Interactions ofRhizobium japonicum strains in soybean nodules and their contribution to nitrogen fixation

Summary Rhizobium japonicum strain 8-0 Str^R applied as inoculum to Clark 63 soybeans formed small ineffective nodules which had very low nitrogenase activity compared to nodules formed by two effective strains, 110 Tet^R and 138 Kan^R. Mean numbers of cells per milligram of nodule tissue for plants up to 34 days old were 7.7×10⁶ for 8-0 Str^R, 4.1×10⁸ for 110 Tet^R and 7.6×10⁸ for 138 Kan^R. Cell counts per unit mass of nodule were independent of plant age for strains 110 Tet^R and 138 Kan^R, however, for strain 8-0 Str^R, 25 and 34 days old plants had fewer viable cells per nodule mass than 18 day old plants. When a mixture of two effective strains was used, the nodules of individual plants were predominantly caused by either 110 Tet^R or 138 Kan^R. In one experiment the predominance was random, but in another, strain 110 Tet^R clearly dominated. Strain 138 Kan^R was absent in some nodules on 18 day old plants, and in others, less than 10² cells per nodule were found. When strains 8-0 Str^R and 138 Kan^R were used as mixed inoculum, most of the nodules had strain 8-0 Str^R but strain 138 Kan^R was detected in many nodules and was generally evident in the largest nodules. Nitrogenase activity by many individual nodules was low except for nodules which had cells of 138 Kan^R. Nitrogenase activity by whole root systems of these plants was relatively high and similar to plants that had only nodules of strain 138 Kan^R. Similar relationships were observed for a mixed inoculum of 8-0 Str^R and 110 Tet^R. In general, mixed inoculations resulted in nodules with a particular strain being dominant for each individual plant. Double infections within individual nodules were not uncommon and such nodules often had disproportionate numbers of cells of two competingR. japonicum strains.Contribution from the Laboratory of Soil Microbiology, Department of Agronomy, Missouri Agricultural Experiment Station. Missouri Journal Series Number 7967.     

Genetic tools for selective labeling of proteins with α-15N-amino acids

Summary A collection of genetic tools that can be used to manipulate amino acid metabolism in Escherichia coli is described. The set comprises 21 strains of bacteria, each containing a different genetic defect that is closely linked to a selectable transposon marker. These tools can be used to construct strains of E. coli with ideal genotypes for residue-specific, selective labeling of proteins with nearly any ¹⁵N-amino acid. By using strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis, dilution of the isotope by endogenous amino acid biosynthesis and scrambling of the label to other types of residues can be avoided.Abbreviations ¹⁵N-amino acid -¹⁵N-amino acid - Cam^R chloramphenicol-resistant - DPA diaminopimelic acid - Hfr high-frequency recombinant - LB Luria broth - Kan^R kanamycin resistant - P1 bacteriophage P1 - pfu plaque-forming units - Str^R streptomycin-resistant - Tet^R tetracycline-resistant     

Decreased extrachromosomal fixation of chimeric plasmid in strain N19 ofHaemophilus influenzae Rd.

Chromosomal DNAcarrying closely-linked genetic marker allelesstrrnovr produces roughly equivalent number of Str^R (streptomycin-resistant) and Nov^R (novobiocin-resistant) transformants from wild type strain but widely differnt ratios (Str^R50: Nov^R1) from a mutant strain N19. Reduction in Amp^R (vector marker) extra chromosomal transformants is observed in N19 with chimeric plasmids carrying chromosomal DNA inserts fromnov region. Thus, Amp^R transformants with chimeric plasmid DNA pJl-8N2 and pJl-8N19 are two to three orders of magnitude lower in N19 than in wild type. However, pJl-8NaI^R33, pKuvrl, p3 and p10 DNA transform N19 and Rd with near-equal efficiency. Reduction in Amp^R transformants is intermediate (30 to 100-fold lower) in strain N19 with pJ18Str^R38 and pD7. These data are interpreted to suggest the presence of a small “aberration” in thenov region.     

The Change in the Entomopathogenic Properties in Streptomycin Resistant <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Streptomycin-resistant strains (Str^R) of the entomopathogenic bacteria Bacillus thuringiensis ssp. galleriae (Btg) have been obtained. Assessment of growth rate of Btg 69–6 colonies revealed significant difference between the initial strain Str^S sensitive to antibiotics and Str^R. Decrease in susceptibility of instar IV larvae of Galleria mellonella to Btg 69–6 Str^R by a factor of eight compared to Btg 69–6 Str^S has also been recorded. In Btg 190 Str^R, the insecticidal activity decreased by a factor of five. In Str^R, the biochemical properties changed after acquisition of resistance compared to the initial strain.     

