Comparative genome analyses of Serratia marcescens FS14 reveals its high antagonistic potential

Abstract

S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens.

Fig 1. Taxonomic classification of Serratia marcescens FS14.

(A) Maximum Likelihood Tree using Poisson correction model [35] and with 100 bootstrap replicates was constructed based on 2 conserved proteins among all bacteria—GyrB and RpoD—by MEGA 5 [34]. (B) The phylogenomic tree was constructed by MrBayes [17] using the random concatenation of 731 aligned core genes as the dataset and GTR + G+ I as the substitution model. The chain length was set to 10,000,000 (1 sample/1000 generations) whilst the burn-in was set as 2000. Posterior probabilities are denoted at nodes.

Fig 2. Venn diagram of five representative Serratia spp..

Five representative genomes, S. marcescens FS14 (CP005927), S. proteamaculans 568 (CP000826), S. plymuthica AS13 (CP002775), S. liquefaciens ATCC27592 (CP006252), and S. fonticola RB-25 (CP007044) were selected to illustrate the Venn diagram. The Venn diagram was not drawn in proportion; it sole purpose is to illustrate the common CDSs shared between the five strains. The overlapping regions represent CDSs shared with respective strains. The number outside the overlapping regions indicates the number of CDSs in each genome without homologs in other Serratia genomes.

Fig 3. Antagonistic activities of Serratia marcescens FS14 against fungi.

(A) Visualization of the FS14- Sclerotinia sclerotiorum (Sscl) and FS14-Fusarium oxysporum (Foxy) confrontation assays, 2–7 days after inoculation of Sscl (1^st and 2^nd rows) and Foxy (3^rd and 4^th rows) in the presence of S. marcescens FS14 (1^st and 3^rd rows) or in its absence (2^nd or 4^th rows); (B) Growth of F. oxysporum and S. sclerotiorum with and without FS14 were closely monitored. Challenging fungi were grown on PDA plates as described in Materials and Methods, the areas of growth of hyphae (in cm²) was measured. Numbers show an average of 3 plates, and error bars show standard errors of the means.

Fig 4. Antagonistic activity of Serratia marcescens FS14 against Ralstonia solanacearum NJ.

(A) Recovery of viable R. solanacearum (RSNJ) cells after 8 hours of co-culture with (initial ratio of 1:1) and without FS14 at 28°C (see Materials and Methods for details); (B) The planktonic culture with quantities of S. marcescens FS14 (FS14) and R. solanacearum NJ (RSNJ) recorded at 0, 2, 4, 6, 8, 10, 12 and 24 hours after incubation. Numbers show an average of three replications, and error bars show standard errors of the means. a: the quantities of FS14 in the samples collected from the planktonic culture containing 1:1 of FS14 and RSNJ. b: the quantities of RSNJ in the samples collected from the planktonic culture containing 1:1 of FS14 and RSNJ.

Fig 5. Genetic organizations of T6SS in Serratia and other bacteria.

(A), (B), (C) and (D) show the genetic organizations of the 11 T6SS clusters found in Serratia, which have different organizations and separate into four families (T6SS-a, T6SS-b, T6SS-c and T6SS-d). T6SS-a was aligned across S. proteamaculans 568, S. fonticola RB-25, S. marcescens Db11, FGI 94, FS14 and WW4 genomes at the corresponding loci. T6SS-b was found in S. marcescens FS14 and WW4. T6SS-c was only found in S. fonticola RB-25. T6SS-d was found in S. proteamaculans 568 and S. marcescens FGI 94. Each T6SS cluster contains conserved T6SS core component genes. Solid blue/green/purple/orange color boxes indicate the conserved T6SS core genes; gray boxes represent variable conserved genes and white boxes are unique genes. Details of the genetic organization of T6SS clusters in Serratia are listed in S4 Table.

Fig 6. Phylogenetic tree of prodigiosin biosynthesis genes from all prodiginine-producing bacteria.

The Maximum Likelihood Tree with 100 bootstrap replicates was constructed based on Poisson correction model [35] with concatenated amino acid sequences of 8 proteins, PigA(RedW), PigC(RedH), PigF(RedI), PigG(RedO), PigH(RedN), PigI(RedM), PigK(RedY) and PigM(RedV) that are involved in prodiginine-biosynthesis and commonly found among 11 prodiginine-producing bacteria. Adjacent colored squares represent different kinds of prodiginines produced by the bacteria: prodigiosin (orange), undecylprodigiosin (red), cycloprodigiosin (purple), cyclononylprodigiosin (green), and butyl-meta-cyclo-heptylprodiginine (blue).

Fig 7. Synteny of the prodigiosin biosynthesis pig clusters among pigmented Serratia spp..

Genes are symbolized by arrows. The white arrows denote condensing enzymes; black arrows represent genes that encode proteins required for the biosynthesis of the monopyrroles; genes encoding proteins required for the biosynthesis of 4-methoxy-2,2-bipyrrole-5-carboxyaldehyde are in thin diagonally striped arrows; checkered arrows are used to highlight the additional pigO gene only found in ATCC39006; vertical striped arrows denote other flanking genes.