Microbial And Extracellular Proteases Diversity And Organic Nitrogen Degradation Mechanism In Sediments Of The South China Sea And Fildes Peninsula, Antarctic

Seventy percentage of the Earth surface is covered by marine sediment and the sediments represent one of the most complex microbial habitats, where there are very rich microorganisms. According to statistics, microbes in deep-sea sediment account for over50%of all microorganisms in our planet. Deep-sea (except Hydrothermal Vent) and polar ocean are extreme with high salinity and low temperature. Microorganism survives in these environments must have a particular mechanism to adapt, with a high diversity of species, genes and ecological functions. To investigate the ecology of microorganisms in sea sediment is crucial to understanding the functioning of global biogeochemical cycles.Degradation of organic nitrogen in sediment is an important part of nitrogen cycling in marine ecosystem. High-Molecular-Weight Organic Nitrogen (HMWON) produced by microorganisms in the seawater settles and accumulates in the sediment in the form of particulate organic nitrogen (PON). Most of PON are degraded into dissolved organic nitrogen (DON) by microorganisms, and then participates into the nitrogen cycling through ammonium regeneration, nitrification and denitrification in the sediment, with nitrification is rate-limiting reaction. Therefore, the recycling of PON in marine sediment would be a considerable part of marine sediment nitrogen cycling. Since proteins are the important component of sedimentary PON and DON, protease-producing bacteria should be the important decomposers on them. Thus research into microorganisms, protease, and amoA gene in marine sediment will provide important implications for marine nitrogen cycling and finding new proteases.We employed454pyrosequencing technology to explore the microbial diversity in the sea sediments of the Antarctic and constructed an amoA gene library. Furthermore, diversity of protease-producing bacteria and proteases in the sediments of the South China Sea and the Antarctic were investigated, and enzymatic properties of a protease produced by Bacillus sp. CF12-9were studied. I Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China SeaTotally78protease-producing bacteria strains were recovered from8sediment samples of the South China Sea with depth of154-2,456m with screening medium. Quantitative statistic by manual count showed that the abundance of cultivated bacteria reached106cells/g in all sediment samples. Based on the sequences of their16S rRNA genes (NCBI GenBank Accession number FJ169962-FJ170039), the phylogenetic affiliation of these strains was analyzed. Except that two strains (E404-10and CF12-9) was a gram positive bacterium belonging to genus Bacillus, the strains were mainly affiliated with the class Gammaproteobacteria and grouped in the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas and Rheinheimera. Alteromonas (34.6%) and Pseudoalteromonas (28.2%) were the predominant groups. The other genera comprised between2.6%and7.7%of all strains. The result also showed that the predominant bacteria in the sediments of different sites and depth were different. In the shallowest sediment sample (E505), the predominant protease-producing bacteria were Shewanella (60%). In contrast, the predominant protease-producing bacteria in the sediment sample deeper than1,000m were Pseudoalteromonas and Alteromonas. A distance-based neighbor-joining tree was constructed with the Gammaproteobacteria-related sequences from this study and reference sequences from the GenBank database. The closest neighbors of most of the strains still lack taxonomic standing or belong to uncultured bacterium clones, and in most case from marine sources. Several isolates, such as CF12-14, E505-2, E505-8and E407-8, exhibited a distant relationship with any of the previously identified species. They may represent potentially new species. Among them CF12-14and E407-8have been classified as new species belong to Idiomarina and Rheinheimera, respectively.The diversity of the bacterial extracellular proteases in the sediments was investigated with protease inhibitors. Phenylmethanesulfonyl fluoride (PMSF, serine protease inhibitor), o-phenanthroline (O-P, metalloprotease inhibitor), E-64(cysteine protease inhibitor) and pepstatin A (P-A, aspartic protease inhibitor) were used to inhibit the activities of the proteases secreted by the screened strains for identification of these proteases. Among the78strains,16strains did not produce enough proteases for inhibition analysis. PMSF inhibited the activities of the proteases from all the detected62strains by23-100%, showing all the strains produce serine proteases in different proportion. Of all62strains, O-P inhibited the activities of the proteases from47strains by20-84%and had a little or no inhibitory effect on the others, showing a majority of the screened strains produce metalloproteases. Moreover, the activities of the proteases from most of the screened strains were inhibited by both PMSF and O-P, indicating these strains simultaneously produce serine proteases and metalloproteases. E-64and pepstatin A only had less than10%or no inhibitory effect on the activities of all proteases, showing that these strains hardly produce cysteine or aspartic protease. Therefore, nearly all the extracellular proteases from the bacteria in the sediment samples are serine proteases or metalloproteases. In addition, the bacterial protease diversity in the sediments was also investigated by detecting the hydrolysis ability of the proteases to different proteins casein, gelatin and elastin, which was analyzed by measuring the ratio of the hydrolytic zone diameter to the colony diameter of a colony on the plates. Of the78strains, only68obviously showed a hydrolytic zone. All the extracellular proteases from these strains could hydrolyze casein and gelatin with obvious hydrolytic zone except for the protease from strain CF14-12which did not hydrolyze gelatin. However, these proteases displayed very different hydrolysis abilities to casein and gelatin. In contrast, only47strains could produce obvious hydrolysis zone around a single colony on elastin plates. A lot of strains produced proteases with high activity to all three proteins. The good examples were strains CF6-2, CF6-14, E505-3, CF12-10, and E407-8. The protease Pseudoalterin secreted by Pseudoalteromonas sp. CF6-2has been classified as a new M23elastase with low identities (<40%) to the known M23proteases. These results indicated that the proteases produced by these strains may belong to different kinds of serine proteases or metalloproteases and there may be some new kinds of proteases.Ⅱ Microbial diversity in the costal sediments of the Maxwell Bay, AntarcticTo explore the diversity of microbial communities, qPCR and454pyrosequencing were employed to investigate8costal sediment samples of King George Island, Antarctic. The number of bacterial16S rDNA was much (at least13times) more than that of archaea, indicating that bacteria were predominant in sediment. A total of151,475and91,098valid reads for bacteria and archaea respectively were obtained from the eight samples through454pyrosequencing analyses. These sequences are available through the NCBI Sequence Read Archive database (accession number SRP017144). Bacterial and archaeal OTUs of the8communities ranged from2516to4278and403to1221, respectively. Bacteria richness was much higher than the archaea for97%definition of OTU with Ace and Chao for bacteria obviously greater than that for archaea. Good’s coverage estimations revealed that87.6%to92.5%of the bacteria species and94.2%to98.8%of the archaea species were obtained in all of the samples. The libraries showed very dissimilar16S rRNA profiles even in phylum level distributions.In total, twenty-three bacteria phyla and two archaea phyla were identified from these samples. Bacteria assemblage was dominated by Firmicutes (61.3%) and Proteobacteria (17.9%). The most17families or groups comprised over92%of all the reads and represented the most abundant common to the8libraries. For archaeal communities, Crenarchaeota and Euryarchaeota were found in all libraries and Euryarchaeota was predominant over Crenarchaeota except for SS9library. We identified Methanomicrobia (40.2%), Halobacteria (27.7%), Group C3(12.1%) and Marine Benthic Group B (11.4%) as the predominant archaeal group.It’s remarkable that lots of reads could not be classified to any order even in this taxonomic level, especially in SS8, SS9and SS13library with a percentage of38.6%,70.1%and45.8%of all reads. These reads mainly affiliated to Marine Benthic Group B and Group C3, indicating that there’s lots of archaea belong to Marine Benthic Group B and Group C3in sediment. The quite different pattern of weighted UniFrac PCoA analysis revealed that the bacterial and archaeal communities might be affected by different environment factors.Copy number of bacterial amoA gene was much less than that of16S rRNA gene (10-4-10-5), indicating that ammonia-oxidizing bacteria accounted for a very small part of microorganisms. Ammonia-oxidizing archaea could not be detected by qPCR. This was consistent with the result of454pyrosequencing.By using the bacterial amoA gene-specific primers amoA1F and amoA2R, a bacterial amoA gene clone library was constructed from the sediment samples. A total of120clones were sequenced, and85sequences were obtained. Sequences with more than97%identity were regarded as an OTU, and as a result8OTUs were obtained. Among them,3OTUs contained as much as78sequences. As revealed by phylogenetic analysis, the amoA gene was affiliated to Nitrosomonadaceae, showing a low diversity of amoA genes from the sediments.