This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leaphart, A. B.
Right arrow Articles by Lovell, C. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leaphart, A. B.
Right arrow Articles by Lovell, C. R.
Agricola
Right arrow Articles by Leaphart, A. B.
Right arrow Articles by Lovell, C. R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, January 2003, p. 693-696, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.693-696.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Formyltetrahydrofolate Synthetase Sequences from Salt Marsh Plant Roots Reveal a Diversity of Acetogenic Bacteria and Other Bacterial Functional Groups

A. B. Leaphart,1 M. J. Friez,2 and C. R. Lovell1*

Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208,1 Greenwood Genetic Center, Greenwood, South Carolina 296462

Received 12 August 2002/ Accepted 25 October 2002


arrow
ABSTRACT
 
Sixty-two partial formyltetrahydrofolate synthetase (FTHFS) structural gene sequences were recovered from roots of salt marsh plants, including Spartina alterniflora, Salicornia virginica, and Juncus roemerianus. Only S. alterniflora roots yielded sequences grouping with FTHFS sequences from known acetogens. Most other FTHFS or FTHFS-like sequences grouped with those from sulfate-reducing bacteria. Several sequences that grouped with Sphingomonas paucimobilis ligH were also recovered.


arrow
INTRODUCTION
 
The acetogens are anaerobic bacteria that utilize the acetyl-coenzyme A (CoA) pathway for synthesis of acetyl-CoA from C1 compounds (6, 33). The acetogens are diverse (6, 29, 31), physiologically versatile (3, 7, 33), and apparently ubiquitous in anoxic and suboxic environments (7). It is not unlikely that acetogens are ecologically significant producers of acetate, a key intermediate in terminal carbon metabolism, in many environments.

Salt marshes are among the most productive ecosystems known. Much of salt marsh terminal carbon metabolism occurs in saturated, anoxic sediments and is due to sulfate-reducing bacteria or, in deeper, sulfate-limited sediments, to methanogenic Archaea. The root zones of salt marsh plants are foci of high microbial biomass and intensive activity by bacteria (reviewed in reference 20). These bacteria occur in sediments impacted by the roots (the rhizosphere), on the root surfaces (the rhizoplane), and within the root cortex (the endorhizosphere) (5, 20). Despite intermittent oxygen availability in the rhizospheres of salt marsh plants due to gas transport processes (13, 32), root-associated anaerobes, including sulfate reducers, methanogens, and acetogens, have been previously documented (9, 12, 19-21, 27). Recently, Küsel et al. (17) demonstrated the presence of acetogenic bacteria in the rhizoplane and endorhizosphere of the seagrass Halodule wrightii. With the exception of recovery of a few partial sequences of the structural gene encoding the acetyl-CoA pathway enzyme, formyltetrahydrofolate synthetase (FTHFS), from roots of the dominant cordgrass Spartina alterniflora (19), there has been no investigation of salt-marsh-plant-associated acetogens or acetogenesis. Consequently, we have little knowledge of what types of plants might harbor these organisms or what types of acetogens might be associated with salt marsh plants.

In this study, we have recovered and phylogenetically analyzed partial FTHFS sequences to examine the diversity of acetogenic and other FTHFS-containing bacteria associated with roots of dominant salt marsh plants. We found a clear demarcation between sequences recovered from the low-marsh dominant S. alterniflora (smooth cordgrass; designated Spartina hereafter) and those from two common high-marsh plants, Salicornia virginica (common pickleweed; designated Salicornia hereafter) and Juncus roemerianus (black needlerush; designated Juncus hereafter).

Live roots were collected from Salicornia, Juncus, and the short and tall growth forms of Spartina from the Crab Haul Creek basin of the North Inlet salt marsh (Georgetown, S.C.; 33°22'N,79°12'W) in September 2000. Juncus roots were obtained from a monophyletic stand near the fringe of the terrestrial biome and from a monophyletic patch within the short Spartina zone. Juncus roots were also collected from separate sites on Goat Island, which is also in the North Inlet ecosystem. Additionally, mixed root samples were taken from a mixed stand of Salicornia and short Spartina in the Crab Haul Creek basin. Roots were rinsed free of sediment with deionized water. DNA was purified from the root samples by using the direct lysis method of Lovell and Piceno (22). Residual contaminants were removed by using the Wizard Prep DNA cleanup kit (Promega, Madison, Wis.).

