Abstract
Nitrogen-fixing bacteria were isolated from sediments and water of a saltmarsh lagoon on the west coast of South Africa, and characterized according to factors that regulate nitrogen fixation in the marine environment. The majority of isolates were assigned to the Photobacterium or Vibrio genera on the basis of physiological and biochemical characteristics. One isolate was further assigned to the species Vibrio diazotrophicus. Carbohydrate utilization by each diazotrophic isolate was examined. Abilities of the isolates to utilize a range of mono-, di-, and polysaccharides largely reflected the predicted availability of organic carbon and energy in the lagoon, except that chitin was not utilized. Biochemical tests on the utilization of combined nitrogen showed that one isolate could utilize nitrate, and that this strain was susceptible to full repression of nitrogenase activity by 10mm nitrate. Urease activity was not detected in any of the isolates. In the absence of molybdenum two of the isolates, a Photobacterium spp. and V. diazotrophicus, reduced acetylene to ethylene and ethane, a property frequently associated with the activity of alternative nitrogenases. Addition of 25µM molybdenum inhibited ethane production by V. diazotrophicus, but stimulated ethylene and ethane production by the Photobacterium isolate. Addition of 28µM vanadium did not appear to regulate ethane production by either strain. Assays of nitrogenase activity in sediments from which some isolates were obtained indicated that molybdenum was not limiting nitrogenase activity at naturally-occurring concentrations. Southern hybridizations of the chromosomes of these strains with the anfH and vnfH genes of Azotobacter vinelandii and the nifH gene of Klebsiella pneumoniae indicated the presence of only one nitrogenase in these isolates.
Similar content being viewed by others
References
Andrews WRH, Hutchings L (1980) Upwelling in the southern Benguela current. Prog Oceanogr 9: 1–81
Béguin P (1990) Molecular biology of cellulose degradation. Annu Rev Microbiol 44:219–248
Bush JA, Wilson PW (1959) A non-gummy chromogenic strain of Azotobacter vinelandii. Nature (London) 184:381–382
Cannon FC, Riedel GE, Ausubel FM (1979) Overlapping sequences of Klebsiella pneumoniae nif DNA cloned and characterized. Mol Gen Genet 174:59–66
Casse F, Boucher C, Julliot JS, Michel M, and Denarie J (1979) Identification and characterization of large plasmids in Rhizobium meliloti using agarose gel electrophoresis. J Gen Microbiol 113:229–242
Chisnell JR, Premakumar RR, Bishop PE (1988) Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii. J Bacteriol 170:27–33
Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci 81:1991–1995
Collier RW (1985) Molybdenum in the northeast Pacific Ocean. Limnol Oceanogr 30:1351–1354
Darylo M, Glowacka M, Skorupska A, Lorkiewicz Z (1981) Nif plasmid from Lignobacter. Arch Microbiol 130:322–324
Dicker HJ, Smith DW (1980) Enumeration and relative importance of acetylene-reducing (nitrogen-fixing) bacteria in a Delaware saltmarsh. Appl Environ Microbiol 39:1019–1025
Dicker HJ, Smith DW (1980) Physiological ecology of acetylene reduction (nitrogen fixation) in a Delaware salt marsh. Microb Ecol 6:161–171
Dilworth MJ, Eady RR, Robson RL, Miller RW (1987) Ethane formation from acetylene as a potential test for vanadium nitrogenase in vivo. Nature 327:167–168
Guerinot ML, Colwell RR (1985) Enumeration, isolation, and characterization of N2-fixing bacteria from seawater. Appl Environ Microbiol 50:350–355
Guerinot ML, Patriquin DG (1981) The association of N2-fixing bacteria with sea urchins. Mar Biol 62:197–207
Guerinot ML, West PA, Lee JV, Colwell RR (1982) Vibrio diazotrophicus sp. nov., a marine nitrogen-fixing bacterium. Int J Syst Bacteriol 32:350–357
Hanson RB (1977) Comparison of nitrogen fixation activity in tall and short Spartina alterniflora salt marsh soils. Appl Environ Microbiol 33:596–602
Hattori A (1983) Denitrification and dissimilatory nitrate reduction. In: Carpenter EJ, Capone DG (eds) Nitrogen in the marine environment. Academic Press, New York, pp 191–232
