Viral degradation of marine bacterial exopolysaccharides.

The identification of the mechanisms by which marine dissolved organic matter (DOM) is produced and regenerated is critical to develop robust prediction of ocean carbon cycling. Polysaccharides represent one of the main constituents of marine DOM and their degradation is mainly attributed to polysaccharidases derived from bacteria. Here, we report that marine viruses can depolymerize the exopolysaccharides (EPS) excreted by their hosts using five bacteriophages that infect the notable EPS producer, Cobetia marina DSMZ 4741. Degradation monitorings as assessed by gel electrophoresis and size exclusion chromatography showed that four out of five phages carry structural enzymes that depolymerize purified solution of Cobetia marina EPS. The depolymerization patterns suggest that these putative polysaccharidases are constitutive, endo-acting and functionally diverse. Viral adsorption kinetics indicate that the presence of these enzymes provides a significant advantage for phages to adsorb onto their hosts upon intense EPS production conditions. The experimental demonstration that marine phages can display polysaccharidases active on bacterial EPS lead us to question whether viruses could also contribute to the degradation of marine DOM and modify its bioavailability. Considering the prominence of phages in the ocean, such studies may unveil an important microbial process that affects the marine carbon cycle.

Optimization of exopolysaccharide production from Pseudomonas stutzeri AS22 and examination of its metal-binding abilities.

AIMS: To investigate the effect of culture conditions and medium components on exopolysaccharide (EPS) production by Pseudomonas stutzeri AS22 and to access the EPS performance as a metal-binding exopolysaccharide. METHODS AND RESULTS: The EPS production conditions of Ps. stutzeri AS22 in submerged culture were optimized using two approaches for EPS quantification, and its metal-binding capacity was evaluated using both single and mixed metal ions systems. Maximum EPS level was achieved after 24 h of incubation at 30°C with an initial pH of 8.0, 250 rev min(-1) stirring level and 10% inoculum size. 50 g l(-1) starch, 5 g l(-1) yeast extract, 0.5 g l(-1) NaCl, 1.4 g l(-1) K2 HPO4, 0.4 g l(-1) MgSO4, 0.4 g l(-1) CaCl2 and 1 g l(-1) mannose were found to be the most suitable carbon, nitrogen, mineral and additional carbohydrate sources, respectively. From metal-binding experiments, the crude EPS showed interesting metal adsorption capacity adopting the order Pb >> Co > Fe > Cu >> Cd. Lead was preferentially biosorbed with a maximal uptake of 460 mg g(-1) crude EPS. CONCLUSIONS: Under the optimal culture requirements, EPS level reached 10.2 g l(-1) after 24 h of fermentation, seven times more than the production under initial conditions. According to the metal-binding assay, the crude EPS has potential to be used as a novel biosorbent in the treatment of heavy metals-contaminated water. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results are interesting in terms of yield as well as efficiency for the potential use of the Ps. stutzeri exopolysaccharide as a metal-absorbent polymer in the bioremediation field.

Real-time PCR optimization to identify environmental Vibrio spp. strains.

AIMS: To identify Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus using standardized DNA extraction method and real-time PCR assays, among a large number of bacterial strains isolated from marine environment. METHODS AND RESULTS: Methods for DNA extraction and real-time PCR were standardized to identify a large number of Vibrio spp. strains isolated through regular collection campaigns of environmental samples. Three real-time PCR assays were developed from a multiplex PCR, targeting V. vulnificus, V. cholerae and V. alginolyticus on the dnaJ gene. After testing their specificity, these systems were applied for the identification of 961 strains isolated at 22°C (446 strains) and 37°C (515 strains) in September 2009. The predominance of V. alginolyticus (82·6%) among the Vibrio spp. strains isolated at 37°C was shown. At 22°C, only 1·6% of the strains were identified by PCR and they were V. alginolyticus. CONCLUSIONS: Reproducible and specific real-time PCR assays combined to a DNA extraction method on microplates were used to constitute a large environmental Vibrio strains collection and to identify and detect potential human pathogenic Vibrio isolated at 37°C. For environmental strains isolated at 22°C, because of the higher species diversity, other approaches, like sequencing, should be chosen for identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol developed in this study provides an appropriate and rapid screening tool to identify a large number of bacterial strains routinely isolated from the environment in long-term studies.

