Complete Genome Sequence And Secretion Proteome Of Haloarchaea Natrinema Sp. J7-2

Natrinema sp. J7-2is an extremely haloarchaeon capable of growing on synthetic media without amino acid supplements. Here we report the complete genome sequence of Natrinema sp. J7-2and complementing experiments to investigate the carbon source metabolic and amino acid synthetic pathways. Natrinema sp. J7-2genome is composed of a3,697,626-bp chromosome and a95,989-bp plasmid pJ7-I. This is the first complete genome sequence of a member of the genus Natrinema. Natrinema sp. J7-2has the ability to use gluconate, glycerol or acetate as the sole carbon source, and its genome encodes complete metabolic pathways for assimilating these substrates. Meanwhile, the biosynthetic pathways for all20amino acids have been reconstructed, and there is a possible evolutionary relationship between haloarchaeal arginine synthetic pathway and bacterial lysine synthetic pathway. The genome harbors complete metabolic pathways for ammonium and nitrite but not nitrate, and has a denitrification pathway to reduce nitrite to N2O. Comparative genomic analysis suggests that most sequenced haloarchaea mainly employ the TrkAH system, rather than Kdp system, to actively uptake potassium. The genomic analysis also reveals that one of the three CRISPR loci in the chromosome is located in an integrative genetic element, probably being propagated via horizontal gene transfer (HGT). Besides, phylogenetic analysis of haloarchaeal genomes has provided some clues about evolutionary relationships and non-vertical evolutionary histories of haloarchaea.Previous genomic and bioinformatic analysis of haloarchaea suggested that a large number of haloarchaeal proteins including many lipoproteins harbored Tat signal peptide, in contrast to the case of bacteria that mainly employ Sec pathway to secret extracellular proteins. The genomic analysis of Natrinema sp. J7-2revealed that this haloarchaeon has a full set of genes encoding the apparatus of both Tat and Sec pathways. Meanwhile, about21Tat substrates and12Sec substrates were prediected in this genome. In addition, we have investigated the proteins secreted into the growth medium and those associated with membrane fraction of Natrinema sp. J7-2at different growth phases by RPLC-ESI-MS/MS and bioinformatic analyses. The extracellular proteomic results showed that the number of real secreted proteins is much fewer than that of the predicted ones in silico. There are21Tat substrates and12Sec substrates detected in the extracellular medium. These extracellular proteins mainly are hydrolases, extracellular solute-binding protens, cell surface proteins, etc. Besides, in the extracellular medium we found4proteins without Tat or Sec signal sequences but harboring the C-terminal signal sequences of type I secretion system. This implies that analogues of bacterial type I secretion system might also function in archaea, and some extracellular proteins might be secreted though archaeal type I secretion system. In the membrane samples,28Tat substrates were detected by MS; and most of them were lipoproteins with Lipobox. Meanwhile28Sec substrates were identified in the membrane samples; they were mainly transmembrane proteins. This result implies that the Sec signal peptide may be involved in insertion and association of proteins to membrane. In addition, the functions and adaptations mechanisms of some proteins detected by MS, such as S-layer proteins, transporter, channels, transducer, and flagellin components, have aso been discussed in this study.For better understanding the role of the Tat and Sec secretion systems in haloarchaea, two typical Tat and Sec extracellular proteases (SptA and SptE) of Natrinema sp. J7-2and their mutants with exchanged signal peptides were expressed in haloarchaeon Haloferax volcanii WFD11. When the Tat signal peptide of SptA was replaced by a Sec signal peptide, no protease activity could be detected in the culture medium or on the cell surface. However, when the Sec signal peptide of SptE was replaced by a Tat signal peptide, protease activity could be detected on the cell surface. This result indicates that haloarchaeal Tat system can transport not only folded proteins but also unfolded proteins.