Primer design to assess bacterial degradation of glyphosate and other phosphonates.

Phosphonates are organic phosphorous (P) compounds frequently detected in the environment due to a very stable CP bond that render them relatively recalcitrant. Glyphosate [N-phosphonomethyl glycine] is the most widely used and best-known synthetic phosphonate, and one of the most concerning herbicides in the world today. Microbial degradation of glyphosate and organophosphonates in general, is the main dissipation mechanism operating in most environments. One microbial metabolic pathway in this process is the CP lyase pathway, entailing an enzymatic complex encoded by about 14 genes (the Phn operon). Our goal was to develop a quantitative polymerase chain reaction (qPCR) assay for a key enzyme, the CP lyase that breaks down the CP bond, via quantification of the codifying phnJ gene. The primers designed in this study fulfill the requirements for a successful qPCR assay, with high efficiency and sensitivity, as well as specific detection of the target sequence in a wide range of taxonomic groups. This is, to our knowledge, the first report of primers designed to target phnJ in both pure cultures and metagenomic DNA from different environmental sources. Direct quantification of phnJ may be a cost-effective proxy to determine glyphosate degradation potential in different matrixes.

A most effective method for selecting a broad range of short and medium-chain-length polyhidroxyalcanoate producing microorganisms

A molecular approach was used for selecting polyhydroxyalcanoate (PHA)-accumulating potential Gram-negative bacteria from different genera by colony polymerase chain reaction (PCR). Three degenerate primers were designed for amplifying a fragment from PHA synthase gene (phaC) (Class I), phaC1 and phaC2 (Class II) genes for detecting PHA-producing bacteria. Thirty-four out of 55 bacterial strains from the old collection selected using Sudan black B staining were phaC+. PCR was used for directly selecting 35 new collection bacterial strains; these strains were phaC+ and their ability to produce PHA was confirmed by Sudan black B staining. Four specific primers were designed on genes of Class II PHA biosynthesis operon. These primers were used for evaluating 9 strains from the old phaC+ collection; 6 showed Class II PHA synthase organisation. 34 from the old and new bacterial isolation were characterised by 16S ribosomal gene (16S rDNA) gene partial sequencing. The tool proposed here can be used for better directing PHA production based on PHA biosynthesis genes and bacterial genera. Class I or II phaC genes were detected in 9 different genera and were able to infer the type of polymer produced.