Structural data on a bacterial exopolysaccharide produced by a deep-sea Alteromonas macleodii strain.

Some marine bacteria collected around deep-sea hydrothermal vents are able to produce, in laboratory conditions, complex and innovative exopolysaccharides. In a previous study, the mesophilic strain Alteromonas macleodii subsp. fijiensis biovar deepsane was collected on the East Pacific Rise at 2600 m depth. It was isolated from a polychaete annelid Alvinella pompejana and is able to synthesise and excrete the exopolysaccharide deepsane. Biological activities have been screened and some protective properties have been established. Deepsane is commercially available in cosmetics under the name of Abyssine(®) for soothing and reducing irritation of sensitive skin against chemical, mechanical and UVB aggression. This study presents structural data for this original and complex bacterial exopolysaccharide and highlights some structural similarities with other known EPS produced by marine Alteromonas strains.

Degradation of mannan I and II crystals by fungal endo-beta-1,4-mannanases and a beta-1,4-mannosidase studied with transmission electron microscopy.

We have used the endo-beta-1,4-mannanase from Trichoderma reesei (Tr Man5A), the endo-beta-1,4-mannanase from Aspergillus niger (An Man5A) and the exo-beta-1,4-mannosidase from A. niger (An Mnd2A) to follow the enzymatic degradation of mannan I and II crystals. The degradation process was studied by transmission electron microscopy and also followed by analysis of the released soluble reducing sugars. The mannan crystals were degraded by the endo-beta-1,4-mannanases and to a lesser extent by the exo-beta-1,4-mannosidase. The observed hydrolysis pattern on mannan I crystals is fully consistent with the current view of the molecular structure of these crystals. The molecular organization of the mannan chains in mannan II crystals is less clear and the digestion results give some further information about the ultrastructure of mannan II. In addition, insight is provided into the mode of the enzymatic attack on the crystals of mannan I and mannan II.

Optimized mixtures of recombinant Humicola insolens cellulases for the biodegradation of crystalline cellulose.

The digestion of bacterial cellulose ribbons by ternary mixtures of enzymes consisting of recombinant cellulases (two cellobiohydrolases, Cel6A and Cel7A, and the endoglucanase Cel45A) from Humicola insolens was investigated over a wide range of mixture composition. The extent of digestion was followed by soluble sugar release (saccharification) analysis together with transmission electron microscopy (TEM) observations. It was found that the addition of minute quantities of Cel45A induced a spectacular increase in saccharification of the substrate with either Cel7A or the mixture of Cel6A and Cel7A. Conversely, only a moderate saccharification resulted from the mixing of Cel45A and Cel6A. This difference is believed to originate from (1) the occasional endo character of Cel6A and (2) the competition of Cel6A and Cel45A for the substrate sites that are sensitive to endo activity. Interestingly, the mixture of enzymes giving rise to the highest saccharification rate did not always correspond to mixtures of enzymes generating the highest synergy. TEM images revealed that the bacterial cellulose ribbons became at the same time cut and narrowed down under the action of an optimized mixture of the three enzymes.

Digestion of single crystals of mannan I by an endo-mannanase from Trichoderma reesei.

The enzymatic degradation of single crystals of mannan I with the catalytic core domain of a beta-mannanase (EC 3.2.1.78 or Man5A) from Trichoderma reesei was investigated by transmission electron microscopy and electron diffraction. The enzyme attack took place at the edge of the crystals and progressed towards their centres. Quite remarkably the crystalline integrity of the crystals was preserved almost to the end of the digestion process. This behaviour is consistent with an endo-mechanism, where the enzyme interacts with the accessible mannan chains located at the crystal periphery and cleaves one mannan molecule at a time. The endo mode of digestion of the crystals was confirmed by an analysis of the soluble degradation products.

Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A.

