Background: Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used γ-proteobacteria, such as Escherichia coli or Pseudomonas species. To this end, we have sought to develop a pair of inducible expression vectors for use in the α-proteobacterium Methylobacterium extorquens, a model methylotroph. Findings: We found that the P _R promoter from rhizobial phage 16-3 was active in M. extorquens and engineered the promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybrid promoters, P _R/cmtO and P _R/tetO , were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively. Compared to an existing cumate-inducible (10-fold range), high-level expression system for M. extorquens, P _R/cmtO and P _R/tetO have 33% of the maximal activity but were able to repress gene expression 3 and 8-fold greater, respectively. Both promoters were observed to exhibit homogeneous, titratable activation dynamics rather than on-off, switch-like behavior. The utility of these promoters was further demonstrated by complementing loss of function of ftfL - essential for growth on methanol - where we show P _R/tetO is capable of not only fully complementing function but also producing a conditional null phenotype. These promoters have been incorporated into a broad-host-range backbone allowing for potential use in a variety of bacterial hosts. Conclusions: We have developed two novel expression systems for use in M. extorquens. The expression range of these vectors should allow for increased ability to explore cellular physiology in M. extorquens. Further, the P _R/tetO promoter is capable of producing conditional null phenotypes, previously unattainable in M. extorquens. As both expression systems rely on the use of membrane permeable inducers, we suspect these expression vectors will be useful for ectopic gene expression in numerous proteobacteria.

Ecosystem functionality depends on interactions among populations, of the same or different taxa, and these are not just the sum of pairwise interactions. Thus, know-how of the social interactions occurring in mixed-populations are of high interest, however they are commonly unknown due to the limitations posed in tagging each population. The limitations include costs/time in tediously fluorescent tagging, and the number of different fluorescent tags. Tag-free strategies exist, such as high-throughput sequencing, but ultimately both strategies require the use of expensive machinery. Our work appoints social behaviours on individual strains in mixed-populations, offering a web-tool (BSocial http://m4m.ugr.es/BSocial.html) for analysing the community framework. Our quick and cheap approach includes the periodic monitoring of optical density (OD) from a full combinatorial testing of individual strains, where number of generations and growth rate are determined. The BSocial analyses then enable us to determine how the addition/absence of a particular species affects the net productivity of a microbial community and use this to select productive combinations, i.e. designate their social effect on a general community. Positive, neutral or negative assignations are applied to describe the social behaviour within the community by comparing fitness effects of the community against the individual strain. The usefulness of this tool for selection of optimal inoculum in biofilm-based methyl tert-butyl ether (MTBE) bioremediation was demonstrated. The studied model uses 7 bacterial strains with diverse MTBE degradation/growth capacities. Full combinatorial testing of 7 individual strains (triplicate tests of 127 combinations) were implemented, along with MTBE degradation as the desired function. Sole observation of highest species fitness did not render the best functional outcome, and only when strains with positive and neutral social assignations were mixed (Rhodococcus ruber EE6, Agrobacterium sp. MS2 and Paenibacillus etheri SH7), was this obtained. Furthermore, the use of positive and neutral strains in all its combinations had a significant higher degradation mean (x1.75) than exclusive negative strain combinations. Thus, social microbial processes benefit bioremediation more than negative social microbial combinations. The BSocial webtool is a great contributor to the study of social interactions in bioremediation processes, and may be used in other natural or synthetic habitats.

Swine manure management poses significant environmental and economic challenges, mainly due to the contribution of traditional practices resulting in antibiotic soil contamination and the spread of antibiotic-resistant bacteria. This research aims to comprehensively characterize swine manure physicochemical and resistome phenotype profiles (including minimum inhibitory concentrations) from several locations with different farm management techniques, providing the foundation for more sustainable and effective treatment strategies The analyses revealed the presence of a wide range of antimicrobial compounds, including amoxicillin, ciprofloxacin, erythromycin, oxytetracycline, tetracycline, sulfamethoxazole and trimethoprim, with concentrations ranging from 7.2 to 5395 µg/kg of dry weight of swine manure. A total of 73 antibiotic-resistant bacteria were isolated, 44 % of which exhibited resistance to three or more antibiotics, i.e., were multidrug resistant bacteria. The highest resistance frequencies were observed against the sulfamethoxazole/trimethoprim combination (62 %), erythromycin (55 %), and amoxicillin (52 %), whereas oxytetracycline (7 %) had the lowest resistance incidence. Notably, several of the environmental strains identified, such as Psychrobacter spp., Denicitrifimonas spp., Brevundimonas spp., Achromobacter spp., and Glutamicibacter spp., have not previously been reported as antibiotic-resistant or multidrug-resistant, highlighting emerging risks within the microbial community associated with swine manure.