By screening with the fluorescent stain Calcofluor, we have isolated 26 independent transposon TnS insertion mutants ofRhizobium meliloti that are deficient in the production of a known extracellular polysaccharide (Exo-). The mutants belonged to six distinct genetic groups based on the ability of their Exo-phenotype to be complemented by different recombinant plasmids from a R. melilot clone bank. With few exceptions, all of the mutants formed ineffective (non-nitrogen-fixing) nodules on alfalfa. For all but one group, the complementing plasmids restored effective nodulation. These results establish a firm and extensive correlation between the ability ofRhizobium to produce a particular polysaccharide and symbiotic proficiency. The ineffective nodules appeared to contain no bacteroids and to form without shepherds' crooks or infection threads; this symbiotic phenotype matches that described for a set of independently isolated mutants that belong phenotypically and genetically to the group B exopolysaccharide mutants described previously [Finan et al. (1985) Cell 40, 869-877]. Apparently the exopolysaccharide, although not required for nodule formation, is involved in wild-type nodule invasion.The interactions between rhizobia and legumes that result in the development of nitrogen-fixing root nodules entail a complex series of events (1-3). Bacteria attach to a root hair, which curls to form a shepherd's crook. Bacterial penetration of the root hair occurs and a tubular infection thread forms, which carries the invading bacteria toward the base of the root hair. Cells deeper in the root cortex divide, and the bacteria are eventually released from the infection thread into the newly formed nodule cells. Nitrogen fixation occurs in the mature intracellular bacteria, called "bacteroids. " Recently it has become clear that the events of nodule formation (the development of root cortical tissue into a differentiated nodule structure) can be uncoupled from the events of nodule invasion (shepherd's crook formation, infection thread formation, and entry of bacteria into host cell cytoplasm). Strains ofAgrobacterium tumefaciens and Escherichia coli containing Rhizobium meliloti nodulation genes (4-7) and a set of R. meliloti mutants at a locus that is also involved in extracellular polysaccharide synthesis (this paper; ref. 8) all form empty nodules (containing no intracellular bacteroids) without (or before) the development of shepherd's crooks or infection threads. It was recently reported that root cortical cell division in soybean can occur independent of infection thread formation, root hair curling, or even bacterial attachment (9, t).A possible role for Rhizobium polysaccharides in the nodulation process has been a subject of great interest for a number of years (11. 12) but has not been demonstrated conclusively (13), although a single mutant that produced no water-soluble polysaccharide and formed ineffective (nonnitrogen-fixing) nodules was reported recently (14). In this paper we describe the isolat...

The rate of production of methane in many environments depends upon mutualistic interactions between sulfate-reducing bacteria and methanogens. To enhance our understanding of these relationships, we took advantage of the fully sequenced genomes of Desulfovibrio vulgaris and Methanococcus maripaludis to produce and analyze the first multispecies stoichiometric metabolic model. Model results were compared to data on growth of the co-culture on lactate in the absence of sulfate. The model accurately predicted several ecologically relevant characteristics, including the flux of metabolites and the ratio of D. vulgaris to M. maripaludis cells during growth. In addition, the model and our data suggested that it was possible to eliminate formate as an interspecies electron shuttle, but hydrogen transfer was essential for syntrophic growth. Our work demonstrated that reconstructed metabolic networks and stoichiometric models can serve not only to predict metabolic fluxes and growth phenotypes of single organisms, but also to capture growth parameters and community composition of simple bacterial communities.

Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans.

A wide range of Bacteria and Archaea sense cellular 2-oxoglutarate (2OG) as an indicator of nitrogen limitation. 2OG sensor proteins are varied, but most of those studied belong to the PII superfamily. Within the PII superfamily, GlnB and GlnK represent a widespread family of homotrimeric proteins (GlnB-K) that bind and respond to 2OG and ATP. In some bacterial phyla, GlnB-K proteins are covalently modified, depending on enzymes that sense cellular glutamine as an indicator of nitrogen sufficiency. GlnB-K proteins are central clearing houses of nitrogen information and bind and modulate a variety of nitrogen assimilation regulators and enzymes. NifI(1) and NifI(2) comprise a second widespread family of PII proteins (NifI) that are heteromultimeric, respond to 2OG and ATP, and bind and regulate dinitrogenase in Euryarchaeota and many Bacteria.

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