1. The finding that the plant is the genetic determinant of leghaemoglobin production in legume nodules was further tested by inoculating snake beans with two strains of Rhizobium selected to give large genetic differences. Carbohydrate requirement patterns, immunological techniques and DNA base ratio determinations were used to demonstrate genetic differences between the two rhizobial strains. 2. Partially purified preparations of the haemoglobins from the nodules produced by the two strains showed no differences when examined by electrophoresis, isoelectric focusing or ion-exchange chromatography. 3. Two different leghaemoglobins from each type of nodule were separated by chromatography on DEAE-cellulose. One of these was isolated in the Fe3+ form and accounted for two-thirds of the total leghaemoglobin. When it was examined in the analytical ultracentrifuge and by amino acid analysis, this major component did not vary with the inoculant rhizobial strain. The molecule had an s20,w of 1.88S, a diffusion coefficient of 10.7×10-7cm2·s-1 and a mol. wt. of 16700. 4. These results strongly support the hypothesis that the mRNA for leghaemoglobin is transcribed from plant DNA.

No abstract

S U M M A R YThe enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.3. I . 2), glutamate synthase (Lglutamine : a-oxoglutarate amino transferase) and glutamate dehydrogenase (EC I . 4 . I .4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slowgrowing R. japonicum and R. lupini.Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M . sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system. I N T R O D U C T I O NThe assimilation of ammonia by bacteria proceeds via either glutamate dehydrogenase or the glutamine synthetase/glutamate synthase system, depending upon the organism and the ammonia concentration of the environment (Tempest, Meers & Brown, 1973; Brown, Macdonald-Brown & Meers, 1974). Dainty (1972) reported the presence of glutamate synthase in a strain of Clostridium pasteurianum which lacked glutamate dehydrogenase, while Nagatani, Shimizu & Valentine (1971) demonstrated the presence of glutamate synthase in a range of nitrogen-fixing bacteria and in bacteroids of Rhizobium japonicum prepared from Glycine max (soya bean) root nodules. Nagatani et al. (1971) claimed that the glutamine synthetase/glutamate synthase system was operative in ammonia assimilation

* Office of theCassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptll (encoding kanamycin resistance) or aacCl (encoding gentamicin resistance) genes were equipped with the tac promoter (PtaC) and the trpA terminator ( T , ) and then cloned between Not1 sites to construct the CAS-Nm (Ptac-npt//-TtpA) and CAS-Gm ( Pt , CP, , , c/ -aacC/ -T, )cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncPl plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a Not1 fragment into the Not1 site of a PUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-npt//-TtwA) and mTn5-Gm (containing P, CP, , , , -aacC/ -TtwA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-P,,-npt / / -T, ) and mTn5-GGm (containing gusA-Pt,~P,,,-dacC/-TtpA) can be used for transcription signal localization or insertional inactivation. The TAC-31 R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid-or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other Gramnegative bacteria.CAS-G Nm (gUSA-Pt,,-npt//-TtpA) or CAS-GGm (gUSA-Pt,CP,,,c/-da~CI-TtpA)

In contrast to the wild-type MoFe protein, neither the alpha-195(Asn) nor the alpha-191(Lys) MoFe protein catalyzed N(2) reduction to NH(3), when complemented with wild-type Fe protein. However, N(2) was bound by the alpha-195(Asn) MoFe protein and inhibited the reduction of both protons and C(2)H(2). The alpha-191(Lys) MoFe protein did not interact with N(2). With the alpha-195(Asn) MoFe protein, the N(2)-induced inhibition of substrate reduction was reversed by removing the N(2). Surprisingly, even though added H(2) also relieved N(2) inhibition of substrate reduction, the alpha-195(Asn) MoFe protein did not catalyze HD formation under a N(2)/D(2) atmosphere. This observation is the first indication that these two reactions have different chemical origins, prompting a revision of the current hypothesis that these two reactions are consequences of the same nitrogenase chemistry. A rationale that accounts for the dichotomy of the two reactions is presented. The two altered MoFe proteins also responded quite differently to azide. It was a poor substrate for both but, in addition, azide was an electron-flux inhibitor with the 195(Asn) MoFe protein. The observed reactivity changes are correlated with likely structural changes caused by the amino acid substitutions and provide important details about the interaction(s) of N(2,) H(2), D(2), and azide with Mo-nitrogenase.

