The effect of toxic malachite green on the bacterial community in Antarctic soil and the physiology of malachite green-degrading Pseudomonas sp. MGO.

The effects of malachite green (MG) on the bacterial community in Antarctic soil were assessed. Culture-independent community analysis using 16S rRNA gene pyrosequencing showed that, in the presence of MG, the relative abundance of Pseudomonas dramatically increased from 2.2 % to 36.6 % (16.6-fold), and Pseudomonas became the predominant genus. The reduction in bacterial biodiversity was demonstrated by diversity indices and rarefaction curves. MG-degrading Pseudomonas sp. MGO was isolated from Antarctic soil. MG tolerance and decolorization activity were confirmed by growth, spectrophotometric, high-performance liquid chromatography, and thin-layer chromatography analyses in high MG concentrations. Our data showed that the decolorization process occurred via biodegradation, while biosorption also occurred after some time during the fed-batch decolorization process. Significant inductions in laccase, nicotinamide adenine dinucleotide-2,6 dichlorophenol indophenol reductase, and MG reductase activities suggested their involvement in the decolorization process. We also showed that the high tolerance of strain MGO to toxic MG might be mediated by upregulation of oxidative stress defense systems such as superoxide dismutase and protease. Collectively, these results demonstrated the response of the Antarctic soil bacterial community to MG and provided insight into the molecular mechanism of MG-tolerant Pseudomonas strains isolated from Antarctic soil.

Complexity of cell-cell interactions between Pseudomonas sp. AS1 and Acinetobacter oleivorans DR1: metabolic commensalism, biofilm formation and quorum quenching.

Acinetobacter oleivorans DR1 lacks an upper pathway for naphthalene degradation and cannot grow using naphthalene as sole carbon source; however, it is capable of growing under naphthalene-amended conditions in the presence of naphthalene-degrading Pseudomonas sp. AS1. ¹H-NMR spectroscopy, high-performance liquid chromatography and gene expression analyses showed that salicylate is a major secreted metabolic intermediate during naphthalene degradation by strain AS1 and that, in turn, it supports the growth of strain DR1. Interspecies biofilm formation, monitored using confocal laser scanning microscopy and microtiter assays, demonstrated that biofilm formation by strain AS1 increased dramatically in the presence of strain DR1 because of the exopolysaccharides generated by the latter. Furthermore, the metabolic commensal interaction of the two strains altered the initial attachment behavior of strain DR1 during biofilm formation. When this strain was cultivated alone under naphthalene-amended conditions, the cells immediately attached to the surface, probably due to the absence of usable substrates, whereas similar behavior was not observed in the mixed culture. This interspecies cell-cell interaction became more complex due to quenching of the quorum-sensing signal of strain DR1 by strain AS1. These complex metabolic and physiological interactions observed in mixed cultures suggest that interspecies interaction is more complicated than previously surmised.

Detection of genetically modified microorganisms in soil using the most-probable-number method with multiplex PCR and DNA dot blot.

The principal objective of this study was to detect genetically modified microorganisms (GMMs) that might be accidentally released into the environment from laboratories. Two methods [plate counting and most-probable-number (MPN)] coupled with either multiplex PCR or DNA dot blots were compared using genetically modified Escherichia coli, Pseudomonas putida, and Acinetobacter oleivorans harboring an antibiotic-resistance gene with additional gfp and lacZ genes as markers. Alignments of sequences collected from databases using the Perl scripting language (Perl API) and from denaturing gradient gel electrophoresis analysis revealed that the gfp, lacZ and antibiotic-resistance genes (kanamycin, tetracycline, and ampicillin) in GMMs differed from the counterpart genes in many sequenced genomes and in soil DNA. Thus, specific multiplex PCR primer sets for detection of plasmid-based gfp and lacZ antibiotic-resistance genes could be generated. In the plate counting method, many antibiotic-resistant bacteria from a soil microcosm grew as colonies on antibiotic-containing agar plates. The multiplex PCR verification of randomly selected antibiotic-resistant colonies with specific primers proved ineffective. The MPN-multiplex PCR method and antibiotic-resistant phenotype could be successfully used to detect GMMs, although this method is quite laborious. The MPN-DNA dot blot method screened more cells at a time in a microtiter plate containing the corresponding antibiotics, and was shown to be a more efficient method for the detection of GMMs in soil using specific probes in terms of labor and accuracy.

Molecular characterization of the extracellular matrix in a Pseudomonas putida dsbA mutant: implications for acidic stress defense and plant growth promotion.

The Pseudomonas putida dsbA mutant displays enhanced extracellular matrix production, which promotes biofilm formation. Here we confirmed that the extracellular matrix consists of both capsular polysaccharides and exopolysaccharides. However, the carbohydrate composition of the P. putida dsbA mutant matrix was shown to be similar to that of the wild-type strain. Our data indicate that the overproduced matrix itself, rather than alterations in the matrix composition, promotes biofilm formation in the P. putida dsbA mutant. Moreover, the mutant was more sensitive than the wild-type to alkali stress (pH 9.0 to 10.0), but not to acidic stress (pH 5.0). Interestingly, acidic stress stimulated polysaccharide production and pellicle formation, while these changes were recovered to the level of the wild-type under alkali conditions in the P. putida dsbA mutant. Enhanced biofilm formation of the dsbA mutant increased the efficiency with which P. putida attached to tomato and pepper seeds, which have longer germinated roots than the wild-type strain. This phenomenon could not be observed in the cucumber plant, which suggests that each plant seed has a different effect on the attachment of P. putida. Interestingly, this increased attachment to plant seeds resulted in more root colonization and plant growth promotion. The findings of this study suggested that the overproduced extracellular matrix caused by deletion of the dsbA gene could have pleiotropic effect on P. putida phenotypes, including acidic stress defense and plant growth promotion.