By TnS mutagenesis of Rhizobium loti PN184 (NZP2037 str-1) and selection for nonfluorescence of colonies on Calcofluor agar, eight independently generated exopolysaccharide (EPS) mutants (three smooth and five rough) were isolated. The parent strain, PN184, was found to produce an acidic EPS. This EPS was produced, with reduced 0 acetylation, by the smooth EPS mutants but not by the rough EPS mutants. Lipopolysaccharide was isolated from all mutants and was identical to that of PN184 as defined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. All (26) is a group of broad-host-range (26,41,43), fast-growing rhizobia, subdivided into two broad groups differentiated by their capacity to form effective (Nod' Fix') or ineffective (Nod' Fix-) nodules on different Lotus species (42, 44). The first group, represented by type strain NZP2037, forms effective nodules on a wide range of Lotus species, while the second group, represented by type strain NZP2213, forms effective nodules only on Lotus tenuis and Lotus corniculatus var. cree and ineffective nodules on other Lotus species examined (42,43). In addition to Lotus species, which form determinate nodules, Pankhurst et al. (43) have shown that R. loti can also nodulate legumes such as Leucaena leucocephala (NZP2037, effective; NZP2213, ineffective), which form indeterminate nodules, thus allowing specific mutations to be tested on hosts differing in nodule morphology.The extracellular and surface polysaccharides produced by Rhizobium species constitute a complex macromolecular interface between the bacterial cell and its environment. Work to date has shown that several of these polysaccharides are involved in the complex series of interactions leading to the establishment of an effective Rhizobiumlegume symbiosis. These include the acidic Calcofluorbinding exopolysaccharide (EPS) of Rhizobium meliloti (14, 34), acidic EPSs or capsular polysaccharides from other Rhizobium species (3,5,6,8), a cryptically synthesized EPS of R. meliloti (19, 58), lipopolysaccharide (LPS) (40), ,B-1,2-glucan (18), and a peptidoglycan-bound polysaccharide (27).The relationship between polysaccharide requirement and effective nodulation is, however, far from simple. For example, current evidence suggests that acidic EPS is required for the establishment of effective nodules on indeterminate * Corresponding author. nodulating legumes but not on determinate nodulating legumes. Hence, it has been shown that acidic EPS is required for the following indeterminate-type symbioses: R. meliloti and alfalfa (14, 34), Rhizobium leguminosarum bv. viciae and peas (3), R. leguminosarum bv. trifolii and clover (5), and Rhizobium sp. strain NGR234 and L. leucocephala (6). In contrast, acidic EPS is not required for the following determinate-type symbioses: Rhizobiumfredii and soybeans (28) and R. leguminosarum bv. phaseoli and Phaseolus beans (3,8). However, acidic EPS is apparently required for the effective nodulation of siratro by Rhizobium sp. strain NGR234 (6, 11). In addition, acidic ...

The unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids. Recent work in several laboratories has established that the Mycobacterium tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus plays a major role in PDIM biosynthesis and secretion and that PDIM is required for virulence. Here we describe two independent transposon mutants (WAg533 and WAg537) of Tuberculosis, caused by closely related members of the Mycobacterium tuberculosis complex, continues to have a major impact on human and animal health worldwide and is responsible for the death of approximately two million people each year, primarily in developing nations (14). Mycobacterium bovis, the pathogen responsible for bovine tuberculosis, is a broad-host-range member of the M. tuberculosis complex, and its transmission to humans is probably responsible for some 5% of human tuberculosis deaths (15). The current tuberculosis vaccine, M. bovis bacillus Calmette-Guérin (BCG), has shown highly variable efficacy, and a significantly better vaccine is urgently required.In New Zealand, traditional test and slaughter approaches to eradication of bovine tuberculosis from domestic livestock have been frustrated by the presence of introduced wildlife, particularly the Australian brushtail possum (Trichosurus vulpecula), which maintains a reservoir of infection (23). Extensive wildlife culling operations over many years have failed to eliminate infected possums from many parts of the country, and vaccination of wildlife against tuberculosis is being investigated. Any vaccine developed for tuberculosis control in the New Zealand environment must be compatible with largescale vaccination of animals, and this requirement has directed research towards the development of rationally attenuated strains of M. bovis with vaccine efficacy (9,11,12). Moredetailed investigation of the attenuation of some of these strains (10, 38) is also contributing to our understanding of the molecular determinants required for tuberculosis pathogenesis.Among the known determinants required for virulence in pathogenic mycobacteria are complex lipid components of the mycobacterial cell wall that act as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids produced by members of the M. tuberculosis complex and closely related species (17). PDIMs are built upon polyketide scaffolds and comprise multimethyl-branched long-chain mycocerosic acids diesterified with long-chain phthiocerol or phthiodiolone diols (28) (Fig. 1). Additional PDIM variants include the phenol-and glycosylphenol-PDIMs (Fig. 1). Recent work in several laboratories has established that proteins encoded by genes at the M. tuberculosis fadD26-mmpL7 locus (fa...

