Davide Zannoni

Microbial degradation of chloroform

Chloroform (CF) is largely produced by both anthropogenic and natural sources. It is detected in ground and surface water sources and it represents the most abundant halocarbon in the atmosphere. Microbial CF degradation occurs under both aerobic and anaerobic conditions. Apart from a few reports describing the utilization of CF as a terminal electron acceptor during growth, CF degradation was mainly reported as a cometabolic process. CF aerobic cometabolism is supported by growth on short-chain alkanes (i.e., methane, propane, butane, and hexane), aromatic hydrocarbons (i.e., toluene and phenol), and ammonia via the activity of monooxygenases (MOs) operatively divided into different families. The main factors affecting CF cometabolism are (1) the inhibition of CF degradation exerted by the growth substrate, (2) the need for reductant supply to maintain MO activity, and (3) the toxicity of CF degradation products. Under anaerobic conditions, CF degradation was mainly associated to the activity of methanogens, although some examples of CF-degrading sulfate-reducing, fermenting, and acetogenic bacteria are reported in the literature. Higher CF toxicity levels and lower degradation rates were shown by anaerobic systems in comparison to the aerobic ones. Applied physiological and genetic aspects of microbial cometabolism of CF will be presented along with bioremediation perspectives.

The high potential iron-sulfur protein (HiPIP) from Rhodoferax fermentans is competent in photosynthetic electron transfer

The functional role of the High Potential Iron‐sulfur Protein (HiPIP) from the photosynthetic bacterium Rhodoferax fermentans was investigated. We demonstrated that the HiPIP increased the rate of light‐induced oxygen reduction mediated by the photosynthetic reaction center (RC); this stimulation reached half‐saturation at [HiPIP]/[RC] ca. 15. The capability of the HiPIP in delivering electrons to the reaction center of Rhodoferax fermentans was demonstrated through kinetic spectrophotometry of cytochrome c‐556 oxidation in the presence or in the absence of HiPIP. It is concluded that the HiPIP is competent in the photosynthetic electron transfer chain of Rhodoferax fermentans.

Electrochemical proton gradient and phosphate potential in bacterial chromatophores

One of the obligate and basic requirements of Mitchell’s chemiosmotic hypothesis [ 1 ] is the consistency of the extent of the electrochemical potential difference of protons across the membrane and the amount of energy available for ATP synthesis. An estimation of this amount of energy has been obtained by measuring the maximal affinity of the phosphorylation reaction at which no net ATP synthesis occurs (phosphate potential in state 4). Experiments of this kind, performed in membrane systems as different as mitochondria [2], chloroplasts [3] and membrane fragments from aerobic [4] and photosynthetic bacteria [5], have all yielded values ranging between 14 and 16 Kcal/mole. These values are considered by several authors to be inconsistent with a stoichiometry of 2H’ translocated per ATP synthesized [ 1 ] and with the size of the proton electrochemical gradient, as estimated on the basis of the distribution of permeable ionic and protonable molecules across the energized membrane, for example [6,7]. Fluorimetric and spectrophotometric methods for the evaluation of pH or potential differences across the membranes of some photosynthetic bacteria have been propos d. Specifically, work in this laboratory [8] has indicated that ApH in bacterial chromatophores can be estimated from the extent of the fluorescence quenching of 9-aminoacridine, employing the technique originally proposed by Shuldiner et al. [9] for chloroplasts. In addition, the presence in photosynthetic membranes of pigments undergoing spectral shifts in response to a transmembrane electric field [lo] allows an evaluation of the membrane potential,

Analyses of both the alkB gene transcriptional start site and alkB promoter-inducing properties of Rhodococcus sp. strain BCP1 grown on n-alkanes.

