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. 2008 Dec 3:9:581.
doi: 10.1186/1471-2164-9-581.

Comparative genomic analysis of carbon and nitrogen assimilation mechanisms in three indigenous bioleaching bacteria: predictions and validations

Gloria Levicán  1 , Juan A Ugalde, Nicole Ehrenfeld, Alejandro Maass, Pilar Parada
Affiliations

Comparative genomic analysis of carbon and nitrogen assimilation mechanisms in three indigenous bioleaching bacteria: predictions and validations

Gloria Levicán et al. BMC Genomics. .

Abstract

Background: Carbon and nitrogen fixation are essential pathways for autotrophic bacteria living in extreme environments. These bacteria can use carbon dioxide directly from the air as their sole carbon source and can use different sources of nitrogen such as ammonia, nitrate, nitrite, or even nitrogen from the air. To have a better understanding of how these processes occur and to determine how we can make them more efficient, a comparative genomic analysis of three bioleaching bacteria isolated from mine sites in Chile was performed. This study demonstrated that there are important differences in the carbon dioxide and nitrogen fixation mechanisms among bioleaching bacteria that coexist in mining environments.

Results: In this study, we probed that both Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans incorporate CO2 via the Calvin-Benson-Bassham cycle; however, the former bacterium has two copies of the Rubisco type I gene whereas the latter has only one copy. In contrast, we demonstrated that Leptospirillum ferriphilum utilizes the reductive tricarboxylic acid cycle for carbon fixation. Although all the species analyzed in our study can incorporate ammonia by an ammonia transporter, we demonstrated that Acidithiobacillus thiooxidans could also assimilate nitrate and nitrite but only Acidithiobacillus ferrooxidans could fix nitrogen directly from the air.

Conclusion: The current study utilized genomic and molecular evidence to verify carbon and nitrogen fixation mechanisms for three bioleaching bacteria and provided an analysis of the potential regulatory pathways and functional networks that control carbon and nitrogen fixation in these microorganisms.