A better tuberculosis vaccine is urgently required to control the continuing epidemic. Molecular techniques are now available to produce a better live vaccine than BCG by producing avirulent strains of the Mycobacterium tuberculosis complex with known gene deletions. In this study, 1000 illegitimate recombinants of Mycobacterium bovis were produced by illegitimate recombination with fragments of mycobacterial DNA containing a kanamycin resistance gene. Eight recombinant strains were selected on the basis of their inability to grow when stationary-phase cultures were inoculated into minimal medium. Five of these recombinants were found to be avirulent when inoculated into guinea pigs. Two of the avirulent recombinants produced vaccine efficacy comparable to BCG against an aerosol challenge in guinea pigs with M. bovis. One of these recombinants had an inactivated glnA2 gene encoding a putative glutamine synthetase. Transcriptional analysis showed that inactivation of glnA2 did not affect expression of the downstream glnE gene. The other recombinant had a block of 12 genes deleted, including the sigma factor gene sigG. Two avirulent recombinants with an inactivated pckA gene, encoding phosphoenolpyruvate carboxykinase which catalyses the first step of gluconeogenesis, induced poor protection against tuberculosis. It is clear that live avirulent strains of the M. tuberculosis complex vary widely in their ability as vaccines to protect against tuberculosis. Improved models may be required to more clearly determine the difference in protective effect between BCG and potential new tuberculosis vaccines.

Campylobacter jejuni ST-474 is the most important human enteric pathogen in New Zealand, and yet this genotype is rarely found elsewhere in the world. Insight into the evolution of this organism was gained by a whole genome comparison of two ST-474, flaA SVR-14 isolates and other available C. jejuni isolates and genomes. The two isolates were collected from different sources, human (H22082) and retail poultry (P110b), at the same time and from the same geographical location. Solexa sequencing of each isolate resulted in 1.659 Mb (H22082) and 1.656 Mb (P110b) of assembled sequences within 28 (H22082) and 29 (P110b) contigs. We analysed 1502 genes for which we had sequences within both ST-474 isolates and within at least one of 11 C. jejuni reference genomes. Although 94.5% of genes were identical between the two ST-474 isolates, we identified 83 genes that differed by at least one nucleotide, including 55 genes with non-synonymous substitutions. These covered 101 kb and contained 672 point differences. We inferred that 22 (3.3%) of these differences were due to mutation and 650 (96.7%) were imported via recombination. Our analysis estimated 38 recombinant breakpoints within these 83 genes, which correspond to recombination events affecting at least 19 loci regions and gives a tract length estimate of 2 kb. This includes a 12 kb region displaying non-homologous recombination in one of the ST-474 genomes, with the insertion of two genes, including ykgC, a putative oxidoreductase, and a conserved hypothetical protein of unknown function. Furthermore, our analysis indicates that the source of this recombined DNA is more likely to have come from C. jejuni strains that are more closely related to ST-474. This suggests that the rates of recombination and mutation are similar in order of magnitude, but that recombination has been much more important for generating divergence between the two ST-474 isolates.

