Initial community evenness favours functionality under selective stress.

Owing to the present global biodiversity crisis, the biodiversity-stability relationship and the effect of biodiversity on ecosystem functioning have become major topics in ecology. Biodiversity is a complex term that includes taxonomic, functional, spatial and temporal aspects of organismic diversity, with species richness (the number of species) and evenness (the relative abundance of species) considered among the most important measures. With few exceptions (see, for example, ref. 6), the majority of studies of biodiversity-functioning and biodiversity-stability theory have predominantly examined richness. Here we show, using microbial microcosms, that initial community evenness is a key factor in preserving the functional stability of an ecosystem. Using experimental manipulations of both richness and initial evenness in microcosms with denitrifying bacterial communities, we found that the stability of the net ecosystem denitrification in the face of salinity stress was strongly influenced by the initial evenness of the community. Therefore, when communities are highly uneven, or there is extreme dominance by one or a few species, their functioning is less resistant to environmental stress. Further unravelling how evenness influences ecosystem processes in natural and humanized environments constitutes a major future conceptual challenge.

Response of ammonia oxidizing bacteria and archaea to acute zinc stress and different moisture regimes in soil.

Ammonia oxidation has been intensively studied for its sensitivity to environmental shifts and stresses. However, acute stress effects on the occurrence and composition of ammonia oxidizing bacteria (AOB) and archaea (AOA) based on expression of related molecular markers in complex soil environments have been to an extent overlooked, particularly concerning transient but commonly occurring environmental changes like soil moisture shifts. The present study investigates the responses of AOB and AOA to moisture shifts and high Zn soil content. AmoA gene copies and transcripts of AOB and AOA along with potential nitrification activity were measured in a soil microcosm approach for investigating the referred environmental shifts. Moisture change from 87 to 50 % of the water holding capacity caused a ~99 % reduction of AOB but not of AOA amoA transcripts that did not change significantly. Increasing applied zinc concentrations resulted in a reduction of potential nitrification rates and negatively affected studied gene expressions of both AOB and AOA, with AOB being more responsive. Both 16 S rRNA and amoA transcripts of AOB had an inverse relation to the applied zinc, indicating a gradual loss in total cell activity. Our results suggest the existence of pronounced differences between AOB and AOA concerning ammonia oxidation activity.

Bacterial diversity and reductive dehalogenase redundancy in a 1,2-dichloroethane-degrading bacterial consortium enriched from a contaminated aquifer.

BACKGROUND: Bacteria possess a reservoir of metabolic functionalities ready to be exploited for multiple purposes. The use of microorganisms to clean up xenobiotics from polluted ecosystems (e.g. soil and water) represents an eco-sustainable and powerful alternative to traditional remediation processes. Recent developments in molecular-biology-based techniques have led to rapid and accurate strategies for monitoring and identification of bacteria and catabolic genes involved in the degradation of xenobiotics, key processes to follow up the activities in situ. RESULTS: We report the characterization of the response of an enriched bacterial community of a 1,2-dichloroethane (1,2-DCA) contaminated aquifer to the spiking with 5 mM lactate as electron donor in microcosm studies. After 15 days of incubation, the microbial community structure was analyzed. The bacterial 16S rRNA gene clone library showed that the most represented phylogenetic group within the consortium was affiliated with the phylum Firmicutes. Among them, known degraders of chlorinated compounds were identified. A reductive dehalogenase genes clone library showed that the community held four phylogenetically-distinct catalytic enzymes, all conserving signature residues previously shown to be linked to 1,2-DCA dehalogenation. CONCLUSIONS: The overall data indicate that the enriched bacterial consortium shares the metabolic functionality between different members of the microbial community and is characterized by a high functional redundancy. These are fundamental features for the maintenance of the community's functionality, especially under stress conditions and suggest the feasibility of a bioremediation treatment with a potential prompt dehalogenation and a process stability over time.

Diverse Reductive Dehalogenases Are Associated with Clostridiales-Enriched Microcosms Dechlorinating 1,2-Dichloroethane.

The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales. Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community.

