Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites

Abstract

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.

FIG. 1.

Cluster analysis of genomic fingerprint patterns of 29 TPH-degrading strains of P. citronellolis. The genomic fingerprints were generated by PCR amplification of the whole-cell suspension with the Rep (ERIC, REP, and BOXAIR) primer sets. The UPGMA algorithm was applied to the similarity matrix with at and above mean Jaccard coefficient (standard deviation) value of 75%. The 29 strains were delineated into 12 genotypic groups.

FIG. 2.

REP-PCR genomic fingerprints of the different Pseudomonas species. Lane M is size standards (100-bp ladder). Lane 1, Pseudomonas putida MTCC 978. Lane 2, Pseudomonas citronellolis MTCC 1191. Lane 3, Pseudomonas aeruginosa MTCC 1034. Lane 4, Pseudomonas aeruginosa MTCC 2642.

FIG. 3.

PCR-based ribotype patterns of 29 strains of P. citronellolis. The gel profiles have been arranged so that in general strains of similar ribotype patterns are grouped together. Lane M contained an external size standard, HindIII-digested λ DNA, and lanes 2 to 30 are P. citronellolis strains TERIDB2, TERIDB3, TERIDB4, TERIDB5, TERIDB6, TERIDB7, TERIDB8, TERIDB9, TERIDB10, TERIDB11, TERIDB12, TERIDB13, TERIDB14, TERIDB15, TERIDB16, TERIDB17, TERIDB18, TERIDB19, TERIDB20, TERIDB21, TERIDB22, TERIDB23, TERIDB24, TERIDB25, TERIDB26, TERIDB27, TERIDB28, TERIDB29, and TERIDB30, respectively.