Use of gene probes to aid in recovery and identification of functionally dominant 2,4-dichlorophenoxyacetic acid-degrading populations in soil

• Published in: Applied and Environmental Microbiology 1994-04, Vol.60 (4), p.1116-1120
• Main Authors: Ka, J.O, Holben, W.E, Tiedje, J.M
• Format: Article
• Language: English
• Subjects: 2,4-D; 2,4-Dichlorophenoxyacetic Acid - metabolism; ADN; BACTERIA; BIODEGRADACION; BIODEGRADATION; Biodegradation, Environmental; Biological and medical sciences; Biology; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Blotting, Southern; DNA, Bacterial - genetics; Environment; FLORA DEL SUELO; FLORE DU SOL; Fundamental and applied biological sciences. Psychology; GENE; GENES; Genes, Bacterial; Gram-Negative Aerobic Bacteria - genetics; Gram-Negative Aerobic Bacteria - isolation & purification; Gram-Negative Aerobic Bacteria - metabolism; Herbicides; HIBRIDACION DE ADN; HYBRIDATION D'ADN; Isolation and description; MICHIGAN; Mission oriented research; Oligonucleotide Probes; Plasmids - genetics; PSEUDOMONACEAE; Pseudomonas - genetics; Pseudomonas - isolation & purification; Pseudomonas - metabolism; SASKATCHEWAN; Soil Microbiology; Soils; TECHNIQUE ANALYTIQUE; TECNICAS ANALITICAS
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.60.4.1116-1120.1994

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to soils in microcosms, and degradation was monitored after each of five repeated additions. Total DNAs were isolated from soil bacterial communities after each 2,4-D treatment. The DNA samples were analyzed on slot blots and Southern blots by using a tfdA gene probe subcloned from plasmid pJP4 and a Spa probe derived from a different 2,4-D-degrading isolate, a Sphingomonas paucimobilis strain. 2,4-D applied to soil was quickly degraded by indigenous microbial populations. As determined by slot blot analyses of DNA from a Michigan soil, the increase in hybridization signal in response to 2,4-D treatments was greater with the Spa probe than with the tfdA probe. In contrast, the DNA from a Saskatchewan soil exhibited an increase in hybridization signal with the tfdA probe. This indicated that a population with 2,4-D-degradative gene sequences different from the tfd4 gene sequence was dominant in the Michigan site, but not in the Saskatchewan site. A Southern blot analysis of DNA from Michigan soil showed that the dominant 2,4-D-degrading population was S. paucimobilis 1443. A less dominant 2,4-D-degrading population was detected with the tfdA probe; further analysis revealed that this population was a Pseudomonas pickettii 712. These gene probe analyses revealed that an important population carrying out 2,4-D degradation was not detected when the canonical tfdA gene probe was used. After a series of new strains were isolated, we identified a probe to detect and identify the dominant members of this new group