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471300.9430Characterization and Preliminary Safety Evaluation of Akkermansia muciniphila PROBIO. In addition to providing certain health advantages to the host, a bacterial strain must possess a clearly defined safety profile to be regarded as a probiotic. In this study, we present a thorough and methodical assessment of the safety of a novel strain of bacteria, Akkermansia muciniphila PROBIO, which was isolated from human feces. Firstly, we examined the strain's overall features, such as its gastrointestinal tolerance and its physiological and biochemical traits. Next, we verified its genotoxic properties through bacterial reverse mutation and in vitro mammalian cell micronucleus assays. The drug sensitivity of A. muciniphila PROBIO was subsequently examined through an analysis of its antibiotic resistance genes. Additionally, the toxicological impact was verified through acute and sub-chronic toxicity studies. A genome-based safety assessment was conducted to gain further insights into gene function, including potential virulence factors and pathogenic properties. Finally, we assessed whether moxifloxacin resistance in A. muciniphila PROBIO is transferred using in vitro conjugation experiments. A. muciniphila PROBIO exhibited superior gastrointestinal tolerance, with no observed hematological or histopathological abnormalities. Moreover, the outcomes pertaining to mutagenic, clastogenic, or toxic impacts were found to be negative, even at exceedingly high dosages. Moreover, no adverse effects associated with the test substance were observed during the examination of acute and sub-chronic toxicity. Consequently, it was plausible to estimate the no-observed-adverse-effect level (NOAEL) to be 6.4 × 10(11) viable bacteria for an average individual weighing 70 kg. Additionally, only three potential drug resistance genes and one virulence factor gene were annotated. A. muciniphila PROBIO is naturally resistant to moxifloxacin, and resistance does not transfer. Collectively, the data presented herein substantiate the presumed safety of A. muciniphila PROBIO for its application in food.202438338577
908510.9419Mode of action and resistance studies unveil new roles for tropodithietic acid as an anticancer agent and the γ-glutamyl cycle as a proton sink. While we have come to appreciate the architectural complexity of microbially synthesized secondary metabolites, far less attention has been paid to linking their structural features with possible modes of action. This is certainly the case with tropodithietic acid (TDA), a broad-spectrum antibiotic generated by marine bacteria that engage in dynamic symbioses with microscopic algae. TDA promotes algal health by killing unwanted marine pathogens; however, its mode of action (MoA) and significance for the survival of an algal-bacterial miniecosystem remains unknown. Using cytological profiling, we herein determine the MoA of TDA and surprisingly find that it acts by a mechanism similar to polyether antibiotics, which are structurally highly divergent. We show that like polyether drugs, TDA collapses the proton motive force by a proton antiport mechanism, in which extracellular protons are exchanged for cytoplasmic cations. The α-carboxy-tropone substructure is ideal for this purpose as the proton can be carried on the carboxyl group, whereas the basicity of the tropylium ion facilitates cation export. Based on similarities to polyether anticancer agents we have further examined TDA's cytotoxicity and find it to exhibit potent, broad-spectrum anticancer activities. These results highlight the power of MoA-profiling technologies in repurposing old drugs for new targets. In addition, we identify an operon that confers TDA resistance to the producing marine bacteria. Bioinformatic and biochemical analyses of these genes lead to a previously unknown metabolic link between TDA/acid resistance and the γ-glutamyl cycle. The implications of this resistance mechanism in the context of the algal-bacterial symbiosis are discussed.201626802120
604620.9417Safety Evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI. Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, Bifidobacterium and Lactobacillus species are probiotic strains that are most commonly commercially produced and actively studied. Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of B. bifidum BGN4 and B. longum BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). B. bifidum BGN4 and B. longum BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). B. bifidum BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of B. bifidum BGN4 was not transferred via conjugation with L. acidophilus ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of B. bifidum BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of B. bifidum BGN4. Tetracycline-resistant genes are prevalent among B. longum strains; B. longum BORI has a tet(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with L. fermentum AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of B. bifidum BGN4 and B. longum BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.201829747442
