Rhizobium sp. strain NGR234 possesses a remarkable number of secretion systems

Abstract

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.

FIG. 1.

(A) Map of the genome of Rhizobium sp. strain NGR234 chromosome (cNGR234, 3.93 Mbp), megaplasmid (pNGR234b, 2.43 Mbp), and sym plasmid (pNGR234a, 0.54 Mbp). Circles are described from outside to innermost circle. The outer circle shows the coordinates and the position of the secretion systems and several other selected genes; the second and the third outer circles indicate the ORFs on the leading and the lagging strands (yellow and green). The next inner circle indicates the sncRNAs (in light red), followed by a circle indicating the positions of the repeats (in light blue). The next inner circle shows the G+C content (dark yellow and purple). The following inner circles marked in red show the putative orthologues that are present on the NGR234 and on the strain 1021 chromosome. The circle marked in blue shows the putative orthologues that are present on the NGR234 and MAFF303099 chromosomes. This analysis is refined on the plasmids. The outer circle in red represents the putative orthologues shared with the chromosome of strain 1021, the next inner circle indicates the putative orthologues shared with pSymA, and the next red circle indicates the putative orthologues shared with pSymB. Similarly, the blue circles indicate the putative orthologues shared with the chromosome of MAFF303099, and the next inner circles indicate the putative orthologues shared with pMLa and pMLb, respectively. Larger views of the different replicons can be found in Fig. S1 to S3 in the supplemental material. (B) Hypothetical structures of the T2SS, T3SS, and T4SS and T4P of NGR234. Individual genes and proteins identified in NGR234 and their locus tags are indicated in Table S2 in the supplemental material. The positions of the various secretion systems are indicated on the outer circle; the GspC, GspS, and GspO proteins in light gray have not yet been identified in NGR234, whereas NGR_c23060 and NGR_c23050 share weak similarity to GspM and GspN, respectively. All essential genes and components of the various secretion apparatuses, which have been identified in NG234, are indicated in brown or dark gray.

FIG. 2.

Conserved clusters of genes linked to the synthesis of AHL-based quorum sensing molecules. (A) Comparison of the TraI/TraR system, together with conserved clusters of genes identified in other rhizobial species. The AHL synthase TraI (NGR_a04220, blue) directs the synthesis of 3-oxo-C8 homoserine lactone, which associates with the response regulator TraR (NGR_a04090, red) and activates transcription. TraM (NGR_a04080, green) functions as a suppressor, preventing TraR from activating target genes under noninducing conditions. The trb genes (trbB to trbI) (NGR_a04210 to NGR_a04100) involved in the conjugal plasmid transfer are shaded dark gray. (B) An additional quorum-sensing system identified on the Rhizobium sp. strain NGR234 chromosome, composed of NgrI (NGR_c16900, blue)/NgrR (NGR_c16890, red) (LuxI/LuxR homologs) and a hypothetical protein (NGR_c16910, light red), possibly linked to quorum sensing and autoinducer synthesis, is also shown. The yellow box highlights the regions with conserved gene organization.

FIG. 3.

Physical maps of the gene clusters of the single type II secretion systems (A), as well as the two copies of the type III pili (B) that have been identified in NGR234 and related bacteria. The yellow box highlights the regions with conserved gene organizations.

FIG. 4.

BiBlast comparison (each single strain against the other two strains) of the complete genomes of strains NGR234, 1021, and CFN42. The Venn diagram shows the numbers of proteins shared or unique within a particular relationship for the three microbes. NGR-specific proteins are indicated in orange, strain 1021 proteins are indicated in green, and strain CFN42 proteins are indicated in blue. The three strains share a core genome of ∼3,200 orthologous genes; all genomes contain about 1,800 genes that are unique for each strain. The 930 putative orthologous genes shared between strains 1021 and NGR234 (but which are absent in R. etli) are compared to 499 orthologues shared by R. etli and NGR234 (but which are absent in strain 1021) indicate that strain NGR234 is more closely related to strain 1021.