Nature Vol, 277 15 February 1979 PE ee aire Ente ea 1 Matowistaya. Mi Soran, [Tr dnt Bit Vadol. 4 262-272 (1961 & Teouks, ¥. Jap. £ Bol 21, 127-134 0971) ret atm ert cari ne eee Pease Ce een sass Pees ees 18. Pore KG. Am. S68, 159-170 197. Cee eae 21 Berk § G.-Coleet, RA Sell EB. Trent Am Eee re Sac 95, 6-520 197) ew Yon, 1957. Low levels of fixed nitrogen required for isolation of free-living N,-fixing organisms from rice roots ISOLATION and counting of Na-fixing bacteria from the natural ‘environment have until now been conducted on selective N-free media. Iti likely that some Nz-fixing bacteria are overlooked by using this procedure. We now report that most of the bacteria present in rice roots are Nz-fixers, but that they require a supply ‘of mineral or organic N for growth. In the presence of organic nitrogen, nitrogenase activity is detected in these organisms, ‘The requirement for organic nitrogen for the detection of nitrogenase is also true for Nz-fixing cultures of free-living thizobia™ Rice plants (IR26) were dug out from an unfertilised wetland rice field and the roots were washed to remove soil. The washed roots were cut into segments and shaken vigorously with glass beads to remove rhizoplane bacteria. After several washings the root fragments were macerated to provide a suspension of inner thizoplane’ bacteria. The lower portion of the stem was also blended to make a suspension of bacteria living in or on the stem. Suspensions were made in sterile tap water and diluted without exclusion of oxygen. Diluted suspensions were spread ‘on tryptic soy agar (0.1% Difco tryptic soy broth and 1.5% Noble agar), a medium used for counting aerobic heterotrophs* ‘The population of heterotrophic aerobic bacteria per g fresh weight sample was 2.3%10" organisms for rhizosphere soil, 1.2% 10" for stem, 1.5% 10" for thizoplane, and 1.1% 10" for inner rhizoplane. Colonies from cach fraction were selected and tested for nitrogenase activity using the acetylene reduc Acetylene reduction activity (ARA) was measured for a 24-h period after 3 days growth at 35 °C on semi-solid glucose yeast extract medium (in g1-': 5.0 glucose, 0.1 Difeo yeast extract, 0.5 K,HPO., 0.04 FeSO, 7H.0, 0.2 MgSO,:,7H.0, 0.0059 NaMoO, : 2H,0, 0.2 CaCla: 2H:0; in mg|"!: 0.15 HsBOs, 0.11 ZnSO, 7H,0, 0.07 CoSO, * 7H:0, 0.005 CuSO, 7H:0, 0.004 MnCl * 4H,0, pH 7.0, and Difco Noble agar 1.75 g! ‘The percentage of bacterial isolates proving nitrogenase positive (more than 6 nmol CH, per tube) was 81% (95/117) for inner thizoplane bacteria, 76% (19/25) for thizoplane bacteria, 37.5% (9/24) for bacteria from the stem, and 2.4% (1/41) for rhizosphere soil bacter ‘Because of the high percentage of inner rhizoplane isolates showing nitrogenase activity, the nutritional requirement of bacteria of the most predominant colony type (about 90% of nitrogenase positive isolates) was studied further. None of the isolates could grow on nitrogen-free basal medium (glucose + 927705550100, S sos mineral salts). The majority (31 of 40) grew on basal-+ ammonium medium (NH,CI 61 mgI"), but the growth was ‘much poorer than on basal medium supplemented with ‘casamino acids (vitamin-free, Nutritional Biochemicals) or yeast extract (0.1 g1"), For isolates that grew best on basal medium plus easamino acids, nitrogenase activities were measured after 3 days of ‘growth on various semi-solid media (Table 1). Activities were high in the presence of casamino acids but a vitamin mixture only slightly stimulated activity. No activity was detected in N-free medium or ammonium-supplemented medium. Each amino acid was tested individually and ali could support growth and nitrogenase actvitity. Table 1 Acetylene-reducing activity of inner rhizoplane isolates in ‘semi-solid media CH produced (nmol per Medium tube per day) 1 (28 isolates tested) Basal (lucose-+ mineral salts) ° ) Basal+ yeast extract (0.1 g1 7211 Basal +NH{Ci (16 mgNi~) o Basal + casamino acid (0.1 g1) 96-18 Basal glutamic acid (0.1 g1-') 3948 Expt 2 (29 isolates tested) Basal + yeast extract 21917 Basal +casamino acid 148213 Basal + glutamic acid 120422 Basal + yeattextract+ succinic acid (10 mM) 202414 Expt 3 (29 isolates tested) Basal + casamino acid 22027 Basal + vitamin mixture” Seed Basal + vitamin mixture +casamino acid 251413 *Vitamin mixture: vitamin B12, 0.015 wM; p-aminobenzoie acid, nicotinic aid, biotin, riboflavin, pyrodoxine, folie acid, and Ca-panto= thenate, all 0.8 uM. ‘The extent of inhibition of nitrogenase activity in the presence ‘of oxygen was determined. When initial po, was 0.01 atm, nitrogenase activity was maximal while at po, 0.2atm and 0.05 atm, nitrogenase activity was not detected. In anaerobic (Po, 0.001) culture, slight activity of nitrogenase was detected Like other aerobic N.-fixing bacteria, such as Azospirillum, these bacteria require microacrophilie conditions for the expression of nitrogenase. ‘To confirm Nz-fixng activity gas mixture composed of 20% 25N, (98 atom % excess), 1% Oz, 79% Ar was introduced into 4-day statie bacterial cultures on basal medium plus glutamate (0.1 81") and grovin for the following 24h at 35°C. The two isolates used for the analysis incorporated 0.18-0.25 atom % excess into cellular nitrogen. Azospirillum lipoferum (SpBr17), ‘which was used as a positive check strain, showed a higher rate of incorporation (1.16 atom % excess). Excretion of fixed nitrogen into the medium was negligible in ail cases. Nitrogenase-positive isolates from the inner rhizoplane were all small rods (0.5 x 2.6 4m), Gram-negative, catalase and oxi- dase positive, lacked cellular motility, and produced acid from lactose, sucrose, and glucose without gas formation and reduced nitrate. Thus these isolates resemble Achromobacter**, The only known No-fixing isolate of this genus was reclassified as Klebsiella pnewnoniae’ ‘A possible explanation forthe inability ofthe isolates to grow fon nitrogen-free media is that the nitrogenase activity (as ‘measured by "N experiments sto0 low. A low oxygen concen- tration produced in the presence of greater cell mass might be more favourable for nitrogenase production, however. The (© Macros Lid 1979566 oxygen factor is another possible explanation of the growth characteristics. Free-living Nr-fixing thizobia" are unable to grow on N-free media because they cannot assimilate fixed nitrogen: this is probably not the case for our root isolates as ‘excretion of fixed nitrogen into the medium was negligible. We have not yet studied the role of amino acids in nitrogenase induction. The use of nitrogen-containing media for the isolation of nitrogenase-positive bacteria could reveal a much higher incidence of nitrogenase genes in prokaryotes than was pre- viously suspected This work is supported by UNDP fund. We thank Dr O. Ito for °N analysis and Dr J. Dobereiner for the Azospirillum strain, IWAO WATANABE WILFREDO L. BARRAQUIO Soil Microbiology Deparment, Ineemational Rice Research Institute, Los Bavios, Laguna, Philippines Rese 12 Segtemer sce 11 Deceer 178 1 Patan 1 C3. Scone W. R.A Gion, AH, Nae 24, 406-407 (195, 4 O'Gara F-& Shang K:T Biochim bop, Act 492, 313-321 (1978, Postnatal cerebellar cells of staggerer mutant mice express immature components on their surface ‘THE histogenesis ofthe central nervous system is controlled, in part, by a defined sequence of cell-surface interactions". Cell- surface carbohydrates are prominent constituents of the exter- ral faces of plasma membranes" and are thought to be important to biological recognition”. Recently, it has also been shown that some cell-surface carbohydrates change during the first 2d after birth, an important period in development of the mouse cerebellum*®, Certain neurological mutations have been shown