Philip Breugelmans

Architecture and spatial organization in a triple-species bacterial biofilm synergistically degrading the phenylurea herbicide linuron

Members of a triple-species 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea (linuron)-mineralizing consortium, i.e. the linuron- and 3,4-dichloroaniline-degrading Variovorax sp. WDL1, the 3,4-dichloroaniline-degrading Comamonas testosteroni WDL7 and the N,O-dimethylhydroxylamine-degrading Hyphomicrobium sulfonivorans WDL6, were cultivated as mono- or multi-species biofilms in flow cells irrigated with selective or nonselective media, and examined with confocal laser scanning microscopy. In contrast to mono-species biofilms of Variovorax sp. WDL1, the triple-species consortium biofilm degraded linuron completely through apparent synergistic interactions. The triple-species linuron-fed consortium biofilm displayed a heterogeneous structure with an irregular surface topography that most resembled the topography of linuron-fed mono-species WDL1 biofilms, indicating that WDL1 had a dominating influence on the triple-species biofilm architecture. This architecture was dependent on the carbon source supplied, as the biofilm architecture of WDL1 growing on alternative carbon sources was different from that observed under linuron-fed conditions. Linuron-fed triple-species consortium biofilms consisted of mounds composed of closely associated WDL1, WDL7 and WDL6 cells, while this association was lost when the consortium was grown on a nonselective carbon source. In addition, under linuron-fed conditions, microcolonies displaying associated growth developed rapidly after inoculation. These observations indicate that the spatial organization in the linuron-fed consortium biofilm reflected the metabolic interactions within the consortium.

A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16

ABSTRACT The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the phenylurea herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a Km and a V max for linuron of 5.8 μM and 0.16 nmol min−1, respectively. This novel member of the amidase signature family is unrelated to phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested phenylurea herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a phenylurea herbicide and the first report on a linuron hydrolase in Gram-negative bacteria.

Proteomic study of linuron and 3,4-dichloroaniline degradation by Variovorax sp WDL1: evidence for the involvement of an aniline dioxygenase-related multicomponent protein

A proteomic approach was used to explore the metabolism of the phenylurea herbicide linuron and 3,4-dichloroaniline (3,4-DCA) in Variovorax sp. WDL1. This bacterium grows on linuron as sole source of carbon, nitrogen and energy, while it transiently accumulates 3,4-DCA as a metabolite. Differential protein expression analysis of Variovorax sp. WDL1 grown in a heterotrophic medium in the presence and absence of linuron or 3,4-DCA was conducted using 2-D PAGE. Selected up- and downregulated proteins were identified with nanoLC-ESI-MS/MS. In the 3,4-DCA-supplemented culture, upregulation of several proteins showing high amino acid sequence similarity to different components of the multicomponent aniline dioxygenase in aniline-degrading Proteobacteria was observed. For one of the components, multiple variant proteins were detected, suggesting that strain WDL1 harbors several copies of the aniline dioxygenase (AD) gene cluster which are simultaneously expressed in the presence of 3,4-DCA. A number of unidentifiable proteins, which were upregulated in the linuron- and/or 3,4-DCA-supplemented cultures, might represent up to now uncharacterized proteins with a role in linuron and/or 3,4-DCA degradation in strain WDL1. In addition, several stress-related proteins were differentially expressed.

A molecular toolbox to estimate the number and diversity of Variovorax in the environment: application in soils treated with the phenylurea herbicide linuron

Real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE) approaches that specifically target the Variovorax 16S rRNA gene were developed to estimate the number and diversity of Variovorax in environmental ecosystems. PCR primers suitable for both methods were selected as such that the enclosed sequence showed maximum polymorphism. PCR specificity was maximized by combining PCR with a targeted endonuclease treatment of template DNA to eliminate 16S rRNA genes of the closely related Acidovorax. DGGE allowed the grouping of PCR amplicons according to the phylogenetic grouping within the genus Variovorax. The toolbox was used to assess the Variovorax community dynamics in agricultural soil microcosms (SMs) exposed to the phenylurea herbicide linuron. Exposure to linuron resulted in an increased abundance within the Variovorax community of a subgroup previously linked to linuron degradation through cultivation-dependent isolation. SMs that were treated only once with linuron reverted to the initial community composition 70 days after linuron exposure. In contrast, SMs irrigated with linuron on a long-term base showed a significant increase in Variovorax number after 70 days. Our data support the hypothesis that the genus Variovorax is involved in linuron degradation in linuron-treated agricultural soils.

Catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH) detection of Dehalococcoides.

Members of the genus Dehalococcoides are well-known for their capacity to reductively dechlorinate chlorinated organic pollutants. The availability of quantitative and sensitive detection methods is of major interest for research on the ecology of those environmentally important micro-organisms. In this paper we describe the development of a Catalyzed Reporter Deposition-Fluorescent In Situ Hybridization (CARD-FISH) for detection of Dehalococcoides cells in enrichment cultures using two oligonucleotide sequences which target two different lineages of Dehalococcoides as probes. Both sequences were previously applied in conventional FISH as probes. Conjugation of the probe to horseradish peroxidase (HRP) did not change the specificity of the probes and bright fluorescent signals were obtained. Despite the use of higher concentrations of probe and the application of longer exposure times in the conventional FISH procedure, CARD-FISH resulted in more intense signals. The CARD-FISH method was applied to a vinyl chloride (VC)-reductively-dechlorinating enrichment culture. Only the probe targeting the CBDB1 lineage of Dehalococcoides reacted with the sample which was in agreement with previous nucleic acid based analysis. The culture consisted of 51%+/-8% of Dehalococcoides cells. Furthermore, the CARD-FISH probes for detecting Dehalococcoides were combined with FISH probes for simultaneous detection of either Bacteria or Archaea which should allow rapid insight into the relative dynamics of the different members of dechlorinating communities as a response to environmental changes. Overall, CARD-FISH proved to be a rapid, reliable and convenient method to detect and quantify Dehalococcoides cells.

Exposure to Solute Stress Affects Genome-Wide Expression but Not the Polycyclic Aromatic Hydrocarbon-Degrading Activity of Sphingomonas sp. Strain LH128 in Biofilms

ABSTRACT Members of the genus Sphingomonas are important catalysts for removal of polycyclic aromatic hydrocarbons (PAHs) in soil, but their activity can be affected by various stress factors. This study examines the physiological and genome-wide transcription response of the phenanthrene-degrading Sphingomonas sp. strain LH128 in biofilms to solute stress (invoked by 450 mM NaCl solution), either as an acute (4-h) or a chronic (3-day) exposure. The degree of membrane fatty acid saturation was increased as a response to chronic stress. Oxygen consumption in the biofilms and phenanthrene mineralization activities of biofilm cells were, however, not significantly affected after imposing either acute or chronic stress. This finding was in agreement with the transcriptomic data, since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to nonstressed conditions. The transcriptomic data suggest that LH128 adapts to NaCl stress by (i) increasing the expression of genes coping with osmolytic and ionic stress such as biosynthesis of compatible solutes and regulation of ion homeostasis, (ii) increasing the expression of genes involved in general stress response, (iii) changing the expression of general and specific regulatory functions, and (iv) decreasing the expression of protein synthesis such as proteins involved in motility. Differences in gene expression between cells under acute and chronic stress suggest that LH128 goes through changes in genome-wide expression to fully adapt to NaCl stress, without significantly changing phenanthrene degrading activity.

