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Journal of Bacteriology, March 2002, p. 1759-1766, Vol. 184, No. 6
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.6.1759-1766.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Two-Component Regulator Mediates Population-Density-Dependent Expression of the Bradyrhizobium japonicum Nodulation Genes

John Loh, Dasharath P. Lohar, Brett Andersen, and Gary Stacey^*

Center for Legume Research and Department of Microbiology, The University of Tennessee, Knoxville, Tennessee 37996

Received 28 August 2001/ Accepted 4 December 2001

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bradyrhizobium japonicum nod gene expression was previously shown to be population density dependent. Induction of the nod genes is highest at low culture density and repressed at high population densities. This repression involves both NolA and NodD2 and is mediated by an extracellular factor found in B. japonicum conditioned medium. NolA and NodD2 expression is maximal at high population densities. We demonstrate here that a response regulator, encoded by nwsB, is required for the full expression of the B. japonicum nodYABC operon. In addition, NwsB is also required for the population-density-dependent expression of both nolA and nodD2. Expression of nolA and nodD2 in the nwsB mutant remained at a basal level, even at high culture densities. The nwsB defect could be complemented by overexpression of a second response regulator, NodW. Consistent with the fact that NolA and NodD2 repress nod gene expression, the expression of a nodY-lacZ fusion in the nwsB mutant was unaffected by culture density. In plant assays with GUS fusions, nodules infected with the wild type showed no nodY-GUS expression. In contrast, nodY-GUS expression was not repressed in nodules infected with the nwsB mutant. Nodule competition assays between the wild type and the nwsB mutant revealed that the addition of conditioned medium resulted in a competitive advantage for the nwsB mutant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The bacterial nodulation genes (nod, nol, and noe) are required for rhizobial species to nodulate their host plants. The protein products of these genes are involved in the biosynthesis of a lipochitooligosaccharide molecule (i.e., the Nod signal) that initiates early developmental stages of nodule formation (reviewed in reference 10). The nodulation genes are induced in response to plant-produced flavonoids. Central to the regulation of the nodulation genes are the rhizobial nodD gene products (12, 19, 22, 38). The NodD proteins belong to the LysR family of prokaryotic transcriptional regulators (37). In Bradyrhizobium japonicum, members of two other families of global regulators (NodVW and NolA) are also involved in modulating the expression of the nodulation genes. NodVW belongs to the two-component regulatory family and positively activates expression of the nodulation genes by isoflavonoids (20, 28, 36). NodVW is essential for the nodulation of cowpea (Vigna unguiculata), mung bean (Vigna radiata), and siratro (Macroptilium atropurpureum) but not soybean (20). This host-specific requirement for NodVW may be explained by the fact that soybean plants produce isoflavones that interact with NodD1 or NodVW, thus allowing for the nodulation of soybean. The inability of a NodVW mutant to nodulate cowpea, siratro, and mung bean suggests that these plants may produce inducers, different from that of soybean, which interact specifically with NodVW and not NodD1. Alternatively, the levels of Nod signals required for nodulation may vary according to the host plant. Thus, while NodD1-mediated Nod signal production is sufficient for soybean nodulation, the level of Nod signal production necessary for cowpea, siratro, and mung bean nodulation requires the concerted effort of both NodVW and NodD1. Two-component systems commonly consist of a membrane-bound sensor kinase and a downstream response regulator (reviewed in reference 43). Typical of two-component systems, NodV and NodW function via a series of phosphorelay steps (28). Initiation of the phosphorylation cascade is triggered in response to the isoflavonoid genistein, resulting in the phosphorylation of the sensor kinase NodV. NodV subsequently phosphorylates its cognate response regulator, NodW. Sequence alignments with other two-component regulators suggest that NodW is probably phosphorylated at the conserved aspartate residue (Asp70) at residue 70. Consistent with this, a mutant NodW (i.e., NodWD70N), containing an aspartate-to-asparagine substitution, was not phosphorylated in vivo or in vitro (28). Isoflavonoid-mediated phosphorylation of NodW plays a key role in modulating nod gene expression and nodulation. For instance, B. japonicum strains defective in nodW demonstrated wild-type levels of isoflavonoid-induced nod gene (i.e., nodYABCSUIJ operon) expression and nodulation when complemented with wild-type nodW but not by the nodW gene encoding the NodWD70N mutant protein (28). NwsAB forms a second two-component system in B. japonicum (21). NwsB, the response regulator, was identified by its ability, when overexpressed from an external promoter, to complement the nodulation defects of a NodW mutant. This complementation is thought to occur as a result of cross talk between the NodVW and NwsAB two-component systems (21). NwsB and NodW share 65% amino acid identity over their entire lengths, as well as a conserved helix-turn-helix DNA-binding motif. These observations strongly suggest that the NwsB protein is likely a homolog of NodW. However, an nwsB mutant was still able to nodulate cowpea, siratro, and mung bean (21).