to express defects in positioning of particular cell types at various stages during cerebellar histogenesis™'", One of the ‘most interesting of these is the staggerer (sg) mutant". In this disorder granule cells degenerate after they have migrated into position in the granular layer. Before this expression of the disorder, the sites of synapse formation between the parallel fibre axons of granule cells and the dencrites of Purkinje cell neurones fal to form; in fact, even the tertiary branchlet spines, the potential postsynaptic sites, do not form'=”"*. Even at birth, before obvious expression of the Purkinje cell defect, external granule cells of sg exhibit a reduced proliferative rate which results in a reduced aumber of external granule cells". In this report, antibodies specifically directed against micro- bial polysaccharides have been applied as carbohydrate probes to developing cerebellar cell populations in vitro. Anti-menin- ‘ococcus type-B polysaccharide antibodies (anti-B) (antigenic determinant neuraminic acid) preferentially react with pre-and neonatal cerebellar cells of CS7BL/6J mice, but do not react with 7-d-old (P7) cells, Antise-1,6-mannan antibodies, on the other hand, react with both prenatal and postnatal cerebellar cells. Comparable results have been obtained with lectins as surface carbohydrate probes". Agglutination of cerebellar cells, with concanavalin A and wheatgerm agglutinin decreased from Nature Vol. 277, 15 February 1979 é 2 ¢ + i i Antibody dilution (10°) Fig, 1 Inhibition of fibre formation in microwelleutures of Td-old sa/sg cerebellum in the presence of various dilutions of anticmeningococcus type-B antibodies (ant-salic acd) (Wl). The Inhibition wae overcome when antibodies were incubated over- right with 10 ug of meningococcus type-B polysaccharide before Adding the antigen-antibody complex to the culture (2). Normal rabbit serum did not inhibit bre formation (@). The numbers plotted represent the mean of three experiments of the total ‘numberof ses in six microwells, ‘embryonic day 13 (E13) to postnatal day 7 (P7). Using anti-B and anti-a~1,6-mannan antibodies to determine the dis- tribution of their respective antigens on the surface of +/+ and sg/sg cerebellar cells in vitro, we found that P7 sg/sg cell ‘cultures express immature cell-surface carbohydrates. ‘The present work is one step in a study to determine whether the defect in some of these neurological mutants might be in cell-surface constituents and whether their alterations might affect the sequence of cell-cell interactions required to generate functional connections in the developing cerebellum, A microwell tissue culture system, in which cells how certain behaviours reminiscent of in vivo cerebellar development’, was used to determine anti-carbohydrate antibody reactivity. This system allows the assay of differential cell assembly, migration and fibre outgrowth. The large number of very reproducible ‘Table 1 Fibre outgrowth in microwell cultures of 7-d-old sg/sg and +/+ cerebellum inthe presence of anti-carbohydrate antibodies [No.of fibres in 6 microwells Se Ye Expt Antibody Dilution +/+ Inhibition sg/ag Inhibition 1 Rabbit serum 1:50 180 193 2 178 169) 3 22 176 i AntiB 1:200 1621018 2 we 8S 3 WS 1288 1 Antio-16- 1:30 169212933 2 2 9 us 0 3 ne) ‘A single-cell suspension (1% 107 cellsmi*) of T-d-old +/+ and se/se cerebellum, respectively, were plated at 5 ul per microwell, and ‘Siu! of antibody dilution in medium then added. Fibre formation was scored in six microwells after 2 in vitro using @ phase contrast light ‘microscope. The variation among cultures was determined as +20%. The data represent the total numberof fibres in six microwells obtained in three different experiments. The numberof fibres obtained in cultures containing rabbit serum were used as standard for 100% fibre formation per experiment