Genetic analysis of the het and nif genes in the blue-green alga Nostoc muscorum

Summary Two multiply marked complementary strains namely Het ⁺ Nif⁺ Str-R and Het ^- Nif^- Ery-R MSO-R were constructed and crossed under conditions counterselective for the Het ⁺ Nif⁺ Str-R parent and selective only for recombinants of Str-R and Ery-R or Str-R and MSO-R constitution. The results of the recombinant analysis with regard to the selected and unselected markers suggested that the Het ^- Nif^- Ery-R MSO-R parent acted as a recipient and the Het ⁺ Nif⁺ Str-R parent as donor of the genetic markers in the cross. The joint inheritance of Het ⁺ and Nif ⁺ unselected markers among the recombinants was found to occur more frequently than the inheritance of the Het ⁺ or Nif ⁺ markers alone. The observed joint inheritance of Het ⁺ and Nif ⁺ markers among the recombinants probably results from the inheritance of the regulatory gene(s) required for the activation of latent het and nif genes. This interpretation is fully supported by (a) the frequency distribution of unselected Het ⁺ and Nif ⁺ markers and (b) the reversion frequency of Het ^- Nif ^- strains to Het ⁺ Nif⁺ prototrophy. Accordingly the apparent close genetic linkage of het and nif genes is not due to their organization in a single operon but to their common regulation by regulatory gene(s) of a positive control nature. The Het ⁺ Nif⁺ wild type, mutant, revertant, and recombinant strains all appear similar in their NO ₃ ^- repression of both heterocyst and nitrogenase. The Het ⁺ Nif^- and Het ^- Nif⁺ recominants also show similar NO ₃ ^- repression of their heterocyst and nitrogenase respectively. The presence of only microaerobic acetylene reducing activity in Het ^- Nif⁺ recombinants clearly indicates the heterocyst to be an organ for protection of nitrogenase against oxygen toxicity.Abbreviations CFU Colony forming units - Ery erythromycin - Ery-R erythromycin resistance - het genotypic designation of genes required for heterocyst differentiation - Het phenotype designation of genes required for heterocyst differentiation - MSO l-Methionine-dl-sulfoximine - MSO-R MSO-resistance - N₂ medium Chu 10 medium without combined nitrogen - NH ₄ ⁺ medium basic mineral medium with ammonium nitrogen - nif genotype designation of genes required for N₂ fixation - Nif phenotype designation of genes required for N₂ fixation - NO ₃ ^- medium Chu 10 medium supplemented with KNO₃ - NTG N-methyl-N-nitro-N-nitrosoguanidine - r gene(s) regulatory gene(s) - Str streptomycin - Str-R streptomycin resistance - Str-S streptomycin sensitive     

Genetic control of heterocyst formation in the blue-Green algae Nostoc muscorum and nostoc linckia

Non-heterocystous, non-nitrogenfixing (het ^- nif^-), heterocystous, non-nitrogenfixing (het ⁺ nif^-) and multiple heterocystous, nitrogen-fixing (M-het ⁺ nif⁺) mutants of heterocystous, nitrogen-fixing (het ⁺ nif⁺) wild-type Nostoc muscorum and Nostoc linckia were isolated and characterized with respect to (a) nitrogenfixing activity, (b) reversion frequency, (c) ammonium repressibility of heterocyst formation, (d) heterocyst spacing pattern, and (e) action of L-methionine-DL-sulphoximine (MSO), an inhibitor of glutamine synthetase (GS), on heterocyst regulation. The mutant and revertant results suggest: (i) either involvement of a common genetic determinant in the formation of heterocyst and nitrogenase or the organization of het genes and nif genes in a single operon prone to complete inactivation by a single polar mutation, (ii) non-participation of active nitrogenase in regulation of heterocyst spacing; (iii) involvement of genetic factor(s) in the control of heterocyst spacing pattern in N. linckia, and (iv) apparently different nature of the mechanism of heterocyst inhibition by proheterocyst from that of heterocyst inhibition by NO ₃ ^- or NH ₄ ⁺ . L-Methionine-DL-sulphoximine inhibits growth and causes heterocyst formation in chains in N. linckia growing in nitrogen-free, NO ₃ ^- , NO ₂ ^- or NH ₄ ⁺ medium, thus indicating a close physiological linkage between heterocyst and inorganic nitrogen metabolism regulation.     

