, would encode only the carboxy terminus of a citrate utilization protein. The fldA-fur arrangement is also present in Escherichia coli. However, the fur-citA arrangement found in K. pneumoniae is novel. It appears that the chromosomal region downstream from the fur gene is unstable and, thus, variable even in closely related bacterial lineages. To assess the ability of the Fur protein sequence to reflect organismal phylogeny, the Fur protein tree was compared to the tree of 16S rRNA (ribosomal RNA). The Fur dataset comprises almost an order of magnitude fewer characters than the 16S rRNA but is nonetheless able to track the phylogenetic signal reasonably well, suggesting that the fur gene, like the 16S rDNA, may not be subject to horizontal gene transfer in these bacteria.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.