Philippe Catala

Phylogenetic and structural response of heterotrophic bacteria to dissolved organic matter of different chemical composition in a continuous culture study.

Dissolved organic matter (DOM) and heterotrophic bacteria are highly diverse components of the ocean system, and their interactions are key in regulating the biogeochemical cycles of major elements. How chemical and phylogenetic diversity are linked remains largely unexplored to date. To investigate interactions between bacterial diversity and DOM, we followed the response of natural bacterial communities to two sources of phytoplankton-derived DOM over six bacterial generation times in continuous cultures. Analyses of total hydrolysable neutral sugars and amino acids, and ultrahigh resolution mass spectrometry revealed large differences in the chemical composition of the two DOM sources. According to 454 pyrosequences of 16S ribosomal ribonucleic acid genes, diatom-derived DOM sustained higher levels of bacterial richness, evenness and phylogenetic diversity than cyanobacteria-derived DOM. These distinct community structures were, however, not associated with specific taxa. Grazing pressure affected bacterial community composition without changing the overall pattern of bacterial diversity levels set by DOM. Our results demonstrate that resource composition can shape several facets of bacterial diversity without influencing the phylogenetic composition of bacterial communities, suggesting functional redundancy at different taxonomic levels for the degradation of phytoplankton-derived DOM.

Evaluation of ChemChrome V6 for bacterial viability assessment in waters

The efficiency of ChemChrome B (CB) and ChemChrome V6 (CV6) dyes to stain viable bacterial cells in water was compared. Both dyes are fluorogenic esters converted to free fluorescein by esterase activity. The dyes were applied to a wide variety of bacterial species, including those poorly stained by CB, and to natural waters. Some species tested gave unacceptable low fluorescence intensities by being inefficiently or non‐labelled with the CB. In contrast, CV6‐stained bacteria were easily detected by both flow cytometry and solid‐phase cytometry. As a consequence, higher viable cell counts were found with CV6 compared with CB in natural waters. Viable counts determined by CV6 staining were always higher than cfu counts. In constrast, respiring cell counts (CTC) were always lower than CV6 counts and, in the case of tap and mineral waters, they were lower than cfu counts.

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

ABSTRACT A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.

Spatial comparison of total vs. active bacterial populations by coupling genetic fingerprinting and clone library analyses in the NW Mediterranean Sea

Spatial distributions of both total (i.e. 16S rDNA-based fingerprints) and active (i.e. 16S rRNA-based fingerprints) bacterial populations, together with total bacterial activity measured by 3H-leucine incorporation, were studied along a 98 km transect in the NW Mediterranean Sea. Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) fingerprinting was coupled to a clone library, allowing CE-SSCP peaks identification and the monitoring of the spatial variation of bacterial phylotypes. Up to 80% of the community peaks matched those obtained from clone library sequences, accounting for 86.7% of the total fingerprinting area. A good agreement was found between the relative abundance of Prochlorococcus in the CE-SSCP fingerprints and flow cytometry counts (r2=0.66, P<0.05). The largest differences between total and active bacterial populations distribution were found at depths with higher bacterial activity (i.e. surface and deep chlorophyll maximum, DCM). SAR11 at the surface and Gammaproteobacteria at the DCM were the most abundant groups on the 16S rDNA-based fingerprints. However, their ratio of relative importance between rRNA : rDNA was <1 in most cases. Conversely, ratios observed for Prochlorococcus, were consistently >1 both at the surface and at the DCM. These results emphasize the need for combining rDNA- and rRNA-based analyses to better understand the functional role of individual bacterial populations in situ.

Rapid Detection and Enumeration of Naegleria fowleri in Surface Waters by Solid-Phase Cytometry

ABSTRACT A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both R-phycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.

Pelagic food web patterns: do they modulate virus and nanoflagellate effects on picoplankton during the phytoplankton spring bloom?