Physiological alterations and regulation of heterocyst and nitrogenase formation in Het(-) Fix(-) mutant strain of Anabaena variabilis

Physiological alterations and regulation of heterocyst and nitrogenase formation have been studied in Het⁻ Fix⁻ mutant strain of diazotrophic cyanobacterium Anabaena variabilis. Het⁻ Fix⁻ mutant strain of A. variabilis has been isolated by N-methyl-N′-nitro-N″-nitrosoguanidine (NTG) mutagenesis and was screened with the penicillin enrichment (500 μg ml⁻¹). Growth, heterocyst differentiation, nitrogenase and glutamine synthetase (biosynthetic and transferase), ¹⁴CO₂-fixation, nitrate reductase (NR), nitrite reductase (NiR), glucose-6-phosphate dehydrogenase (G6PDH), and isocitrate dehydrogenase (IDH) activities, and NO₃ ⁻, NO₂ ⁻, and NH₄ ⁺ uptake and whole cell protein profile in different metabolic conditions were studied in the Het⁻ Fix⁻ mutant strain taking wild-type A. variabilis as reference. Het⁻ Fix⁻ mutant strain was incapable of assimilating elemental nitrogen (N₂) due to its inability to form heterocysts and nitrogenase and this was the reason for its inability to grow in BG-11₀ medium (free from combined nitrogen). In contrast, wild-type strain grew reasonably well in the absence of combined nitrogen sources and also showed heterocyst differentiation (8.5%) and nitrogenase activity (10.8 ηmol C₂H₄ formed μg⁻¹ Chl a h⁻¹) in N₂-medium. Wild-type strain also exhibited higher NR, NiR, and GS activities compared to its Het⁻ Fix⁻ mutant strain, which may presumably be due to acquisition of high uptake of NO₃ ⁻, NO₂ ⁻, and NH₂ ⁺. Wild-type strain in contrast to its Het⁻ Fix⁻ mutant strain also exhibited high level of G6PDH, IDH, and ¹⁴CO₂ fixation activities. Low levels of G6PDH and IDH activities in Het⁻ Fix⁻ mutant strain further confirmed the lack of heterocyst differentiation and nitrogenase activity in the Het⁻ Fix⁻ mutant strain. NR, NiR, and GS activities in both the strains were energy-dependent and the energy required is mainly derived from photophosphorylation. Furthermore, it was found that de novo protein synthesis is necessarily required for the activities of NR, NiR, and GS in both wild-type and its Het⁻ Fix⁻ mutant strain. Received: 21 December 2001 / Accepted: 28 January 2002     

An azide-resistant mutant of the blue-green algaNostoc muscorum producing heterocyst and nitrogenase in the presence of fixed inorganic nitrogen source

The N₂, NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine growing cultures of parentNostoc muscorum are found more or less equally sensitive to azide inhibition of growth. A mutant strain resistant to sodium azide was isolated from the parent strain in NO ₃ ^– medium and the two strains were compared with regard to their heterocyst formation and nitrogenase activity in NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine media. While the parent strain stops production of both heterocyst and nitrogenase in all the fixed nitrogen media, the azide resistant strain forms both in the fixed inorganic nitrogen media but only heterocyst and no nitrogenase in the glutamine medium. Clearly a single genetic determinant of regulatory nature appears to mediate azide-resistance as well as relief of heterocyst and nitrogenase formation from inhibition by the fixed inorganic nitrogen source. The results of glutamine effect on the heterocyst and nitrogenase formation of the two strains indicate the operation of two levels of glutamine-sensitive regulation, one which operates through the common genetic determinant of heterocyst and nitrogenase regulation and the other exclusive to nitrogenase regulation. The in vivo functional nitrogenase does not appear to be the reason for azide-resistance and neither ammonia nor glutamine or its close metabolic product seems to function in the control of heterocyst spacing.     

Response of two strains of Nostoc muscorum to metal stress and salinity

Two strains of a cyanobacterium Nostoc muscorum, wild-type N. muscorum (Cd^s) and an isolate having resistance to the heavy metal cadmium (Cd^r), were selected for characterisation of their growth potential and physiological assays in certain defined stress environments. The chosen determinants were copper (heavy metal) and NaCl (salt stress). The observations on growth, heterocyst frequency, chlorophyll and nucleic acid contents, photosynthetic O₂ evolution, ¹⁴C incorporation and acetylene reduction suggested that the strain Cd^r was also resistant to copper. This strain, however, was found to be more sensitive to NaCl in comparison to its wild-type counterpart. NaCl was found to enhance sugar accumulation in Cd^s and was more inhibitory to acetylene reduction rates than to the photosynthetic activities. The interaction between Cu and NaCl appeared to be antagonistic as the depression of growth and physiological activities by a mixture of the two was lesser than that caused by either of these. These observations form the first report on the response of a metal resistant strain of cyanobacterium to salinity.     