A third global regulatory component, NolA, is involved in mediating the repression of the nodulation genes. NolA, a member of the MerR family of regulatory proteins, positively activates the repressor NodD2, which then represses nod gene expression (18, 19). nolA was first identified as a genotype-specific gene since it conferred upon members of B. japonicum serogroup 123 the ability to nodulate certain soybean genotypes (34). The nolA gene possesses the unique capacity to encode three functionally distinct proteins, derived from the translation of three in-frame ATG initation sites (29). Recently, we showed that NolA plays a key regulatory role in the feedback repression of nod gene transcription in response to intracellular Nod signal production (26). Interestingly, expression of nolA is regulated in a population-density-dependent manner by an extracellular factor that accumulates in the supernatant of B. japonicum cultures (27). The population density expression of nolA is reminiscent of that observed with genes under quorum-sensing control. In quorum sensing, the bacteria respond to the accumulation of a self-generated, diffusible signal. The quorum signal functions as a means to monitor population levels, allowing for appropriate gene expression when the quorum signal exceeds a threshold (for a review, see reference 46). Quorum sensing appears to be a widespread phenomenon among many plant-associated microbes (see, for example, references 3, 8, 16, 24, 32, 33, and 44). General differences occur in the mode by which this cell-cell communication occurs among gram-negative and gram-positive bacteria. For instance, the quorum-sensing molecules of gram-negative bacteria are generally N-acylhomoserine lactones (acyl-HSLs) (17), while in gram-positive bacteria, small peptides usually serve an equivalent role (25). In gram-negative bacteria, the specific receptors for the acyl-HSLs are members of the LuxR family of transcriptional regulators (17). In contrast, two-component systems perceive the quorum signals in gram-positive bacteria (25). Studies by Bassler and coworkers showed that two-component systems regulate genes in the gram-negative bacterium Vibrio harveyi in response to quorum signals (4, 5, 15). In the present study, we sought to further characterize the role of the response regulator NwsB in both nod gene regulation and the nodulation process. The results demonstrate that mutations to nwsB decreased nod gene expression in B. japonicum in response to genistein. In addition, we also show that NwsB is involved in mediating the population-density-dependent expression of the nodulation genes.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and culture conditions. The bacterial strains and plasmids used in this study are shown in Table 1. For routine growth, B. japonicum strains were cultured in RDY medium at 30°C (42). HM salt medium (9) supplemented with 0.1% arabinose was used for growth of B. japonicum for matings and for extracting nucleic acids. Escherichia coli strains were maintained in Luria-Bertani medium at 37°C (35). When appropriate, antibiotics were added to the culture medium at the following concentrations: for B. japonicum we used 100 µg of streptomycin, spectinomycin, kanamycin, or tetracycline/ml and 30 µg of chloramphenicol/ml, and for E. coli we used 50 µg of ampicillin, spectinomycin, or kanamycin/ml and 25 µg of tetracycline/ml.

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TABLE 1. Strains and plasmids used in this study

B. japonicum strain JNWS21, harboring a nwsB interposon mutation, was generated as follows: PCR was used to amplify the coding regions of the B. japonicum nwsB gene. The amplified DNA included regions 5' and 3' of the nwsB gene (21). The primers used were 5'-GGAGACCGGGCCGGATCACC-3' and 5'-CGTTACAACTTGGCTTCATCC-3'. The PCR product (2.2 kb) was ligated into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.). The resultant plasmid (pCR2.1-NwsB) was digested with SspI and filled in with Klenow. An Sm^r-Sp^r cassette obtained by digesting plasmid pHP45 with SmaI was inserted into blunt-ended pCR2.1-NwsB. The resultant plasmid, pCR2.1-NWSB, was digested with EcoRI, and the 4.2-kb fragment containing nwsB was cloned into the EcoRI site of the suicide vector pSUP202 (39). The resultant plasmid was transformed into E. coli S17-1 (39) and mobilized by biparental mating into B. japonicum USDA110. Transconjugants were selected based on Sp^r-Sm^r and Tc^s, the latter being an indication of a double-crossover event. Confirmation of these mutations was verified by Southern blot analyses (data not shown). To generate an nwsB mutation in strain JLA6 (an Sm^r-Sp^r USDA110 strain harboring a chromosomal nolA-lacZ fusion [27]), pCR2.1-NWSB was digested with EcoRI and the 4.2-kb nwsB fragment cloned into the vector pGEM-T (Promega, Madison, Wis.), creating pGEMT-NwsB. pGEMT-NwsB was then digested with HindIII to release the Sm^r-Sp^r cassette, and the nwsB-containing fragment was filled in with Klenow. A Km^r cassette from pBSL15 (1) was then blunt-end cloned into the nwsB gene, yielding pGEMT-NWSB-Km. The nwsB fragment was digested with EcoRI, cloned into pSUP202, and conjugally transferred to strain JLA6 as described above. Transconjugants were selected on the basis of kanamycin resistance and tetracycline sensitivity. The mutations generated were confirmed by Southern hybridization (data not shown).