11630.9406The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor. The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpP(ADEP)) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpP(ADEP) in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpP(ADEP)) as an ADEP resistance gene. ClpP(ADEP) constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.201931399403
843940.9403Comparative genomics analysis and virulence-related factors in novel Aliarcobacter faecis and Aliarcobacter lanthieri species identified as potential opportunistic pathogens. BACKGROUND: Emerging pathogenic bacteria are an increasing threat to public health. Two recently described species of the genus Aliarcobacter, A. faecis and A. lanthieri, isolated from human or livestock feces, are closely related to Aliarcobacter zoonotic pathogens (A. cryaerophilus, A. skirrowii, and A. butzleri). In this study, comparative genomics analysis was carried out to examine the virulence-related, including virulence, antibiotic, and toxin (VAT) factors in the reference strains of A. faecis and A. lanthieri that may enable them to become potentially opportunistic zoonotic pathogens. RESULTS: Our results showed that the genomes of the reference strains of both species have flagella genes (flaA, flaB, flgG, flhA, flhB, fliI, fliP, motA and cheY1) as motility and export apparatus, as well as genes encoding the Twin-arginine translocation (Tat) (tatA, tatB and tatC), type II (pulE and pulF) and III (fliF, fliN and ylqH) secretory pathways, allowing them to secrete proteins into the periplasm and host cells. Invasion and immune evasion genes (ciaB, iamA, mviN, pldA, irgA and fur2) are found in both species, while adherence genes (cadF and cj1349) are only found in A. lanthieri. Acid (clpB), heat (clpA and clpB), osmotic (mviN), and low-iron (irgA and fur2) stress resistance genes were observed in both species, although urease genes were not found in them. In addition, arcB, gyrA and gyrB were found in both species, mutations of which may mediate the resistance to quaternary ammonium compounds (QACs). Furthermore, 11 VAT genes including six virulence (cadF, ciaB, irgA, mviN, pldA, and tlyA), two antibiotic resistance [tet(O) and tet(W)] and three cytolethal distending toxin (cdtA, cdtB, and cdtC) genes were validated with the PCR assays. A. lanthieri tested positive for all 11 VAT genes. By contrast, A. faecis showed positive for ten genes except for cdtB because no PCR assay for this gene was available for this species. CONCLUSIONS: The identification of the virulence, antibiotic-resistance, and toxin genes in the genomes of A. faecis and A. lanthieri reference strains through comparative genomics analysis and PCR assays highlighted the potential zoonotic pathogenicity of these two species. However, it is necessary to extend this study to include more clinical and environmental strains to explore inter-species and strain-level genetic variations in virulence-related genes and assess their potential to be opportunistic pathogens for animals and humans.202235761183
602050.9398Safety evaluation of Lactococcus lactis IDCC 2301 isolated from homemade cheese. For applications of microorganisms as probiotics in the food industry, safety evaluation has increasingly become important to ensure the health of consumers. Although people have been using various lactic acid bacteria for different purposes, some studies have reported that certain lactic acid bacteria exhibit properties of virulence and produce toxic compounds. Thus, it is necessary to examine the characteristics associated with lactic acid bacteria that are safe for use as probiotics. This research aimed to assess the safety of Lactococcus lactis IDCC 2301 isolated from homemade cheese using in vitro and in vivo assays, including antibiotic resistance, hemolytic activity, toxin production, infectivity, and metabolic activity in immune-compromised animal species. The results demonstrated that the strain was susceptible to nine antibiotics suggested by the European Food Safety Authority (EFSA). Whole-genome analysis revealed that L. lactis IDCC 2301 neither has toxigenic genes nor harbors antibiotic resistance. Moreover, L. lactis IDCC 2301 showed neither hemolytic nor β-glucuronidase activity. Furthermore, none of the D-lactate and biogenic amines were produced by L. lactis IDCC 2301. Finally, it was demonstrated that there was no toxicity and mortality using single-dose oral toxicity tests in rats. These results indicate that L. lactis IDCC 2301 can be safely used as probiotics for human consumption.202235035910