ABSTRACT Rhodococcus sp. strain BCP1, known for its capacity to grow on short-chain n-alkanes (C2 to C7) and to cometabolize chlorinated solvents, was found to also utilize medium- and long-chain n-alkanes (C12 to C24) as energy and carbon sources. To examine this feature in detail, a chromosomal region which includes the alkB gene cluster encoding a non-heme di-iron monooxygenase (alkB), two rubredoxins, and one rubredoxin reductase was cloned from the BCP1 genome. Furthermore, the activity of the alkB gene promoter (P alkB ) was examined in the presence of gaseous, liquid, and solid n-alkanes along with intermediates of the putative n-alkane degradation pathway. A recombinant plasmid, pTPalkBLacZ, was constructed by inserting the lacZ gene downstream of P alkB , and it was used to transform Rhodococcus sp. strain BCP1. Measurements of β-galactosidase activity showed that P alkB is induced by C6 to C22 n-alkanes. Conversely, C2 to C5 and >C22 n-alkanes and alkenes, such as hexene, were not inducers of alkB expression. The effects on P alkB expression induced by alternative carbon sources along with putative products of n-hexane metabolism were also evaluated. This report highlights the great versatility of Rhodococcus sp. strain BCP1 and defines for the first time the alkB gene transcriptional start site and the alkB promoter-inducing capacities for substrates different from n-alkanes in a Rhodococcus strain.

A kinetic study of biohydrogen production from glucose, molasses and cheese whey by suspended and attached cells of Thermotoga neapolitana.

Batch tests of H2 production from glucose, molasses and cheese whey by suspended and immobilized cells of Thermotoga neapolitana were conducted to develop a kinetic model of the process. H2 production was inhibited by neither H2 (up to 0.7 mg L(-1)) nor O2 (up to 0.2 mg L(-1)). The H2 specific rates obtained at different substrate concentrations were successfully interpolated with Andrews inhibition model. With glucose and molasses, biofilms performed better than suspended cells. The suspended-cell process was successfully scaled-up to a 19-L bioreactor. Assays co-fed with molasses and cheese whey led to higher H2 productivities and H2/substrate yields than the single-substrate tests. The simulation of the suspended-cell continuous-flow process indicated the potential attainment of H2 productivities higher than those of the batch tests (up to 3.6 mmol H2 h(-1) L(-1) for molasses and 0.67 mmol H2 h(-1) L(-1) for cheese whey) and allowed the identification of the optimal dilution rate.

Acetate Permease (ActP) Is Responsible for Tellurite (TeO32−) Uptake and Resistance in Cells of the Facultative Phototroph Rhodobacter capsulatus

ABSTRACT The highly toxic oxyanion tellurite has to enter the cytoplasm of microbial cells in order to fully express its toxicity. Here we show that in the phototroph Rhodobacter capsulatus, tellurite exploits acetate permease (ActP) to get into the cytoplasm and that the levels of resistance and uptake are linked.

Reduction of chalcogen oxyanions and generation of nanoprecipitates by the photosynthetic bacterium Rhodobacter capsulatus

The facultative photosynthetic bacterium Rhodobacter capsulatus is characterized in its interaction with the toxic oxyanions tellurite (Te(IV)) and selenite (Se(IV)) by a highly variable level of resistance that is dependent on the growth mode making this bacterium an ideal organism for the study of the microbial interaction with chalcogens. As we have reported in the past, while the oxyanion tellurite is taken up by R. capsulatus cells via acetate permease and it is reduced to Te(0) in the cytoplasm in the form of splinter-like black intracellular deposits no clear mechanism was described for Se(0) precipitation. Here, we present the first report on the biotransformation of tellurium and selenium oxyanions into extracellular Te(0) and Se(0)nanoprecipitates (NPs) by anaerobic photosynthetically growing cultures of R. capsulatus as a function of exogenously added redox-mediator lawsone, i.e. 2-hydroxy-1,4-naphthoquinone. The NPs formation was dependent on the carbon source used for the bacterial growth and the rate of chalcogen reduction was constant at different lawsone concentrations, in line with a catalytic role for the redox mediator. X-ray diffraction (XRD) analysis demonstrated the Te(0) and Se(0) nature of the nanoparticles.

Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus.

The toxic oxyanion tellurite (TeO3(2-)) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te(0) in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te(0) nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600-700 nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te(0) to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents.

Genome Sequence of the Polychlorinated-Biphenyl Degrader Pseudomonas pseudoalcaligenes KF707

Pseudomonas pseudoalcaligenes KF707 is a soil polychlorinated biphenyl (PCB) degrader, able to grow both planktonically and as a biofilm in the presence of various toxic metals and metalloids. Here we report the genome sequence (5,957,359 bp) of P. pseudoalcaligenes KF707, which provides insights into metabolic degradation pathways, flagellar motility, and chemotaxis.

Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.