Environmental conditions and community evenness determine the outcome of biological invasion.

Biological invasion is widely studied, however, conclusions on the outcome of this process mainly originate from observations in systems that leave a large number of experimental variables uncontrolled. Here using a fully controlled system consisting of assembled bacterial communities, we evaluate the degree of invasion and the effect on the community functionality in relation to the initial community evenness under specific environmental stressors. We show that evenness influences the level of invasion and that the introduced species can promote functionality under stress. The evenness-invasibility relationship is negative in the absence and neutral in the presence of stress. Under these conditions, the introduced species is able to maintain the functionality of uneven communities. These results indicate that communities, initially having the same genetic background, in the presence of the same invader, react in a different way with respect to invasibility and functionality depending on specific environmental conditions and community evenness.

Microbial symbionts of honeybees: a promising tool to improve honeybee health.

Among pollinators, honeybees are the most important ones and exert the essential key ecosystem service of pollination for many crops, fruit and wild plants. Indeed, several crops are strictly dependent on honeybee pollination. Since few decades, honeybees are facing large-scale losses worldwide, the causes of which are found in the interaction of several biotic and abiotic factors, such as the use of pesticides, the habitat loss, the spread of pathogens and parasites and the occurrence of climate changes. Insect symbionts are emerging as a potential tool to protect beneficial insects, ameliorating the innate immune homeostasis and contributing to the general insect wellbeing. A review about the microbial symbionts associated to honeybees is here presented. The importance of the honeybee microbial commensals for the maintenance and improvement of honeybee health is discussed. Several stressors like infestations of Varroa mites and the use of pesticides can contribute to the occurrence of dysbiosis phenomena, resulting in a perturbation of the microbiocenosis established in the honeybee body.

Genetic and biochemical diversity of Paenibacillus larvae isolated from Tunisian infected honey bee broods.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a virulent disease of honeybee (Apis mellifera) larvae. In Tunisia, AFB has been detected in many beekeeping areas, where it causes important economic losses, but nothing is known about the diversity of the causing agent. Seventy-five isolates of P. larvae, identified by biochemical tests and 16S rRNA gene sequencing, were obtained from fifteen contaminated broods showing typical AFB symptoms, collected in different locations in the northern part of the country. Using BOX-PCR, a distinct profile of P. larvae with respect to related Paenibacillus species was detected which may be useful for its identification. Some P. larvae-specific bands represented novel potential molecular markers for the species. BOX-PCR fingerprints indicated a relatively high intraspecific diversity among the isolates not described previously with several molecular polymorphisms identifying six genotypes on polyacrylamide gel. Polymorphisms were also detected in several biochemical characters (indol production, nitrate reduction, and methyl red and oxidase tests). Contrary to the relatively high intraspecies molecular and phenotypic diversity, the in vivo virulence of three selected P. larvae genotypes did not differ significantly, suggesting that the genotypic/phenotypic differences are neutral or related to ecological aspects other than virulence.

A Chloroflexi bacterium dechlorinates polychlorinated biphenyls in marine sediments under in situ-like biogeochemical conditions.

We investigated the reductive dechlorination of Aroclor 1254 PCBs by a coplanar PCB-dechlorinating microbial community enriched from an actual site contaminated marine sediment of the Venice lagoon in sterile slurry microcosms of the same sediment suspended in its site water, i.e., under biogeochemical conditions that closely mime those occurring in situ. The culture dechlorinated more than 75% of the penta- through hepta-chlorinated biphenyls to tri- and tetra-chlorinated congeners in 30 weeks. The dechlorination rate was reduced by the addition of H(2) and short chain fatty acids, which stimulated sulfate-reduction and methane production, and markedly increased by the presence of vancomycin or ampicillin. DGGE analysis of 16S rRNA genes on PCB-spiked and PCB-free cultures ruled out sulfate-reducing and methanogenic bacteria and revealed the presence of a single Chloroflexi phylotype closely related to the uncultured bacteria m-1 and SF1 associated to PCB dechlorination. These findings suggest that a single dechlorinator is responsible for the observed extensive dechlorination of Aroclor 1254 and that a Chloroflexi species similar to those already detected in freshwater and estuarine contaminated sediments mediates PCB dechlorination in the marine sediment adopted in this study under biogeochemical conditions resembling those occurring in situ in the Brentella Canal of Venice Lagoon.

