Calcium regulates the mycophagous ability of Burkholderia gladioli strain NGJ1 in a type III secretion system-dependent manner

Abstract

Background: A rice associated bacterium Burkholderia gladioli strain NGJ1 demonstrates mycophagy, a phenomenon wherein bacteria feed on fungi. Previously, we have reported that NGJ1 utilizes type III secretion system (T3SS) to deliver a prophage tail-like protein (Bg_9562) into fungal cells to establish mycophagy. Results: In this study, we report that calcium ion concentration influences the mycophagous ability of NGJ1 on Rhizoctonia solani, an important fungal pathogen. The calcium limiting condition promotes mycophagy while high calcium environment prevents it. The expression of various T3SS apparatus encoding genes of NGJ1 was induced and secretion of several potential T3SS effector proteins (including Bg_9562) into extracellular milieu was triggered under calcium limiting condition. Using LC-MS/MS proteome analysis, we identified several calcium regulated T3SS effector proteins of NGJ1. The expression of genes encoding some of these effector proteins was upregulated during mycophagous interaction of NGJ1 with R. solani. Further, mutation of one of these genes (endo-β-1, 3-glucanase) rendered the mutant NGJ1 bacterium defective in mycophagy while complementation with full length copy of the gene restored its mycophagous activity. Conclusion: Our study provides evidence that low calcium environment triggers secretion of various T3SS effectors proteins into the extracellular milieu and suggests the importance of cocktail of these proteins in promoting mycophagy.

The alternative sigma factors, rpoN1 and rpoN2 are required for mycophagous activity of Burkholderia gladioli strain NGJ1

“…We have previously reported that T3SS plays an important role during mycophagy, the T3SS mutant NGJ1 bacterium being completely defective in mycophagy (Swain et al ., 2017). The wild‐type NGJ1 deploys various T3SS effectors, including Bg_9562, a prophage tail‐like protein (Swain et al ., 2017), and endo‐β‐1, 3‐ glucanase (Yadav et al ., 2020) to potentially lyse fungal cells to feed on them. The data presented in the current study reflect that the expression of Bg_9562 , endo‐β‐1 , 3‐glucanase as well as T3SS apparatus ( hrcC and hrcN ) encoding genes are upregulated during mycophagous interaction of wild‐type NGJ1 with R. solani and the upregulation is dependent on RpoN1 and RpoN2.…”

“…Considering this, we anticipate them to be under indirect regulation of RpoNs in NGJ1. This is further supported by our observation that rpoN1 and rpoN2 genes (upregulated during 24 h of mycophagous interaction) exhibit different temporal expression with that of T3SS genes (upregulated during 48 h, but not during 24 h of mycophagy interaction; Yadav et al ., 2020). In this regard, it is to be noted that RpoN modulates T3SS functions in Erwinia amylovora (a plant pathogenic bacteria) via regulation of HrpL (an alternative sigma factor; sigma 70), the master regulator of T3SS (Ancona et al ., 2014; Lee et al ., 2016).…”

“…Certain Burkholderia sp. are opportunistic human and plant pathogens (Suárez‐Moreno et al ., 2012; Syed and Wooten, 2021), while many others exhibit plant growth promotion (Castanheira et al ., 2016), antimicrobial (Yadav et al ., 2020), and symbiotic nitrogen fixation ability (De Meyer et al ., 2016). Moreover, many Burkholderia sp.…”

“…We have previously demonstrated that a rice‐associated B. gladioli strain NGJ1 utilizes T3SS to forage over Rhizoctonia solani (a necrotrophic fungal pathogen having wide host range) (Swain et al ., 2017). Further, we have reported Bg_9562, a prophage tail‐like protein (Swain et al ., 2017) and a endo‐β‐1,3‐glucanase (Yadav et al ., 2020) to be novel T3SS effectors of NGJ1, being important for mycophagy on R. solani .…”

Effect of metal ions on lipopeptide secretion from Bacillus subtilis strain FJAT-4: Negative regulation by Ca2+

“…For example, Ca 2+ could influence the adhesion of Bacillus sp. presumably by regulating aggregation and formation of extracellular polymeric substances (He et al, 2016), changes in Ca 2+ concentration could affect pectinolytic enzyme production in Pectobacterium carotovorum and biofilm formation in Xylella fastidiosa (Flego et al, 1997;Parker et al, 2016); the swarming motility of Pseudomonas syringae grown on agar medium supplemented with Ca 2+ was reduced, due to the precipitation of calcium phosphate on the surface of cells (Fishman et al, 2018); and the mycophagous ability of Burkholderia gladioli strain NGJ1 against Rhizoctonia solani was regulated by Ca 2+ in a type III secretion system-dependent manner (Yadav et al, 2020). It has also been shown that Ca 2+ modulates bacterial gene expression (Fishman et al, 2018;Gode-Potratz et al, 2010), suggesting its regulatory properties.…”