ß-Galactosidase activity assays. ß-Galactosidase activity was assayed as described by Banfalvi et al. (2). The induction of nod-lacZ expression by genistein was measured as described previously (41, 47). For induction with conditioned medium (CM), assays were performed as described by Loh et al. (27). CM was obtained from supernatants of B. japonicum cultures grown to an optical density at 600 nm (OD₆₀₀) of 2.0. Under the conditions used, an OD₆₀₀ unit corresponded to ca. 10⁹ cells/ml. The CM was concentrated 100-fold by rotary evaporation and added to induction assays (10 µl of concentrated CM per ml of induction medium [27]). Induction assays were performed in minimal medium (6). ß-Galactosidase activity was measured 14 h after induction as detailed by Banfalvi et al. (2).

Plant nodulation assays. Seeds of Glycine max (soybean) cv. Essex and Vigna unguiculata (cowpea) cv. Caloona were surface sterilized as described by Nieuwkoop et al. (31). After germination for 48 h, the seedlings were transferred to either plastic growth pouches (Mega International, Minneapolis, Minn.) or plastic Leonard jars containing vermiculite. The seedlings were then inoculated with 5 x 10⁷ cells of the appropriate bacterial culture. Inoculation of the bacterial cultures was performed in the presence or absence of CM (10 µl of concentrated CM per ml of culture). Plants were grown in a growth chamber under a 16-h photoperiod. The plants were watered with a nitrogen-free nutrient solution (45).

GUS assays. For GUS (ß-glucuronidase) assays, 10⁷ B. japonicum cells harboring the plasmid pVKNodYGUS4 or pVKGUS (27) were inoculated onto soybean or cowpea roots. At 24 days postinoculation, nodules were harvested and examined for GUS expression. GUS assays were performed as described by Jefferson et al. (23). Nodules were sectioned and prepared as described in Loh et al. (27). Digital images were taken on a stereomicroscope (Olympus SZX12; Olympus, Tokyo, Japan) fitted with an Olympus DP10 camera or a Nikon E600 microscope fitted with Nomarski optics.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of nolA and nodD2 is significantly reduced in an nwsB mutant. Previous work (27) demonstrated that the nolA and nodD2 genes were expressed in a population density fashion. Consistent with this, the expression of both nolA-lacZ and nodD2-lacZ (i.e., in strains Bj110-42 [18] and Bj110-1248 [19], respectively) were found to increase with culture density (Fig. 1A). In contrast, analyses of the same fusions in an nwsB mutant strain revealed a significant reduction in both nolA-lacZ (i.e., in strain JNWS24) and nodD2-lacZ (i.e., strain JNWS41) expression, even at high population densities (Fig. 1A). Since a quorum factor found in CM was previously shown to modulate the population density expression of nolA and nodD2, CM derived from cultures of either USDA110 or the nwsB mutant (i.e., JNWS21) were tested for their ability to induce nolA-lacZ and nodD2-lacZ expression (Table 2). While the addition of CM from USDA110 cultures resulted in induction of nolA-lacZ and nodD2-lacZ expression in the wild type (i.e., Bj110-42 and Bj110-1248, respectively), significantly less expression was detected in the nwsB mutant strains JNWS24 (i.e., nolA-lacZ) and JNWS44 (nodD2-lacZ). Similarly, the addition of CM derived from the nwsB mutant strain JNWS21 resulted in strong induction of nolA-lacZ and nodD2-lacZ expression in the wild type, with reduced expression in both strains JNWS24 and JNWS41. This latter result suggests that the nwsB mutant strain retains the capacity to synthesize the quorum signal but is defective in its response to this signal as measured by nolA and nodD2 induction.