Ⅲ Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the Maxwell Bay, AntarcticTotally203bacteria strains were recovered from8sediment samples of the Maxwell Bay, Antarctic with depth of15-55m by using the same method as that for the samples from the South China Sea. The abundance of cultivated bacteria reached105cells/g in all sediment samples. Their16S rRNA genes were cloned, sequenced and deposited in GenBank under the accession number KC160632-KC160834. Ninety-eight strains could form colony but without clear hydrolytic zone in screening plats. Therefore105strains were used for subsequent analysis. Totally23genus and5unknown Bacteroidetes strains were identified from these samples. The unknown Bacteroidetes strains were SS9.12, SS9.38, SS14.29, SS14.30and SS14.31which clustered together with a very distant relationship with other known Bacteroidetes species. These strains may affiliate to a new genus belonging to Bacteroidetes. The communities showed very dissimilar16S rRNA profiles in genus level distributions. The strains were mainly affiliated with the phyla Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria. In summary, genera Bacillus (22.9%), Flavobacterium (21.0%) and Lacinutrix (16.2%) were the predominant groups and were the most frequently recovered isolates (recovered from6,6,5sediments, respectively), formed the largest groups in term of abundance (63of105isolates). There were up to14genera recovered with only one strain, comprising16.2%of all strains totally. Moreover, the result also showed that the predominant bacteria in the sediments of different sites were different. As many as73strains could not produce enough proteases for inhibition analysis. The inhibitor assay of the other35strains showed that nearly all the extracellular proteases from the bacteria in the sediment samples from Maxwell Bay were serine proteases or metalloproteases, which was consistent with the result of the South China Sea. Of the105strains, only48showed an obvious hydrolytic zone and these proteases displayed very different hydrolysis abilities to casein and gelatin, indicating these proteases may belong to different kinds of serine proteases or metalloproteases. Overall, the bacteria from Antarctic coastal sediment had a relatively low ability to produce extracellular protease compared to the bacteria from the South China Sea.Ⅳ Enzymatic property of serine protease Ba-1produced by Bacillus sp. CF12-9A serine protease Ba-1produced by Bacillus sp. CF12-9was purified and its substrate specificity was studied. Ba-1could hydrolyze different proteins including casein, gelatin, collagen and elastin. The protease kept35%activity at0℃and its optimum temperature was30℃, indicating that the protease Ba-1from Bacillus sp. CF12-9was a typical cold-adapted enzyme. The protease was an alkaline protease with an optimum pH of8.5. The inhibitor assay indicated the protease Ba-1was a serine protease. Its activity could be promoted slightly by2mM Co2+or Cu2+by4%, and inhibited by K+, Li+, Zn2+and Fe2+by20%,20%,30%and80%, respectively. Other detected ions inhibited its activity slightly. The protease showed the highest activity in a buffer without NaCl, and～20%activity was reduced by0.5M NaCl, revealing the low tolerance of Ba-1to NaCl. The N-terminal sequence of Ba-1was obtained by Edman degradation and the resulting sequence was WTPNDYYYQGYQYGP. This sequence was subjected to NCBI Blastp, which showed that Ba-1might be a S8A protease. Based on the conserved sequence of S8A family and the sequenced N-terminal amino acid sequence of Ba-1, the conserved sequence of Ba-1was obtained by PCR. Then the upstream and downstream sequences were amplified by TAIL-PCR. The obtained ORF contains1215bp, and sequence analysis showed that the gene encodes a proenzyme, containing a signal peptide (M1-A27), an19peptidase inhibitor (E55-T124) and mature enzyme (W125-Y404). Conserved domain analysis indicated that Ba-1is an S8A family serineprotease with a single catalytic domain. Sequence homology analysis showed that the maximum identity was72%between Ba-1and Ak.l protease produced by B. sp. Ak.l and the protease produced by Geobacillus stearothermophilus. Ba-1lost all the activity at35℃for only30min. However, Ak.1protease and the protease produced by Geobacillus stearothermophilus were stable at60-70℃, reflecting differences between Ba-1and them. The above results would offer theoretical basis for illustration of the role of Ba-1in deep sea nitrogen cycling and for its application.In summary, microbial diversity in Antarctic marine sediment was investigated and bacterial amoA gene clone library was constructed. Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea and the Maxwell Bay, Antarctic were investigated. Furthermore, enzymatic proterties of a protease Ba-1produced by strain Bacillus sp. CF12-9were analized. This paper offers an important theoretical basis for the study of nitrogen cycling in marine sediment.