51960.9397The Ruegeria pomeroyi acuI gene has a role in DMSP catabolism and resembles yhdH of E. coli and other bacteria in conferring resistance to acrylate. The Escherichia coli YhdH polypeptide is in the MDR012 sub-group of medium chain reductase/dehydrogenases, but its biological function was unknown and no phenotypes of YhdH(-) mutants had been described. We found that an E. coli strain with an insertional mutation in yhdH was hyper-sensitive to inhibitory effects of acrylate, and, to a lesser extent, to those of 3-hydroxypropionate. Close homologues of YhdH occur in many Bacterial taxa and at least two animals. The acrylate sensitivity of YhdH(-) mutants was corrected by the corresponding, cloned homologues from several bacteria. One such homologue is acuI, which has a role in acrylate degradation in marine bacteria that catabolise dimethylsulfoniopropionate (DMSP) an abundant anti-stress compound made by marine phytoplankton. The acuI genes of such bacteria are often linked to ddd genes that encode enzymes that cleave DMSP into acrylate plus dimethyl sulfide (DMS), even though these are in different polypeptide families, in unrelated bacteria. Furthermore, most strains of Roseobacters, a clade of abundant marine bacteria, cleave DMSP into acrylate plus DMS, and can also demethylate it, using DMSP demethylase. In most Roseobacters, the corresponding gene, dmdA, lies immediately upstream of acuI and in the model Roseobacter strain Ruegeria pomeroyi DSS-3, dmdA-acuI were co-regulated in response to the co-inducer, acrylate. These observations, together with findings by others that AcuI has acryloyl-CoA reductase activity, lead us to suggest that YdhH/AcuI enzymes protect cells against damaging effects of intracellular acryloyl-CoA, formed endogenously, and/or via catabolising exogenous acrylate. To provide "added protection" for bacteria that form acrylate from DMSP, acuI was recruited into clusters of genes involved in this conversion and, in the case of acuI and dmdA in the Roseobacters, their co-expression may underpin an interaction between the two routes of DMSP catabolism, whereby the acrylate product of DMSP lyases is a co-inducer for the demethylation pathway.201222563425
846970.9395Probiogenomic analysis of Lactiplantibacillus plantarum SPS109: A potential GABA-producing and cholesterol-lowering probiotic strain. Lactiplantibacillus plantarum SPS109, an isolated strain of lactic acid bacteria (LAB) from fermented foods, showed remarkable potential as a probiotic with dual capabilities in γ-aminobutyric acid (GABA) production and cholesterol reduction. This study employs genomic and comparative analyses to search into the strain's genetic profile, safety features, and probiotic attributes. The safety assessment reveals the absence of virulence factors and antimicrobial resistance genes, while the genome uncovers bacteriocin-related elements, including sactipeptides and a cluster for putative plantaricins, strengthening its ability to combat diverse pathogens. Pangenome analysis revealed unique bacteriocin-related genes, specifically lcnD and bcrA, distinguishing SPS109 from four other L. plantarum strains producing GABA. In addition, genomic study emphasizes SPS109 strain distinctive features, two GABA-related genes responsible for GABA production and a bile tolerance gene (cbh) crucial for cholesterol reduction. Additionally, the analysis highlights several genes of potential probiotic properties, including stress tolerance, vitamin production, and antioxidant activity. In summary, L. plantarum SPS109 emerges as a promising probiotic candidate with versatile applications in the food and beverage industries, supported by its unique genomic features and safety profile.202439044985
