Piet J. M. Nuijten

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.

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.

DNA Rearrangements in the Flagellin Locus of an flaA Mutant of Campylobacter jejuni during Colonization of Chicken Ceca

ABSTRACT Campylobacter jejuni is an enteropathogen for humans but commensal for chickens. In both hosts, the flagella and motility are important colonization factors. The flagellin gene is duplicated inCampylobacter, but only one flagellin gene,flaA, is sufficient for motility. We found that, during colonization of the chicken intestine, a nonmotile flaAmutant of C. jejuni underwent rearrangements within its flagellin locus, thereby regaining its motility and colonization capacity. In contrast, in vitro motile revertants isolated from liquid culture showed different flagellin DNA rearrangements than after reversion in the chicken.

Analysis of flagellin gene expression in flagellar phase variants ofCampylobacter jejuni 81116

Flagella production inCampylobacter jejuni 81116 is subject to phase variation; the bacterium is able to switch its flagellum synthesis, and thereby its motility, on and off. Under standard laboratory growth conditions flagellar phase variants can be maintained as stable, pure cultures. We found conditions that efficiently induced a phase shiftin vitro. TheflaA gene but not theflaB gene is subject to the on and off switch. Minor amounts of FlaB are still present in aflagellate cells. We previously showed that flagellin gene expression in phase variants was regulated at the transcriptional level. Here, sequence data prove that abolishment offlaA transcription is not caused by DNA rearrangements or mutations within the flagellin locus. SinceflaA is preceeded by a typical σ28 promoter aC. jejuni σ28 homolog could play a role in regulation offlaA gene expression but such a gene or protein could not be detected. However,in vitro transcription could be detected using σ28-holoenzyme preparations fromBacillus subtilis. Possible regulatory mechanisms that may control flagellar phase variation in Campylobacter are discussed.