Insights Into The Physiology And Evolution Of Miscellaneous Crenarchaeota Group (MCG) And β-galactosidases From The Sediment From The South China Sea Based On Metagenomic Analyses

Based on 16S rRNA gene sequences, Carl Woese et al. (1990) proposed that archaea were classified into two major lineages, Crenarchaeota and Euryarchaeota. Crenarchaeota are specifically for hyperthermophilic organisms. Euryarchaeota have diverse physiologies such as halophilic, methanogenic and thermophilic traits. With the discovery of mesophilic crenarchaeota, representative of ammonia-oxidizing archaeal group MG-I (Marine Group I), the archaeal phylogenetic tree is found to be composed of more than two major lineages. Phylogenetic analyses of 16S-23S rRNA genes, ribosomal proteins and core informational processing genes of three MG-I crenarchaeota, Cenarchaenum symbiosum, Nitrosopumilus maritimus, and Nitrososphaera gargensis showed that mesophilic crenarchaeota were separated from hyperthermophilic crenarchaeota and formed a distinct group, suggesting that mesophilic crenarchaeota is a new archaeal phylum, Thaumarchaeota, proposed by Brochier-Armanet et al. (2008).MCG (Miscellaneous Crenarchaeota Group) archaea, another group of mesophilic crenarchaeota, may also possess unique genomic and evolutionary traits. MCG is a group of phylogenetically diverse archaea, with the label "miscellaneous" referring to its wide range of habitats, including soil, freshwater, marine hydrothermal vents, and surface and deep marine subsurface sediments. Up to date, there is no cultivated representative of MCG, and little is known about the physiology of this group. Current knowledge about MCG is limited to the phylogenetic analysis of the 16S rRNA gene sequences. Based on 16S rRNA genes, most studies foucus on the phylogenetic analysis of MCG and uncultivated archaeal groups, and phylogenetic affiliation of MCG compared to cultivated archaea is poorly studied. Therefore, more studies are necessary to gain insights into the genome, physiology and evolution of MCG. Now, metagenomics is frequently used to gain insights into the physiology and evolution of uncultivated microorganisms, which therefore can be used to study the MCG. The sediment sample E505 was collected from 0-5 cm surface sediment of 154 m deep from the South China Sea. In this study, metagenomics was used to gain insights into the physiology and evolution of MCG from the sediment sample E505 from the South China Sea. In addition, through functional screening, theβ-galactosidases in E505 sediment were studied.I. Archaeal diversity analysis of a marine sediment sample E505 from the South China SeaBy using the archaea-specific primers Arch21F and Arch958R, an archaeal 16S rRNA gene clone library was constructed from a surface sediment sample E505 from the South China Sea. A total of 144 clones were sequenced, and 121 sequences were obtained. Sequences with more than 97% identity were regarded as identical phylotypes, and as a result 64 unique archaeal phylotypes were obtained. The resultant archaeal sequences fall into 8 groups including MG-I (54.5%), MCG (24.0%), Marine Benthic Group A (MBGA,2.5%), Marine Benthic Group B (MBGB, 3.3%), Deep-Sea Hydrothermal Vent Euryarchaeotal Group 6 (DHVE6,13.2%), Marine Benthic Group E (MBGE,0.8%), Marine Benthic Group D (MBGD,0.8%), and other unclassified euryarchaeota (0.8%). The 0-5 cm sample represents the mixture of the seawater and sediment. As revealed by phylogenetic analysis, the archaeal community in E505 reflected this, which was a mixture of water column archaea, MG-I, and marine subsurface sediment archaea, most abundantly MCG. Phylogenetic analysis of representative MCG phylotypes from different environments showed that most MCG phylotypes from E505 sample cluster with the sequences from deep subsurface sediment in tropical western Pacific and estuary sediments in Mandovi and Zuari.II. Insights into the physiology of MCG archaea based on metagenomic analysisA metagenomic library was constructed from the E505 sediment, which contains 10,652 fosmids. The fosmid library was then PCR-scrcened for archaeal fosmids with Arch21F and Arch958R primers, and three fosmids carrying archaeal 16S rRNA genes were obtained, E6-3G, E37-7F and E48-1C. BLAST results showed that the 16S rRNA genes on the three fosmids are closely related to reported MCG archaeal 16S rRNA gene sequences from marine sediments (identities=98%), suggesting that they are all from MCG archaea. The 16S rRNA sequence identities between the fosmids E6-3G, E37-7F