Qi-Long Qin

A Proposed Genus Boundary for the Prokaryotes Based on Genomic Insights

Genomic information has already been applied to prokaryotic species definition and classification. However, the contribution of the genome sequence to prokaryotic genus delimitation has been less studied. To gain insights into genus definition for the prokaryotes, we attempted to reveal the genus-level genomic differences in the current prokaryotic classification system and to delineate the boundary of a genus on the basis of genomic information. The average nucleotide sequence identity between two genomes can be used for prokaryotic species delineation, but it is not suitable for genus demarcation. We used the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance. A comprehensive genomic survey indicated that the POCP can serve as a robust genomic index for establishing the genus boundary for prokaryotic groups. Basically, two species belonging to the same genus would share at least half of their proteins. In a specific lineage, the genus and family/order ranks showed slight or no overlap in terms of POCP values. A prokaryotic genus can be defined as a group of species with all pairwise POCP values higher than 50%. Integration of whole-genome data into the current taxonomy system can provide comprehensive information for prokaryotic genus definition and delimitation.

Comparative genomics reveals a deep-sea sediment-adapted life style of Pseudoalteromonas sp. SM9913

Deep-sea sediment is one of the most important microbial-driven ecosystems, yet it is not well characterized. Genome sequence analyses of deep-sea sedimentary bacteria would shed light on the understanding of this ecosystem. In this study, the complete genome of deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913 (SM9913) is described and compared with that of the closely related Antarctic surface sea-water ecotype Pseudoalteromonas haloplanktis TAC125 (TAC125). SM9913 has fewer dioxygenase genes than TAC125, indicating a possible sensitivity to reactive oxygen species. Accordingly, experimental results showed that SM9913 was less tolerant of H2O2 than TAC125. SM9913 has gene clusters related to both polar and lateral flagella biosynthesis. Lateral flagella, which are usually present in deep-sea bacteria and absent in the related surface bacteria, are important for the survival of SM9913 in deep-sea environments. With these two flagellar systems, SM9913 can swim in sea water and swarm on the sediment particle surface, favoring the acquisition of nutrients from particulate organic matter and reflecting the particle-associated alternative lifestyle of SM9913 in the deep sea. A total of 12 genomic islands were identified in the genome of SM9913 that may confer specific features unique to SM9913 and absent from TAC125, such as drug and heavy metal resistance. Many signal transduction genes and a glycogen production operon were also present in the SM9913 genome, which may help SM9913 respond to food pulses and store carbon and energy in a deep-sea environment.

The complete genome of Zunongwangia profunda SM-A87 reveals its adaptation to the deep-sea environment and ecological role in sedimentary organic nitrogen degradation

BackgroundZunongwangia profunda SM-A87, which was isolated from deep-sea sediment, is an aerobic, gram-negative bacterium that represents a new genus of Flavobacteriaceae. This is the first sequenced genome of a deep-sea bacterium from the phylum Bacteroidetes.ResultsThe Z. profunda SM-A87 genome has a single 5 128 187-bp circular chromosome with no extrachromosomal elements and harbors 4 653 predicted protein-coding genes. SM-A87 produces a large amount of capsular polysaccharides and possesses two polysaccharide biosynthesis gene clusters. It has a total of 130 peptidases, 61 of which have signal peptides. In addition to extracellular peptidases, SM-A87 also has various extracellular enzymes for carbohydrate, lipid and DNA degradation. These extracellular enzymes suggest that the bacterium is able to hydrolyze organic materials in the sediment, especially carbohydrates and proteinaceous organic nitrogen. There are two clustered regularly interspaced short palindromic repeats in the genome, but their spacers do not match any sequences in the public sequence databases. SM-A87 is a moderate halophile. Our protein isoelectric point analysis indicates that extracellular proteins have lower predicted isoelectric points than intracellular proteins. SM-A87 accumulates organic osmolytes in the cell, so its extracelluar proteins are more halophilic than its intracellular proteins.ConclusionHere, we present the first complete genome of a deep-sea sedimentary bacterium from the phylum Bacteroidetes. The genome analysis shows that SM-A87 has some common features of deep-sea bacteria, as well as an important capacity to hydrolyze sedimentary organic nitrogen.

Comparative genomics of the marine bacterial genus Glaciecola reveals the high degree of genomic diversity and genomic characteristic for cold adaptation

To what extent the genomes of different species belonging to one genus can be diverse and the relationship between genomic differentiation and environmental factor remain unclear for oceanic bacteria. With many new bacterial genera and species being isolated from marine environments, this question warrants attention. In this study, we sequenced all the type strains of the published species of Glaciecola, a recently defined cold-adapted genus with species from diverse marine locations, to study the genomic diversity and cold-adaptation strategy in this genus.The genome size diverged widely from 3.08 to 5.96 Mb, which can be explained by massive gene gain and loss events. Horizontal gene transfer and new gene emergence contributed substantially to the genome size expansion. The genus Glaciecola had an open pan-genome. Comparative genomic research indicated that species of the genus Glaciecola had high diversity in genome size, gene content and genetic relatedness. This may be prevalent in marine bacterial genera considering the dynamic and complex environments of the ocean. Species of Glaciecola had some common genomic features related to cold adaptation, which enable them to thrive and play a role in biogeochemical cycle in the cold marine environments.

