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Riboflavin (vitamin B 2 ) is essential for human beings and it has to be provided by healthy nutrition. The use of fermentation with riboflavin-overproducing lactic acid bacteria (LAB) represents an ideal strategy to generate, by in situ biofortification, functional drinks. These beverages can positively contribute to consumer health and address nutritional deficiencies. In the present work, the functional capabilities of Weissella cibaria BAL3C-5 C120T for riboflavin-overproduction and dextran-production during fermentation of oat-, rice-, soybean- and almond-based drinks have been evaluated. It was confirmed that the strain was capable of producing riboflavin and dextran in the analysed drinks. This property was especially pronounced in the oat-based drink, where after 24 h of fermentation the strain was able to increase riboflavin and dextran levels up to 3.4 mg/L and 3.2 g/L, respectively. Moreover, under optimized conditions the strain was able to enrich the fermented oat-based drinks with the prebiotic oligosaccharide panose (up to 6.6 g/L). In addition, in the oat-based drinks BAL3C-5 C120T showed a good pH-lowering ability (from 7.0 to 3.8) as well as a high 80 % cell viability after one month of storage. Rheological analysis of the resulting fermented oat-based beverages revealed a thixotropic structure related to a gel-like behaviour which was not observed in the non-fermented control drinks. In summary, these results confirmed the unique characteristics of W. cibaria BAL3C-5 C120T strain for the development of biofortified and functional plant-based beverages with improved nutritional and rheological properties. Analysis of the BAL3C-5 C120T strain survival under gastrointestinal conditions and its autoaggregation properties, also indicated its potential use as a probiotic delivered in an oat-based fermented beverage. In this context, this study also promotes the utilization of W. cibaria species in health and food industries where it has not yet been used as a starter or adjunct culture., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The digestive physiology of house dust mites (HDMs) is particularly relevant for their allergenicity since many of their allergens participate in digestion and are excreted into faecal pellets, a main source of exposure for allergic subjects. To gain insight into the mite dietary digestion, the genome of the HDM Dermatophagoides pteronyssinus was screened for genes encoding peptidases (n = 320), glycosylases (n = 77), lipases and esterases (n = 320), peptidase inhibitors (n = 65) and allergen-related proteins (n = 52). Basal gene expression and transcriptional responses of mites to dietary cystatin A, a cysteine endopeptidase inhibitor with previously shown antinutritional effect on mites, were analysed by RNAseq. The ingestion of cystatin A resulted in significant regulation of different cysteine endopeptidase and glycosylase genes. One Der p 1-like and two cathepsin B-like cysteine endopeptidase genes of high basal expression were induced, which suggests their prominent role in proteolytic digestion together with major allergen Der p 1. A number of genes putatively participating in the interaction of mites with their microbiota and acquired by horizontal gene transfer were repressed, including genes encoding the peptidase Der p 38, two 1,3-beta-glucanases, a lysozyme and a GH19 chitinase. Finally, the disruption of mite digestion resulted in the regulation of up to 17 allergen and isoallergen genes. Altogether, our results shed light on the putative role of specific genes in digestion and illustrate the connection between the digestive physiology of HDM and allergy., (© 2024 The Author(s). Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

C-REPEAT BINDING FACTORS (CBFs) are highly conserved plant transcription factors that promote cold tolerance. In Arabidopsis (Arabidopsis thaliana), three CBFs (CBF1 to CBF3) play a critical role in cold acclimation, and the expression of their corresponding genes is rapidly and transiently induced during this adaptive response. Cold induction of CBFs has been extensively studied and shown to be tightly controlled, yet the molecular mechanisms that restrict the expression of each CBF after their induction during cold acclimation are poorly understood. Here, we present genetic and molecular evidence that the decline in the induction of CBF3 during cold acclimation is epigenetically regulated through the Polycomb Repressive Complex (PRC) 2. We show that this complex promotes the deposition of the repressive mark H3K27me3 at the coding region of CBF3, silencing its expression. Our results indicate that the cold-inducible long noncoding RNA SVALKA is essential for this regulation by recruiting PRC2 to CBF3. These findings unveil a SVALKA-PRC2 regulatory module that ensures the precise timing of CBF3 induction during cold acclimation and the correct development of this adaptive response., Competing Interests: Conflict of interest statement: All authors declare that they have no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Arabidopsis (Arabidopsis thaliana) PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) post-translationally modifies RNA-binding proteins by arginine (R) methylation. However, the impact of this modification on the regulation of RNA processing is largely unknown. We used the spliceosome component, SM-LIKE PROTEIN 4 (LSM4), as a paradigm to study the role of R-methylation in RNA processing. We found that LSM4 regulates alternative splicing (AS) of a suite of its in vivo targets identified here. The lsm4 and prmt5 mutants show a considerable overlap of genes with altered AS raising the possibility that splicing of those genes could be regulated by PRMT5-dependent LSM4 methylation. Indeed, LSM4 methylation impacts AS, particularly of genes linked with stress response. Wild-type LSM4 and an unmethylable version complement the lsm4-1 mutant, suggesting that methylation is not critical for growth in normal environments. However, LSM4 methylation increases with abscisic acid and is necessary for plants to grow under abiotic stress. Conversely, bacterial infection reduces LSM4 methylation, and plants that express unmethylable-LSM4 are more resistant to Pseudomonas than those expressing wild-type LSM4. This tolerance correlates with decreased intron retention of immune-response genes upon infection. Taken together, this provides direct evidence that R-methylation adjusts LSM4 function on pre-mRNA splicing in an antagonistic manner in response to biotic and abiotic stress., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Six lactic acid bacteria (LAB) isolated from Algerian sheep's milk, traditional butter, date palm sap and barley, which produce dextran, mannitol, oligosaccharides and vitamin B 2 have been characterized. They were identified as Leuconostoc mesenteroides (A4X, Z36P, B12 and O9) and Liquorilactobacillus mali (BR201 and FR123). Their exopolysaccharides synthesized from sucrose by dextransucrase (Dsr) were characterized as dextrans with (1,6)-D-glucopyranose units in the main backbone and branched at positions O-4, O-2 and/or O-3, with D-glucopyranose units in the side chain. A4X was the best dextran producer (4.5 g/L), while the other strains synthesized 2.1-2.7 g/L. Zymograms revealed that L. mali strains have a single Dsr with a molecular weight (Mw) of ~ 145 kDa, while the Lc. mesenteroides possess one or two enzymes with 170-211 kDa Mw. As far as we know, this is the first detection of L. mali Dsr. Analysis of metabolic fluxes from sucrose revealed that the six LAB produced mannitol (~ 12 g/L). The co-addition of maltose-sucrose resulted in the production of panose (up to 37.53 mM), an oligosaccharide known for its prebiotic effect. A4X, Z36P and B12 showed dextranase hydrolytic enzymatic activity and were able to produce another trisaccharide, maltotriose, which is the first instance of a dextranase activity encoded by Lc. mesenteroides strains. Furthermore, B12 and O9 grew in the absence of riboflavin (vitamin B 2 ) and synthesized this vitamin, in a defined medium at the level of ~ 220 μg/L. Therefore, these LAB, especially Lc. mesenteroides B12, are good candidates for the development of new fermented food biofortified with functional compounds., (© 2024. The Author(s).)

