Calcium Determines Lactiplantibacillus plantarum Intraspecies Competitive Fitness.

The importance of individual nutrients for microbial strain robustness and coexistence in habitats containing different members of the same species is not well understood. To address this for Lactiplantibacillus plantarum in food fermentations, we performed comparative genomics and examined the nutritive requirements and competitive fitness for L. plantarum strains B1.1 and B1.3 isolated from a single sample of teff injera fermentation batter. Compared to B1.1 and other L. plantarum strains, B1.3 has a smaller genome, limited biosynthetic capacities, and large mobilome. Despite these differences, B1.3 was equally competitive with B1.1 in a suspension of teff flour. In commercially sourced, nutrient-replete MRS (cMRS) medium, strain B1.3 reached 3-fold-higher numbers than B1.1 within 2 days of passage. Because B1.3 growth and competitive fitness were poor in mMRS medium (here called mMRS), a modified MRS medium lacking beef extract, we used mMRS to identify nutrients needed for robust B1.3 growth. No improvement was observed when mMRS was supplemented with nucleotides, amino acids, vitamins, or monovalent metals. Remarkably, the addition of divalent metal salts increased the growth rate and cell yields of B1.3 in mMRS. Metal requirements were confirmed by inductively coupled plasma mass spectrometry, showing that total B1.3 intracellular metal concentrations were significantly (up to 2.7-fold) reduced compared to B1.1. Supplemental CaCl2 conferred the greatest effect, resulting in equal growth between B1.1 and B1.3 over five successive passages in mMRS. Moreover, calcium supplementation reversed a B1.3 strain-specific, stationary-phase, flocculation phenotype. These findings show how L. plantarum calcium requirements affect competitive fitness at the strain level. IMPORTANCE Ecological theory states that the struggle for existence is stronger between closely related species. Contrary to this assertion, fermented foods frequently sustain conspecific individuals, in spite of their high levels of phylogenetic relatedness. Therefore, we investigated two isolates of Lactiplantibacillus plantarum, B1.1 and B1.3, randomly selected from a single batch of teff injera batter. These strains spanned the known genomic and phenotypic range of the L. plantarum species, and in laboratory culture medium used for strain screening, B1.3 exhibited poor growth and was outcompeted by the more robust strain B1.1. Nonetheless, B1.1 and B1.3 were equally competitive in teff flour. This result shows how L. plantarum has adapted for coexistence in that environment. The capacity for the single macronutrient calcium to restore B1.3 competitive fitness in laboratory culture medium suggests that L. plantarum intraspecies diversity found in food systems is fine-tuned to nutrient requirements at the strain level.

Metabolism of 4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone by Retinal Pigmented Epithelial Cells.

4-Hydroxy-7-oxo-5-heptenic acid (HOHA)-lactone is a biologically active oxidative truncation product released (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation from docosahexaenoate lipids. We now report that HOHA-lactone readily diffuses into retinal pigmented epithelial (RPE) cells where it is metabolized. A reduced glutathione (GSH) Michael adduct of HOHA-lactone is the most prominent metabolite detected by LC-MS in both the extracellular medium and cell lysates. This molecule appeared inside of ARPE-19 cells within seconds after exposure to HOHA-lactone. The intracellular level reached a maximum concentration at 30 min and then decreased with concomitant increases in its level in the extracellular medium, thus revealing a unidirectional export of the reduced GSH-HOHA-lactone adduct from the cytosol to extracellular medium. This metabolism is likely to modulate the involvement of HOHA-lactone in the pathogenesis of human diseases. HOHA-lactone is biologically active, e.g., low concentrations (0.1-1 µM) induce secretion of vascular endothelial growth factor (VEGF) from ARPE-19 cells. HOHA-lactone is also a precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives of primary amino groups in proteins and ethanolamine phospholipids that have significant pathological and physiological relevance to age-related macular degeneration (AMD), cancer, and wound healing. Both HOHA-lactone and the derived CEP can contribute to the angiogenesis that defines the neovascular "wet" form of AMD and that promotes the growth of tumors. While GSH depletion can increase the lethality of radiotherapy, because it will impair the metabolism of HOHA-lactone, the present study suggests that GSH depletion will also increase levels of HOHA-lactone and CEP that may promote recurrence of tumor growth.