Environmental Dissolved Organic Matter Governs Biofilm Formation and Subsequent Linuron Degradation Activity of a Linuron-Degrading Bacterial Consortium

ABSTRACT It was examined whether biofilm growth on dissolved organic matter (DOM) of a three-species consortium whose members synergistically degrade the phenylurea herbicide linuron affected the consortiums integrity and subsequent linuron-degrading functionality. Citrate as a model DOM and three environmental DOM (eDOM) formulations of different quality were used. Biofilms developed with all DOM formulations, and the three species were retained in the biofilm. However, biofilm biomass, species composition, architecture, and colocalization of member strains depended on DOM and its biodegradability. To assess the linuron-degrading functionality, biofilms were subsequently irrigated with linuron at 10 mg liter−1 or 100 μg liter−1. Instant linuron degradation, the time needed to attain maximal linuron degradation, and hence the total amount of linuron removed depended on both the DOM used for growth and the linuron concentration. At 10 mg liter−1, the final linuron degradation efficiency was as high as previously observed without DOM except for biofilms fed with humic acids which did not degrade linuron. At 100 μg liter−1 linuron, DOM-grown biofilms degraded linuron less efficiently than biofilms receiving 10 mg liter−1 linuron. The amount of linuron removed was more correlated with biofilm species composition than with biomass or structure. Based on visual observations, colocalization of consortium members in biofilms after the DOM feed appears essential for instant linuron-degrading activity and might explain the differences in overall linuron degradation. The data show that DOM quality determines biofilm structure and composition of the pesticide-degrading consortium in periods with DOM as the main carbon source and can affect subsequent pesticide-degrading activity, especially at micropollutant concentrations.

Physiological and Transcriptome Response of the Polycyclic Aromatic Hydrocarbon Degrading Novosphingobium sp. LH128 after Inoculation in Soil

Soil bioaugmentation involves the inoculation of pollutant-degrading bacteria to accelerate pollutant degradation. Often the inoculum shows a dramatic decrease in Colony Forming Units (CFU) upon soil inoculation but this behavior is not well-understood. In this study, the physiology and transcriptomic response of a GFP tagged variant of Novosphingobium sp. LH128 was examined after inoculation into phenanthrene spiked soil. Four hours after inoculation, strain LH128-GFP showed about 99% reduction in CFU while microscopic counts of GFP-expressing cells were identical to the expected initial cell density, indicating that the reduction in CFU number is explained by cells entering into a Viable But Non-Culturable (VBNC)-like state and not by cell death. Transcriptome analysis showed a remarkably higher expression of phenanthrene degradation genes 4 h after inoculation, compared to the inoculum suspension concomitant with an increased expression of genes involved in stress response. This indicates that the cells were active in phenanthrene degradation while experiencing stress. Between 4 h and 10 days, CFU numbers increased to numbers comparable to the inoculated cell density. Our results suggest that strain LH128-GFP enters a VBNC-like state upon inoculation into soil but is metabolically active and that VBNC cells should be taken into account in evaluating bioaugmentation approaches.

Response to mixed substrate feeds of the structure and activity of a linuron-degrading triple-species biofilm

We sought to determine whether the pesticide-degrading performance of a multi-species bacterial biofilm is affected by co-occurrence of multiple nutrient sources. Thus, the 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea (linuron)-degrading activity of a triple-species linuron-degrading consortium, cultivated in continuous flow biofilm systems, was monitored when exposed to mixed substrate feeds which contained, in addition to linuron, readily assimilated carbon (i.e. citrate and trypticase soy broth) and/or nitrogen (i.e. ammonium) sources. The addition of alternative carbon sources at different concentrations resulted in diminished linuron degradation efficiency. In addition, the efficiency of removal of the linuron metabolite 3,4-dichloroaniline was affected. These effects might be attributed to catabolic repression of the linuron metabolic pathway in the presence of alternative carbon substrates. Moreover, each nutrient condition resulted in a particular biofilm composition and a particular spatial and structural organization, which might also be related to the performance of the biofilm community. Results show that the activity of pesticide-degrading biofilms strongly depends on prevailing nutrient conditions and that the ideal biofilm configuration and activity, as observed under selective conditions, does not exist in real-life environmental conditions where mixtures of substrates are often present.

Identification of opsA, a Gene Involved in Solute Stress Mitigation and Survival in Soil, in the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Novosphingobium sp. Strain LH128

ABSTRACT The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading Novosphingobium sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that opsA is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing. opsA appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability.