The Type 3 Protein Secretion System of Cupriavidus taiwanensis Strain LMG19424 Compromises Symbiosis with Leucaena leucocephala

Abstract

Cupriavidus taiwanensis forms proficient symbioses with a few Mimosa species. Inactivation of a type III protein secretion system (T3SS) had no effect on Mimosa pudica but allowed C. taiwanensis to establish chronic infections and fix nitrogen in Leucaena leucocephala. Unlike what was observed for other rhizobia, glutamate rather than plant flavonoids mediated transcriptional activation of this atypical T3SS.

TEXT

In the absence of reduced nitrogen, most legume species form nitrogen-fixing associations with soil bacteria collectively known as rhizobia. Symbioses between rhizobia and legumes come in many forms and shapes (11) but always culminate with the formation on roots (or stems) of specialized organs called nodules. To limit risks of detrimental infections as well as maximize nitrogen fixation, plants restrict the intracellular colonization by rhizobia to specialized nodule cells. Within these nodule cells, rhizobia differentiate into nitrogen-fixing bacteroids that exchange ammonia for carbohydrates derived from photosynthates (13). Colonization of plant tissues by rhizobia involves the exchange between both symbionts of molecular codes such as flavonoids, nodulation factors, and surface polysaccharides (6, 14). Rhizobia are phylogenetically disparate bacteria distributed in many genera of alpha- and betaproteobacteria, referred to as α- and β-rhizobia. In several distantly related α-rhizobia such as Sinorhizobium fredii strain NGR234 (16) or Bradyrhizobium japonicum strain 110 (8), type III protein secretion systems (T3SS) were shown to promote or impair symbiosis with host plants (4); the effects were sometimes specific at the cultivar level (18). Whether β-rhizobia also use T3SS to modulate their host range has not been investigated so far.

The T3SS of Cupriavidus taiwanensis is an atypical rhizobial secretion system.

The β-rhizobium Cupriavidus taiwanensis LMG19424 is a specific symbiont of Mimosa species, including Mimosa pudica. Genome sequencing revealed that LMG19424 carries genes for a T3SS cluster of unknown function on its chromosome 2 (1). Genes required for type III secretion and cellular translocation (sct) are clustered into two groups of genes divergently transcribed, the largest of which covers 12.2 kb and includes sctVSCDJKLNTU. Interestingly this genetic organization differs markedly from that found in α-rhizobia but resembles that of the human opportunist Burkholderia cenocepacia (1). In most rhizobia, transcription of both nodulation (nod, nol, noe) and T3SS (sct) genes is controlled via flavonoid-dependent regulatory cascades (4, 7). To study the expression of C. taiwanensis T3SS genes, the promoter of sctV (PsctV) was amplified and fused to lacZ of pCZ388 (3), yielding pCZ-PsctV. Once pCZ-PsctV was mobilized into CBM832, a streptomycin-resistant derivative of LMG19424 (10), its activity was monitored in free-living cells grown in quarter-strength minimal medium (MM/4-S) supplemented with 10 mM succinate as the carbon source and vitamins (10). In these growth conditions, the PsctV-lacZ fusion was not induced by luteolin or apigenin, two inducers of LMG19424 nodulation genes such as nodB (Table 1). In contrast, the addition of glutamate triggered the activity of PsctV but not that of a nodB-lacZ fusion (pCBM01). Although it has been reported that the presence of glutamate induces the expression of T3SS functions in cells of Pseudomonas aeruginosa and Ralstonia solanacearum grown in vitro in minimal media (see, e.g., reference 3), signals responsible for the in vivo activation of sct loci in both pathogens remain unknown. Thus, our data confirmed that pCZ-PsctV was functional and that, unlike what was found for α-rhizobia, regulation of T3SS genes in C. taiwanensis was not mediated by flavonoids capable of inducing nod gene expression.

Table 1.

Glutamate rather than flavonoids activates T3SS genes of C. taiwanensis^a

Construct	β-Galactosidase activity (±SD) of construct in MM/4-S medium containing:
	No inducer	Apigenin	Luteolin	Glutamate
pCZ388	25.8 (±7.3)	18.5 (±8.8)	20.2 (±10.1)	28.9 (±3.9)
pCBM01	89.6 (±8.8)	2,689 (±322)	2,287 (±125)	128.0 (±34.1)
pCZ-PsctV	35.4 (±3.9)	17.3 (±9.7)	32.4 (±4.1)	591.4 (±53.9)

^aThe promoter region of sctV was amplified using primers PsctV_F (5′-CCAAGCTTATCAGGCTCCATATGCGG-3′) and PsctV_R (5′-AACTGCAGATCACGGCAAACAGCAGCA-3′), cloned into pGEM-T Easy (Promega) as one HindIII-PstI fragment, and further subcloned into pCZ388 using the same restriction enzymes. The resulting PsctV-lacZ transcriptional fusion (pCZ-PsctV) was introduced into CBM832 by conjugation. β-Galactosidase activities of pCZ-PsctV, a nodB-lacZ fusion (pCBM01) (10), and empty vector pCZ388 (3) were measured in transconjugants of CBM832 grown for 24 h in MM/4-S supplemented with vitamins and 10 mM succinate using apigenin (5 μM), luteolin (15 μM), or glutamate (90 mM) as the inducer. Values are reported as Miller's units and represent the means of at least 3 independent experiments.

The T3SS of C. taiwanensis has no effect on M. pudica.

To examine the role of C. taiwanensis T3SS in symbiosis, a polar mutation was introduced in sctN (RALTA_B1253), which encodes an ATPase required for T3SS-dependent secretion of proteins (4). A fragment internal to sctN was amplified by PCR using CBM832 genomic DNA and primers HindIII-F_SctN (5′-CCAAGCTTGATCCGGTGGACAACGAAC-3′) and BamHI-R_SctN (5′-CGGGATCCCGATATGGCCGTCGAGGATG-3′). The 647-bp amplicon was cloned into suicide vector pVO155 (12) digested with BamHI and HindIII, yielding pVOsctN. Once pVOsctN was mobilized into CBM832 by triparental mating, single-reciprocal recombination of pVOsctN with chromosome 2 was selected using resistance to neomycin. Southern hybridization and PCR amplifications confirmed the genotype of the sctN mutant strain CBM312, in which pVO155 separates sctN into two truncated fragments of 1,035 and 348 bp.

The phenotype of mutant strain CBM312 was assessed first on Mimosa pudica, the primary host of C. taiwanensis (2). Plants were grown in Magenta jars containing vermiculite, watered using nitrogen-free B&D solution, and inoculated with 2 × 10⁸ bacteria per germinated seedling (5). As shown in Fig. 1, both CBM312 and CBM832 elicited root nodules in which cells of the central nitrogen fixation zone were massively infected with bacteria. Bacteria reisolated from nodules were found to be resistant to neomycin and to carry a copy of pVO155 inserted in sctN. Electron micrographs of nodule sections also confirmed that bacteroids of CBM312 and CBM832 appeared similar in size and shape (Fig. 1B and D). At 45 days postinoculation the average numbers of nodules, nodule fresh weights, and shoot dry weights of plants inoculated with either CBM312 or CBM832 were similar, indicating that a functional T3SS was not required for proficient symbiosis on M. pudica (Table 2). Additional nodulation tests confirmed that both CBM832 and CBM312 failed to form nodules on roots of Vigna unguiculata and Pachyrhizus tuberosus and induced nonfixing pseudonodule-like structures on Tephrosia vogelii and Crotalaria juncea roots.

Fig 1.

Sections of nodules formed by CBM832 (A, B) or CBM312 (C, D) on roots of M. pudica 35 days postinoculation. (A and C) Cross sections of nodules seen at low magnification (bars, 500 μm) using light microscopy. (B and D) Electron micrographs of nodule cells containing bacteroids (bars, 2 μm).

Table 2.

