Discovery and genome analysis of the plant growth-promoting endophytic bacterium Enterobacter cloacae ENHK

Abstract

Enterobacter cloacae strain ENHK is a Gram-negative endophyte belonging to the family Enterobacteriacae. Initially considered as an unknown bacterium, E. cloacae ENHK was first isolated from a diseased pepper (Capsicum annuum) plant infected by the bacterial plant pathogen Ralstonia solanacearum in Hong Kong in 2010. A pure isolate was obtained for whole genome sequencing. De novo shortgun and paired-end sequencing by 454 GS Junior platform were applied to obtain a draft genome sequence of E. cloacae ENHK containing 36 contigs in one scaffold. Gaps were closed by PCR and primer walking using Sanger sequencing to produce the first complete genome sequence of a plant-associated strain of E. cloacae. The genome size of E. cloacae ENHK consists of a single chromosome of 4,726,582kb, with a GC content of 55.1%. Gene annotation and analysis was performed using NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) and further annotation and comparative genome analysis was performed by the SEED-based automated annotation system provided by the RAST server. Comparative genome analysis indicated that E. cloacae ENHK shares major genomic features with Enterobacter sp.638 that is characterized for its plant-growth promoting and endophytic behaviors. Further genome analysis revealed antagonistic potentials of E. cloacae ENHK against other microbes by possessing antagonistic mechanisms which involve microbial competition, production of antimicrobial compounds and induction of plant defense response. Candidates of Chitinases and type VI secretion system associated rhs-related genetic element were identified and their potential antibacterial activity were investigated. E. cloacae ENHK was further demonstrated to suppress the growth of several plant pathogenic fungal species Alternaria sp., Choanephora infundibulifera, Colletotrichum capsici, Didymella bryoniae, Fusarium oxysporum, Sclerotinia sclerotiorum and Sclerotinia rolfsii and a plant pathogenic bacterial species Ralstonia solanacearum. Among the publicly available completed genome sequences of the Enterobacter species complex, E. cloacae ENHK is most closely related to E. cloacae subsp. cloacae ATCC13047, an opportunistic human pathogen. A comparative genome analysis showed that critical factors involving human pathogenesis in terms of virulence and specific adhesion were identified in the variable genomic regions in E. cloacae subsp. cloacae ATCC 13047 and are absent in E. cloacae ENHK. In addition, two microbial competition related type VI secretion systems (T6SS) were found conserved in both E. cloacae strains. Phylogenetic analysis revealed that the two systems were associated with other plant-associated and human/animal-associated species respectively in the Enterobacteriaceae. The results indicated that T6SSs may provide the E. cloacae strains fitness advantages for microbial competition in the microflora of a diverse environment. In short, comparative genome analysis suggested that the conserved chromosomal regions retain the general physiological and survival of the species, while variable genomic regions play a critical role in determining the functional differences of the pathogenic E. cloacae subsp. cloacae ATCC13047 and the endophytic E. cloacae ENHK. Finally, significant findings derived from the current thesis research are summarized and potential applications of E. cloacae ENHK in agricultural, medical and industrial areas, as well as future prospectus are discussed.