Dirk Springael

The role of mobile genetic elements in bacterial adaptation to xenobiotic organic compounds

Retrospective studies clearly indicate that mobile genetic elements (MGEs) play a major role in the in situ spread and even de novo construction of catabolic pathways in bacteria, allowing bacterial communities to rapidly adapt to new xenobiotics. The construction of novel pathways seems to occur by an assembly process that involves horizontal gene transfer: different appropriate genes or gene modules that encode different parts of the novel pathway are recruited from phylogenetically related or distant hosts into one single host. Direct evidence for the importance of catabolic MGEs in bacterial adaptation to xenobiotics stems from observed correlations between catabolic gene transfer and accelerated biodegradation in several habitats and from studies that monitor catabolic MGEs in polluted sites.

Catabolic mobile genetic elements and their potential use in bioaugmentation of polluted soils and waters

Genes that encode the degradation of both naturally occurring and xenobiotic organic compounds are often located on plasmids, transposons or other mobile and/or integrative elements. The list of published reports of such mobile genetic elements (MGEs) keeps growing as researchers continue to isolate and characterize new degrading bacteria and their corresponding degradative genes. There is also growing evidence that horizontal exchange of catabolic (degradative) genes among bacteria in microbial communities plays an important role in the evolution of catabolic pathways. Around 10 years ago the hypothesis was raised that we might be able to accelerate this natural gene exchange and pathway construction by introducing and subsequently spreading degradative genes, located on MGEs, into well established, competitive indigenous microbial populations as a means of bioaugmentation of polluted soils and waters. During the last decade, only a few reports on successful MGE- mediated bioaugmentation have been published. After summarizing the diversity of degradative MGEs, this review presents an overview of studies that have monitored the transfer of degradative genes in soil microcosms and in activated sludge and other wastewater treatment reactors, with emphasis on those that have clearly shown a direct effect of gene transfer on accelerated biodegradation. A few successful cases suggest that the strategy could indeed work under specific conditions, such as when the in situ degradation potential is absent and the pollutant degrading transconjugants can grow and become numerically dominant populations in the bacterial community. Further studies in this area are obviously needed to improve our current knowledge on the efficiency of gene dissemination as a tool in bioremediation.

Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance. An omega-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained. A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined. Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified. The predicted gene products of czrCBA show a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc. The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds. DNA-DNA hybridization indicated strong conservation of czr in other environmental P. aeruginosa isolates and in the P. aeruginosa type strain PAO1, a clinical isolate. This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1. A high sequence identity (till 99% at the nt level) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs. The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.

Bacteria, not archaea, restore nitrification in a zinc-contaminated soil

Biological ammonia oxidation had long been thought to be mediated solely by discrete clades of β- and γ-proteobacteria (ammonia-oxidizing bacteria; AOB). However, ammonia-oxidizing Crenarchaeota (ammonia-oxidizing archaea; AOA) have recently been identified and proposed to be the dominant agents of ammonia oxidation in soils. Nevertheless, the dynamics of AOB versus AOA, and their relative contribution to soil ammonia oxidation and ecosystem functioning on stress and environmental perturbation, remain unknown. Using a 3-year longitudinal field study and the amoA gene as a molecular marker, we demonstrate that AOB, but not AOA, mediate recovery of nitrification after zinc (Zn) contamination. Pristine soils showed approximately equal amoA gene copy numbers and transcript levels for AOB and AOA. At an intermediate Zn dose (33.7 mmol Zn per kg), ammonia oxidation was completely inhibited, and the numbers of AOB and AOA amoA gene copies and gene transcripts were reduced. After 2 years, ammonia oxidation in the field soils was fully restored to preexposure levels, and this restoration of function was concomitant with an increase of AOB amoA gene copy and gene transcript numbers. Analysis of the restored community revealed domination by a phylogenetically distinct Zn-tolerant Nitrosospira sp. community. In contrast, the numbers of AOA amoA gene copies and gene transcripts remained 3- and 104-fold lower than recovered AOB values, respectively. Thus, although recent findings have emphasized a dominant role of archaea in soil-borne ammonia oxidation, we demonstrate that a phylogenetic shift within the AOB community drives recovery of nitrification from Zn contamination in this soil.

Influence of the carbon/nitrogen/phosphorus ratio on polycyclic aromatic hydrocarbon degradation by Mycobacterium and Sphingomonas in soil

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in the environment is often limited due to unfavorable nutrient conditions for the bacteria that use these PAHs as sole source of carbon and energy. Mycobacterium and Sphingomonas are 2 PAH-degrading specialists commonly present in PAH-polluted soil, but not much is known about their specific nutrient requirements. By adding different inorganic supplements of nitrogen (N) and phosphorus (P), affecting the overall carbon/nitrogen/phosphorus ratio of soil in soil slurry degradation tests, we investigated the impact of soil inorganic N and P nutrient conditions on PAH degradation by PAH-degrading Sphingomonas and Mycobacterium strains. The general theoretically calculated C/N/P ratio of 100/10/1 (expressed in moles) allowed rapid PAH metabolization by Sphingomonas and Mycobacterium strains without limitation. In addition, PAH-degradation rate and extent was not affected when ca. ten times lower concentrations of N and P were provided, indicating that Sphingomonas and Mycobacterium strains are capable of metabolizing PAHs under low nutrient conditions. Nor does PAH-degradation seem to be affected by excesses of N and P creating an imbalanced C/N/P ratio. However, supplements of N and P salts increased the salinity of soil slurry solutions and seriously limited or even completely blocked biodegradation.

