Enrico Tatti

Metabolomic Investigation of the Bacterial Response to a Metal Challenge

ABSTRACT Pseudomonas pseudoalcaligenes KF707 is naturally resistant to the toxic metalloid tellurite, but the mechanisms of resistance are not known. In this study we report the isolation of a KF707 mutant (T5) with hyperresistance to tellurite. In order to characterize the bacterial response and the pathways leading to tolerance, we utilized Phenotype MicroArray technology (Biolog) and a metabolomic technique based on nuclear magnetic resonance spectroscopy. The physiological states of KF707 wild-type and T5 cells exposed to tellurite were also compared in terms of viability and reduced thiol content. Our analyses showed an extensive change in metabolism upon the addition of tellurite to KF707 cultures as well as different responses when the wild-type and T5 strains were compared. Even in the absence of tellurite, T5 cells displayed a “poised” physiological status, primed for tellurite exposure and characterized by altered intracellular levels of glutathione, branched-chain amino acids, and betaine, along with increased resistance to other toxic metals and metabolic inhibitors. We conclude that hyperresistance to tellurite in P. pseudoalcaligenes KF707 is correlated with the induction of the oxidative stress response, resistance to membrane perturbation, and reconfiguration of cellular metabolism.

Comparison of 16S rRNA and 16S rDNA T-RFLP Approaches to Study Bacterial Communities in Soil Microcosms Treated with Chromate as Perturbing Agent

Transcripts of ribosomal RNA have been used for assessing the structure and dynamics of active bacterial populations; however, it remains unclear whether the information provided by community profiling derived from RNA is different from that derived from DNA, particularly when a selective pressure is applied on the bacterial community.In the present work, terminal-restriction fragment length polymorphism (T-RFLP) community profiles based on DNA and RNA extracted from soil microcosms treated with a toxic concentration of chromate were compared.Microcosms of a nonpolluted agricultural soil and of a heavy-metal-rich soil (serpentine) were treated with chromate and DNA and RNA were extracted. T-RFLP analysis was performed on amplified and retro-amplified 16SrRNA gene sequences, and band profiles obtained from samples of DNA and of RNA were compared. Some of the T-RFLP bands, identified as peculiar peaks in the profiles, were cloned and sequenced for taxonomic interpretation.Results indicated that: (1) community profiles derived from RNA and DNA were partly overlapping; (2) there was a strong correlation between the dynamics shown by RNA- and DNA-based T-RFLP profiles; (3) chromate addition exerted a clear effect on both agricultural and serpentine soil bacterial communities, either at the DNA and at the RNA level; however, the profiles derived from RNA showed sharper differences between treated and control samples than that of DNA-based profiles.

Effects of temperatures near the freezing point on N2O emissions, denitrification and on the abundance and structure of nitrifying and denitrifying soil communities

Climate warming in temperate regions may lead to decreased soil temperatures over winter as a result of reduced snow cover. We examined the effects of temperatures near the freezing point on N(2)O emissions, denitrification, and on the abundance and structure of soil nitrifiers and denitrifiers. Soil microcosms supplemented with NO3 - and/or NO3 - plus red clover residues were incubated for 120 days at -4 °C, -1 °C, +2 °C or +5 °C. Among microcosms amended with residues, N(2)O emission and/or denitrification increased with increasing temperature on Days 2 and 14. Interestingly, N(2)O emission and/or denitrification after Day 14 were the greatest at -1 °C. Substantial N(2) O emissions were only observed on Day 2 at +2 °C and +5 °C, while at -1 °C, N(2)O emissions were consistently detected over the duration of the experiment. Abundances of ammonia oxidizing bacteria (AOB) and archaea (AOA), Nitrospira-like bacteria and nirK denitrifiers were the lowest in soils at -4 °C, while abundances of Nitrobacter-like bacteria and nirS denitrifiers did not vary among temperatures. Community structures of nirK and nirS denitrifiers and Nitrobacter-like bacteria shifted between below-zero and above-zero temperatures. Structure of AOA and AOB communities also changed but not systematically among frozen and unfrozen temperatures. Results indicated shifts in some nitrifier and denitrifier communities with freezing and a surprising stimulation of N(2)O emissions at -1 °C when NO3 - and C are present.

