Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C. necator JMP134 could be isolated after soil enrichment. In native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5alpha (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.
Plant peptidases are involved in protein degradation in the rumen of grazing animals, but little is known about the variation in their activity under anaerobic conditions at 39°C. We investigated the activity of endogenous peptidases in 342 accessions of grass and legume forages and the molecular weight and class of peptidases in a subsample of them. Proteolytic activity was measured by in vitro incubation of tissue over a gelatin substrate. Molecular weight and class of peptidases were assessed by zymograms. A wide range in proteolytic activity index (PAI) was detected (mean PAI = 0.35, ranged from 0.00 to 1.30). More than 88% of the observed variance was due to differences in genus, species, and cultivar. Most of the species had one main peptidase, and their molecular weights ranged from 54 to 130 kDa. In the six species tested, proteases present corresponded to the serine class. The wide variation in proteolysis observed and the predominant occurrence of one peptidase of the same enzyme class support the idea that a reduction of protease activity in forages can be achieved by genetic improvement.
We have mutagenized a clinical strain of Salmonella enterica sv. typhi with mini-transposon Tn10dTet (T-POP) to obtain conditional lethal (tetracycline-dependent) mutants with T-POP insertions upstream of essential genes. Generalized transducing phage P22 was used to introduce T-POP from a S. typhimurium donor into a S. typhi recipient. Chromosomal DNA was purified from the mutagenized donor strains, fragmented, and then electroporated into S. typhi to backcross the original T-POP insertions. Four tetracycline-dependent mutants with two distinct terminal phenotypes were found among 1700 mutants with T-POP insertions. When grown in the absence of tetracycline, two of the four tetracycline-dependent mutants arrest at a late stage in the cell cycle, can be rescued by outgrowth in media with tetracycline, and define a reversible checkpoint late in the cell cycle. One of these insertions creates an operon fusion with a gene, yqgF, that is conserved among gram-negative bacteria and likely encodes an essential Holliday junction resolvase. T-POP insertions can be used not only to identify essential S. typhi genes but also to reveal novel phenotypes resulting from the depletion of their products. Thus, the construction of S. typhi mutants with deletions generalized transducing phage for S. typhi has yet to be of nonessential genes and assays of their phenotypes in found. Although P22 can adsorb to S. typhi and inject human cells will identify many new virulence determiits DNA into a S. typhi recipient, P22 cannot develop nants. However, because one can make deletions only lytically in S. typhi. Therefore, P22 cannot be used to of nonessential genes, this PCR-based method for gene disruption does not yield information about the roles that essential genes play in virulence.
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