Structure Revision of a Widespread Marine Sulfonolipid Class Based on Isolation and Total Synthesis.

The cosmopolitan marine Roseobacter clade is of global biogeochemical importance. Members of this clade produce sulfur-containing amino lipids (SALs) involved in biofilm formation and marine surface colonization processes. Despite their physiological relevance and abundance, SALs have only been explored through genomic mining approaches and lipidomic studies based on mass spectrometry, which left the relative and absolute structures of SALs unresolved, hindering progress in biochemical and functional investigations. Herein, we report the structural revision of a new group of SALs, which we named cysteinolides, using a combination of analytical techniques, isolation and degradation experiments and total synthetic efforts. Contrary to the previously proposed homotaurine-based structures, cysteinolides are composed of an N,O-acylated cysteinolic acid-containing head group carrying various different (α-hydroxy)carboxylic acids. We also performed the first validated targeted-network based analysis, which allowed us to map the distribution and structural diversity of cysteinolides across bacterial lineages. Beyond offering structural insight, our research provides SAL standards and validated analytical data. This information holds significance for forthcoming investigations into bacterial sulfonolipid metabolism and biogeochemical nutrient cycling within marine environments.

Aminolipids elicit functional trade-offs between competitiveness and bacteriophage attachment in Ruegeria pomeroyi.

Lipids play a crucial role in maintaining cell integrity and homeostasis with the surrounding environment. Cosmopolitan marine roseobacter clade (MRC) and SAR11 clade bacteria are unique in that, in addition to glycerophospholipids, they also produce an array of amino acid-containing lipids that are conjugated with beta-hydroxy fatty acids through an amide bond. Two of these aminolipids, the ornithine aminolipid (OL) and the glutamine aminolipid (QL), are synthesized using the O-acetyltransferase OlsA. Here, we demonstrate that OL and QL are present in both the inner and outer membranes of the Gram-negative MRC bacterium Ruegeria pomeroyi DSS-3. In an olsA mutant, loss of these aminolipids is compensated by a concurrent increase in glycerophospholipids. The inability to produce aminolipids caused significant changes in the membrane proteome, with the membrane being less permeable and key nutrient transporters being downregulated while proteins involved in the membrane stress response were upregulated. Indeed, the import of 14C-labelled choline and dimethylsulfoniopropionate, as a proxy for the transport of key marine nutrients across membranes, was significantly impaired in the olsA mutant. Moreover, the olsA mutant was significantly less competitive than the wild type (WT) being unable to compete with the WT strain in co-culture. However, the olsA mutant unable to synthesize these aminolipids is less susceptible to phage attachment. Together, these data reveal a critical role for aminolipids in the ecophysiology of this important clade of marine bacteria and a trade-off between growth and avoidance of bacteriophage attachment.

Trade-offs of lipid remodeling in a marine predator-prey interaction in response to phosphorus limitation.

Phosphorus (P) is a key nutrient limiting bacterial growth and primary production in the oceans. Unsurprisingly, marine microbes have evolved sophisticated strategies to adapt to P limitation, one of which involves the remodeling of membrane lipids by replacing phospholipids with non-P-containing surrogate lipids. This strategy is adopted by both cosmopolitan marine phytoplankton and heterotrophic bacteria and serves to reduce the cellular P quota. However, little, if anything, is known of the biological consequences of lipid remodeling. Here, using the marine bacterium Phaeobacter sp. MED193 and the ciliate Uronema marinum as a model, we sought to assess the effect of remodeling on bacteria-protist interactions. We discovered an important trade-off between either escape from ingestion or resistance to digestion. Thus, Phaeobacter grown under P-replete conditions was readily ingested by Uronema, but not easily digested, supporting only limited predator growth. In contrast, following membrane lipid remodeling in response to P depletion, Phaeobacter was less likely to be captured by Uronema, thanks to the reduced expression of mannosylated glycoconjugates. However, once ingested, membrane-remodeled cells were unable to prevent phagosome acidification, became more susceptible to digestion, and, as such, allowed rapid growth of the ciliate predator. This trade-off between adapting to a P-limited environment and susceptibility to protist grazing suggests the more efficient removal of low-P prey that potentially has important implications for the functioning of the marine microbial food web in terms of trophic energy transfer and nutrient export efficiency.

The Proteobacterial Methanotroph Methylosinus trichosporium OB3b Remodels Membrane Lipids in Response to Phosphate Limitation.