513880.9394Diversity and role of plasmids in adaptation of bacteria inhabiting the Lubin copper mine in Poland, an environment rich in heavy metals. The Lubin underground mine, is one of three mining divisions in the Lubin-Glogow Copper District in Lower Silesia province (Poland). It is the source of polymetallic ore that is rich in copper, silver and several heavy metals. Black shale is also significantly enriched in fossil organic matter in the form of long-chain hydrocarbons, polycyclic aromatic hydrocarbons, organic acids, esters, thiophenes and metalloporphyrins. Biological analyses have revealed that this environment is inhabited by extremophilic bacteria and fungi. Kupfershiefer black shale and samples of water, bottom and mineral sediments from the underground (below 600 m) Lubin mine were taken and 20 bacterial strains were isolated and characterized. All exhibited multi-resistant and hypertolerant phenotypes to heavy metals. We analyzed the plasmidome of these strains in order to evaluate the diversity and role of mobile DNA in adaptation to the harsh conditions of the mine environment. Experimental and bioinformatic analyses of 11 extrachromosomal replicons were performed. Three plasmids, including a broad-host-range replicon containing a Tn3 family transposon, carried genes conferring resistance to arsenic, cadmium, cobalt, mercury and zinc. Functional analysis revealed that the resistance modules exhibit host specificity, i.e., they may increase or decrease tolerance to toxic ions depending on the host strain. The other identified replicons showed diverse features. Among them we identified a catabolic plasmid encoding enzymes involved in the utilization of histidine and vanillate, a putative plasmid-like prophage carrying genes responsible for NAD biosynthesis, and two repABC-type plasmids containing virulence-associated genes. These findings provide an unique molecular insight into the pool of extrachromosomal replicons and highlight their role in the biology and adaptation of extremophilic bacteria inhabiting terrestrial deep subsurface.201526074880
602290.9394Bile Salt Hydrolase Degrades β-Lactam Antibiotics and Confers Antibiotic Resistance on Lactobacillus paragasseri. Bile salt hydrolase (BSH) is a well-characterized probiotic enzyme associated with bile detoxification and colonization of lactic acid bacteria in the human gastrointestinal tract. Here, we isolated a putative BSH (LpBSH) from the probiotic bacterium Lactobacillus paragasseri JCM 5343(T) and demonstrated its bifunctional activity that allows it to degrade not only bile salts but also the antibiotic (penicillin). Although antibiotic resistance and bile detoxification have been separately recognized as different microbial functions, our findings suggest that bifunctional BSHs simultaneously confer ecological advantages to host gut bacteria to improve their survival in the mammalian intestine by attaining a high resistance to bile salts and β-lactams. Strain JCM 5343(T) showed resistance to both bile salts and β-lactam antibiotics, suggesting that LpBSH may be involved in this multi-resistance of the strain. We further verified that such bifunctional enzymes were broadly distributed among the phylogeny, suggesting that the bifunctionality may be conserved in other BSHs of gut bacteria. This study revealed the physiological role and phylogenetic diversity of bifunctional enzymes degrading bile salts and β-lactams in gut bacteria. Furthermore, our findings suggest that the hitherto-overlooked penicillin-degrading activity of penicillin acylase could be a potential new target for the probiotic function of gut bacteria.202235733973
8471100.9394Effects of Klebsiella michiganensis LDS17 on Codonopsis pilosula growth, rhizosphere soil enzyme activities, and microflora, and genome-wide analysis of plant growth-promoting genes. Codonopsis pilosula is a perennial herbaceous liana with medicinal value. It is critical to promote Codonopsis pilosula growth through effective and sustainable methods, and the use of plant growth-promoting bacteria (PGPB) is a promising candidate. In this study, we isolated a PGPB, Klebsiella michiganensis LDS17, that produced a highly active 1-aminocyclopropane-1-carboxylate deaminase from the Codonopsis pilosula rhizosphere. The strain exhibited multiple plant growth-promoting properties. The antagonistic activity of strain LDS17 against eight phytopathogenic fungi was investigated, and the results showed that strain LDS17 had obvious antagonistic effects on Rhizoctonia solani, Colletotrichum camelliae, Cytospora chrysosperma, and Phomopsis macrospore with growth inhibition rates of 54.22%, 49.41%, 48.89%, and 41.11%, respectively. Inoculation of strain LDS17 not only significantly increased the growth of Codonopsis pilosula seedlings but also increased the invertase and urease activities, the number of culturable bacteria, actinomycetes, and fungi, as well as the functional diversity of microbial communities in the rhizosphere soil of the seedlings. Heavy metal (HM) resistance tests showed that LDS17 is resistant to copper, zinc, and nickel. Whole-genome analysis of strain LDS17 revealed the genes involved in IAA production, siderophore synthesis, nitrogen fixation, P solubilization, and HM resistance. We further identified a gene (koyR) encoding a plant-responsive LuxR solo in the LDS17 genome. Klebsiella michiganensis