Symbiotic properties of CBM312, CBM832, and NGR234^a

Host plant	Inoculant	mNN	mNFW (mg)	mSDW (mg)
M. pudica (45 dpi)	CBM312	48.5 (±15.6)	58.7 (±12.9)	157.5 (±83.8)
	CBM832	49.0 (±16.1)	62.4 (±19.3)	151.8 (±40.1)
	NGR234	0.0	0.0	14.7 (±4.8)
L. leucocephala (50 dpi)	CBM312	23.3 (±6.1)	226.7 (±71.8)b	209.2 (±53.7)b
	CBM832	21.9 (±6.1)	77.1 (±21.0)	95.9 (±20.5)
	NGR234	19.5 (±4.4)	170.1 (±39.1)c	283.0 (±68.8)c

^aSymbiotic properties of inoculated strains are reported as the mean nodule number (mNN), nodule fresh weight (mNFW), and shoot dry weight (mSDW) per inoculated plant, 45 or 50 days postinoculation (dpi). Results are the means for at least 10 plants per inoculant, with the standard deviations shown in parentheses. mSDWs of noninoculated control plants were 79.6 mg (±17.5 mg) and 10.8 mg (±1.7 mg) for L. leucocephala and M. pudica, respectively.

^bValue obtained with CBM312 that is significantly different from that for CBM832 at the α level of 5%.

^cValue obtained with NGR234 that is significantly different from that for CBM312 at the α level of 5%.

A functional T3SS restricts the host range of C. taiwanensis.

Native to southern Mexico and northern Central America, L. leucocephala is a species of small trees that belongs to the same Mimoseae tribe as M. pudica. L. leucocephala (Lam.) de Wit forms nitrogen-fixing associations with various rhizobial species (17), including the promiscuous Sinorhizobium fredii strain NGR234 (15), but not with the closely related S. fredii strain USDA257, which lacks a functional copy of the nodSU locus (9). NodS and NodU, which add, respectively, methyl and carbamoyl groups onto Nod factors, are present in both strains NGR234 and C. taiwanensis LMG19424 (1). In addition, a number of α-rhizobial strains that were isolated from nodules of acacia but that nodulate L. leucocephala were shown to synthesize Nod factors that were structurally similar to those made by C. taiwanensis (1). On L. leucocephala, the parent strain CBM832 formed small nodules that were poorly infected, however (Fig. 2A). Inter- as well as intracellular structures resembling infection threads or pockets were observed (see Fig. S1 in the supplemental material), but overall few nodule cells contained intracellular CBM832 symbiosomes and these appeared often to be in the process of being degraded (Fig. 2B). Nodules also lacked leghemoglobin and failed to fix nitrogen, and ultimately plants were starved for nitrogen (Table 1). In contrast, nodules formed by mutant strain CBM312 fixed nitrogen and had well-defined meristematic zones (Fig. 2C), as well as mature nodule cells with densely packed bacteroids (Fig. 2D). This indicated that a functional T3SS impaired the persistent colonization of nodule cells rather than the processes of nodulation and infection on plant tissues per se. Compared to NGR234, CBM312 seemed a less efficient symbiont, as the shoot dry weights of plants were significantly lower (Table 1). Interestingly, the T3SS of NGR234 was reported to have no measurable effect on L. leucocephala (4, 16).

Fig 2.

Sections of nodules formed by CBM832 (A, B) or CBM312 (C, D) on roots of L. leucocephala 35 days postinoculation. (A and C) Light micrographs of nodule sections at a low magnification (bars, 500 μm). (B and D) Electron micrographs of nodule cells containing intracellular bacteria of CBM832 (B) or nitrogen-fixing bacteroids of CBM312 (D) (bars, 2 μm).

Thus, a functional T3SS prevents symbiosis with L. leucocephala and contributes to restriction of the host range of C. taiwanensis. Rhizobia evolved in many unrelated genera, most probably by acquiring sets of symbiotic genes via lateral transfer followed by a reprogramming of the recipient genome to express and optimize these symbiotic traits (11). In this respect, strain LMG19424 was shown to harbor the most compact symbiotic island described so far (1), suggesting that it evolved recently from a nonsymbiotic ancestor that is closely related to a bacterium capable of infecting humans (2). A genetic organization similar to that of the T3SS of pathogenic bacteria, transcriptional activation not mediated by flavonoids, and activity that compromises colonization of nodule cells by bacteroids suggest that integration of C. taiwanensis T3SS in the symbiotic lifestyle is not complete in a microsymbiont that is still in the making.