Comparative genome analysis of Enterobacter cloacae

Abstract

The Enterobacter cloacae species includes an extremely diverse group of bacteria that are associated with plants, soil and humans. Publication of the complete genome sequence of the plant growth-promoting endophytic E. cloacae subsp. cloacae ENHKU01 provided an opportunity to perform the first comparative genome analysis between strains of this dynamic species. Examination of the pan-genome of E. cloacae showed that the conserved core genome retains the general physiological and survival genes of the species, while genomic factors in plasmids and variable regions determine the virulence of the human pathogenic E. cloacae strain; additionally, the diversity of fimbriae contributes to variation in colonization and host determination of different E. cloacae strains. Comparative genome analysis further illustrated that E. cloacae strains possess multiple mechanisms for antagonistic action against other microorganisms, which involve the production of siderophores and various antimicrobial compounds, such as bacteriocins, chitinases and antibiotic resistance proteins. The presence of Type VI secretion systems is expected to provide further fitness advantages for E. cloacae in microbial competition, thus allowing it to survive in different environments. Competition assays were performed to support our observations in genomic analysis, where E. cloacae subsp. cloacae ENHKU01 demonstrated antagonistic activities against a wide range of plant pathogenic fungal and bacterial species.

Figure 1. Phylogenomic analysis of Enterobacter spp. Bayesian tree with posterior p-values of the genomes of Enterobacter spp. using 1732 core genes of eight Enterobacter and three Pantoea genomes.

Figure 2. Genomic alignment of Enterobacter cloacae.

MAUVE [35] alignment of the genome sequences of E. cloacae subsp. cloacae ATCC13047, E. cloacae subsp. dissolvens SDM, E. cloacae EcWSU1 and E. cloacae subsp. cloacae ENHKU01. Same color boxes represent homologous regions of sequence, without rearrangement (locally collinear blocks or LCB), shared between E. cloacae genomes. Black arrows show the genomic position of the T4SS located in ATCC13047.

Figure 3. Comparison of subsystem features between E. cloacae.

Genome sequences of ATCC13047, SDM, EcWSU1 and ENHKU01 were uploaded to the SEED Viewer server (http://rast.nmpdr.org/seedviewer.cgi) independently. Functional roles of RAST annotated genes were assigned and grouped in subsystem feature categories as shown in the figure [26], and colored bars indicate the number of genes assigned to each category. Details for subsystem and functional role assignment for the genes of each strain are listed in Data S1.

Figure 4. Venn diagram of four E. cloacae strains.

The Venn diagram shows the pan-genome of ATCC13047, SDM, EcWSU1 and ENHKU01 generated using EDGAR [27]. Overlapped regions represent common CDS shared between the E. cloacae genomes. The number outside the overlapped regions indicates the number of CDS in each genome without homologs in other sequenced E. cloacae genomes.

Figure 5. Phylogenetic analysis of chitinase genes.

Neighbor-joining tree with bootstrap values of chitinase genes associated with (A) ECENHK_07430 and (B) ECENHK_08915 constructed by twenty-five representing orthologs from each species using MEGA5. The blue box indicates the corresponding chitinase gene of ENHKU01. Chitinases that have been functionally characterized for their antifungal activities are highlighted in green boxes [87,88,89,90,91].

Figure 6. Genetic organization of T6SS in Enterobacter .

(A) and (B) show genetic organization of the two T6SS clusters commonly found in Enterobacter . ENHKU01 and ATCC13047 contain both T6SS, and EcWSU1, SDM and LF7a have one of the two, and other Enterobacter have none. The two clusters of T6SS in Enterobacter have different genetic organization but are aligned across the Enterobacter genomes at the corresponding loci. Each T6SS cluster is composed of conserved regions formed by conserved T6SS core component genes, which are indicated in solid blue/ green color boxes, and variable regions that are indicated by arrows. The variable regions contain a variable number of conserved genes (solid gray color) and unique genes (white color boxes). Most genes located in the variable regions are described as hypothetical proteins. Genes possibly involved in bacteriocin activity are shown in red. Details of the genetic organization of T6SSs in Enterobacter are listed in Data S4.

Figure 7. Phylogenetic analysis of ClpV.

(A) The neighbor-joining tree of T6SSs using ClpV orthologs from 348 T6SS clusters in 231 species. The 231 species are grouped according to their class, which is indicated using color lines: α for the subdivision of Proteobacteria (Pink), β subdivision (Green), γ subdivision (Blue), δ/α subdivision (Black) and other bacteria unrelated to Proteobacteria (Gray). ClpV orthologs are distributed in five clades and named I-V. Naming of clades is according to Boyer et al [86]. Each clade is contributed by different bacterial families of Proteobacteria. Our result is consistent with a previous phylogenetic analysis of T6SS using 13 T6SS conserved component genes [86]. ClpV of E. cloacae are distributed in clades II and III of the phylogenetic tree and are clustered together with other strains and species in the family of Enterobacteriaceae possessing T6SS, thus forming sub-trees in each clade (indicated by gray circles). A simplified and enlarged version of the neighbor-joining tree with bootstrap values showing (B) the sub-tree of clade III formed by ClpV orthologs associated with ECENHK_13140 and (C) the sub-tree of clade II associated with ECENHK_15865. Color squares indicate the habitats of the corresponding bacterial species: plant/ soil-associated (light green), insect-associated (green) and Human/ animal associated (orange).

Figure 8. Antagonistic activity of E. cloacae subsp. cloacae ENHKU01 against fungi.

(A) Visualization of fungal growth with and without ENHKU01: (from left to right, upper row) Alternaria sp., Colletotrichum capsici , Didymellabryoniae , (from left to right, lower row) Fusarium oxysporum and Sclerotinia sclerotiorum. Photos were taken 7 days after incubation; (B) Growth of Colletotrichum capsici (Col) and Sclerotiniascleotiorum (Scl) were closely monitored with and without ENHKU01. Challenging fungi were grown on PDA plates as described in Methods and Materials, the radius of growth of hyphae (in cm) was measured. Numbers show an average of 10 plates, and error bars represent the S.D. from the mean.

Figure 9. Antagonistic activity of E. cloacae subsp. cloacae ENHKU01 against R. solanacearum.

Figures show results of ENHKU01 – R. solanacearum competition assays: (A) the planktonic culture with the quantities of E. cloacae subsp. cloacae ENHKU01 (ENHK) and R. solanacearum (RSHK) in log C.F.U. per ml recorded at 0, 2, 4, 6 and 24 hours after incubation. Numbers show an average of 4 replications, and error bars represent the S.D. from the mean; (B) Biofilm culture, relative percentage of E. cloacae subsp. cloacae ENHKU01 (ENHK): R. solanacearum (RSHK) at day 0 and day 1 of incubation.