[Show abstract][Hide abstract] ABSTRACT: 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 clarified yet. 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.
[Show abstract][Hide abstract] ABSTRACT: Marine xylanases are rather less studied compared to terrestrial xylanases. In this study, a new xylanase gene, xynB, was cloned from the marine bacterium, Glaciecola mesophila KMM241, and expressed in Escherichia coli. xynB encodes a multi-domain xylanase XynB of glycoside hydrolase (GH) family 8. The recombinant XynB comprises an N-terminal domain (NTD) with unknown function and a catalytic domain, which is structurally novel among the characterized xylanases of GH family 8. XynB has the highest identity (38%) to rXyn8 among the characterized xylanases. The recombinant XynB showed maximal activity at pH 6-7 and 35 °C. It is thermolabile and salt-tolerant. XynB is an endo-xylanase that demands at least five sugar moieties for effective cleavage and to hydrolyze xylohexaose and xylopentaose into xylotetraose, xylotriose and xylobiose. NTD was expressed in Escherichia coli to analyze its function. The recombinant NTD exhibited a high binding ability to insoluble xylan and avicel and little binding ability to chitosan and chitin. Since the NTD shows no obvious homology to any known carbohydrate-binding module (CBM) sequence in public databases, XynB may contain a new type of CBM.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Pseudoalteromonas is an important genus widespread in marine environment, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. By now, there are only a few genetic systems for Pseudoalteromonas reported and no commercial Pseudoalteromonas genetic system is available, which impedes the study of Pseudoalteromonas, especially for psychrophilic strains. The aim of this study is to develop a heterologous expression system for psychrophilic Pseudoalteromonas.
A cryptic plasmid pSM429 isolated from psychrophilic Pseudoalteromonas sp. BSi20429 from the Arctic sea ice, was sequenced and characterized. The plasmid pSM429 is 3874 bp in length, with a G+C content of 28%. Four putative open reading frames (ORFs) were identified on pSM429. Based on homology, the ORF4 was predicted to encode a replication initiation (Rep) protein. A shuttle vector (Escherichia coli, Pseudoalteromonas), pWD, was constructed by ligating pSM429 and pUC19 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. To determine the minimal replicon of pSM429 and to check the functionality of identified ORFs, various pWD derivatives were constructed. All derivatives except the two smallest ones were shown to allow replication in Pseudoalteromonas sp. SM20429, a plasmid-cured strain of Pseudoalteromonas sp. BSi20429, suggesting that the orf4 and its flanking intergenic regions are essential for plasmid replication. Although not essential, the sequence including some repeats between orf1 and orf2 plays important roles in segregational stability of the plasmid. With the aid of pWD-derived plasmid pWD2, the erythromycin resistance gene and the cd gene encoding the catalytic domain of a cold-adapted cellulase were successfully expressed in Pseudoalteromonas sp. SM20429.
Plasmid pSM429 was isolated and characterized, and the regions essential for plasmid replication and stability were determined, helping the development of pSM429-based shuttle vectors. The shuttle vectors pWD and its derivatives could be used as cloning vectors for Pseudoalteromonas, offering new perspectives in the genetic manipulation of Pseudoalteromonas strains. With the aid of pWD-derived vector and its host, the erythromycin resistance gene and the cd gene of a cold-adapted protein were successfully expressed, indicating that the potential use of this system for recombinant protein production, especially for cold-adapted proteins.