Flavonoid Glycosides with a Triazole Moiety for Marine Antifouling Applications: Synthesis and Biological Activity Evaluation.

Over the last decades, antifouling coatings containing biocidal compounds as active ingredients were used to prevent biofouling, and eco-friendly alternatives are needed. Previous research from our group showed that polymethoxylated chalcones and glycosylated flavones obtained by synthesis displayed antifouling activity with low toxicity. In this work, ten new polymethoxylated flavones and chalcones were synthesized for the first time, including eight with a triazole moiety. Eight known flavones and chalcones were also synthesized and tested in order to construct a quantitative structure-activity relationship (QSAR) model for these compounds. Three different antifouling profiles were found: three compounds (1b, 11a and 11b) exhibited anti-settlement activity against a macrofouling species (Mytilus galloprovincialis), two compounds (6a and 6b) exhibited inhibitory activity against the biofilm-forming marine bacteria Roseobacter litoralis and one compound (7b) exhibited activity against both mussel larvae and microalgae Navicula sp. Hydrogen bonding acceptor ability of the molecule was the most significant descriptor contributing positively to the mussel larvae anti-settlement activity and, in fact, the triazolyl glycosylated chalcone 7b was the most potent compound against this species. The most promising compounds were not toxic to Artemia salina, highlighting the importance of pursuing the development of new synthetic antifouling agents as an ecofriendly and sustainable alternative for the marine industry.

Manganese Oxide Biomineralization Provides Protection against Nitrite Toxicity in a Cell-Density-Dependent Manner.

Manganese biomineralization is a widespread process among bacteria and fungi. To date, there is no conclusive experimental evidence for how and if this process impacts microbial fitness in the environment. Here, we show how a model organism for manganese oxidation is growth inhibited by nitrite, and that this inhibition is mitigated in the presence of manganese. We show that such manganese-mediated mitigation of nitrite inhibition is dependent on the culture inoculum size, and that manganese oxide (MnOX) forms granular precipitates in the culture, rather than sheaths around individual cells. We provide evidence that MnOX protection involves both its ability to catalyze nitrite oxidation into (nontoxic) nitrate under physiological conditions and its potential role in influencing processes involving reactive oxygen species (ROS). Taken together, these results demonstrate improved microbial fitness through MnOX deposition in an ecological setting, i.e., mitigation of nitrite toxicity, and point to a key role of MnOX in handling stresses arising from ROS.IMPORTANCE We present here a direct fitness benefit (i.e., growth advantage) for manganese oxide biomineralization activity in Roseobacter sp. strain AzwK-3b, a model organism used to study this process. We find that strain AzwK-3b in a laboratory culture experiment is growth inhibited by nitrite in manganese-free cultures, while the inhibition is considerably relieved by manganese supplementation and manganese oxide (MnOX) formation. We show that biogenic MnOX interacts directly with nitrite and possibly with reactive oxygen species and find that its beneficial effects are established through formation of dispersed MnOX granules in a manner dependent on the population size. These experiments raise the possibility that manganese biomineralization could confer protection against nitrite toxicity to a population of cells. They open up new avenues of interrogating this process in other species and provide possible routes to their biotechnological applications, including in metal recovery, biomaterials production, and synthetic community engineering.

Study of marine bacteria inactivation by photochemical processes: disinfection kinetics and growth modeling after treatment.

The importance of seawater treatment in order to avoid microbiological pollution related to aquaculture or ballast water management has increased during the last few years. Bacterial indicators used for the evaluation of different disinfection treatments are usually related with both waste and drinking water, these standards are not usual microorganisms found in seawater. Thus, it is thought necessary to study the behavior of different marine-specific organisms in regard to improve the disinfection processes in seawater. In this study, three different bacteria have been selected among major groups of bacterial community from marine waters: two water-associated, Roseobacter sp. and Pseudomonas litoralis, and one sediment-associated, Kocuria rhizophila. A kinetic inactivation model together with a post-treatment growth tendency has been obtained after the application of UV-C and UV/H2O2 processes. According to the first kinetic rate constant, different responses were obtained for the different bacterial groups. Once the treatment was applied, modeling of growth curves revealed high recover within the first 3 days after treatment, even when UV/H2O2 was applied. This study introduces a sensitivity index, in which results show different levels of resistance for both treatments, being Roseobacter sp. the most sensitive bacteria, followed by P. litoralis and K. rhizophila.

