Homology and distribution of CO dehydrogenase structural genes in carboxydotrophic bacteria

The 17 (S), 30 (M) and 87 kDa (L) subunits of CO dehydrogenases from the CO-oxidizing bacteria Pseudomonas carboxydoflava, Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans OM5 were isolated and purified. The N-terminal sequences of same subunits from different bacteria showed distinct homologies. Dot blot hybridization employing oligonucleotide probes derived from the sequences of the S-subunit of P. carboxydovorans OM5 and the M-subunit of P. carboxydohydrogena and DNA of the plasmid-containing CO-oxidizing bacteria Alcaligenes carboxydus, Azomonas B1, P. carboxydoflava, P. carboxydovorans OM2, OM4 and OM5 indicated that all genes encoding these subunits reside on plasmids. That in P. carboxydovorans OM5 CO dehydrogenase structural genes are located entirely on plasmid pHCG3 was evident from the absence of hybridization employing DNA from the cured mutant strain OM5-12. CO dehydrogenase structural genes could be identified on the chromosome of the plasmid-free bacteria Arthrobacter 11/x, Bacillus schlegelii, P. carboxydohydrogena and P. carboxydovorans OM3. There was no example of a plasmid-harboring carboxydotrophic bacterium that did not carry CO dehydrogenase structural genes on the plasmid. The N-terminal sequences of CO dehydrogenase structural genes were found to be conserved among carboxydotrophic bacteria of distinct taxonomic position, independent of the presence of plasmids. It is discussed whether this might be the consequence of horizontal gene transfer.     

Genes encoding ribulosebisphosphate carboxylase and phosphoribulokinase are duplicated in Pseudomonas carboxydovorans and conserved in carboxydotrophic bacteria

Heterologous gene probes derived from cfxLp and cfxPp genes of Alcaligenes eutrophus H16 revealed the presence of structural genes encoding ribulosebisphosphate carboxylase (Rubisco) and phosphoribulokinase (PRK) on the genome of carboxydotrophic bacteria. The two genes were found to be rather conserved. In Pseudomonas carboxydovorans OM5 cfx genes reside on the plasmid pHCG3 and the chromosome as well, indicating that they are duplicated. Also in all plasmidharboring carboxydotrophic bacteria cfxL and cfxP structural genes were found to be plasmid-coded. Our results extend the list of carboxydotrophy structural genes residing on the plasmid pHCG3 and strongly support the idea that the components essential for the chemolithoautotrophic utilization of CO by Pseudomonas carboxydovorans OM5 are plasmid-coded. A cfxL gene probe from Rhodospirillum rubrum did not detectably hybridize with DNA from any of the carboxydotrophic bacteria examined.Abbreviations CODH carbon monoxide dehydrogenase - H₂ase hydrogenase - kb kilobase - PRK phosphoribulokinase - Rubisco ribulosebisphosphate carboxylase - SDS sodium dodecylsulfate     

Nitrate respiration,denitrification, and utilization of nitrogen sources by aerobic carbon monoxide-oxidizing bacteria

We describe the ability of carboxydotrophic bacteria for nitrate respiration or denitrification. Four out of fourteen strains examined could denitrify heterotrophically forming N₂ (Pseudomonas carboxydoflava) or N₂O (Pseudomonas carboxydohydrogena, Pseudomonas compransoris, and Pseudomonas gazotropha). Three carried out a heterotrophic nitrate respiration (Arthrobacter 11/x, Azomonas B1, and Azomonas C2). P. carboxydohydrogena could use H₂ as electron donor for nitrate respiration under chemolithoautotrophic growth conditions. CO did not support denitrification or nitrate respiration of carboxydotrophic bacteria, although the free energy changes of the reactions would be sufficiently negative to allow growth. CO at 50 kPa was a weak inhibitor of N₂O-reduction in carboxydotrophic and non-carboxydotrophic bacteria and decelerated denitrifying growth. Carboxydotrophic bacteria could utilize a wide range of N-sources. Results obtained with a plasmid-cured mutant of Pseudomonas carboxydovorans OM5 showed, that genes involved in nitrogen assimilation entirely reside on the chromosome. In the presence of an suitable electron donor, most carboxydotrophic bacteria could carry out a reduction of nitrate to nitrite that did not support growth and did not lead to the formation of ammonia.This article is dedicated to Professor Hans G. Schlegel on the occasion of his 65th birthday and in admiration for his élan and eternal idealism     

