Michael Dilworth

Summary This manual brings together state-of-the-art methods for the study of root-nodule bacteria, both in the free-living state and in symbiosis with legumes. In each chapter, the manual introduces a topic and provides guidance on how study of the symbiosis might best be tackled. Detailed descriptions of the protocols that need to be followed, potential problems and pitfalls are provided. Topics covered include acquiring, recognising, growing and storing rhizobia, experimenting with strains in the laboratory, glasshouse and field, and applying contemporary molecular and genetic methodologies to assist in the study of rhizobia.

Background and Aims: The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. Methods: Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. Key Results: Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. Conclusions: Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.

Strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from nitrogen-fixing nodules of the native legumes Listia angolensis (from Zambia) and Lupinus texensis (from Texas, USA). Phylogenetic analysis of the 16S rRNA gene showed that the novel strains belong to the genus Microvirga, with 96.1 % or greater sequence similarity with type strains of this genus. The closest relative of the representative strains Lut6T and WSM3557T was M. flocculans TFBT, with 97.6-98.0 % similarity, while WSM3693T was most closely related to M. aerilata 5420S-16T, with 98.8 % similarity. Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA, rpoB) and cellular fatty acid profiles confirmed the placement of Lut6T, WSM3557T and WSM3693T within Microvirga. DNA:DNA relatedness values and physiological and biochemical tests allowed genotypic and phenotypic differentiation of Lut6T, WSM3557T and WSM3693T from each other and from other validly published Microvirga species. The nodA sequence of Lut6T was placed in a clade that contained strains of Rhizobium, Mesorhizobium and Sinorhizobium, while the 100 % identical nodA sequences of WSM3557T and WSM3693T clustered with Bradyrhizobium, Burkholderia and Methylobacterium strains. Concatenated sequences for nifD and nifH show that Lut6T, WSM3557T and WSM3693T were most closely related to Rhizobium etli CFN42T nifDH. On the basis of genotypic, phenotypic and DNA relatedness data, three novel species of Microvirga are proposed: Microvirga lupini (Lut6T = LMG26460T, = HAMBI 3236) Microvirga lotononidis (WSM3557T = LMG26455T, = HAMBI 3237) and Microvirga zambiensis (WSM3693T = LMG26454T, = HAMBI 3238).

Steady-state assays of nitrogenases share at least five requirements: an anaerobic environment, a consistent source of magnesium adenosine triphosphate (MgATP), a suitable source of reductant, a buffer system compatible with the product-quantification protocol to be used, and the desired substrate. The assay is initiated by injection of the component protein(s) of the enzyme or MgATP and terminated by injection of either acid or a solution of Na2EDTA. The various nitrogenases catalyze the reduction of a wide variety of substrates. This chapter outlines the methods used to analyze the products of nitrogenase-catalyzed reactions involving nitrogen-nitrogen bonds, nitrogen-oxygen bonds, carbon-nitrogen bonds, carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, and hydrogen only. The usefulness of measurements of residual amounts of other components of nitrogenase assays is also discussed.

Ensifer (Sinorhizobium) medicae is an effective nitrogen fixing microsymbiont of a diverse range of annual Medicago (medic) species. Strain WSM419 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from a M. murex root nodule collected in Sardinia, Italy in 1981. WSM419 was manufactured commercially in Australia as an inoculant for annual medics during 1985 to 1993 due to its nitrogen fixation, saprophytic competence and acid tolerance properties. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first report of a complete genome se-quence for a microsymbiont of the group of annual medic species adapted to acid soils. We reveal that its genome size is 6,817,576 bp encoding 6,518 protein-coding genes and 81 RNA only encoding genes. The genome contains a chromosome of size 3,781,904 bp and 3 plasmids of size 1,570,951 bp, 1,245,408 bp and 219,313 bp. The smallest plasmid is a fea-ture unique to this medic microsymbiont.

The South African legumes Lotononis bainesii, L. listii and L. solitudinis are specifically nodulated by highly effective, pink-pigmented bacteria that are most closely related to Methylobacterium nodulans on the basis of 16S rRNA gene homology. Methylobacterium spp. are characterized by their ability to utilize methanol and other C1 compounds, but 11 Lotononis isolates neither grew on methanol as a sole carbon source nor were able to metabolize it. No product was obtained for PCR amplification of mxaF, the gene encoding the large subunit of methanol dehydrogenase. Searches for methylotrophy genes in the sequenced genome of Methylobacterium sp. 4-46, isolated from L. bainesii, indicate that the inability to utilize methanol may be due to the absence of the mxa operon. While methylotrophy appears to contribute to the effectiveness of the Crotalaria/M. nodulans symbiosis, our results indicate that the ability to utilize methanol is not a factor in the Lotononis/Methylobacterium symbiosis.

The Medicago genus is of global importance to agriculture, with the perennial M. sativa being the most widely cultivated and studied member. After many years of studying this plant along with its microsymbiont Sinorhizobium meliloti, it became clear that another host was required to allow simultaneous study of the genetic determinants of both symbiotic partners.M. sativawas unsuited to this role as it is autotetraploid, allogamous and shows strong in-breeding depression, making the analysis of recessive mutations no easy task. Researchers identified the annual medic M. truncatulaas a viable alternative as this host is diploid, autogamous and possess a rapid generation time, among other traits. Consequently, this organism was chosen for sequencing.

Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021-M. truncatula symbiosis at fixing N2 was evaluated. • N2 fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed. • Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells. • The Sm1021-M. truncatula symbiosis is poorly matched for N2 fixation and the strain could possess broader N2 fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N2 fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.

Biological nitrogen fixation, especially via the legume-Rhizobium symbiosis, is important for world agriculture. The productivity of legume crops and pastures is significantly affected by soil acidity; in some cases it is the prokaryotic partner that is pH sensitive. Growth of Rhizobium is adversely affected by low pH, especially in the acid stress zone. Rhizobia exhibit an adaptive acid tolerance response (ATR) that is influenced by calcium concentration. Using Tn5-mutagenesis, gusA fusions and proteome analysis, we have identified a range of genes that are essential for growth at low pH (such as actA, actP, exoR, actR and actS). At least three regulatory systems exist. The two-component sensor-regulator system, actSR, is essential for induction of the adaptive ATR. Two other regulatory circuits exist that are independent of ActR. One system involves the low pH-induced regulator gene, phrR, which may control other low pH-regulated genes. The other circuit, involving a regulator that is yet unidentified, controls the expression of a pH-regulated structural gene (lpiA). We have used pH-responsive gusA fusions to identify acid-inducible genes (such as lpiA), and then attempted to identify the regulators of these genes. The emerging picture is of a relatively complex set of systems that respond to external pH.

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5-7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WRI101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium metiloti, Rhizobium tropici and Agrobacterium tumetaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated IpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of 1piA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated 1piA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.