MALDI-TOF MS and ESI-LTQ-Orbitrap tandem mass spectrometry reveal specific porphyranase activity from a Pseudoalteromonas atlantica bacterial extract

Mass spectrometry analysis highlighted an unprecedented β-methyl-porphyranase activity in protein extract fromPseudoalteromonas atlantica, which can accommodate the methylated building blocks of porphyran.

Site-Specific Insertion of IS492 in Pseudoalteromonas atlantica

ABSTRACT Reversible insertion of IS492 at a site within epsG on the Pseudoalteromonas atlantica chromosome controls peripheral extracellular polysaccharide production and biofilm formation by P. atlantica. High-frequency precise excision of IS492 from epsG requires 5 and 7 bp of flanking DNA, suggesting that IS492 transposition involves a site-specific recombination mechanism. The site specificity of IS492 insertion was examined in P. atlantica and shown to be specific for a 7-bp target, 5′-CTTGTTA-3′. Characterization of numerous insertion events at the target site in epsG indicated that insertion is also orientation specific. The frequency of IS492 insertion at the epsG target site (2.7 × 10−7/cell/generation), determined by quantitative PCR, is 4 to 5 orders of magnitude lower than the frequency of IS492 precise excision from the same site. Comparison of insertion sites for IS492 and the highly related ISPtu2 from Pseudoalteromonas tunicata suggests DNA sequence and/or structural features that may contribute to site recognition and recombination by the transposase of IS492.

Expression of β-agarase I DagA in prokaryotic cell and its activity identification

The DagA gene and DagA(▽), which is a DagA gene encoding sequence without signal peptide, were cloned from genome DNA ofPseudoalteromonas atlantica19262 by polymerase chain reaction (PCR). After ligation with pET21 vector, DagA and DagA(▽) were respectively expressed inEscherichia coliER2566 using molecular chaperones DsbC and FkpA. A strain of ER2566-pET21a-DagA(▽)-DsbC was screened as a highly effective expressing system in the form of an inclusion body that had the target protein with up to 60% total bacterial protein. DagA protein was renatured and purified by dissolving it in 8 mol/l of urea, using Ni-NTA resin affinity chromatography and refolding using the urea gradient method. DagA with a molecular weight of ~30.8 kDa was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and had the ability to digest agarose. In a pH range of 4.8–6.8, DagA maintained a bioactivity greater than 60%, with 5.8 being the optimum pH, and it exhibited activity at temperatures from 37°C to 60°C, with 55°C being the optimum temperature.

The endo-β-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours

Two β-agarase genes, agaA and agaB, were functionally cloned from the marine bacterium Zobellia galactanivorans. The agaA and agaB genes encode proteins of 539 and 353 amino acids respectively, with theoretical masses of 60 and 40 kDa. These two β-agarases feature homologous catalytic domains belonging to family GH-16. However, AgaA displays a modular architecture, consisting of the catalytic domain (AgaAc) and two C-terminal domains of unknown function which are processed during secretion of the enzyme. In contrast, AgaB is composed of the catalytic module and a signal peptide similar to the N-terminal signature of prokaryotic lipoproteins, suggesting that this protein is anchored in the cytoplasmic membrane. Gel filtration and electrospray MS experiments demonstrate that AgaB is a dimer in solution, while AgaAc is a monomeric protein. AgaAc and AgaB were overexpressed in Escherichia coli and purified to homogeneity. Both enzymes cleave the β-(1→4) linkages of agarose in a random manner and with retention of the anomeric configuration. Although they behave similarly towards liquid agarose, AgaAc is more efficient than AgaB in the degradation of agarose gels. Given these organizational and catalytic differences, we propose that, reminiscent of the agarolytic system of Pseudoalteromonas atlantica, AgaA is specialized in the initial attack on solid-phase agarose, while AgaB is involved with the degradation of agarose fragments.