To identify and understand the presence of metabolites responsible for the variation in the metabolic profile of Vibrio coralliilyticus under extreme conditions.
Multiple batches of V. coralliilyticus were grown under normal conditions. Four samples in one batch were subjected to extreme conditions via a freeze-thaw cycle during lyophilization. Polar metabolites were extracted using a combination of methanol, water and heat. Nuclear magnetic resonance (NMR)-based metabolic profiles indicated significant differences between the normal and stressed samples. Three compounds identified in the stressed metabolome were maltose, ethanolamine, and the bioplastic-type compound (BTC) 2-butenoic acid, 2-carboxy-1-methylethyl ester. This is the first report of the production of this BTC by V. coralliilyticus.
The presence of maltose and ethanolamine indicates a state of acute nutrient limitation; therefore, we hypothesize that the cell's metabolism turned to its own cell wall, or perhaps neighbouring cells, for sources of carbon and nitrogen. The presence of the BTC also supports the acute nutrient limitation idea because of the parallels with polyhydroxyalkanoate (PHA) production in other gram-negative bacteria, including other Vibrio species.
Recent metabolomics research on the temperature-dependent coral pathogen V. coralliilyticus has led to the discovery of several compounds produced by the organism as a response to high density, low nutrient conditions. The three metabolites, along with (1) H NMR metabolic fingerprints of the nutrient limited samples, are proposed to serve as metabolic markers for extremely stressful conditions of V. coralliilyticus.
"Low temperature quenching is accepted by most researchers. The particular quenching temperature and agents range from 0 °C (ice or ice cold 0.9 % NaCl), to –40/–50 °C (60 % methanol), to almost –200 °C (liquid N 2 ) (Boroujerdi et al. 2012; Dietmair et al. 2010; Lodi and Ronen 2011; Sellick et al. 2009; Ye et al. 2012a); (3) metabolites extraction. This step is the most variable among all the sample preparation procedures. "
[Show abstract][Hide abstract] ABSTRACT: Pre-analytical treatments of bacteria are crucial steps in bacterial metabolomics studies. In order to achieve reliable samples that can best represent the global metabolic profile in vivo both qualitatively and quantitatively, many sample treatment procedures have been developed. The use of different methods makes it difficult to compare the results among different groups. In this work, E. coli samples were tested by using NMR spectroscopy. Both liquid N2 and cold methanol quenching procedures reduce the cell membrane integrity and cause metabolites leakage. However, liquid N2 quenching affected the cell viability and the NMR metabolites’ profile less than cold methanol procedure. Samples obtained by metabolite extraction were significantly superior over cell suspensions and cell lysates, with a higher number of detectable metabolites. Methanol/chloroform extraction proved most efficient at extraction of intracellular metabolites from both qualitative and quantitative points of view. Finally, standard operating procedures of bacterial sample treatments for NMR metabolomics study are presented.
[Show abstract][Hide abstract] ABSTRACT: Nuclear magnetic resonance (NMR) spectroscopy has been used to obtain metabolic profiles of the polar diatom Fragilariopsis cylindrus, leading to the identification of a novel metabolite in this organism. Initial results from an ongoing metabolomics study have led to the discovery of isethionic acid (2-hydroxyethanesulfonic acid, CAS: 107-36-8) as a major metabolite in F. cylindrus. This compound is being produced by the organism under normal culture conditions. This finding is the first report of a diatom producing isethionic acid. In addition to isethionic acid, four other metabolites, dimethylsulfoniopropionate (DMSP), betaine, homarine, and proline were present and may serve as osmoprotectants in F. cylindrus. NMR-based metabolite profiles of F. cylindrus were obtained along a growth curve of the organism. The relative concentration levels of the five metabolites were monitored over a growth period of F. cylindrus from 18 to 25 days. All showed an increase in relative concentration with time, except for proline, which began to decrease after day 21.
[Show abstract][Hide abstract] ABSTRACT: Atlantis II and Discovery are two hydrothermal and hypersaline deep-sea pools in the Red Sea rift that are characterised by strong thermo-halo stratification and temperatures steadily peaking near the bottom. We conducted comprehensive vertical profiling of the microbial populations in both pools and highlighted the influential environmental factors. Pyrosequencing of the 16S ribosomal RNA genes (rRNA genes) revealed shifts in community structures vis-à-vis depth. High diversity and low abundance were the features of the deepest convective layers despite the low cell density. Surprisingly, the brine interfaces were significantly higher in cell count, compared with the overlying deep-sea water, yet they were lowest in diversity. Vertical stratification of the bacterial populations was apparent as we move from the Alphaproteobacteria-dominated deep-sea to the Planctomycetacia or Deferribacteres-dominated interfaces to the Gammaproteobacteria-dominated brine layers. The archaeal marine group I was dominant in the deep-sea water and interfaces; whilst several euryarchaeotic groups increased in the brine. Across sites, microbial phylotypes and abundances varied substantially in the brine interface of Discovery compared with Atlantis II; despite the near-identical populations in the overlaying deep-sea waters. The lowest convective layers harboured interestingly similar microbial communities, even though temperature and heavy metal concentrations were very different. Multivariate analysis indicated that temperature and salinity were the major influences shaping the communities. The harsh conditions and the low-abundance phylotypes could explain the observed correlation in the brine pools.
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