Plasmids in carboxydotrophic bacteria: physical and restriction analysis

Twenty species and strains of aerobic CO-oxidizing bacteria were screened for the occurrence of plasmids. Six of them harbored plasmids between 45 and 558kb. Megaplasmids of 428 and 558 kb were resolved in Alcaligenes carboxydus. Restriction digest patterns of plasmids from different carboxydotrophic bacteria were dissimilar. However, the patterns obtained with the plasmids from the strains OM5, OM4 and OM2 of Pseudomonas carboxydovorans were very much the same. The nine cured mutants of P. carboxydovorans OM5, as well as the deletion mutant OM5-29, could not grow chemolithotrophically with CO or H₂ plus CO₂, as they were devoid of CO dehydrogenase, hydrogenase and ribulose bisphosphate carboxylase. The deletion mutant OM5-24 retained the ability to grow with CO. It could not grow with H₂ plus CO₂ and was devoid of H₂ase. The data suggest the residence of structural and/or regulatory genes of CODH, H₂ase and RuBPCx on plasmid pHCG3 of P. carboxydovorans.Abbreviations CODH carbon monoxide dehydrogenase - CRM cross reacting material - EMS ethyl methane sulfonate - H₂ase hydrogenase - kb kilobase - NTG N-methyl-N-nitro-N-nitrosoguanidine - RuBPCx ribulose bisphosphate carboxylase - SDS sodium dodecylsulfate     

CO2 is the first species formed upon CO oxidation by CO dehydrogenase from Pseudomonas carboxydovorans

The active species of CO₂, i.e. CO₂ or HCO ₃ ^- , formed in the CO dehydrogenase reaction was determined using the pure enzyme from the carboxydotrophic bacterium Pseudomonas carboxydovorans. Employing an assay system similar to that used to test for carbonic anhydrase, data were obtained which are quite compatible with those expected if CO₂ is the first species formed. In addition, carbonic anhydrase activity was not detected in P. carboxydovorans.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Reduced pyridine nucleotides in Pseudomonas carboxydovorans are formed by reverse electron transfer linked to proton motive force

In cell suspensions of Pseudomonas carboxydovorans pulsed with lithotrophic substrates (CO or H₂) in the presence of oxygen, formation of reduced pyridine nucleotides and of ATP could be demonstrated using the bioluminescent assay. Experiments employing base-acid transition, an uncoupler and inhibitors of ATPase or electron transport enabled us to propose a model for the formation of NAD(P)H in chemolithotrophically growing P. carboxydovorans.The protonophor FCCP (carbonly-p-trifluormethoxyphenylhydrazon) inhibited both, formation of NAD(P)H and of ATP. In the absence of oxygen, a chemical potential imposed by base-acid transition resulted in the formation of NAD(P)H and ATP when electrogenic substrates (CO or H₂) were present. This suggests proton motive force-driven NAD(P)H formation. The proton motive force was generated by oxidation of substrate, and not by ATP hydrolysis, as obvious from NAD(P)H formation during inhibition of ATP synthesis by oligomycin and N,N-dicyclohexylcarbodiimide.That the CO-born electrons are transferred via the ubiquinone 10-cytochrome b region to NADH dehydrogenase functioning in the reverse direction, was indicated by inhibition of NAD(P)H formation by HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) and rotenone, and by resistance to antimycin A.We conclude that in P. carboxydovorans, growing with CO or H₂, electrons and a proton motive force, generated by respiration, are required to drive an reverse electron transfer for the formation of reduced pyridine nucleotides.Abbreviations CODH carbon monoxide dehydrogenase - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl-p-trifluormethoxyphenylhydrazon - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - pmf proton motive force     

Reisolation of the carbon monoxide utilizing hydrogen bacterium Pseudomonas carboxydovorans (Kistner) comb. nov.

