Identification of an rsh gene from a Novosphingobium sp. necessary for quorum-sensing signal accumulation

Abstract

The stringent response is a mechanism by which bacteria adapt to environmental stresses and nutritional deficiencies through the synthesis and hydrolysis of (p)ppGpp by RelA/SpoT enzymes. Alphaproteobacteria and plants contain a single Rsh enzyme (named for RelA/SpoT homolog) that is bifunctional. Here we report the identification of a new species of bacteria belonging to the genus Novosphingobium and characterization of an rsh mutation in this plant tumor-associated isolate. Isolate Rr 2-17, from a grapevine crown gall tumor, is a member of the Novosphingobium genus that produces the N-acyl-homoserine lactone (AHL) quorum-sensing (QS) signals. A Tn5 mutant, Hx 699, deficient in AHL production was found to have an insertion in an rsh gene. The Rsh protein showed significant percent sequence identity to Rsh proteins of alphaproteobacteria. The Novosphingobium sp. rsh gene (rsh(Nsp)) complemented the multiple amino acid requirements of the Escherichia coli relA spoT double mutant by restoring the growth on selection media. Besides QS signal production, the rsh mutation also affects soluble polysaccharide production and cell aggregation. Genetic complementation of the Hx 699 mutant with the rsh(Nsp) gene restored these phenotypes. This is the first discovery of a functional rsh gene in a member of the Novosphingobium genus.

FIG. 1.

Riesling grapevine nopaline tumor isolate Rr 2-17 is a member of the Novosphingobium genus and produces cell-to-cell communication signals. (A) Phylogenetic position of Rr 2-17 among 18 taxa of the family Sphingomonadaceae based on the 16S rRNA gene. Monophyly of each ingroup genus is confirmed with >90% bootstrap support (Sphingomonas, cluster 1 [C1]; Sphingobium, cluster 2; Novosphingobium, cluster 3; and Sphingopyxis, cluster 4) using parsimony, minimum-evolution, and neighbor-joining methods. These results support the revision of the genus Sphingomonas (60). A member of the Sphingomonadaceae family, Zymomonas mobilis, was used as the outgroup (22). The accession numbers of the sequences used for the construction of the phylogenic tree are presented in Table S3 in the supplemental material. (B) AHL signal profile. Chromatography of extracts and standards with biosensor strain NTL4(pZLR4) overlays was performed with 10 μl of Rr 2-17 supernatant equivalent (lane 3), 1 nmol of C8-HL (lane 1), 10 nmol of C10-HL (lane 1), 250 pmol of C6-HL (lane 2), 3-O-C8-HL produced from A. tumefaciens strain NT1(pTiC58ΔaccR) (1 μl of a 20× preparation) (lane 4), or 10 pmol of 3-O-C6-HL (lane 5). The black dots represent the centers of signals to determine the R_f values. The loading origin is indicated.

FIG. 2.

Identification of a mutant deficient in AHL signal synthesis and the hypomucoid phenotype. (A) Identification of AHL deficiency in extracts of Hx 699. Extracts of Rr 2-17 and Hx 699 (10 μl) were assayed. C−, aEtOAc-only negative control; C+, C6-HSL in aEtOAc at 100 nmol (positive control). Treatments were assayed using the LuxR-dependent E. coli biosensor JM109(pSB401). The experiment was repeated three times, and the mean values of replications and standard errors are shown. (B) Hypomucoid phenotype of Hx 699 compared to Rr 2-17. Rr 2-17 and Hx 699 were grown on PD agar medium for 5 days.

FIG. 3.

