Physiological and Genetic Characterization of a Diazotrophic Pseudomonad

Abstract

A soil isolate, 4B, which had been previously assigned to the genus Pseudomonas and shown to be capable of reducing C2H2 with simple phenolic compounds as sole carbon source, was further characterized in comparison with two other diazotrophs which were identified as pseudomonads. The DNA base composition of 4B was 60.2 mol% G + C. Plasmid DNA was not detected in Y.-K. C H A N A N D OTHERS METHODS Bacterial strains, plasmids and DNA.Strain 4B, which has been tentatively assigned to the genus Pseudomonas, was originally isolated from a forest soil (Chan, 1986) and was recently deposited in the American Type Culture Collection (ATCC 43038). P. delafieldii (ATCC 17505) and P . putida (ATCC 33015) were obtained from the ATCC. Pseudomonas strain H8 (ATCC 35402) was a gift from W. ). Pseudomonas strain DC, originally isolated from the roots of the grass Deschampsia caespitosa (Haahtela et al., 1983), was kindly sent to us by K. Haahtela (University of Helsinki, Finland). Klebsiella pneumoniae M5A1 was obtained from V. N. Iyer, (Carleton University, Ottawa, Canada); Escherichia coli 553 (proA metF A) carrying plasmid RP4 (AprKmr NmrTcr) was from the Plasmid Referencecenter, Stanford University, USA ; E. coli B was from the Department of Microbiology, University of Manitoba, Canada; and E. coli HBlOl(pSA30) was from D. R. Helinski, (University of California, USA).Reference DNAs of Clostridiumperfringens, E. coli B and Micrococcus lysodeikticus were purchased from Sigma.Culture media. The maintenance agar used for the diazotrophic species consisted of 0.1 % (w/v) Difco tryptic soy broth solidified with 1.6% (w/v) Difco Bacto-agar; P. delafieldii was maintained on nutrient agar. Tests for autotrophic growth with Hz, O2 and C 0 2 were done in the defined medium described by De Bont & Leijten (1976).

N2-fixing pseudomonads and related soil bacteria

“…These include simple sugars, polysaccharides, organic acids, polyols, amino acids, hydrocarbons and aromatic compounds. They are expected, and in some cases shown, to support nitrogenase activity [38,39,46,48,63,92,116] as well as saprophytic competitiveness. Interestingly, the well-studied aromatic degradation pathways of pseudomonads also exist in N2-fixing azotobacters [117,118] which are phylogenetically closely related to the authentic Pseudomonas species [14].…”

“…Because of the heterogeneity of the non-fluorescent P. stutzeri species [23], the stability of its nomenclature may still be doubted by the taxonomic purist. Pseudomonas species strain 4B (Table 2) was originally identified as similar to P. delafieldii based on biochemical tests [39]. Its taxonomic status, however, remains elusive based on DNA-rRNA hybridisation data (J.…”

“…They may also be synergistically active in degrading recalcitrant substances. An example of the pseudomonads with such capabilities is Pseudomonas species 4B which was isolated by enrichment with benzoate [39,63]. It was found to have high N 2 fixation activity in co-culture with a cellulolytic bacterium, Cellulomonas sp.…”

“…According to Palleroni [7], pseudomonads lack N2-fixing ability. The ability of the pseudomonads to fix atmospheric N 2 has not been appreciated [7,66,67] usually because of the following problems [39]: (a) mistaken or uncertain identification of the diazotroph; (b) lack of rigorous tests for diazotrophy in pseudomonads; and (c) multigeneric nature of the genus Pseudomonas as discussed above. Early attempts to find an N2-fixing pseudomonad were unsuccessful, due to the failure to recognise the 02 sensitivity or microaerobic character of its N2-fixing system.…”

“…Hence, Southern hybridisation [91], in which the Mo-nitrogenase structural genes nifHDK (usually obtained from the well-studied diazotroph Klebsiella pneumoniae M5AI, eqv. K. oxytoca) are used to probe an organism's genomic DNA, has been employed as a test for diazotrophy [39,90,[92][93][94][95][96][97]. Strictly, such probing is a test complementary to activity assay for indicating the genetic similarity between nitrogenases from different species.…”