Comparative genomics reveals a novel genetic organization of the sad cluster in the sulfonamide-degrader 'Candidatus Leucobacter sulfamidivorax' strain GP

Abstract

Background: Microbial communities recurrently establish metabolic associations resulting in increased fitness and ability to perform complex tasks, such as xenobiotic degradation. In a previous study, we have described a sulfonamide-degrading consortium consisting of a novel low-abundant actinobacterium, named strain GP, and Achromobacter denitrificans PR1. However, we found that strain GP was unable to grow independently and could not be further purified.

Results: Previous studies suggested that strain GP might represent a new putative species within the Leucobacter genus (16S rRNA gene similarity < 97%). In this study, we found that average nucleotide identity (ANI) with other Leucobacter spp. ranged between 76.8 and 82.1%, further corroborating the affiliation of strain GP to a new provisional species. The average amino acid identity (AAI) and percentage of conserved genes (POCP) values were near the lower edge of the genus delimitation thresholds (65 and 55%, respectively). Phylogenetic analysis of core genes between strain GP and Leucobacter spp. corroborated these findings. Comparative genomic analysis indicates that strain GP may have lost genes related to tetrapyrrole biosynthesis and thiol transporters, both crucial for the correct assembly of cytochromes and aerobic growth. However, supplying exogenous heme and catalase was insufficient to abolish the dependent phenotype. The actinobacterium harbors at least two copies of a novel genetic element containing a sulfonamide monooxygenase (sadA) flanked by a single IS1380 family transposase. Additionally, two homologs of sadB (4-aminophenol monooxygenase) were identified in the metagenome-assembled draft genome of strain GP, but these were not located in the vicinity of sadA nor of mobile or integrative elements.

Conclusions: Comparative genomics of the genus Leucobacter suggested the absence of some genes encoding for important metabolic traits in strain GP. Nevertheless, although media and culture conditions were tailored to supply its potential metabolic needs, these conditions were insufficient to isolate the PR1-dependent actinobacterium further. This study gives important insights regarding strain GP metabolism; however, gene expression and functional studies are necessary to characterize and further isolate strain GP. Based on our data, we propose to classify strain GP in a provisional new species within the genus Leucobacter, 'Candidatus Leucobacter sulfamidivorax'.

Keywords: Bacterial consortium; Cryo-transmission electron microscopy; Metagenome-assembled genome; Phylogenetic analysis; Sulfonamides.

Fig. 1

Electron micrographs of frozen hydrated Achromobacter denitrificans strain PR1 (a) and strain GP (b). PM – Plasma membrane; OM – Outer membrane; FG – Flagellum; CW – Cell wall; C – Carbon support grid

Fig. 2

Abundance of strain PR1 and strain GP after 15 h incubation at different pH, salinity (in DLB), temperatures (in MMSY) and media (R2A, TSA, BHI and MMSY). The values for copies of the 16S rRNA gene per ml are plotted in logarithmic scale. Values are the mean values of triplicates and the error bars represent the standard deviation. Significant differences in strain GP abundance are indicated by a, b, c and d (from higher to lower values of the mean) as determined by two-way ANOVA (pH, temperature and salinity) or one-way ANOVA (PR1/GP ratio in R2A, TSA, BHI and MMSY) and the Tukey test at p < 0.05 within each tested condition [33]

Fig. 3

ANI (a), AAI (b) and POCP (c) heatmaps comparing values between strain GP and validly named species of the Leucobacter genus at the time of analysis

Fig. 4

Phylogenomic relationships between the Leucobacter genus and strain GP inferred from concatenated amino acid alignments of 400 universal proteins obtained with PhyloPhlAn [52]. Representative members of genera Microbacterium, Leifsonia, Gulosibacter, Agromyces a n d Arthrobacter were included as outgroup. Leucobacter spp. strains sequenced in this study are marked with an asterisk, and sulfonamide degraders are shown in bold. Node labels indicate local support values obtained with FastTree using the Shimodaira-Hasegawa test [53]. The scale bar represents the number of expected substitutions per site. The tree was rooted at the outgroup node and visualized with FigTree [125]

Fig. 5

Representation of the genetic organization of the sad cluster in sulfonamide degraders: Microbacterium sp. strains BR1 and CJ77, Arthrobacter sp. strains D2 and D4 and strain GP. Contig numbers and locus for each region are shown next to or on top of the DNA backbone. The diagram was designed with Simple Synteny [85]

Fig. 6

Maximum likelihood phylogenetic trees inferred from amino acid alignments with MEGA6 [87] of (a) SadA, (b) SadB, (c) SadC, (d) YceI transporter and (e) IS1380/IS3/IS4 transposases shared between sulfonamide degraders. Strain GP is shown in bold; sulfonamide degraders are marked with an asterisk (*); and structural homologs to these enzymes obtained with SWISS-MODEL [88] are shown in bright blue. Node labels indicate: ML bootstrap support above 50% (in bold) / NJ bootstrap support values above 50% / Bayesian posterior probabilities above 70%. The scale bar represents the number of expected substitutions per site. The tree was rooted at the midpoint and visualized with FigTree [125]

Fig. 7

Pairwise alignment with BLASTp of the regions of the substrate binding pocket of XiaF (accession number 5LVW) and each homolog of SadA (a) and SadB (b) in strains GP, Microbacterium sp. BR1 and Arthrobacter sp. D2 and D4. Conserved regions between the different SadA and SadB homologs are highlighted in green, nonconserved residues are highlighted in red. Residues shared by all sequences are marked with an asterisk. The diagrams were designed with Excel 2013