Simulating CO2 leakage from sub-seabed storage to determine metal toxicity on marine bacteria.

CO2 storage in sub-seabed marine geological formations has been proposed as an adequate strategy to mitigate high CO2 concentration from the atmosphere. The lack of knowledge about the potential risks of this technology on marine bacteria population in presence of metals has lead us to perform laboratory-scale experiments in order to evaluate its consequences. Thus, the effects of Zn and Cd were studied under acid conditions on Roseobacter sp. and Pseudomonas litoralis. Bacterial abundance (cellsmL-1), growth rates (µ, h-1), relative inhibitory effects of CO2 (RICO2), and production of Extracellular Polysaccharides Substances (EPS) (µgGlucosecells-1) were evaluated. A decreasing exopolysaccharides (EPS) production was found under low pH. Bacterial abundance as well as growth rates showed negative effects. Data obtained in this work are useful to determine the potential effects associated with enrichment of CO2 and metals on the marine ecosystem.

Dynamics of amino acid utilization in Phaeobacter inhibens DSM 17395.

Time-resolved utilization of multiple amino acids by Phaeobacter inhibens DSM 17395 was studied during growth with casamino acids. The 15 detected amino acids could be grouped according to depletion rate into four different categories, i.e. from rapid (category I) to nondepletion (category IV). Upon entry into stationary growth phase, amino acids of category I (e.g. glutamate) were (almost) completely depleted, while those of categories II (e.g. leucine) and III (e.g. serine) were further consumed at varying rates and to different extents. Thus, cultures entered stationary growth phase despite the ample presence of organic nutrients, i.e. under nonlimiting conditions. Integrated proteomic and metabolomic analysis identified 1747 proteins and 94 intracellular metabolites. Of these, 180 proteins and 86 metabolites displayed altered abundance levels during growth. Most strikingly, abundance and activity profiles of alanine dehydrogenase concomitantly increased with the onset of enhanced alanine utilization during transition into stationary growth phase. Most enzymes of amino acid and central metabolism, however, displayed unaltered abundances across exponential and stationary growth phases. In contrast, metabolites of the Entner-Doudoroff pathway and gluconeogenesis as well as cellular fatty acids increased markedly in abundance in early stationary growth phase.

A novel inducer of Roseobacter motility is also a disruptor of algal symbiosis.

Silicibacter sp. strain TM1040, a member of the Roseobacter clade, forms a symbiosis with unicellular phytoplankton, which is inextricably linked to the biphasic "swim or stick" lifestyle of the bacteria. Mutations in flaC bias the population toward the motile phase. Renewed examination of the FlaC(-) strain (HG1016) uncovered that it is composed of two different cells: a pigmented type, PS01, and a nonpigmented cell, PS02, each of which has an identical mutation in flaC. While monocultures of PS01 and PS02 had few motile cells (0.6 and 6%, respectively), coculturing the two strains resulted in a 10-fold increase in the number of motile cells. Cell-free supernatants from coculture or wild-type cells were fully capable of restoring motility to PS01 and PS02, which was due to increased fliC3 (flagellin) transcription, FliC3 protein levels per cell, and flagella synthesis. The motility-inducing compound has an estimated mass of 226 Da, as determined by mass spectrometry, and is referred to as Roseobacter Motility Inducer (RMI). Mutations affecting genes involved in phenyl acetic acid synthesis significantly reduced RMI, while defects in tropodithietic acid (TDA) synthesis had marginal or no effect on RMI. RMI biosynthesis is induced by p-coumaric acid, a product of algal lignin degradation. When added to algal cultures, RMI caused loss of motility, cell enlargement, and vacuolization in the algal cells. RMI is a new member of the roseobacticide family of troponoid compounds whose activities affect roseobacters, by shifting their population toward motility, as well as their phytoplankton hosts, through an algicidal effect.

The Jekyll-and-Hyde chemistry of Phaeobacter gallaeciensis.

Emiliania huxleyi, an environmentally important marine microalga, has a bloom-and-bust lifestyle in which massive algal blooms appear and fade. Phaeobacter gallaeciensis belongs to the roseobacter clade of α-Proteobacteria, the populations of which wax and wane with that of E. huxleyi. Roseobacter are thought to promote algal growth by biosynthesizing and secreting antibiotics and growth stimulants (auxins). Here we show that P. gallaeciensis switches its secreted small molecule metabolism to the production of potent and selective algaecides, the roseobacticides, in response to p-coumaric acid, an algal lignin breakdown product that is symptomatic of aging algae. This switch converts P. gallaeciensis into an opportunistic pathogen of its algal host.

