Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons

AIM: The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism. METHODS AND RESULTS: The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture. CONCLUSIONS: This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.     

Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry approach to biomarker discovery in blue mussels (Mytilus edulis) exposed to polyaromatic hydrocarbons and heavy metals under field conditions

Proteomics provide potential in the discovery of new sensitive biomarkers for environmental pollution. To evaluate this potential, we have utilized ProteinChip® technology to analyze the proteomic profile of blue mussels (Mytilus edulis) from polluted marine habitats surrounding the island of Karmøy, Norway. Two different types of contamination, heavy metals and polyaromatic hydrocarbons (PAHs), were compared to a clean reference site. Differentially expressed proteins/peptides were found, which showed a specific induction or a general suppression associated with the field site of origin. By combining sets of protein markers in a tree-building algorithm, we were able to correctly classify samples from these sites with an accuracy of 90%.     

Identification of proteins induced by polycyclic aromatic hydrocarbon in Mycobacterium vanbaalenii PYR-1 using two-dimensional polyacrylamide gel electrophoresis and de novo sequencing methods

Protein profiles of Mycobacterium vanbaalenii PYR-1 grown in the presence of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) were examined by two-dimensional gel electrophoresis (2-DE). Cultures of M. vanbaalenii PYR-1 were incubated with pyrene, pyrene-4,5-quinone (PQ), phenanthrene, anthracene, and fluoranthene. Soluble cellular protein fractions were analyzed and compared, using immobilized pH gradient (IPG) strips. More than 1000 gel-separated proteins were detected using a 2-DE analysis program within the window of isoelectric point (pI) 4-7 and a molecular mass range of 10-100 kDa. We observed variations in the protein composition showing the upregulation of multiple proteins for the five PAH treatments compared with the uninduced control sample. By N-terminal sequencing or mass spectrometry, we further analyzed the proteins separated by 2-DE. Due to the lack of genome sequence information for this species, protein identification provided an analytical challenge. Several PAH-induced proteins were identified including a catalase-peroxidase, a putative monooxygenase, a dioxygenase small subunit, a small subunit of naphthalene-inducible dioxygenase, and aldehyde dehydrogenase. We also identified proteins related to carbohydrate metabolism (enolase, 6-phosphogluconate dehydrogenase, indole-3-glycerol phosphate synthase, and fumarase), DNA translation (probable elongation factor Tsf), heat shock proteins, and energy production (ATP synthase). Many proteins from M. vanbaalenii PYR-1 showed similarity with protein sequences from M. tuberculosis and M. leprae. Some proteins were detected uniquely upon exposure to a specific PAH whereas others were common to more than one PAH, which indicates that induction triggers not only specific responses but a common response in this strain.     

Effectiveness of phytoremediation as a secondary treatment for polycyclic aromatic hydrocarbons (PAHs) in composted soil

A greenhouse study was conducted over a 12-month period to investigate the fate of polycyclic aromatic hydrocarbons (PAHs) in soil using phytoremediation as a secondary treatment. The soil was pretreated by composting for 12 weeks, then planted with tall fescue (Festuca arundinacea), annual ryegrass (Lolium multiflorum), and yellow sweet clover (Melilotus officinalis). Two sets of unvegetated controls also were evaluated, one fertilized and one unfertilized. Total PAH concentrations decreased in the tall fescue, annual ryegrass, and yellow sweet clover treatments by 23.9%, 15.3%, and 9.1%, respectively, whereas the control was reduced by less than 5%. The smaller two- and most of the three-ringed compounds--naphthalene, acenaphthylene, acenaphthene, fluorene, and anthracene--were not found in detectable concentrations in any of the treatments. The most probable number analysis for microbial PAH degraders did not show any statistically significant differences among treatments. There were significant differences among treatments (p < 0.05) for the residual concentrations of five of the target PAHs. Root surface area measurements indicated that tall fescue and annual ryegrass both had significantly higher root surface area than yellow sweet clover, although the two species were not significantly different from each other. The tall fescue treatment resulted in the highest root and shoot biomass, followed by annual ryegrass and yellow sweet clover, and also had the highest percent of contaminant removal after 12 months. These results imply a positive relationship between plant biomass development and PAH biodegradation.     

Phylogeography of two European Reticulitermes (Isoptera) species: the Iberian refugium

Chemical, i.e. cuticular hydrocarbons, and molecular data were used to probe the phylogeography of Reticulitermes termites collected from various parts of France, Spain and Portugal. Phylogenetic relationships were inferred from sequences of the internal transcribed spacer (ITS2) of nuclear ribosomal RNA genes as well as from two partial mitochondrial DNA segments, the cytochrome oxidase II gene and a sequence combining the tRNA-Leu gene and fragments of the NADH dehydrogenase I and ribosomal 16S genes. Two species, namely, R. grassei and R. banyulensis, were identified based on an analysis of cuticular hydrocarbons and the identification was confirmed by ITS2 haplotyping. However, phylogeny based on the analysis of mitochondrial DNA was not completely in agreement with the conclusions drawn from the chemical and nuclear data. An analysis of 56 R. grassei colonies revealed intraspecific differentiation into two major lineages with distinct geographical ranges. Whereas analysis of cuticular hydrocarbons showed that R. banyulensis was chemically distinct from R. grassei, analysis of mitochondrial DNA showed its close kinship with the R. grassei lineage occurring in southern Spain. This kinship could be explained by their evolution from a common polymorphic ancestor species in this ice age refugium.     

