<i>Cupriavidus pinatubonensis</i> AEO106 deals with copper-induced oxidative stress before engaging in biodegradation of the herbicide 4-chloro-2-methylphenoxyacetic acid

Abstract Background Microbial degradation of phenoxy acid (PA) herbicides in agricultural soils is important to minimize herbicide leaching to groundwater reservoirs. Degradation may, however, be hampered by exposure of the degrader bacteria to toxic metals as copper (Cu) in the soil environment. Exposure to Cu leads to accumulation of intracellular reactive oxygen species (ROS) in some bacteria, but it is not known how Cu-derived ROS and an ensuing oxidative stress affect the degradation of PA herbicides. Based on the previously proposed paradigm that bacteria deal with environmental stress before they engage in biodegradation, we studied how the degradation of the PA herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) by the model PA degrader Cupriavidus pinatubonensis AEO106 was affected by Cu exposure. Results Exposure of C. pinatubonensis in batch culture to sublethal concentrations of Cu increased accumulation of ROS measured by the oxidant sensing probe 2,7-dichlorodihydrofluorescein diacetate and flow cytometry, and resulted in upregulation of a gene encoding a protein belong to the Ohr/OsmC protein family. The ohr/osmC gene was also highly induced by H2O2 exposure suggesting that it is involved in the oxidative stress response in C. pinatubonensis. The increased ROS accumulation and increased expression of the oxidative stress defense coincided with a delay in the catabolic performance, since both expression of the catabolic tfdA gene and MCPA mineralization were delayed compared to unexposed control cells. Conclusions The current study suggests that Cu-induced ROS accumulation in C. pinatubonensis activates a stress response involving the product of the ohr/osmC gene. Further, the stress response is launched before induction of the catabolic tfdA gene and mineralization occurs

The biosurfactant viscosin transiently stimulates n-hexadecane mineralization by a bacterial consortium

Pseudomonas produces powerful lipopeptide biosurfactants including viscosin, massetolide A, putisolvin, and amphisin, but their ability to stimulate alkane mineralization and their utility for bioremediation have received limited attention. The four Pseudomonas lipopeptides yielded emulsification indices on hexadecane of 20–31 % at 90 mg/l, which is comparable to values for the synthetic surfactant Tween 80. Viscosin was the optimal emulsifier and significantly stimulated n-hexadecane mineralization by diesel-degrading bacterial consortia but exclusively during the first 2 days of batch culture experiments. Growth of the consortia, as determined by OD(600) measurements and quantification of the alkB marker gene for alkane degradation, was arrested after the first day of the experiment. In contrast, the control consortia continued to grow and reached higher OD(600) values and higher alkB copy numbers during the next days. Due to the short-lived stimulation of n-hexadecane mineralization, the stability of viscosin was analyzed, and it was observed that added viscosin was degraded by the bacterial consortium during the first 2 days. Hence, viscosin has a potential as stimulator of alkane degradation, but its utility in bioremediation may be limited by its rapid degradation and growth-inhibiting properties

Nitrogen regulation of the <i>xyl</i> genes of <i>Pseudomonas putida</i> mt-2 propagates into a significant effect of nitrate on <i>m</i>-xylene mineralization in soil

The nitrogen species available in the growth medium are key factors determining expression of xyl genes for biodegradation of aromatic compounds by Pseudomonas putida. Nitrogen compounds are frequently amended to promote degradation at polluted sites, but it remains unknown how regulation observed in the test tube is propagated into actual catabolism of, e.g. m‐xylene in soil, the natural habitat of this bacterium. To address this issue, we have developed a test‐tube‐to‐soil model system that exposes the end‐effects of remediation practices influencing gene expression of P. putida mt‐2. We found that NO (3) (−) compared with NH (4) (+) had a stimulating effect on xyl gene expression in pure culture as well as in soil, and that this stimulation was translated into increased m‐xylene mineralization in soil. Furthermore, expression analysis of the nitrogen‐regulated genes amtB and gdhA allowed us to monitor nitrogen sensing status in both experimental systems. Hence, for nitrogen sources, regulatory patterns that emerge in soil reflect those observed in liquid cultures. The current study shows how distinct regulatory traits can lead to discrete environmental consequences; and it underpins that attempts to improve bioremediation by nitrogen amendment should integrate knowledge on their effects on growth and on catabolic gene regulation under natural conditions

Genetic rearrangements in <i>Pseudomonas amygdali</i> pathovar <i>aesculi </i>shape coronatine plasmids

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid

Molecular tools in rhizosphere microbiology—from single-cell to whole-community

International audienceIt is the aim of this chapter to present an overview of new, molecular tools that have been developed over recent years to study individual, single cells and composite, complex communities of microorganisms in the rhizosphere. We have carefully focused on culture-independent assays and selected methodologies that have already been or will soon be applicable for rhizosphere microbiology. Emphasis is placed on rhizosphere bacteria and the review first describes a number of the new methodologies developed for detection and localization of specific bacterial populations using modern electron and fluorescence microscopy combined with specific tagging techniques. First half of the chapter further comprises a thorough treatise of the recent development of reporter gene technology, i.e. using specific reporter bacteria to detect microscale distributions of rhizosphere compounds such as nutrients, metals and organic exudates or contaminants. Second half of the chapter devoted to microbial community analysis contains a thorough treatise of nucleotide- and PCR-based technologies to study composition and diversity of indigenous bacteria in the natural rhizosphere. Also included are the most recent developments of functional gene and gene expression analyses in the rhizosphere based on specific mRNA transcript or transcriptome analysis, proteome analysis and construction of metagenomic libraries