Bacillus subtilis is one of the best-studied bacteria and serves as a Gram-positive model system to address fundamental biological processes. Depending on conditions, a B. subtilis cell can initiate one out of various distinct differentiation processes to cope with changing environmental conditions. One of these differentiation processes is natural competence that allows cells to adsorb exogenous DNA and subsequently incorporate it into its chromosome by homologous recombination. Due to competence development, the genome of B. subtilis can be easily manipulated, and this has contributed to B. subtilis being a model system. In this chapter, we describe some of the most common genetic tools that can be used in combination with natural competence to tailor the genome of B. subtilis., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gluten consumption causes several immunological and non-immunological intolerances in susceptible individuals. In this study, the dextran-producing Weissella cibaria BAL3C-5 and its derivative, the riboflavin-overproducing strain BAL3C-5 C120T, together with a commercial bakery yeast, were used to ferment gluten-free (GF)-doughs obtained from corn and rice flours at two different concentrations and supplemented with either quinoa, buckwheat, or chickpea to obtain laboratory-scale GF bread. The levels of dextran, riboflavin, and total flavins were determined in the fermented and breads. Both strains grew in fermented doughs and contributed dextran, especially to those made with corn plus quinoa (~1 g/100 g). The highest riboflavin (350-150 µg/100 g) and total flavin (2.3-1.75 mg/100 g) levels were observed with BAL3C-5 C120T, though some differences were detected between the various doughs or breads, suggesting an impact of the type of flour used. The safety assessment confirmed the lack of pathogenic factors in the bacterial strains, such as hemolysin and gelatinase activity, as well as the genetic determinants for biogenic amine production. Some intrinsic resistance to antibiotics, including vancomycin and kanamycin, was found. These results indicated the microbiological safety of both W. cibaria strains and indicated their potential application in baking to produce GF bread.

Artificial lighting is complementary and single-source lighting for controlled Environment Agriculture (CEA) to increase crop productivity. Installations to control CO 2 levels and luminaires with variable spectrum and intensity are becoming increasingly common. In order to see the net assimilation of CO 2 based on the relationship between the three factors: intensity, spectrum and CO 2 concentration, tests are proposed on tomatoes seedling with combinations of ten spectra (100B, 80B20G, 20B80G, 100G, 80G20R, 20G80R, 100R, 80R20B, 20R80B, 37R36G27B) seven light intensities (30, 90, 200, 350, 500, 700 and 1000 μmol·m -2 s -1 ) and nine CO 2 concentrations (200, 300, 400, 500, 600, 700, 800 and 900 ppm). These tomato seedlings grew under uniform conditions with no treatments applied up to the moment of measurement by a differential gas analyzer. We have developed a model to evaluate and determine under what spectrum and intensity of light photosynthesis the Net assimilation of CO 2 (A n ) is more significant in the leaves of tomato plants, considering the CO 2 concentration as an independent variable in the model. The evaluation of the model parameters for each spectrum and intensity shows that the intensity has a more decisive influence on the maximum A n rate than the spectra. For intensities lower than 350 μmol·m -2 s -1 , it is observed that the spectrum has a greater influence on the variable A n . The spectra with the best behaviour were 80R20B and 80B20R, which maintained A n values between 2 and 4 (μmol CO 2 ·m -2 ·s -1 ) above the spectra with the worst behaviour (100G, 80G20R, 20G80R and 37B36G27R) in practically all situations. Photosynthetic Light-Use Efficiency (PLUE) was also higher for the 80B20R and 20R80B spectra with values of 36,07 and 33,84 mmol CO 2 ·mol photon -1 , respectively, for light intensities of 200 μmol·m -2 s -1 and 400 ppm of CO 2 that increased to values of 49,65 and 48,38 mmol CO 2 ·mol photon -1 for the same light intensity and concentrations of 850 ppm. The choice of spectrum is essential, as indicated by the data from this study, to optimize the photosynthesis of the plant species grown in the plant factory where light intensities are adjusted for greater profitability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Moratiel, Jimenez, Mate, Ibánez, Moreno and Tarquis.)

Resistance to the frontline antibiotic rifampicin constitutes a challenge to the treatment and control of tuberculosis. Here, we analyzed the mutational landscape of Mycobacterium smegmatis during long-term evolution with increasing concentrations of rifampicin, using a mutation accumulation assay combined with whole-genome sequencing. Antibiotic treatment enhanced the acquisition of mutations, doubling the genome-wide mutation rate of the wild-type cells. While antibiotic exposure led to extinction of almost all wild-type lines, the hypermutable phenotype of the Δ nucS mutant strain (noncanonical mismatch repair deficient) provided an efficient response to the antibiotic, leading to high rates of survival. This adaptative advantage resulted in the emergence of higher levels of rifampicin resistance, an accelerated acquisition of drug resistance mutations in rpoB (β RNA polymerase), and a wider diversity of evolutionary pathways that led to drug resistance. Finally, this approach revealed a subset of adaptive genes under positive selection with rifampicin that could be associated with the development of antibiotic resistance. IMPORTANCE Rifampicin is the most important first-line antibiotic against mycobacterial infections, including tuberculosis, one of the top causes of death worldwide. Acquisition of rifampicin resistance constitutes a major global public health problem that makes the control of the disease challenging. Here, we performed an experimental evolution assay under antibiotic selection to analyze the response and adaptation of mycobacteria, leading to the acquisition of rifampicin resistance. This approach explored the total number of mutations that arose in the mycobacterial genomes under long-term rifampicin exposure, using whole-genome sequencing. Our results revealed the effect of rifampicin at a genomic level, identifying different mechanisms and multiple pathways leading to rifampicin resistance in mycobacteria. Moreover, this study detected that an increase in the rate of mutations led to enhanced levels of drug resistance and survival. In summary, all of these results could be useful to understand and prevent the emergence of drug-resistant isolates in mycobacterial infections., Competing Interests: The authors declare no conflict of interest.

