Francisca Diana da Silva Araújo

Direct Protocol for Ambient Mass Spectrometry Imaging on Agar Culture

Herein we describe a new protocol that allows direct mass spectrometry imaging (IMS) of agar cultures. A simple sample dehydration leads to a thin solid agar, which enables the direct use of spray-based ambient mass spectrometry techniques. To demonstrate its applicability, metal scavengers siderophores were imaged directly from agar culture of S. wadayamensis, and well resolved and intense images were obtained using both desorption electrospray ionization (DESI) and easy ambient sonic-spray ionization (EASI) with well-defined selective spatial distributions for the free and the metal-bound molecules, providing clues for their roles in cellular metabolism.

N-Acyl-homoserine Lactones from Enterobacter sakazakii (Cronobacter spp.) and Their Degradation by Bacillus cereus Enzymes

A chemical study of acyl-homoserine lactones (acyl-HSLs) produced by Enterobacter sakazakii resulted in the identification of three molecules: (S)-N-heptanoyl-HSL, (S)-N-dodecanoyl-HSL and (S)-N-tetradecanoyl-HSL. Mixed cultures of E. sakazakii and Bacillus cereus depleted E. sakazakii acyl-HSLs, suggesting acyl-HSL degradation by B. cereus hydrolases (hydrolysis of the lactone or amide moiety). The expression of B. cereus acyl-HSL lactonase and acyl-homoserine acylase was confirmed by monitoring the biotransformation of (S)-N-dodecanoyl-HSL into (S)-N-dodecanoyl-homoserine, dodecanoic acid and homoserine in the presence of B. cereus whole cells, using electrospray-mass spectrometry (ESI-MS).

A Survey of the Peptide Profile in Prato Cheese as Measured by MALDI-MS and Capillary Electrophoresis.

In this study, we describe the characterization of the peptide profile in commercial Prato cheese by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and capillary electrophoresis (CE). Ten commercial Prato cheese brands were characterized via their physicochemical composition and subjected to fractionation according to solubility at pH 4.6. The pH 4.6 insoluble fraction was evaluated by CE, whereas MALDI-MS was applied to the fraction soluble at pH 4.6 and in 70% ethanol. CE revealed a characteristic pattern of hydrolysis, with formation of para-κ-casein, hydrolysis of αs1 -casein at the Phe23 - Phe24 bond, and hydrolysis of β-casein. For the MALDI-MS data, a complex peptide profile was observed, with the identification of 44 peptides previously reported (24 peptides from αs1 -casein, 14 from β-casein, 3 from κ-casein, and 3 from αs2 -casein). It was also observed that cheeses with salt-in-moisture content greater than 5% showed an accumulation of a bitter-tasting peptide (m/z 1536, αs1 -CN f1-13), suggesting a relationship between the higher salt concentration and the abundance of this peptide. In conclusion, the results showed that even commercial cheeses produced with different raw material and processing conditions showed very similar peptide profiles when assessed at the molecular level, and only 9 peptides were responsible for discrimination of cheeses.

Potential of Burkholderia seminalis TC3.4.2R3 as Biocontrol Agent Against Fusarium oxysporum Evaluated by Mass Spectrometry Imaging

AbstractSpecies of genus Burkholderia display different interaction profiles in the environment, causing either several diseases in plants and animals or being beneficial to some plants, promoting their growth, and suppressing phytopathogens. Burkholderia spp. also produce many types of biomolecules with antimicrobial activity, which may be commercially used to protect crops of economic interest, mainly against fungal diseases. Herein we have applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to investigate secondary metabolites produced by B. seminalis TC3.4.2R3 in monoculture and coculture with plant pathogen Fusarium oxysporum. The siderophore pyochelin and the rhamnolipid Rha-Rha-C15-C14 were detected in wild-type B. seminalis strain, and their productions were found to vary in mutant strains carrying disruptions in gene clusters associated with antimicrobial compounds. Two mycotoxins were detected in F. oxysporum. During coculture with B. seminalis, metabolites probably related to defense mechanisms of these microorganisms were observed in the interspecies interaction zone. Our findings demonstrate the effective application of MALDI-MSI in the detection of bioactive molecules involved in the defense mechanism of B. seminalis, and these findings suggest the potential use of this bacterium in the biocontrol of plant diseases caused by F. oxysporum. Graphical Abstractᅟ

Mass spectrometry characterization of endophytic bacterium Curtobacterium sp. strain ER1/6 isolated from Citrus sinensis

The bacteria of the genus Curtobacterium are usually seen as plant pathogen, but some species have been identified as endophytes of different crops and could as such present a potential for disease control and plant growth promotion. We have therefore applied the desorption electrospray ionization mass spectrometry imaging (DESI-MSI) in the direct analysis of living Curtobacterium sp. strain ER1/6 colonies to map the surface metabolites, and electrospray ionization tandem mass spectrometry (ESI-MS/MS) for characterization of these compounds. Several colony-associated metabolites were detected. The ESI-MS/MS showed characteristic fragmentations for phospholipids including the classes of glycerophosphocholine, glycerophosphoglycerol, and glycerophosphoinositol as well as several fatty acids. Although a secure identification was not obtained, many other metabolites were also detected for this bacteria species. Principal component analysis showed that fatty acids were discriminatory for Curtobacterium sp. ER1/6 during inoculation on periwinkle wilt (PW) medium, whereas phospholipids characterize the bacterium when grown on the tryptic soy agar (TSA) medium.

Absolute Configuration of Solenopsis Piperidines is a Tool to Classify Fire Ants (Formicidae:Myrmicinae)

Piperidine alkaloids are predominant in ant venom glands of S. invicta and S. saevissima. These ants are broadly sympatric in Southeastern Brazil, and potentially diagnostic compounds could assist in ant identification. We determined the absolute configuration of 2,6-dialkyl-piperidines of S. invicta and S. saevissima workers and used these data to group Southeastern Brazil species. The monitoring of venom samples by chiral gas chromatography coupled to a flame ionization detector (GC-FID) revealed that S. saevissima produces the four stereoisomers of 2-methyl-6-undecylpiperidines, 2-methyl-6-tridecyl-piperidines and 2-methyl-6-tridecenyl-piperidines, while S. invicta venom consisted primarily of a single enantiomer cis and trans from each piperidine alkaloid. Our findings showed that the absolute configuration of piperidine alkaloids of fire ant venom may be a potential chemical tool to distinguish S. invicta and S. saevissima sympatric species in Southeastern Brazil.

How Important Is the Absolute Configuration to Bacteria Quorum Sensing and Quorum Quenching

Bacteria communicate with each other by producing and detecting small diffusible molecules in a process called quorum sensing (QS). This mechanism allows bacteria to coordinate their activities in response to their populations. For instance, QS regulates diverse phenotypes, such as virulence factors bioluminescence, biofilm formation, and antibiotic production, in various bacteria that survived in plants and mammals, only after reaching critical populations (Waters and Bassler 2005).