Role of quorum sensing in Sinorhizobium meliloti-Alfalfa symbiosis

Abstract

The ExpR/Sin quorum-sensing system of the gram-negative soil bacterium Sinorhizobium meliloti plays an important role in the establishment of symbiosis with its host plant Medicago sativa. A mutant unable to produce autoinducer signal molecules (sinI) is deficient in its ability to invade the host, but paradoxically, a strain lacking the quorum-sensing transcriptional regulator ExpR is as efficient as the wild type. We compared the whole-genome expression profile of the wild-type strain with strains missing one of the quorum-sensing regulatory components to identify genes controlled by the ExpR/Sin system throughout the different phases of the bacterial growth cycle, as well as in planta. Our analyses revealed that ExpR is a highly versatile regulator with a unique ability to show different regulatory capabilities in the presence or absence of an autoinducer. In addition, this study provided us with insight into the plant invasion defect displayed by the autoinducer mutant. We also discovered that the ExpR/Sin quorum-sensing system is repressed after plant invasion. Therefore, quorum sensing plays a crucial role in the regulation of many cell functions that ensures the successful invasion of the host and is inactivated once symbiosis is established.

FIG. 1.

Categories of genes controlled by the ExpR/Sin quorum-sensing system. Microarray analyses allowed us to divide the ExpR/Sin quorum-sensing system-dependent genes into two groups. Expression of the genes in group I depended on the transcriptional regulator ExpR and was independent of the presence of AHLs. Genes in group II required both ExpR and AHLs for proper expression and were subdivided into subgroups A and B. Subgroup A contained genes that were differentially expressed in both the expR and sinI mutants compared to that in the wild type during all phases of the bacterial growth cycle. Subgroup B consisted of genes that were differentially expressed only during early or later phases of growth.

FIG. 2.

Expression of the endoribonuclease L-PSP (SMa0089) gene is ExpR dependent and SinI independent. Gene expression was analyzed at late-log phase by quantitative real-time PCR and represented as a fold change in expression between the indicated strains and the wild type (wt). The expR sinI strain was complemented in trans by the addition of crude AHLs. Activity of SMa0089 is independent of AHLs and requires the presence of the intact expR gene.

FIG. 3.

The transcriptional regulator ExpR is required for activation of the motility genes at a low cell population density. Expression of the motility genes visN, rem, and flbT in the wild-type strain versus the expR mutant and the expR mutant complemented with constitutively expressed visN and visR on a plasmid during the early log phase of growth (OD₆₀₀, 0.2). The relative expression is calculated as the fold change between the wild type and mutant strains. The positive number indicates upregulation of gene expression in the mutant, and the negative number indicates downregulation compared to that in the wild type. Constitutive expression of visN and visR bypasses the need of ExpR for activation of the motility genes.

FIG. 4.

Dependence of gene expression on AHL concentration during the different phases of the bacterial growth cycle. Expression of representative genes from ExpR/SinI-dependent, growth phase-independent (expE2) (A), and growth phase-dependent (rem) (B) subgroups was measured by quantitative real-time PCR in the wild type (wt), the sinI mutant, and the sinI mutant complemented with AHLs. Cultures were grown to early (OD₆₀₀, 0.2) and late log (OD₆₀₀, 1.2) phases. Results were compared to the respective gene expression in the wild-type strain at the early log phase of growth. The addition of AHLs to the sinI mutant restored expE2 expression to the late-log-phase, wild-type level regardless of the growth phase but failed to repress expression of rem at the early log phase. wt, wild type.

FIG. 5.

The inability to shut down flagellum synthesis in the sinI mutant interferes with its invasion efficiency. High levels of expression of the motility genes in the sinI mutant resulted in a reduction of the percentage of invaded nodules compared to that of the wild type. Strains with the expR and the expR sinI mutations showed no invasion defects since the presence of ExpR is necessary for activation of the motility genes. Mutation of the flagellum structural genes flaA and flaB in the sinI background restored the sinI mutant's symbiosis ability to wild-type levels, suggesting that the presence of flagella interferes with plant invasion. Data are significant at a P level of < 0.01.

FIG. 6.

RT-PCR expression analysis of the ExpR/Sin system components and quorum-sensing-dependent genes in free-living bacteria and bacteroids. Expression levels of the sinI, expR, and quorum-sensing-dependent, calcium-binding protein SMc04171 genes were evaluated in the wild type grown to the late-log phase (OD₆₀₀, 1.2) and in bacteroids formed by this strain in nodules 8 days and 4 weeks postinoculation. Expression levels of the previously evaluated genes nifH and ftsZ2 were used as a reference and the 16S rRNA gene as a control. Quorum-sensing regulatory components and genes dependent on them are inactive after the symbiosis establishment.