4-Hydroxy-7-oxo-5-heptenoic Acid Lactone Induces Angiogenesis through Several Different Molecular Pathways.

Oxidative stress and angiogenesis have been implicated not only in normal phenomena such as tissue healing and remodeling but also in many pathological processes. However, the relationships between oxidative stress and angiogenesis still remain unclear, although oxidative stress has been convincingly demonstrated to influence the progression of angiogenesis under physiological and pathological conditions. The retina is particularly susceptible to oxidative stress because of its intensive oxygenation and high abundance of polyunsaturated fatty acyls. In particular, it has high levels of docosahexanoates, whose oxidative fragmentation produces 4-hydroxy-7-oxo-5-heptenoic acid lactone (HOHA-lactone). Previously, we found that HOHA-lactone is a major precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives, which are tightly linked to age-related macular degeneration (AMD). CEPs promote the pathological angiogenesis of late-stage AMD. We now report additional mechanisms by which HOHA-lactone promotes angiogenesis. Using cultured ARPE-19 cells, we observed that HOHA-lactone induces secretion of vascular endothelial growth factor (VEGF), which is correlated to increases in reactive oxygen species and decreases in intracellular glutathione (GSH). Wound healing and tube formation assays provided, for the first time, in vitro evidence that HOHA-lactone induces the release of VEGF from ARPE-19 cells, which promotes angiogenesis by human umbilical vein endothelial cells (HUVEC) in culture. Thus, HOHA-lactone can stimulate vascular growth through a VEGF-dependent pathway. In addition, results from MTT and wound healing assays as well as tube formation experiments showed that GSH-conjugated metabolites of HOHA-lactone stimulate HUVEC proliferation and promote angiogenesis in vitro. Previous studies demonstrated that HOHA-lactone, through its CEP derivatives, promotes angiogenesis in a novel Toll-like receptor 2-dependent manner that is independent of the VEGF receptor or VEGF expression. The new studies show that HOHA-lactone also participates in other angiogenic signaling pathways that include promoting the secretion of VEGF from retinal pigmented epithelial cells.

Strain diversity of plant-associated Lactiplantibacillus plantarum.

Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) is a lactic acid bacteria species found on plants that is essential for many plant food fermentations. In this study, we investigated the intraspecific phenotypic and genetic diversity of 13 L. plantarum strains isolated from different plant foods, including fermented olives and tomatoes, cactus fruit, teff injera, wheat boza and wheat sourdough starter. We found that strains from the same or similar plant food types frequently exhibited similar carbohydrate metabolism and stress tolerance responses. The isolates from acidic, brine-containing ferments (olives and tomatoes) were more resistant to MRS adjusted to pH 3.5 or containing 4% w/v NaCl, than those recovered from grain fermentations. Strains from fermented olives grew robustly on raffinose as the sole carbon source and were better able to grow in the presence of ethanol (8% v/v or sequential exposure of 8% (v/v) and then 12% (v/v) ethanol) than most isolates from other plant types and the reference strain NCIMB8826R. Cell free culture supernatants from the olive-associated strains were also more effective at inhibiting growth of an olive spoilage strain of Saccharomyces cerevisiae. Multi-locus sequence typing and comparative genomics indicated that isolates from the same source tended to be genetically related. However, despite these similarities, other traits were highly variable between strains from the same plant source, including the capacity for biofilm formation and survival at pH 2 or 50°C. Genomic comparisons were unable to resolve strain differences, with the exception of the most phenotypically impaired and robust isolates, highlighting the importance of utilizing phenotypic studies to investigate differences between strains of L. plantarum. The findings show that L. plantarum is adapted for growth on specific plants or plant food types, but that intraspecific variation may be important for ecological fitness and strain coexistence within individual habitats.

Abundance, diversity and plant-specific adaptations of plant-associated lactic acid bacteria.

