Insertion sequence IS6770 modulates potassium symporter kup transcription in Enterococcus faecalis JH2-2 under low pH conditions.

Enterococcus faecalis is a phylogenetically and industrially relevant microorganism associated with Lactic Acid Bacteria. Some strains of this bacterium are employed as probiotics in commercial applications, while others serve as the principal component in starter cultures for artisanal regional cheese production. However, over the last decade, this species has emerged as an opportunistic multiresistant pathogen, raising concerns about its impact on human health. Recently, we identified multiple potassium transporter systems in E. faecalis, including the Ktr systems (KtrAB and KtrAD), Kup, KimA and Kdp complex (KdpFABC). Nevertheless, the physiological significance of these proteins remains not fully understood. In this study, we observed that the kup gene promoter region in the JH2-2 strain was modified due to the insertion of a complete copy of the IS6770 insertion sequence. Consequently, we investigated the influence of IS6770 on the expression of the kup gene. To achieve this, we conducted a mapping of the promoter region of this gene in the E. faecalis JH2-2 strain, employing fluorescence gene reporters. In addition, a transcriptional analysis of the kup gene was executed in a strain derived from E. faecalis V583 that lacks the IS30-related insertion element, facilitating the identification of the transcriptional start site. Next, the expression of the kup gene was evaluated via RT-qPCR under different pH stressful conditions. A strong upregulation of the kup gene was observed at an initial pH of 5.0 in the strain derived from E. faecalis V583. However, the activation of transcription was not observed in the E. faecalis JH2-2 strain due to the hindrance caused by the presence of IS6770. Besides that, our computational analysis of E. faecalis genomes elucidates a plausible association between transposition and the regulation of the kup gene. Remarkably, the ubiquitous presence of IS6770 throughout the phylogenetic tree implies its ancient existence within E. faecalis. Moreover, the recurrent co-occurrence of IS6770 with the kup gene, observed in 30 % of IS6770-positive strains, alludes to the potential involvement of this genomic arrangement in the adaptive strategies of E. faecalis across diverse niches.

Study of volatile compounds profiles in milk matrices using Enterococcus faecalis EstA and Rhizomucor miehei lipase.

The use of esterase/lipase enzymes of different origins in food industry is a widely employed strategy to enhance the formation of characteristic aromatic compounds derived from fat and diversify flavour. In the present work, we studied EstA enzyme of Enterococcus faecalis and a high purity Rhizomucor miehei lipase (Palatase). EstA was obtained recombinantly in Escherichia coli BL21 (DE3), and optimum esterase activity was detected at pH 6.75 and 40 °C. We evaluated the effect of the enzymes on milk mixtures prepared with different fat contents (2.8 and 6%) and structure (native or homogenized) on volatile compounds profiles. The milk fat structure before and after the application of low homogenization was characterized by dynamic light dispersion and microscopy. Native milk fat mixtures presented particles of 4.6 µm and 184 nm and homogenized mixtures had particles of 1.4 µm and 258 nm; microscopy images were in concordance with these results. Fifteen volatile compounds were identified, including ketones, esters, alcohols, and acids. We showed the key role of milk fat levels and microstructure in the nature of the volatile compounds produced by the R. miehei enzyme. Both in native or homogenized states, the highest content of fat favored a higher production of acids whereas the lowest fat level favored a higher esters production along with a more balanced volatile profile. For EstA enzyme, results showed a limited action on fat, as biosynthesis of esters only increased with the highest fat level homogenized.

Redundant potassium transporter systems guarantee the survival of Enterococcus faecalis under stress conditions.

Enterococcus is able to grow in media at pH from 5.0 to 9.0 and a high concentration of NaCl (8%). The ability to respond to these extreme conditions requires the rapid movement of three critical ions: proton (H+), sodium (Na+), and potassium (K+). The activity of the proton F0F1 ATPase and the sodium Na+ V0V1 type ATPase under acidic or alkaline conditions, respectively, is well established in these microorganisms. The potassium uptake transporters KtrI and KtrII were described in Enterococcus hirae, which were associated with growth in acidic and alkaline conditions, respectively. In Enterococcus faecalis, the presence of the Kdp (potassium ATPase) system was early established. However, the homeostasis of potassium in this microorganism is not completely explored. In this study, we demonstrate that Kup and KimA are high-affinity potassium transporters, and the inactivation of these genes in E. faecalis JH2-2 (a Kdp laboratory natural deficient strain) had no effect on the growth parameters. However, in KtrA defective strains (ΔktrA, ΔkupΔktrA) an impaired growth was observed under stress conditions, which was restored to wild type levels by external addition of K+ ions. Among the multiplicity of potassium transporters identify in the genus Enterococcus, Ktr channels (KtrAB and KtrAD), and Kup family symporters (Kup and KimA) are present and may contribute to the particular resistance of these microorganisms to different stress conditions. In addition, we found that the presence of the Kdp system in E. faecalis is strain-dependent, and this transporter is enriched in strains of clinical origin as compared to environmental, commensal, or food isolates.

Effects of Cholesterol Extraction Process and Fat and Whey Protein Additions on Ice Cream Mixes.

The aim of this work was to evaluate the cholesterol extraction process in ice cream mixes (ICMs) by using ß-cyclodextrin (ßCD) and to analyze the effect of this extraction on the ICM rheological, stability, and sensory characteristics. The effects of fat and whey protein (WP) additions on ICM stability were also evaluated. The maximum percentage obtained for cholesterol extraction was 93.6%. The flow curves indicated that ICM showed a thixotropic behavior before and after cholesterol extraction, which was enhanced when the fat content and/or percentage of ßCD increased. The stability of ICM with cholesterol-reduced content (RCho-ICM) was influenced by the fat content and/or the presence of WP. The RCho-ICM with the highest fat and/or WP addition showed less tendency to melt and had the smallest amount of accumulated molten liquid. These latter ICMs presented the slowest melting rates. Also, RCho-ICMs proved to be more stable than ICMs. RCho-ICM samples obtained with a ratio of ßCD/fat content of 1% w/w were evaluated by a trained sensory panel. In addition, an acceptability test of the sample with better sensory attributes was conducted. PRACTICAL APPLICATION: The effects of a cholesterol extraction process and fat and whey protein additions on the rheological and stability characteristics of ice cream mixes were evaluated. The extraction of cholesterol from an ice cream mix is interesting from a nutritional point of view and the extraction process of cholesterol itself may also help to improve the mix stability by controlling the fat and/or whey protein contents. These findings may prove useful as a starting point for the rational design of new functional ice cream mixes.