As agents of mortality, viruses and nanoflagellates impact on picoplankton populations. We examined the differences in interactions between these compartments in two French Atlantic bays. Microbes, considered here as central actors of the planktonic food web, were first monitored seasonally in Arcachon (2005) and Marennes-Oléron (2006) bays. Their dynamics were evaluated to categorize trophic periods using the models of Legendre and Rassoulzadegan as a reference framework. Microbial interactions were then compared through 48 h batch culture experiments performed during the phytoplankton spring bloom, identified as herbivorous in Marennes and multivorous in Arcachon. Marennes was spatially homogeneous compared with Arcachon. The former was potentially more productive, featuring a large number of heterotrophic pathways, while autotrophic mechanisms dominated in Arcachon. A link was found between viruses and phytoplankton in Marennes, suggesting a role of virus in the regulation of autotroph biomass. Moreover, the virus-bacteria relation was weaker in Marennes, with a bacterial lysis potential of 2.6% compared with 39% in Arcachon. The batch experiments (based on size-fractionation and viral enrichment) revealed different microbial interactions that corresponded to the spring-bloom trophic interactions in each bay. In Arcachon, where there is a multivorous web, flagellate predation and viral lysis acted in an opposite way on picophytoplankton. When together they both reduced viral production. Conversely, in Marennes (herbivorous web), flagellates and viruses together increased viral production. Differences in the composition of the bacterial community composition explained the combined flagellate-virus effects on viral production in the two bays.

Hellea balneolensis gen. nov., sp. nov., a prosthecate alphaproteobacterium from the Mediterranean Sea

A novel aerobic, heterotrophic, prosthecate bacterium, designated 26III/A02/215(T), was isolated from surface water of the north-western Mediterranean Sea. Cells stained Gram-negative and were straight to slightly curved rods, forming red colonies on agar plates. The strain grew at 15-37 degrees C inclusive (optimum 30 degrees C) and grew optimally at seawater salinity. Growth was observed on organic acids, amino acids and complex organic substrates. The fatty acids (>5 %) detected in strain 26III/A02/215(T) were C(17 : 1)omega6c, C(18 : 1)omega7c and C(17 : 0). The lipid pattern indicated the presence of phosphatidylglycerol, glucuronopyranosyldiglyceride, monoglycosyldiglyceride, an unidentified glycolipid and three unidentified phospholipids. Phosphatidylethanolamine and diphosphatidylglycerol were absent. Ubiquinone Q-10 was the only respiratory lipoquinone. The G+C content of the genomic DNA was 46.8 mol%. Comparative 16S rRNA gene sequence analysis indicated that strain 26III/A02/215(T) belonged to the Hyphomonas-Hirschia-Robiginitomaculum branch of the order Caulobacterales. This affiliation was consistent with the results of polar lipid analyses. Among this group, the novel isolate was most closely related to Robiginitomaculum antarcticum (93.9 % 16S rRNA gene sequence similarity to the type strain). On the basis of genotypic, chemotaxonomic and phenotypic distinctness, we propose a novel genus, Hellea gen. nov., with Hellea balneolensis sp. nov. as the type species. The type strain of Hellea balneolensis is 26III/A02/215(T) (=DSM 19091(T) =CIP 109500(T) =OOB 269(T)).

Different marine heterotrophic nanoflagellates affect differentially the composition of enriched bacterial communities