Type 1 diabetes mellitus (T1DM) has been increasing in prevalence in the last decades and has become a global burden. Autoantibodies against human glutamate decarboxylase (GAD65) are among the first to be detected at the onset of T1DM. Diverse viruses have been proposed to be involved in the triggering of T1DM because of molecular mimicry, i.e., similarity between parts of some viral proteins and one or more epitopes of GAD65. However, the possibility that bacterial proteins might also be responsible for GAD65 mimicry has been seldom investigated. To date, many genomes of Streptococcus pneumoniae (the pneumococcus), a prominent human pathogen particularly prevalent among children and the elderly, have been sequenced. A dataset of more than 9000 pneumococcal genomes was mined and two different (albeit related) genes (gadA and gadB), presumably encoding two glutamate decarboxylases similar to GAD65, were found. The various gadA Spn alleles were present only in serotype 3 pneumococci belonging to the global lineage GPSC83, although some homologs have also been discovered in two subspecies of Streptococcus constellatus (pharyngis and viborgensis), an isolate of the group B streptococci, and several strains of Lactobacillus delbrueckii. Besides, gadB Spn alleles are present in > 10% of the isolates in our dataset and represent 16 GPSCs with 123 sequence types and 20 different serotypes. Sequence analyses indicated that gadA- and gadB-like genes have been mobilized among different bacteria either by prophage(s) or by integrative and conjugative element(s), respectively. Substantial similarities appear to exist between the putative pneumococcal glutamate decarboxylases and well-known epitopes of GAD65. In this sense, the use of broader pneumococcal conjugate vaccines such as PCV20 would prevent the majority of serotypes expressing those genes that might potentially contribute to T1DM. These results deserve upcoming studies on the possible involvement of S. pneumoniae in the etiopathogenesis and clinical onset of T1DM., (© 2023. The Author(s).)

Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources and ecological conditions, including the infected host. Given this situation, it is conceivable that P. aeruginosa virulence might be, at least in part, under metabolic control, in such a way that virulence determinants are produced just when needed. Indeed, it has been shown that the catabolite repression control protein Crc, which together with the RNA chaperon Hfq regulates the P. aeruginosa utilization of carbon sources at the post-transcriptional level, also regulates, directly or indirectly, virulence-related processes in P. aeruginosa . Among them, Crc regulates P. aeruginosa cytotoxicity, likely by modulating the activity of the Type III Secretion System (T3SS), which directly injects toxins into eukaryotic host cells. The present work shows that the lack of Crc produces a Type III Secretion-defective phenotype in P. aeruginosa . The observed impairment is a consequence of a reduced expression of the genes encoding the T3SS, together with an impaired secretion of the proteins involved. Our results support that the impaired T3SS activity of the crc defective mutant is, at least partly, a consequence of a defective protein export, probably due to a reduced proton motive force. This work provides new information about the complex regulation of the expression and the activity of the T3SS in P. aeruginosa . Our results highlight the need of a robust bacterial metabolism, which is defective in the ∆crc mutant, to elicit complex and energetically costly virulence strategies, as that provided by the T3SS.

Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. This is the first P-AT resistance mechanism to be reported., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fermentative processes by lactic acid bacteria can produce metabolites of interest to the health and food industries. Two examples are the production of B-group vitamins, and of prebiotic and immunomodulatory dextran-type exopolysaccharides. In this study, three riboflavin- and dextran-producing Weissella cibaria strains (BAL3C-5, BAL3C-7 and BAL3C-22) were used to develop a new method for selection and isolation of spontaneous riboflavin-overproducing W. cibaria mutants. This method was based on the selection of strains resistant to roseoflavin. The DNA sequencing of the FMN riboswitch of bacterial cell populations treated with various roseoflavin concentrations, revealed the existence of at least 10 spontaneous and random point mutations at this location. Folding and analysis of the mutated FMN riboswitches with the RNA fold program predicted that these mutations could result in a deregulation of the rib operon expression. When the roseoflavin-treated cultures were plated on medium supporting dextran synthesis, the most promising mutants were identified by the yellow color of their mucous colonies, exhibiting a ropy phenotype. After their isolation and recovery in liquid medium, the evaluation of their riboflavin production revealed that the mutant strains synthesized a wide range of riboflavin levels (from 0.80 to 6.50 mg/L) above the wild-type level (0.15 mg/L). Thus, this was a reliable method to select spontaneous riboflavin-overproducing and dextran-producing strains of W. cibaria. This species has not yet been used as a starter or adjunct culture, but this study reinforces the potential that it has for the food and health industry for the production of functional foods or as a probiotic. Furthermore, analysis of the influence of FMN present in the growth medium, on rib mRNA and riboflavin levels, revealed which mutant strains produce riboflavin without flavin regulation. Moreover, the BAL3C-5 C120T mutant was identified as the highest riboflavin-overproducer. Determination of its chromosomal DNA sequence and that of BAL3C-5, revealed a total identity between the 2 strains except for the C120T mutation at the FMN riboswitch. To our knowledge, this work is the first demonstration that only a single alteration in the genome of a lactic acid bacteria is required for a riboflavin-overproducing phenotype., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Diez-Ozaeta, Martín-Loarte, Mohedano, Tamame, Ruiz-Masó, del Solar, Dueñas and López.)

RNA viruses have recently been detected in association with house dust mites, including laboratory cultures, dust samples, and mite-derived pharmaceuticals used for allergy diagnosis. This study aimed to assess the incidence of viral infection on Dermatophagoides pteronyssinus physiology and on the allergenic performance of extracts derived from its culture. Transcriptional changes between genetically identical control and virus-infected mite colonies were analysed by RNAseq with the support of a new D. pteronyssinus high-quality annotated genome (56.8 Mb, 108 scaffolds, N50 = 2.73 Mb, 96.7% BUSCO-completeness). Extracts of cultures and bodies from both colonies were compared by inspecting major allergen accumulation by enzyme-linked immunosorbent assay (ELISA), allergen-related enzymatic activities by specific assays, airway inflammation in a mouse model of allergic asthma, and binding to allergic patient's sera IgE by ImmunoCAP. Viral infection induced a significant transcriptional response, including several immunity and stress-response genes, and affected the expression of seven allergens, putative isoallergens and allergen orthologs. Major allergens were unaffected except for Der p 23 that was upregulated, increasing ELISA titers up to 29% in infected-mite extracts. By contrast, serine protease allergens Der p 3, 6 and 9 were downregulated, being trypsin and chymotrypsin enzymatic activities reduced up to 21% in extracts. None of the parameters analysed in our mouse model, nor binding to human IgE were significantly different when comparing control and infected-mite extracts. Despite the described physiological impact of viral infection on the mites, no significant consequences for the allergenicity of derived extracts or their practical use in allergy diagnosis have been detected., (© 2022 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Many microorganisms produce and excrete acetoin (3-hydroxy-2-butanone) when growing in environments that contain glucose or other fermentable carbon sources. This excreted compound can then be assimilated by other bacterial species such as pseudomonads. This work shows that acetoin is not a preferred carbon source of Pseudomonas putida, and that the induction of genes required for its assimilation is down-modulated by different, independent, global regulatory systems when succinate, glucose or components of the LB medium are also present. The expression of the acetoin degradation genes was found to rely on the RpoN alternative sigma factor and to be modulated by the Crc/Hfq, Cyo and PTS Ntr regulatory elements, with the impact of the latter three varying according to the carbon source present in addition to acetoin. Pyruvate, a poor carbon source for P. putida, did not repress acetoin assimilation. Indeed, the presence of acetoin significantly improved growth on pyruvate, revealing these compounds to have a synergistic effect. This would provide a clear competitive advantage to P. putida when growing in environments in which all the preferred carbon sources have been depleted and pyruvate and acetoin remain as leftovers from the fermentation of sugars by other microorganisms., (© 2022 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

