Aerobic endospore-forming bacteria (Bafes) are gram-positive microorganisms with low guanine-cytosine content in their genetic material. They can form spores as a survival strategy when faced with unfavorable environmental conditions. The spore consists of the vegetative cellular unit, with repressed metabolism and higher resistance to temperature, radiation, chemical agents, and predation of superior organisms. Bafes have several biotechnological applications: they produce bioinsecticides, detergents, textile materials, biofuels, and bioremediation of heavy metals. Bioremediation translates into removing, transforming, or reducing polluting agents from the environment, such as petroleum. Petroleum consists of a complex mixture of hydrocarbons mainly used asfuel, and, due to the high demand, accidents that contaminate soils and seas are frequent, disturbing the life cycle of these ecosystems. Given this scenario, finding bacteria capable of biodegrading petroleum and diesel oil is essential for building an effective bioremediation system. This work aims to select Bafes that can use hydrocarbons to obtain energy when petroleum and diesel S10 are offered as the only carbon source. The bacteria used in this study are Bafes isolated from the Distrito Federal soil (SDF) that are part of the CBafes collection of the Microbiology Laboratory/LaBafes of the University of Brasília. 50 SDF strains were selected for cultivation. After the first screening, 46 SDF strains were cultivated in a solid mineral medium supplemented with diesel oil as the sole carbon source. Out of 46, 9 strains grew in S10 diesel oil, and five strains were able to grow in the petroleum-containing medium. Two strains that showed more significant growth in the media used were chosen for quantification tests for total intracellular and secreted proteins, qualitative tests for bacterial oil consumption, tests for lipases and biosurfactants production, and the ability to adhere to hydrocarbons. Disparities were observed in the concentration of proteins in the different media tested, as well as the differences between intracellular and secreted proteins. The absence of the biosurfactant formation motivated the search for another test that could reveal the device used by the strains for contact between the oil and the cell, confirmed by the adhesion test of the strains to diesel oil. The robust and extensive search for diesel and petroleum degrading bacteria and their preliminary studies offers several promising possibilities for further research on microorganisms that bioremediate petroleum and derivatives.

Phospholipids are fundamental molecules for the structure and function of the plasma membrane of eukaryotic and prokaryotic cells, in addition to being involved in the process of adaptation to the environment and cell signaling. Currently, several studies explore phospholipid biosynthesis pathways as a potential therapeutic target against pathogenic fungi, therefore, this study aimed to investigate the role of phosphatidylcholine biosynthesis pathways in the virulence of the fungus Cryptococcus neoformans, through the construction and characterization of knockout mutants for the genes encoding putative enzymes of two biosynthetic pathways for this phospholipid. The mutants were obtained through molecular gene deletion techniques using Double Joint-Polymerase Chain Reaction (DJ-PCR) and bioballistics, and were subjected to phenotypic tests to evaluate different attributes associated with their virulence. The mutant of the OPI3 gene, from the de novo pathway, was significantly affected on growth in the absence of choline in the culture medium, while the opi3Δpct1Δ double mutant for both pathways can grow only in the presence of yeast extract, or on egg yolk agar. Both mutants induce larger polysaccharide capsules than wild-type KN99α, while in antifungal susceptibility tests, the mutant of the PCT1 gene, from the Kennedy alternative pathway, showed greater susceptibility to antifungals from the azoles class, such as fluconazole and itraconazole. The results indicate that these pathways are associated with other cellular mechanisms, in addition to maintaining the integrity of the plasma membrane

Plastics are fundamental materials for the daily life of human beings. In 2021, the production of plastics reached 390.7 million tons, the most produced plastics were polyethylene, polypropylene and polyvinyl chloride. Due to high production and inadequate disposal, environmental contamination by plastics has become one of the most relevant environmental problems, impacting the lives of animals, plants, and humans. Biodegradation of plastics using microorganisms or parts of them, such as enzymes, is one of the most sustainable ways of dealing with this waste. Although several studies with microorganisms have already been carried out, a plastic degradation profile has not yet been described and established. In this work, two enzymes, peroxiredoxin (BCP) and coniferyl aldehyde dehydrogenase (CalB) were selected from previous genomic and transcriptomic data of Delftia sp. when grown with high-density polyethylene. The main objective of this work is to perform the purification and characterization of these enzymes and to study their effects on polyethylene degradation, and other types of plastics. Furthermore, as a series of proteins known in the literature for being lignin-degrading were found in the genome and transcriptome data of Delftia sp., this bacterium was cultivated with synthetic dyes that mimic lignin structures to evaluate the biotechnological potential of this bacterium in lignin degradation. The enzyme BCP was purified using his-tag affinity chromatography and size exclusion chromatography and displayed activity in hydrogen peroxide removal. The protein CalB is on purification process. Delftia sp. cultured with synthetic dyes showed the ability to decolorize the dyes, indicating a possible lignin-degrading activity. The next study steps will be the purification of the CalB protein and characterization of the two proteins, identification of optimal pH and temperature, the effect of pH and temperature difference on the secondary structure of the enzymes, and the thermostability of the enzymes. Then, the experiments of the enzymes with different plastics will be carried out and the analysis of the plastic structure using FTIR and GC-MS to infer the occurrence of modification on the structure by the activity of the enzymes.

