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The pha1 gene cluster (pha 1A'-G) of Sinorhizobium meliloti has previously been characterized as a necessary component for proper invasion into plant root tissue. It has been suggested to encode a multi-subunit [K.sup.+]/[H.sup.+] antiporter, since mutations in the pha1 region rendered S. meliloti cells sensitive to [K.sup.+] and alkali, and because there is high amino acid sequence similarity to previously characterized multi-subunit cation/[H.sup.+] antiporters (Mrp antiporters). However, the detailed transport properties of the Phal system are yet to be determined. Interestingly, most of the Mrp antiporters are highly selective for [Na.sup.+], unlike the Phal system. Here, we report the functional expression of the Phal system in Escherichia coil and the measurement of cation/[H.sup.+] antiport activity. We showed that the Phal system is indeed a [K.sup.+]/[H.sup.+] antiporter with a pH optimum under mildly alkaline conditions. Moreover, we found that the Phal system can transport [Na.sup.+]; this was unexpected based on previous phenotypic analyses of pha1 mutants. Furthermore, we demonstrated that the cation selectivity of the Phal system was altered when the pH was lowered from the optimum. The downregulation of [Na.sup.+]/[H.sup.+] and [K.sup.+]/[H.sup.+] antiport activities upon acidic shift appeared to occur via different processes, which might indicate the presence of distinct mechanisms for the regulation of the [K.sup.+]/[H.sup.+] and [Na.sup.+]/[H.sup.+] antiport activities of the Phal system.

Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group across cis double bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes, cfa1 and cfa2, proposed to code for CFA synthases in Sinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type and cfa1 and cfa2 mutant strains grown under Pi starvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wild-type cells and the cfa1 mutant, total cyclopropane fatty acids (CFAs) increased by 10% and 15% under [P.sub.i] starvation and acidic conditions, respectively; whereas in the cfa2 mutant, CFAs were less than 0.1% of wild-type under both growth conditions. Reporter gene fusion experiments revealed that cfa1 and cfa2 were expressed at similar levels in free-living cells. Thus under the conditions we examined, cfa2 was required for the cyclopropanation of lipids in S. meliloti whereas the role of cfa1 remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred on cis-11-octadecenoic acid located in either the sn-1 or the sn-2 position in phospholipids and that cyclopropanation in the sn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under [P.sub.i] starvation. The cfa2 gene was also required for cyclopropanation of non-phosphorus-containing lipids. Principal components analysis revealed no differences in the cyclopropanation of four lipid classes. We concluded that cyclopropanation occurred independently of the polar head group. Neither cfa1 nor cfa2 was required for symbiotic nitrogen fixation.

The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expGlexpD1 and expE1 promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG[(His).sub.6] fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG[(His).sub.6] to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG-DNA interaction. Abbreviations: AFM, atomic force microscopy; EMSA, electrophoretic mobility shift assay; EPS, exopolysaccharide; EPS I, succinoglycan; EPS II, galactoglucan; HTH, helix-turn-helix; [k.sub.on], on-rate; [k.sub.off], off-rate; [K.sub.d], dissociation constant.

Results show that two DNA segments from Sinorhizobium meliloti exhibit sequence similarity to rpoH genes, encoding the sigma(sup)32 RpoH heat -shock transcription factor. Of the two genes, rpoH1 and rpoH2, the former upon mutation impairs growth and affect nitrogen fixation during symbiosis with alfalfa.

Exopolysaccharide galactoglucan (EPS II) production in the wild-type strain of the soil bacterium Sinorhizobium meliloti is induced by phosphate-limiting conditions or by extra copies of the exp gene cluster. Based on similarities to the transcriptional regulators of the MarR family, an additional putative regulatory gene, expG, was determined in the exp gene cluster. Employing exp-lacZ transcriptional fusions, a stimulating effect of extra copies of this expG on the transcription of all exp complementation groups was determined.

Diphosphopyridine-nucleotide-dependent malic enzyme (DME) of Rhizobium meliloti is needed for symbiotic nitrogen fixation since it forms a pathway in which malate is converted to acetyl-CoA in bacteroids. This enzyme shows maximal activity with NAD+ as cofactor but has reduced activity when NADP+ is used. Acetyl-CoA strongly inhibited DME activity but not TME activity. The dme gene was expressed in free-living cells and in nitrogen-fixing bacteroids while tme gene was repressed in bacteroids.

The cloning and sequencing of the poly-beta-hydroxybutyrate (PHB) biosynthetic genes phaA, phaB and ORF1, in Rhizobium meliloti 41, can be done using heterologus hybridization on a cosmid library. Another gene, phaC, clone is obtained from a R. meliloti PHB-Tn5 mutant. Escherichia coli transducted with these clones expresses phaA and phaB and synthesize PHB. The defect in PHB-mutant of R. meliloti can be removed by the phaC clone. Details of a study conducted on the genes and an analysis of the results are given.

The Rhizobium (R.) meliloti strain 042B, from alfalfa grown in the Xinjiang Autonomous Region of China can nodulate and fix nitrogen with soybean plants indicating that the symbiotic capabilities of R. meliloti are not restricted to alfalfa and other legumes. R. strains generally form nodules only on a selected range of host plants though the host specificity varies in different strains. The R. meliloti strain can fix more nitrogen per hectare than other rhizobia including soybean rhizobia.

Analysis of rhizopine scylloinosamine (SI) synthesis gene (mos) shows that the Rhizobium meliloti strain RM220-3 producing rhizopine SI in nodules contains three ORFs namely, ORF1, mosB and mosC arranged in a 3.9 kb operon unit. The L5-30 mos operon contains four ORFs and a construct developed from the L5-30 mos locus having truncated mosA gene produces SI instead of 3-O-MSI.

A study of the role of osmotic up- and downshocks in glycerophosphorylated cyclic (1,2)-beta-glucan synthesis in growing cultures of Rhizobium meliloti through pulse-chased experiments with (3H) glycerol and (14C) glucose reveals that the R. meliloti phosphoglycerol transferase is constitutive. Hyperosmotic stress decreases the rate of phosphoglycerol transfer to cyclic (1,2)-beta-glucan. Growth in a 0.4 molar NaCl-containing medium does not exhibit any transfer, while cultures in low-osmolarity medium exhibit the transfer. Both non-ionic and ionic solutes suppress the activity of phosphoglycerol transferase.

Examination of the characteristics of luteolin uptake by the wild-type Rhizobium meliloti strains Rm41 reveals the existence of several steps in luteolin uptake. The first step, a pH-dependent reversible absorption consisting of a passive dissolution of flavonoid, occurs more in bacteria killed by high temperature. The second step is a pH-independent stable accumulation step, and low-temperature metabolic inhibition studies suggest an active extrusion process. Most of the luteolin exists in the outer membrane under physiological conditions, and is kept there by R. meliloti to protect the respiratory chain against the toxic effect of flavonoids.

Sensitivity of Rhizobium meliloti to pH that ranges from 5.5 to 6.0 is further affected by organic acids such as monocaryboxylic and acetic acids. Growth is inhibited when the internal pH value goes below 7.15. The coupled effect of organic acids and low pH is determined to be bacteriostatic.

The study of Rhizobium meliloti CXM1-105 and CMX-188 strain Tn5-induced mutants reveals that the mutants enhance higher shoot dry mass upon injection into plants. The Tn5 cotransducible symbiotic effectiveness is due to insertions in megaplasmids 1 and 2. The most efficient mutants increase host shoot dry mass by 23-26% and plant nitrogen by 23-27%.