Characteristics of nitrogen-fixingKlebsiella oxytoca isolated from wheat roots

Summary Of 45 fermentative gram negative bacterial isolates examined from wheat roots, three were capable of fixing atmospheric nitrogen as determined by the acetylene reduction technique and by protein contents of cells. A gram negative non-motile facultatively anaerobic bacterial strain capable of N₂ fixation was identified asKlebsiella oxytoca ZMK-2.Optimal growth and N₂ fixation occurred at pH 6.5. The optimum temperatures for growth under anaerobic conditions ranged between 30°–37°C. Acetylene reduction by intact cells was strikingly inhibited by 0.1 atm. or greater partial pressure of O₂. Furthermore, the accumulation of H₂ in the gas phase over cultures ofKlebsiella oxytoca ZMK-2 at partial pressures greater than 0.02 atm. resulted in a striking inhibition in the rate of C₂H₂ reduction. The addition of suspensions of eitherKlebsiella oxytoca ZMK-2 orAzotobacter vinelandii or a mixed culture of these two organisms to axenic cultures of wheat plants produced no significant increase in plant growth as measured by plant dry weight or nitrogen content of plants.     

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus

Summary The structural genes (hup) of the H₂ uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization with the structural genes of the H₂ uptake hydrogenase of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup^-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68108 dalton) and by alignment with the NH₂ amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34 256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology, but to a lesser extent, with the hydrogenase of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues, (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe–4S] ferredoxins.     

DNA sequence and genetic analysis of the Rhodobacter capsulatus nifENX gene region: homology between NifX and NifB suggests involvement of NifX in processing of the iron-molybdenum cofactor

Summary Rhodobacter capsulatus genes homologous to Klebsiella pneumoniae nifE, nifN and nifX were identified by DNA sequence analysis of a 4282 bp fragment of nif region A. Four open reading frames coding for a 51188 (NifE), a 49459 (NifN), a 17459 (NifX) and a 17472 (ORF4) dalton protein were detected. A typical NifA activated consensus promoter and two imperfect putative NifA binding sites were located in the 377 bp sequence in front of the nifE coding region. Comparison of the deduced amino acid sequences of R. capsulatus NifE and NifN revealed homologies not only to analogous gene products of other organisms but also to the and subunits of the nitrogenase iron-molybdenum protein. In addition, the R. capsulatus nifE and nifN proteins shared considerable homology with each other. The map position of nifX downstream of nifEN corresponded in R. capsulatus and K. pneumoniae and the deduced molecular weights of both proteins were nearly identical. Nevertheless, R. capsulatus NifX was more related to the C-terminal end of NifY from K. pneumoniae than to NifX. A small domain of approximately 33 amino acid residues showing the highest degree of homology between NifY and NifX was also present in all nifB proteins analyzed so far. This homology indicated an evolutionary relationship of nifX, nifY and nifB and also suggested that NifX and NifY might play a role in maturation and/or stability of the iron-molybdenum cofactor. The open reading rame (ORF4) downstream of nifX in R. capsulatus is also present in Azotobacter vinelandii but not in K. pneumoniae. Interposon-induced insertion and deletion mutants proved that nifE and nifN were necessary for nitrogen fixation in R. capsulatus. In contrast, no essential role could be demonstrated for nifX and ORF4 whereas at least one gene downstream of ORF4 appeared to be important for nitrogen fixation.     

Gnotobiotic cultures of rice plants up to ear stage in the absence of combined nitrogen source but in the presence of free living nitrogen fixing bacteriaAzotobacter vinelandii andRhodopseudomonas capsulata

Summary An all glass tight growth chamber, entirely sterilizable, has been constructed to carry out axenic and gnotobiotic cultures of rice plants (Oryza sativa L.). When grown in liquid medium and in the absence of combined nitrogen but in the presence of the diazotrophsAzotobacter vinelandii andRhodopseudomonas capsulata, rice plants exhibited a complete biological cycle from germination up to ear stage, during a period of time similar to the one encountered in french paddy soil of Camargue. In one experiment, mannitol was given to rice culture medium together withAzotobacter vinelandii andRhodopseudomonas capsulata. In another experiment, mannitol was not given together with Rhodopseudomonas, and still positive nitrogen gain was obtained, although it was less than culture with mannitol. When¹⁵N labeled cells of Rhodopseudomonas were added in rice culture medium,¹⁵N was partly transferred to rice plant. Among the nitrogen substances excreted from the bacteria in the rhizosphere medium, large organic molecules were shown to be the most abundant in our experimental conditions. Moreover, the concentration of free ammonia or aminoacids present in the rice rhizosphere were always compatible with a bacterial nitrogenase activity.     

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c ₂ from mutant H41 demonstrated at the same time the participation of the c ₂-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl bacteriochlorophyll - CIE crossed immuno-electrophoresis - DMSO dimethyl sulfoxide     

Growth and survival ofAzospirillum brasilense andArthrobacter giacomelloi in binary continuous culture

Summary Azospirillum brasilense andArthrobacter giacomelloi were grown in single and mixed succinate-limited continuous cultures at a partial oxygen pressure of 0.01atm. Growth, viability and survival during nutrient starvation were examined at various dilution rates. At D=0.05 h^–1, K_s values for succinate consumed were calculated.Arthrobacter giacomelloi viability was inversely related to dilution rate whereasAzo. brasilense was directly related. Slightly lower values of viability were obtained in mixed culture, but the ratio between the microorganisms was constant. The survival ofArth. giacomelloi in single culture decreased with increasing growth rate while survival ofAzo. brasilense was directly related to dilution rate. Acetylene reduction activity was generally very low in both single and mixed cultures. Respiration rate was also determined and the mixed culture showed an oxygen uptake rate higher than that of single cultures.Research work supported by CNR, Italy. Special grant I.P.R.A. Sub-project 1. Paper N. 317.     

Use of starter cultures containingStreptococcus diacetilactis,Lactobacillus brevis andSaccharomyces cerevisiae for fermenting milk for production of Nigeriannono

Starter cultures consisting ofStreptococcus diacetilactis, Strept. cremoris, Lactobacillus brevis andSaccharomyces cerevisiae were tested singly and in mixtures for ability to ferment milk to producenono with organoleptically acceptable qualities. Only mixed cultures containing eitherStrept. diacetilactis orStrept. cremoris andL. brevis were suitable. Presence of yeast adversely affected either acid formation or diacetyl production.Nono containingStrept. diacetilactis was acceptable, even in the presence ofSacch. cerevisiae, because of the high diacetyl production. A mixed starter containing two of these organisms,Strept. diacetilactis orStrept. cremoris andL. brevis, is recommended fornono production.Sacch. cerevisiae is not essential.R. N. Okagbue and M. O. Bankole were formerly with the Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria.     

Biological species ofArmillaria and their mycoparasitic associations withRhodophyllus abortivus in Hokkaido

Specimens of basidiomes and/or rhizomorphs ofArmillaria mellea complex and basidiomes ofRhodophyllus abortivus, developing on the same decaying stumps or stems of forest trees, were collected in three forests in Hokkaido. Normal basidiomes ofR. abortivus were found near to, but free from, the rhizomorphs and/or basidiomes ofArmillaria, while abnormal basidiomes, as carpophoroid forms, were developed on the rhizomorphs ofArmillaria. Of three mycoparasiticArmillaria isolates found withR. abortivus, one was identified asA. gallica and two asA. jezoensis. The isolates ofR. abortivus showed excellent mycelial growth and rhizomorph formation on PDA. However, on MDA, RMDA and BMDA, they showed poor aerial mycelia growth and no rhizomorphs. In the contrapositional cultures, the growth ofA. gallica was completely inhibited byR. abortivus on PDA but only slightly inhibited on MDA and RMDA. On the other hand, mutual inhibition at a distance was observed on BMDA. The mycelial growth and rhizomorph formation inA. jezoensis were severely inhibited by the colony ofR. abortivus on PDA, but only slightly inhibited on MDA. On RMDA and BMDA, the colonies of twoArmillaria species andR. abortivus showed mutual inhibition at a distance and apparent rhizomorph formation by bothArmillaria species.     

Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii

A part of the gene encoding cbb ₃-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c ₄ and c ₅. Thus, the A. vinelandii respiratory chain is shown to contain cbb ₃-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.     

Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria

1. Dry weight yields from mixed cultures ofProsthecochloris aestuarii orChlorobium limicola with the sulfur reducingDesulfuromonas acetoxidans were determined on different growth limiting amounts of acetate, ethanol or propanol. The obtained yields agreed well with values predicted from stoichiometric calculations. 2. From mixed cultures of twoChlorobium limicola strains withDesulfovibrio desulfuricans orD. gigas on ethanol as the growth limiting substrate, dry weight yields were obtained as calculated for the complete utilization of the ethanol by the mixed cultures. 3. Dry weight yield determinations for two pure cultures ofChlorobium limicola with different growth limiting amounts of sulfide in the absence and presence of excess acetate confirmed that acetate is incorporated byChlorobium in a fixed proportion to sulfide; compared to the yield in the absence of acetate the yield is increased two to threefold in the presence of acetate. 4. The lowest possible sulfide concentrations necessary for optimal growth of mixed cultures of eitherProsthecochloris orChlorobium withDesulfuromonas on acetate were 7–8 mg H₂S per liter of medium. 5. Doubling times at the growth rate limiting light intensities of 5, 10, 20, 50, 100 and 200 lux were determined under optimal growth conditions for the following phototrophic bacteria:Prosthecochloris aestuarii, Chlorobium phaeovibriodes, Chromatium vinosum andRhodopseudomonas capsulata. Reasonably good growth was still obtained withProsthecochloris at 10 and 5 lux light intensity at which no growth of the purple bacteria could be observed.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

Azotobacter vinelandii strains of disparate origin produce azotobactin siderophores with identical structures

Summary The yellow green fluorescent siderophore, azotobactin, was purified from cultures of twoAzotobacter vinelandii strains. Structural analysis of azotobactin from the North AmericanA. vinelandii strains O and its capsule negative variant UW (also called OP) revealed that both strains produced azotobactins with identical structures. Moreover, azotobactin produced by these two strains was structurally identical to azotobactin D, the fluorescent siderophore previously isolated from the EuropeanA. vinelandii strain D (CCM 289). Unlike strains of fluorescentPseudomonas which produce structurally diverse pyoverdins, strains ofA. vinelandii of disparate origin produced azotobactins of identical structure. Lactonization of azotobactin did not interfere with the ability of this compound to function as a siderophore.     

Effect of O2 limitation on growth and respiration of the wild type and an ascorbate-tetramethyl-p-phenylenediamine-oxidase-negative mutant strain ofAzotobacter vinelandii

Azotobacter vinelandii strain AVOP (wild type) and an ascorbate-N,N,N,N-tetramethylene-p-phenylenediamine oxidase-negative mutant (AV11) were each grown in O₂-limited chemostat cultures. The results showed that the mutant strain grew and used O₂ less efficiently than the wild-type strain. Respiration rates of membrane particles with NADH or malate as the substrate were similar for each strain. Succinate oxidase activity was about fourfold lower in membrane particles prepared from mutant than from wild-type strain. Cyanide at a concentration that completely inhibited ascorbate-TMPD oxidase activity resulted in a 50% inhibition of NADH oxidase activity in membrane particles of AVOP. These data suggest that the cytochromeo,a ₁, oxidase branch of the respiratory chain may be important in the physiology ofA. vinelandii under O₂-limiting growth conditions.     