NucS/EndoMS-dependent noncanonical mismatch repair (MMR) ensures the stability of genomic DNA in mycobacteria and acts as a guardian of the genome by preventing the accumulation of point mutations. In order to address whether the inactivation of noncanonical MMR could increase the acquisition of drug resistance by mutation, a Δ nucS strain was constructed and explored in the emerging pathogen Mycobacterium abscessus. Deletion of nucS resulted in a mutator phenotype with increased acquisition of resistance to macrolides and aminoglycosides, the two main groups of antimycobacterial agents for M. abscessus treatment, and also to second-line drugs such as fluoroquinolones. Inactivation of the noncanonical MMR in M. abscessus led to increases of 10- to 22-fold in the appearance of spontaneous mutants resistant to the macrolide clarithromycin and the aminoglycosides amikacin, gentamicin, and apramycin, compared with the wild-type strain. Furthermore, emergence of fluoroquinolone (ciprofloxacin) resistance was detected in a nucS -deficient strain but not in a wild-type M. abscessus strain. Acquired drug resistance to macrolides and aminoglycosides was analyzed through sequencing of the 23S rRNA gene rrl and the 16S rRNA gene rrs from independent drug-resistant colonies of both strains. When the acquisition of clarithromycin resistance was examined, a different mutational profile was detected in the M. abscessus Δ nucS strain compared with the wild-type one. To summarize, M. abscessus requires the NucS-dependent noncanonical MMR pathway to prevent the emergence of drug-resistant isolates by mutation. To our knowledge, this is the first report that reveals the role of NucS in a human pathogen, and these findings have potential implications for the treatment of M. abscessus infections. IMPORTANCE Chronic infections caused by M. abscessus are an emerging challenge in public health, posing a substantial health and economic burden, especially in patients with cystic fibrosis. Treatment of M. abscessus infections with antibiotics is particularly challenging, as its complex drug resistance mechanisms, including constitutive resistance through DNA mutation, lead to high rates of treatment failure. To decipher the evolution of antibiotic resistance in M. abscessus, we studied NucS-dependent noncanonical MMR, a unique DNA repair pathway involved in genomic maintenance. Inactivation of NucS is linked to the increase of DNA mutations (hypermutation), which can confer drug resistance. Our analysis detected increased acquisition of mutations conferring resistance to first-line and second-line antibiotics. We believe that this study will improve the knowledge of how this pathogen could evolve into an untreatable infectious agent, and it uncovers a role for hypermutators in chronic infectious diseases under antibiotic pressure.

The coordination of cell division with stress response is essential for maintaining genome stability in plant meristems. Proteins involved in pre-mRNA splicing are important for these processes in animal and human cells. Based on its homology to the splicing factor SART1, which is implicated in the control of cell division and genome stability in human cells, we analyzed if MDF has similar functions in plants. We found that MDF associates with U4/U6.U5 tri-snRNP proteins and is essential for correct splicing of 2,037 transcripts. Loss of MDF function leads to cell division defects and cell death in meristems and was associated with up-regulation of stress-induced genes and down-regulation of mitotic regulators. In addition, the mdf-1 mutant is hypersensitive to DNA damage treatment supporting its role in coordinating stress response with cell division. Our analysis of a dephosphomutant of MDF suggested how its protein activity might be controlled. Our work uncovers the conserved function of a plant splicing factor and provides novel insight into the interplay of pre-mRNA processing and genome stability in plants., (© 2022 de Luxán-Hernández et al.)

This work describes a method for deriving riboflavin overproducing strains of Weissella cibaria by exposing three strains (BAL3C-5, BAL3C-7, and BAL3C-22) isolated from dough to increasing concentrations of roseoflavin. By this procedure, we selected one mutant overproducing strain from each parental strain (BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2, respectively). Quantification of dextran and riboflavin produced by the parental and mutant strains in a defined medium lacking riboflavin and polysaccharides confirmed that riboflavin was only overproduced by the mutant strains, whereas dextran production was similar in both mutant and parental strains. The molecular basis of the riboflavin overproduction by the mutants was determined by nucleotide sequencing of their rib operons, which encode the enzymes of the riboflavin biosynthetic pathway. We detected a unique mutation in each of the overproducing strains. These mutations, which map in the sensor domain (aptamer) of a regulatory element (the so-called FMN riboswitch) present in the 5' untranslated region of the rib operon mRNA, appear to be responsible for the riboflavin-overproducing phenotype of the BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2 mutant strains. Furthermore, the molecular basis of dextran production by the six W. cibaria strains has been characterized by ( i ) the sequencing of their dsr genes encoding dextransucrases, which synthesize dextran using sucrose as substrate, and ( ii ) the detection of active Dsr proteins by zymograms. Finally, the parental and mutant strains were analyzed for in situ production of riboflavin and dextran during experimental bread making. The results indicate that the mutant strains were able to produce experimental wheat breads biofortified with both riboflavin and dextran and, therefore, may be useful for the manufacture of functional commercial breads., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hernández-Alcántara, Chiva, Mohedano, Russo, Ruiz-Masó, del Solar, Spano, Tamame and López.)