Mineral-microbe interactions: biotechnological potential of bioweathering.

Mineral-microbe interaction has been a key factor shaping the lithosphere of our planet since the Precambrian. Detailed investigation has been mainly focused on the role of bioweathering in biomining processes, leading to the selection of highly efficient microbial inoculants for the recovery of metals. Here we expand this scenario, presenting additional applications of bacteria and fungi in mineral dissolution, a process with novel biotechnological potential that has been poorly investigated. The ability of microorganisms to trigger soil formation and to sustain plant establishment and growth are suggested as invaluable tools to counteract the expansion of arid lands and to increase crop productivity. Furthermore, interesting exploitations of mineral weathering microbes are represented by biorestoration and bioremediation technologies, innovative and competitive solutions characterized by economical and environmental advantages. Overall, in the future the study and application of the metabolic properties of microbial communities capable of weathering can represent a driving force in the expanding sector of environmental biotechnology.

Microbial symbionts: a resource for the management of insect-related problems.

Microorganisms establish with their animal hosts close interactions. They are involved in many aspects of the host life, physiology and evolution, including nutrition, reproduction, immune homeostasis, defence and speciation. Thus, the manipulation and the exploitation the microbiota could result in important practical applications for the development of strategies for the management of insect-related problems. This approach, defined as 'Microbial Resource Management' (MRM), has been applied successfully in various environments and ecosystems, as wastewater treatments, prebiotics in humans, anaerobic digestion and so on. MRM foresees the proper management of the microbial resource present in a given ecosystem in order to solve practical problems through the use of microorganisms. In this review we present an interesting field for application for MRM concept, i.e. the microbial communities associated with arthropods and nematodes. Several examples related to this field of applications are presented. Insect microbiota can be manipulated: (i) to control insect pests for agriculture; (ii) to control pathogens transmitted by insects to humans, animals and plants; (iii) to protect beneficial insects from diseases and stresses. Besides, we prospect further studies aimed to verify, improve and apply MRM by using the insect-symbiont ecosystem as a model.

Identification of molecular markers to follow up the bioremediation of sites contaminated with chlorinated compounds.

The use of microorganisms to clean up xenobiotics from polluted ecosystems (soil and water) represents an ecosustainable and powerful alternative to traditional remediation processes. Recent developments in molecular-biology-based techniques have led to rapid and sensitive strategies for monitoring and identifying bacteria and catabolic genes involved in the degradation of xenobiotics. This chapter provides a description of recently developed molecular-biology-based techniques, such as PCR with degenerate primers set, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), southern blot hybridization, and long-range PCR, used to give a picture of the catabolically relevant microorganisms and of the functional genes present in a polluted system. By using a case study of a groundwater aquifer contaminated with 1,2-dichloroethane (1,2-DCA), we describe the identification of microorganisms potentially involved in the 1,2-DCA dehalorespiration (Dehalobacter sp. and Desulfitobacterium sp.) and a complete new gene cluster encoding for a 1,2-DCA reductive dehalogenase. The application of these techniques to bioremediation can improve our understanding of the inner mechanisms to evaluate the feasibility of a given treatment and provide us with a method to follow up bacteria and catabolic genes involved in the degradation of contaminants during the activities in situ.

Suppurative adenitis of preputial glands associated with Corynebacterium mastitidis infection in mice.