Halophile organisms grow in environments with high concentrations of salts and are distributed in the three domains of life: Bacteria, Eukarya and Archaea. Such environments include salt waters, soils, fermented salt foods and food salts. The presence of halophilic prokaryotes in food salts is common and has been described in several scientific works, however, there is no information about their occurrence in salts sold in Brazil. Thus, this work had as main objective the characterization of extreme halophilic prokaryotes present in food salts, initially through culture-dependent approaches. Aliquots of four commercial salts were inoculated into two culture media for halophiles (ATCC and MGM), with the addition of 3 or 4 M NaCl, in search of extreme halophile organisms. The results obtained suggest the presence of both bacteria and archaea in the analyzed salts, since microbial growth was observed in all analyzed media. Most of the colonies obtained had a reddish coloration, typical of halophilic archaea, while microscopic analysis revealed the presence of both cocci and bacilli, predominantly Gram negative. All cultures were subjected to DNA extraction, followed by PCR, with specific primers for Archaea and Bacteria. Amplicons obtained from some of the salts were then ligated into cloning vectors and transformed into Escherichia coli XL1-Blue cells. Recombinant clones were analyzed by DNA sequencing and the results reveal the presence of archaea whose 16S rRNA genes exhibit a high degree of identity with the respective genes of archaea of the genera Halobacterium and Halolamina.

Urinary tract infections (UTIs) are the most frequent cause of bacterial infections in humans. It is estimated that each year, approximately 150 million people are affected by this pathology worldwide. Responsible for 80%-90% of cases, the Gram-negative enterobacteria known as Uropathogenic Escherichia coli (UPEC), is the most common causative agent of community-acquired UTIs. The ability of UPEC to survive in the urinary tract depends as much on its physiology and metabolism as on its virulence determinants. Recently, it has been demonstrated that there is a link between the transport and metabolism of various carbohydrates and virulence in enterobacteria. However, as the precise role of carbohydrate utilization in the expression and regulation of bacterial virulence has not yet been determined, we seek to understand the impact that the metabolism of different carbon sources has on the expression of fitness and virulence factors in the pathogenesis of UPECs resistant to the multiple drugs (MDR), with emphasis on the expression of type 1 fimbriae. The objective of this work is to understand the effect that the use of different carbon sources has on the expression of genes and mechanisms of virulence of UPECs MDR strains. For this purpose, four different strains of MDR UPECs (representatives of the group A, B1, B2 and D) were submitted to the determination of the profile of resistance to antibiotics, the analysis of the capacity of survival and reproduction (bacterial fitness), of expressing type fimbriae 1 functional, to form biofilm using different carbon sources and to survive in blood and serum. In anaerobic conditions similar to those found along the urinary tract, all UPECs analyzed used D-(-)-Sorbitol as a high-yield carbon source. Three of the four analyzed strains do not use D-(-)-Fructose as a carbon source for their growth. Data obtained from the Yeast Agglutination Assay suggest that D-(-)-Fructose metabolism is related to the expression of functional type 1 fimbriae on the cell surface of three of the four strains analyzed. UPEC 76 did not use D-(-)-Fructose as a carbon source for its growth or to express functional type 1 fimbriae. N-acetyl-D-glucosamine metabolism promotes biofilm formation in UPECs MDR. When grown in the presence of N-acetyl-D-glucosamine as the sole carbon source, three of the four UPECs showed an increased ability to survive in blood and serum. Taken together, our data suggest that the metabolism of different carbon sources (sugars) modulate the expression of virulence factors such as exacerbated growth, expression of functional type 1 fimbriae, biofilm production and serum resistance in different strains of Uropathogenic Escherichia coli resistant to multiple drugs of clinical origin.