From enrichment cultures four carbon monoxide utilizing bacteria were isolated; strain OM5 isolated from waste water was studied in detail. The cells are Gram-negative, slightly curved rods, motile by a single subpolarly inserted flagellum. The colonies are smooth, translucent and not slimy.The cells are able to grow autotrophically in mineral medium under an atmosphere of 40% CO, 5% O₂ and 55% N₂ at a doubling time of 20h (30°C) or of 85% H₂, 5% O₂ and 10% CO₂ at a doubling time of 7h. Heterotrophic growth occurrd on organic acids such as acetate (t _d =8h), pyruvate (t _d =8h), lactate, crotonate, malate, succinate (t _d =8h), formate (t _d =35h) and glyoxylate as substrates.The enzyme system for carbon monoxide utilization is formed only during growth on CO; hydrogenase is present in cells grown on CO or on H₂+CO₂ as well as grown on pyruvate. The rate of oxygen reduction by intact CO-grown cells is 3.7-fold higher in the presence of hydrogen than in the presence of carbon monoxide. During growth the stoichiometry of gas uptake was 6.1 CO+2.8 O₂+H₂O CH₂O+5.1 CO₂. For the new isolate the name Pseudomonas carboxydovorans (Kistner) comb. nov. has been proposed.Part of this work was presented at the Second International Symposium on Microbial Growth on C₁-Compounds in Puschino, USSR, September 12th–16th, 1977.     

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,growth, and protein synthesis of Pseudomonas halodurans and Alteromonas luteo-violaceus during sulfate limitation

Sulfate concentration in the growth medium exerted a strong influence on the sulfur content of protein in two marine bacteria, Pseudomonas halodurans and Alteromonasluteo-violaceus, but the distribution of sulfur in major biochemical fractions was not affected. 90% of the total cellular sulfur was contained in low molecular weight organic compounds and protein; inorganic sulfate was not an important component. The sulfur content of isolated protein and total cellular sulfur increased in proportion to the external sulfate concentration for both bacteria, reaching a maximum at about 100–250 M. The growth rate of P. halodurans only was dependent on the sulfate concentration.Sulfur starvation of cells labeled to equilibrium with ³⁵S-sulfate resulted in a rapid decrease in low molecular weight organic S with a concommitant increase in alcohol soluble (P. halodurans) or residue protein (A. luteo-violaceus). Although cell division was prevented, total protein increased in both bacteria, resulting in synthesis of sulfur-deficient protein. This effect was most pronounced in P. halodurans.Addition of ³⁵S-sulfate to sulfur-starved A. luteo-violaceus further demonstrated that sulfur metabolism was restricted primarily to the synthesis and utilization of sulfurcontaining protein precursors. The low molecular weight organic S pool was replenished rapidly, and the pool size per cell reached twice the normal value before cell division resumed. Incorporation into protein was very rapid.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid     

Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida

Summary Toluate 1,2-dioxygenase is the first enzyme of a meta-cleavage pathway for the oxidative catabolism of benzoate and substituted benzoates to Krebs cycle intermediates that is specified by TOL plasmid pWW0 of Pseudomonas putida. A collection of derivatives harbouring Tn1000 insertions and defective in toluate dioxygenase have been isolated from pPL392, a pBR322-based hybrid plasmid carrying the TOL plasmid meta-cleavage pathway operon. In parallel, a series of N-methyl-N-nitro-N-nitrosoguanidine-induced mutant plasmids defective in this enzyme activity were isolated from pNM72, a pKT231-based hybrid plasmid carrying the same operon. Pairs of mutant plasmids, consisting of one Tn1000 derivative and one nitrosoguanidine-induced derivative, were used for complementation analysis of toluate dioxygenase in Escherichia coli recA bacteria, in which the formation of 2-hydroxymuconic semialdehyde from benzoate was examined. Four cistrons for toluate 1,2-dioxygenase were thus identified. DNA fragments containing nitrosoguanidine-induced mutant cistrons plus the other meta-cleavage operon genes were cloned into pOT5, an R388-based vector, and complementation tests between different nitrosoguanidine-induced mutant cistrons were carried out in Pseudomonas putida cells, this time scoring for growth on p-toluate. This analysis also identified four cistrons. Examination of the products of these cistrons, by means of E. coli minicells containing pPL392 or its Tn1000 insertion derivatives, indicated that the first two cistrons of the operon comprise a single gene, xylX, which encodes a 57 kilodalton protein, and that the third cistron, xy/Y, encodes a 20 kilodalton protein.     