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Figures

Figure 1
Figure 1
Schematic diagram of the reductive tricarboxylic acid cycle of L. ferriphilum DSM 17947. Catalytic enzymes are indicated by numbers: 1, malate dehydrogenase (EC 1.1.1.37); 2, fumarate hydratase (EC 4.2.1.2); 3, fumarate reductase (EC 6.2.1.5); 4, succinyl-CoA synthetase (EC 6.2.1.5); 5, 2-oxoglutarate ferredoxin oxidoreductase (EC 1.2.7.3); 6, isocitrate dehydrogenase (EC 1.1.1.42); 7, aconitase hydratase 1 (EC 4.2.1.3); 8, citryl-CoA synthetase (EC 6.2.1.18); 9, citryl-CoA lyase (EC 4.1.3.34); 10, pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). Key enzymes are indicated by asterisks. Fdxred, reduced ferredoxin.
Figure 2
Figure 2
Structure and genetic organization of the L. ferriphilum DSM 17947 genes in cluster 1 and cluster 2 predicted to be involved in reductive tricarboxylic acid (RTCA) cycle. A. Schematic map of the RTCA locus. Genes located in Cluster 1: ccsA (citryl-CoA synthetase, subunit A), ccsB (citryl-CoA synthetase, subunit B), acnA (Aconitase A), ccl (citryl-CoA lyase), orf1, frdA (fumarate reductase, subunit A), fdrB (fumarate reductase, subunit B), sucC (succinyl-CoA synthetase, beta subunit), sucD (succinyl-CoA synthetase, alpha subunit) and trx (thioredoxin). Genes located in Cluster 2: forA (2-oxoglutarate ferredoxin oxidoreductase, alpha subunit), forB (2-oxoglutarate ferredoxin oxidoreductase, beta subunit), forG (2-oxoglutarate ferredoxin oxidoreductase, gamma subunit), forE (2-oxoglutarate ferredoxin, epsilon subunit), orf2 (hypothetical protein), orf3 (hypothetical protein), porA (pyruvate ferredoxin oxidoreductase, alpha subunit), porB (pyruvate ferredoxin oxidoreductase, beta subunit), porG (pyruvate ferredoxin oxidoreductase, gamma subunit), porE (pyruvate ferredoxin oxidoreductase, epsilon subunit), porD (pyruvate ferredoxin oxidoreductase, delta subunit). B. RT-PCR amplification (RT) of intergenic. -, RT-PCR amplification control without reverse transcriptase; +, standard PCR amplification control. Primer pairs used for amplification are indicated at the top of each panel and amplification product sizes are indicated at the bottom of each panel.
Figure 3
Figure 3
Proposed models of the metabolic direction of the Embden-Meyerhof-Parnas (EMP) and TCA cycle pathways in the three microorganisms examined in this study. A) Model representing the pathways utilized by L. ferriphilum DSM 17947. PK, pyruvate kinase (EC 2.7.1.40); PEPS, phosphoenolpyruvate syntethase (EC 2.7.9.2); PC, pyruvate carboxylase (6.4.1.1); PEPC, phosphoenolpyruvate carboxylase (4.1.1.3.1); POR, pyruvate ferredoxin oxidoreductase (1.2.7.1); PDH, pyruvate dehydrogenase (EC 1.2.4.1.); CS, citrate synthase (EC2.3.3.1).B) Model representing the pathways utilized by A. ferrooxidans DSM 16786 and A. thiooxidans DSM 17318. In A. ferrooxidans, the A, B and G subunits of the Por enzyme (encoded by porABG genes) do not have amino acid identity with those from L. ferriphilum. In addition, por genes from L. ferriphilum DSM 17947 were not detected in either A. ferrooxidans DSM 16786 or A. thiooxidans DSM 17318.
Figure 4
Figure 4
Schematic diagram of the A. ferrooxidans DSM 16786 genomic region containing putative nitrogen metabolism genes. A. ferrooxidans DSM 16786 genes implicated in nitrogen fixation (nifHDK), assembly of the nitrogenase protein (fdxD-fdx-nifN-nifE) and regulation of nitrogen assimilation (nifA, draGT) are indicated.
Figure 5
Figure 5
Schematic diagram of the A. thiooxidans DSM17318 genomic region containing putative nitrate assimilation genes. The following genes are indicated: ntrA encodes the periplasmic component of the nitrate transport system; narK encodes a nitrate/nitrite transporter; nirB encodes a nitrite reductase, which is interrupted by the transposase tnpA; nirD encodes the nitrite reductase small subunit; narB encodes a nitrate reductase; cysG encodes an uroporphyrin-III C-methyltransferase; and nasT encodes a nitrate assimilation system regulator.
Figure 6
Figure 6
Phylogenetic tree based on NifA protein sequences. The tree was inferred using the Neighbor-Joining method, with 1000 replicates. Only those branches that appear in more than 50% of the boostrap replicates are considered. Evolutionary distances were computed using the JTT matrix. Analysis was conducted using the software MEGA4 [110].
Figure 7
Figure 7
Regulatory model of nitrogen assimilation proposed for A. ferrooxidans DSM 16786. Proteins (boxes) and their resulting functions (lines) are indicated in red for activation during low nitrogen conditions and in blue during high nitrogen condition. In green are identified the principal effectors and nitrogen source that trigger the activation/inactivation of the enzymes involved in the model: ammonia (NH3), molecular nitrogen (N2), glutamine and 2-oxoglutarate. In orange are represented the signal transducer proteins: GlnD (uridylyltransferase), NtrB (Nitrogen regulation protein ntrB) and GlnE (Glutamate-ammonia-ligase adenylyltransferase). It is important to mention that this model considers the role of NifA in activating target genes under low nitrogen conditions.
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