RESUMO
Bacteria of the species Oceanotoga teriensis belong to the family Petrotogaceae, are Gram-negative bacilli, are moderately thermophilic and are included in the group of thiosulfate-reducing bacteria, being capable of significantly accelerating corrosion in metallic structures. However, no in-depth study on the genome, antibiotic resistance and mobile elements has been carried out so far. In this work, the isolation, phenotypic and genotypic characterization of the multi-resistant O. teriensis UFV_LIMV02 strain was carried out, from water samples from an offshore oil extraction platform in Rio de Janeiro (Brazil). We determined that the isolate has a genome of 2 812 778 bp in size, with 26â% GC content, organized into 34 contigs. Genomic annotation using Rapid Annotation using Subsystem Technology revealed the presence of genes related to resistance to antibiotics and heavy metals. By evaluating the antimicrobial resistance of the isolate using the disc diffusion technique, resistance was verified for the classes of antibiotics, beta-lactams, fluoroquinolones, aminoglycosides, sulfonamides, lincosamides and rifamycins, a total of 14 antibiotics. The search for genomic islands, prophages and defence systems against phage infection revealed the presence of five genomic islands in its genome, containing genes related to resistance to heavy metals and antibiotics, most of which are efflux pumps and several transposases. No prophage was found in its genome; however, nine different defence systems against phage infection were detected. When analysing the clustered regularly interspaced short palindromic repeat (CRISPR) systems, four CRISPR arrays, classified as types I-B and III-B, with 272 spacers, can provide the strain with immunity to different mobile genetic elements and bacteriophage infection. The results found in this study show that the isolate UFV_LIVM02 is an environmental bacterium, resistant to different classes of antibiotics, and that the proteins encoded by the predicted genomic islands may be associated with the development of greater resistance to antibiotics and heavy metals. They provide evidence that environmental bacteria found in offshore oil exploration residues may pose a risk for the spread of antibiotic resistance genes. More comprehensive studies on the microbial community present in oil waste are needed to assess the risks of horizontal gene transfer.
RESUMO
Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µgâ¯ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µgâ¯ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µgâ¯ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µgâ¯ml-1) and P. aeruginosa P2307 (65.00 µgâ¯ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at â MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.
Assuntos
Antibacterianos , Endopeptidases , Glucanos , Polimixina B , Fagos de Salmonella , Endopeptidases/farmacologia , Endopeptidases/química , Endopeptidases/metabolismo , Polimixina B/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Fagos de Salmonella/genética , Fagos de Salmonella/fisiologia , Fagos de Salmonella/química , Glucanos/química , Glucanos/farmacologia , Animais , Testes de Sensibilidade Microbiana , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/virologia , Camundongos , Salmonella typhimurium/virologia , Salmonella typhimurium/efeitos dos fármacos , Bacteriófagos/fisiologia , Bacteriófagos/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia , Proteínas Virais/químicaRESUMO
OBJECTIVES: Bovine seminal plasma proteins perform several functions related to sperm function. Changes in the expression pattern or abundance of seminal proteins are related to changes in the fertilizing capacity of bulls. Considering the role of seminal plasma proteins in sperm function and animal reproduction, we investigated changes in the protein abundance profile in response to sperm morphological changes using a proteomic approach. DATADESCRIPTION: In our present investigation, we employed liquid chromatography coupled with mass spectrometry to elucidate the proteomic composition of seminal plasma obtained from Nellore bulls exhibiting varying percentages of sperm abnormalities. Following semen collection, seminal plasma was promptly isolated from sperm, and proteins were subsequently precipitated, enzymatically digested using porcine trypsin, and subjected to analysis utilizing the Acquity nano UHPLC System in conjunction with a mass spectrometer. This dataset encompasses a total of 297 proteins, marking the inaugural instance in which a comparative profile of seminal plasma proteins in young Nellore bulls, categorized by their sperm abnormality percentages, has been delineated using LC-MS/MS. The comprehensive nature of this dataset contributes pivotal proteomic insights, representing a noteworthy advancement in our understanding of the reproductive biology of the Nellore breed.
