Preparation of Hard Palm Seeds for Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry Analysis.

Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is applied to identify compounds in their native environments. Currently, MALDI-IMS is frequently used in clinical analysis. Still, an excellent perspective exists for better applying this technique to understand chemical compounds' physiological information in plant tissues. However, preparation may be challenging for specific samples from botanical materials, as MALDI-IMS requires thin slices (12-20 µm) for appropriate data acquisition and successful analysis. In this sense, previously, we developed a sample preparation protocol to obtain thin sections of Euterpe oleracea (açaí palm) hard seeds, enabling their molecular mapping by MALDI-IMS. Here, we show that the developed protocol is suitable for preparing other seeds from the same genus. Briefly, the protocol was based on submerging the seeds in deionized water for 24 h, embedding samples with gelatin, and sectioning them in an acclimatized cryostat. Then, for matrix deposition, an xy motion platform was coupled to an electrospray ionization (ESI) needle spray using a 1:1 (v/v) 2,5-dihydroxybenzoic acid (DHB) and methanol solution with 0.1% trifluoroacetic acid at 30 mg/mL. E. precatoria and E. edulis seed data were processed using software to map their metabolite patterns. Hexose oligomers were mapped within sample slices to prove the adequacy of the protocol for those samples, as it is known that those seeds contain large amounts of mannan, a polymer of the hexose mannose. As a result, peaks of hexose oligomers, represented by [M + K]+ adducts of (Δ = 162 Da), were identified. Thus, the sample preparation protocol, previously developed tailor-made for E. oleracea seeds, also enabled MALDI-IMS analysis of two other hard palm seeds. In short, the method could constitute a valuable tool for research in the morpho-anatomy and physiology of botanical materials, especially from cut-resistant samples.

Accumulation of ornithine lipids in Vibrio cholerae under phosphate deprivation is dependent on VC0489 (OlsF) and PhoBR system.

Ornithine lipids (OLs) are phosphorus-free lipids found in many bacteria grown under phosphate deprivation, a condition that activates the PhoBR system and leads to phosphate uptake and metabolism. Two OL synthesis pathways have already been described. One depends on OlsB and OlsA acyltransferases to add, respectively, the first and second acyl chains to an ornithine molecule. The other pathway is carried out by OlsF, a bifunctional enzyme responsible for both acylation steps. Although Vibrio cholerae lacks olsBA genes, an olsF homologue (vc0489) was identified in its genome. In this work we demonstrated that V. cholerae produces OLs and expresses vc0489 in response to phosphate depletion, in a PhoBR-dependent manner. In Escherichia coli, under similar condition, vc0489 expression leads to OL accumulation. These results indicate a strong connection between OL synthesis and VC0489 from V. cholerae and, for the first time, a direct regulation of an olsF homologue by the PhoBR system.

Transcriptional and post-transcriptional regulation of pst2 operon expression in Vibrio cholerae O1.

One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions that are known to block 3'-5' exonucleolytic attacks.

Catalases and PhoB/PhoR system independently contribute to oxidative stress resistance in Vibrio cholerae O1.

All cells are subjected to oxidative stress, a condition under which reactive oxygen species (ROS) production exceeds elimination. Bacterial defences against ROS include synthesis of antioxidant enzymes like peroxidases and catalases. Vibrio cholerae can produce two distinct catalases, KatB and KatG, which contribute to ROS homeostasis. In this study, we analysed the mechanism behind katG and katB expression in two V. cholerae O1 pandemic strains, O395 and N16961, of classical and El Tor biotypes, respectively. Both strains express these genes, especially at stationary phase. However, El Tor N16961 produces higher KatB and KatG levels and is much more resistant to peroxide challenge than the classical strain, confirming a direct relationship between catalase activity and oxidative stress resistance. Moreover, we showed that katG and katB expression levels depend on inorganic phosphate (Pi) availability, in contrast to other bacterial species. In N16961, katB and katG expression is reduced under Pi limitation relative to Pi abundance. Total catalase activity in N16961 and its phoB mutant cells was similar, independently of growth conditions, indicating that the PhoB/PhoR system is not required for katB and katG expression. However, N16961 cells from Pi-limited cultures were 50-100-fold more resistant to H2O2 challenge and accumulated less ROS than phoB mutant cells. Together, these findings suggest that, besides KatB and KatG, the PhoB/PhoR system is an important protective factor against ROS in V. cholerae N16961. They also corroborate previous results from our and other groups, suggesting that the PhoB/PhoR system is fundamental for V. cholerae biology.