Metabolic Capacity of Sinorhizobium (Ensifer) meliloti Strains as Determined by Phenotype MicroArray Analysis

ABSTRACT Sinorhizobium meliloti is a soil bacterium that fixes atmospheric nitrogen in plant roots. The high genetic diversity of its natural populations has been the subject of extensive analysis. Recent genomic studies of several isolates revealed a high content of variable genes, suggesting a correspondingly large phenotypic differentiation among strains of S. meliloti. Here, using the Phenotype MicroArray (PM) system, hundreds of different growth conditions were tested in order to compare the metabolic capabilities of the laboratory reference strain Rm1021 with those of four natural S. meliloti isolates previously analyzed by comparative genomic hybridization (CGH). The results of PM analysis showed that most phenotypic differences involved carbon source utilization and tolerance to osmolytes and pH, while fewer differences were scored for nitrogen, phosphorus, and sulfur source utilization. Only the variability of the tested strain in tolerance to sodium nitrite and ammonium sulfate of pH 8 was hypothesized to be associated with the genetic polymorphisms detected by CGH analysis. Colony and cell morphologies and the ability to nodulate Medicago truncatula plants were also compared, revealing further phenotypic diversity. Overall, our results suggest that the study of functional (phenotypic) variability of S. meliloti populations is an important and complementary step in the investigation of genetic polymorphism of rhizobia and may help to elucidate rhizobial evolutionary dynamics, including adaptation to diverse environments.

Characterization of Chromate-Resistant and -Reducing Bacteria by Traditional Means and by a High-Throughput Phenomic Technique for Bioremediation Purposes

To select strains for the bioremediation of Cr(VI)‐polluted environments, four highly Cr(VI)‐resistant bacterial isolates were identified and characterized using both traditional techniques and a novel approach called phenotype microarrays. The isolates were identified as members of Pseudomonas mendocina (strains 34 and 56) and members of Pseudomonas corrugata (strains 22 and 28). Results showed that it was possible, by varying the carbon/energy source, to decouple bacterial growth and Cr(VI) reduction, inasmuch as some carbon/energy sources were more effective electron donors for chromate reduction, whereas other sources supported growth but not an effective chromate reduction. The isolates were characterized by a novel high‐throughput technique, phenotype microarrays (PM)‐Biolog, which can test up to 2000 cellular phenotypes simultaneously. The isolates belonging to P. corrugata had PM profiles different from those of the isolates belonging to P. mendocina. Such differences were related to the capacity of the isolates to resist various chemicals, pH values, and osmolytic substances. With the PM technique a very large amount of information about the fitness of isolates in the presence of different stressors could be obtained.

Involvement of the oscA gene in the sulphur starvation response and in Cr(VI) resistance in Pseudomonas corrugata 28.

Pseudomonas corrugata 28 is a Cr(VI)-hyper-resistant bacterium. A Cr(VI)-sensitive mutant was obtained by insertional mutagenesis using EZ-Tn5 Tnp. The mutant strain was impaired in a gene, here named oscA (organosulphur compounds), which encoded a hypothetical small protein of unknown function. The gene was located upstream of a gene cluster that encodes the components of the sulphate ABC transporter, and it formed a transcriptional unit with sbp, which encoded the periplasmic binding protein of the transporter. The oscA-sbp transcriptional unit was strongly and quickly overexpressed after chromate exposure, suggesting the involvement of oscA in chromate resistance, which was further confirmed by means of a complementation experiment. Phenotype MicroArray (PM) analysis made it possible to assay 1536 phenotypes and also indicated that the oscA gene was involved in the utilization of organosulphur compounds as a sole source of sulphur. This is believed to be the first evidence that oscA plays a role in activating a sulphur starvation response, which is required to cope with oxidative stress induced by chromate.