DNA was extracted from cell pellets of authentic sulfate-reducing bacteria by using the Wizard DNA purification kit (Promega). The sulfate-reducing bacteria were kindly provided by Richard Devereux and included the following: Desulfoarculus baarsii, Desulfomicrobium baculatum, Desulfovibrio desulfuricans, Desulfovibrio piger, Desulfovibrio salexigens, an unnamed Desulfovibrio isolate from H. wrightii roots designated summer lac-1, and unnamed isolates BG8 and BG14. We also recovered partial FTHFS sequences from two acetogenic Treponema strains, provided by John Breznak; Eubacterium limosum ATCC 8486, provided by Ralph Tanner; and Acetobacterium psammolithicum, provided by Harry Beller.

PCR amplifications, cloning, sequencing, translation and alignments were performed according to the methods of Leaphart and Lovell (19). All root samples yielded strong, specific amplification products, although products from the mixed Salicornia-Spartina roots and from short Spartina were noticeably less intense than the others. The translated, partial FTHFS sequences were examined for key, highly conserved amino acids important to structure and/or function of this protein (19, 23, 26). Residues that differentiated FTHFS sequences of known acetogens from those from other sources included Lys 187, Lys 256, Thr 266, Leu 314, Tyr 339, Ile 370, Pro 385, Val 406, Ala 407, Val 411, Lys 414, Gly 419, Ile 447, Ala 470, Leu 480, and Lys 484 (Asn 484 in the Moorella thermoacetica sequence).

Thirty-nine partial FTHFS sequences were recovered from Crab Haul Creek basin plant roots. Ten sequences were from short Spartina, 15 from tall Spartina, 10 from Salicornia, 2 from the Salicorni-Spartina mixed zone, 1 from the Juncus patch, and 1 from the Juncus terrestrial fringe main stand. Another 22 sequences were recovered from the Juncus patch, and 1 was recovered from the Juncus main stand from Goat Island. In total, 62 new sequences from salt marsh plant roots were included. Eight sequences were recovered from pure cultures of sulfate-reducing bacteria, two from acetogenic treponemes, and one each from E. limosum ATCC 8486 and A. psammolithicum. Sequences recovered from the roots of Goat Island short Spartina in a previous study (19) were included in this analysis and were designated Goat Island short Spartina.

Phylogenetic trees were constructed by neighbor joining, with complete deletion of gaps and missing data and Poisson correction for multiple substitutions (see reference 19). Use of other distance estimation parameters and algorithms resulted in no significant effect on dendrogram topology (data not shown). The dendrogram (Fig. 1) contained three major FTHFS sequence clusters. Cluster A consisted of sequences from known pure culture acetogens as well as divergent sequences from known FTHFS-containing but nonacetogenic organisms. Three new tall and two new short Spartina sequences were included within Cluster A. Short Spartina 44 sequence was affiliated with Clostridium formicoaceticum and Clostridium aceticum (89.1% and 91.8% similar, respectively), as was tall Spartina 4 (87.1% similar to short Spartina 44). Short Spartina 4 and tall Spartina 34 were 83.9% similar to each other and were most similar to C. formicoaceticum and C. aceticum among the known acetogen sequences. Tall Spartina 32 grouped with Sporomusa ovata (78.1% similarity) and Sporomusa termitida (79.6% similarity). A separate small cluster fell between major clusters A and B and contained sequences short Spartina 3 and 45 and tall Spartina 25. These sequences all had similarities greater than 80%.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 1. Phylogenetic analysis of partial FTHFS and FTHFS-like sequences. The dendrogram was generated by using neighbor joining- and Poisson-corrected distances. Thermoplasma acidophilum and Thermoplasma volcanicum FTHFS sequences were used as outgroup taxa. The values at the nodes are the percentages of 1,000 bootstrap replicates supporting the branching order. Bootstrap values below 50% are not shown.

It seems significant that FTHFS sequences with strong affiliations to those from authentic acetogens were only recovered from roots of Spartina. This plant inhabits low marsh elevations where the frequency and duration of tidal flooding are greater than at higher elevations. Due to the slow rate of gas diffusion into water-saturated sediments and the high oxygen demand, bulk sediments in the short and tall Spartina zones are anoxic below the upper few millimeters (25). However, Spartina ventilates its roots and rhizosphere (13, 32), introducing oxygen. In light of recent findings that some, possibly most, acetogens can reduce free oxygen (16), ventilation would not be expected to hinder acetogen colonization of the roots. Salicornia lacks aerenchyma and is apparently unable to ventilate its rhizosphere (1). Ventilation by J. roemerianus has not been described, though some Juncus species can ventilate (4). The selective advantages enjoyed by acetogens on Spartina roots relative to roots of the other salt marsh plants are presently unknown, but the differences in gas transport among these plants are intriguing.

Cluster B included the majority and greatest diversity of root FTHFS sequences. Subcluster B1 contained sequences from D. baarsii and sulfate-reducing isolate BG8, four sequences from short Spartina, and three sequences from the Goat Island Juncus patch. Subcluster B2 contained five sequences from Salicornia, four from the Goat Island Juncus patch, and two from short Spartina. Subcluster B3 contained a tight grouping of two tall Spartina sequences with the D. baculatus sequence. The tall Spartina 15 sequence was 99.4% similar to the D. baculatus sequence. This grouping was also allied with a group containing 10 sequences from the Goat Island Juncus patch, 3 from short Spartina, and 1 from the Spartina-Salicornia mixed zone. Subcluster B4 contained a tight grouping of sulfate reducer sequences with tall Spartina 20. The Desulfovibrio sp. summer lac-1 sequence was 99.7% similar to tall Spartina 20, while the D. desulfuricans sequence was 99.4% and 99.5% similar to summer lac-1 and tall Spartina 20, respectively.

Cluster B sequences were ubiquitous and diverse, and a number of them were very similar to FTHFS sequences recovered from known sulfate-reducing bacteria. Acetate is considered to be quantitatively the principal substrate for sulfate reducers in natural ecosystems (11, 18, 30), and catabolism of acetate has been shown to occur through the tricarboxylic acid cycle (2, 10) and via the acetyl-CoA pathway, functioning in reverse (8, 28). In addition, D. baarsii is capable of autotrophic acetogenesis by using FTHFS and the acetyl-CoA pathway (14, 15). It seems likely, on the basis of the broad distribution of sulfate reducer FTHFS sequences within this cluster and the very high similarities between FTHFS sequences from known sulfate reducers and those from roots, that many of the root sequences within this cluster originated from sulfate-reducing bacteria.

Cluster C was relatively small and contained another grouping of a sulfate reducer sequence with a sequence from tall Spartina. The D. salexigens sequence was 99.7% similar to tall Spartina 21. This group was allied with short Spartina 25 and 40. Branching from this grouping were tall Spartina 35 and another grouping containing the D. piger sequence and short Spartina 42 (99.7% similar). Partial FTHFS sequences from Mesorhizobium loti and Sinorhizobium meliloti were also allied with these sequences, as were Goat Island Juncus patch 49 and the sequence for the Sphingomonas paucimobilis o-demethylating enzyme encoded by ligH, which has 60% similarity to the FTHFS sequence from Moorella thermoacetica (24). The grouping of the S. paucimobilis LigH sequence with this cluster is of interest, but none of the sequences from plant roots was highly similar to that of LigH.

The diversity of partial FTHFS sequences recovered from salt-marsh-plant-associated acetogens and other FTHFS-containing organisms was substantial. The environmental FTHFS sequences within the true acetogen cluster included only sequences recovered from roots of Spartina. The other salt marsh plants inhabit somewhat different marsh zones, and the differences among the plant species and their habitats may provide the ecological basis for this finding. Further study of the rates of acetogenesis and FTHFS expression on the rhizoplane and in the endorhizosphere of these plants is needed to further clarify the meaning and significance of these results.


arrow
Nucleotide sequence accession numbers.
 
The nucleotide sequences determined in this study have been deposited in the GenBank database under accession numbers AJ494749 to AJ494825.


arrow
ACKNOWLEDGMENTS
 
We acknowledge Henry Beller, John Breznak, Richard Devereux, and Ralph Tanner for providing pure reference cultures of acetogens and sulfate-reducing bacteria and Christopher Bagwell for assistance with field sampling and DNA purifications.

