Bernard A.M. van der Zeijst

Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site

Abstract The nucleotide sequence of the peplomer (E2) gene of MHV-A59 was determined from a set of overlapping cDNA clones. The E2 gene encodes a protein of 1324 amino acids including a hydrophobic signal peptide. A second large hydrophobic domain is found near the COOH terminus and probably represents the membrane anchor. Twenty glycosylation sites are predicted. Cleavage of the E2 protein results in two different 90K species, 90A and 90B (L. S. Sturman, C. S. Ricard, and K. V. Holmes (1985) J. Virol. 56, 904–911), and activates cell fusion. Protein sequencing of the trypsin-generated N-terminus revealed the position of the cleavage site. 90A and 90B could be identified as the C-terminal and the N-terminal parts, respectively. Amino acid sequence comparison of the A59 and 1HM E2 proteins showed extensive homology and revealed a stretch of 89 amino acids in the 90B region of the A59 E2 protein that is absent in JHM.

Capsular polysaccharide synthesis in Streptococcus pneumoniae serotype 14: molecular analysis of the complete cps locus and identification of genes encoding glycosyltransferases required for the biosynthesis of the tetrasaccharide subunit

We have reported previously on seven genes (cps14B–H ) of Streptococcus pneumoniae serotype 14, which are part of the type 14 capsular polysaccharide synthesis (cps14 ) locus. This study describes the cloning and sequencing of the remaining part of the cps14 locus. The entire cps14 gene cluster consists of 12 open reading genes (cps14A to cps14L ), which appear to be arranged as a single transcriptional unit. The flanking regions of the cps14 locus contain vestiges of insertion elements. Moreover, a 115‐bp‐long repeated DNA element, which is also present in several other intergenic regions on the pneumococcal chromosome, has been identified upstream of cps14AAll 12 open reading frames (ORFs) were inactivated by the insertion of a tetracycline resistance cassette. The cps14A to cps14J and cps14L mutants were unencapsulated, whereas only a limited amount of capsular polysaccharide was expressed by a cps14K insertion mutant. Comparison with DNA and protein sequences available in databases allowed us to predict functions for four out of the five new cps14 gene products. The biosynthetic function of Cps14I was determined experimentally by analysis of intermediates in the synthesis of the type 14 tetrasaccharide subunit, catalysed by membrane preparations of Escherichia coli expressing pneumococcal glycosyltransferases. The cps14I gene encodes the β‐1,3‐N‐acetylglucosaminyltransferase activity necessary for the addition of the third sugar in the synthesis of the type 14 repeating unit. The activity encoded by cps14J was established using a synthetic glycosyltransferase acceptor: cps14J encodes a β‐1,4‐galactosyltransferase, which requires β‐linked GlcNAc as an acceptor. Thus, Cps14J is responsible for the addition of the last (fourth) sugar in the synthesis of the type 14 subunit.

Genetic manipulation of Campylobacter: evaluation of natural transformation and electro-transformation

Two methods, natural transformation and electro-transformation, for the introduction of DNA into nine strains of Campylobacter jejuni were compared. Both methods were successful with a limited number of strains. Natural transformation was efficient only for the introduction of C. jejuni chromosomal DNA, while electro-transformation was also applicable for the introduction of Escherichia coli-derived vector DNA into at least one C. jejuni strain. The efficiency of DNA recombination after entry was determined using C. jejuni chromosomal DNA containing disrupted flagellin genes of C. jejuni or suicide vectors containing a portion of these genes. In the latter case, DNA recombination occurred with as little as 200-bp homology present, indicating that only short homologous DNA segments are required.

