Adaptive mutation and pellicle formation in static culture of Burkholderia glumae causing bacterial grain rot

Abstract

Bacteria under unfavorable environment make necessary adjustments to survive. The morphological and genetic diversification in bacteria involve changes including lifestyle switch from motile to sessile cells in pellicle initiation and development. The pellicle as a thin buoyant multicellular layer at the air-liquid interface in Gram-negative bacteria have received little attention compared to the solid surface colonizing biofilm produced from Gram-negative bacteria. Though, there remain questions to be answered in functional role and the trigger of bacterial cellulose biosynthesis as pellicles, here the aerobic rice pathogen Burkholderia glumae BGR1 biosynthesizing cellulase-sensitive pellicles in bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP)- and flagellum-dependent, yet in quorum sensing independent manner with colony morphological variation and reduced virulence is shown. The elevated level of c-di-GMP was detected in pellicle forming colony variants. Since c-di-GMP is involved in adhesin production and motility associated biofilm formation inhibition, the PAS/GGDEF motif possessing diguanylate cyclase gene for c-di-GMP diosynthesis, the pelI from B. glumae and the adapted GGDEF motif carrying response regulator, pleD, from Agrobacterium tumefaciens was taken to investigate a role in facilitated pellicle formation in B. glumae under unfavorable in vitro condition. A cluster of genes involved in bacterial cellulose biosynthesis (BCS) was found to harbor bcsD, bcsR, bcsQ, bcsA, bcsB, bcsZ, and bcsC in B. glumae. An inverse relationship between the mutations in BCS genes and virulence of BCS mutants in rice plants was observed that the mutants had significantly reduced virulence. The bcsB upregulated PAS/PAC domain carrying bspP (BGLU_RS28885) mutant strain in B. glumae had facilitated pellicle formation. The quicker pellicle formation was observed from bspP spontaneous deletion or point mutation strains of B. glumae. The spontaneous mutations in bspP was considered as genetic sacrifice to better adapt to unfavorable environment and to survive. By possessing relatively flexible genome structures as survival tactics in bacteria, the aerobic BGR1 genetically alters genotype resulting phenotypic changes that fits the environment better. The colonies with various morphology and quicker pellicle formation as more fitted individuals were able to survive a week in static culture growth condition. The unfavorable bacterial niche such as omitted aeration from in vitro culture condition for an aerobe, triggered colony variants with diverse genetic mutations in bspP to facilitate pellicle formation for aerobic cells to gain excess to available oxygen. The cells in static culture were able to survive as they avoid cell death due to alkalization via the obcA upregulation for keeping extracellular pH close to neutral through oxalate production. Hence, the extended survivorship in bspP mutant was supported by bcsB gene upregulation, and oxalate-mediated detoxification of the alkaline static environment. Though bspP as adaptive genetic provided successful extension in viability in unfavorable environment, had adverse effects on B. glumae colonization and virulence in the host.

세균벼알마름병원균은 호기성 식물 병원균으로써 호의적이지 않은 환경에서 생존하기 위해 운동성 세균 생활방식을 고착성 세균 생활방식으로 전환하고 유전자 희생을 통한 세균막을 형성 후 생존한다. 유전 및 표현형의 환경 적응을 위한 변이는 환경에 가장 적합하게 변이 되는데, 호기성인 세균벼알마름병원균 야생형 BGR1은 공기 순환 없는 액체 배양 조건에서 편모 의존적 운동성으로 대기와 가까운 액체의 경계 면으로 이동하여 생존하고 빠르게 세균막을 이룬다. 대기와 배양액의 경계 면에 도달한 BGR1은 운동성을 잃고 세균막을 이루는 세균 군집의 모양은 고체 배지 배양 시 각기 다르게 나타난다. 촉진된 세균막은 bis-(3′-5′)-cyclic dimeric guanosine monophosphate(c-di-GMP)의 생합성이 증가하여 이루어진 현상으로 호의적이지 않은 생체외 시험관적 조건에서 생존하기 위한 적응이다. 생체외 시험관 적 조건에서 생존하기위한 적응이 성공적으로 BGR1이 살 수 있도록 도움이 되었지만 생체내, 벼에 접종시 병원성이 현저히 감소되거나 사라진 것이 확인되었다.