Compost Effect on Plant Growth-Promoting Rhizobacteria and Mycorrhizal Fungi Population in Maize Cultivations

The composting process allows the conversion of organic waste into organic matter that can be used in counteracting organic matter soil depletion. Moreover, agricultural use of compost increases plants defense abilities against pathogens, leading to a higher crop yield. Key plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhial fungi (AMF) populations were evaluated after incorporating high quality composts in both conventional and organic maize production in order to determine if compost application affects the presence of important PGPR and AMF. Results obtained indicate that while the use of the compost as an amendment may exert a limited influence on AMF population, it can significantly modulate the composition of PGPR in the rhizosphere of maize plants.

Aerobic Reduction of Chromium(VI) by Pseudomonas corrugata 28: Influence of Metabolism and Fate of Reduced Chromium

Pseudomonas corrugata 28 represents a microorganism that can potentially be applied for in situ bioremediation of Cr(VI) contaminated sites. This strain combines a high resistance toward toxic Cr(VI) with the ability to reduce Cr(VI) to Cr(III) under oxic conditions. In this study, the aerobic reduction of Cr(VI) by Pseudomonas corrugata 28 was examined under different carbon and sulfur supply conditions to assess the influence of microbial carbon and sulfur metabolism on Cr(VI) reduction. The fate of reduced chromium was elucidated by investigating the speciation of chromium in solution as well as the interaction of chromium with bacterial surfaces. Reduction of Cr(VI) was found to be a metabolic process resulting mainly in the formation of dissolved organic Cr(III)-complexes. Small amounts of reduced chromium were weakly associated with bacterial surfaces. The formation of inorganic Cr(III)-precipitates was not indicated.

Characterization of two genes involved in chromate resistance in a Cr(VI)-hyper-resistant bacterium

Mechanisms underlying chromate resistance in Cr(VI)-hyper-resistant Pseudomonas corrugata strain 28, isolated from a highly Cr(VI) polluted soil, were studied by analyzing its two Cr(VI)-sensitive mutants obtained by insertion mutagenesis. The mutants, namely Crg3 and Crg96, were characterized by the identification of disrupted genes, and by the high-throughput approach called Phenotype MicroArray (PM), which permitted the assay of 1,536 phenotypes simultaneously. Crg3 and Crg96 mutants were affected in a malic enzyme family gene and in a gene encoding for a RecG helicase, respectively. The application of PM provided a wealth of new information relating to the disrupted genes and permitted to establish that chromate resistance in P. corrugata strain 28 also depends on supply on NADPH required in repairing damage induced by chromate and on DNA integrity maintenance.

Tillage Management and Seasonal Effects on Denitrifier Community Abundance, Gene Expression and Structure over Winter

Tillage effects on denitrifier communities and nitrous oxide (N2O) emissions were mainly studied during the growing season. There is limited information for the non-growing season, especially in northern countries where winter has prolonged periods with sub-zero temperatures. The abundance and structure of the denitrifier community, denitrification gene expression and N2O emissions in fields under long-term tillage regimes [no-tillage (NT) vs conventional tillage (CT)] were assessed during two consecutive winters. NT exerted a positive effect on nirK and nosZ denitrifier abundance in both winters compared to CT. Moreover, the two contrasting managements had an opposite influence on nirK and nirS RNA/DNA ratios. Tillage management resulted in different denitrifier community structures during both winters. Seasonal changes were observed in the abundance and the structure of denitrifiers. Interestingly, the RNA/DNA ratios were greater in the coldest months for nirK, nirS and nosZ. N2O emissions were not influenced by management but changed over time with two orders of magnitude increase in the coldest month of both winters. In winter of 2009–2010, emissions were mainly as N2O, whereas in 2010–2011, when soil temperatures were milder due to persistent snow cover, most emissions were as dinitrogen. Results indicated that tillage management during the growing season induced differences in denitrifier community structure that persisted during winter. However, management did not affect the active cold-adapted community structure.