Alexandra Jeudy

Cell walls of brown algae are complex supramolecular assemblies containing various original, sulfated, and carboxylated polysaccharides. Among these, the major marine polysaccharide component, alginate, represents an important biomass that is successfully turned over by the heterotrophic marine bacteria. In the marine flavobacterium Zobellia galactanivorans, the catabolism and uptake of alginate are encoded by operon structures that resemble the typical Bacteroidetes polysaccharide utilization locus. The genome of Z. galactanivorans contains seven putative alginate lyase genes, five of which are localized within two clusters comprising additional carbohydrate-related genes. This study reports on the detailed biochemical and structural characterization of two of these. We demonstrate here that AlyA1PL7 is an endolytic guluronate lyase, and AlyA5 cleaves unsaturated units, α-L-guluronate or β-D-manuronate residues, at the nonreducing end of oligo-alginates in an exolytic fashion. Despite a common jelly roll-fold, these striking differences of the mode of action are explained by a distinct active site topology, an open cleft in AlyA1(PL7), whereas AlyA5 displays a pocket topology due to the presence of additional loops partially obstructing the catalytic groove. Finally, in contrast to PL7 alginate lyases from terrestrial bacteria, both enzymes proceed according to a calcium-dependent mechanism suggesting an exquisite adaptation to their natural substrate in the context of brown algal cell walls.

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.

Laminarinase is commonly used to describe β-1,3-glucanases widespread throughout Archaea, bacteria, and several eukaryotic lineages. Some β-1,3-glucanases have already been structurally and biochemically characterized, but very few from organisms that are in contact with genuine laminarin, the storage polysaccharide of brown algae. Here we report the heterologous expression and subsequent biochemical and structural characterization of ZgLamAGH16 from Zobellia galactanivorans, the first GH16 laminarinase from a marine bacterium associated with seaweeds. ZgLamAGH16 contains a unique additional loop, compared with other GH16 laminarinases, which is composed of 17 amino acids and gives a bent shape to the active site cleft of the enzyme. This particular topology is perfectly adapted to the U-shaped conformation of laminarin chains in solution and thus explains the predominant specificity of ZgLamAGH16 for this substrate. The three-dimensional structure of the enzyme and two enzyme-substrate complexes, one with laminaritetraose and the other with a trisaccharide of 1,3-1,4-β-d-glucan, have been determined at 1.5, 1.35, and 1.13 Å resolution, respectively. The structural comparison of substrate recognition pattern between these complexes allows the proposition that ZgLamAGH16 likely diverged from an ancestral broad specificity GH16 β-glucanase and evolved toward a bent active site topology adapted to efficient degradation of algal laminarin.

The genomic data on heterotrophic marine bacteria suggest the crucial role that microbes play in the global carbon cycle. However, the massive presence of hypothetical proteins hampers our understanding of the mechanisms by which this carbon cycle is carried out. Moreover, genomic data from marine microorganisms are essentially annotated in the light of the biochemical knowledge accumulated on bacteria and fungi which decompose terrestrial plants. However marine algal polysaccharides clearly differ from their terrestrial counterparts, and their associated enzymes usually constitute novel protein families. In this study, we have applied a combination of bioinformatics, targeted activity screening and structural biology to characterize a hypothetical protein from the marine bacterium Zobellia galactanivorans, which is distantly related to GH43 family. This protein is in fact a 1,3-α-3,6-anhydro-l-galactosidase (AhgA) which catalyses the last step in the degradation pathway of agars, a family of polysaccharides unique to red macroalgae. AhgA adopts a β-propeller fold and displays a zinc-dependent catalytic machinery. This enzyme is the first representative of a new family of glycoside hydrolases, especially abundant in coastal waters. Such genes of marine origin have been transferred to symbiotic microbes associated with marine fishes, but also with some specific human populations.

BACKGROUND: The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. RESULTS: We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. CONCLUSIONS: The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.