Challenges and opportunities for overcoming dog use in agrochemical evaluation and registration.

Progress in developing new tools, assays, and approaches to assess human hazard and health risk provides an opportunity to re-evaluate the necessity of dog studies for the safety evaluation of agrochemicals. A workshop was held where partic­ipants discussed the strengths and limitations of past use of dogs for pesticide evaluations and registrations. Opportunities were identified to support alternative approaches to answer human safety questions without performing the required 90-day dog study. Development of a decision tree for determining when the dog study might not be necessary to inform pesticide safety and risk assessment was proposed. Such a process will require global regulatory authority participation to lead to its acceptance. The identification of unique effects in dogs that are not identified in rodents will need further evaluation and determination of their relevance to humans. The establishment of in vitro and in silico approaches that can provide critical data on relative species sensitivity and human relevance will be an important tool to advance the decision process. Promising novel tools including in vitro comparative metabolism studies, in silico models, and high-throughput assays able to identify metabolites and mechanisms of action leading to development of adverse outcome pathways will need further development. To replace or eliminate the 90-day dog study, a collaborative, multidisciplinary, international effort that transcends organi­zations and regulatory agencies will be needed in order to develop guidance on when the study would not be necessary for human safety and risk assessment.

Identification and characterization of the intracellular poly-3-hydroxybutyrate depolymerase enzyme PhaZ of Sinorhizobium meliloti.

BACKGROUND: S. meliloti forms indeterminate nodules on the roots of its host plant alfalfa (Medicago sativa). Bacteroids of indeterminate nodules are terminally differentiated and, unlike their non-terminally differentiated counterparts in determinate nodules, do not accumulate large quantities of Poly-3-hydroxybutyrate (PHB) during symbiosis. PhaZ is in intracellular PHB depolymerase; it represents the first enzyme in the degradative arm of the PHB cycle in S. meliloti and is the only enzyme in this half of the PHB cycle that remains uncharacterized. RESULTS: The S. meliloti phaZ gene was identified by in silico analysis, the ORF was cloned, and a S. meliloti phaZ mutant was constructed. This mutant exhibited increased PHB accumulation during free-living growth, even when grown under non-PHB-inducing conditions. The phaZ mutant demonstrated no reduction in symbiotic capacity; interestingly, analysis of the bacteroids showed that this mutant also accumulated PHB during symbiosis. This mutant also exhibited a decreased capacity to tolerate long-term carbon starvation, comparable to that of other PHB cycle mutants. In contrast to other PHB cycle mutants, the S. meliloti phaZ mutant did not exhibit any decrease in rhizosphere competitiveness; however, this mutant did exhibit a significant increase in succinoglycan biosynthesis. CONCLUSIONS: S. meliloti bacteroids retain the capacity to synthesize PHB during symbiosis; interestingly, accumulation does not occur at the expense of symbiotic performance. phaZ mutants are not compromised in their capacity to compete for nodulation in the rhizosphere, perhaps due to increased succinoglycan production resulting from upregulation of the succinoglycan biosynthetic pathway. The reduced survival capacity of free-living cells unable to access their accumulated stores of PHB suggests that PHB is a crucial metabolite under adverse conditions.

Influence of the poly-3-hydroxybutyrate (PHB) granule-associated proteins (PhaP1 and PhaP2) on PHB accumulation and symbiotic nitrogen fixation in Sinorhizobium meliloti Rm1021.

Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.

Methods for the Isolation of Genes Encoding Novel PHA Metabolism Enzymes from Complex Microbial Communities.

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bio-plastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti and Pseudomonas putida, allows the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates the functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Role of a conserved membrane glycine residue in a dicarboxylate transporter from Sinorhizobium meliloti.

Nitrogen-fixing rhizobial bacteroids import dicarboxylates by using the DctA transporter. G114 of DctA is highly conserved. A G114D mutant is inactive, but DctA with a small amino acid (G114A) or a helix disrupter (G114P) retains significant activity. G114 probably interacts with other membrane helices in stabilizing a substrate-binding pocket.

The role of PHB metabolism in the symbiosis of rhizobia with legumes.

The carbon storage polymer poly-beta-hydroxybutyrate (PHB) is a potential biodegradable alternative to plastics, which plays a key role in the cellular metabolism of many bacterial species. Most species of rhizobia synthesize PHB but not all species accumulate it during symbiosis with legumes; the reason for this remains unclear, although it was recently shown that a metabolic mutant of a nonaccumulating species retains the capacity to store PHB in symbiosis. Although the precise roles of PHB metabolism in these bacteria during infection, nodulation, and nitrogen fixation are not determined, the elucidation of these roles will influence our understanding of the metabolic nature of the symbiotic relationship. This review explores the progress that was made in determining the biochemistry and genetics of PHB metabolism. This includes the elucidation of the PHB cycle, variations in PHB metabolism among rhizobial species, and the implications of these variations, while proposing a model for the role of PHB metabolism and storage in symbiosis.