Corynebacterium glutamicum is a non-pathogenic bacterium widely used in the industrial production of amino acids, which ranks third worldwide among the most produced molecules through fermentation, reaching millions of tons with increasing numbers in the global market. C. glutamicum has been extensively studied as traditional methods used for amino acid production are cost-intensive, and alternatives are being sought to reduce production costs and increase productivity. In this context, proteomics has played a crucial role in understanding and improving the efficiency of amino acid production, as the understanding of biosynthetic pathways relies on the interpretation of proteomic analyses. Proteomics offers the possibility of describing and characterizing the functioning of metabolic pathways, including the identification of post-translational modifications (PTMs) and protein degradation. Thus, the present study aimed to identify and characterize proteins and protein complexes involved in the amino acid biosynthetic pathways of this bacterium, particularly in glutamate production with the influence of Tween 40, using a bottom-up proteomic approach with mass spectrometry, analyzing the intracellular and extracellular proteomic profiles. Analysis of amino acids secreted by the microorganism resulted in a glutamate concentration of 0.70 ± 0.14 mM after 18 hours of cultivation in the Tween 40 condition, while in the control condition, it was 0.04 ± 0.02 mM (t-test p<0.05). Protein complexes were separated via BN-PAGE electrophoresis, revealing intracellular protein complexes ranging from 20 kDa to 720 kDa. Main proteins involved in C. glutamicum metabolism were identified, such as enolase and glyceraldehyde-3-phosphate dehydrogenase, belonging to the glycolytic pathway, and glutamine synthetase, involved in glutamate biosynthesis. Analysis of the extracellular proteomic profile by SDS-PAGE showed proteins ranging from 10 to 80 kDa, suggesting differences in the abundance of some proteins between the conditions. Bottom-up analysis of the secretome by LC-MS/MS identified some more abundant regulated proteins in the Tween 40 condition, such as 2-oxoglutarate dehydrogenase, which is a precursor for L-glutamate synthesis. These results contribute to a better understanding of the biochemical machinery involved in the production and secretion of amino acids by Corynebacterium glutamicum can aid in the improvement of its scientific manipulation towards biotechnological interests.

The global energy demand and the concern with the development of a sustainable economy have encouraged the search for processes to obtain chemical compounds with the potential to replace, at least in part, those derived from non-renewable fossil sources. In this context, lignocellulosic biomass presents great potential as an alternative and renewable raw material for the production of high value-added compounds through bioprocesses. One of the main challenges in these processes refers to the presence of inhibitors in lignocellulosic hydrolysate, which are formed or released during the stages of pretreatment and hydrolysis of biomass. These are compounds that can inhibit microbial metabolism and, consequently, the yield and productivity of bioprocesses. The inhibitors are gathered in 3 main groups: organic acids (e.g., acetic, formic and levulinic acids), furaldehydes (furfural and 5-hydroxymethyl-2-furaldehyde) and phenolic compounds. Previous studies demonstrate the potential of the yeast Komagataella phaffii to remain active in the presence of these inhibitors. Thus, physiological and transcriptomic analyses of K. phaffii revealed genes with potential to increase the yeast tolerance to furaldehyde, acetic acid and sugarcane bagasse hydrolysate. This work aims to validate the expression of five putative genes - YJR096W, YDL124W, SAP30, FLR1 and 2661 - in the response of K. phaffii M12 in the presence of different inhibitors present in different fermentative culture conditions. Initially, each of the genes was amplified by PCR and cloned into the pGEM®-T Easy cloning vector. For cloning into the pKLD expression vector, under control of the PGK1 promoter, the BamHI/NotI and BcuI cloning sites were employed. Until then, K. phaffii strains expressing the YJR096W gene have been successfully constructed. Microplate and Erlenmeyer flask assays were performed to evaluate the responses of the recombinant strain to the inhibitors furfural, HMF and lignocellulosic hydrolysate. The control and recombinant strains showed the same growth profiles, substrate consumption and product formation in the presence of HMF 2 g/L, furfural 3 g/L and lignocellulosic hydrolysate 30%. Thus, under the culture conditions evaluated so far, overexpression of the YJR096W gene in K. phaffii M12 did not result in increased tolerance to the presence of the inhibitors furfural, HMF and lignocellulosic hydrolysate. The next steps involve overexpression of the remaining genes and validation of the recombinant strains.