This research was supported by NSF grants MCB-9873606 and DEB-9903623 to C.R.L.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, 715 Sumter St., University of South Carolina, Columbia, SC 29208. Phone: (803) 777-7036. Fax: (803) 777-4002. E-mail: lovell{at}biol.sc.edu. Back


arrow
REFERENCES
 
    1
  1. Anderson, C. E. 1974. A review of structure in several North Carolina salt marsh plants, p. 307-344. In R. J. Reimold and W. H. Queens (ed.), Ecology of halophytes. Academic Press, New York, N.Y.
    2. 2
  2. Brandis, A., N. A. Gerhart, R. K. Thauer, F. Widdel, and N. Pfennig. 1983. Anaerobic oxidation to CO2 by Desulfobacter postgatei. 1. Demonstration of all enzymes required for the operation of the citric acid cycle. Arch. Microbiol. 136:222-229.[CrossRef]
    3. 3
  3. Braun, M., S. Schoberth, and G. Gottschalk. 1979. Enumeration of bacteria forming acetate from H2 and CO2 in anaerobic habitats. Arch. Microbiol. 120:201-204.[CrossRef][Medline]
    4. 4
  4. Brix, H., B. K. Sorrell, and P. T. Orr. 1992. Internal pressurization and convective gas flow in some emergent freshwater macrophytes. Limnol. Oceanogr. 37:1420-1433.
    5. 5
  5. Campbell, R., and M. P. Greaves. 1990. Anatomy and community structure of the rhizosphere, p. 11-34. In J. M. Lynch (ed.), The rhizosphere. John Wiley & Sons, Chichester, England.
    6. 6
  6. Drake, H. L. 1994. Acetogenesis, acetogenic bacteria, and the acetyl-CoA "Wood/Ljungdahl" pathway: past and current perspectives, p. 3-60. In H. L. Drake (ed.), Acetogenesis. Chapman and Hall, New York, N.Y.
    7. 7
  7. Drake, H. L., S. L. Daniel, K. Küsel, C. Matthies, C. Kuhner, and S. Braus-Stromeyer. 1997. Acetogenic bacteria: what are the in situ consequences of their diverse metabolic versatilities? BioFactors 6:13-24.[Medline]
    8. 8
  8. Fuchs, G. 1986. CO2 fixation in acetogenic bacteria: variations on a theme. FEMS Microbiol. Rev. 39:181-213.[CrossRef]
    9. 9
  9. Gandy, E. L., and D. C. Yoch. 1988. Relationship between nitrogen-fixing sulfate reducers and fermenters in salt marsh sediments and roots of Spartina alterniflora. Appl. Environ. Microbiol. 54:2031-2036.[Abstract/Free Full Text]
    10. 10
  10. Gebhart, N. A., D. Linder, and R. K. Thauer. 1983. Anaerobic acetate oxidation to CO2 by Desulfobacter postgatei. 2. Evidence from 14C-labeling studies for the operation of the citric acid cycle. Arch. Microbiol. 136:230-233.[CrossRef]
    11. 11
  11. Gibson, G. R. 1990. Physiology and ecology of the sulphate-reducing bacteria. J. Appl. Bacteriol. 69:769-797.[Medline]
    12. 12
  12. Hines, M. E., R. S. Evans, B. R. Sharak Genthner, S. G. Willis, S. Friedman, J. N. Rooney-Varga, and R. Devereux. 1999. Molecular phylogenetic and biogeochemical studies of sulfate-reducing bacteria in the rhizosphere of Spartina alterniflora. Appl. Environ. Microbiol. 65:2209-2216.[Abstract/Free Full Text]
    13. 13
  13. Hwang, Y. H., and J. T. Morris. 1991. Evidence for hygrometric pressurization in the internal gas space of Spartina alterniflora. Plant Physiol. 96:166-171.[Abstract/Free Full Text]
    14. 14
  14. Jansen, K., G. Fuchs, and R. K. Thauer. 1985. Autotrophic CO2 fixation by Desulfovibrio baarsii: demonstration of enzyme activities characteristic for the acetyl-CoA pathway. FEMS Microbiol. Lett. 28:311-315.
    15. 15
  15. Jansen, K., R. K. Thauer, F. Widdel, and G. Fuchs. 1984. Carbon assimilation pathways in sulfate reducing bacteria. Formate, carbon dioxide, carbon monoxide, and acetate assimilation by Desulfovibrio baarsii. Arch. Microbiol. 138:257-262.[CrossRef]
    16. 16
  16. Karnholz, A., K. Küsel, A. Gossner, A. Schramm, and H. L. Drake. 2002. Tolerance and metabolic response of acetogenic bacteria toward oxygen. Appl. Environ. Microbiol. 68:1005-1009.[Abstract/Free Full Text]
    17. 17
  17. Küsel, K., H. C. Pinkart, H. L. Drake, and R. Devereux. 1999. Acetogenic and sulfate-reducing bacteria inhabiting the rhizoplane and deep cortex cells of the sea grass Halodule wrightii. Appl. Environ. Microbiol. 65:5117-5123.[Abstract/Free Full Text]