and E48-1C range from 81% to 83%, indicating they belong to different MCG subdivisions. The three MCG fosmids were sequenced by 454 methods. Open reading frames (ORFs) were predicted using GLIMMER, and were further annotated by searching NCBI non-redundant protein database (nr) and InterPro database. The E6-3G fosmid is 38,277 bp long, and contains one 16S-5.8S-23S rRNA operon and 30 ORFs. ORFs 3-10 on E6-3G are likely to be involved in the biosynthesis of N-glycan of cell wall/membrane. The E37-7F fosmid is 42,618 bp long, and contains one 16S-5.8S-23S rRNA operon, two tRNAs and 41 ORFs. ORFs 22,23 and 27 on E37-7F fosmid encode three putative methyltransferases related to the biosynthesis of menaquinone/ubiquinone, which function as electron carriers in the prokaryotic electron-transport chain. The existence of three putative phosphatidylethanolamine N-methyltransferase genes (ORFs 29,31, and 32) and one putative choline kinase gene (ORF 24) suggest that choline might exist in the cell wall/membrane of E37-7F archaea. The E48-1C fosmid is 34,738 bp long, and contains a single 16S rRNA, one tRNA and 37 ORFs. E48-1C archaea may be resistant to toxic metal ions, since ORF 4 on E48-1C fosmid encodes a homologous gene of toxic metal defense regulator (ArsR).Ⅲ. Insights into the molecular evolution of MCG archaeaBased on the genomic fragments of E6-3G, E37-7F and E48-1C, analyses of phylogenies, co-linearity, and tetranucleotide frequencies were carried out to reveal the evolution of MCG archaea.16S rRNA gene phylogeny showed that the mesophilic crenarchaeota form a monophyletic lineage, and within the monophyly of mesophilic crenarchaeota, MCG and MG-I plus relatives form two mutually exclusive branches, suggesting that MCG archaea may represent a new lineage of the mesophilic crenarchaeota. Phylogenetic trees based on several informational processing genes are also in agreement with the 16S rRNA gene tree. By calculating the internal correlation of tetranucleotide frequencies, MCG were compared to other reported mesophilic crenarchaeal genomic fragments, and results showed that MCG genomes are unstable. Further analyses of tetranucleotide frequencies revealed multiple DNA fragments with aberrant tetranucleotide frequencies in the MCG fosmids. Phylogenetic analysis of all the genes located in the aberrant regions of tetranucleotide frequencies suggested that some fragments in the MCG fosmids are probably derived from non-crenarchaeal or non-archaeal genomes, which might cotribute to the low genome homogeneity of MCG archaea. Based on correlation values of stable tetranucleotide frequencies (z-scores), mesophilic crenarchaeota were compared to hyperthermophilic crenarchaeota and euryarchaeota, and results showed that MCG and MG-I plus relatives have different tetranucleotide frequencies, also suggesting that MCG is distinct from MG-I plus relatives within the mesophilic crenarchaeota. In addition, comparative genomics revealed no colinear regions between MCG fosmids and reported archaeal genomic fragments (213) or genomes (91), suggesting that the MCG archaea are quite different from the sequenced archaea in gene arrangement.IV. Screening ofβ-galactosidases from the marine sediment sample E505 from the South China Sea based on functional metagenomic analysisFunctional assayes were carried out to screen activeβ-galactosidases in the E505 metagenomic library. A total of 11 positive clones were identified, and 4 (E13-9A, E14-1H, E14-5B and E109-6D) of these were completely sequenced. Annotation revealed no genes on the four fosmids encodingβ-galactosidases. Howerer, the four genomic fragments encode a few of hypothetical proteins and have relatively low coding regions, indicating that theirβ-galactosidase genes may be novel. Based on transposon insertion mutagenesis, analysis of fosmid sequences, and subsequentβ-galactosidase activity assays of recombinant proteins, the reason for theβ-galactosidase activities from the fosmid clones were determined. The observedβ-galactosidase activity is caused by the a-complementation between the EPI300 host chromosomal DNA lacZ△M15 and a combined a-peptide from different DNA inserts and pCC 1 FOS vector. Authough there are previous reports that novel P-galactosidases have been isolated by this method, our results show that this method has disadvantages. When functional metagenomics are used to screen environmental P-galactosidases, some advice on the hosts and vectors used in the construction of metagenomic libraries is proposed to avoid false positive clones.