A Novel Coenzyme A Analogue in the Anaerobic, Sulfate-Reducing, Marine Bacterium Desulfobacula toluolica Tol2T.

Coenzyme A (CoA) thioesters are formed during anabolic and catabolic reactions in every organism. Degradation pathways of growth-supporting substrates in bacteria can be predicted by differential proteogenomic studies. Direct detection of proposed metabolites such as CoA thioesters by high-performance liquid chromatography coupled with high-resolution mass spectrometry can confirm the reaction sequence and demonstrate the activity of these degradation pathways. In the metabolomes of the anaerobic sulfate-reducing bacterium Desulfobacula toluolica Tol2T grown with different substrates various CoA thioesters, derived from amino acid, fatty acid or alcohol metabolism, have been detected. Additionally, the cell extracts of this bacterium revealed a number of CoA analogues with molecular masses increased by 1 dalton. By comparing the chromatographic and mass spectrometric properties of synthetic reference standards with those of compounds detected in cell extracts of D. toluolica Tol2T and by performing co-injection experiments, these analogues were identified as inosino-CoAs. These CoA thioesters contain inosine instead of adenosine as the nucleoside. To the best of our knowledge, this finding represents the first detection of naturally occurring inosino-CoA analogues.

Suspect screening and targeted analysis of acyl coenzyme A thioesters in bacterial cultures using a high-resolution tribrid mass spectrometer.

Analysis of acyl coenzyme A thioesters (acyl-CoAs) is crucial in the investigation of a wide range of biochemical reactions and paves the way to fully understand the concerned metabolic pathways and their superimposed networks. We developed two methods for suspect screening of acyl-CoAs in bacterial cultures using a high-resolution Orbitrap Fusion tribrid mass spectrometer. The methods rely on specific fragmentation patterns of the target compounds, which originate from the coenzyme A moiety. They make use of the formation of the adenosine 3',5'-diphosphate key fragment (m/z 428.0365) and the neutral loss of the adenosine 3'-phosphate-5'-diphosphate moiety (506.9952) as preselection criteria for the detection of acyl-CoAs. These characteristic ions are generated either by an optimised in-source fragmentation in a full scan Orbitrap measurement or by optimised HCD fragmentation. Additionally, five different filters are included in the design of method. Finally, data-dependent MS/MS experiments on specifically preselected precursor ions are performed. The utility of the methods is demonstrated by analysing cultures of the denitrifying betaproteobacterium "Aromatoleum" sp. strain HxN1 anaerobically grown with hexanoate. We detected 35 acyl-CoAs in total and identified 24 of them by comparison with reference standards, including all 9 acyl-CoA intermediates expected to occur in the degradation pathway of hexanoate. The identification of additional acyl-CoAs provides insight into further metabolic processes occurring in this bacterium. The sensitivity of the method described allows detecting acyl-CoAs present in biological samples in highly variable abundances. Graphical abstract.

Ontogenetic resource utilization and migration reconstruction with δ13C values of essential amino acids in the Cynoscion acoupa otolith.

With the increasing anthropogenic impacts on fish habitats, it has become more important to understand which primary resources sustain fish populations. This resource utilization can differ between fish life stages, and individuals can migrate between habitats in search of resources. Such lifetime information is difficult to obtain due to the large spatial and temporal scales of fish behavior. The otolith organic matrix has the potential to indicate this resource utilization and migration with δ13C values of essential amino acids (EAAs), which are a direct indication of the primary producers. In a proof-of-concept study, we selected the Acoupa weakfish, Cynoscion acoupa, as a model fish species with distinct ontogenetic migration patterns. While it inhabits the Brazilian mangrove estuaries during juvenile stages, it moves to the coastal shelf as an adult. Thus, we expected that lifetime resource utilization and migration would be reflected in δ13CEAA patterns and baseline values in C. acoupa otoliths. By analyzing the C. acoupa otolith edges across a size range of 12-119 cm, we found that baseline δ13CEAA values increased with size, which indicated an estuarine to coastal shelf distribution. This trend is highly correlated with inorganic δ13C values. The δ13CEAA patterns showed that estuarine algae rather than mangrove-derived resources supported the juvenile C. acoupa populations. Around the juvenile size of 40 cm, resource utilization overlapped with those of adults and mean baseline δ13CEAA values increased. This trend was confirmed by comparing otolith core and edges, although with some individuals potentially migrating over longer distances than others. Hence, δ13CEAA patterns and baseline values in otoliths have great potential to reconstruct ontogenetic shifts in resource use and habitats. The insight could aid in predictions on how environmental changes affect fish populations by identifying the controlling factors at the base of the food web.