In the present review the latest research studies on Kombucha tea are summarized. Special attention has been paid on microbial population, chemical parameters, biocellulose production, and mainly, on the latest evidences of the biological activities of Kombucha tea. Kombucha tea is a fermented sweetened black or green tea which is obtained from a fermentative process driven by a symbiotic culture of yeast, acetic acid bacteria and lactic acid bacteria. In the last years, its consumption has increasingly grown due to its multiple and potential benefits on human health. This fact has motivated a significant increase in the number of research studies that are focused on the biological activities of this beverage. In this context, this review gathers the main studies that have analyzed the different properties of Kombucha tea (as antioxidant, antimicrobial, antidiabetic, antitumoral, anti-inflammatory, antihypertensive, hepatoprotective, hypocholesterolemic, and probiotic activities). It is highlighted that nowadays few human-based evidences are available to prove the beneficial effect of Kombucha tea on humans' health. In conclusion, further work on Kombucha tea is needed since nowadays few information is available on both clinical studies and the characterization of bioactive compounds and their properties., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Three-component ParABS partition systems ensure stable inheritance of many bacterial chromosomes and low-copy-number plasmids. ParA localizes to the nucleoid through its ATP-dependent nonspecific DNA-binding activity, whereas centromere-like parS -DNA and ParB form partition complexes that activate ParA-ATPase to drive the system dynamics. The essential parS sequence arrangements vary among ParABS systems, reflecting the architectural diversity of their partition complexes. Here, we focus on the pSM19035 plasmid partition system that uses a ParB pSM of the ribbon-helix-helix (RHH) family. We show that parS pSM with four or more contiguous ParB pSM -binding sequence repeats is required to assemble a stable ParA pSM -ParB pSM complex and efficiently activate the ParA pSM -ATPase, stimulating complex disassembly. Disruption of the contiguity of the parS pSM sequence array destabilizes the ParA pSM -ParB pSM complex and prevents efficient ATPase activation. Our findings reveal the unique architecture of the pSM19035 partition complex and how it interacts with nucleoid-bound ParA pSM -ATP., Competing Interests: AV, JA, KM No competing interests declared

In recent years, an unstoppable trend toward minimally processed foods has increased the popularity of fermented foods as a beneficial nutritional and functional strategy. Within food fermentations, complex microbial communities trigger different biochemical reactions that result in the release of multiple bioactive compounds with beneficial effects on human health. In the present review the latest studies on fermented foods are summarized. Special attention has been paid on the health benefits of main fermented foods available nowadays, the principal bioactive compounds responsible for such properties as well as the future trends of research studies regarding their potentialities. This review emphasizes the need of clinical evidence to ensure that fermented foods may entail a significant improvement on well-being. Fermented foods may represent a non-invasive strategy to face multiple disorders, as hypertension, diabetes, hyperlipidemia, oxidative stress and multiple cognitive disordes, among others. Release of bioactive compounds, microbial enzymatic conversions or probiotic activities are the main responsible for such interesting properties. However, the need of well-designed clinical trials is a must in order to obtain conclusive results. Bioavailability and biodisponibility of bioactive compounds as well as the design of precision probiotics are also another focus of interest in which it must be deepen., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Plants are concurrently exposed to biotic and abiotic stresses, including infection by viruses and drought. Combined stresses result in plant responses that are different from those observed for each individual stress. We investigated compensatory effects induced by virus infection on the fitness of hosts grown under water deficit, and the hypothesis that water deficit improves tolerance, estimated as reproductive fitness, to virus infection. Our results show that infection by Turnip mosaic virus (TuMV) or Cucumber mosaic virus (CMV) promotes drought tolerance in Arabidopsis thaliana and Nicotiana benthamiana . However, neither CMV nor TuMV had a positive impact on host reproductive fitness following withdrawal of water, as determined by measuring the number of individuals producing seeds, seed grains, and seed germination rates. Importantly, infection by CMV but not by TuMV improved the reproductive fitness of N. benthamiana plants when exposed to drought compared to watered, virus-infected plants. However, no such conditional phenotype was found in Arabidopsis plants infected with CMV. Water deficit did not affect the capacity of infected plants to transmit CMV through seeds. These findings highlight a conditional improvement in biological efficacy of N. benthamiana plants infected with CMV under water deficit, and lead to the prediction that plants can exhibit increased tolerance to specific viruses under some of the projected climate change scenarios.

Introduction and Aims: Viral hepatitis, which appears most frequently at birth or during childhood, is a disease whose transmission routes include tears, bile, sexual fluids, sweat, milk, urine, feces, and saliva. The aim of the present study was to analyze the specificity of the immunochromatographic and ELISA diagnostic tests for hepatitis B surface antigen and compare them with PCR testing., Materials and Methods: The study sample was made up of 140 men and 60 women referred to the Urmia Medical University hospital to undergo PCR testing for HBV diagnosis. The ELISA test was performed using the Pioneer Medicine Company kit (Tehran, Iran)., Results: The results of the HBs-Ag rapid test and the ELISA test were compared with the PCR test. The HBs-Ag rapid test had 97% sensitivity and 91% specificity, whereas the ELISA test had 78% sensitivity and 76% specificity., Discussion and Conclusion: According to our results, the immunochromatographic test was accurate for diagnosing HBs-Ag in blood and the ELISA test had acceptable sensitivity and specificity, compared with PCR testing., (Copyright © 2021 Asociación Mexicana de Gastroenterología. Published by Masson Doyma México S.A. All rights reserved.)

Listeria monocytogenes is a saprophytic gram-positive bacterium, and an opportunistic foodborne pathogen that can produce listeriosis in humans and animals. It has evolved an exceptional ability to adapt to stress conditions encountered in different environments, resulting in a ubiquitous distribution. Because some food preservation methods and disinfection protocols in food-processing environments cannot efficiently prevent contaminations, L. monocytogenes constitutes a threat to human health and a challenge to food safety. In the host, Listeria colonizes the gastrointestinal tract, crosses the intestinal barrier, and disseminates through the blood to target organs. In immunocompromised individuals, the elderly, and pregnant women, the pathogen can cross the blood-brain and placental barriers, leading to neurolisteriosis and materno-fetal listeriosis. Molecular and cell biology studies of infection have proven L. monocytogenes to be a versatile pathogen that deploys unique strategies to invade different cell types, survive and move inside the eukaryotic host cell, and spread from cell to cell. Here, we present the multifaceted Listeria life cycle from a comprehensive perspective. We discuss genetic features of pathogenic Listeria species, analyze factors involved in food contamination, and review bacterial strategies to tolerate stresses encountered both during food processing and along the host's gastrointestinal tract. Then we dissect host-pathogen interactions underlying listerial pathogenesis in mammals from a cell biology and systemic point of view. Finally, we summarize the epidemiology, pathophysiology, and clinical features of listeriosis in humans and animals. This work aims to gather information from different fields crucial for a comprehensive understanding of the pathogenesis of L. monocytogenes.

