Identification of a major cyclic-di-GMP synthase gene involved in pellicle formation in Burkholderia glumae

Abstract

셀룰로스가 주성분인 pellicle은 액체 배지와 공기 사이에 형성되는 얇은 막으로 여러 가지 환경적인 스트레스로부터 생물체를 보호하는 생물막의 일종이다. 세균이 형성하는 생물막은 주로 밀도인식기작인 quorum sensing과 second messenger인 cyclic-di-GMP (c-di-GMP)의 조절을 받는 것으로 알려져 있는데, 세포 내의 c-di-GMP 농도는 GG(D/E)EF와 EAL domain을 통해 합성, 분해되어 조절된다. 본 연구에서는 세균성벼알마름병을 일으키는 병원균인 Burkholderia glumae의 주요 c-di-GMP 합성유전자가 과발현되었을 때 pellicle 형성이 촉진되는 동시에 flagella의 기능에 영향을 미침으로써 motility를 저해함을 밝혔다. B. glumae의 wild-type strain인 BGR1을 Luria-Bertani (LB) 배지에 접종하여 28℃에서 4일 동안 배양하면 배지표면에 pellicle이 형성되는 것을 관찰하였다. Tn3-gusA fusion을 통해 B. glumae의 셀룰로스 합성유전자들이 pellicle 형성과 관련이 있고, GG(D/E)EF와 EAL domain을 encoding하는 여러 유전자 중 pelI가 IPTG inducible promoter로 과발현되었을 때 pellicle 형성이 촉진되는 것을 관찰하였으며, swimming assay와 bacterial cell의 속도 측정을 통해 과발현된 pelI가 flagella function에 영향을 주는 것을 확인하였다.

Cellulose is important for protection of bacteria from environmental stresses and consequently makes them live longer and persist their virulence. The wild-type strain of Burkholderia glumae forms pellicle composed of cellulose at surface in Luria-Bertani (LB) medium. B. glumae is a Gram-negative bacterium that causes rice grain rot at the flowering stage. The wild-type strain of B. glumae forms pellicle at 28℃ whereas the quorum sensing (QS) -defective mutants failed to produce pellicle in LB. However, the QS-defective mutants formed pellicle in a buffered LB with 100 mM HEPES. The mutational analysis of genes that are involved in cellulose biosynthesis in B. glumae confirmed that cellulose biosynthetic genes are involved in pellicle formation. In many bacteria, bacterial multi-cellular behaviors including pellicle formation is often regulated by cyclic di-GMP (c-di-GMP), a second messenger. The GGDEF protein synthesizes c-di-GMP, while EAL and HD-GYP proteins hydrolyze c-di-GMP. In the B. glumae BGR1, 21 genes which encode GGDEF and/or EAL domains were identified. All 21 genes were individually mutated with Tn3-gusA or over-expressed under inducible promoter to determine which gene is the most important for pellicle formation. Mutational analysis did not give any clue to identify the most important gene for c-di-GMP synthesis and pellicle formation. Instead, when bglu_2g07220 called pelI was over-expressed in the wild-type strain, pellicle weas formed much faster than the wild-type strain carrying the empty vector. Moreover, the swimming motility was reduced under over-expression of pelI. The swimming velocity decreased and direction of swimming became irregular when pelI was over-expressed. In conclusion, QS does not directly regulate cellulose biosynthetic genes. Failure of pellicle formation by the QS-defective mutant is probably due to indirect influences by QS. Among 21 genes that encode GGDEF and/or EAL domains, pelI plays a major role in cellulose biosynthesis and pellicle formation. The results of swimming assay and velocity measurement in the wild type strain under over-expression of pelI indicate that elevated production of c-di-GMP influences both cellulose biosynthesis and flagella functions in B. glumae.