Isolation and identification of virus-specific mRNAs in cells infected with mouse hepatitis virus (MHV-A59)

Abstract We have determined the kinetics of virus production and virus-specific RNA synthesis in Sac(−) cells infected with mouse hepatitis virus strain A59 (MHV-A59). Immunofluorescence showed that all cells became infected at a multiplicity of 10 PFU/cell. The virus was concentrated and purified to obtain the high titered stocks needed for these one-step growth experiments. Release of virus into the culture medium started 4 hr after infection (pi) and was complete at 10 hr pi. Synthesis of virus-specific RNA, measured by the incorporation of [3H]uridine in the presence of 1 μg/ml actinomycin D, also started at 4 hr pi and its maximum rate occurred between 6 and 8 hr pi. RNA labeled during this period was isolated from infected cells. About 50% of this RNA bound to oligo(dT)-cellulose; this material was denatured with glyoxal-dimethyl sulfoxide and analyzed by electrophoresis in 1% agarose gels. Seven RNA species with the following molecular weights were present: 5.6 × 106 (RNA1), 4.0 × 106 (RNA2), 3.0 × 106 (RNA3), 1.4 × 106 (RNA4), 1.2 × 106 (RNA5), 0.9 × 106 (RNA6), and 0.6 × 106 (RNA7). RNA1 comigrated with the viral genome. Artifacts caused by defective interfering particles or breakdown of RNA were excluded. To determine whether these RNA species were functional as messengers in infected cells, virus-specific RNAs present in polyribosomes were analyzed. EDTA treatment was used to discriminate between RNA present in polyribosomes and in EDTA-resistant, presumably ribonucleoprotein, particles. Most (91%) of RNA1 was present in EDTA-resistant particles; the remainder and all other RNAs synthesized between 6 and 8 hr pi were present in polyribosomes. We conclude that MHV-A59 has six subgenomic mRNAs. Since the total molecular mass (11.1 × 106 daltons) of these messengers is about twice that of the viral genome, sequence homologies must exist between the mRNAs. The position of these homologous regions and the translation products of each of the mRNAs remain to be determined.

Genetic Basis for the Structural Difference between Streptococcus pneumoniae Serotype 15B and 15C Capsular Polysaccharides

ABSTRACT In a search for the genetic basis for the structural difference between the related Streptococcus pneumoniae capsular serotypes 15B and 15C and for the reported reversible switching between these serotypes, the corresponding capsular polysaccharide synthesis (cps) loci were investigated by keeping in mind that at the structural level, the capsules differ only in O acetylation. The cps locus of a serotype 15B strain was identified, partially PCR amplified with primers based on the related serotype 14 sequence, and sequenced. Sequence analysis revealed, among other open reading frames, an intact open reading frame (designated cps15bM) whose product, at the protein level, exhibited characteristics of previously identified acetyltransferases. Genetic analysis of the corresponding region in a serotype15C strain indicated that the same gene was present but had a premature stop in translation. Closer analysis indicated that the serotype 15B gene contained a short tandem TA repeat consisting of eight TA units. In serotype 15C, this gene contained nine TA units that resulted in a frameshift and a truncated product. Genetic analysis of 17 serotype 15B and 15C clinical isolates revealed a perfect correlation between the serotype and the length of the short tandem repeat in the putative O-acetyltransferase gene. The number of TA repeating units varied between seven and nine in the various isolates. Together, the data strongly suggest that the structural difference between serotypes 15B and 15C is based on variation in the short tandem TA repeat in the O-acetyltransferase gene and that the transition between serotypes is due to slipped-strand mispairing with deletion or insertion of TA units in the cps15bM gene.

Expression of CFA/I fimbriae is positively regulated

Production of the plasmid-coded fimbrial antigen CFA/I of Escherichia coli requires both CFA/I region 1 and CFA/I region 2, which are separated by about 40 kb on the wildtype plasmid. The nucleotide sequence of region 2 was determined and contains an open reading frame (cfa d), encoding a protein of 265 amino acids. The protein has no signal sequence and upon sequence analysis appeared to be a DNA-binding protein. A plasmid was constituted, with a promoterless beta-galactosidase gene preceded by the promoter of region 1. Introduction of a plasmid, carrying the cfa d gene, into a strain containing this construct enhanced expression of beta-galactosidase by at least five-fold indicating that the cfa d protein was enhancing expression from the promoter of region 1. The cfa d gene sequence differed at 28 positions from the Rns gene, which encodes a protein that is a positive regulator of the expression of CS1 or CS2 fimbriae. It was shown that the cfa d gene and the Rns gene can functionally substitute each other in regulating fimbrial synthesis.