LDS17 may therefore be useful in microbial fertilizers for Codonopsis pilosula. The identification of genes related to plant growth and HM resistance provides an important foundation for future analyses of the molecular mechanisms underlying the plant growth promotion and HM resistance of LDS17. IMPORTANCE: We comprehensively evaluated the plant growth-promoting characteristics and heavy metal (HM) resistance ability of the LDS17 strain, as well as the effects of strain LDS17 inoculation on the Codonopsis pilosula seedling growth and the soil qualities in the Codonopsis pilosula rhizosphere. We conducted whole-genome analysis and identified lots of genes and gene clusters contributing to plant-beneficial functions and HM resistance, which is critical for further elucidating the plant growth-promoting mechanism of strain LDS17 and expanding its application in the development of plant growth-promoting agents used in the environment under HM stress.202438563743
6026110.9391Probiotic Characteristics and Whole Genome Analysis of Lactiplantibacillus plantarum PM8 from Giant Panda (Ailuropoda melanoleuca) Milk. Milk is a rich source of probiotics, particularly lactic acid bacteria (LAB), which have been shown to promote gut health, support the immune system, enhance digestion, and prevent pathogen colonization. This study aimed to isolate and identify LAB strains from giant panda (Ailuropoda melanoleuca) milk, evaluate their probiotic properties, and analyze the genomic characteristics of a promising strain. Thirteen LAB strains were isolated from 12 samples of giant panda milk. Among all LAB strains, Lactiplantibacillus plantarum PM8 (PM8) demonstrated probiotic properties and safety features. It exhibited strong growth performance, high antipathogenic activity against four pathogens, and strong survival rates under simulated gastrointestinal conditions. PM8 also showed excellent adhesion capabilities to Caco-2 cells. Additionally, safety assessment revealed no hemolysin production and minimal antibiotic resistance, making it a promising candidate for probiotic applications. The genome of PM8 consists of 3,227,035 bp with a GC content of 44.60% and contains 3171 coding sequences, including 113 carbohydrate-active enzyme genes and genes related to exopolysaccharides synthesis, vitamin B biosynthesis, adhesion, antioxidant activity, and bile salt hydrolysis. Notably, it contains genes involved in nonribosomally synthesized secondary metabolite and bacteriocin production. The genomic safety analysis confirmed that PM8 lacks the capacity to transmit bacterial antimicrobial resistance and is non-pathogenic to both humans and animals. These findings suggest that PM8 holds considerable potential for enhancing gut health and supporting the development of safe probiotic products.202539900880
193120.9390Screening of metagenomic and genomic libraries reveals three classes of bacterial enzymes that overcome the toxicity of acrylate. Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate resistance, likely, again, through the removal of the toxic product acrylyl-CoA.201424848004
8835130.9390Resistance Mechanism and Physiological Effects of Microcin Y in Salmonella enterica subsp. enterica Serovar Typhimurium. Salmonella bacteria pose a significant threat to animal husbandry and human health due to their virulence and multidrug resistance. The lasso peptide MccY is a recently discovered antimicrobial peptide that acts against various serotypes of Salmonella. In this study, we further explore the resistance mechanism and activity of MccY. Mutants of Ton system genes, including tonB, exbB, and exbD, in Salmonella enterica subsp. enterica serovar Typhimurium were constructed, and the MICs to MccY exhibited significant increases in these deletion mutants compared to the MIC of the parent strain. Subsequently, MccY resistance was quantitatively analyzed, and these mutants also showed greatly reduced rates of killing, even with a high concentration of MccY. In addition, a minimal medium with low iron environment enhanced the sensitivity of these mutants to MccY. Measurements of a series of physiological indicators, including iron utilization, biofilm formation, and motility, demonstrated that MccY may decrease the virulence of S. Typhimurium. Transcriptomic analysis showed that iron utilization, biofilm formation, flagellar assembly, and virulence-related genes were downregulated to varying degrees when S. Typhimurium was treated with MccY. In conclusion, deletion of Ton system genes resulted in resistance to MccY and the susceptibility of these mutants to MccY was increased and differed under a low-iron condition. This lasso peptide can alter multiple physiological properties