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,protein synthesis,and growth of Pseudomonas halodurans and Alteromonas luteo-violaceus during unperturbed batch growth

Analysis of the distribution of ³⁵S-sulfate and ¹⁴C-glutamate in major biochemical components of the two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus, was compared with cell density and total cellular protein during exponential growth in batch culture. For both organisms, the sulfur distribution was restricted principally to the low molecular weight organic and protein fractions, which together accounted for over 90% of the total sulfur. Carbon was more widely distributed, with these two fractions containing only 70% of the total label.Growth rate constants calculated from increases in cell numbers, protein, and ³⁵S and ¹⁴C in the various fractions indicated nearly balanced growth in A. luteo-violaceus, with constants derived from all biosynthetic parameters agreeing within 5% during the exponential phase. In contrast, protein synthesis and ³⁵S incorporation into residue protein constants were 30% higher than constants derived from cell counts and incorporation of ¹⁴C in P. halodurans. Therefore the cellular protein content P. halodurans varied over a two-fold range, with maximum protein per cell in the late exponential phase. A distinct reduction in the rate constants for total protein and ³⁵S incorporation into residue protein foreshadowed entry into the stationary phase more than one generation before other parameters.Incorporation of ³⁵S-sulfate into residue protein paralleled protein synthesis in both bacteria. The weight percent S in protein agreed well with the composition of an average protein derived from the literature. Sulfur incorporation into protein may be a useful measurement of marine bacterial protein synthesis.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid - CFU colony forming unit     

Studies on the aerobic utilization of synthesis gas (syngas) by wild type and recombinant strains of Ralstonia eutropha H16

The biotechnical platform strain Ralstonia eutropha H16 was genetically engineered to express a cox subcluster of the carboxydotrophic Oligotropha carboxidovoransOM5, including (i) the structural genes coxM, ‐S and ‐L, coding for an aerobic carbon monoxide dehydrogenase (CODH) and (ii) the genes coxD, ‐E, ‐F and ‐G, essential for the maturation of CODH. The cox_Oc genes expressed under control of the CO₂‐inducible promoter P_L enabled R. eutropha to oxidize CO to CO₂ for the use as carbon source, as demonstrated by ¹³CO experiments, but the recombinant strains remained dependent on H₂ as external energy supply. Therefore, a synthetic metabolism, which could be described as ‘carboxyhydrogenotrophic’, was established in R. eutropha. With this extension of the bacterium's substrate range, growth in CO‐, H₂‐ and CO₂‐containing artificial synthesis gas atmosphere was enhanced, and poly(3‐hydroxybutyrate) synthesis was increased by more than 20%.     

Transfer and expression of a hydrocarbon-degrading plasmid pHCL from Pseudomonas putida to marine bacteria

Transfer of a catabolic plasmid from Pseudomonas putida to indigenous marine bacteria and obligate halophilic bacteria was carried out under both in vitro and in situ conditions. The marine recipients, which could not otherwise grow on hydrocarbon substrates, were able to degrade them after the horizontal transfer of the catabolic plasmid from P. putida. Mating conducted on nutrient plates yielded comparatively more transconjugants than in broth mating under laboratory conditions (10⁶ c.f.u./ml). The transconjugants stably maintained the plasmid when they were maintained in seawater amended with selective pressure (antibiotics/Hg (25 g/l) even after 30 days, whereas under non-selective conditions they progressively lost the plasmid after 24 days. The expression of the plasmid in the marine recipients was investigated by gas chromatographic analysis. The overall objective of this study is to evolve a novel strategy for bioremediation of oil spills and the results of the present study suggest that the present approach would offer a better solution for the removal of harmful substances from the environment by avoiding serious interference with the microbial flora of the ecosystem.     

