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. 2006 Mar 28;103(13):5230-5.
doi: 10.1073/pnas.0600912103. Epub 2006 Mar 17.

Eukaryotic Control on Bacterial Cell Cycle and Differentiation in the Rhizobium-legume Symbiosis

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Free PMC article

Eukaryotic Control on Bacterial Cell Cycle and Differentiation in the Rhizobium-legume Symbiosis

Peter Mergaert et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Symbiosis between legumes and Rhizobium bacteria leads to the formation of root nodules where bacteria in the infected plant cells are converted into nitrogen-fixing bacteroids. Nodules with a persistent meristem are indeterminate, whereas nodules without meristem are determinate. The symbiotic plant cells in both nodule types are polyploid because of several cycles of endoreduplication (genome replication without mitosis and cytokinesis) and grow consequently to extreme sizes. Here we demonstrate that differentiation of bacteroids in indeterminate nodules of Medicago and related legumes from the galegoid clade shows remarkable similarity to host cell differentiation. During bacteroid maturation, repeated DNA replication without cytokinesis results in extensive amplification of the entire bacterial genome and elongation of bacteria. This finding reveals a positive correlation in prokaryotes between DNA content and cell size, similar to that in eukaryotes. These polyploid bacteroids are metabolically functional but display increased membrane permeability and are nonviable, because they lose their ability to resume growth. In contrast, bacteroids in determinate nodules of the nongalegoid legumes lotus and bean are comparable to free-living bacteria in their genomic DNA content, cell size, and viability. Using recombinant Rhizobium strains nodulating both legume types, we show that bacteroid differentiation is controlled by the host plant. Plant factors present in nodules of galegoid legumes but absent from nodules of nongalegoid legumes block bacterial cell division and trigger endoreduplication cycles, thereby forcing the endosymbionts toward a terminally differentiated state. Hence, Medicago and related legumes have evolved a mechanism to dominate the symbiosis.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Size, shape, and DNA content of free-living, cultured S. meliloti bacteria and S. meliloti bacteroids isolated from nitrogen-fixing M. truncatula nodules. (A) Nomarski (Upper) and fluorescence (Lower) microscopy of DAPI-stained bacteria and bacteroids. (B) DNA content of DAPI-stained bacteria and bacteroids measured by flow cytometry. (C) Fluorescence microscopy of bacteria and bacteroids stained with DAPI, propidium iodide (PI), or 5-cyano-2,3-di-4-tolyl tetrazolium chloride (CTC). “Heat-killed” indicates 10-min treatment at 70°C. (Scale bars, 10 μm.)
Fig. 2.
Fig. 2.
Detection of genome changes in S. meliloti bacteria and bacteroids by CGH. (A) Comparison of bacteroids and cultured Sm1021. (B) Self-comparison of cultured Sm1021. The broader signal in A is due to higher experimental variability. (C) Comparison of S. meliloti strains 1021 and 41, differing in geographical origin but >99% similar at the nucleotide level. (D) Comparison of Sm41 and derivative ZB138. Each dot on the scatter plots is the Cye5/Cye3 hybridization ratio for the two compared samples (on the ordinate) for an individual gene (on the abscissa). A ratio of 1 indicates equal copy numbers in the two samples, whereas a ratio deviating from 1 indicates lower copy numbers or absence in one of the samples. The 6208 Sm1021 genes are ordered along the abscissa as they appear in pSymA (dark gray), pSymB (light gray), and chromosome (black).
Fig. 3.
Fig. 3.
Bacteroid differentiation is dissimilar in the indeterminate and determinate nodules and is controlled by the host plant. Bacteria and bacteroids for different symbiotic combinations were visualized by fluorescence microscopy after DAPI and PI staining. The DNA content is indicated below each sample as well as the nodule type from which bacteroids were purified. (Scale bar, 10 μm.)

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