Methane is a potent greenhouse gas in the atmosphere, and its concentration has continued to increase in recent decades. Aerobic methanotrophs, bacteria that use methane as the sole carbon source, are an important biological sink for methane, and they are widely distributed in the natural environment. However, relatively little is known on how methanotroph activity is regulated by nutrients, particularly phosphorus (P). P is the principal nutrient constraining plant and microbial productivity in many ecosystems, ranging from agricultural land to the open ocean. Using a model methanotrophic bacterium, Methylosinus trichosporium OB3b, we demonstrate here that this bacterium can produce P-free glycolipids to replace membrane phospholipids in response to P limitation. The formation of the glycolipid monoglucuronic acid diacylglycerol requires plcP-agt genes since the plcP-agt mutant is unable to produce this glycolipid. This plcP-agt-mediated lipid remodeling pathway appears to be important for M. trichosporium OB3b to cope with P stress, and the mutant grew significantly slower under P limitation. Interestingly, comparative genomics analysis shows that the ability to perform lipid remodeling appears to be a conserved trait in proteobacterial methanotrophs; indeed, plcP is found in all proteobacterial methanotroph genomes, and plcP transcripts from methanotrophs are readily detectable in metatranscriptomics data sets. Together, our study provides new insights into the adaptation to P limitation in this ecologically important group of bacteria. IMPORTANCE Methane is a potent greenhouse gas in the atmosphere, and its concentration has continued to increase steadily in recent decades. In the natural environment, bacteria known as methanotrophs help mitigate methane emissions at no cost to human beings. However, relatively little is known regarding how methane oxidation activity in methanotrophs is regulated by soil nutrients, particularly phosphorus. Here, we show that methanotrophs can modify their membrane in response to phosphorus limitation and that the ability to change membrane lipids is important for methanotroph activity. Genome and metatranscriptome analyses suggest that such an adaptation strategy appears to be strictly conserved in all proteobacterial methanotrophs and is used by these bacteria in the natural environment. Together, our study provides a plausible molecular mechanism for better understanding the role of phosphorus on methane oxidation in the natural environment.

Acrylate protects a marine bacterium from grazing by a ciliate predator.

Cleavage of dimethylsulfoniopropionate (DMSP) can deter herbivores in DMSP-producing eukaryotic algae; however, it is unclear whether a parallel defence mechanism operates in marine bacteria. Here we demonstrate that the marine bacterium Puniceibacterium antarcticum SM1211, which does not use DMSP as a carbon source, has a membrane-associated DMSP lyase, DddL. At high concentrations of DMSP, DddL causes an accumulation of acrylate around cells through the degradation of DMSP, which protects against predation by the marine ciliate Uronema marinum. The presence of acrylate can alter the grazing preference of U. marinum to other bacteria in the community, thereby influencing community structure.

A novel class of sulfur-containing aminolipids widespread in marine roseobacters.

Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated salA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modeling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. salA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

Marine Bacteria Display Different Escape Mechanisms When Facing Their Protozoan Predators.

Free-living amoeba are members of microbial communities such as biofilms in terrestrial, fresh, and marine habitats. Although they are known to live in close association with bacteria in many ecosystems such as biofilms, they are considered to be major bacterial predators in many ecosystems. Little is known on the relationship between protozoa and marine bacteria in microbial communities, more precisely on how bacteria are able survive in environmental niches where these bacterial grazers also live. The objective of this work is to study the interaction between the axenized ubiquitous amoeba Acanthamoeba castellanii and four marine bacteria isolated from immersed biofilm, in order to evaluate if they would be all grazed upon by amoeba or if they would be able to survive in the presence of their predator. At a low bacteria-to-amoeba ratio, we show that each bacterium is phagocytized and follows a singular intracellular path within this host cell, which appears to delay or to prevent bacterial digestion. In particular, one of the bacteria was found in the amoeba nucleolar compartment whereas another strain was expelled from the amoeba in vesicles. We then looked at the fate of the bacteria grown in a higher bacteria-to-amoeba ratio, as a preformed mono- or multi-species biofilm in the presence of A. castellanii. We show that all biofilms were subjected to detachment from the surface in the presence of the amoeba or its supernatant. Overall, these results show that bacteria, when facing the same predator, exhibit a variety of escape mechanisms at the cellular and population level, when we could have expected a simple bacterial grazing. Therefore, this study unravels new insights into the survival of environmental bacteria when facing predators that they could encounter in the same microbial communities.

Multispecies Biofilm Development of Marine Bacteria Implies Complex Relationships Through Competition and Synergy and Modification of Matrix Components.

Microbial communities composition is largely shaped by interspecies competition or cooperation in most environments. Ecosystems are made of various dynamic microhabitats where microbial communities interact with each other establishing metabolically interdependent relationships. Very limited information is available on multispecies biofilms and their microhabitats related to natural environments. The objective of this study is to understand how marine bacteria isolated from biofilms in the Mediterranean Sea interact and compete with each other when cultivated in multispecies biofilms. Four strains (Persicivirga mediterranea TC4, Polaribacter sp. TC5, Shewanella sp. TC10 and TC11) with different phenotypical traits and abilities to form a biofilm have been selected from a previous study. Here, the results show that these strains displayed a different capacity to form a biofilm in static versus dynamic conditions where one strain, TC11, was highly susceptible to the flux. These bacteria appeared to be specialized in the secretion of one or two exopolymers. Only TC5 seemed to secrete inhibitory molecule(s) in its supernatant, with a significant effect on TC10. Most of the strains negatively impacted each other, except TC4 and TC10, which presented a synergetic effect in the two and three species biofilms. Interestingly, these two strains produced a newly secreted compound when grown in dual-species versus mono-species biofilms. TC5, which induced a strong inhibition on two of its partners in dual-species biofilms, outfitted the other bacteria in a four-species biofilm. Therefore, understanding how bacteria respond to interspecific interactions should help comprehending the dynamics of bacterial populations in their ecological niches.