Fitness-Conditional Genes for Soil Adaptation in the Bioaugmentation Agent Pseudomonas veronii 1YdBTEX2.

Strain inoculation (bioaugmentation) is a potentially useful technology to provide microbiomes with new functionalities. However, there is limited understanding of the genetic factors contributing to successful establishment of inoculants. This work aimed to characterize the genes implicated in proliferation of the monoaromatic compound-degrading Pseudomonas veronii 1YdBTEX2 in nonsterile polluted soils. We generated two independent mutant libraries by random minitransposon-delivered marker insertion followed by deep sequencing (Tn-seq) with a total of 5.0 × 105 unique insertions. Libraries were grown in multiple successive cycles for up to 50 generations either in batch liquid medium or in two types of soil microcosms with different resident microbial content (sand or silt) in the presence of toluene. Analysis of gene insertion abundances at different time points (passed generations of metapopulation growth), in comparison to proportions at start and to in silico generated randomized insertion distributions, allowed to define ~800 essential genes common to both libraries and ~2,700 genes with conditional fitness effects in either liquid or soil (195 of which resulted in fitness gain). Conditional fitness genes largely overlapped among all growth conditions but affected approximately twice as many functions in liquid than in soil. This indicates soil to be a more promiscuous environment for mutant growth, probably because of additional nutrient availability. Commonly depleted genes covered a wide range of biological functions and metabolic pathways, such as inorganic ion transport, fatty acid metabolism, amino acid biosynthesis, or nucleotide and cofactor metabolism. Only sparse gene sets were uncovered whose insertion caused fitness decrease exclusive for soils, which were different between silt and sand. Despite detectable higher resident bacteria and potential protist predatory counts in silt, we were, therefore, unable to detect any immediately obvious candidate genes affecting P. veronii biological competitiveness. In contrast to liquid growth conditions, mutants inactivating flagella biosynthesis and motility consistently gained strong fitness advantage in soils and displayed higher growth rates than wild type. In conclusion, although many gene functions were found to be important for growth in soils, most of these are not specific as they affect growth in liquid minimal medium more in general. This indicates that P. veronii does not need major metabolic reprogramming for proliferation in soil with accessible carbon and generally favorable growth conditions. IMPORTANCE Restoring damaged microbiomes is still a formidable challenge. Classical widely adopted approaches consist of augmenting communities with pure or mixed cultures in the hope that these display their intended selected properties under in situ conditions. Ecological theory, however, dictates that introduction of a nonresident microbe is unlikely to lead to its successful proliferation in a foreign system such as a soil microbiome. In an effort to study this systematically, we used random transposon insertion scanning to identify genes and possibly, metabolic subsystems, that are crucial for growth and survival of a bacterial inoculant (Pseudomonas veronii) for targeted degradation of monoaromatic compounds in contaminated nonsterile soils. Our results indicate that although many gene functions are important for proliferation in soil, they are general factors for growth and not exclusive for soil. In other words, P. veronii is a generalist that is not a priori hindered by the soil for its proliferation and would make a good bioaugmentation candidate.

PHO1 family members transport phosphate from infected nodule cells to bacteroids in Medicago truncatula.

Legumes play an important role in the soil nitrogen availability via symbiotic nitrogen fixation (SNF). Phosphate (Pi) deficiency severely impacts SNF because of the high Pi requirement of symbiosis. Whereas PHT1 transporters are involved in Pi uptake into nodules, it is unknown how Pi is transferred from the plant infected cells to nitrogen-fixing bacteroids. We hypothesized that Medicago truncatula genes homologous to Arabidopsis PHO1, encoding a vascular apoplastic Pi exporter, are involved in Pi transfer to bacteroids. Among the seven MtPHO1 genes present in M. truncatula, we found that two genes, namely MtPHO1.1 and MtPHO1.2, were broadly expressed across the various nodule zones in addition to the root vascular system. Expressions of MtPHO1.1 and MtPHO1.2 in Nicotiana benthamiana mediated specific Pi export. Plants with nodule-specific downregulation of both MtPHO1.1 and MtPHO1.2 were generated by RNA interference (RNAi) to examine their roles in nodule Pi homeostasis. Nodules of RNAi plants had lower Pi content and a three-fold reduction in SNF, resulting in reduced shoot growth. Whereas the rate of 33Pi uptake into nodules of RNAi plants was similar to control, transfer of 33Pi from nodule cells into bacteroids was reduced and bacteroids activated their Pi-deficiency response. Our results implicate plant MtPHO1 genes in bacteroid Pi homeostasis and SNF via the transfer of Pi from nodule infected cells to bacteroids.

Prediction of regulatory long intergenic non-coding RNAs acting in trans through base-pairing interactions.

BACKGROUND: Long intergenic non-coding RNAs (lincRNAs) can act as regulators of expression of protein-coding genes. Trans-natural antisense transcripts (trans-NATs) are a type of lincRNAs that contain sequence complementary to mRNA from other loci. The regulatory potential of trans-NATs has been poorly studied in eukaryotes and no example of trans-NATs regulating gene expression in plants are reported. The goal of this study was to identify lincRNAs, and particularly trans-NATs, in Arabidopsis thaliana that have a potential to regulate expression of target genes in trans at the transcriptional or translational level. RESULTS: We identified 1001 lincRNAs using an RNAseq dataset from total polyA+ and polysome-associated RNA of seedlings grown under high and low phosphate, or shoots and roots treated with different phytohormones, of which 550 were differentially regulated. Approximately 30% of lincRNAs showed conservation amongst Brassicaceae and 25% harbored transposon element (TE) sequences. Gene co-expression network analysis highlighted a group of lincRNAs associated with the response of roots to low phosphate. A total of 129 trans-NATs were predicted, of which 88 were significantly differentially expressed under at least one pairwise comparison. Five trans-NATs showed a positive correlation between their expression and target mRNA steady-state levels, and three showed a negative correlation. Expression of four trans-NATs positively correlated with a change in target mRNA polysome association. The regulatory potential of these trans-NATs did not implicate miRNA mimics nor siRNAs. We also looked for lincRNAs that could regulate gene expression in trans by Watson-Crick DNA:RNA base pairing with target protein-encoding loci. We identified 100 and 81 with a positive or negative correlation, respectively, with steady-state level of their predicted target. The regulatory potential of one such candidate lincRNA harboring a SINE TE sequence was validated in a protoplast assay on three distinct genes containing homologous TE sequence in their promoters. Construction of networks highlighted other putative lincRNAs with multiple predicted target loci for which expression was positively correlated with target gene expression. CONCLUSIONS: This study identified lincRNAs in Arabidopsis with potential in regulating target gene expression in trans by both RNA:RNA and RNA:DNA base pairing and highlights lincRNAs harboring TE sequences in such activity.