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FIG. 1. (A) Population-density-dependent expression of nolA-lacZ and nodD2-lacZ. B. japonicum wild type or NwsB mutant harboring either nolA-lacZ or nodD2-lacZ was grown in minimal medium to various ODs, and the ß-galactosidase activities of these fusions were determined (2). (B) Analysis of chromosomal nolA-lacZ fusion expression in nwsB transconjugants harboring either pTE3, pTE3WWT3, or pTE3Wmut10. pTE3WWT3 expresses NodW from the trp promoter of pTE3, while pTE3Wmut10 expresses NodWD70N.

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TABLE 2. Effect of CM on nolA-lacZ and nodD2-lacZ expression in B. japonicum wild type and nwsB mutant

Population density expression of nolA is restored by overexpression of NodW. A closer examination of the expression of nolA-lacZ expression in strain JNWS24 revealed that, while nolA expression was reduced in this strain, slightly elevated levels of nolA expression could still be detected in this strain at high culture densities (cf. wild type [400 U] and JNWS24 [75 U]) (Fig. 1A). Since cross talk between NwsB and NodW had been previously reported (21), we suspected that NodW could account for the residual nolA activity in strain JNWS24. Based on this assumption, we reasoned that overexpression of NodW would complement the quorum-defective phenotype observed with the nwsB mutant. As shown in Fig. 1B, the expression of nodW in strain JNWS82, containing an nwsB mutation and a chromosomal nolA-lacZ fusion, restored the ability of this mutant to express nolA in a population density manner. Interestingly, complementation was also observed in the nwsB mutant (i.e., strain JNWS83) expressing the nonphosphorylated form of NodW (i.e., NodWD70N) (28). Control transconjugants with the vector pTE3 (i.e., JNWS81) did not exhibit population density expression of nolA-lacZ (Fig. 1B).

Effects of nwsB mutation on nodY expression in vitro and in planta. To determine the effects of the nwsB mutation on nodY expression, the ability of genistein to induce a nodY-lacZ fusion at low and high ODs was assayed. At a low OD (i.e., OD₆₀₀ = 0.1), genistein induction of nodY-lacZ expression in the nwsB mutant (i.e., JNWS31) was reduced compared to that of the wild type (i.e., ZB977) (Fig. 2A). However, unlike the wild type, nodY-lacZ induction in the nwsB mutant was not affected by cell population density (cf. OD₆₀₀ = 0.1 versus OD₆₀₀ = 1.8). In agreement with this, the addition of CM derived from B. japonicum cultures grown to an OD₆₀₀ of 2.0 had little effect on nod gene expression in JNWS31 (Fig. 2B). In contrast, addition of CM drastically reduced the ability of genistein to induce nodY-lacZ expression in the wild type. To examine whether the nwsB mutation would affect nodY expression in planta, we inoculated soybean and cowpea plants with nwsB mutant transconjugants harboring a nodY-GUS fusion (i.e., strain JNN41). As shown in Fig. 3, little or no nodY-GUS expression was observed in nodules infected with the wild-type nodY-GUS transconjugants. This result is similar to that reported previously (27), where nodY-GUS expression was not detected in nodules harvested at time intervals ranging from 6 to 30 days postinoculation. In contrast, nodules infected with strain JNN41 exhibited significant GUS staining at 24 days postinoculation (Fig. 3). As a positive control, plants were infected with the nolA mutant BjB3 harboring the nodY-GUS fusion. Nodules derived from this sample showed strong GUS staining, as previously reported (27).

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FIG. 2. (A) Comparison of nodY-lacZ expression in B. japonicum USDA110 or JNWS21 (nwsB) mutant at a low or high population density. B. japonicum cells were grown to an OD600 of 0.1 or 1.8, and the ability of 1 µM genistein to induce the nodY-lacZ expression was determined after 12 h of incubation. (B) Effect of CM on nodY-lacZ expression in USDA110 or JNWS21 (nwsB mutant). B. japonicum cultures (OD600 = 0.1) were induced for 5 h with CM. The CM was obtained from B. japonicum cultures grown to an OD600 of 2.0. The results represent the means of three separate experiments ± the standard deviation. Under the conditions used, an OD600 unit corresponded to ca. 109 cells/ml.