Growth and acetylene reduction by Gloeotrichia echinulata (Smith) Richter in axenic culture

Gloeotrichia echinulata, isolated in axenic culture, grows in medium free of combined nitrogen but better growth occurs in the presence of low levels of combined nitrogen (5–12 mm NO^- ₃; 0·1–2·0 mm NH⁺ ₄). The addition of 5–40 mm glucose and the addition of soil extract also stimulate growth. Heterocyst frequency declines in the presence of high levels of combined nitrogen, but the effect of added glucose on heterocyst frequency is equivocal. Soil extract inhibits heterocyst production. Significant acetylene reduction activity is found only in the light with rates in the presence of NO^- ₃ and in the presence of exogenous glucose being higher than on N₂. NH⁺ ₄ inhibited nitrogenase activity.     

Isolation and Partial Characterization of Het− Fix− Mutant Strain of the Diazotrophic Cyanobacterium Anabaena variabilis Showing Chromatic Adaptation

We propose a model to describe the changes taking place in biochemical processes/events to explain the development of heterocyst and nitrogenase in a diazotrophic cyanobacterium Anabaena variabilis. For this purpose, a mutant strain of A. variabilis lacking heterocyst differentiation and incapable of growth with dinitrogen as the sole source of nitrogen has been isolated after nitrosoguanidine (NTG) mutagenesis and selection by penicillin enrichment. The mutant strain (Het⁻ Fix⁻) thus isolated has morphological variation and was incapable of reducing acetylene under anaerobic conditions, indicating its mutational loss of the process of nitrogen fixation. The Het⁻ Fix⁻ mutant strain had reduced glutamine synthetase (transferase) activity compared with its wild-type counterpart, suggesting a link between nif gene expression and the expression of gln A, the structural gene of GS. The Het⁻ Fix⁻ mutant strain compared with its wild-type strain also had an extremely high level of phycobiliprotein and a low level of carotenoids. Furthermore, the coiling of vegetative filaments in the Het⁻ Fix⁻ mutant strain, which reduced the surface area to be exposed to light, was a direct indication of the chromatic adaptation, because the mutant strain was found to be photosensitive, showing bleaching of the cells under high light intensity. Received: 13 December 2000 / Accepted: 9 February 2001     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria

Summary Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N₂-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N₂ fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het^- mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N₂-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het^- heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.     

Mutational Engineering of the Cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis> for Resistance to Growth-Inhibitory Action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N₂ fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li⁺-R and Na⁺-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O₂-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol m⁻³ proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline. Received: 10 July 2002 / Accepted: 13 August 2002     

Effect of vesicular-arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria on growth and nutrition of soybean in a neutral-calcareous soil amended with32P-45Ca-tricalcium phosphate

Summary The optimum conditions for growth ofFrankia sp. HFPCcI3 isolated fromCasuarina cunninghamiana, were studied in batch culture using defined media. Maximum growth (doubling time was 24 h)_was achieved at 33°C and at pH 6.3 with pyruvate and NH ₄ ⁺ as the sole C and N sources, respectively. Removal of NH ₄ ⁺ from the culture medium resulted in vesicle differentiation which was paralleled by induction of acetylene reduction activity. Growth on atmospheric N₂ was optimal with combined pyruvate and glucose as the carbon sources and displayed a doubling time of about 48 h. Comparisons in growth and N₂-fixing activity ofFrankia strains grown in a variety of cultural conditions demonstrate the range of behavior among the strains.     

Evidence for the involvement of a genetic determinant controlling functional specificity of group VI B elements in the metabolism of N2 and NO-3 in the blue-green alga Nostoc muscorum.

The parent $\text{N. muscorum}$ is Mo-requiring for growth on N₂ or NO₃^? as nitrogen source. W and Cr both are observed to competitively inhibit the function of Mo in growth on N₂ and NO₃^? media in parent strain. Spontaneous mutants growing in the presence of W or Cr were isolated and when examined, found to be W- or Cr-requiring for growth with both N₂ and NO₃^? as nitrogen source. The results of the characterization of the three strains with respect to mutation frequency, interaction among Mo, W and Cr in growth on N₂ as nitrogen source, and requirement of W or Cr for NO₃^? inhibition of heterocyst formation in W- or Cr-requiring strain growing in NO₃^? medium, all suggest the operation of a single genetic determinant in specifying whether Mo, W or Cr (group VI B elements) is required for growth with both N₂ and NO₃^? as nitrogen sources. They also further suggest that this single genetic determinant is common to nitrogenase and nitrate reductase.     