The metabolism of formyl-substituted benzanthracenes to hydroxymethyl metabolites in rat liver in vitro and in vivo

Hydroxylation of benzylic methyl carbon atoms on drugs and carcinogenic polycyclic aromatic hydrocarbons (PAHs) forms benzylic alcohols. Many carcinogenic and mutagenic PAHs bear a primary or secondary benzylic hydroxyl group attached to the meso-region of the molecule. According to the unified theory, PAHs bearing a benzylic hydroxyl group are proximate carcinogenic metabolites. This paper demonstrates that carcinogenic benz[a]anthracenes bearing a formyl group at the meso-region undergo enzymatic reductive metabolism to the corresponding carcinogenic benzylic alcohol in vitro and in vivo. The unified theory would then predict sulfuric acid esterification of such benzylic alcohols as the final common step in their metabolic activation to generate ultimate electrophilic benzylic carbocations. Finally, oxidative metabolism of 7-formylbenz[a]anthracenes gives rise to corresponding carboxylic acids and other oxygenated metabolites that are carcinogenically inert. Thus, oxidative metabolism of meso-region formyl compounds represents an avenue for the elimination of the carcinogen in a detoxified form.     

Hydrocarbon distribution and colony odour homogenisation in <Emphasis Type="Italic">Pachycondyla apicalis</Emphasis>

Summary Within and between individuals hydrocarbon (HC)-circulation was studied in Pachycondyla apicalis workers, using radioactive labeling. Newly synthesized HCs occurred both in the PPG and on the epicuticle in appreciable amounts, lesser quantities were found in the crop. The front basitarsal brush contained a greater amount of radiolabeled HCs than could be predicted from its surface area, suggesting preferential secretion to these organs. We propose that the newly synthesized HCs are secreted primarily to the front basitarsal brushes and are thereafter either distributed throughout the body surface, or cleared via the PPG and the alimentary canal.Using labeled HCs as a model, we tracked the time-dependent dispersion of cuticular lipids among 11 workers, one of which was prelabeled for 24 hours. Distribution among the recipients became progressively uniform, reaching near homogenization between 5–10 days. The mean HCs transfer of P. apicalis to the PPG was substantially lower compared to that of Camponotus fellah or Aphaenogaster senilis. In contrast, transfer to the cuticle in this species was superior. We attribute the low transfer to the PPG to the inefficacy of passive body contact characteristic of P. apicalis, as opposed to trophallaxis and/or allogrooming that typify the other two species. The higher occurrence of radiolabeled HCs in P. apicalis cuticle can be attributed to their accumulation in the basitarsal brushes. The impact of cuticular lipid transfer and formation of uniform colony odour, as opposed to the maintenance of an idiosyncratic caste-specific composition, are discussed.Received 5 September 2002; revised 17 January 2003; accepted 10 February 2003.     

Sesquiterpene constituents in Petasites hybridus

The essential oil of the rhizomes of Petasites hybridus (Asteraceae) was investigated by gas chromatography, mass spectrometry, 1- and 2-dimensional NMR techniques and chemical correlations. Two new sesquiterpene hydrocarbons, petasitene and pethybrene, could be identified. Petasitene is the parent sesquiterpene hydrocarbon to the known norsesquiterpene albene. The absolute configuration of petasitene could be assigned by conversion of natural albene to petasitene by partial synthesis. Pethybrene is a tricyclic sesquiterpene hydrocarbon, which rearranges to the structurally related alpha-isocomene under acidic conditions. Several sesquiterpenes were isolated from the hydrodistillation products of Petasites hybridus and investigated by spectroscopic methods and chemical correlations     

Sesquiterpene constituents from the liverwort Bazzania japonica

The hydrodistillation products of the liverwort Bazzania japonica were separated by preparative gas chromatography (GC) and investigated by spectroscopic methods. Seven unknown compounds were isolated and identified by GC-MS and NMR. Four of them, the norsesquiterpene hydrocarbons 4-epi-11-nor-aristola-1(10),11-diene (1), 4-epi-11-nor-aristola-1,9,11-triene (2), 4-epi-11-nor-aristola-9,11-diene (3), and one oxygenated sesquiterpene, (-)-aristol-1(10)-en-12-ol (5) are new natural compounds, and one, (+)-himachala-2,4-diene (7), has for the first time been isolated from liverworts. The absolute configurations of 5 and 7 were derived by chemical correlation reactions and/or enantioselective GC using cyclodextrin phases. 1, 2 and 3 have identical absolute configuration.     

Microbial degradation of high and low molecular weight polyaromatic hydrocarbons in a two-phase partitioning bioreactor by two strains of Sphingomonas sp.

A mixture of six polyaromatic hydrocarbons (naphthalene, phenanthrene, fluoranthene, pyrene, chyrysene and benzo[a]pyrene), varying in size from 2 to 5 rings, was dissolved in dodecane, and used as the delivery phase of a partitioning bioreactor. Two species of Sphingomonas were then used individually, and as a consortium, to determine which of the PAHs were degraded. Only low molecular weight PAHs (naphthalene, phenanthrene and fluoranthene) were degraded by the individual strains, but the consortium degraded all substrates either to completion or near completion.