Howarth RW, Cole JJ (1985) Molybdenum availability, nitrogen limitation, and phytoplankton growth in natural waters. Science 229:653–655
Jacobson MR, Brigle KE, Bennett LT, Setterquist RA, Wilson MS, Cash VL, Benyon J, Newton WE, Dean DR (1989) Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J Bacteriol 171:1071–1077
Joerger RD, Bishop PE (1988) Bacterial alternative nitrogen fixation systems. Crit Rev Microbiol 16:1–14
Joerger RD, Jacobson MR, Premakumar R, Wolfinger ED, Bishop PE (1989) Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii. J Bacteriol 171:1075–1086
Joerger RD, Loveless TM, Pau RN, Mitchenall LA, Simon BH, Bishop PE (1990) Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii. J Bacteriol 172:3400–3408
Kentemich T, Danneberg G, Hundeshagen B, Bothe H (1988) Evidence for the occurrence of the alternative, vanadium-containing nitrogenase in the cyanobacterium Anabaena variabilis. FEMS Microbiol Lett 51:19–24
Kimble LK, Madigan MT (1992) Evidence for an alternative nitrogenase in Heliobacterium gestii. FEMS Microbiol Lett 100:255–260
Lehman LJ, Roberts GP (1991) Identification of an alternative nitrogenase system in Rhodospirillum rubrum. J Bacteriol 173:5705–5711
Maruyama Y, Taga N, Matsuda O (1970) Distribution of nitrogen-fixing bacteria in the central Pacific ocean. J Oceanogr Soc (Jpn) 26:360–366
Murray MG, Thompson WF (1980) Rapid isolation of high-molecular-weight plant DNA. Nucl Acids Res 8:4321–4325
O'Donohue MJ, Moriarty DJW, Mac Rae IC (1991) Nitrogen fixation in sediments and the rhizosphere of the seagrass Zostera capricorni. Microb Ecol 22:53–64
Pau RN, Mitchenall LA, Robson RL (1989) Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium. J Bacteriol 171:124–129
Paulsen DM, Paerl HW, Bishop PE (1991) Evidence that molybdenum-dependent nitrogen fixation is not limited by high sulfate concentrations in marine environments. Limnol Oceanogr 36:1325–1334
Plazinski J, Dart PJ, Rolfe BG (1983) Plasmid visualization and nif gene location in nitrogen-fixing Azospirillum strains. J Bacteriol 155:1429–1433
Robson RL, Eady RR, Richardson TH, Miller RW, Hawkins M, Postgate JR (1986) The alternative nitrogenase of Azotobacter chroococcum is a vanadium enzyme. Nature 322:388–390
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbour Laboratory Press, New York
Scherer P (1989) Vanadium and molybdenum requirement for the fixation of molecular nitrogen by two Methanosarcina strains. Arch Microbiol 151:44–48
Shah VK, Brill WJ (1977) Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci USA 74:3249–3253
Shieh WY, Simidu U, Maruyama Y (1989) Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed. Microb Ecol 18:249–259
Smibert RM, Krieg NR (1981) General characterization. In: Gerhardt et al. (eds) Manual of methods for general bacteriology. ASM, Washington DC, pp 409–443
Strandberg GW, Wilson PW (1968) Formation of the nitrogen fixing system in Azotobacter vinelandii. Can J Microbiol 14:25–31
Thayer GW, Wolfe DA, Williams RB (1975) The impact of man on seagrass systems. Am Sci 63:288–296
Urdaci MC, Stal LJ, Marchand M (1988) Occurrence of nitrogen fixation among Vibrio spp. Arch Microbiol 150:224–229
Valiela IV, Teal JM (1979) The nitrogen budget of a saltmarsh ecosystem. Nature 280:652–656
Watanabe I, Barraquio WL (1979) Low levels of fixed nitrogen required for isolation of free-living, N2-fixing organisms from rice roots. Nature 277:565–566
Wilson K (1989) Large scale CsCl preparation of bacterial genomic DNA, unit 2.4.3. In: Ausubel FM et al. (eds) Current protocols in molecular biology, vol 1. John Wiley and Sons, New York
Author information
Authors and Affiliations
Additional information
Correspondence to: B.J. Tibbles.
Rights and permissions
About this article
Cite this article
Tibbles, B.J., Rawlings, D.E. Characterization of nitrogen-fixing bacteria from a temperate saltmarsh lagoon, including isolates that produce ethane from acetylene. Microb Ecol 27, 65–80 (1994). https://doi.org/10.1007/BF00170115
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00170115