Assuntos
Proteoma , Sêmen , Espermatozoides , Animais , Masculino , Bovinos , Sêmen/metabolismo , Sêmen/química , Proteoma/metabolismo , Espermatozoides/metabolismo , Espectrometria de Massas em Tandem , Proteômica/métodos , Proteínas de Plasma Seminal/metabolismo , Proteínas de Plasma Seminal/genética , Cromatografia LíquidaRESUMO
Aluminum (Al) toxicity limits crop production worldwide. Although studies have identified genes associated with Al tolerance in crops, a large amount of data remains unexplored using other strategies. Here, we searched for single substitutions and InDels across differentially expressed genes (DEGs), linked DEGs to Al-tolerance QTLs reported in the literature for common maize, and investigated the alternative splicing regulated by Al3+ toxicity. We found 929 substitutions between DEGs in Al-tolerant and 464 in Al-sensitive inbred lines, of which 165 and 80 were non-synonymous, respectively. Only 12 NS variants had deleterious predicted effect on protein function in Al-tolerant and 13 in Al-sensitive. Moreover, 378 DEGs were mapped in Al-QTL regions for the Al-tolerant and 213 for the Al-sensitive. Furthermore, Al stress is primarily regulated at the transcriptional level in popcorn. Important genes identified, such as HDT1, SWEET4a, GSTs, SAD9, PIP2-2, CASP-like 5, and AGP, may benefit molecular assisted popcorn breeding or be useful in biotechnological approaches. These findings offer insights into the mechanisms of Al tolerance in popcorn and provide a 'hypothesis-free' strategy for identifying and prioritizing candidate genes that could be used to develop molecular markers or cultivars resilient to acidic soils.
Assuntos
Alumínio , Transcriptoma , Alumínio/toxicidade , Zea mays/genética , Produtos Agrícolas , Processamento AlternativoRESUMO
This study aimed to evaluate the proteomic profile of seminal plasma from young Nellore bulls. We used 20 bulls aged between 19.8 and 22.7 months, divided into two groups according to the results of the Breeding Soundness Evaluation (BSE): approved (FIT n = 10) and not approved (UNFIT n = 10). The scrotal perimeter was measured and a semen collection was performed through electroejaculation. The percentage of sperm motility, mass motility, and sperm vigor were calculated using conventional microscopy, and the percentage of sperm abnormalities was calculated using phase-contrast microscopy of all ejaculates. Seminal plasma was separated from spermatozoa using centrifugation and processed for proteomic analysis by LC-MS/MS. Seminal plasma proteins were identified using MASCOT Daemon software v.2.4.0 and label-free quantification analysis was carried out by SCAFFOLD Q+ software v.4.0 using the Exponentially Modified Protein Abundance Index (emPAI) method. Functional classification of proteins was performed based on their genetic ontology terms using KOG. Functional cluster analysis was performed on DAVID. There were no differences in scrotal perimeter and physical semen characteristics between FIT and UNFIT groups of bulls. The percentage of sperm abnormalities was higher (p < 0.05) in the UNFIT group of bulls. A total of 297 proteins were identified for the two groups. There were a total of 11 differentially abundant proteins (p < 0.05), two of them more abundant in FIT bulls (Spermadhesin-1 and Ig gamma-1 chain C region) and nine in UNFIT bulls (Vasoactive intestinal peptide, Metalloproteinase inhibitor 2, Ig lambda-1 chain C regions, Protein FAM3C, Hemoglobin beta, Seminal ribonuclease, Spermadhesin 2, Seminal plasma protein BSP-30kDa, and Spermadhesin Z13). Spermadhesin-1 was the protein with the highest relative abundance (36.7%) in the seminal plasma among all bulls, corresponding to 47.7% for the FIT bulls and 25,7% for the UNFIT bulls. Posttranslational modification, protein turnover, and chaperones were the functional categories with the highest number of classified proteins. Protein functional annotation clusters were related to Phospholipid efflux, ATP binding, and chaperonin-containing T-complex. The differentially abundant proteins in the group of FIT bulls were related to sperm capacitation and protection against reactive species of oxygen. In contrast, differentially expressed proteins in the group of UNFIT bulls were related to motility inhibition, intramembrane cholesterol removal and oxidative stress. In conclusion, the proteomic profile of the seminal plasma of FIT bulls presents proteins with participation in several biological processes favorable to fertilization, while the proteins of the seminal plasma of UNFIT bulls indicate a series of alterations that can compromise the fertilizing capacity of the spermatozoa. In addition, the relative abundance of spermadhesin-1 found in the seminal plasma of young Nellore bulls could be studied as a reproductive parameter for selection.