Effect of four heavy metals on the biology of Nostoc muscorum

Summary This study presents the effects of Cr, Pb, Ni and Ag on growth, pigments, protein, DNA, RNA, heterocyst frequency, uptake of NH₄ ⁺ and N0₃ ^–, loss of electrolytes (Na⁺ and K⁺), nitrate reductase and glutamine synthetase activities ofNostoc muscorum. The statistical tests revealed a direct positive correlation between the metal concentration and inhibition of different processes. Ni was found to be more toxic against growth, pigments and heterocyst differentiation compared to the other metals. Inhibition of pigment showed the following trend: chlorophyll > phycocyanin > carotenoid. No generalized trend for inhibition of macromolecules was observed. The loss of K⁺ and Na⁺ as affected by Cr, Ni and Pb was similar but more pronounced for K⁺ than Na⁺. The inhibition of physiological variables depicted the following trend: Na⁺ loss > K⁺ loss > glutamine synthetase > NH₄ uptake > growth > N0₃ ^– uptake > nitrate reductase > heterocyst frequency. This study therefore suggests that loss of electrolytes can be used as a first signal of metal toxicity in cyanobacteria. However, further study is needed to confirm whether the abnormality induced by nickel (branch formation) is a physiological or genetic phenomenon.     

Regulation of methylammonium transport inParacoccus denitrificans

Depending on the growth conditionsParacoccus denitrificans synthesizes two different carriers mediating uptake of methylamine. When used as a nitrogen source, methylamine is transported via a NH ₄ ⁺ carrier, and its transport is inhibited by NH ₄ ⁺ but not by ethylamine. When used as a carbon source, methylamine is transported by a specific alkylamine carrier, and its transport is inhibited by ethylamine but not by NH ₄ ⁺ . The NH ₄ ⁺ carrier is under nitrogen control, the alkylamine carrier under carbon control.Abbreviations MA Methylamine - FCCP p-trifluormethoxycarbonylcyanide-phenylhydrazone     

Host-controlled modification and restriction in Bacillus subtilis: Bsu 168-system and BsuR-system in B. subtilis 168.

Summary A Bsu168-specific restriction deficient (r ₁₆₈ ^- ) mutant of Bacillus subtilis Marburg 168 was transformed to be BsuR-specific restriction proficient (r _R ⁺ ) with B. subtilis R DNA as efficiently as the Bsu168-specific restriction proficient (r ₁₆₈ ⁺ ) parental strain (hsrM ⁺, hsdR ^-).We constructed r _R ⁺ m _R ⁺ r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain (ISMR 4), r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ⁺ strain (ISR 11) and r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ^- strain (ISR 6) from strain 101 (r ₁₆₈ ⁺ m ₁₆₈ ⁺ ), strain 1012 (r ₁₆₈ ^- m ₁₆₈ ⁺ ) and strain RM125 (r ₁₆₈ ^- m ₁₆₈ ^- ), respectively by transformation with B. subtilis R DNA, and tested their restriction and modification activities on phage 105C. The results show that the sites recognized by Bsu168-specific restriction and modification enzymes and the sites recognized by BsuR-specific ones are not overlapping.We conclude that the Bsu168-modification and restriction system and the BsuR-modification and restriction system are controlled independently by two distinct sets of genes in the r _R ⁺ m _R ⁺ transformant of r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain B. subtilis 168.     

Genetic control of ammonium transport in nitrogen fixing cyanobacterium Nostoc muscorum

Summary Ammonium (NH ₄ ⁺ ) transport was investigated in Nostoc muscorum ISU (wild type) and spontaneous mutants resistant to cyanophage N-1 (Nm/N-1), streptomycin (Nm/Sm) and methylamine (Nm/MA). N₂-fixing wild-type cells transported NH ₄ ⁺ via two transport systems: the high-affinity (K _m 11 M) and low-affinity (K _m 66 M), which formed 10 and 50-fold concentration gradients, respectively. The high-affinity system of Nm/MA (K _m 11 M) was similar to the wild type but the low-affinity system had reduced affinity for NH ₄ ⁺ (K _m 125 M), while Nm/N-1 and Nm/Sm mutants had only a high-affinity transport system (K _m 20 and 28 M, respectively). The growth of mutant Nm/N-1 was more sensitive to 1 mM NH ₄ ⁺ or methylamine than other strains, and also glutamine-synthetase activity was most reduced in NH ₄ ⁺ -grown cells. l-methionine-d, l-sulfoximine (20 M) treatment of N₂-grown Nm/N-1 cells resulted in a higher rate of NH ₄ ⁺ efflux. The apparent alterations in kinetic constants of NH ₄ ⁺ transport in mutants and glutamine synthetase activity suggested that NH ₄ ⁺ in N. muscorum is transported by specific carrier(s) and the transport is genetically controlled.     

