Timtchenko, T. | bioRxiv Science

bioRxiv2023

Transposon sequencing reveals the essential gene set and genes enabling gut symbiosis in the insect symbiont Caballeronia insecticola

Caballeronia insecticola is a bacterium belonging to the Burkholderia genus sensu lato, able to colonize multiple environments like soils and the gut of the bean bug Riptortus pedestris. To identify the essential genome of a bacterium is a first step in the understanding of its lifestyles. We constructed a saturated Himar1 mariner transposon library and revealed by transposon-sequencing (Tn-seq) that 498 protein-coding genes constitute the essential genome of C. insecticola for growth in free-living conditions. By comparing essential gene sets of C. insecticola and seven related Burkholderia s.l. strains, only 120 common genes were identified indicating that a large part of the essential genome is strain-specific. In order to reproduce specific nutritional conditions that are present in the gut of R. pedestris, we grew the mutant library in minimal media supplemented with candidate gut nutrients and identified several condition-dependent fitness-defect genes by Tn-seq. To validate the robustness of the approach, insertion mutants in six fitness genes were constructed and their growth-deficiency in media supplemented with the corresponding nutrient was confirmed. The mutants were further tested for their efficiency in R. pedestris gut colonization, confirming that gluconeogenic carbon sources, taurine and inositol, are nutrients consumed by the symbiont in the gut.

microbiology↗

bioRxiv2020

Sinorhizobium meliloti functions required for resistance to antimicrobial NCR peptides and bacteroid differentiation

Legumes of the Medicago genus form symbiosis with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of the intracellular symbionts. Members of the Nodule-specific Cysteine Rich peptide (NCRs) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts, including an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes, lipopolysaccharide modifications mediated by lpsB and lpxXL as well as rpoH1, encoding a stress sigma factor. Mutants of these genes show in vitro a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions including peptide transporters, the bacterial envelope structures and stress response regulators to resist the aggressive assault of NCR peptides in the nodule cells. ImportanceThe nitrogen fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plant needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane composition and the stress response regulator RpoH1.

microbiology↗