Antibiotic production improvement in the rare actinomycete Planobispora rosea by selection of mutants resistant to the Aminoglycosides Streptomycin and Gentamycin and to Rifamycin

During a strain improvement program, spontaneous mutants with single or combined resistance to streptomycin (Str^r), gentamycin (Gen^r) or rifamycin (Rif^r) were selected from the industrial strain of Planobispora rosea, which is the producer of thiazolylpeptide GE2270. Among the mutants resistant to each single antibiotic, higher producers occurred more frequently (60%) among Gen^r than in Rif^r (10%) and Str^r (24%) populations. Two Gen^r mutants showed up to 1.5-fold improvement in GE2270 production while single resistant mutants Str^r and Rif^r produced slightly more than the parental strains. The combination of Str^r and Rif^r in the same strain improved GE2270 yield up to 1.7-fold. Finally, a higher GE2270 producing strain (1.8-fold improvement with respect to the parental strain) was selected among those mutants with triple resistance to streptomycin, rifamycin and gentamycin. A hierarchical increase in aerial mycelium and spore formation was observed which paralleled GE2270 production improvement.     

Competitive growth of slow growingRhizobium japonicum against fast growingEnterobacter andPseudomonas species at low concentrations of succinate and other substrates in dialysis culture

A cultivation system with simultaneous growth of six bacterial cultures in separate bags in dialysis culture was developed. In a medium with no added carbon source (one half concentrated Hoagland solution, water deionized and distilled), cell number ofRhizobium japonicum increased during a 7 day period by a factor of 35, whereas the number ofEnterobacter aerogenes cells decreased to one half. With a concentration of 100 nM succinate as an additional carbon source in the inflow,Rhizobium japonicum 61-A-101 cell number increased by a factor of 50 during an 8 day period, whereas cell number ofEnterobacter cloacae NCTC 10005 only doubled and ofEnterobacter aerogenes NCTC 10006 decreased. At 10 mM concentration of succinate in the inflow, doubling time the twoEnterobacter strains was about 12 h, compared to about 24 h for theRhizobium japonicum strain. Varying the succinate concentration from 10 mM to 100 nM in the inflow,Rhizobium japonicum 61-A-101 surpassed theEnterobacter aerogenes strains in the growth rate between 1 mM and 100 M succinate in the inflowing medium. Three otherRhizobium japonicum strains (fix⁺ and fix^-) did grow with a similar rate as strain 61-A-101 at very low concentrations of substrate. Growth rates for the strains were confirmed by protein data per culture. Growing in competition with twoPseudomonas strains,Rhizobium japonicum RH 31 Marburg (fix^-) did overgrow alsoPseudomonas fluorescens, was however outgrown byPseudomonas putida. In utilizing low concentrations of a¹⁴C labelled organic acid (malonate), three strains ofRhizobium japonicum left 2–4 times smaller amounts of¹⁴C in the medium than two species ofPseudomonas and two species ofArthrobacter.On sabbatical leave at ANU     

Saprophytic competence of acid tolerant strains ofRhizobium trifolii in acid soil

Summary Laboratory prescreening ofRhizobium trifolii for acid tolerance, based upon the ability of rhizobia to grow in acid media (pH 4.2) containing Al (15 M), was successful for the selection of strains capable of survival in acid soil.Both sterile and non-sterile soils of varying acidity were inoculated with several strains ofR. trifolii.Acid tolerant strains generally had significantly higher populations at every sample period than an acid sensitive strain. Amelioration of soil acidity by liming improved persistence of all strains. Soil sterilization by autoclaving adversely affected survival of all strains at each soil acidity level.Paper Number 8766 of the Journal Series, North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

Revertants of a streptomycin-resistant, oligosporogenous mutant ofBacillus subtilis

Summary Revertants of a streptomycin-resistant (Str^R), oligosporogenous (Spo^-) mutant ofBacillus subtilis were selected for the ability to sporulate. The revertants obtained fell into two phenotypic classes: Str^S Spo⁺ (streptomycin-sensitive, sporeforming), which arose by reversion of the streptomycin resistance mutations of the parent strain; and Str^R Spo⁺, which arose by the acquisition of additional mutations, some of which were shown to affect ribosomal proteins. Alterations of ribosomal proteins S4 and S16 in the 30S subunit and L18 in the 50S subunit were detected in Str^R Spo⁺ revertants by polyacrylamide gel electrophoresis. Streptomycin resistance of the parental strain and the Str^R revertants was demonstrated to reside in the 30S ribosomal subunit. The second site mutations of the revertants depressed the level of streptomycin resistance in vivo and in the in vitro translation of phage SP01 messenger ribonucleic acid (mRNA) relative to the resistance exhibited by the Str^R parental strain. The Str^R parent grew slowly and sporulated at approximately 1% of the wild type level. The Str^S revertants closely resembled the wild type strain with regard to growth and sporulation. The Str^R revertants grew at rates intermediate between those of the Str^R parent and wild type, and sporulated at wild type levels.     