Background: Allergy to house dust mites (HDM), the most important source of indoor allergens worldwide, is diagnosed and treated using natural extracts from cultures that can contain immunoactive components from the HDM microbiome, including mite-infecting viruses. This study aimed to contribute to the discovery and characterization of RNA viruses from Dermatophagoides pteronyssinus, followed by their detection in different mite-derived sources., Methods: Viruses were assembled after in silico metatranscriptomic analysis of D. pteronyssinus RNA samples, visualized by electron microscopy, and RNA detected by direct RT-PCR or data mining. Mite culture performance was evaluated in vivo., Results: Seven RNA viruses were identified in our laboratory stock colony. Picornavirus-like viral particles were detected in epithelial cells of the digestive system and in fecal pellets. Most of these viruses could be persistently transmitted to an inbred virus-free colony by inoculating fecal material from the stock colony. Upon viral infection, no significant effect could be seen on mite population growth. Transcriptomic screening confirmed the presence of homolog sequences to these viruses in independent laboratory stocks of D. pteronyssinus and in other Astigmata mites. Noteworthy, RNA from most of the viruses could be detected by RT-PCR on house dust samples, reference standards, and/or commercial diagnostic D. pteronyssinus extracts., Conclusions: Our results show that viral infections are common and widespread in D. pteronyssinus, both in natural and culture-based growth conditions. Potential effects on the mites themselves and consequences toward allergenicity in humans whether exposed naturally or after immunotherapy are discussed., (© 2021 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Bacteriophages (phages) are viruses that infect bacteria. They are the most abundant biological entity on Earth (current estimates suggest there to be perhaps 10 31 particles) and are found nearly everywhere. Temperate phages can integrate into the chromosome of their host, and prophages have been found in abundance in sequenced bacterial genomes. Prophages may modulate the virulence of their host in different ways, e.g., by the secretion of phage-encoded toxins or by mediating bacterial infectivity. Some 70% of Streptococcus pneumoniae (the pneumococcus)-a frequent cause of otitis media, pneumonia, bacteremia and meningitis-isolates harbor one or more prophages. In the present study, over 4000 S. pneumoniae genomes were examined for the presence of prophages, and nearly 90% were found to contain at least one prophage, either defective (47%) or present in full (43%). More than 7000 complete putative integrases, either of the tyrosine (6243) or serine (957) families, and 1210 full-sized endolysins (among them 1180 enzymes corresponding to 318 amino acid-long N -acetylmuramoyl-L-alanine amidases [LytA PPH ]) were found. Based on their integration site, 26 different pneumococcal prophage groups were documented. Prophages coding for tRNAs, putative virulence factors and different methyltransferases were also detected. The members of one group of diverse prophages (PPH090) were found to integrate into the 3' end of the host lytA Spn gene encoding the major S. pneumoniae autolysin without disrupting it. The great similarity of the lytA Spn and lytA PPH genes (85-92% identity) allowed them to recombine, via an apparent integrase-independent mechanism, to produce different DNA rearrangements within the pneumococcal chromosome. This study provides a complete dataset that can be used to further analyze pneumococcal prophages, their evolutionary relationships, and their role in the pathogenesis of pneumococcal disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martín-Galiano and García.)

The prefoldin complex (PFDc) was identified in humans as a co-chaperone of the cytosolic chaperonin T-COMPLEX PROTEIN RING COMPLEX (TRiC)/CHAPERONIN CONTAINING TCP-1 (CCT). PFDc is conserved in eukaryotes and is composed of subunits PFD1-6, and PFDc-TRiC/CCT folds actin and tubulins. PFDs also participate in a wide range of cellular processes, both in the cytoplasm and in the nucleus, and their malfunction causes developmental alterations and disease in animals and altered growth and environmental responses in yeast and plants. Genetic analyses in yeast indicate that not all of their functions require the canonical complex. The lack of systematic genetic analyses in plants and animals, however, makes it difficult to discern whether PFDs participate in a process as the canonical complex or in alternative configurations, which is necessary to understand their mode of action. To tackle this question, and on the premise that the canonical complex cannot be formed if one subunit is missing, we generated an Arabidopsis (Arabidopsis thaliana) mutant deficient in the six PFDs and compared various growth and environmental responses with those of the individual mutants. In this way, we demonstrate that the PFDc is required for seed germination, to delay flowering, or to respond to high salt stress or low temperature, whereas at least two PFDs redundantly attenuate the response to osmotic stress. A coexpression analysis of differentially expressed genes in the sextuple mutant identified several transcription factors, including ABA INSENSITIVE 5 (ABI5) and PHYTOCHROME-INTERACTING FACTOR 4, acting downstream of PFDs. Furthermore, the transcriptomic analysis allowed assigning additional roles for PFDs, for instance, in response to higher temperature., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Plants react to environmental challenges by integrating external cues with endogenous signals to optimize survival and reproductive success. However, the mechanisms underlying this integration remain obscure. While stress conditions are known to impact plant development, how developmental transitions influence responses to adverse conditions has not been addressed. Here, we reveal a molecular mechanism of stress response attenuation during the onset of flowering in Arabidopsis (Arabidopsis thaliana). We show that Arabidopsis MORF-RELATED GENE (MRG) proteins, components of the NuA4 histone acetyltransferase complex that bind trimethylated-lysine 36 in histone H3 (H3K36me3), function as a chromatin switch on the floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) to coordinate flowering initiation with plant responsiveness to hostile environments. MRG proteins are required to activate SOC1 expression during flowering induction by promoting histone H4 acetylation. In turn, SOC1 represses a broad array of genes that mediate abiotic stress responses. We propose that during the transition from vegetative to reproductive growth, the MRG-SOC1 module constitutes a central hub in a mechanism that tunes down stress responses to enhance the reproductive success and plant fitness at the expense of costly efforts for adaptation to challenging environments., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Many lactic acid bacteria (LAB) produce metabolites with applications in the food industry, such as dextran-type exopolysaccharides (EPS) and riboflavin (vitamin B 2 ). Here, 72 bacteria were isolated from sourdoughs made by Spanish bread-makers. In the presence of sucrose, colonies of 22 isolates showed a ropy phenotype, and NMR analysis of their EPS supported that 21 of them were dextran producers. These isolates were identified by their random amplified polymorphic DNA (RAPD) patterns and their rrs and pheS gene sequences as LAB belonging to four species ( Weissella cibaria , Leuconostoc citreum , Leuconostoc falkenbergense and Leuconostoc mesenteroides ). Six selected strains from the Leuconostoc (3) and Weissella (3) genera grew in the absence of riboflavin and synthesized vitamin B 2 . The EPS produced by these strains were characterized as dextrans by physicochemical analysis, and the L. citreum polymer showed an unusually high degree of branching. Quantification of the riboflavin and the EPS productions showed that the W. cibaria strains produce the highest levels (585-685 μg/and 6.5-7.4 g/L, respectively). Therefore, these new LAB strains would be good candidates for the development of fermented foods bio-fortified with both dextrans and riboflavin. Moreover, this is the first report of riboflavin and dextran production by L. falkenbergense .

Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Hypericum perforatum L. (St. John's wort) is a well-known medicinal plant that possesses secondary metabolites with beneficial pharmacological properties. However, improvement in the production of secondary metabolites via genetic manipulation is a challenging task as H. perforatum remains recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, the transcripts of key genes involved in several plant defence responses (secondary metabolites, RNA silencing, reactive oxygen species (ROS) and specific defence genes) were investigated in H. perforatum suspension cells inoculated with A. tumefaciens by quantitative real-time PCR. Results indicated that key genes from the xanthone, hypericin and melatonin biosynthesis pathways, the ROS-detoxification enzyme HpAOX, as well as the defence genes Hyp-1 and HpPGIP, were all upregulated to rapidly respond to A. tumefaciens elicitation in H. perforatum. By contrast, expression levels of genes involved in hyperforin and flavonoid biosynthesis pathways were markedly downregulated upon A. tumefaciens elicitation. In addition, we compared the expression patterns of key genes in H. perforatum leaf tissues with and without dark glands, a major site of secondary metabolite production. Overall, we provide evidence for the upregulation of several phenylpropanoid pathway genes in response to elicitation by Agrobacterium, suggesting that production of secondary metabolites could modulate H. perforatum recalcitrance to A. tumefaciens-mediated transformation.

Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a comprehensive view of lysins' structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G-) (higher net charge and amphipathic helices) are responsible for improved interaction with the G- envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecule design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G- surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages' host bacteria, and sequence-predicted physicochemical properties. Based on such classifiers, an investigation of the differential appearance of certain features was conducted. This analysis revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. In particular, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment, and aliphatic index) were associated with G- phage lysins, appearing specifically at their C-terminal end. Information on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts is provided, specifically supporting the nowadays-common hypothesis that lysins from G- usually contain AMP-like regions. IMPORTANCE Phage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The potential of lysins as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysin diversity landscape by examining a set of phage lysin genes. We have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures and, thus, the reach of their specialization regarding cell wall structures. These results provide clarity and evidence to sustain some of the common hypotheses in current literature, as well as making available an updated and characterized database of lysins sequences for further developments.

The interplay between the phytohormone abscisic acid (ABA) and the gasotransmitter nitric oxide (NO) regulates seed germination and post-germinative seedling growth. We show that GAP1 (germination in ABA and cPTIO 1) encodes the transcription factor ANAC089 with a critical membrane-bound domain and extranuclear localization. ANAC089 mutants lacking the membrane-tethered domain display insensitivity to ABA, salt, and osmotic and cold stresses, revealing a repressor function. Whole-genome transcriptional profiling and DNA-binding specificity reveals that ANAC089 regulates ABA- and redox-related genes. ANAC089 truncated mutants exhibit higher NO and lower ROS and ABA endogenous levels, alongside an altered thiol and disulfide homeostasis. Consistently, translocation of ANAC089 to the nucleus is directed by changes in cellular redox status after treatments with NO scavengers and redox-related compounds. Our results reveal ANAC089 to be a master regulator modulating redox homeostasis and NO levels, able to repress ABA synthesis and signaling during Arabidopsis seed germination and abiotic stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In an article in this issue of Environmental Microbiology, Segura et al. report the identification of an unusual global regulator in Novosphingobium sp. HR1a, a metabolically versatile bacterial strain isolated from the rhizosphere able to assimilate a wide range of polyaromatic hydrocarbons (PAHs). Physiological and transcriptomic assays suggest that this regulator, named PahT, activates the expression of genes involved in the assimilation of PAHs, and of compounds such as sugars and acetate, facilitating their co-metabolism. This effect is the opposite to the carbon catabolite repression strategy that allows metabolically versatile bacteria to favour the use of some compounds over others. PahT was found to stimulate sugar uptake and metabolization in the presence and absence of PAHs and to facilitate microaerobic respiration if PAHs were present. A survey of the genomes of several Sphingomonadaceae members showed that PahT is not present in all strains of this family, but that it is strongly associated with PAH degradation genes. Since not all PAH-degrading strains contain pahT, it seems that PahT is not essential for PAH degradation but likely provides a selective advantage to PAH-degrading strains in environments such as the rhizosphere where other potential carbon sources are available., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Phage (endo)lysins are nowadays one of the most promising ways out of the current antibiotic resistance crisis. Either as sole therapeutics or as a complement to common antibiotic chemotherapy, lysins are already entering late clinical phases to get regulatory agencies' authorization. Even the old paradigm of the inability of lysins to attack Gram-negative bacteria from without has already been overcome in a variety of ways: either by engineering approaches or investigating the natural mechanisms by which some wild-type lysins are able to interact with the bacterial surface. Such inherent ability of some lysins has been linked to antimicrobial peptide (AMP)-like regions, which are, on their own, a significant source for novel antimicrobials. Currently, though, many of the efforts for searching novel lysin-based antimicrobial candidates rely on experimental screenings. In this work, we have bioinformatically analyzed the C-terminal end of a collection of lysins from phages infecting the Gram-negative genus Pseudomonas . Through the computation of physicochemical properties, the probability of such regions to be an AMP was estimated by means of a predictive k -nearest neighbors ( k NN) model. This way, a subset of putatively membrane-interacting lysins was obtained from the original database. Two of such candidates (named Pae87 and Ppl65) were prospectively tested in terms of muralytic, bacteriolytic, and bactericidal activity. Both of them were found to possess an activity against Pseudomonas aeruginosa and other Gram-negative bacterial pathogens, implying that the prediction of AMP-like regions could be a useful approach toward the mining of phage lysins to design and develop antimicrobials or antimicrobial parts for further engineering., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vázquez, Blanco-Gañán, Ruiz and García.)

L3 larvae of anisakid nematodes are an important problem for the fisheries industry and pose a potential risk for human health by acting as infectious agents causing allergies and as potential vectors of pathogens and microrganisms. In spite of the close bacteria-nematode relationship very little is known of the anisakids microbiota. Fresh fish could be contaminated by bacteria vectored in the cuticle or in the intestine of anisakids when the L3 larvae migrate through the muscles. As a consequence, the bacterial inoculum will be spread, with potential effects on the quality of the fish, and possible clinical effects cannot be discarded. A total of 2,689,113 16S rRNA gene sequences from a total of 113 L3 individuals obtained from fish captured along the FAO 27 fishing area were studied. Bacteria were taxonomically characterized through 1803 representative operational taxonomic units (OTUs) sequences. Fourteen phyla, 31 classes, 52 orders, 129 families and 187 genera were unambiguously identified. We have found as part of microbiome an average of 123 OTUs per L3 individual. Diversity indices (Shannon and Simpson) indicate an extraordinary diversity of bacteria at an OTU level. There are clusters of anisakids individuals (samples) defined by the associated bacteria which, however, are not significantly related to fish hosts or anisakid taxa. This suggests that association or relationship among bacteria in anisakids, exists without the influence of fishes or nematodes. The lack of relationships with hosts of anisakids taxa has to be expressed by the association among bacterial OTUs or other taxonomical levels which range from OTUs to the phylum level. There are significant biological structural associations of microbiota in anisakid nematodes which manifest in clusters of bacteria ranging from phylum to genus level, which could also be an indicator of fish contamination or the geographic zone of fish capture. Actinobacteria, Aquificae, Firmicutes, and Proteobacteria are the phyla whose abundance value discriminate for defining such structures.

