Whole genome sequencing and analysis reveal insights into the genetic structure, diversity and evolutionary relatedness of luxI and luxR homologs in bacteria belonging to the Sphingomonadaceae family

Abstract

Here we report the draft genomes and annotation of four N-acyl homoserine lactone (AHL)-producing members from the family Sphingomonadaceae. Comparative genomic analyses of 62 Sphingomonadaceae genomes were performed to gain insights into the distribution of the canonical luxI/R-type quorum sensing (QS) network within this family. Forty genomes contained at least one luxR homolog while the genome of Sphingobium yanoikuyae B1 contained seven Open Reading Frames (ORFs) that have significant homology to that of luxR. Thirty-three genomes contained at least one luxI homolog while the genomes of Sphingobium sp. SYK6, Sphingobium japonicum, and Sphingobium lactosutens contained four luxI. Using phylogenetic analysis, the sphingomonad LuxR homologs formed five distinct clades with two minor clades located near the plant associated bacteria (PAB) LuxR solo clade. This work for the first time shows that 13 Sphingobium and one Sphingomonas genome(s) contain three convergently oriented genes composed of two tandem luxR genes proximal to one luxI (luxR-luxR-luxI). Interestingly, luxI solos were identified in two Sphingobium species and may represent species that contribute to AHL-based QS system by contributing AHL molecules but are unable to perceive AHLs as signals. This work provides the most comprehensive description of the luxI/R circuitry and genome-based taxonomical description of the available sphingomonad genomes to date indicating that the presence of luxR solos and luxI solos are not an uncommon feature in members of the Sphingomonadaceae family.

Keywords: Novosphingobium; Sphingomonadaceae; luxI/R; luxR solos; phylogenetic; quorum-sensing; whole genome sequencing.

Figure 1

Flowchart of the systematic and stringent bioinformatics methodology used in this study for the identification of luxI/R in sphingomonads and large-scale phylogenetic/phylogenomic tree construction.

Figure 2

Phylogenomic tree depicting the evolutionary relationship of currently sequenced sphingomonads based on approximately 400 conserved single-copy genes. The four whole genomes sequenced in study were shown in rectangle boxes. Selected members from the genera Rhodospirillum, Agrobacterium and Rhodobacter were designated as outgroup. Bootstrap support of less than 50% was not shown.

Figure 3

Unrooted phylogenetic tree of functionally validated LuxR homologs, Plant associated bacteria (PAB) LuxR Solos (Gonzalez and Venturi, 2013) and Identified sphingomonad LuxR Homologs. Clades highlighted in green and pink represent PAB LuxR solos and sphingomonad double LuxR, LuxI, respectively. Branches colored in brown, blue, red, green, and purple represent the Sphingomonadaceae, Sphingomonas, Novosphingobium, Sphingobium, and Sphingopyxis lineages, respectively. Black star next to taxa name indicates LuxR homologs from strains sequenced in this study. Accession numbers and aligned sequences are available in Supplemental Data 1.

Figure 4

Alignment of plant associated bacteria solos. LuxR solos and selected sphingomonad LuxR homologs. Number above the alignment corresponds to the residue number of the TraR protein. Regions highlighted in yellow indicate the invariant sites of canonical LuxR homologs (Fuqua and Greenberg, 2002) while variation from the conserved site was highlighted in green. The conserved sites corresponding to autoinducer binding and DNA binding were indicated by blue and purple triangles, respectively.

Figure 5

Gene organization of luxR Solos and luxR-luxR-luxI in the four whole genome sequenced sphingomonads. (A) EasyFig generated linear comparison of luxR solos in the genomic region of selected sphingomonads. Approximately 5000 bp of genomic region flanking the luxR solos is shown. (B) Gene orientation of the identified convergent double luxR, luxI group and its gene neighborhood variation. T1 to T6 denotes different gene neighborhoods identified in the convergent double luxR, luxI. Arrows without label represent gene coding for hypothetical protein. Please see Table 2 for topology variation present in sphingomonad genomes. The numbers “1,” “2,” and “3” represent virB1, virB2, and virB3 genes respectively. Additional abbreviations include: lcmT, Isoprenylcysteine carboxyl methyltransferase; metB, Cystathionine gamma-synthase; phyH, phytanoly dioxygenase.

Figure 6

Pairwise identity matrix of identified convergent luxR-luxR-luxI (LuxR-A and LuxR-B) in sphingomonads. The letters A and B correspond to different partner luxRs in the luxR-luxR-luxI. The genes coding for LuxR homologs with the same symbol and color were convergently oriented with respect to each other.

Figure 7

Genomic and genetic evidence for the presence of luxI solos in sphingomonads. (A) Gene neighborhood showing non-LuxR genes located in the vicinity of the putative LuxI genes (arrow with blue diamond). Analysis of the translated protein sequence for the gene upstream and convergently oriented to the putative luxI gene in Sphingobium chinhatense IP26 and Sphingobium sp. KK2 indicates that it may be an N-terminal truncated LuxR protein (arrow with black star). (B) A representative Interproscan analysis domain analysis of the N-terminal truncated LuxR protein noted in Figure 7A. (C) Protein alignment of the putative LuxI solos. Number above the alignment corresponds to the amino acid residue of TraI. Amino acid residues are conserved in all LuxI-type proteins (Fuqua and Greenberg, 2002) are highlighted in yellow.