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FIG. 3. Comparison of nodY-GUS expression in cowpea and soybean nodules infected with the wild type, nwsB mutant JNWS21, and nolA mutant BjB3. nodY-GUS staining was determined in nodules 24 days postinoculation. (A to C) Soybean nodules; (D to F) cowpea nodules. In panels A and D, the nodules were infected with USDA110 (nodY-GUS) transconjugants. In panels B and E, the nodules were infected with JNWS21 (nodY-GUS) transconjugants. In panel C and F, the nodules were infected with BjB3 (nodY-GUS). Nodules were hand sectioned and stained with X-Gluc (5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid). Digital images were obtained with a stereomicroscope.

Effect of CM on nodulation. We next sought to determine the effect of CM addition on the nodulation ability of an nwsB mutant. The nodulation tests were performed in plastic growth pouches that allowed us to monitor nodule development spatially and temporally. As a reference mark, the root tip at the time of inoculation was noted on the outside of the plastic pouches. In previous studies, it was shown that the zone of emergent root hairs (just above the root tip) was the region of the root most susceptible to rhizobial infection (see, for example, reference 7). This mark also allowed us to distinguish between root tissue that was present at the time of bacterial inoculation and root tissue that formed after inoculation. Using this method, we were able to separate nodules that formed on root tissue already present (i.e., no delay in nodulation) versus nodules that were colonized by bacteria that were inhibited or delayed in their ability to infect the soybean root (i.e., nodules formed below the root tip mark). In samples where no CM was added, the nwsB mutant showed a slight delay in its ability to nodulate soybean (Fig. 4). This is reflected in fewer nodules found above the root tip mark and a corresponding increase in nodules formed below the root tip mark. The addition of CM to wild-type cultures drastically reduced the ability to nodulate soybean, resulting in very few nodules formed above the root tip mark. This result is consistent with that reported by Loh et al. (27). In contrast to what was observed with the wild type, the addition of CM had little or no effect on the ability of the nwsB mutant (i.e., JNWS21) to nodulate soybean.

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FIG. 4. Effect of CM on the ability of B. japonicum wild type and nwsB mutant JNWS21 to nodulate soybean plants. B. japonicum cells, untreated or incubated with CM for 1 h, were inoculated onto soybean plants (107 cells per root). At 21 days postinoculation, the numbers of nodules were determined. The standard error is also presented. Nodules were scored based on their location, either above or below the root tip mark at the time of inoculation (see Materials and Methods).

To further characterize the ability of the JNWS21 to nodulate, we compared the ability of this strain to nodulate soybean in competition with the wild type. JNWS21 and wild-type cells were mixed in different ratios and inoculated onto soybean or cowpea plants in the presence or absence of CM. The nodules formed above the root tip mark were selected and streaked on agar plates. Single colonies were picked and tested for streptomycin resistance, the antibiotic marker for mutant JNWS21. Table 3 clearly shows that the addition of CM to the bacterial inoculant samples increased the ability of the nwsB mutant strain to nodulate both soybean and cowpea.

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TABLE 3. Effect of CM on the ability of B. japonicum strains USDA110 and JNWS21 (nwsB mutant) to nodulate soybean

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous work had demonstrated that NodW was essential for the full expression of the B. japonicum nodulation genes. The requirement for NodW stems from the fact that NodVW, in addition to NodD, forms a second isoflavonoid recognition system in B. japonicum (28). Hence, mutations to NodVW reduce the ability of this bacterium to perceive and respond to the plant-produced isoflavonoids. In the present study, we demonstrated that a mutation to nwsB also results in decreased nod gene expression. Given the high similarity to NodW, it is likely that in the presence of genistein, NwsB is activated by a cellular sensor kinase, contributing to the activation of the nodulation genes. In this regard, Grob et al. (21) suggested that, in addition to its cognate kinase NwsA, activation of NwsB may also occur via a second unidentified sensor kinase. This view stems from the fact that a nodW nwsA double mutant was still able to nodulate cowpea, siratro, and mung bean. A likely candidate for this would be the sensor kinase NodV, which responds to the isoflavonoid genistein (28).

In agreement with the fact that expression of the nodulation genes is essential for nodulation, the nwsB mutant was found to be slightly delayed in its ability to nodulate both cowpea and soybean. This delay was reflected in the increased number of nodules found in root tissue that was not present at the time of inoculation (i.e., below the root tip mark, Fig. 4). These nodulation results for cowpea differ slightly from those published by Grob et al. (21), who noted that an nwsB mutation did not affect the ability of the bacterium to infect cowpea. This discrepancy may reflect differences both in the way that the plants were grown and in the criteria by which nodules were scored. For instance, our growth assays utilized plastic growth pouches, whereas Grob et al. performed their plant infection tests in glass jars containing vermiculite. In addition, our infection tests scored nodule numbers in relation to the root tip mark at the time of inoculation. This is in contrast to work by Grob et al. (21), where total nodules per plant were counted 21 days postinoculation.