Symbiotic efficiency of Hup+ and Hup− strains ofRhizobium japonicum

Summary Relative efficiency of Hup⁺ and Hup^– R. japonicum strains with Pusa 16 cultivar of soybean was studied. Inoculation with the Hup⁺ strain (A 1014) reduced the protein content in grain as compared to uninoculated control.     

Siderophore and organic acid production in root nodule bacteria

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 M added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2–5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron.All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 M added iron).Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 M added iron) during the stationary phase of growth.Abbreviations CAS chrome azurol S - HDTMA hexadecyltrime-thylammonium bromide     

Early Infection and Competition for Nodulation of Soybean by Bradyrhizobium japonicum 123 and 138

Interactions of soybean with Bradyrhizobium japonicum 123 (serogroup 123) and 138 (serogroup c1) were used to examine the relationship between early infection rates, competition for nodulation, and patterns of nodule occupancy. Both strains formed more infections in autoclaved soil (sterile soil) than in untreated soil (unsterile soil). Inoculation did not increase numbers of infection threads in unsterile soil-grown plants, where infection of proximal portions of primary roots was complete by 5 days after planting. Both strains infected and nodulated at similar rates in sterile soil. Nodules were always clustered on the upper root system, regardless of inoculation and soil treatment. Sixty-seven percent of the nodules of uninoculated plants grown in unsterile soil were occupied by rhizobia belonging to serogroups other than 123 or c1. Inoculation with strain 123 or 138 increased occupancy by that strain at the expense of residency by other rhizobia. Eighty-three percent of all nodules on plants dually inoculated with both strains in sterile soil contained strain 138. The corresponding value for plants inoculated in unsterile soil was 31%. Neither inoculum strain dominated occupancy of first-formed nodules in unsterile soil. It appears that north central Missouri soil may not have populations of highly competitive serogroup 123 and that early infection and nodulation rates do not contribute to the competitive success of strain 138.     

Inoculum Density and Population Dynamics of Suppressive and PathogenicStreptomycesStrains and Their Relationship to Biological Control of Potato Scab

SeveralStreptomycesstrains are capable of suppressing potato scab caused byStreptomyces scabies.Although these strains have been successful in the biocontrol of potato scab in the field, little is known about how populations of pathogenicStreptomycesin the potato rhizosphere are influenced by inoculation of the suppressive strains. The effects of inoculum densities of pathogenic and suppressiveStreptomycesstrains on their respective populations on roots and in rhizosphere soil were examined during the growing season. The relationships between inoculum density or rhizosphere population densities and disease severity were also investigated. Populations of suppressiveStreptomycesstrain 93 increased significantly on roots with increasing inoculum dose. At its highest inoculum dose, the suppressive strain reached a population density greater than 10⁶CFU/g root 14 weeks after planting. The ability of the suppressive strain to increase its populations with increasing inoculum density was hindered at high inoculum doses of the pathogen, suggesting that density-dependent competitive interactions may be occurring between the two antagonists. Strain 93 was most effective at preventing scab early in the growing season (8 weeks after planting), when tubers were most susceptible to the scab disease. Population densities of the suppressive strain in soil were more highly negatively correlated with scab severity than were populations on roots, suggesting that rhizosphere soil rather than potato roots may be the primary source of inoculum of the suppressive strain for tubers.     

A new perfusion system for the measurement and characterization of potential rates of soil nitrification

The effect of two isoflavonoids, coumestrol and daidzein which are present in aseptically grown roots and root exudates of soybean, was tested on some rhizospheric microorganisms. It was found that coumestrol promotes the growth ofR. japonicum USDA 138 (about 30%) andR. leguminosarum (about 15%) whereas it inhibits the growth ofAgrobacterium tumefaciens (about 50%) andPseudomonas sp. (about 20%). The following microorganisms were unaffected by this molecule:R. japonicum W505,Agrobacterium radiobacter, Micrococcus luteus andCryptococcus laurentii. It was found that daidzein promotesR. japonicum USDA 138 growth (about 20%) and inhibitsPseudomonas sp. growth (about 20%); other microorganisms were unaffected. In addition, coumestrol favoured the formation of ‘coccoids’ cells byRhizobium japonicum USDA 138 which could be the infective state of this strain. It seems that this compound is able to help nodulation of soybean by aRhizobium strain. This result supports the work of Peterset al. (1986) and Redmondet al. (1986) who show that flavones present in plant exudates induces expression of nodulation genes in Rhizobium.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Enhancing Soybean Rhizosphere Colonization by Rhizobium japonicum