A resistência antimicrobiana (RAM) acontece quando bactérias não respondem às terapias ofertadas tornando as infecções mais difíceis de serem tratadas podendo levar à morte do paciente. Serratia marcescens é uma enterobactéria responsável por causar infecções relacionadas à assistência à saúde (IRAS) e, frequentemente, apresentam RAM. Esta espécie é considerada um agente causador de infecções oportunistas de tratamentos desafiadores devido à presença de vários tipos de mecanismos promotores de RAM. Neste contexto, é fundamental que se amplie o estudo dos mecanismos de disseminação de RAM visando o diagnóstico rápido e preciso, além do rastreamento epidemiológico para interromper a cadeia de transmissão de RAM dentro do ambiente. No presente trabalho, Quatro (4) isolados de Serratia marcescens causadores de bacteremia em pacientes internos em uma Unidade de Terapia Intensiva (UTI) foram submetidos à determinação do perfil de susceptibilidade antimicrobiana e de epidemiologia molecular. A determinação do perfil de susceptibilidade antimicrobiana foi determinada por testes fenotípicos (antibiograma por métodos automatizados, disco difusão e imunocromatografia), genotípico para a detecção de beta-lactamases (PCR) e por genômica (sequenciamento WGS). O delineamento da cadeia de transmissão dos isolados foi determinado por epidemiologia molecular através da análise de fingerprinting de sequencias repetitivas intergênicas para enterobactérias (ERIC-PCR) e por pangenoma. As análises fenotípicas e de genômica para não susceptibilidade demonstram que todos os isolados se apresentaram resistentes a todos antimicrobianos disponíveis sendo classificados como pan-resistentes (PDR). A imunocromatografia identificou as carbapenemases KPC e NDM em todos os isolados, contudo a genotipagem por PCR identificou KPC apenas em 2 isolados e a genômica não identificou a enzima em nenhum deles. As análises de similaridade genética por ERIC-PCR e pangenomica determinou que os isolados se apresentaram filogeneticamente idênticos e, portanto, clonais. Em sua totalidade os resultados indicam que análises genômicas por WGS são ferramentas precisas e robustas para o diagnóstico de RAM, contudo a mesma deve ser realizada imediatamente após o isolamento bacteriano para evitar diagnósticos imprecisos devido a perdas de elementos genéticos instáveis, como no caso de blaKPC que codifica a enzima KPC. A análise de pan-genoma, também, demonstrou-se precisa para a identificação de clo

The study of viruses is extremely important for society, as it comprises the study of organisms responsible for causing numerous diseases. Regular monitoring of viruses isolated from cultures is very important with regard to plant virology. Fast and accurate identification of potentially dangerous viruses can prevent serious outbreaks in economically important crops. Currently, High Throughput Sequencing (HTS) technologies have become an affordable and powerful tool for this purpose. In this study, we evaluated the use of wastewater samples to monitor plant viruses using HTS analysis and RT-qPCR. Plant viruses belonging to 12 virus families were found, of which Virgaviridae, Solemoviridae, Tymoviridae, Alphaflexiviridae, Betaflexiviridae, Closteroviridae and Secoviridae were the most abundant with more than 20 species. In addition, a quarantine virus and a new species of tobamovirus were also discovered. The relevance of processed foods as a source of these viruses in wastewater was also maintained. Pepper tobamo mild mottle virus (PMMoV) was detected in abundance in samples of processed pepper-based foods and sewage samples, and latent common garlic carlavirus (GarCLV) was detected in smaller amounts in samples of dried and fresh garlic and sewage sample. This showed a high explanation between viral abundance in sewage and in processed food sources. The potential use of wastewater for virus research is discussed in this study. In addition to virus monitoring, it is also of great importance to study known viruses that may also have economic importance not only in the phytosanitary aspect, but also technologically, such as the use of expression vectors. Pepper ringpot virus (PepRSV) is the only tobravirus reported in Brazil so far. It has been used as a viral vector for the production of recombinant proteins. However, as it is a virus restricted to Brazil, it is still a little studied virus when compared to other species also used for this purpose. This work also aims to study a putative new ORF N1 of PepRSV, for which mutant constructions of ORF N1 were agroinfiltrated in leaves of Nicotiana benthamiana and its intracellular location, from the fusion of the protein with flowering proteins to be observed by confocal microscope. With the mutant constructions, we can observe distinct symptoms such as the activation of local lesions, wilting and dropping, previously not seen in the original infectious clones. ORFN1/GFP showed localization associated with the endoplasmic reticulum.

Lignocellulosic biomass is the most abundant renewable resource hence its utilization is fundamental to achieving a sustainable economy. Two of the main challenges in lignocellulose utilization by microorganisms are the efficient assimilation of sugars and microbial tolerance towards inhibitory molecules formed and released during pretreatment and hydrolysis of the material. Among them, acetic acid is the main acid released by hemicellulose deacetylation and exerts strong inhibitory effect on microbial growth, consequently hampering sugar assimilation and bioproducts yield. Transcriptional factor Haa1 previously described in S. cerevisiae is the main regulator of cellular response to physiological stress caused by acetic acid and therefore its overexpression is a potential strategy to improve yeast tolerance in the presence of such inhibitor. In this work, the performance of recombinant K. phaffii strains overexpressing homologous Haa1 transcription factor was evaluated in the presence of acetic acid and lignocellulosic hydrolysate. Additionally, the effect of Haa1 overexpression on xylonic acid by K. phaffii was also evaluated. Haa1 overexpression allowed higher tolerance to K. phaffii under 4 g·L-1 and 6 g·L-1 acetic acid reducing time for detoxification in 12 hours and increasing biomass production in 37%. The effect of Haa1 overexpression did not show significative improvement in xylonic acid production by K. phaffii when this was cultivated em fed batch with glucose and xylose. Results demonstrate efficiency of Haa1 overexpression towards acetic acid tolerance and open possibilities to enhance yeast performance on xylonic acid production.