Chemotaxis of Burkholderia sp. Strain SJ98 towards chloronitroaromatic compounds that it can metabolise

Janmejay Pandey 1 2 Narinder K Sharma 0 2 Fazlurrahman Khan 2 Anuradha Ghosh 0 2 John G Oakeshott 3 Rakesh K Jain 2 Gunjan Pandey 3 0 Kansas State University , Manhattan, KS 66506 , USA 1 Georgia Health Science University , Augusta GA 30912 , USA 2 Institute of Microbial Technology , Sector 39A, Chandigarh 160036 , India 3 CSIRO Ecosystem Sciences , GPO Box 1700, Canberra ACT 2601 , Australia Background: Burkholderia sp. strain SJ98 is known for its chemotaxis towards nitroaromatic compounds (NACs) that are either utilized as sole sources of carbon and energy or co-metabolized in the presence of alternative carbon sources. Here we test for the chemotaxis of this strain towards six chloro-nitroaromatic compounds (CNACs), namely 2-chloro-4-nitrophenol (2C4NP), 2-chloro-3-nitrophenol (2C3NP), 4-chloro-2-nitrophenol (4C2NP), 2chloro-4-nitrobenzoate (2C4NB), 4-chloro-2-nitrobenzoate (4C2NB) and 5-chloro-2-nitrobenzoate (5C2NB), and examine its relationship to the degradation of such compounds. Results: Strain SJ98 could mineralize 2C4NP, 4C2NB and 5C2NB, and co-metabolically transform 2C3NP and 2C4NB in the presence of an alternative carbon source, but was unable to transform 4C2NP under these conditions. Positive chemotaxis was only observed towards the five metabolically transformed CNACs. Moreover, the chemotaxis was induced by growth in the presence of the metabolisable CNAC. It was also competitively inhibited by the presence of nitroaromatic compounds (NACs) that it could metabolise but not by succinate or aspartate. Conclusions: Burkholderia sp. strain SJ98 exhibits metabolic transformation of, and inducible chemotaxis towards CNACs. Its chemotactic responses towards these compounds are related to its previously demonstrated chemotaxis towards NACs that it can metabolise, but it is independently inducible from its chemotaxis towards succinate or aspartate. - Background Microbial bioremediation can be an efficient, economic and environmentally friendly alternative to other physico-chemical approaches used for the cleanup of contaminated soils [1-3]. However, in situ bioremediation trials show that this approach is not as successful under natural environmental conditions as would be expected from in vitro experiments [4,5]. One of the major reasons for this is the limited bioavailability of the pollutant, which in turn is a function of its hydrophobicity, solubility and persistence in the environmental matrix [4,5]. Increasingly, however, it has been recognized that microbial chemotaxis towards the pollutant can also be a major determinant [6-9]. Chloro-nitroaromatic compounds (CNACs) are a new class of toxic xenobiotic compounds that have been extensively used over the last few decades in the synthesis of pesticides, herbicides, dyes etc. Because of their stability, toxicity, mutagenicity and potential carcinogenicity, many CNACs, including chloro-nitrophenols (CNPs), chloro-nitrobenzenes (CNs) and chloro-nitrobenzoates (CNBs), have been listed as priority pollutants by organizations such as the United States Environment Protection Agency [10-13]. Microbial degradation could in theory be used to restore sites contaminated with CNACs but these compounds have proven to be extremely stable and recalcitrant to metabolic degradation [14] and there are very few reports of pure microbial isolates which are capable of degrading them [15-18]. We have recently shown that Burkholderia sp. strain SJ98 can degrade 2-chloro-4-nitrophenol (2C4NP) and utilize it as sole source of carbon and energy [19]. This strain was previously shown to mount a chemotactic response towards a number of nitroaromatic compounds (NACs) that it can either completely metabolize or cometabolically transform in the presence of an alternative carbon source [20-23]. Here we show that strain SJ98 is also chemotactic towards certain CNACs which it is able to metabolise. To the best of our knowledge, this is the first report of microbial chemotaxis towards CNACs. Methods Bacterial strain, media and culture conditions Burkholderia sp. SJ98 was previously isolated by a chemotactic enrichment technique from a pesticide-contaminated soil sample [22]. Initially this strain was identified as Ralstonia sp. strain SJ98 but it has now been re-classified as a Burkholderia sp. [24]. During the present study, strain SJ98 was grown in minimal medium (MM) supplemented with the test CNACs. CNACs were added as filter-sterilized solutions in MM to obtain working concentrations of 50-500 M. Filter-sterilized succinate (10 mM) was added as an alternative carbon source to the MM where necessary. The composition of the medium was as described earlier [25]. Incubations were carried out at 30C under shaking conditions (180 rpm) and growth was monitored spectrophotometrically at 600 nm. For culture maintenance, strain SJ98 was routinely grown on nutrient agar (NA) or nutrient broth (NB) prepared according to the manufacturers recommendations and as described earlier [19]. Metabolic activity of strain SJ98 on tested CNACs In tandem with the chemotactic assays (see below), the metabolic activity of strain SJ98 on the tested CNACs was also determined by growth studies, resting cell assays and biochemical analyses of the growth medium to detect transformation products. The purpose of, and methods for each of these studies are indicated below: Growth studies The initial screening of the metabolic activity of strain SJ98 on test CNACs was performed with growth studies using MM supplemented with 50-500 M of each CNAC as the sole sources of carbon and energy. Metabolic activity was determined by growth, monitored spectrophotometrically. For CNACs that could not be utilized as sole sources of carbon and energy during the initial screening, the culture medium for subsequent growth studies was supplemented with 10 mM of sodium succinate. Resting cell studies Resting cell studies were carried out to identify some of the degradation intermediates and elucidate the catabolic pathways of those CNACs that were completely mineralized by strain SJ98 (described below). These studies were performed according to procedures described earlier [19,20,26]; briefly, cells of strain SJ98 grown in 250 ml of nutrient broth (Sigma-Aldrich (GmbH, Germany)) medium up to mid-exponential phase (OD600 0.45-0.60) were harvested by centrifugation at 3500 rpm for 8-10 min at ambient temperature, washed twice with 10 mM sodium phosphate buffer (pH 7.2) and then resuspended in 50 ml of MM supplemented with 300 M of the test CNAC (2C4NP or 4C2NB) and incubated at 30C. Induction of CNAC degradation was monitored via visible decolorization of the induction medium. (Since most CNACs are yellow colored in aqueous growth medium and turn colorless upon microbial catabolic activities, the decolorization of the culture medium is used as an important indicator for induction of the degradation mechanism). After induction, the cells were harvested, washed and re-suspended in 2 (...truncated)