Chromate resistance and biofilm development in Pseudomonas alcaliphila 34: molecular bases

Hexavalent chromium [Cr(VI)] is considered an environmental priority pollutant for both its high dangerousness towards human health and its wide diffusion. In contrast, the trivalent form of chromium [Cr(III)] is much less toxic and insoluble. Hence, the basic process for chromium detoxification is the transformation of Cr(VI) to Cr(III). Bioremediation, exploiting microorganisms' ability to reduce Cr(VI) to Cr(III), represents a considerable alternative to traditional physic-chemical technologies, which are uneconomical, mainly for treatment of wide contaminated sites, and can create, in turn, environmental problems. A noticeable contribution to develop an efficient bioremediation approach can be provided by the use of microbial biofilm. Biofilms, the form commonly taken by microorganisms in environment, are able to contrast environmental stress (nutrient limitation, sudden pH changes, toxic compounds, etc.) and to detoxify contaminants more efficiently than microorganisms grown under planktonic conditions. Pseudomonas alcaliphila 34 is a chromate hyper-resistant and biofilm-producing bacterium, previously characterized in terms of hundreds of biochemical attributes and its chromate-reducing capability in the presence of different carbon/energy sources, and proposed for bioremediation processes. In this work Pseudomonas alcaliphila 34 was investigated in order to give a significant contribution to the development of an efficient biological system for the remediation of chromate contaminated soils. The general aim of the project was reached with the achievement of the following intermediate objectives: i. The development of a procedure, combining the Calgary Biofilm Device (MBEC device) and Phenotype MicroArray (PM) technology, for a wide-scale analysis of toxic chemicals susceptibility of biofilm and planktonic culture. ii. The sequencing, assembling and annotation of P. alcaliphila 34 genome. iii. The investigation of molecular bases implicated in chromate resistance and biofilm development in P. alcaliphila 34 strain by transcriptome analysis. The development of an integrated system combining two high-throughput technologies, MBEC device/PM, offered the possibility to obtain an extensive characterization of toxic compounds susceptibility of P. alcaliphila 34 biofilm and planktonic culture. The common assumption that biofilms are more tolerant than planktonic cells was confuted and was showed that cultures in the stationary phase were often more tolerant than biofilms in presence of the majority of chemicals used. Therefore, any conclusions regarding biofilm and planktonic culture resistance should take into account the growth phase of planktonic cultures and the nature of chemicals. Annotation of P. alcaliphila 34 genome allowed the identification of 4,983 protein-coding sequences and 61 tRNAs. Genome analysis indicated that P. alcaliphila 34 possesses a putative chrBACF operon that is involved in the high chromate resistance of the bacterium. Mercuric and arsenic resistance operons and many genes encoding putative multidrug resistance efflux systems were also identified on the genome. From temporal genomic profiling of P. alcaliphila 34 planktonic cultures exposed to an acute chromate stress the overexpression of genes involved in sulfur metabolism was highlighted, as well as the activation of oxidative stress response system and mechanism related to DNA repair. The chromate shock response of P. alcaliphila 34 was also characterized by the enhanced expression of genetic pathways relative to iron acquisition and metabolism, and the down-regulation of copper metabolism, suggesting a correlation between chromate exposure and the pathways of these two metals. The analysis of differentially expressed genes related to the early developing P. alcaliphila 34 biofilm revealed the induction of the pathway involved in flagellar motility and the down-regulation of a type IV pilus metabolic system. Interestingly, from the transcriptomic analysis of P. alcaliphila 34 response to an acute chromate challenge, the presence of pathways involved in biofilm formation has emerged. This observation indicates that biofilm formation may be a survival strategy to deal with chromate. In conclusion, obtained data have shown that : • the MBEC device/PM approach is a reliable, repeatable, accurate, and quick method to evaluate the effect of toxic chemicals (i.e., antibiotics, biocides, heavy metals) on metabolic activity of microbial biofilms; • the acquired know-how indicated that it is necessary to determine at what stage of growth the microorganism shows the best fitness in presence of a given pollutants to plan bioremediation processes; • the transcriptomic analysis provided insights into the molecular mechanisms related to biofilm development and chromate resistance. Furthermore, the obtained information may be used to design a biological system for bioremediation of chromate contaminated soils using biofilm of the highly Cr(VI)-resistant P. alcaliphila 34 strain.