Trimethylamine N -oxide (TMAO) is a well-known naturally occurring osmolyte in animals that counteracts the effect of different denaturants related to environmental stress and has recently been associated with severe human chronic diseases. In plants, however, the presence of TMAO has not yet been reported. In this study, we demonstrate that plants contain endogenous levels of TMAO, that it is synthesized by flavin-containing monooxygenases, and that its levels increase in response to abiotic stress conditions. In addition, our results reveal that TMAO operates as a protective osmolyte in plants, promoting appropriate protein folding and as an activator of abiotic stress-induced gene expression. Consistent with these functions, we show that TMAO enhances plant adaptation to low temperatures, drought, and high salt. We have thus uncovered a previously unidentified plant molecule that positively regulates abiotic stress tolerance., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Pseudomonas aeruginosa is an important opportunistic human pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under different conditions and from diverse cellular compartments have been joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1, the highest coverage among bacterial PeptideAtlas datasets. The proteins included cover 89% of metabolic proteins, 72% of proteins involved in genetic information processing, 83% of proteins responsible for environmental information processing, more than 88% of the ones related to quorum sensing and biofilm formation, and 89% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. The manuscript describes the building of the PeptideAtlas and the contribution of the different proteomic data used. SIGNIFICANCE: Pseudomonas aeruginosa is among the most versatile human bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants represents an attractive alternative to transgene-based silencing approaches. However, improvement of dsRNA stability in natural conditions is required in order to provide long-term protection against the targeted virus. Here, we tested the protective effect of topical application of Escherichia coli -encapsulated dsRNA compared to naked dsRNA against single and dual infection by Potato virus X expressing the green fluorescent protein (PVX-GFP) and Potato virus Y (PVY) in Nicotiana benthamiana . We found that, in our conditions, the effectiveness of E. coli -encapsulated dsRNA in providing RNAi-mediated protection did not differ from that of naked dsRNA. dsRNA vaccination was partly effective against a dual infection by PVX-GFP and PVY, manifested by a delay in the expression of the synergistic symptoms at early times after inoculation. Using PVX-GFP as a reporter virus together with a suite of RNAi knockdown transgenic lines, we have also shown that RNA-directed RNA polymerase 6 and the combined activities of DICER-like 2 (DCL2) and DCL4 act to promote efficient resistance to virus infection conferred by topical application of dsRNA in N. benthamiana . Our results provide evidence that exogenous dsRNA molecules are processed by the RNA silencing pathways commonly used by the host in response to virus infection.

The recruitment and activation of polymorphonuclear neutrophils (PMNs) are of central importance for the elimination of pathogens in bacterial infections. We investigated the Streptococcus pneumoniae -dependent induction of the transcription factor Krüppel-like factor (KLF) 4 in PMNs as a potential regulator of PMN activation. We found that KLF4 expression is induced in human blood-derived PMNs in a time- and dose-dependent manner by wild-type S. pneumoniae and capsule knockout mutants. Unencapsulated knockout mutants induced stronger KLF4 expression than encapsulated wild types. The presence of autolysin LytA-competent (thus viable) pneumococci and LytA-mediated bacterial autolysis were required for KLF4 induction in human and murine PMNs. LyzMcre-mediated knockdown of KLF4 in murine blood-derived PMNs revealed that KLF4 influences pneumococci killing and increases the release of the proinflammatory cytokines tumor necrosis factor α and keratinocyte chemoattractant and decreases the release of the anti-inflammatory cytokine interleukin-10. Thus, S. pneumoniae induces KLF4 expression in PMNs, which contributes to PMN activation in S. pneumoniae infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bhattacharyya, Herta, Conrad, Frey, García, Suttorp, Hippenstiel and Zahlten.)

The appearance of carbapenem-resistant Klebsiella pneumoniae has increased the use of colistin as a last-resort antibiotic for treating infections by this pathogen. A consequence of its use has been the spread of colistin-resistant strains, in several cases carrying colistin resistance genes. In addition, when susceptible strains are confronted with colistin during treatment, mutation is a major cause of the acquisition of resistance. To analyze the mechanisms of resistance that might be selected during colistin treatment, an experimental evolution assay for 30 days using as a model the clinical K. pneumoniae kp52145 isolate in the presence of increasing amounts of colistin was performed. All evolved populations presented a decreased susceptibility to colistin, without showing cross-resistance to antibiotics belonging to other structural families. We did not find any common mutation in the evolved mutants, neither in already known genes, previously known to be associated with the resistance phenotype, nor in new ones. The only common genetic change observed in the strains that evolved in the presence of colistin was the amplification of a 34 Kb sequence, homologous to a prophage (Enterobacteria phage Fels-2). Our data support that gene amplification can be a driving force in the acquisition of colistin resistance by K. pneumoniae .

Background: The sustainable control of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), is compromised by the development of resistance to malathion and lambda-cyhalothrin in Spanish field populations. At present, field populations remain susceptible to spinosad. However, the resistant strain JW-100s has been obtained under laboratory selection with spinosad, and resistance has been associated with the presence of different mutations causing truncated transcripts of the α6 subunit of the nicotinic acetylcholine receptor (nAChRα6)., Results: An F1 screen assay followed by the molecular characterization of surviving flies has been used to search for spinosad-resistant alleles in field populations. Two different resistant alleles giving rise to truncated isoforms of Ccα6 have been identified, which corresponds to an estimated allelic frequency of at least 0.0023-0.0046. The fitness values of the resistant nAChRα6 alleles found in the laboratory strain JW-100s were estimated to be 0.4 for RR and 0.2 for SR. Mathematical modelling predicted that spinosad-resistant alleles will rapidly decline over time in field populations if their fitness cost was the same as estimated for laboratory-resistant alleles. However, they are predicted to increase in the field if their fitness cost is lower and resistance management strategies are not implemented., Conclusion: Spinosad-resistant alleles have been detected in field populations for the first time. Our modelling simulations indicate that the best option to delay the appearance of spinosad resistance would be its rotation with other insecticides without cross-resistance. The integrated F1 screen/molecular genetic analysis presented here can be used for future monitoring studies. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)