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Summary The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 by in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 by in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84 — Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.     

The Effect of Lowered Concentrations of Carbon,Nitrogen and Phosphorus Sources on the Growth Dynamics of the R,S, and M Dissociants of Pseudomonas aeruginosa

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosaK-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants have virtually identical . The maximal values of were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in in the nitrogen-deficient medium containing 0.55% NaNO₃. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus- deficient medium containing 0.028% NaH₂PO₄· 2H₂O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.     

Interfamilial transfer of amber suppressor gene for the isolation of amber mutants of mycobacteriophage I3

A suppressor-containing strain of Mycobacterium smegmatis SN₂ was isolated by transferring an amber suppressor carried on the plasmid of Pseudomonas pseudoalcaligenes ERA through transformation. Amber mutants of mycobacteriophage I3 were isolated.Non-Common Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - moi Multiplicity of infection An abstract of this communication was presented at the IV International Congress of Genetics, Moscow, U.S.S.R., August 21–30, 1978     

Characterization and expression of genes from the RubisCO gene cluster of the chemoautotrophic symbiont of <Emphasis Type="Italic">Solemya velum</Emphasis>: <Emphasis Type="Italic">cbbLSQO</Emphasis>

Chemoautotrophic endosymbionts residing in Solemya velum gills provide this shallow water clam with most of its nutritional requirements. The cbb gene cluster of the S. velum symbiont, including cbbL and cbbS, which encode the large and small subunits of the carbon-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), was cloned and expressed in Escherichia coli. The recombinant RubisCO had a high specific activity, 3 mol min^–1 mg protein ^–1, and a K _CO2 of 40.3 M. Based on sequence identity and phylogenetic analyses, these genes encode a form IA RubisCO, both subunits of which are closely related to those of the symbiont of the deep-sea hydrothermal vent gastropod Alviniconcha hessleri and the photosynthetic bacterium Allochromatium vinosum. In the cbb gene cluster of the S. velum symbiont, the cbbLS genes were followed by cbbQ and cbbO, which are found in some but not all cbb gene clusters and whose products are implicated in enhancing RubisCO activity post-translationally. cbbQ shares sequence similarity with nirQ and norQ, found in denitrification clusters of Pseudomonas stutzeri and Paracoccus denitrificans. The 3 region of cbbO from the S. velum symbiont, like that of the three other known cbbO genes, shares similarity to the 3 region of norD in the denitrification cluster. This is the first study to explore the cbb gene structure for a chemoautotrophic endosymbiont, which is critical both as an initial step in evaluating cbb operon structure in chemoautotrophic endosymbionts and in understanding the patterns and forces governing RubisCO evolution and physiology.     

The Evolutionary History of <Emphasis Type="Italic">Shigella</Emphasis> and Enteroinvasive <Emphasis Type="Italic">Escherichia coli</Emphasis> Revised

In Shigella and enteroinvasive Escherichia coli (EIEC), the etiologic agents of shigellosis in humans, the determinants responsible for entry of bacteria into and dissemination within epithelial cells are encoded by a virulence plasmid. To understand the evolution of the association between the virulence plasmid and the chromosome, we performed a phylogenetic analysis using the sequences of four chromosomal genes (trpA, trpB, pabB, and putP) and three virulence plasmid genes (ipaB, ipaD, and icsA) of a collection of 51 Shigella and EIEC strains. The phylogenetic tree derived from chromosomal genes showed a typical star phylogeny, indicating a fast diversification of Shigella and EIEC groups. Phylogenetic groups obtained from the chromosomal and plasmidic genes were similar, suggesting that the virulence plasmid and the chromosome share similar evolutionary histories. The few incongruences between the trees could be attributed to exchanges of fragments of different plasmids and not to the transfer of an entire plasmid. This indicates that the virulence plasmid was not transferred between the different Shigella and EIEC groups. These data support a model of evolution in which the acquisition of the virulence plasmid in an ancestral E. coli strain preceded the diversification by radiation of all Shigella and EIEC groups, which led to highly diversified but highly specialized pathogenic groups.     

Diversity ofFrankia strains isolated from a single alder stand

One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined at random. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates.