Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely spread mechanisms for osmoadaptation in the Halobacteriales.

We investigated the mechanisms of osmoadaptation in the order Halobacteriales, with special emphasis on Haladaptatus paucihalophilus, known for its ability to survive in low salinities. H. paucihalophilus genome contained genes for trehalose synthesis (trehalose-6-phosphate synthase/trehalose-6-phosphatase (OtsAB pathway) and trehalose glycosyl-transferring synthase pathway), as well as for glycine betaine uptake (BCCT family of secondary transporters and QAT family of ABC transporters). H. paucihalophilus cells synthesized and accumulated ∼1.97-3.72 µmol per mg protein of trehalose in a defined medium, with its levels decreasing with increasing salinities. When exogenously supplied, glycine betaine accumulated intracellularly with its levels increasing at higher salinities. RT-PCR analysis strongly suggested that H. paucihalophilus utilizes the OtsAB pathway for trehalose synthesis. Out of 83 Halobacteriales genomes publicly available, genes encoding the OtsAB pathway and glycine betaine BCCT family transporters were identified in 38 and 60 genomes, respectively. Trehalose (or its sulfonated derivative) production and glycine betaine uptake, or lack thereof, were experimentally verified in 17 different Halobacteriales species. Phylogenetic analysis suggested that trehalose synthesis is an ancestral trait within the Halobacteriales, with its absence in specific lineages reflecting the occurrence of gene loss events during Halobacteriales evolution. Analysis of multiple culture-independent survey data sets demonstrated the preference of trehalose-producing genera to saline and low salinity habitats, and the dominance of genera lacking trehalose production capabilities in permanently hypersaline habitats. This study demonstrates that, contrary to current assumptions, compatible solutes production and uptake represent a common mechanism of osmoadaptation within the Halobacteriales.

Diversity of benzyl- and alkylsuccinate synthase genes in hydrocarbon-impacted environments and enrichment cultures.

Hydrocarbon-degrading microorganisms play an important role in the natural attenuation of spilled petroleum in a variety of anoxic environments. The role of benzylsuccinate synthase (BSS) in aromatic hydrocarbon degradation and its use as a biomarker for field investigations are well documented. The recent discovery of alkylsuccinate synthase (ASS) allows the opportunity to test whether its encoding gene, assA, can serve as a comparable biomarker of anaerobic alkane degradation. Degenerate assA- and bssA-targeted PCR primers were designed in order to survey the diversity of genes associated with aromatic and aliphatic hydrocarbon biodegradation in petroleum-impacted environments and enrichment cultures. DNA was extracted from an anaerobic alkane-degrading isolate (Desulfoglaeba alkenexedens ALDC), hydrocarbon-contaminated river and aquifer sediments, a paraffin-degrading enrichment, and a propane-utilizing mixed culture. Partial assA and bssA genes were PCR amplified, cloned, and sequenced, yielding several novel clades of assA genes. These data expand the range of alkane-degrading conditions for which relevant gene sequences are available and indicate that considerable diversity of assA genes can be found in hydrocarbon-impacted environments. The detection of genes associated with anaerobic alkane degradation in conjunction with the in situ detection of alkylsuccinate metabolites was also demonstrated. Comparable molecular signals of assA/bssA were not found when environmental metagenome databases of uncontaminated sites were searched. These data confirm that the assA gene is a useful biomarker for anaerobic alkane metabolism.