Interaction of the Bordetella pertussis filamentous hemagglutinin with heparin

Heparin, a glycosaminoglycan synthesized in connective tissue-mast cells, appeared to inhibit the hemagglutination of rabbit erythrocytes induced by the filamentous hemagglutinin (FHA), a major adhesin of Bordetella pertussis. This inhibition suggested an interaction of heparin with the FHA region responsible for the hemagglutination activity. FHA-heparin interactions may play a role in bacterial attachment and persistence in the lungs during human pertussis. To confirm a direct FHA-heparin interaction, heparin was used as ligand in an affinity chromatography procedure. This technique allowed to purify FHA directly from the bacterial culture medium in a single-step using heparin-Sepharose CL-6B or Zetaffinity heparin 60 disks. The purified FHA was highly immunoreactive with anti-FHA monoclonal antibodies and showed no signs of degradation after 15 successive cycles of freezing-thawing. The described purification method is simple, and suitable for the rapid preparation of FHA.     

Identification of immunodominant epitopes in the filamentous hemagglutinin of Bordetella pertussis

The filamentous hemagglutinin (FHA) of Bordetella pertussis is a principal adhesin, which plays a key role in the colonization of the upper respiratory tract. FHA is also a protective antigen, which has been incorporated in the new generation of acellular vaccines against whooping cough. The protein is synthesized as a large 367-kDa precursor, which is then processed into a 220-kDa secreted polypeptide. To optimize the use of this protein for vaccine purposes it would be helpful to define the regions encompassing immunodominant epitopes. Twelve recombinant plasmids have been generated encoding fusion proteins between fragments of the matured-secreted 220-kDa form of FHA and the vector-encoded phage MS2 polymerase. Protein extracts of the resulting recombinant clones have been tested for reactivity with sera from 20 patients convalescent from whooping cough, and two human standard sera. The results indicate the presence of an immunodominant B cell epitope in the polypeptide coded by a 1-kb DNA fragment encompassing positions 5781-6800 of the published sequence. These results suggest that the identified fragment should be conserved in the formulation of vaccines against pertussis.     

Purification and Characterization of Alkaline Serine Proteinase from Photosynthetic Bacterium,Rubrivivax gelatinosus KDDS1

In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture. An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35°C. Molecular weight of the purified enzyme was estimated to be 32.5 kDa. The enzyme showed the highest activity at 45°C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA. The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei. Thus, the enzyme from Rvi. gelatinosus KDDS1 was thought to be a serine-type proteinase. This was the first serine proteinase characterized from photosynthetic bacteria.     

Common accessory genes for the Bordetella pertussis filamentous hemagglutinin and fimbriae share sequence similarities with the papC and papD gene families.

The Bordetella pertussis filamentous hemagglutinin (FHA) is a major virulence factor responsible for attachment, one of the early events in bacterial pathogenesis. Deletion of its structural gene, fhaB, or a Tn5 insertion in fhaA, downstream of fhaB, resulted in a FHA- and fimbriae- phenotype, although fhaB and the fim genes are not linked. The fhaB downstream region therefore most likely encodes accessory proteins required for the biosynthesis of FHA and fimbriae, despite the lack of sequence similarities between these two proteins. The nucleotide sequence of this area contains the open reading frames fhaD and fhaA, whose products share sequence similarities with the papD and papC gene products, respectively. PapD is a periplasmic chaperone protein able to bind to the Escherichia coli P pilin subunits and to transport them towards the outer membrane protein PapC which is responsible for pilus membrane translocation. An additional open reading frame, fhaE, is located downstream of fhaA. Its amino acid sequence shares similarities with those of the fimbrial subunits. Deletion analyses suggest that fhaB and the downstream genes can be transcribed as a polycistronic operon, and primer extension analysis revealed the presence of a second promoter between fhaB and fhaD.     