Lactic acid bacteria (LAB) are essential for many fruit, vegetable and grain food and beverage fermentations. However, the numbers, diversity and plant-specific adaptions of LAB found on plant tissues prior to the start of those fermentations are not well understood. When measured, these bacteria have been recovered from the aerial surfaces of plants in a range from <10 CFU g-1 to over 108.5 CFU g-1 of plant tissue and in lower quantities from the soil and rhizosphere. Plant-associated LAB include well-known generalist taxa such as Lactobacillus plantarum and Leuconostoc mesenteroides, which are essential for numerous food and beverage fermentations. Other plant-associated LAB encompass specialist taxa such as Lactobacillus florum and Fructobacillus, many of which were discovered relatively recently and their significance on plants and in foods is not yet recognized. LAB recovered from plants possess the capacity to consume plant sugars, detoxify phenolic compounds and tolerate the numerous biotic and abiotic stresses common to plant surfaces. Although most generalist and some specialist LAB grow rapidly in food and beverages fermentations and can cause spoilage of fresh and fermented fruits and vegetables, the importance of living plants as habitats for these bacteria and LAB contributions to plant microbiomes remain to be shown.

Genome Sequence of Lactiplantibacillus plantarum ATCC 202195, a Probiotic Strain That Reduces Sepsis and Other Infections during Early Infancy.

The draft genome of probiotic Lactiplantibacillus plantarum ATCC 202195, with the trademark PPLP-217, is comprised of 3,368,305 bp with a G+C content of 44.3% and no plasmids. The strain is able to grow on lactose, raffinose, and fructooligosaccharides.

4-Hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone induces apoptosis in retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cell dysfunction and death play vital roles in age-related macular degeneration (AMD) pathogenesis. Previously we showed that oxidative cleavage of docosahexenoate (DHA) phospholipids generates an α,ß-unsaturated aldehyde, 4-hydroxy-7-oxohept-4-enoic acid (HOHA) lactone, that forms ω-carboxyethylpyrrole (CEP) derivatives through adduction to proteins and ethanolamine phospholipids. CEP derivatives and autoantibodies accumulate in the retinas and blood plasma of individuals with AMD and are a biomarker of AMD. They promote the choroidal neovascularization of "wet AMD". Immunization of mice with CEP-modified mouse serum albumin induces "dry AMD"-like lesions in their retinas as well as interferon-gamma and interleukin-17 production by CEP-specific T cells that promote inflammatory M1 polarization of macrophages. The present study confirms that oxidative stress or inflammatory stimulus produces CEP in both the primary human ARPE-19 cell line and hRPE cells. Exposure of these cells to HOHA lactone fosters production of reactive oxygen species. Thus, HOHA lactone participates in a vicious cycle, promoting intracellular oxidative stress leading to oxidative cleavage of DHA to produce more HOHA lactone. We now show that HOHA lactone is cytotoxic, inducing apoptotic cell death through activation of the intrinsic pathway. This suggests that therapeutic interventions targeting HOHA lactone-induced apoptosis may prevent the loss of RPE cells during the early phase of AMD. We also discovered that ARPE-19 cells are more susceptible than hRPE cells to HOHA lactone cytotoxicity. This is consistent with the view that, compared to normal RPE cells, ARPE-19 cells exhibit a diseased RPE phenotype that also includes elevated expression of the mesenchymal indicator vimentin, elevated integrin a5 promotor strength and deficient secretion of the anti-VEGF molecule pigment-epithelium-derived factor fostering weaker tight junctions.

The Lactococcus lactis KF147 nonribosomal peptide synthetase/polyketide synthase system confers resistance to oxidative stress during growth on plant leaf tissue lysate.

Strains of Lactococcus lactis isolated from plant tissues possess adaptations that support their survival and growth in plant-associated microbial habitats. We previously demonstrated that genes coding for a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) system involved in production of an uncharacterized secondary metabolite are specifically induced in L. lactis KF147 during growth on plant tissues. Notably, this NRPS/PKS has only been identified in plant-isolated strains of L. lactis. Here, we show that the L. lactis KF147 NRPS/PKS genes have homologs in certain Streptococcus mutans isolates and the genetic organization of the NRPS/PKS locus is conserved among L. lactis strains. Using an L. lactis KF147 mutant deficient in synthesis of NrpC, a 4'-phosphopantetheinyl transferase, we found that the NRPS/PKS system improves L. lactis during growth under oxidative conditions in Arapidopsis thaliana leaf lysate. The NRPS/PKS system also improves tolerance of L. lactis to reactive oxygen species and specifically H2 O2 and superoxide radicals in culture medium. These findings indicate that this secondary metabolite provides a novel mechanism for reactive oxygen species detoxification not previously known for this species.