A cell wall elicitor preparation from the needle pathogen Dothistroma pini was used to induce defence responses in Pinus radiata cell suspension cultures. Addition of elicitor to cell suspensions induced a rapid, transient burst in the accumulation of H2O2, with maximal response between 20 and 40 min post-elicitation. The protein kinase inhibitors staurosporine and K252a inhibited H2O2 accumulation showing that protein phosphorylation is required in the signal transduction pathway leading to the oxidative burst. Over a more extended time period elicitation of suspension cells lead to the activation of phenylpropanoid biosynthesis. The activity of phenylalanine ammonia-lyase (EC 4.3.1.5), the first enzyme in the phenylpropanoid biosynthetic pathway, increased 8-fold following elicitation with maximal activity 36 h post-elicitation. The activity of cinnamyl alcohol dehydrogenase (EC 1.1.1.195), an enzyme involved in lignin biosynthesis, increased 2.5-fold with maximal response 48–72 h post-elicitation. Thioglycolic acid extractable material increased 2-fold with maximal response 48–72 h post-elicitation, and phloroglucinol–HCl-positive material increased over the same time course. These data show that P. radiata suspension cells are an excellent model system for investigating the biochemistry and enzymology of pathogen defence responses in P. radiata.

A repeated cross-sectional study was conducted to determine the prevalence of Campylobacter spp. and the population structure of C. jejuni in European starlings and ducks cohabiting multiple public access sites in an urban area of New Zealand. The country's geographical isolation and relatively recent history of introduction of wild bird species, including the European starling and mallard duck, create an ideal setting to explore the impact of geographical separation on the population biology of C. jejuni, as well as potential public health implications. A total of 716 starling and 720 duck fecal samples were collected and screened for C. jejuni over a 12 month period. This study combined molecular genotyping, population genetics and epidemiological modeling and revealed: (i) higher Campylobacter spp. isolation in starlings (46%) compared with ducks (30%), but similar isolation of C. jejuni in ducks (23%) and starlings (21%), (ii) significant associations between the isolation of Campylobacter spp. and host species, sampling location and time of year using logistic regression, (iii) evidence of population differentiation, as indicated by FST, and host-genotype association with clonal complexes CC ST-177 and CC ST-682 associated with starlings, and clonal complexes CC ST-1034, CC ST-692, and CC ST-1332 associated with ducks, and (iv) greater genetic diversity and genotype richness in ducks compared with starlings. These findings provide evidence that host-associated genotypes, such as the starling-associated ST-177 and ST-682, represent lineages that were introduced with the host species in the 19th century. The isolation of sequence types associated with human disease in New Zealand indicate that wild ducks and starlings need to be considered as a potential public health risk, particularly in urban areas.We applied molecular epidemiology and population genetics to obtain insights in to the population structure, host-species relationships, gene flow and evolution of Campylobacter jejuni in urban ducks and starlings.

Healthy bobby calves are an asymptomatic reservoir of E. coli O157 in New Zealand and may represent an important source of infection for humans. Carriage was not associated with concentration of IgG in serum, carcass weight or gender.

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.Tuberculosis exacts enormous human suffering and death on a global basis and is also a widespread cause of animal morbidity and mortality. The primary human pathogen is Mycobacterium tuberculosis, although in many regions a significant amount of human disease results from infection with the very closely related organisms Mycobacterium africanum and the major animal pathogen Mycobacterium bovis. Together with a few less important species, these organisms are collectively referred to as the M. tuberculosis complex. The high degree of similarity in the pathogenesis of disease caused by these pathogens and the recent findings from genomic studies that the vast majority of genes within these species are identical or nearly so (16,22) clearly indicate that the majority of their virulence genes are shared in common. There is particular interest in discovering the identity of these genes, so that new strategies including improved vaccines and antibacterials can be developed for combating tuberculosis.Over the last 15 years, there has been continual development of molecular genetic techniques that can be applied to the M. tuberculosis complex (2). This enabled the discovery of individual tuberculosis virulence genes from 1995 onwards (7, 38) and led to the application over the last 5 years of a number of global techniques for identifying large numbers of genes that are essential for virulence or whose expression is altered during different stages of infection and disease. Among these global techniques, signature tag mutagenesis (18) is widely recognized as a powerful tool for investigating pathogenic organisms because of its ability to directly identify mutants that have reduced virulence (24). This identific...