Altered MoFe proteins of Azotobacter vinelandii Mo-nitrogenase, with amino acid substitutions in the FeMo-cofactor environment, were used to probe interactions among C(2)H(2), C(2)H(4), CO, and H(2). The altered MoFe proteins used were the alpha-195(Asn) or alpha-195(Gln) MoFe proteins, which have either asparagine or glutamine substituting for alpha-histidine-195, and the alpha-191(Lys) MoFe protein, which has lysine substituting for alpha-glutamine-191. On the basis of K(m) determinations, C(2)H(2) was a particularly poor substrate for the nitrogenase containing the alpha-191(Lys) MoFe protein. Using C(2)D(2), a correlation was shown between the stereospecificity of proton addition to give the products, cis- and trans-C(2)D(2)H(2), and the propensity of nitrogenase to produce ethane. The most extensive loss of stereospecificity occurred with nitrogenases containing either the alpha-195(Asn) or the alpha-191(Lys) MoFe proteins, which also exhibited the highest rate of ethane production from C(2)H(2). These data are consistent with the presence of a common ethylenic intermediate on the enzyme, which is responsible for both ethane production and loss of proton-addition stereochemistry. C(2)H(4) was not a substrate of the nitrogenase with the alpha-191(Lys) MoFe protein and was a poor substrate of the nitrogenases incorporating either the wild-type or the alpha-195(Gln) MoFe protein, both of which had a low V(max) and high K(m) (120 kPa). Ethylene was a somewhat better substrate for the nitrogenase with the alpha-195(Asn) MoFe protein, which exhibited a K(m) of 48 kPa and a specific activity for C(2)H(6) formation from C(2)H(4) 10-fold higher than the others. Neither the wild-type nitrogenase nor the nitrogenase containing the alpha-195(Asn) MoFe protein produced cis-C(2)D(2)H(2) when turned over under trans-C(2)D(2)H(2). These results suggest that the C(2)H(4)-reduction site is affected by substitution at residue alpha-195, although whether the effect is related to the substrate-reduction site directly or is mediated through disturbance of the delivery of electrons/protons is unclear. Ethylene inhibited total electron flux, without uncoupling MgATP hydrolysis from electron transfer, to a similar extent for all four A. vinelandii nitrogenases. This observation indicates that this C(2)H(4) flux-inhibition site is remote from the C(2)H(4)-reduction site. Added CO eliminated C(2)H(4) reduction but did not fully relieve its electron-flux inhibition with all four A. vinelandii nitrogenases, supporting the suggestion that electron-flux inhibition by C(2)H(4) is not directly connected to C(2)H(4) reduction. Thus, C(2)H(4) has two binding sites, and the presence of CO affects only the site at which it binds as a substrate. When C(2)H(2) was added, it also eliminated C(2)H(6) production from C(2)H(4) and also did not relieve electron-flux inhibition fully. Thus, C(2)H(2) and C(2)H(4) are likely reduced at the same site on the MoFe protein. Two schemes are presented to integrate the results of the interactions of C(2)H(2) an...

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 % sequence similarity with type strains of this genus. The closest relative of the representative strains Lut6(T) and WSM3557(T) was Microvirga flocculans TFBT, with 97.6-98.0% similarity, while WSM3693(T) was most closely related to Microvirga aerilata 5420S-16(T), with 98.8 % similarity. Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA and rpoB) and cellular fatty acid profiles confirmed the placement of Lut6(T), WSM3557(T) and WSM3693(T) within the genus Micro virga. DNA-DNA relatedness values, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of Lut6(T), W5M3557(T) and WSM3693(T) from each other and from other Micro virga species with validly published names. 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 WSM3557(T) and WSM3693(T) clustered with Bradyrhizobium, Burkholderia and Methylobacterium strains. Concatenated sequences for nifD and nifH show that the sequences of Lut6(T), W5M3557(T) and W5M3693(T) were most closely related to that of Rhizobium etli CFN42(T) nifDH. On the basis of genotypic, phenotypic and DNA relatedness data, three novel species of Micro virga are proposed: Micro virga lupini sp. nov. (type strain Lut6(T) = LMG 26460(T) = HAMBI 3236(T)), Micro virga lotononidis sp. nov. (type strain W5M3557(T) = LMG 26455(T) = HAMBI 3237(T)) and Micro virga zambiensis sp. nov. (type strain WSM3693(T) = LMG 26454(T) = HAMBI 3238(T))

Summary• 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 N 2 was evaluated.• N 2 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 N 2 fixation and the strain could possess broader N 2 fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N 2 fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.New Phytologist (2008) 179: 62-66