We studied the effects of predation on the cytometric and phylogenetic features of two enriched bacterial communities obtained from two cultures of marine heterotrophic nanoflagellates: Jakoba libera and a mixed culture of Cafeteria sp. and Monosiga sp. Protists were harvested by flow cytometric cell sorting and eight different treatments were prepared. Each bacterial community was incubated with and without protists, and we added two treatments with protists and the bacteria present after the sorting procedure (cosorted bacteria). The bacterial community derived from the culture of Jakoba libera had higher green fluorescence per cell (FL1) than that derived from the mixed culture of Cafeteria sp. and Monosiga sp. When the experiment began all treatments presented bacterial communities that increase in fluorescence per bacterium (FL1); after that the FL1 decreased when bacteria attained maximal concentrations; and, finally, there was a new increase in FL1 toward the end of the experiment. Cosorted bacteria of Jakoba libera had the same fluorescence as the bacterial community derived from this protist, while the bacteria derived from the mixed culture of Cafeteria sp. and Monosiga sp. was nearly twice as fluorescent than that of the parental community. All treatments presented a general decline of SSC along the incubation. Therefore, there was a small influence of protists on the cytometric signature of each bacterial community. However, each bacterial community preyed by Jakoba libera or the mixed culture of Cafeteria sp. and Monosiga sp. led to four different phylogenetic fingerprint. Besides, the final Communities were different from the fingerprint of controls without protists, and most of them diverge from the fingerprint of cosorted bacteria. Our results confirm that changes in the phylogenetic composition of marine bacterial communities may depend on the initial communities of both bacteria and protists.

Phylogenetic and functional diversity of Bacteria and Archaea in a unique stratified lagoon, the Clipperton atoll (N Pacific)

The Clipperton lagoon in the North Pacific Ocean has been isolated from the surrounding sea for c. 160 years. It has a stratified water column that comprises an oxic and brackish upper water layer (mixolimnion) and a deep sulfuric anoxic saline layer (monimolimnion), separated by a steep pycnocline. Here, we test whether the Clipperton lagoon with its distinctive physico-chemical features, geographic isolation, recent water column stratification, and large nutrient input harbors original microbial communities. The combination of capillary electrophoresis single-strand polymorphism (CE-SSCP) fingerprinting and sequencing of cloned bacterial and archaeal 16S rRNA genes, and functional genes for methanogenesis (mcrA), methanotrophy (pmoA), and sulfate reduction (dsrAB), revealed that microbial communities and pathways were highly stratified down the water column. The mixolimnion contained ubiquitous freshwater clades of Alpha- and Betaproteobacteria, while the pycnocline contained mostly green sulfur bacteria (phylum Chlorobi). Sequences of the upper layers were closely related to sequences found in other aquatic ecosystems, suggesting that they have a strong potential for dispersal and colonization. In contrast, the monimolimnion contained new deeply branching bacterial divisions within the OP11 cluster and the Bacteroidetes, and was the most diverse of the layers. The unique environmental conditions characterizing the deep layers of the lagoon may explain the novelty of the microbial communities found at the Clipperton atoll.

Potential Effect of Freshwater Virus on the Structure and Activity of Bacterial Communities in the Marennes-Oléron Bay (France)

Batch culture experiments using viral enrichment were conducted to test the response of a coastal bacterial community to autochthonous (i.e., co-existing) or allochthonous riverine viruses. The effects of viral infections on bacterial dynamics and activity were assessed by epifluorescence microscopy and thymidine incorporation, respectively, whereas the effect of viral infection on bacterial community composition was examined by polymerase chain reaction-single strand conformation polymorphism 16S ribosomal RNA fingerprinting. The percentages of high nucleic acid-containing cells, evaluated by flow cytometry, were significantly correlated (r2 = 0.91, n = 12, p < 0.0001) to bacterial production, making this value a good predictor of active cell dynamics along the study. While confinement and temperature were the two principal experimental factors affecting bacterial community composition and dynamics, respectively, additions of freshwater viruses had significant effects on coastal bacterial communities. Thus, foreign viruses significantly reduced net bacterial population increase as compared to the enrichment treated with inactivated virus. Moreover, freshwater viruses recurrently and specifically affected bacterial community composition, as compared to addition of autochthonous viruses. In most cases, the combined treatment viruses and freshwater dissolved organic matter helped to maintain or even enhance species richness in coastal bacterial communities in agreement to the ‘killing the winner’ hypothesis. Thus, riverine virus input could potentially influence bacterial community composition of the coastal bay albeit with modest modification of bulk bacterial growth.