RESUMO
Spathaspora passalidarum is a xylose-fermenting microorganism promising for the fermentation of lignocellulosic hydrolysates. This yeast is more sensitive to ethanol than Saccharomyces cerevisiae for unclear reasons. An RNA-seq experiment was performed to identify transcriptional changes in S. passalidarum in response to ethanol and gain insights into this phenotype. The results showed the upregulation of genes associated with translation and the downregulation of genes encoding proteins involved in lipid metabolism, transporters, and enzymes from glycolysis and fermentation pathways. Our results also revealed that genes encoding heat-shock proteins and involved in antioxidant response were upregulated, whereas the osmotic stress response of S. passalidarum appears impaired under ethanol stress. A pseudohyphal morphology of S. passalidarum colonies was observed in response to ethanol stress, which suggests that ethanol induces a misperception of nitrogen availability in the environment. Changes in the yeast fatty acid profile were observed only after 12 h of ethanol exposure, coinciding with the recovery of the yeast xylose consumption ability. These findings suggest that the lack of fast membrane lipid adjustments, the halt in nutrient absorption and cellular metabolism, and the failure to induce the expression of osmotic stress-responsive genes are the main aspects underlying the low ethanol tolerance of S. passalidarum. KEY POINTS: ⢠Ethanol stress halts Spathaspora passalidarum metabolism and fermentation ⢠Genes encoding nutrient transporters showed downregulation under ethanol stress ⢠Ethanol induces a pseudohyphal cell shape, suggesting a misperception of nutrients.
RESUMO
Sexual rest is a transient condition, which compromises conception rates, characterized by large volumes of ejaculate with high percentages of dead sperm observed in bulls. The biochemical mechanisms leading to this ejaculate pattern are not fully understood. Six adult resting Nellore bulls were submitted to Breeding Soundness Evaluation by four consecutive semen collections through the electroejaculation method during a 30 min period. Each ejaculate had its semen phenotypic parameters; morphology and physical aspects were evaluated. To assess enzymatic activity (superoxide dismutase, catalase, and glutathione S-transferase), lipid peroxidation (concentrations of malondialdehyde and nitric oxide), fatty acid, and proteomic profile aliquots of spermatozoa from the first and fourth ejaculates were used. All sperm parameters differed between the first and fourth ejaculates. Spermatozoa from the first ejaculate showed lower enzymatic activity and a higher concentration of lipid peroxidation markers. Among the 19 identified fatty acids, 52.7% are polyunsaturated. Relative abundance analysis showed that C12:0 and C18:0 fatty acids differed between the first and fourth ejaculates, being the fourth ejaculate richer in spermatozoa. The proteomics analysis identified a total of 974 proteins in both sample groups (first and fourth ejaculates). The majority of identified proteins are related to cellular processes and signaling. Quantitative proteomics showed 36 differentially abundant proteins, 6 up-regulated proteins in the first ejaculate, and 30 up-regulated proteins in the fourth ejaculate. Spermatozoa from bulls at sexual rest have less antioxidant capacity, causing changes in their fatty acid composition and protein profile, which generates the observed sperm pattern and lower fertilization capacity.