Antigenicity of the peplomer protein of infectious bronchitis virus

Abstract To study the antigenic structure of the peplomer protein of the avian coronavirus infectious bronchitis virus, fragments from the peplomer gene were generated by restriction-enzyme cleavage or by limited DNase digestion and inserted in the Escherichia coli expression plasrnid pEX (Stanley and Luzio, 1984). The antigenicity of the expression products was tested using a number of polyclonal antisera and monoclonal antibodies. The polyclonal antisera recognized different sets of epitopes in the 1162-residue sequence. The N-terminal region of one of the two subunits, S2, was recognized by all polyclonal sera and by two monoclonal antibodies. This clearly immunodominant region contains at least two adjacent or overlapping epitopes, one of which has been localized within 18 residues. The epitopes found as antigenic pEX expression products do not coincide with the regions in the S1 subunit that have been found to contain hypervariable sequences. We suggest that these regions constitute conformation dependent neutralization epitopes that cannot be detected in the pEX system. The relevance of our finclings for vaccine development is discussed.

The nucleotide sequence of the peplomer gene of porcine transmissible gastroenteritis virus (TGEV) : Comparison with the sequence of the peplomer protein of feline infectious peritonitis virus (FIPV)

Abstract The amino acid sequence of the peplomer protein of transmissible gastroenteritis virus (TGEV) has been derived from the cloned cDNA sequence. The gene encodes a protein of 1447 amino acids with a molecular weight of 159 574. Comparison with the primary structure of the peplomer protein of feline infectious peritonitis virus (FIPV) (de Groot et al., 1987b) revealed one domain, from amino acids 1 to 274, in which the nucleotide homology was 39%, whereas in the second domain (from residues 275 to 1447) the homology was 93%.

Functional Analysis of Glycosyltransferases Encoded by the Capsular Polysaccharide Biosynthesis Locus of Streptococcus pneumoniae Serotype 14

Bacteria belonging to the speciesStreptococcus pneumoniae vary in their capsule. Presently, 90 capsular serotypes are known, all possessing their own specific polysaccharide structure. Little is known about the biosynthesis of these capsular polysaccharides. The cps locus of S. pneumoniae serotype 14 was cloned. So far, 7 open reading frames have been sequenced, cps14B to cps14H. The gene products are similar to proteins involved in bacterial polysaccharide biosynthesis, both of Gram-negative and -positive micro-organisms. Gene-specific mutants were created for cps14D tocps14H by insertional mutagenesis. All mutants no longer agglutinated with a monoclonal antibody against type 14 capsule polysaccharides. The biosynthetic function of cps14E andcps14G was determined by analysis of the intermediates in the synthesis of the oligosaccharide subunit, formed in membrane preparations of the wild-type and mutant strains and in membrane preparations of Escherichia coli expressing the pneumococcal glycosyltransferases. The enzyme encoded bycps14E is a glucosyl-1-phosphate transferase that links glucose to a lipid carrier, the first step in the biosynthesis of the type 14 repeating unit. The gene product of cps14G encodes a β-1,4-galactosyltransferase, the enzyme responsible for the second step in the subunit synthesis, the transfer of galactose to lipid-linked glucose.

Phylogenetic position of Cowdria ruminantium (Rickettsiales) determined by analysis of amplified 16S ribosomal DNA sequences

The 16S ribosomal DNA sequence of Cowdria ruminantium, the causative agent of heartwater disease in ruminants, was determined. An analysis of this sequence showed that C. ruminantium forms a tight phylogenetic cluster with the canine pathogen Ehrlichia canis and the human pathogen Ehrlichia chaffeensis. Although a close relationship between the genus Cowdria and several members of the tribe Ehrlichieae has been suspected previously, the tight phylogenetic cluster with E. canis and E. chaffeensis is surprising in view of known differences in host preference and target cells.