Segregation characteristics of multiple chromosomes ofAzotobacter vinelandii

The genome ofAzotobacler vinelandii has been taggedin vivo with transposons. The cells have then been allowed to divide and the pattern of segregation of the genomes has been studied. The results suggest the presence of multiple (possibly identical) copies of the genome inA. vinelandii. Only a fraction of the total number of genomes seem to have been tagged with transposon and an equilibrium between alleles of the same gene with and without the transposon was evident during random segregation.     

Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Summary Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H₂ at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H₂ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H₂ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H₂ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H₂ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H₂ases.     

Frontiers in mycoplasma pathogenicity

Four different strains ofLactobacillus delbrueckii subsp.bulgaricus (Ss₁ and Yop₁₂) andStreptococcus salivarius subsp.thermophilus (Ss₂ and Yop₉) were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yop₁₂+Ss₂) shows stimulation ofS. thermophilus and inhibition ofL. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated.     

Influence of nitrogen source on growth and nitrogenase activity inAzotobacter vinelandii

Growth and nitrogenase activity were studied in cultures ofAzotobacter vinelandii growing with dinitrogen, ammonium sulfate, aspartic acid or yeast extract. Nitrogenase activity was measured by means of the C₂H₂ reduction test.In the presence of ammonium sulfate nitrogenase is completely repressed. After exhaustion of ammonia its activity is restored following a diauxic lag period of 30 min. With aspartic acid nitrogenase activity is partially repressed, and growth yield is higher than in the culture growing with N₂ only. This is due to simultaneous use of dinitrogen and aspartate. Fluctuations of nitrogenase activity occurring during exponential growth and the mechanism of their regulation are discussed.Abbreviations NA nitrogenase activity - BNF Burk's nitrogen free medium     

Cloning of ferredoxin I gene fromAzotobacter vinelandii using synthetic oligonucleotide probes

Two synthetic oligonucleotide probe mixtures, whose sequences were inferred from two separate stretches of amino acids, one closer to the carboxy terminal and the other closer to the amino terminal, of ferredoxin I protein ofAzotobacter vinelandii, were used to select ferredoxin I gene clones from a cosmid gene library ofAzotobacter vinelandii. Restriction analysis revealed that 7 out of 10 selected clones were of the same type. All these clones were found to hybridize withfixABCX genes ofRhizobium meliloti.     

Production and harvesting of ionically wall-bound extensin from living cell suspension cultures

Cell-suspension cultures ofSpinacia andRosa accumulated a cell wall protein, extensin, in a form that was amenable to leaching from the surface of the living cells by a brief treatment with non-toxic salts. Cultures ofLycopersicon, Capsicum, Acer andFestuca did not accumulate this class of extensin. InSpinacia andRosa, optimum yields of leachable extensin were achieved from young cultures, in media at relatively low pH, by leaching with 0.1 M CaCl₂. Older cultures, pH values >6.5, and LaCl₃ or higher concentrations of CaCl₂ were less effective.Abbreviation TCA trichloroacetic acid     

Attachment,colonization and proliferation ofAzospirillum brasilense andEnterobacter spp. on root surface of grasses

Root colonization studies, employing immunofluorescence and using locally isolated strains, showed thatEnterbacter sp. QH7 andEnterobacter agglomerans AX12 attached more readily to the roots of most plants compared withAzospirillum brasilense JM82. Heat treatment of either root or inoculum significantly decreased the adsorption of bacteria to the root surface. Kallar grass and rice root exudates sustained the growth ofA. brasilense JM82,Enterobacter sp. QH7 andE. agglomerans AX12 in Hoagland and Fahraeus medium. All the strains colonized kallar grass and rice roots in an axenic culture system. However, in studies involving mixed cultures,A. brasilense JM82 was inhibited byEnterobacter sp. QH7 in kallar grass rhizosphere and the simultaneous presence ofEnterobacter sp. QH7 andE. agglomerans AX12 suppressed the growth ofA. brasilense JM82 in rice rhizosphere. The bacterial colonization pattern changed from dispersed to aggregated within 3 days of inoculation. The colonization sites corresponded mainly to the areas where root mucigel was present. The area around the point of emergence of lateral roots usually showed maximum colonization.