Suppuration of the preputial gland in mice occurs as a septic complication of fight wounds around the external genitalia. Currently reported bacterial isolates from these lesions are limited to Staphylococcus aureus, Pasteurella pneumotropica, and Klebsiella oxytoca. In the context of a pilot experiment aimed at defining the aging phenotype of estrogen receptor beta knockout (BERKO) mice, 2 male mice (1 of the BERKO line and the other from the age- and sex-matched wild-type control group) were discovered at necropsy to have preputial gland lesions. In both cases, histopathologic examination confirmed severe suppuration and abscesses of the preputial glands associated with systemic reactive (secondary) amyloidosis. Both Gram staining and Bacillus Calmette-Guérin immunohistochemistry highlighted the presence of numerous bacillary to rod-shaped bacteria within the preputial lesions. Subsequent PCR analysis coupled with denaturing gradient gel electrophoresis identified Corynebacterium mastitidis in the preputial gland abscesses. This organism is isolated infrequently from the milk of sheep with subclinical mastitis and was identified as part of the normal microflora of the human ocular surface. No information regarding the epidemiology and pathogenesis of C. mastitidis infection in laboratory animals is currently available, and to our knowledge this report is the first description of C. mastitidis infection in mice.

Characterization of the microbial community from the marine sediment of the Venice lagoon capable of reductive dechlorination of coplanar polychlorinated biphenyls (PCBs).

The native microbial community of a contaminated sediment from Brentella Canal (Venice Lagoon, Italy) was enriched in slurry microcosms consisting of sterile sediment suspended in sterile site water in the presence of 3,3',4,4'-tetrachlorobiphenyl, 3,3',4,4',5- and 2,3',4,4',5-pentachlorobiphenyls, 3,3',4,4',5,5'- and 2,3,3',4,4',5-hexachlorobiphenyls. The enrichment cultures were characterized at each subculturing step by 16S rRNA gene Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE) analysis. About 90% of spiked polychlorinated biphenyls (PCBs) were stoichiometrically converted into di- and tri-chlorinated congeners by each enriched culture via dechlorination of flanked para chlorines and ortho-flanked meta chlorines. A 2-fold increase in PCB-dechlorination rate, the disappearance of lag phase, as well as a remarkable increase of sulfate consumption and a decline of methanogenic activity, were observed throughout subculturing. A reduction of complexity of the archaeal community, which was composed by Methanomicrobiales and Methanosarcinales, was also observed as a result of culture enrichment. The bacterial community included members of the Alpha, Gamma, Delta and Epsilon divisions of Proteobacteria, Firmicutes and Chloroflexi. Two sequence phylotypes related to the genus Sulforovum and the species Desulfococcus multivorans and two Chloroflexi enriched throughout subculturing, thus suggesting that these bacteria were involved in PCB dechlorination in the marine sediments of Brentella canal.

A novel Bacteroidetes symbiont is localized in Scaphoideus titanus, the insect vector of Flavescence dorée in Vitis vinifera.

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, "Candidatus Phytoplasma vitis," which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the "Candidatus Cardinium hertigii" group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of "Ca. Cardinium hertigii." This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of "Ca. Phytoplasma vitis" were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and "Ca. Phytoplasma vitis" have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.

Novel natural ligands for Drosophila olfactory receptor neurones.

Due to its well-defined genome, the fruitfly Drosophila melanogaster has become a very important model organism in olfactory research. Despite all the research invested, few natural odour ligands have been identified. By using a combined gas chromatographic-single receptor neurone recording technique (GC-SC), we set out to identify active odour molecules in head space-collected volatiles from preferred food sources, i.e. different overripe or rotting fruit. In total, we performed 101 GC-SC experiments on 85 contacted sensilla. Using GC-mass spectrometry, we identified 24 active compounds. Synthetic samples of these compounds were used to establish dose-response curves for several of the receptor neurone types encountered. The response patterns of individual neurones were repeatable, and neurones were found to reside in stereotyped pairs. In total, we identified eight distinct sensillum types based on response profiles of 12 olfactory receptor neurone types. In most recordings, a single GC peak would produce a strong response, whereas a few other, often chemically related, compounds would produce weaker responses. The GC-SC recordings revealed that the olfactory receptor neurones investigated were often selective and could be divided into distinct functional types with discrete characteristics. Dose-response investigations revealed very low response thresholds to the tested compounds. Six of the novel ligands were also tested for their behavioural effect in a T-maze set up. Of these, five elicited attraction and one elicited repulsion.