Elucidation of the metabolic pathway of fluorene and cometabolic pathways of phenanthrene, fluoranthene, anthracene and dibenzothiophene by Sphingomonas sp. LB126.

The metabolic pathway of the PAH fluorene and the cometabolic pathway of the PAHs phenanthrene, fluoranthene, anthracene and dibenzothiophene in Sphingomonas sp. LB126 were examined. To our knowledge this is the first study on the cometabolic degradation of the three-ring PAHs phenanthrene, anthracene and the four-ring PAH fluoranthene by a fluorene-utilizing species. Metabolism of fluorene was shown to proceed via the 9-fluorenone pathway to form o-phthalic acid and protocatechuic acid. The cometabolic mono-hydroxylation found for phenanthrene, fluoranthene and anthracene shows similarity with the hydroxylation of fluorene. Several mono- and dihydroxy products and ring-cleavage products were identified for phenanthrene, fluoranthene and anthracene. It appeared that the cometabolism of those three compounds is a non-specific process, in contrast to the metabolism of fluorene. For dibenzothiophene the metabolites dibenzothiophene-5-oxide and dibenzothiophene-5,5-dioxide were identified; these compounds appeared to be the products of a dead-end pathway. Since apart from dibenzothiophene no metabolites were found in very high concentrations for any of the other substrates, complete degradation is suggested, even for the cometabolic degradation of phenanthrene, fluoranthene and anthracene.

Microbial community structure of a heavy fuel oil-degrading marine consortium: linking microbial dynamics with polycyclic aromatic hydrocarbon utilization.

A marine microbial consortium obtained from a beach contaminated by the Prestige oil spill proved highly efficient in removing the different hydrocarbon families present in this heavy fuel oil. Seawater cultures showed a complete removal of all the linear and branched alkanes, an extensive attack on three to five-ring polycyclic aromatic hydrocarbons [PAHs; including anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, and benzo(a)pyrene] (30-100%), and a considerable depletion of their alkyl derivatives. Community dynamics analysis revealed that Alcanivorax species, known alkane degraders, predominated in the initial stages. This was followed by an increase in Alphaproteobacteria (i.e. Maricaulis, Roseovarius), which coincided with the depletion of low molecular PAHs. Finally, these were succeeded by Gammaproteobacteria (mainly Marinobacter and Methylophaga), which were involved in the degradation of the high molecular-weight PAHs. The role of these populations in the removal of the specific components was confirmed by the analysis of subcultures established using the aliphatic or the aromatic fraction of the fuel oil, or single PAHs, as carbon sources. The genus Marinobacter seemed to play a major role in the degradation of a variety of hydrocarbons, as several members of this group were isolated from the different enrichment cultures and grew on plates with hexadecane or single PAHs as sole carbon sources.

The Biphenyl- and 4-Chlorobiphenyl-Catabolic Transposon Tn4371, a Member of a New Family of Genomic Islands Related to IncP and Ti Plasmids

ABSTRACT The nucleotide sequence of the biphenyl catabolic transposon Tn4371 has been completed and analyzed. It confirmed that the element has a mosaic structure made of several building blocks. In addition to previously identified genes coding for a tyrosine recombinase related to phage integrases and for biphenyl degradation enzymes very similar to those of Achromobacter georgiopolitanum KKS102, Tn4371 carries many plasmid-related genes involved in replication, partition, and other, as-yet-unknown, plasmid functions. One gene cluster contains most of the genes required to express a type IV secretion-mating pair formation apparatus coupled with a TraG ATPase, all of which are related to those found on IncP and Ti plasmids. Orthologues of all Tn4371 plasmid-related genes and of the tyrosine recombinase gene were found, with a very similar organization, in the chromosome of Ralstonia solanacearum and on the yet-to-be-determined genomic sequences of Erwinia chrysanthemi and Azotobacter vinelandii. In each of these chromosomal segments, conserved segments were separated by different groups of genes, which also differed from the Tn4371 bph genes. The conserved blocks of genes were also identified, in at least two copies, in the chromosome of Ralstonia metallidurans CH34. Tn4371 thus appears to represent a new family of potentially mobile genomic islands with a broad host range since they reside in a wide range of soil proteobacteria, including plant pathogens.

Dynamics of an Oligotrophic Bacterial Aquifer Community during Contact with a Groundwater Plume Contaminated with Benzene, Toluene, Ethylbenzene, and Xylenes: an In Situ Mesocosm Study

ABSTRACT An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.

Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway, through o-phthalic acid.

ABSTRACT Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.