Dataset Information

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

ABSTRACT: Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR.

SUBMITTER: de Lima-Morales D

PROVIDER: S-EPMC4702652 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

de Lima-Morales Daiana D Chaves-Moreno Diego D Wos-Oxley Melissa L ML Jáuregui Ruy R Vilchez-Vargas Ramiro R Pieper Dietmar H DH

Applied and environmental microbiology 20151016 1

Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydrox ...[more]

PMID: 26475106

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Project description:Strain inoculation (bioaugmentation) is a potentially useful technology to provide microbiomes with new functionalities. However, there is limited understanding of the genetic factors contributing to successful establishment of inoculants. This work aimed to characterize the genes implicated in proliferation of the monoaromatic compound-degrading Pseudomonas veronii 1YdBTEX2 in nonsterile polluted soils. We generated two independent mutant libraries by random minitransposon-delivered marker insertion followed by deep sequencing (Tn-seq) with a total of 5.0 × 105 unique insertions. Libraries were grown in multiple successive cycles for up to 50 generations either in batch liquid medium or in two types of soil microcosms with different resident microbial content (sand or silt) in the presence of toluene. Analysis of gene insertion abundances at different time points (passed generations of metapopulation growth), in comparison to proportions at start and to in silico generated randomized insertion distributions, allowed to define ~800 essential genes common to both libraries and ~2,700 genes with conditional fitness effects in either liquid or soil (195 of which resulted in fitness gain). Conditional fitness genes largely overlapped among all growth conditions but affected approximately twice as many functions in liquid than in soil. This indicates soil to be a more promiscuous environment for mutant growth, probably because of additional nutrient availability. Commonly depleted genes covered a wide range of biological functions and metabolic pathways, such as inorganic ion transport, fatty acid metabolism, amino acid biosynthesis, or nucleotide and cofactor metabolism. Only sparse gene sets were uncovered whose insertion caused fitness decrease exclusive for soils, which were different between silt and sand. Despite detectable higher resident bacteria and potential protist predatory counts in silt, we were, therefore, unable to detect any immediately obvious candidate genes affecting P. veronii biological competitiveness. In contrast to liquid growth conditions, mutants inactivating flagella biosynthesis and motility consistently gained strong fitness advantage in soils and displayed higher growth rates than wild type. In conclusion, although many gene functions were found to be important for growth in soils, most of these are not specific as they affect growth in liquid minimal medium more in general. This indicates that P. veronii does not need major metabolic reprogramming for proliferation in soil with accessible carbon and generally favorable growth conditions. IMPORTANCE Restoring damaged microbiomes is still a formidable challenge. Classical widely adopted approaches consist of augmenting communities with pure or mixed cultures in the hope that these display their intended selected properties under in situ conditions. Ecological theory, however, dictates that introduction of a nonresident microbe is unlikely to lead to its successful proliferation in a foreign system such as a soil microbiome. In an effort to study this systematically, we used random transposon insertion scanning to identify genes and possibly, metabolic subsystems, that are crucial for growth and survival of a bacterial inoculant (Pseudomonas veronii) for targeted degradation of monoaromatic compounds in contaminated nonsterile soils. Our results indicate that although many gene functions are important for proliferation in soil, they are general factors for growth and not exclusive for soil. In other words, P. veronii is a generalist that is not a priori hindered by the soil for its proliferation and would make a good bioaugmentation candidate.

Project description:BackgroundBioaugmentation aims to use the capacities of specific bacterial strains inoculated into sites to enhance pollutant biodegradation. Bioaugmentation results have been mixed, which has been attributed to poor inoculant growth and survival in the field, and, consequently, moderate catalytic performance. However, our understanding of biodegradation activity mostly comes from experiments conducted under laboratory conditions, and the processes occurring during adaptation and invasion of inoculants into complex environmental microbiomes remain poorly known. The main aim of this work was thus to study the specific and different cellular reactions of an inoculant for bioaugmentation during adaptation, growth and survival in natural clean and contaminated non-sterile soils, in order to better understand factors limiting bioaugmentation.ResultsAs inoculant we focused on the monoaromatic compound-degrading bacterium Pseudomonas veronii 1YdBTEX2. The strain proliferated in all but one soil types in presence and in absence of exogenously added toluene. RNAseq and differential genome-wide gene expression analysis illustrated both a range of common soil responses such as increased nutrient scavenging and recycling, expression of defense mechanisms, as well as environment-specific reactions, notably osmoprotection and metal homeostasis. The core metabolism of P. veronii remained remarkably constant during exponential growth irrespective of the environment, with slight changes in cofactor regeneration pathways, possibly needed for balancing defense reactions.ConclusionsP. veronii displayed a versatile global program, enabling it to adapt to a variety of soil environments in the presence and even in absence of its target pollutant toluene. Our results thus challenge the widely perceived dogma of poor survival and growth of exogenous inoculants in complex microbial ecosystems such as soil and provide a further basis to developing successful bioaugmentation strategies.

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Dataset Information

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

Publications

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

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