The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense

The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth. In this work, we report on chemoheterotrophic growth of R. sodomense in media containing either acetate or succinate supplemented with 0.3–0.5% yeast extract. Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with E _m,7 of +171 ± 10, +62 ± 10 and –45 ± 13 mV (561–575 nm), and +268 ± 6, +137 ± 10 and –43 ± 12 mV (551–540 nm). A small amount of a soluble c-type cytochrome with a mol. mass of 15 kDa (E _m,7≥ +150 mV) was identified. Spectroscopic and immunological methods excluded the presence of cytochrome of the c ₂ class and high-potential iron-sulfur proteins. Inhibitory studies indicated that only 60–70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 μM, respectively). These results were interpreted to show that the oxidative electron transport chain of R. sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 μM cyanide. These features taken together suggest that R. sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c. Received: 8 June 1998 / Accepted: 25 August 1998     

The construction of a cloning vector designed for gene replacement in Bordetella pertussis

We report here the construction of a plasmid cloning vector, pRTP1, designed to facilitate exchange of cloned and chromosomal alleles of the human bacterial pathogen Bordetella pertussis. pRTP1 provides the ability to successively select two homologous recombination events within the cloned sequences. The first is by selection for maintenance of the ampicillin-resistance gene on the plasmid which is unable to replicate autonomously after transfer via conjugation. The second selection, via streptomycin (Sm) selection, is against the maintenance of vector sequences which contain a gene encoding the Sm-sensitive allele of the gene for ribosomal protein S12 thus rendering an otherwise Sm-resistant strain Sm-sensitive. We demonstrate the use of this vector to introduce an unmarked mutation, constructed in vitro, into the chromosomal locus encoding pertussis toxin.     

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir.

The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of Tn5 insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the fha and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only fha or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by Tn5 mutagenesis in E. coli and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA.     

Characterization of a Bordetella pertussis fimbrial gene cluster which is located directly downstream of the filamentous haemagglutinin gene

The biosynthesis of fimbriae is a complex process requiring multiple genes which are generally found clustered on the chromosome. In Bordetella pertussis, only major fimbrial subunit genes have been identified, and no evidence has yet been found that they are located in a fimbrial gene cluster. To locate additional genes involved in the biosynthesis of B. pertussis fimbriae, we used TnphoA mutagenesis. A PhoA+ mutant (designated B176) was isolated which was affected in the production of both serotype 2 and 3 fimbriae. Cloning and sequencing of the DNA region harbouring the transposon insertion revealed the presence of at least three additional fimbrial genes, designated fimB, fimC and fimD. The transposon was found to be located in fimD. Analysis of PhoA activity indicated that the fimbrial gene cluster was positively regulated by the bvg locus. A potential binding site for BvgA was observed upstream of fimB. FimB showed homology with the so-called chaperone-like fimbrial proteins, while FimC was homologous with a class of fimbrial proteins located in the outer membrane and presumed to be involved in transport and anchorage of fimbrial subunits. An insertion mutation in fimB abolished the expression of fimbrial subunits, implicating this gene in the biosynthesis of both serotype 2 and 3 fimbriae. Upstream of fimB a pseudogene (fimA) was observed which showed homology with the three major fimbrial subunit genes, fim2, fim3 and fimX. The construction of a phylogenetic tree suggested that fimA may be the primordial major fimbrial subunit gene from which the other three were derived by gene duplication. Interestingly, the fimbrial gene cluster was found to be located directly downstream from the gene coding for the filamentous haemagglutinin, an important B. pertussis adhesin, possibly suggesting co-operation between the two loci in the pathogenesis of pertussis.     

Calculability analysis in underdetermined metabolic networks illustrated by a model of the central metabolism in purple nonsulfur bacteria

Metabolite balancing has turned out to be a powerful computational tool in metabolic engineering. However, the linear equation systems occurring in this analysis are often underdetermined. If it is difficult or impossible to find the missing constraints, it is nevertheless feasible in some cases to determine the values of a subset of the unknown rates. Here, a procedure for finding out which reaction rates can be uniquely calculated in underdetermined metabolic networks and computing these rates is given. The method is based on the null space to the stoichiometry matrix corresponding to the reactions with unknown rates. It is shown that this method is considerably easier to handle than an algorithm given previously (Van der Heijden et al., 1994a). Furthermore, a useful elementary representation of the null space is presented which is closely related with the elementary flux modes. This unique representation is central to a more general approach to observability/calculability analysis. In particular, it allows one to find, in an easy way, those sets of measurable rates that enable a calculation of a certain unknown rate. Besides, rates which are never calculable by metabolite balancing may be easily detected by this method. The applicability of these methods is illustrated by a model of the central metabolism in purple nonsulfur bacteria. The photoheterotrophic growth of these representatives of anoxygenic photosynthetic bacteria is stoichiometrically analyzed. Interesting metabolic constraints caused by the necessary balancing of NADPH can be detected in a highly underdetermined system. This is, to our knowledge, the first application of stoichiometric analysis to the metabolic network in this bacteria group using metabolite balancing techniques. A new software tool, the FluxAnalyzer, is introduced. It allows quantitative and structural analysis of metabolic networks in a graphical user interface.     

Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin

Recombinant plasmids encoding a Vibrio cholerae haemagglutinin were isolated from the highly virulent V. cholerae strain C5 by cosmid cloning. Both Escherichia coli HB101 containing the recombinant plasmids and V. cholerae C5 were able to agglutinate a variety of erythrocytes from human and animal origin; this haemagglutination was not inhibited by D-mannose or L-fucose. Subcloning of the recombinant cosmid DNA revealed that a 1.3 kb DNA fragment was sufficient for haemagglutinin production in E. coli HB101. Under direction of this 1.3 kb Vibrio DNA fragment, two proteins were made in E. coli minicells, of 27 and 10 kDa. Haemagglutinin-encoding sequences were not detected in every V. cholerae strain.     

Structure of the Bordetella pertussis gene coding for the serotype 3 fimbrial subunit

Bordetella pertussis strains contain at least three distinct genes coding for fimbrial subunits, designated fim2, fim3, and fimX. The sequences of the fim2 and fimX genes have been published. Here we present the sequence of the fim3 gene. Proximal and distal to the fim3 gene, regions were observed that could function as rho-independent terminators, suggesting that the gene is not part of a larger operon. Comparison of the putative promoter regions of the fim2 and fim3 genes revealed a conserved region containing a stretch of approximately 13 C's. This region may be involved in fimbrial phase variation. A comparison of the deduced amino acid sequences of the three fimbrial subunits revealed conserved, variable, and hypervariable regions. The hypervariable regions coincided with predicted antigenic determinants. Peptides derived from the conserved regions may be incorporated into a future pertussis vaccine to induce antibodies which confer protection against strains producing different fimbrial serotypes.     

Structural factors which control the position of the Qy absorption band of bacteriochlorophyll a in purple bacterial antenna complexes

This paper presents a concise review of the structural factors which control the energy of the Q_y absorption band of bacteriochlorophyll a in purple bacterial antenna complexes. The energy of these Q_y absorption bands is important for excitation energy transfer within the bacterial photosynthetic unit. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular cloning and nucleotide sequence of a cDNA clone coding for rat brain myelin proteolipid

A cDNA library from rat brain was constructed in pBR322 and screened with a 14-mer mixed oligonucleotide probe based on residues 231-235 of bovine proteolipid (PLP). A positive clone was isolated: it contained a 1334-base-pair cDNA insert and was subjected to DNA sequence analysis. The cDNA encoded information for the 276 amino acids of rat PLP. Comparison with bovine PLP sequence showed a complete amino acid sequence homology except for 4 amino acid residues.     

Molecular cloning and characterization of a Candida albicans gene (EFB1) coding for the elongation factor EF-1β

Abstract The formation of H₂ by chemolithoautrophically growing Oligotropha carboxidovorans has been identified as the result of the oxidation of CO mediated by the cytoplasmic species of the molybdenum-containing CO dehydrogenase multienzyme complex as follows: CO + H ₂ O → CO ₂+ H ₂. Purified CO dehydrogenase was shown to carry hydrogen uptake and formation activities in addition to its catabolic function which is the oxidation of CO. Among the electron donors supporting H₂ formation were CO, NADH, reduced flavins and reduced viologen dyes. The reduction of protons to H₂ by cytoplasmic CO dehydrogenase is interpreted as a detoxification reaction for electrons to prevent cell damage in O. carboxidovorans .     

Molecular cloning and nucleotide sequence of a variant wheat histone H4 gene

To determine whether there is structural variation among histone H4 genes in wheat, one (TH091) of the H4 genes that had been cloned from a wheat genomic DNA library was sequenced and compared with another H4 gene (TH011) which we had described previously [Tabata et al., Nucl. Acids Res. 11 (1983) 5865-5865]. Nucleotide sequence analysis revealed that there are 17 nucleotide replacements in the protein-coding region of two H4 genes, causing only one amino acid substitution: a glycine at position 4 (from the N terminus) in TH011 was replaced by an aspartic acid in TH091. S1 mapping, using total nuclear RNA from germinated seeds, indicated that the H4 gene was transcribed in vivo.