Luciana Giovannetti

Characterization of Cr(VI)-Resistant Bacteria Isolated from Chromium-Contaminated Soil by Tannery Activity

Bacterial strains, previously isolated from a chromium-polluted soil, were identified on the basis of Gram reaction and biochemical characteristics (Biolog system). Moreover, chromate MICs, chromate reduction capability, multiple heavy metal tolerance, and antibiotic susceptibility were tested for each isolate. All strains but one were Gram-positive and resistant to high concentrations of chromate. The most Cr(VI)-resistant isolate (22mM) was identified as Corynebacterium hoagii. All Cr(VI)-resistant strains except the isolate ChrC20 were capable of catalyzing the reduction of Cr(VI) to Cr(III), a less toxic and less water-soluble form of chromium. The only isolate Cr(VI)-sensitive, attributed to the Pseudomonas genus, also exhibited Cr(VI)-reduction. Isolates were also screened for the presence of plasmid DNA. The strains ChrC20 and ChrB20 harbored one and two plasmids of high molecular mass, respectively. This approach permitted selection of some bacterial strains, which could be used for bioremediation of Cr(VI)-polluted environments.

Bacterial communities associated with the rhizosphere of transgenic Bt 176 maize (Zea mays) and its non transgenic counterpart

The effect of transgenic Bt 176 maize on the rhizosphere bacterial community has been studied with a polyphasic approach by comparing the rhizosphere of Bt maize cultivated in greenhouse with that of its non transgenic counterpart grown in the same conditions. In the two plants the bacterial counts of the copiotrophic, oligotrophic and sporeforming bacteria, and the community level catabolic profiling, showed no significant differences; differences between the rhizosphere and bulk soil bacterial communities were evidenced. Automated ribosomal intergenic spacer analysis (ARISA) showed differences also in the rhizosphere communities at different plant ages, as well as between the two plant types. ARISA fingerprinting patterns of soil bacterial communities exposed to root growth solutions, collected from transgenic and non transgenic plants grown in hydroponic conditions, were grouped separately by principal component analysis suggesting that root exudates could determine the selection of different bacterial communities.

Deciphering the role of RND efflux transporters in Burkholderia cenocepacia

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in Gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4–D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.

DNA restriction fingerprint analysis of the soil bacterium Azospirillum

Total DNAs of 18 strains of Azospirillum from different sources and geographical areas were compared by restriction endonuclease pattern analysis. Fragments obtained with HindIII or BglII were separated by PAGE and stained with silver nitrate. Each strain possessed a unique and reproducible fingerprint with each enzyme, thereby facilitating strain recognition. UPGMA analysis recovered clusters of band patterns that were compared to the distribution of species within the genus Azospirillum.

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.

Relationships between the olive fly and bacteria

The relationship between the olive fly population and epiphytic bacteria of the olive tree was investigated by carrying out a 1‐year survey in the field. The olive fly population affected the number of bacteria present on the olive surface. Scanning electron microscope observations demonstrated that bacteria may be ingested by the fly’s mouth apparatus through the midline of the pseudotracheae. DNA amplification of the oesophageal bulb content using 16S bacteria universal primers and DNA sequencing evidenced that Candidatus Erwinia dacicola was the predominant species present. The role of bacteria in olive fly biology is discussed.

Important discoveries from analysing bacterial phenotypes

The ability to test hundreds to thousands of cellular phenotypes in a single experiment has opened up new avenues of investigation and exploration and led to important discoveries in very diverse applications of microbiological research and development. The information provided by global phenotyping is complementary to, and often more easily interpretable than information provided by global molecular analytical methods such as gene chips and proteomics. This report summarizes advances presented by scientists brought together to share their experiences and knowledge gained with high‐throughput phenotyping.

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.

Species attribution and strain typing of Oenococcus oeni (formerly Leuconostoc oenos) with restriction endonuclease fingerprints

In several wines, malolactic fermentation is required to improve the organoleptic characters and to stabilize the final product. In order to establish a controlled malolactic fermentation in wine, easy identification and sensitive typing of strains of Oenococcus oeni (new name of the malolactic bacterium Leuconostoc oenos) used as starter cultures are necessary. To accomplish these tasks, several strains of Oenococcus oeni isolated from wines of the Chianti region (Italy), along with reference strains and strains of L. mesenteroides subsp. mesenteroides, L. carnosum, L. fallax, L. pseudomesenteroides, L. lactis and Weisella paramesenteroides, were studied with RFLP of ribosomal genes and ultrasensitive total DNA restriction pattern analysis performed on polyacrylamide gel. With each of four restriction endonucleases used, identical restriction profiles of ribosomal genes were obtained for all strains of O. oeni. These ribopatterns, being strongly dissimilar to profiles of the other lactic acid bacteria tested, appear to be well suited for the attribution of wine lactic acid bacteria to the species O. oeni. Cluster analysis performed on two total DNA restriction profile data sets showed that the species O. oeni possesses a good degree of genomic homogeneity. Very sensitive typing of strains of O. oeni was obtained with total DNA restriction profiles. The potential of an integrated approach using restriction profiles for species assignment and typing of selected malolactic bacteria is demonstrated.

Molecular and phenotypic characterization of Acinetobacter strains able to degrade diesel fuel

Characterization of bacterial communities in oil-contaminated soils and evaluation of their degradation capacities may serve as a guide for improving remediation of such environments. Using physiological and molecular methods, the aim of this work was to characterize 17 Acinetobacter strains (13 species) able to use diesel fuel oil as sole carbon and energy source. The strains were first tested for their ability to grow on different alkanes on minimal medium containing high NaCl concentrations. The envelope hydrophobicity of each strain was assessed by microbial adhesion to the hydrocarbon test (MATH) when grown in LB medium or minimal medium containing succinate or diesel fuel. Most strains were hydrophobic both in LB and minimal medium, except for strain Acinetobacter venetianus VE-C3 that was hydrophobic only in minimal medium. Furthermore, two A. venetianus strains, RAG-1(T) and LUH 7437, and strain ATCC 17905 (genomic species 13BJ) displayed biosurfactant activity. The alkM gene encoding alkane hydroxylase was detected in the chromosome of the 15 strains by PCR amplification, sequencing and Southern blot analysis. Phenotype microarray analysis performed on the five A. venetianus strains revealed that they differentially used purines as N-source and confirmed that they are unable to use carbohydrates.