Genomic analyses of two Alteromonas stellipolaris strains reveal traits with potential biotechnological applications

Abstract

The Alteromonas stellipolaris strains PQQ-42 and PQQ-44, previously isolated from a fish hatchery, have been selected on the basis of their strong quorum quenching (QQ) activity, as well as their ability to reduce Vibrio-induced mortality on the coral Oculina patagonica. In this study, the genome sequences of both strains were determined and analyzed in order to identify the mechanism responsible for QQ activity. Both PQQ-42 and PQQ-44 were found to degrade a wide range of N-acylhomoserine lactone (AHL) QS signals, possibly due to the presence of an aac gene which encodes an AHL amidohydrolase. In addition, the different colony morphologies exhibited by the strains could be related to the differences observed in genes encoding cell wall biosynthesis and exopolysaccharide (EPS) production. The PQQ-42 strain produces more EPS (0.36 g l^-1) than the PQQ-44 strain (0.15 g l^-1), whose chemical compositions also differ. Remarkably, PQQ-44 EPS contains large amounts of fucose, a sugar used in high-value biotechnological applications. Furthermore, the genome of strain PQQ-42 contained a large non-ribosomal peptide synthase (NRPS) cluster with a previously unknown genetic structure. The synthesis of enzymes and other bioactive compounds were also identified, indicating that PQQ-42 and PQQ-44 could have biotechnological applications.

Figure 1

Comparison of the genomes of Alteromonas stellipolaris strains PQQ-42, PQQ-44, LMG 21856 and LMG 21861^T. Venn diagram of orthologous and specific genes in each strain (a). BRIG visualization of chromosomal sequences of strains PQQ-42, PQQ-44, LMG 21856 and LMG 21861^T using PQQ-42 or PQQ-44 as reference sequences (b). Gaps in the diagram indicate areas of the genome present in strains PQQ-42 and/or PQQ-44 but absent in the other strains.

Figure 2

Phylogenetic tree based on concatenated sequences of Alteromonas species. The 16S ribosomal RNA, gyrase subunit B gene (gyrB) and RNA polymerase subunit beta gene (rpoB) were used. The tree was generated using the neighbor-joining method with 1,000 bootstrap replications and Pseudomonas syringae CC 1557 as the outgroup.

Figure 3

Growth differences in Alteromonas stellipolaris PQQ-42 and PQQ-44 in solid and liquid medium. Colony morphology and growth appearance of strains PQQ-42 (a,b) and PQQ-44 (c,d) after 24 hours of incubation in MA and MB.

Figure 4

Budding division of strains PQQ-42 and PQQ-44. Observation by transmission electron microscopy of emerging buds (1), bud chains (2) and prosthecae (3) in strains PQQ-42 (a,d) and PQQ-44 (b,c), as indicated by arrows.

Figure 5

Exopolysaccharide production in strains PQQ-42 and PQQ-44. Uranyl acetate staining of strains PQQ-42 (a) and PQQ-44 (b) as observed by transmission electron microscopy. Ruthenium red staining of strains PQQ-42 (c) and PQQ-44 (d) as observed by transmission electron microscopy. Strains PQQ-42 (e,g) and PQQ-44 (f,h) as observed by scanning electron microscopy after critical point drying. Arrows indicate exopolysaccharide accumulation in each strain. Monosaccharide composition of exopolysaccharides produced by strains PQQ-42 and PQQ-44 determined by ion chromatography (i).

Figure 6

Time-dependent AHL-degradation of synthetic AHLs by strains PQQ-42 and PQQ-44. The graph shows the percentage of signal molecules remaining after 16, 24 and 48 hours of incubation with strain PQQ-42 (a) or PQQ-44 (b). Error bars represent standard deviations.

Figure 7

Phylogenetic analysis of the penicillin acylase of PQQ-42 and PQQ-44. Tree generated by Neighbor-Joining method with 1,000 bootstrap replications and based on known acylase and lactonase aminoacid sequences.

Figure 8

PQQ-42 non-ribosomal peptide synthase gene cluster overview. Biosynthetic, transport and regulation genes (a) and predicted structure of the non-ribosomal peptide encoded in the PQQ-42 genome (b). Gene number and associated predicted function are as follows: 1, α-galactosidase, 2, tetratricopeptide; 3, acetylhydrolase/lipoprotein signal peptide; 4, major facilitator superfamily MFS1; 5, dihydrodipicolinate synthase; 6, hypothetical protein; 7, ATP-binding region, ATPase-like protein/hybrid sensor histidine kinase/response regulator; 8, response regulator receiver protein; 9, hypothetical protein; 10, hypothetical protein; 11, ABC-type siderophore export system, fused ATPase and permease/cyclic peptide transporter; 12, SyrP-like protein; 13, non-ribosomal peptide synthase; 14, non-ribosomal peptide synthetase; 15, long-chain-fatty-acid-CoA ligase/non-ribosomal peptide synthase; 16, hypothetical protein; 17, transcriptional regulator, CadC; 18, hypothetical protein; 19, membrane fusion protein of RND family multidrug efflux pump; 20, acriflavin resistance protein; 21, thioesterase; 22, vulnibactin synthase, phosphopantetheinyl transferase component; 23, hypothetical protein; 24, hypothetical protein; 25, ISXo8 transposase; 26, single-stranded DNA-binding protein; 27, putative sodium-dependent galactose transporter; 28, galactokinase; 29, galactose-1-phosphate uridylyltransferase; 30, galactose mutarotase; 31, galactose operon repressor, GalR-LacI family of transcriptional regulators.

Figure 9

Flagella observed by transmission electron microscopy. Absence of flagellum in strain PQQ-42 (a) and polar flagellum in strain PQQ-44 (b), as indicated by arrow.