Dispersed cellulose ribbons from bacterial cellulose were subjected to digestion with cloned Cel7A (cellobiohydrolase [CBH] I) and Cel6A (CBH II) from Humicola insolens either alone or in a mixture and in the presence of an excess of beta-glucosidase. Both Cel7A and Cel6A were effective in partially converting the ribbons into soluble sugars, Cel7A being more active than Cel6A. In combination, these enzymes showed substantial synergy culminating with a molar ratio of approximately two-thirds Cel6A and one-third Cel7A. Ultrastructural transmission electron microscopy (TEM) observations indicated that Cel7A induced a thinning of the cellulose ribbons, whereas Cel6A cut the ribbons into shorter elements, indicating an endo type of action. These observations, together with the examination of the digestion kinetics, indicate that Cel6A can be classified as an endo-processive enzyme, whereas Cel7A is essentially a processive enzyme. Thus, the synergy resulting from the mixing of Cel6A and Cel7A can be explained by the partial endo character of Cel6A. A preparation of bacterial cellulose ribbons appears to be an appropriate substrate, superior to Valonia or bacterial cellulose microcrystals, to visualize directly by TEM the endo-processivity of an enzyme such as Cel6A.

Digestion of crystalline cellulose substrates by the clostridium thermocellum cellulosome: structural and morphological aspects.

The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valonia ventricosa was investigated. The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation. The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly. Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes. The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact. This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch. Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Ialpha/Ibeta composition throughout the degradation process. Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes. The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its 'commitment' towards complete degradation.

Molecular directionality of beta-chitin biosynthesis.

The molecular packing in beta-chitin unit cells was experimentally determined by a combination of unidirectional degradation by Bacillus circulans chitinase A1 and microdiffraction electron crystallography using highly crystalline beta-chitin microfibrils from the protective tubes secreted by Lamellibrachia satsuma. The mode of chain packing was found to be identical with that of the previously published crystal model for beta-chitin, despite a controversial definition of the unit cell parameters. Here, a "parallel-down" packing was determined, where the reducing ends of chains point in an opposite direction to the crystallographic c-axis. Microdiffraction analyses of nascent beta-chitin microfibrils generated from diatom Thalassiosira sp. showed that the c-axis of the crystal was directed toward the diatoms, and therefore the reducing end of a growing chain pointed away from the locus of biosynthesis. This mechanism agreed well with what we found recently in the cellulose biosynthesis system, and provides strong evidence that the polymerization by the processive glycosyl transferase takes place at the non-reducing end of the growing polysaccharide chains.

Unidirectional processive action of cellobiohydrolase Cel7A on Valonia cellulose microcrystals.

On the basis of the 'parallel-up' structure of the cellulose crystal, a crystallographic approach to study the mode of action of cellobiohydrolase Cel7A on Valonia cellulose microcrystal has been carried out. After incubation with Cel7A, most of the initially smooth and well defined Valonia microcrystals displayed fibrillation. However, as the hydrolysis reaction was rather heterogeneous, some microcrystals remained superficially intact. Close investigation on such crystals revealed polar morphology: one end was narrowed extremely or pointed. Electron microdiffraction analysis of these crystals evidenced that the narrowing of the microcrystals occurs at their reducing end side. This was also confirmed by the visualization of selective reducing end labeling at the pointed ends of microcrystals. These lines of investigation are the first demonstration that the processivity of Cel7A action against insoluble highly crystalline celluloses is unambiguously toward non-reducing ends from reducing ends.

Dynamic light scattering study of the two-domain structure of Humicola insolens endoglucanase V.

Endoglucanase V (EG V) of HUmicola insolens is composed of a catalytic domain and of a cellulose-binding domain linked by a 33 amino acid long peptide rich in Ser, Thr and Pro residues. This work describes the dynamic behavior of the two-domain structure of EG V as revealed by quasi-elastic light scattering experiments. For both the full-length and the isolated catalytic domain, the autocorrelation function is essentially described by a single relaxation mode. The equivalent hydrodynamic radius of the catalytic domain was found to correspond precisely to the dimensions measured from the previously determined three-dimensional structure. The results obtained with the full-length protein allow a description of the two domain structure of EG V similar to that resulting from earlier studies using small angle X-ray scattering on cellulases from Trichoderma reesei. The hydrodynamic dimensions of the entire enzyme can be approximated as an ellipsoid with dimensions of 42 x 133.6 A.