Comparative Genomics of Cyclic di-GMP Metabolism and Chemosensory Pathways in Shewanella algae Strains: Novel Bacterial Sensory Domains and Functional Insights into Lifestyle Regulation

Abstract

Shewanella spp. play important ecological and biogeochemical roles, due in part to their versatile metabolism and swift integration of stimuli. While Shewanella spp. are primarily considered environmental microbes, Shewanella algae is increasingly recognized as an occasional human pathogen. S. algae shares the broad metabolic and respiratory repertoire of Shewanella spp. and thrives in similar ecological niches. In S. algae, nitrate and dimethyl sulfoxide (DMSO) respiration promote biofilm formation strain specifically, with potential implication of taxis and cyclic diguanosine monophosphate (c-di-GMP) signaling. Signal transduction systems in S. algae have not been investigated. To fill these knowledge gaps, we provide here an inventory of the c-di-GMP turnover proteome and chemosensory networks of the type strain S. algae CECT 5071 and compare them with those of 41 whole-genome-sequenced clinical and environmental S. algae isolates. Besides comparative analysis of genetic content and identification of laterally transferred genes, the occurrence and topology of c-di-GMP turnover proteins and chemoreceptors were analyzed. We found S. algae strains to encode 61 to 67 c-di-GMP turnover proteins and 28 to 31 chemoreceptors, placing S. algae near the top in terms of these signaling capacities per Mbp of genome. Most c-di-GMP turnover proteins were predicted to be catalytically active; we describe in them six novel N-terminal sensory domains that appear to control their catalytic activity. Overall, our work defines the c-di-GMP and chemosensory signal transduction pathways in S. algae, contributing to a better understanding of its ecophysiology and establishing S. algae as an auspicious model for the analysis of metabolic and signaling pathways within the genus Shewanella. IMPORTANCE Shewanella spp. are widespread aquatic bacteria that include the well-studied freshwater model strain Shewanella oneidensis MR-1. In contrast, the physiology of the marine and occasionally pathogenic species Shewanella algae is poorly understood. Chemosensory and c-di-GMP signal transduction systems integrate environmental stimuli to modulate gene expression, including the switch from a planktonic to sessile lifestyle and pathogenicity. Here, we systematically dissect the c-di-GMP proteome and chemosensory pathways of the type strain S. algae CECT 5071 and 41 additional S. algae isolates. We provide insights into the activity and function of these proteins, including a description of six novel sensory domains. Our work will enable future analyses of the complex, intertwined c-di-GMP metabolism and chemotaxis networks of S. algae and their ecophysiological role.

Keywords: Shewanella; c-di-GMP; chemotaxis; sensing; signal transduction; whole-genome sequencing.

FIG 1

WGS-based distance and taxonomic comparisons using MinHash. An all-by-all pairwise comparison of the MinHash genome sketch for each S. algae strain. The clustered distance matrix depicts the JSI, also referred to as Jaccard distance, for each unique strain pairing based on comparisons of WGS-assembly nucleotide composition. JSI values closer to 1 (darker) showcase higher WGS similarity, while values approaching 0 (lighter) reflect greater dissimilarity. The column annotation bar indicates each strain’s isolation source. Row annotation box colors reflect the S. algae reference genome from the GTDB with the highest JSI value to each of the 42 strains included in the study. Row annotation bar plot values represent the JSI between the query and GTDB subject strains.

FIG 2

Pangenome analysis of S. algae strains. (A) Pangenome rarefaction curves illustrating the growth in pangenome size (top, purple) and stabilization of the core genome (bottom, green) after subsequent genome additions during construction. (B) Pie chart showcasing relative proportions of the core, shell, and cloud pangenome categories. (C) Gene frequency bar plot reflecting the number of homologous gene clusters present (y axis) in respective proportions of the isolate population used to construct the S. algae pangenome (x axis). Bars to the far right indicate gene clusters comprising the core genome. (D) Clustered pangenome dendrogram based on the respective accessory genome (gene presence/absence for shell and cloud genomic categories) profiles of each independent S. algae genome. Each column represents a homologous gene cluster with blue indicating presence and white indicating absence. Dendrogram tip point colors designate the isolation source. (E) Summary of S. algae pangenome KEGG pathway annotations. Bar colors correspond to the respective pangenome category (red, cloud; blue, core; green, shell), and bar heights indicate the percentage of total gene clusters comprising each category that received KEGG pathway annotations.

FIG 3

Phylogenetic tree of full-length GGDEF domains of S. algae CECT 5071 and domain architectures of the corresponding proteins. Experimentally characterized GGDEF domain proteins AdrA (STM0385), YfiN (STM2672), YciR (STM1703), and YfgF (STM2503) from Salmonella Typhimurium and PleD (PDB ID 2V0N) from Caulobacter vibrioides were chosen as reference points. The circles on the right indicate GGDEF domains with (filled blue circles) or without (open blue circles) the active-site GG(D/E)EF motif and the presence of the autoinhibitory RxxD motif (black circles) and/or the EAL domain with (filled green circles) or without (open green circles) the eponymous EAL motif. Node support values above 30% are indicated. Protein names are followed by their domain architectures, determined by searches with SMART, ScanProsite, HHPred, and CDVist. Domains identified by HHSearch as implemented in CDVist are displayed if the probability was ≥90.0%. Protein lengths (in amino acid residues) are indicated at the bottom. The domain names and their Pfam database (47) entries are as follows: GGDEF, PF00990; EAL, PF00563; HAMP, PF00672; GAF, PF01590; PAS (or PAS+PAC), PF00989; CHASE, PF03924; TPR, PF00515; DUF, PF11849, Reg_prop, PF07494; YYY, PF07495; NIT, PF08376; GAPES4, PF17157; SGL, PF08450; REC, Response_reg, PF00072; SpoVT_C, PF15714; dCache, PF02743; 5TM-5TMR_LYT, PF07694; PBPb, SBP_bac_3, PF00497; 7TMR-DISMED2, PF07696; 7TMR-DISM_7TM, PF07695; MASE1, PF05231; MASE2, PF05230; MASE3, PF17159; ANAPC5. PF12862; FleQ, PF06490; ECF-ribofla_trS, PF07155; TOM20_plant, PF06552; Protoglobin, PF11563; DivIC, PF04977. For MASE6 and MASE7, see Fig. S5; for DgcCoil, see Fig. S6.

FIG 4

Phylogenetic tree of the EAL domains of S. algae CECT 5071 and domain architectures of the corresponding proteins. EAL domains of experimentally characterized proteins YciR (STM1703), YfgF (STM2503), YhjH (STM3611), and YjcC (STM4264) from Salmonella Typhimurium, YahA from E. coli, Tbd1265 from Thiobacillus denitrificans (PDB ID 3N3T), and BlrP1 from Klebsiella pneumoniae (PDB ID 3GG1) were chosen as reference points. Other details and domain shapes are as in Fig. 2, except for the BLUF (PF04940), CBS (PF00571), LapD_MoxY_N (PF16448), CSS-motif (PF12792), and DUF3369 (PG11849) domains.

FIG 5

Phylogenetic tree of HD-GYP domains of S. algae CECT 5071 and domain architectures of the corresponding proteins. The experimentally characterized HD-GYP domain proteins from Vibrio cholerae (VCA0681, PDB ID 5Z7C), Pseudomonas aeruginosa PAO1 (PA4108 and PA4781; PDB ID 4R8Z), Persephonella marina (PDB ID 4MDZ), and Xanthomonas campestris (RpfG) were chosen as reference points for HD-GYP domains. Other details and domain shapes are as in Fig. 2, except for the HD-GYP domain (COG2206 in the COG database [72]) and DUF3391 (PF11871 in Pfam).

FIG 6

Cyclic di-GMP turnover proteome of Shewanella algae strains. (A) Distribution of GGDEF, EAL, GDDEF+EAL, and HD-GYP domains in S. algae genomes (bars, left axis) and total putative c-di-GMP turnover protein density per genome (dots, right axis). (B) Heat map showing the degree of conservation of putative orthologs of CECT 5071 c-di-GMP turnover proteins in other 41 S. algae genomes. Green, proteins that are ≥90% identical in length (P_align ≥ 90%) and have ≥95% amino acid sequence identity (P_ident ≥ 95%); yellow, proteins with either P_align > 90% and P_ident > 75% or P_align > 50% and P_ident > 90%; red, proteins with P_align < 50% and P_ident < 75%; orange, annotated proteins showing single nucleotide changes disrupting the open reading frames or split across contigs.

FIG 7

Phylogenies of the GGDEF, EAL, and HD-GYP domains of S. algae c-di-GMP turnover proteins not encoded by the type strain. The phylogenies of the GGDEF (A), EAL (B), and HD-GYP (C) domains identified in the 41 S. algae genomes in the context of type strain domains are displayed by GrapeTree representations (146). Nodes representing CECT 5071 proteins are in red, reference c-di-GMP turnover proteins (listed in the legends to Fig. 1 and 3) are in magenta, and distinct c-di-GMP turnover proteins from other strains are in turquoise. S. algae proteins are shown under their Prokka gene tags (see Table S2 for GenBank accession numbers).

FIG 8

Shewanella algae chemoreceptors. (A) Domain architectures of S. algae chemoreceptors. Shown are the topologies of the reference chemoreceptor proteome of the type strain CECT 5071 (black lettering), as well as chemoreceptors identified in other sequenced S. algae strains with distinct topologies (blue lettering). Chemoreceptors that were not annotated in databases but identified through homology modeling are indicated with the suffix “-like”. (B) Heat map showing conserved (green), divergent (yellow) or absent (red) S. algae CECT 5071 chemoreceptors in all other 41 S. algae genomes. Color criteria are as in Fig. 6.