Plant-microbe-microbe interactions influence the faba bean nodule colonization by diverse endophytic bacteria.

Legume root nodules harbor rhizobia and other non-nodulating endophytes known as nodule-associated bacteria (NAB) whose role in the legume symbiosis is still unknown. We analysed the genetic diversity of 34 NAB isolates obtained from the root nodules of faba bean grown under various soil conditions in Egypt using 16S rRNA and concatenated sequences of three housekeeping genes. All isolates were identified as members of the family Enterobacteriaceae belonging to the genera Klebsiella, Enterobacter and Raoultella. We identified nine enterobacterial genospecies, most of which have not been previously reported as NAB. All isolated strains harbored nifH gene sequences and most of them possessed plant growth-promoting (PGP) traits. Upon co-inoculation with an N2 fixing rhizobium (Rlv NGB-FR128), two strains (Enterobacter sichanensis NGB-FR97 and Klebsiella variicola NGB-FR116) significantly increased nodulation, growth and N-uptake of faba bean plants over the single treatments or the uninoculated control. The presence of these enterobacteria in nodules was significantly affected by the host plant genotype, symbiotic rhizobium genotype and endophyte genotype, indicating that the nodule colonization process is regulated by plant-microbe-microbe interactions. This study emphasizes the importance of nodule-associated enterobacteria and suggests their potential role in improving the effectiveness of rhizobial inoculants.

Boosting isoprene production via heterologous expression of the Kudzu isoprene synthase gene (kIspS) into Bacillus spp. cell factory.

Isoprene represents a key building block for the production of valuable materials such as latex, synthetic rubber or pharmaceutical precursors and serves as basis for advanced biofuel production. To enhance the production of the volatile natural hydrocarbon isoprene, released by plants, animals and bacteria, the Kudzu isoprene synthase (kIspS) gene has been heterologously expressed in Bacillus subtilis DSM 402 and Bacillus licheniformis DSM 13 using the pHT01 vector. As control, the heterologous expression of KIspS in E. coli BL21 (DE3) with the pET28b vector was used. Isoprene production was analyzed using Gas Chromatography Flame Ionization Detector. The highest isoprene production was observed by recombinant B. subtilis harboring the pHT01-kIspS plasmid which produced 1434.3 µg/L (1275 µg/L/OD) isoprene. This is threefold higher than the wild type which produced 388 µg/L (370 µg/L/OD) isoprene, when both incubated at 30 °C for 48 h and induced with 0.1 mM IPTG. Additionally, recombinant B. subtilis produced fivefold higher than the recombinant B. licheniformis, which produced 437.2 µg/L (249 µg/L/OD) isoprene when incubated at 37 °C for 48 h induced with 0.1 mM IPTG. This is the first report of optimized isoprene production in B. licheniformis. However, recombinant B. licheniformis showed less isoprene production. Therefore, recombinant B. subtilis is considered as a versatile host for heterologous production of isoprene.

Genetic improvement of n-butanol tolerance in Escherichia coli by heterologous overexpression of groESL operon from Clostridium acetobutylicum.

Strain tolerance to toxic metabolites remains an important issue in the production of biofuels. Here we examined the impact of overexpressing the heterologous groESL chaperone from Clostridium acetobutylicum to enhance the tolerance of Escherichia coli against several stressors. Strain tolerance was identified using strain maximum specific growth rate (µ) and strain growth after a period of solvent exposure. In comparison with control strain, the groESL overexpressing strain yielded a 27 % increase in growth under 0.8 % (v/v) butanol, a 9 % increase under 1 % (v/v) butanol, and a 64 % increase under 1.75 (g/l) acetate. Moreover, after 10 h, groESL overexpression resulted in increase in relative tolerance of 58 % compared with control strain under 0.8 % (v/v) butanol, 56 % increase under 1 % (v/v) butanol, 42 % increase under 1 % (v/v) isobutanol, 36 % increase under 4 % (v/v) ethanol, 58 % increase under 1.75 (g/l) acetate. These data demonstrate that overexpression of the groESL from C. acetobutylicum in E. coli increased tolerance to several stressors. Solvent tolerant strain of E. coli was developed to be used as a basic strain for biofuel production.

Genetic determinants for n-butanol tolerance in evolved Escherichia coli mutants: cross adaptation and antagonistic pleiotropy between n-butanol and other stressors.

Cross-tolerance and antagonistic pleiotropy have been observed between different complex phenotypes in microbial systems. These relationships between adaptive landscapes are important for the design of industrially relevant strains, which are generally subjected to multiple stressors. In our previous work, we evolved Escherichia coli for enhanced tolerance to the biofuel n-butanol and discovered a molecular mechanism of n-butanol tolerance that also conferred tolerance to the cationic antimicrobial peptide polymyxin B in one specific lineage (green fluorescent protein [GFP] labeled) in the evolved population. In this work, we aim to identify additional mechanisms of n-butanol tolerance in an independent lineage (yellow fluorescent protein [YFP] labeled) from the same evolved population and to further explore potential cross-tolerance and antagonistic pleiotropy between n-butanol tolerance and other industrially relevant stressors. Analysis of the transcriptome data of the YFP-labeled mutants allowed us to discover additional membrane-related and osmotic stress-related genes that confer n-butanol tolerance in E. coli. Interestingly, the n-butanol resistance mechanisms conferred by the membrane-related genes appear to be specific to n-butanol and are in many cases antagonistic with isobutanol and ethanol. Furthermore, the YFP-labeled mutants showed cross-tolerance between n-butanol and osmotic stress, while the GFP-labeled mutants showed antagonistic pleiotropy between n-butanol and osmotic stress tolerance.