Studies on two classes of rifampicin-resistant mutants of the nitrogen-fixing cyanobacteriumNostoc muscorum

Two types of nitrosoguanidine-induced rifampicin-resistant mutants ofNostoc muscorum were isolated and characterized. Compared with the wild type, the strainrif-1 (rif ^r het ⁺ nif ⁺ blu) revealed high growth rate, heterocyst frequency (10%–12%), nitrogenase activity, phycocyanin pigment, photosynthetic O₂ evolution, and higher activities of phosphoribulokinase and Fd-NADP⁺-oxidoreductase. The heterocyst spacing pattern in the mutant was altered and did not respond to 7-azatryptophan, -2-thienylalanine, rifampicin, andl-methionine-dl-sulfoximine treatment. The second type of mutantrif ^r -2 (rif ^r het ⁺ nif ^–) did not show nitrogenase activity and was unable to grow on molecular nitrogen even under microaerobic conditions, although it produced heterocysts (6%–7%) under these conditions of incubation. The pattern of macromolecular synthesis, particularly of RNA and protein, the rate of acetylene reduction, and photosynthetic O₂ evolution were not affected in the mutant strains with the treatment of drug. The characteristics of the mutants reflected the possibility of pleiotropic mutation and also suggested that therif marker is most likely associated in close genetic proximity with regulatory gene(s) ofhet andnif system.     

Identification and cloning of nodulation genes from the stem-nodulating bacterium ORS571

Summary After random Tn5 mutagenesis of the stem-nodulating Sesbania rostrata symbiont strain ORS571, Nif^-, Fix^- and Nod^- mutants were isolated. The Nif^- mutants had lost both free-living and symbiotic N₂ fixation capacity. The Fix^- mutants normally fixed N₂ in the free-living state but induced ineffective nodules on S. rostrata. They were defective in functions exclusively required for symbiotic N₂ fixation. A further analysis of the Nod^- mutants allowed the identification of two nod loci. A Tn5 insertion in nod locus 1 completely abolished both root and stem nodulation capacity. Root hair curling, which is an initial event in S. rostrata root nodulation, was no longer observed. A 400 bp region showing weak homology to the nodC gene of Rhizobium meliloti was located 1.5 kb away from this nod Tn5 insertion. A Tn5 insertion in nod locus 2 caused the loss of stem and root nodulation capacity but root hair curling still occurred. The physical maps of a 20.5 kb DNA region of nod locus 1 and of a 40 kb DNA region of nod locus 2 showed no overlaps. The two nod loci are not closely linked to nif locus 1, containing the structural genes for the nitrogenase complex (Elmerich et al. 1982).     

Akinetes of the cyanobacteriumNostoc PCC 7524: morphological changes during synchronous germination

Following dilution into fresh medium in the light, akinetes ofNostoc PCC 7524 germinated synchronously. Synchrony was maintained at a high level during the first 24 h, at which time the young filaments were composed either of three cells (with N₂ as nitrogen source) or four cells (with NO ₃ ^- or NH ₄ ⁺ ), and at a slightly lower level during the next 24 h of growth. The pattern of cell division was similar in media containing the different nitrogen sources although the timing of the major events varied. In the presence of N₂ or NO ₃ ^- , heterocysts differentiated synchronously; the first developed invariably from a terminal cell of the young filament at approximately 19 h, the second from the other terminal cell after further vegetative cell division. Heterocyst differentiation did not occur in the presence of NH ₄ ⁺ . In the absence of nitrogen (gas phase argon: CO₂) akinete germination initially followed the same pattern as that observed in N₂, this early stage probably occurring at the expense of intracellular reserve materials.During germination, a new laminated layer, similar in structure and position to that found in the heterocyst envelope, appeared in the akinete envelope. This layer was not present in the germinating akinetes of a mutant which was incapable of forming heterocysts.