NwsB is also involved in the regulation of the nodulation genes in response to cell population density. B. japonicum strains mutated in nwsB no longer demonstrated culture-density-dependent expression of nolA and nodD2. Consistent with the fact that nolA and nodD2 negatively regulate the expression of the nodYABC operon, nodY-lacZ expression was not repressed in the nwsB mutant at high cell population densities. The inability of the nwsB mutant to respond to cell population densities was further examined in nodulation assays. The results suggest that quorum regulation does play a significant role in the ability of a bacterium to infect its host plant. While the addition of CM derived from cells grown to a high OD (OD₆₀₀ = 2.0) significantly delayed the ability of the wild-type strain to nodulate soybean and cowpea, the quorum-insensitive nwsB mutant showed no delay when similarly treated. In addition, the application of CM to inoculants containing a mixture of both the wild type and the nwsB mutant affected the occupancy of nodules formed above the root tip mark at the time of inoculation. With CM, the nwsB mutant represented a greater proportion of the nodule population than that found in the untreated inoculants. Therefore, at high cell population densities, the nwsB mutant is able to better compete with the wild type for nodule occupancy. A mutant defective in the quorum regulation of the nodulation genes is important in view of the fact that high population densities are found in commercial inoculants. Indeed, Loh et al. (27) demonstrated that inducers of nolA and nodD2 were present in commercial inoculants and could ultimately reduce the efficacy of these inoculants. Consistent with this observation, Lohrke et al. (30) demonstrated that soybean nodulation was reduced when inoculants with a high population density were utilized.

Finally, the results demonstrated that the overexpression of NodW can compensate for the loss of NwsB. As discussed above, this result is probably due to the fact that NodW is likely a homolog of NwsB. Garcia et al. (18) showed that nolA expression was not induced by the isoflavonoid genistein. This result can be explained by the fact that nolA is regulated by unphosphorylated forms of NodW and NwsB. For example, expression of NodWD70N, a mutant NodW that is not phosphorylated, restored quorum-sensing regulation of nolA and the nodulation genes. Previous work showed that phospho-NodW was required for nod gene induction (28). The cross talk between NodVW and NwsAB and the fact that unphosphorylated and phosphorylated forms of the response regulators have different effects on nolA and nod gene expression suggests that control of phosphorylation is likely a critical switch between activation and repression of nod gene expression.

Our results clearly indicate that a second response regulator, NwsB, plays a key role in nod gene regulation and the ability of the bacterium to effectively infect its host plants. It is intriguing that B. japonicum utilizes a two-component system to mediate induction of the nodulation genes, as well as to mediate the repression of the nodulation genes. NwsB therefore forms a key junction, by which the bacterium integrates both plant- and bacterium-derived signals, leading to the appropriate expression of the nodulation genes.

As mentioned above, quorum sensing among plant-associated microbes generally involves the production of acyl-HSLs that interact with a LuxR-type transcriptional regulator (16, 17, 24, 32, 33), leading to the transcription of quorum-regulated genes required for infection. Unlike these systems, the B. japonicum quorum system does not appear to be AHL-LuxR based (27). Given that two-component systems are known to regulate target genes in response to environmental stimuli, the possibility exists that NwsAB may sense the quorum molecule and, therefore, function as an interface between the quorum signal and its target genes. Indeed, this role has been described in both Vibrio harveyi and the plant pathogens Ralstonia solanacearum and Xanthomonas campestris, in which non-AHL quorum signals are detected by sensor proteins of two-component regulator systems (5, 8, 13, 14, 40). Finally, the fact that NwsB affects the expression of the MerR-type regulator NolA illustrates the cellular integration of members of different global families in the control of B. japonicum gene expression.

 
This work was supported by grant MCB-9728281 from the National Science Foundation and by Liphatech Corp. (Milwaukee, Wis.).

 
* Corresponding author. Mailing address: M409 Walters Life Sciences Building, University of Tennessee, Knoxville, TN 37996. Phone: (865) 974-4041. Fax: (865) 974-4007. E-mail: gstacey{at}utk.edu.

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Journal of Bacteriology, March 2002, p. 1759-1766, Vol. 184, No. 6
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.6.1759-1766.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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