of S. Typhimurium. Our study will contribute to improve the knowledge and understanding of the mechanism of MccY resistance in Salmonella strains. IMPORTANCE The resistance of Salmonella to traditional antibiotics remains a serious challenge. Novel anti-Salmonella drugs are urgently needed to address the looming crisis. The newly identified antimicrobial peptide MccY shows broad prospects for development and application because of its obvious antagonistic effect on various serotypes of Salmonella. However, our previous study showed that the peptide could confer resistance to Salmonella by disrupting the receptor gene fhuA. In this study, we further explored the potential resistance mechanism of MccY and demonstrated the importance of the Salmonella Ton complex for MccY transport. Disruption in Ton system genes resulted in S. Typhimurium resistance to this peptide, and MccY could alter multiple bacterial physiological properties. In summary, this study further explored the resistance mechanism and antibacterial effect of MccY in S. Typhimurium and provided a scientific basis for its development and application.202236453909
6016140.9390Investigating human-derived lactic acid bacteria for alcohol resistance. BACKGROUND: Excessive alcohol consumption has been consistently linked to serious adverse health effects, particularly affecting the liver. One natural defense against the detrimental impacts of alcohol is provided by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which detoxify harmful alcohol metabolites. Recent studies have shown that certain probiotic strains, notably Lactobacillus spp., possess alcohol resistance and can produce these critical enzymes. Incorporating these probiotics into alcoholic beverages represents a pioneering approach that can potentially mitigate the negative health effects of alcohol while meeting evolving consumer preferences for functional and health-centric products. RESULTS: Five lactic acid bacteria (LAB) isolates were identified: Lactobacillus paracasei Alc1, Lacticaseibacillus rhamnosus AA, Pediococcus acidilactici Alc3, Lactobacillus paracasei Alc4, and Pediococcus acidilactici Alc5. Assessment of their alcohol tolerance, safety, adhesion ability, and immunomodulatory effects identified L. rhamnosus AA as the most promising alcohol-tolerant probiotic strain. This strain also showed high production of ADH and ALDH. Whole genome sequencing analysis revealed that the L. rhamnosus AA genome contained both the adh (encoding for ADH) and the adhE (encoding for ALDH) genes. CONCLUSIONS: L. rhamnosus AA, a novel probiotic candidate, showed notable alcohol resistance and the capability to produce enzymes essential for alcohol metabolism. This strain is a highly promising candidate for integration into commercial alcoholic beverages upon completion of comprehensive safety and functionality evaluations.202438659044
8754150.9390Detoxifying bacterial genes for deoxynivalenol epimerization confer durable resistance to Fusarium head blight in wheat. Fusarium head blight (FHB) and the presence of mycotoxin deoxynivalenol (DON) pose serious threats to wheat production and food safety worldwide. DON, as a virulence factor, is crucial for the spread of FHB pathogens on plants. However, germplasm resources that are naturally resistant to DON and DON-producing FHB pathogens are inadequate in plants. Here, detoxifying bacteria genes responsible for DON epimerization were used to enhance the resistance of wheat to mycotoxin DON and FHB pathogens. We characterized the complete pathway and molecular basis leading to the thorough detoxification of DON via epimerization through two sequential reactions in the detoxifying bacterium Devosia sp. D6-9. Epimerization efficiently eliminates the phytotoxicity of DON and neutralizes the effects of DON as a virulence factor. Notably, co-expressing of the genes encoding quinoprotein dehydrogenase (QDDH) for DON oxidation in the first reaction step, and aldo-keto reductase AKR13B2 for 3-keto-DON reduction in the second reaction step significantly reduced the accumulation of DON as virulence factor in wheat after the infection of pathogenic Fusarium, and accordingly conferred increased disease resistance to FHB by restricting the spread of pathogenic Fusarium in the transgenic plants. Stable and improved resistance was observed in greenhouse and field conditions over multiple generations. This successful approach presents a promising avenue for enhancing FHB resistance in crops and reducing mycotoxin contents in grains through detoxification of the virulence factor DON by exogenous resistance genes from microbes.202438593377