Assuntos
Proteômica , Sêmen , Masculino , Bovinos , Animais , Espermatozoides , Análise do Sêmen/veterinária , Estresse Oxidativo , Ácidos Graxos , Motilidade dos EspermatozoidesRESUMO
Introduction: Bacteriophages infecting human pathogens have been considered potential biocontrol agents, and studying their genetic content is essential to their safe use in the food industry. Tequatrovirus ufvareg1 is a bacteriophage named UFV-AREG1, isolated from cowshed wastewater and previously tested for its ability to inhibit Escherichia coli O157:H7. Methods: T. ufvareg1 was previously isolated using E. coli O157:H7 (ATCC 43895) as a bacterial host. The same strain was used for bacteriophage propagation and the one-step growth curve. The genome of the T. ufvareg1 was sequenced using 305 Illumina HiSeq, and the genome comparison was calculated by VIRIDIC and VIPTree. Results: Here, we characterize its genome and compare it to other Tequatrovirus. T. ufvareg1 virions have an icosahedral head (114 x 86 nm) and a contracted tail (117 x 23 nm), with a latent period of 25 min, and an average burst size was 18 phage particles per infected E. coli cell. The genome of the bacteriophage T. ufvareg1 contains 268 coding DNA sequences (CDS) and ten tRNA genes distributed in both negative and positive strains. T. ufvareg1 genome also contains 40 promoters on its regulatory regions and two rho-independent terminators. T. ufvareg1 shares an average intergenomic similarity (VIRIDC) of 88.77% and an average genomic similarity score (VipTree) of 88.91% with eight four reference genomes for Tequatrovirus available in the NCBI RefSeq database. The pan-genomic analysis confirmed the high conservation of Tequatrovirus genomes. Among all CDS annotated in the T. ufvareg1 genome, there are 123 core genes, 38 softcore genes, 94 shell genes, and 13 cloud genes. None of 268 CDS was classified as being exclusive of T. ufvareg1. Conclusion: The results in this paper, combined with other previously published findings, indicate that T. ufvareg1 bacteriophage is a potential candidate for food protection against E. coli O157:H7 in foods.
Assuntos
Bacteriófagos , Escherichia coli O157 , Humanos , Escherichia coli O157/genética , Bacteriófagos/genética , Genoma , Genômica , Sequência de BasesRESUMO
Mastitis, mainly caused by bacterial intramammary infections, is the main problem in the breeding of dairy animals. The inflammations of the mammary gland is separated by types of mastitis, being subclinical, clinical, and the most severe, gangrenous mastitis. Here, we used 16S rRNA amplicon sequencing to characterize the bacterial microbiota of goat milk in the different types of goat mastitis caused by bacteria. We used 72 goat milk samples from a region of the state of Minas Gerais in Brazil, of which 12 were from clinically healthy animals, 42 from animals diagnosed with subclinical mastitis, 16 from animals with clinical mastitis, and 2 from animals with gangrenous mastitis. The group related to gangrenous mastitis was the most divergent in terms of alpha and beta diversity. The most abundant genus among samples of the groups was Staphylococcus spp., and we found a high abundance of Mycoplasma sp. in the milk of animals diagnosed with clinical mastitis. The most statistically relevant microorganisms among the groups were Prevotella sp., Ruminococcaceae, Prevotella ruminicola sp., and Providencia sp. We highlight a new association of bacterial agents in gangrenous mastitis among Escherichia sp./Shigella sp. and Enterococcus sp. and provide the second report of the genus Alkalibacterium sp., in milk samples. Only the taxa Staphylococcus sp., Bacteroides sp., Enterococcus, and Brevidabacterium sp., were present in all groups. The superpathway of L-tryptophan biosynthesis metabolites and the sucrose degradation III (sucrose invertase) pathway were the most prominent ones among the groups. In this study, we demonstrate how a rich microbiota of goat milk from healthy animals can be altered during the aggravation of different types of mastitis, in addition to demonstrating new bacterial genera in milk not previously detected in other studies as well as new associations between agents.
RESUMO
Endolysins are bacteriophage-derived lytic enzymes with antimicrobial activity. The action of endolysins against Gram-negative bacteria remains a challenge due to the physical protection of the outer membrane. However, recent research has demonstrated that signal-anchor-release (SAR) endolysins permeate the outer membrane of Gram-negative bacteria. This study investigates 2628 putative endolysin genes identified in 183,298 bacteriophage genomes. Previously, bioinformatic approaches resulted in a database of 66 SAR endolysins. This manuscript almost doubles the list with 53 additional SAR endolysin candidates. Forty-eight of the putative SAR endolysins described in this study contained one muramidase catalytic domain, and five included additional cell wall-binding domains at the C-terminus. For the moment, SAR domains are found in four protein families: glycoside hydrolase family 19 (GH19), glycoside hydrolase family 24 (GH24), glycoside hydrolase family 25 (GH25), and glycoside hydrolase family 108 (GH108). These SAR lysis are clustered in eight groups based on biochemical properties and domain presence/absence. Therefore, in this study, we expand the arsenal of endolysin candidates that might act against Gram-negative bacteria and develop a consult database for antimicrobial proteins derived from bacteriophages.