Sinorhizobium meliloti: Genetics and Genomics

[Summary] [Rhizobium] [Medicago]

Nod genes

Much of our work concerns the bacterial genes used to stimulate plant nodule formation, called nodgenes. The nodgenes are only expressed when the bacteria are exposed to a flavonoid signal from the plant host root. A Rhizobiumprotein, NodD, is responsible for activating transcription of the Nod genes in response to plant inducers. Over 40 diverse variants of NodD exist in various Rhizobiumsymbionts, whether as members of multigene families within a species (which is the case for S. meliloti)or as the single NodD form for diverse Rhizobiumspecies. Each form encodes a DNA binding protein, but a number of the variants confer responsiveness to slightly different plant inducers. This provides an extraordinary natural experiment in protein engineering. The structural basis for flavonoid-protein interaction, and the mechanism for transcriptional activation, are twin goals of the NodD biochemistry part of our project. We have established a computer-based model for NodD structure and are using this as a basis for studying flavone-protein interactions. The activity of NodD appears to depend on interaction with the chaperone GroEL (see Figure 1), and we are assessing this via in vitro reconstitution experiments. Our goal is to understand in detail how the host signal controls the bacterial partner.

Bacterial biochemistry

The Rhizobium nodgenes encode enzymes, which act together to create a novel lipochito-oligosaccharide signal called a Nod Factor (NF) (see Figure 2). Products of the common nod genes synthesize the lipo-oligosaccharide backbone, while host specific proteins carry out modifications that add side groups to the backbone. Some nodgene encoded enzymes have side effects, carrying out biochemical actions on other substrates. The Sinorhizobium melilotigene products for NF sulfate activation and sulfate transfer, NodPQ and NodH, play a role in modifying the cell surface as well. We have are studying the formation of multi-protein complexes that may govern the fate of sulfate into one metabolic pathway vs another. We have also discovered a novel activity, a sulfotransferase enzyme that appears to be membrane bound. Through study of this and other enzymes, we will study sulfate as an example of a metabolite used in both housekeeping and specialized pathways.

Infection, differentiation and cell division

Bacteria invade plants via a novel process that provokes the host to rearrange its extracellular matrix synthesis. Following invasion, the bacteria are released into target host cells, where they differentiate to a form called the bacteroid. While early stages of nodule development are governed by the exchange of signals that induce and result from nodgene expression, the subsequent stages of infection may require additional functions/factors that are not well known. In previous work, we have used genetic and expression approaches to study bacterial behavior in infection threads, and to identify the genes that are active during invasion and bacteroid differentiation (Gage et al., 1996; Oke and Long. 1999). We are interested in the control of bacterial cell division within the host plant, and in a collaboration with Dr. Lucy Shapiro's laboratory, we are studying the regulatory protein CtrA and the genetic circuits that interact with it. We have also analyzed the contribution of the S. meliloti relAhomolog to bacterial symbiosis: initial studies show that relAis not required for infection, but is required for symbiotic nitrogen fixation.

Genome

Our goal is to establish the complete genome sequence for pSymA, the 1.4 Megabase symbiosis megaplasmid of Sinorhizobium meliloti. We established markers for pSym-A from a library of purified pSym-A DNA which has been used by F. Galibert and colleagues at UPR41-CNRS(Rennes, France), to establish a high resolution map of the S. meliloti pSym-A replicon (Barloy Huber et al., 2000). An international consortium is determining the sequence of the entire Sinorhizobium meliloti genome.

Work with Dr. Nancy Federspiel and colleagues at the Stanford DNA sequencing and technology center, we have constructed a large new library of pSym-A DNA in M13. We have carried out random sequencing of this library, assembling a total of 10X sequence. Assembly, gap closing, editing and annotation are now in progress. We anticipate finishing the sequence by mid summer 2000, and hope to publish by the end of calendar year 2000.

We are using array approaches and global mutagenesis to move from genome definition to functional genomics. We especially hope to use the emerging data on the genome sequence of S. melilotito define genes for intermediate stages of symbiosis and for bacterial behavior and cell division.

Figure 1.

Figure 2.

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