6043160.9388Histamine and cholesterol lowering abilities of lactic acid bacteria isolated from artisanal Pico cheese. AIMS: This study was designed to select lactic acid bacteria with histamine- and cholesterol-reducing abilities to be used as potential probiotics. METHODS AND RESULTS: Thirty strains of lactic acid bacteria isolated from an artisanal raw milk cheese were screened for their abilities to degrade histamine, reduce cholesterol and hydrolyse bile salts. Strains were also screened for safety and probiotic traits, such as resistance to gastrointestinal conditions, adhesion to Caco-2 cells, resistance to antibiotics and presence of virulence genes. Two Lactobacillus paracasei strains presented high cholesterol- and histamine-lowering abilities, tested negative for the presence of virulence genes and showed susceptibility to most important antibiotics. These strains were also shown to possess desirable in vitro probiotic properties, revealed by tolerance to gastrointestinal conditions and high adhesion to intestinal cells. CONCLUSIONS: Among the screened strains, Lb. paracasei L3C21M6 revealed the best cholesterol and histamine reducing abilities together with desirable probiotic and safety features to be used in food applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain L3C21M6 is a good candidate for use as a probiotic with histamine-degrading activity and cholesterol lowering effect. In addition, this strain could be use in dairy foods to prevent histamine food poisoning.202032500572
8731170.9387Isolation of Potato Endophytes and Screening of Chaetomium globosum Antimicrobial Genes. Antimicrobial peptides (AMPs) have natural antibacterial activities that pathogens find difficult to overcome. As a result of this occurrence, AMPs can act as an important substitute against the microbial resistance. In this study, we used plate confrontation tests to screen out 20 potential endophytes from potato tubers. Among them, endophyte F5 was found to significantly inhibit the growth of five different pathogenic fungi. Following that, phylogenetic analysis revealed that the internal transcribed spacer (ITS) sequences were 99% identical to Chaetomium globosum corresponding sequences. Thereafter, the Bacillus subtilis expression system was used to create a C. globosum cDNA library in order to isolate the resistance genes. Using this approach, the resistance gene screening technology in the indicator bacteria built-in library was used to identify two antimicrobial peptides, CgR2150 and CgR3101, with broad-spectrum antibacterial activities. Furthermore, the results showed that CgR2150 and CgR3101 have excellent UV, thermal, and enzyme stabilities. Also, these two peptides can significantly inhibit the growth of various bacteria (Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Clavibacter michiganensis, and Clavibacter fangii) and fungi (Fusarium graminearum, Rhizoctonia solani, and Botrytis cinerea). Scanning electron microscopy (SEM) observations revealed that CgR2150 and CgR3101 peptides act against bacteria by disrupting bacterial cell membranes. Moreover, hemolytic activity assay showed that neither of the two peptides exhibited significant hemolytic activity. To conclude, the antimicrobial peptides CgR2150 and CgR3101 are promising in the development of a new antibacterial agent and for application in plant production.202235563004
194180.9387Possible Role of CHAD Proteins in Copper Resistance. Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.202438399813
8193190.9386Sinorhizobium meliloti Functions Required for Resistance to Antimicrobial NCR Peptides and Bacteroid Differentiation. Legumes of the Medicago genus have a symbiotic relationship with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of intracellular symbionts. Members of the nodule-specific cysteine-rich peptide (NCR) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont, but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts; these include an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes and lipopolysaccharide modifications mediated by lpsB and lpxXL, as well as rpoH1, encoding a stress sigma factor. Strains with mutations in these genes show a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids, although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions, including peptide transporters, the bacterial envelope structures, and stress response regulators, to resist the aggressive assault of NCR peptides in the nodule cells. IMPORTANCE The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants' needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia, but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen-fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane, and the stress response regulator RpoH1.202134311575