A study was conducted to seek means to increase the colonization of the rhizosphere of soybeans (Glycine max L. Merrill) by Rhizobium japonicum. For this purpose, a strain of R. japonicum that was resistant to benomyl, streptomycin, and erythromycin was used. The numbers of R. japonicum rose quickly in the first 2 days after soybean seeds were planted in soil and then rapidly fell. The decline was slower if the seeds were coated with benomyl. This fungicide reduced the numbers of bacteria and protozoa in the rhizosphere, but the effect became less or disappeared as the plants grew. In sterile soil inoculated with R. japonicum and a mixture of microorganisms, the numbers of R. japonicum were usually lower if protozoa were present than if they were absent. Nodulation and plant yield were increased by the addition of benomyl to soybean seeds sown in sterile soil inoculated with R. japonicum and a mixture of microorganisms. The addition of streptomycin and erythromycin to soil stimulated the growth of R. japonicum but inhibited other bacteria in the presence or absence of soybeans. The data indicate that colonization can be increased by the use of antimicrobial agents and R. japonicum strains resistant to those inhibitors.     

Monohybrid and dihybrid segregations in the progenies of tobacco transformed for kanamycin resistance with a Ti-vector system

A chimeric DNA construction having nopaline synthase promoter, coding sequences of neomycin phosphotransferase gene conferring resistance to antibiotic kanamycin and OCS (octopine synthase) polyadenylation sequences bracketed by T-DNA ends was transferred to tobacco. Leaf discs were infected withA. tumefaciens containing disarmed, cointegrate plasmid pGV3850:: 1103 and allowed to form a callus in the presence of kanamycin. Shoots regenerated from infected leaf discs either through the callus or arising directly were further selected for their ability to root in kanamycin-containing media. Among the nine transgenic plants that were progeny tested, the transferred bacterial gene segregated as monohybrid ratio (3 Kan^R: 1 Kan^s) in seven. Segregation data of two plant progenies indicated the presence of two independent loci of Kan^R DNA insertion (15 Kan^R: 1 Kan ^s ). Back-cross segregation data were consistent with the monohybrid or independent assortment of duplicate factors. Thus in the two cases, a minimum independent integration of two copies of T-DNA each with a Kan^R marker is inferred.     

Response of soybean-Rhizobium symbioses to mineral nitrogen

Summary The effect of mineral nitrogen on establishment and activity of symbioses between soybean and several strains ofRhizobium japonicum and on the establishment of nodules ofR. japonicum isolated from nodules of field crops is studied. All strains were highly susceptible to the effects of 200 ppm NO₃–N on the establishment of symbiosis; 50 ppm NO₃–N had little effect. Response of symbioses establishhed in the absence of mineral N to short term exposure to nitrate or ammonium varied significantly between strains. Nodule isolates from soybean crops growing in nitrifying soil were no less susceptible to the inhibitory effects of mineral N on nodule formation than a laboratory culture of the commercial inoculant strain.     

Competition studies withRhizobium trifolii in laboratory experiments

Summary Competition studies were carried out in soil cores by comparing the nodule forming ability of antibiotic resistance marked strains, inoculated at three inoculum levels onTrifolium repens versus an effective indigenous Rhizobium population of 1.5×10⁵/gm. It was seen that the indigenous marked isolate G1067 formed a high proportion of nodules sampled (>90%) at all three inoculum levels (10⁵, 10⁷ and 10⁹ cells/seed) wherein the introduced foreign strain G1032 formed >50% of the nodules at the highest inoculum level.In a test tube experiment, competition for nodule sites was examined by inoculating mixtures of twoR. trifolii strains at different input levels on two cultivars ofTrifolium repens var. Huia and Titan. It was seen that G1032 was less competitive than G1006 and G1067 on cv. Huia but was more competitive on cv. Titan than the other two strains.     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

Identification and characterization of plasmids in hydrogen uptake positive and hydrogen uptake negative strains ofRhizobium japonicum

Modifications were made of published procedures to allow routine isolation of plasmids fromRhizobium japonicum. The plasmid profiles of a series of H₂ uptake positive and H₂ uptake negative strains were compared. None of the strains ofR. japonicum with high H₂ uptake activities exhibited discernible plasmids, while most of the strains, with little or no H₂ uptake activity, showed plasmids with molecular weights ranging from approximately 49–290 x10⁶. An examination of H₂ uptake negative mutants derived from an H₂ uptake positive parent revealed two discernible plasmid bands in nonrevertible mutants but no detectable plasmids in revertible mutants or in the parent strain from which mutants were derived.