Contribution of Pentose Catabolism to Molecular Hydrogen Formation by Targeted Disruption of Arabinose Isomerase (araA) in the Hyperthermophilic Bacterium Thermotoga maritima

Abstract

Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H₂). In this organism, the catabolism of pentose sugars such as arabinose depends on the interaction of the pentose phosphate pathway with the Embden-Myerhoff and Entner-Doudoroff pathways. Although the values for H₂ yield have been determined using pentose-supplemented complex medium and predicted by metabolic pathway reconstruction, the actual effect of pathway elimination on hydrogen production has not been reported due to the lack of a genetic method for the creation of targeted mutations. Here, a spontaneous and genetically stable pyrE deletion mutant was isolated and used as a recipient to refine transformation methods for its repair by homologous recombination. To verify the occurrence of recombination and to assess the frequency of crossover events flanking the deleted region, a synthetic pyrE allele, encoding synonymous nucleotide substitutions, was used. Targeted inactivation of araA (encoding arabinose isomerase) in the pyrE mutant was accomplished using a divergent, codon-optimized Thermosipho africanus pyrE allele fused to the T. maritima groES promoter as a genetic marker. Mutants lacking araA were unable to catabolize arabinose in a defined medium. The araA mutation was then repaired using targeted recombination. Levels of synthesis of H₂ using arabinose-supplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H₂ production that was dependent on arabinose consumption. Development of a targeted recombination system for genetic manipulation of T. maritima provides a new strategy to explore H₂ formation and life at an extremely high temperature in the bacterial domain.

Importance: We describe here the development of a genetic system for manipulation of Thermotoga maritima T. maritima is a hyperthermophilic anaerobic bacterium that is well known for its efficient synthesis of molecular hydrogen (H₂) from the fermentation of sugars. Despite considerable efforts to advance compatible genetic methods, chromosome manipulation has remained elusive and hindered use of T. maritima or its close relatives as model hyperthermophiles. Lack of a genetic method also prevented efforts to manipulate specific metabolic pathways to measure their contributions to H₂ yield. To overcome this barrier, a homologous chromosomal recombination method was developed and used to characterize the contribution of arabinose catabolism to H₂ formation. We report here a stable genetic method for a hyperthermophilic bacterium that will advance studies on the basic and synthetic biology of Thermotogales.

Keywords: anaerobes; biohydrogen; extremophiles; genetic systems; homologous recombination.

FIG 1

Genotypic analysis, DNA sequence, and growth curve of the pyrE-129 mutant. (A) DNA sequence of the pyrE-64 mutant, its revertants, and the pyrE-129 mutant. The highlighted and boxed nucleotide sequence indicates sites of the deletion and insertion events in mutant strains. The numbers indicate the location of the deletion and insertion within the pyrE gene in all five strains. (B) PCR amplification of the pyrE allele using genomic DNA from the pyrE-129 mutant (lane 1) and the wild type (lane 2). (C) Growth of the pyrE-129 mutant, the wild type, and the repaired pyrE-129 mutant in a defined medium (DM) with or without uracil supplementation. The image of the gel was modified by cropping intervening lanes.

FIG 2

Schematic representation of the pyrE locus and wild-type pyrE DNAs for repair of the pyrE-129 mutant. (A) Genomic environment of the pyrE-129 mutation. The gray bar indicates the location of the 129-nt deletion in pyrE. The scale bar indicates 500-bp increments across the pyrE locus and the various lengths of wild-type DNA fragments used for allele replacement. (B) PCR analysis of pyrE⁺ recombinants with a forward primer complementary to wild-type sequence absent in pyrE-129. Lane 1, wild type; lane 2, pyrE-129; lane 3, no DNA; lanes 4 to 8, pyrE⁺ recombinants.

FIG 3

Recombination at the pyrE locus. The genomic region of the pyrE-129 mutation is indicated by tick marks with coordinates. The gray box indicates the location of the 129-nt deletion. The small black tick lines within the three lines indicate the locations of the synonymous codon changes in the pyrE locus of the recombinants. The three X symbols indicate the recombination events resulting in three recombination outcomes when using the synthetic pyrE allele.

FIG 4

Disruption of araA. (A) Schematic representation of homologous recombination at the araA locus with the expected size for the araA mutant and the wild type using primers within or external to the araA locus. The genetic marker was P_groES::pyrE_Taf (shown as P_groES TafpyrE in panel A and P_groES::Taf_pyrE in panel D). (B) PCR amplification of the disrupted araA allele. Lane MW, molecular weight standards; lane 1, wild-type araA locus; lanes 2 to 4, araA mutant locus. (C) PCR amplification of the repaired araA allele. Lane MW, molecular weight standards; lane 1, araA mutant; lane 2, wild type; lanes 3 to 7, repaired mutant araA loci. (D) Growth curve of the araA mutant, wild-type, and araA WT repair strains in defined medium with maltose or arabinose. Maltose was used as a positive control for growth.

FIG 5

Analysis of H₂ production in the araA mutant. H₂ production by the araA mutant and parental strain cultivated with various amounts of arabinose was measured. The solid straight line with the closed circles indicates H₂ production by the araA mutant. The solid line with the open circle indicates H₂ production by the wild type. The H₂ values are shown as the means of the results from three replicates, with the error bars representing the standard deviations. The dashed line without symbols indicates the theoretical H₂ yield for growth on pentose sugar.