Functional genomics analysis of carbohydrate conversion to biohydrogen by pure and mixed cultures of hyperthermophilic Thermotoga species

The genus Thermotoga is comprised of fermentative anaerobes with optimal growth temperatures as high as 80°C. To understand the genetic and physiological diversity within this genus, the genome sequences of five Thermotoga species (T. maritima, T. neapolitana, T. sp. RQ2, T. petrophila, T. lettingae) were compared using bioinformatics tools. Except for T. lettingae, the genomes exhibited high degrees of homology and shared organizational traits. The in silico comparison was supported by genomic DNA cross-hybridization to a T. maritima cDNA microarray, where 83--94% of the probes in the three other Thermotoga species were recognized. These results indicated that the four Thermotoga species share a core genome (∼1470 ORFs); ORFs unique to particular species likely reflect the influence of specific environmental or evolutionary factors. The significant homology among the four Thermotoga species facilitated development of a multi-species cDNA microarray for use in pure and mixed culture transcriptional response studies.;The Thermotoga multi-species cDNA microarray was used to examine pure and mixed culture transcriptomes for growth on glucose and on a polysaccharide mixture. The multi-species array was used to estimate species composition of the mixed culture; composition varied from 6:1.5:1:1 for glucose batch culture to 2.3:2:2:1 in glucose continuous culture for T. sp. RQ2: T. maritima: T. petrophila: T. neapolitana, respectively. Composition in polysaccharide batch culture was similar to glucose continuous culture. Transcriptional response analysis provided clues to interspecies interactions. In glucose mixed culture, the ORFs encoding a phage tail-like bacteriocin (TM0785), lon proteases (TM1633, TM1869), sigmaE (TM1598), and a putative bacteriocin (TM1300) related to subtilosin A from Bacillus subtilis, were up-regulated relative to pure cultures. Differential regulation of several ORFs encoding HicAB Toxin-Antitoxin pairs (TM1310a-1313, TM1320-21) was noted, suggesting a potential role in interspecies interactions. Comparisons of growth on glucose and polysaccharides revealed changes in both core and non-core ORF transcription. All cultures exhibited upregulation of core genome xylan (TM0056-61, TM0070-77) and beta-mannan utilization operons (TM1218-1223) on polysaccharide culture. An unclassified ABC transporter operon found only in T. neapolitana and T. sp RQ2 (TRQ2_0970-75) and a beta-linked exopolysaccharide operon found only in T. maritima (TM0622-30) were up-regulated on glucose. A beta-mannan utilization operon (TM1746-51), found only in T. maritima and T. sp RQ2, was up-regulated on polysaccharide culture.;To investigate the potential of Thermotoga for biofuels production, biohydrogen generation through carbohydrate fermentation was examined for both pure and mixed Thermotoga cultures. T. maritima showed that, unlike the hyperthermophilic archaeon, Pyrococcus furiosus, which uses similar fermentative metabolism, sulfur had minimal effect on transcription. Furthermore, T. maritima preferred cellobiose over maltose, perhaps related to superior bioenergetics mediated by a cellobiose phosphorylase (TM1848) encoded in its genome. Volumetric H2 production rates (∼1.2x10-3 mol H 2 liter-1 hour-1) were similar for pure and mixed cultures, perhaps related to the function of metabolic pathways comprising the core genome.;This work demonstrates the usefulness of multi-species arrays for examining closely related Thermotoga. The results indicted that, while differences in transcription were noted among pure and mixed cultures, culture growth and H2 production levels are not affected by species, substrate, or competition.