    18. 18
  18. Laanbroek, H. J., and N. Pfennig. 1981. Oxidation of short-chain fatty acids by sulfate-reducing bacteria in freshwater and in marine sediments. Arch. Microbiol. 128:330-335.[CrossRef][Medline]
    19. 19
  19. Leaphart, A. B., and C. R. Lovell. 2001. Recovery and analysis of formyltetrahydrofolate synthetase gene sequences from natural populations of acetogenic bacteria. Appl. Environ. Microbiol. 67:1392-1395.[Abstract/Free Full Text]
    20. 20
  20. Lovell, C. R. 2002. Plant-microbe interactions in the marine environment, p. 2539-2554. In G. Bitton (ed.), Encyclopedia of environmental microbiology, vol. 5. John Wiley & Sons, New York, N.Y.
    21. 21
  21. Lovell, C. R., Y. M. Piceno, J. M. Quattro, and C. E. Bagwell. 2000. Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass Spartina alterniflora. Appl. Environ. Microbiol. 66:3814-3822.[Abstract/Free Full Text]
    22. 22
  22. Lovell, C. R., and Y. M. Piceno. 1994. Purification of DNA from estuarine sediments. J. Microbiol. Methods 20:161-174.[CrossRef]
    23. 23
  23. Lovell, C. R., A. Przbyla, and L. G. Ljungdahl. 1990. Primary structure of the thermostable formyltetrahydrofolate synthetase from Clostridium thermoaceticum. Biochemistry 29:5687-5694.[CrossRef][Medline]
    24. 24
  24. Nishikawa, S., T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama. 1998. Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate. Appl. Environ. Microbiol. 64:836-842.[Abstract/Free Full Text]
    25. 25
  25. Pinckney, J., and R. G. Zingmark. 1993. Biomass and production of benthic microalgal communities in estuarine habitats. Estuaries 16:887-897.[CrossRef]
    26. 26
  26. Radfar, R., R. Shin, G. M. Sheldrick, W. Minor, C. R. Lovell, J. D. Odom, R. B. Dunlap, and L. Lebioda. 2000. The crystal structure of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica. Biochemistry 39:3920-3926.[CrossRef][Medline]
    27. 27
  27. Rooney-Varga, J. N., R. Devereux, R. S. Evans, and M. E. Hines. 1997. Seasonal changes in the relative abundance of uncultivated sulfate-reducing bacteria in a salt marsh sediment and in the rhizosphere of Spartina alterniflora. Appl. Environ. Microbiol. 63:3895-3901.[Abstract]
    28. 28
  28. Schauder, R., B. Eikmanns, R. K. Thauer, F. Widdel, and G. Fuchs. 1986. Acetate oxidation to CO2 in anaerobic bacteria via a novel pathway not involving reactions of the citric acid cycle. Arch. Microbiol. 145:162-172.[CrossRef]
    29. 29
  29. Schink, B. 1994. Diversity, ecology, and isolation of acetogenic bacteria, p. 197-235. In H. L. Drake (ed.) Acetogenesis. Chapman and Hall, New York, N.Y.
    30. 30
  30. Sorensen, J., D. Christensen, and B. B. Jorgensen. 1981. Volatile fatty acids and hydrogen as substrates for sulfate-reducing bacteria in anaerobic marine sediment. Appl. Environ. Microbiol. 42:5-11.[Abstract/Free Full Text]
    31. 31
  31. Tanner, R. S., and C. R. Woese. 1994. A phylogenetic assessment of the acetogens, p. 254-272. In H. L. Drake (ed.), Acetogenesis. Chapman and Hall, New York, N.Y.
    32. 32
  32. Teal, J. M., and J. W. Kanwisher. 1966. Gas transport in the marsh grass Spartina alterniflora. J. Exp. Bot. 17:355-361.[Abstract/Free Full Text]
    33. 33
  33. Wood, H. G., and L. G. Ljungdahl. 1991. Autotrophic character of the acetogenic bacteria, p. 201-250. In L. L. Barton and J. Shively (ed.), Variations in autotrophic life. Academic Press, San Diego, Calif.

Applied and Environmental Microbiology, January 2003, p. 693-696, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.693-696.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leaphart, A. B.
Right arrow Articles by Lovell, C. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leaphart, A. B.
Right arrow Articles by Lovell, C. R.
Agricola
Right arrow Articles by Leaphart, A. B.
Right arrow Articles by Lovell, C. R.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»