Site of Tn5 insertion; distinct domains of Rsh_Nsp; partial alignment of conserved motifs I, II, and IV of the histidine-aspartate domain of the superfamily of metal-dependent phosphohydrolases (1); and phylogenetic analysis. (A) The site of Tn5 insertion is between the proline codon CCT (residue 249) and the tyrosine codon TAC near the beginning of the synthase domain (vertical stripes in panel B). (B) The full-length Rr 2-17 RelA/SpoT homolog (Rsh) contains four conserved superfamily domains, i.e., the histidine-aspartate (HD) or hydrolase (horizontally striped box), synthase (vertically striped box), TGS (vertically dashed box), and ACT (bricked box) domains. The arrow indicates the approximate site of Tn5 insertion in Hx 699. (C) Partial amino acid alignment of the Rsh proteins from Rr 2-17 (line 1; accession number EU984514), Sphingomonas wittichii (line 2; accession number ABQ69873), Sphingopyxis alaskensis (line 3; accession number ABF54382), Agrobacterium tumefaciens (line 4; accession number AAK86838), Rhizobium etli (line 5; accession number ABC90188), and E. coli SpoT (line 6, accession number AAC76674) and RelA (line 7; accession number AAC75826). The alignment was constructed using the program MegAlign (DNAstar) with the Clustal W algorithm. This alignment was against the conserved motifs (I, II, and IV) of the entire superfamily of metal-dependent phosphohydrolases (1). The number before the first amino acid in each sequence indicates the amino acid residue at the beginning of the partial alignments. Motif I shows the conserved region potentially involved in the ppGpp degradation activity (residues RASGKPYF) flanked by conserved histidine (H) residues. Motif II shows the conserved histidine and aspartate (HD) and glutamate and aspartate (ED) signatures, and motif IV shows the conserved aspartate (D) residue. No conserved residues in the Rsh (lines 1 to 5) and SpoT (line 6) proteins are conserved in the RelA protein of E. coli (line 7). (D) Phylogenetic tree based on the nucleotide sequences of relA/spoT and relA/spoT-like homologs (rsh genes), showing relationships of rsh sequences from 20 taxa, including the Novosphingobium sp. strain Rr 2-17 Rsh protein. The rsh gene sequences from the higher plants Arabidopsis thaliana and Oryza sativa were used as outgroups to root the tree. The new sequence is confidently (100% bootstrap) nested within the alphaproteobacterial group. The results confirm a close sister relationship of the rsh sequence of the new isolate Rr 2-17 to that from N. aromaticivorans using parsimony, minimum-evolution, and neighbor-joining methods with 100% bootstrap support. The accession numbers of the sequences used for the construction of the phylogenetic tree are presented in Table S2 in the supplemental material.

FIG. 4.

Functional analysis of Rsh_Nsp in E. coli mutants. (A) Complementation analysis. E. coli relA mutant CF1652, E. coli relA spoT double mutant CF1693, and E. coli wild-type strain CF1648 were used. Each strain is with rsh (pHG3) and without rsh (pHG2, empty vector). (B) Growth of the same strains as in panel A on rich undefined LB agar medium. (C) Restoration of growth of the E. coli relA spoT double mutant transformed with pHG3 on M9-glucose medium supplemented with the amino acids serine, methionine, and glycine at 1 mM each (SMG medium). (D) Restoration of growth of the E. coli relA spoT double mutant transformed with pHG3 on AT medium.

FIG. 5.

Quantification of soluble polysaccharides and cell aggregation by Rr 2-17 and Hx 699. (A) Quantification of soluble polysaccharides from Rr 2-17 and Hx 699 with and without rsh. Each strain was grown for 3 days on PD agar medium. Cells were harvested in sterile water, and total soluble glucose was determined. Rr 2-17(pHG4), Rr 2-17 containing extra copies of rsh; Hx 699(pHG4), Hx 699 containing rsh. The data are means from three replications, and bars represent standard deviations. (B) Quantification of cell aggregation. The dilution of the overnight cultures at the beginning of the experiments is indicated. Cultures were grown in each experiment for 7 days on TYE medium.

FIG. 6.

(A to C) Analysis of QS molecules from Rr 2-17 and Hx 699 grown on rich PD agar (A and B) and estimation of total accumulated AHL signals detectable with a LuxR-dependent biosensor (C). (A) AHL signal accumulation in Rr 2-17(pRK290) and Rr 2-17 with extra copies of rsh on pHG4 at 0, 3, 6, and 9 days. The culture extract applied per lane is equal to 40 μl of culture supernatant. AHL standards of C6-HL (100 pmol) and C8-HL (33 pmol) are included in the standard lane (Std). The origin of loading is indicated. (B) AHL signal accumulation in Hx 699(pRK290) and complemented Hx 699(pHG4) at 3, 6, and 9 days. (C) Quantification of AHL signal accumulation in aEtOAc extracts of Rr 2-17(pRK290) (open circles), Rr 2-17(pHG4) (solid circles), Hx 699(pRK290) (open squares), and Hx 699(pHG4) (solid squares) at 0, 3, 6, and 9 days. The same samples were used in the TLC assays (A and B). The extract assayed was equivalent to 400 μl of culture supernatant. The experiment was repeated three times, and the mean values and standard errors are shown. (D) Alignment of gene context/neighborhoods around luxI homologs in the sequenced genomes of the Sphingomonadales order. Genes that have conserved neighborhoods are shown by boxed arrows, and the direction of the arrow indicates the transcriptional direction. Names of representative encoded protein are given, as are the species in which it is present and the approximate nucleotide location number of each LuxI (solid black arrows). Broken cross lines in the S. wittichii RW1(pSWIT02) LuxI gene context indicate intervening regions contain additional genes.