Structural basis for dimerization and catalysis of a novel esterase from the GTSAG motif subfamily of the bacterial hormone-sensitive lipase family

Background: Catalytic mechanisms of GTSAG motif subfamily enzymes of the bacterial hormone-sensitive lipases (HSLs) family are largely unknown. Results: E25, a GTSAG motif subfamily esterase, adopts a novel dimerization pattern. Dimerization keeps the catalytic Asp282 orientation for E25 catalysis. Conclusion: Dimerization and some catalytic profiles of E25 are distinctive from other HSLs. Significance: Our study sheds light on protein folding and evolution of HSLs. Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 Å. E25 is mesophilic (optimum temperature at 50 °C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2–C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical α/β hydrolase-fold domain. Residues Ser186, Asp282, and His312 comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp282 within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family.

Molecular insight into bacterial cleavage of oceanic dimethylsulfoniopropionate into dimethyl sulfide

Significance DMS is an important participant in the global sulfur and carbon cycles. DMS oxidation products cause the formation of cloud condensation nuclei and hence may influence weather and climate. DMS is produced through the cleavage of dimethylsulfoniopropionate (DMSP) mainly by marine bacterial DMSP lyases. The molecular mechanism of DMSP cleavage to generate DMS remains unclear. In this study, the crystal structure of DddQ, a DMSP lyase, was solved, and detailed biochemical and structural analyses were performed. Our results also provided a foremost insight into the catalytic mechanism of the DMSP cleavage reaction. This study offers a better understanding of how marine bacteria cleave DMSP to generate the climatically important gas DMS. The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a β-barrel fold structure and contains a Zn2+ ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the β-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between “open” and “closed” states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction.

Genetic structure of three fosmid‐fragments encoding 16S rRNA genes of the Miscellaneous Crenarchaeotic Group (MCG): implications for physiology and evolution of marine sedimentary archaea

Archaea of the Miscellaneous Crenarchaeotic Group (MCG) exist widely in soil, freshwater and marine sediments of both surface and subsurface. However, current knowledge about this group is limited to its phylogenetic diversity. An archaeal 16S library was constructed from a sediment sample from the South China Sea, which was dominated by MCG and Marine Group I (MG-I). A metagenomic library was constructed from the same sediment sample, and three MCG fosmids (E6-3G, E37-7F and E48-1C) containing 16S rRNA genes were screened. Annotation showed that the three genomic fragments encode a variety of open reading frames (ORFs) that are potentially homologous to important functional genes related to lipid biosynthesis, energy metabolism, and resistance to oxidants. No colinear regions were found between MCG fosmids and reported archaeal genomic fragments or genomes, suggesting that the MCG archaea are quite different from the sequenced archaea in gene arrangement. Analyses of both the phylogenies of 16S rRNA genes and several informational processing genes and nucleotide frequencies showed that MCG archaea are distinct from MG-I plus relatives. In addition, tetranucleotide frequency analysis in combination with phylogenetic analysis suggested that some fragments in the MCG fosmids are probably derived from non-MCG or non-archaeal genomes.

Interdomain hydrophobic interactions modulate the thermostability of microbial esterases from the hormone-sensitive lipase family.

Background: The effect of interdomain interactions on the thermostability of microbial hormone-sensitive lipases (HSLs) remains unclear. Results: The absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40, a thermolabile HSL esterase. Conclusion: Interdomain hydrophobic interactions are a key element for the thermostability of microbial HSLs. Significance: Our study is helpful for protein engineering of thermolabile HSLs. Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp202 and Phe203 in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability.

Diversity of Both the Cultivable Protease-Producing Bacteria and Bacterial Extracellular Proteases in the Coastal Sediments of King George Island, Antarctica

Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 105 cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%), Flavobacterium (21.0%) and Lacinutrix (16.2%). Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea.

Genome Sequences of Six Pseudoalteromonas Strains Isolated from Arctic Sea Ice

Yu et al. (Polar Biol. 32:1539-1547, 2009) isolated 199 Pseudoalteromonas strains from Arctic sea ice. We sequenced the genomes of six of these strains, which are affiliated to different Pseudoalteromonas species based on 16S rRNA gene sequences, facilitating the study of physiology and adaptation of Arctic sea ice Pseudoalteromonas strains.