Resistance and tolerance to tropodithietic acid, an antimicrobial in aquaculture, is hard to select.

The antibacterial compound tropodithietic acid (TDA) is produced by bacteria of the marine Roseobacter clade and is thought to explain the fish probiotic properties of some roseobacters. The aim of the present study was to determine the antibacterial spectrum of TDA and the likelihood of development of TDA resistance. A bacterial extract containing 95% TDA was effective against a range of human-pathogenic bacteria, including both Gram-negative and Gram-positive bacteria. TDA was bactericidal against Salmonella enterica serovar Typhimurium SL1344 and Staphylococcus aureus NCTC 12493 and killed both growing and nongrowing cells. Several experimental approaches were used to select mutants resistant to TDA or subpopulations of strains with enhanced tolerance to TDA. No approach (single exposures to TDA extract administered via different methods, screening of a transposon library for resistant mutants, or prolonged exposure to incremental concentrations of TDA) resulted in resistant or tolerant strains. After more than 300 generations exposed to sub-MIC and MIC concentrations of a TDA-containing extract, strains tolerant to 2× the MIC of TDA for wild-type strains were selected, but the tolerance disappeared after one passage in medium without TDA extract. S. Typhimurium mutants with nonfunctional efflux pump and porin genes had the same TDA susceptibility as wild-type strains, suggesting that efflux pumps and porins are not involved in innate tolerance to TDA. TDA is a promising broad-spectrum antimicrobial in part due to the fact that enhanced tolerance is difficult to gain and that the TDA-tolerant phenotype appears to confer only low-level resistance and is very unstable.

Reclassification of Roseobacter gallaeciensis Ruiz-Ponte et al. 1998 as Phaeobacter gallaeciensis gen. nov., comb. nov., description of Phaeobacter inhibens sp. nov., reclassification of Ruegeria algicola (Lafay et al. 1995) Uchino et al. 1999 as Marinovum algicola gen. nov., comb. nov., and emended descriptions of the genera Roseobacter, Ruegeria and Leisingera.

A heterotrophic, antibiotic-producing bacterium, strain T5(T), was isolated from the German Wadden Sea, located in the southern region of the North Sea. Sequence analysis of the 16S rRNA gene of this strain demonstrated close affiliation with Roseobacter gallaeciensis BS107(T) (99 % similarity), but the results of genotypic (DNA-DNA hybridization and DNA G + C content) and phenotypic characterization revealed that strain T5(T) represents a novel species. The novel organism is strictly aerobic, Gram-negative, rod-shaped, motile and forms brown-pigmented colonies. Strain T5(T) produces the antibiotic tropodithietic acid throughout the exponential phase which inhibits the growth of bacteria from different taxa, as well as marine algae. Strain T5(T) requires sodium ions and utilizes a wide range of substrates, including oligosaccharides, sugar alcohols, organic acids and amino acids. The DNA G + C content is 55.7 mol%. Comparative 16S rRNA gene sequence analysis revealed that strains T5(T) and Roseobacter gallaeciensis BS107(T) group with Leisingera methylohalidivorans as their closest described relative within the Roseobacter clade (97.9 and 97.6 % sequence similarity, respectively) and with Ruegeria algicola (96.6 and 96.5 % similarity, respectively) of the Alphaproteobacteria. Comparison of strains T5(T) and Roseobacter gallaeciensis BS107(T) with Roseobacter denitrificans and Roseobacter litoralis showed striking differences in 16S rRNA gene sequence similarities, chemical composition, pigmentation, presence of bacteriochlorophyll a and antibiotic production. On the basis of these results, it is proposed that Roseobacter gallaeciensis is reclassified as the type species of a new genus, Phaeobacter, as Phaeobacter gallaeciensis comb. nov. (type strain BS107(T) = CIP 105210(T) = ATCC 700781(T) = NBRC 16654(T) = DSM 17395(T)). Strain T5(T) (=LMG 22475(T) = DSM 16374(T)) is proposed as the type strain of a novel species of this genus, Phaeobacter inhibens sp. nov. At the same time, emended descriptions are provided of the genera Roseobacter, Ruegeria and Leisingera, as well as reclassifying Ruegeria algicola as the type species of a new genus, Marinovum, with the name Marinovum algicola comb. nov.