Taurine (2-aminoethanesulfonic acid) is an amino acid-like compound widely distributed in animals and an essential nutrient in some species. Targeted metabolomics of marine and freshwater microalgae combined with medium supplementation identified biosynthetic pathway intermediates and necessary catalytic activities. Genomic analysis was then used to predict the first taurine biosynthetic pathway in these organisms. MRM-based electrospray ionization (ESI) LC-MS/MS analysis demonstrated that taurine is synthesized using a carbon backbone from L-serine combined with sulfur derived from sulfate. Metabolite analysis showed a nonuniform pattern in levels of pathway intermediates that were both species and supplement dependent. While increased culture salinity raised taurine levels modestly in marine alga, taurine levels were strongly induced in a freshwater species implicating taurine as an organic osmolyte. Conservation of the synthetic pathway in algae and metazoans together with a pattern of intermittent distribution in other lineages suggests that it arose early in 0 1 5 ) 2 eukaryotic evolution. Elevated levels of cell-associated taurine in algae could offer a new and biorenewable source of this unusual bioactive compound.

Extremely thermoacidophilic members of the Archaea such as the lithoautotroph, Metallosphaera sedula, are among the most acid resistant forms of life and are of great relevance in bioleaching. Here, adaptive laboratory evolution was used to enhance the acid resistance of this organism while genomics and transcriptomics were used in an effort to understand the molecular basis for this trait. Unlike the parental strain, the evolved derivative, M. sedula SARC-M1, grew well at pH of 0.90. Enargite (Cu3AsS4) bioleaching conducted at pH 1.20 demonstrated SARC-M1 leached 23.78 % more copper relative to the parental strain. Genome re-sequencing identified two mutations in SARC-M1 including a nonsynonymous mutation in Msed_0408 (an amino acid permease) and a deletion in pseudogene Msed_1517. Transcriptomic studies by RNA-seq of wild type and evolved strains at various low pH values demonstrated there was enhanced expression of genes in M. sedula SARC-M1 encoding membrane complexes and enzymes that extrude protons or that catalyze proton-consuming reactions. In addition, M. sedula SARC-M1 exhibited reduced expression of genes encoding enzymes that catalyze proton-generating reactions. These unique genomic and transcriptomic features support a model for increased acid resistance arising from enhanced control over cytoplasmic pH.

Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.

Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H 2 ). 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 H 2 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 H 2 using arabinosesupplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H 2 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 H 2 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 (H 2 ) 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 H 2 yield. To overcome this barrier, a homologous chromosomal recombination method was developed and used to characterize the contribution of arabinose catabolism to H 2 formation. We report here a stable genetic

Crenarchaeota include extremely thermoacidophilic organisms that thrive in geothermal environments dominated by sulfidic ores and heavy metals such as mercury. Mercuric ion, Hg(II), inactivates transcription in the crenarchaeote Sulfolobus solfataricus and simultaneously derepresses transcription of a resistance operon, merHAI, through interaction with the MerR transcription factor. While mercuric reductase (MerA) is required for metal resistance, the role of MerH, an adjacent small and predicted product of an ORF, has not been explored. Inactivation of MerH either by nonsense mutation or by in-frame deletion diminished Hg(II) resistance of mutant cells. Promoter mapping studies indicated that Hg(II) sensitivity of the merH nonsense mutant arose through transcriptional polarity, and its metal resistance was restored partially by single copy merH complementation. Since MerH was not required in vitro for MerA-catalysed Hg(II) reduction, MerH may play an alternative role in metal resistance. Inductively coupled plasma-mass spectrometry analysis of the MerH deletion strain following metal challenge indicated that there was prolonged retention of intracellular Hg(II). Finally, a reduced rate of mer operon induction in the merH deletion mutant suggested that the requirement for MerH could result from metal trafficking to the MerR transcription factor.