Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing

DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [¹³C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase α subunits (BphA) from bacteria that incorporated [¹³C]into DNA in 3-day incubations of the soils with [¹³C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.Polychlorinated biphenyls (PCBs) are very stable chloroorganic compounds with the general formula C₁₂H_10-xCl_x. Mixtures of PCBs have been used as coolants and lubricants in transformers, capacitors, and other electrical equipment as they do not burn easily and are good insulators. It is estimated that some 1.5 million tons of PCBs were produced up to 1988 worldwide (11; http://www.atsdr.cdc.gov/cercla; http://www.epa.gov/epawaste/hazard/tsd/pcbs/pubs/about.htm). Although production of these compounds was stopped, due to their long-term persistence, many sites all over the world are still contaminated with PCBs. Moreover, not only do PCBs threaten human health in the vicinity of the contaminated area, but lower PCB congeners volatilize and migrate to places far from where they were originally released (2, 3, 16). Also, their metabolic products have environmental significance; activities of both plants and microorganisms result in formation of different intermediates and final products whose toxicity can in some cases be even higher than that of the original toxicant (24, 26; http://www.atsdr.cdc.gov/cercla).Physical-chemical methods used for the removal of PCBs often cause further natural disturbance and pollution; in contrast, biological methods of removal (i.e., bioremediation) are less expensive and more environmentally sound and thus have aroused much interest (7). These methods include the use of microorganisms and also exploitation of plants (i.e., phytoremediation) (19) and the cooperation of plants with microorganisms in the rhizosphere (i.e., rhizoremediation) (21). These bioremediation options also include the use of genetically modified bacteria (6) and/or plants (18, 23). PCBs were only recently introduced into the environment, and no completely efficient pathways for the aerobic bacterial degradation of all of these compounds have evolved (34); however, lower chlorinated PCB congeners can be degraded via the pathway that is used by aerobic bacteria to degrade biphenyl (35). Therefore, metabolism of biphenyl as a potential cometabolite of PCBs was the subject of this study.The biphenyl degradation pathway is the same in all aerobic bacteria, and enzymes of this pathway degrade biphenyl in four steps into benzoate and 2-hydroxypenta-2,4-dienoate (21). The first enzyme of the pathway, biphenyl dioxygenase, has broad substrate specificity and thus permits degradation of biphenyl-related compounds (9). Substrates for biphenyl dioxygenase comprise, in addition to biphenyl itself, other diphenyl or benzene skeletons with several substituents, including halogens and bicyclic or tricyclic fused heterocyclic aromatics (35). These substrates also include certain natural compounds, including some plant flavonoids, phenols, or terpenes (10). Bacteria capable of metabolizing biphenyl are thus pervasive members of many microbial communities in vegetated soil.As reported previously (20), there are two main problems with introduction of a new population of degrading or genetically modified microorganisms to enhance the biodegradation of PCBs in a contaminated environment: legislative barriers and the inability of strains added to the soil to survive. Therefore, the use of microorganisms for bioremediation of contaminated sites is not likely to be successful. Hence, understanding the biodegradative processes in the natural communities is necessary for planning remediation strategies. Identification of members of the community potentially responsible for the degradative process has recently been enabled by DNA-based stable isotope probing (SIP), as reviewed previously; therefore, this technique has become an efficient tool in microbial ecology (33). In this study, by tracking the transfer of ¹³C from [¹³C]biphenyl into bacterial DNA, it was possible to identify biphenyl-metabolizing bacteria in PCB-contaminated soil. To analyze how the bacterial diversity can be changed by introduction of a plant and subsequent cultivation in a greenhouse, bacteria in the rhizosphere of horseradish (Armoracia rusticana) cultivated in a contaminated soil were studied.     

Evolution of middle-late Pleistocene human cranio-facial form: A 3-D approach

The classification and phylogenetic relationships of the middle Pleistocene human fossil record remains one of the most intractable problems in paleoanthropology. Several authors have noted broad resemblances between European and African fossils from this period, suggesting a single taxon ancestral to both modern humans and Neanderthals. Others point out ‘incipient’ Neanderthal features in the morphology of the European sample and have argued for their inclusion in the Neanderthal lineage exclusively, following a model of accretionary evolution of Neanderthals. We approach these questions using geometric morphometric methods which allow the intuitive visualization and quantification of features previously described qualitatively. We apply these techniques to evaluate proposed cranio-facial ‘incipient’ facial, vault, and basicranial traits in a middle-late Pleistocene European hominin sample when compared to a sample of the same time depth from Africa. Some of the features examined followed the predictions of the accretion model and relate the middle Pleistocene European material to the later Neanderthals. However, although our analysis showed a clear separation between Neanderthals and early/recent modern humans and morphological proximity between European specimens from OIS 7 to 3, it also shows that the European hominins from the first half of the middle Pleistocene still shared most of their cranio-facial architecture with their African contemporaries.     

Application of methyl jasmonate to grey willow (<Emphasis Type="Italic">Salix cinerea</Emphasis>) attracts insectivorous birds in nature

It has been suggested that insectivorous birds may be guided by herbivore-induced plant volatiles (HIPVs) to herbivore-rich trees with herbivorous damage. The HIPV production in plants is partly mediated by jasmonic acid signalling pathway. Methyl jasmonate (MeJA) was proved to be a suitable agent for induction of HIPVs similar to those induced by herbivorous insects in many plant species. We studied the effects of methyl jasmonate on volatile emission and natural enemy attraction using mature grey willow (Salix cinerea) under natural conditions in Czech Republic. We treated 12 experimental shrubs with 30 mM MeJA and completed the experiment with 12 control shrubs. We monitored attacks by natural predators with artificial plasticine caterpillars which were checked daily. Birds most often pecked the caterpillars exposed on MeJA-treated shrubs and this attractiveness differed significantly from control. Attractiveness of MeJA-treated shrubs did not differ significantly from control shrubs for arthropod predators. Spraying MeJA on grey willows resulted in significantly higher production of α-pinene, β-pinene, 3-carene, limonene and β-ocimene. There was a marginally significant positive correlation between the predation rate by birds and relative change in α-pinene emissions.     

Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor

The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with L-alanine or L-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of L-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either L-alanine or L-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to D-alanine do not respond to D-alanine but respond to the same L-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these L-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via L-alanine and GerB or GerB* was not inhibited by D-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to L-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to L-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to L-amino acids, and GerA interacts only with L-valine, L-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to L-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination.     

LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation

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