SummaryThe molecular genetic basis of high-frequency serotype 3 capsule phase variation in Streptococcus pneumoniae (the pneumococcus) was investigated. Pneumococci were grown in sorbarod biofilms at 348C to mimic nasopharyngeal carriage. Different type 3 pneumococci commonly associated with invasive disease generated apparently random tandem duplications of 11-239 bp segments within the cap3A gene of the type 3 capsule locus. These duplications alone were found to be responsible for high-frequency capsule phase variation, in which (phase off) acapsular variants possessed duplications within cap3A, and (phase on) capsular revertants possessed wildtype cap3A genes, indicating the precise excision of the duplication. Additionally, the frequency of phase reversion (off to on) was found to exhibit a linear relationship between (log) frequency of reversion and (log) length of duplication. This apparently random duplication giving rise to phase variation is in stark contrast to the 'preprogrammed' contingency genes in many Gram-negative organisms that possess homopolymeric sequence repeats or motifs for sitespecific recombination.

We examined the ability of a soil bacterium, Agrobacterium radiobacter J14a, to degrade the herbicide atrazine under a variety of cultural conditions, and we used this bacterium to increase the biodegradation of atrazine in soils from agricultural chemical distribution sites. J14a cells grown in nitrogen-free medium with citrate and sucrose as carbon sources mineralized 94% of 50 μg of [14C-U-ring]atrazine ml−1 in 72 h with a concurrent increase in the population size from 7.9 × 105 to 5.0 × 107 cells ml−1. Under these conditions cells mineralized the [ethyl-14C]atrazine and incorporated approximately 30% of the 14C into the J14a biomass. Cells grown in medium without additional carbon and nitrogen sources degraded atrazine, but the cell numbers did not increase. Metabolites produced by J14a during atrazine degradation include hydroxyatrazine, deethylatrazine, and deethyl-hydroxyatrazine. The addition of 105 J14a cells g−1 into soil with a low indigenous population of atrazine degraders treated with 50 and 200 μg of atrazine g−1soil resulted in two to five times higher mineralization than in the noninoculated soil. Sucrose addition did not result in significantly faster mineralization rates or shorten degradation lag times. However, J14a introduction (105 cells g−1) into another soil with a larger indigenous atrazine-mineralizing population reduced the atrazine degradation lag times below those in noninoculated treatments but did not generally increase total atrazine mineralization.

SUMMARYNine serological techniques were compared by monitoring the response to infection with Rift Valley fever (RVF) virus in three sheep. Antibodies were monitored daily for the first 14 days after infection, then weekly and later fortnightly up to week 24. The earliest antibody response was detected in one sheep on day 3 by a plaque reduction neutralization test, and by day 6 antibodies were demonstrable in all three sheep by haemagglutination-inhibition, reversed passive haemagglutination-inhibition, immunodiffusion, indirect immunofluorescence (IF), enzyme-linked immunosorbent assay and neutralization ofcytopathic effect in cell cultures. Antibodies were demonstrable by complement fixation on day 8 at the earliest. IF and the two neutralization techniques produced the highest titres, but all tests could be used satisfactorily for the serological diagnosis of RVF. Inactivated antigen could be used for all except the neutralization tests. A radioimmunoassay technique using l251-labelled staphylococcal protein A detected antibodies on day 8 at the earliest and produced lower mean titres than some of the other techniques. This was probably because sheep immunoglobulins bind protein A poorly.

Capsule phase variants were isolated from serotype 8 and serotype 37 pneumococcal sorbarods. Sequence duplications within the essential capsule genes -cap8E (type 8) and tts (type 37) -were found to introduce frameshifts and generate acapsular phenotypes. Capsular revertants possessed wild-type cap8E and tts genes, indicating the precise excision of these duplications. Reversion frequencies (OFF-ON) fit a linear relationship between log(frequency of reversion) and log(length of duplication), previously found for serotype three pneumococci [Waite, R. D., Struthers, J. K. & Dowson, C. G. (2001). Mol Microbiol 42, 1223Microbiol 42, -1232. This study provides evidence that capsule phase variation can occur in pneumococcal serotypes with either simple (one to three genes) or complex capsule-encoding loci (12 genes). Given the key role of CapE (the first monosaccharide transferase) in other clinically important pneumococci, such as serotypes 14 and 19F with complex capsular loci, the observed duplication within cap8E suggests that capsule phase variation could be controlled by tandem sequence duplication in capE homologues in other pneumococcal serotypes that construct their capsules through polymerization of lipid-linked intermediates. INTRODUCTIONThe worldwide increase in antibiotic resistance (Butler et al., 1998) and suboptimal efficacy of existing capsular vaccines (Musher, 1992) has resulted in Streptococcus pneumoniae (the pneumococcus) remaining a major cause of human mortality and morbidity. The pneumococcus is the cause of serious diseases such as pneumonia, bacteraemia, sepsis and meningitis, and less-severe infections such as otitis media and sinusitis (Paton et al., 1993). The groups most at risk from pneumococcal disease are young children, the elderly and the immunocompromised, such as AIDS patients (World Health Organization, 1999). Asymptomatic carriage, with bacteria adhering to the throat or nasopharynx, maintains a large reservoir of pneumococci for human infection (Appelbaum et al., 1996;Austrian, 1986;Sung et al., 1995).There are currently 90 recorded antigenically distinct capsular serotypes of pneumococci (Henrichsen, 1995). These are composed of repeating polysaccharide units (van Dam et al., 1990) and help confer resistance to complement-mediated opsonophagocytosis (Moxon & Kroll, 1990); hence, they play a key role in survival during systemic infection in vivo and in animal models (Watson & Musher, 1990). Recently, it has been reported that the presence of pneumococcal capsule is also important for murine carriage (Magee & Yother, 2001). Although the majority of pneumococci from clinical isolates are capsulate, a previous population genetic study of pneumococcal carriage identified two pneumococcal isolates from pernasal swabs of children and one from a pernasal swab of a female human immunodeficiency virus-positive patient that were non-serotypable (Muller-Graf et al., 1999). In addition, acapsulate pneumococci have been associated with outbreaks of conjunctivitis (Ertugrul et al., 1997).Th...