The Evolution of the Genus Bacteroides in the House Mouse Species Complex

The mammalian intestinal tract harbors a complex community of microorganisms that share an ancient evolutionary history and establish mutually beneficial relationships with their hosts. Despite inter-individual variation in gut microbial community composition, the major bacterial phyla remain conserved over the time of mammalian evolution. However, much less is known about evolutionary processes in the mammalian gut. Bacteroides are dominant intestinal bacteria and linked to many health-related traits of the host. Despite the importance of this genus, only a few species are well studied. Thus, there is still a lack of information regarding the patterns of within-species diversity across different host species, which could be linked to potential local adaption to different host environments. Using the house mouse species complex as a model, I first aimed to identify potential signatures of differentiation in Bacteroides abundance according to host subspecies. By performing a geographical survey of Mus musculus musculus and M. m. domesticus gut microbiota using 16S rRNA gene sequencing, I found host subspecies to play minor role in gut community structure compared to the impact of geography. Nevertheless, indicator species analysis of the Bacteroides genus identified consistent host subspecies-Bacteroides associations across different geographic locations. Next, I aimed to characterize candidate Bacteroides taxa [strain level amplicon sequence variants (ASVs)] and identify the differences in their genomes that might contribute to bacterial adaptation to the different mouse subspecies. For this, a combination of culturing and genomic analysis methods was used, which yielded fully sequenced genomes of 146 Bacteroides isolates. Taxonomic classification indicates that two potentially new Bacteroides species were isolated, along with B. acidifaciens, B. caecimuris and B. sartorii strains. Furthermore, a perfect match between a candidate indicator Bacteroides ASV, which strongly associates to M. m. musculus, and both unclassified isolates was detected, suggesting the involvement of this potentially new Bacteroides species in the intriguing host-microbe association. Finally, I aimed to identify contact-independent antagonistic interactions between gut-associated Bacteroides strains among these two house mouse subspecies. I found some Bacteroides isolates to engage in antagonistic interactions, and the observed inhibitory interactions seem to occur mostly between isolates belonging to different Bacteroides species (inter-species antagonism) and to different mouse populations than between strains isolated from different host subspecies. In conclusion, the present work is the first study systematically investigating gut-associated Bacteroides among two house mouse subspecies. Strong host-Bacteroides associations were identified to be consistent across different geographic locations. Whole genome sequencing of the isolated strains sheds light on the Bacteroides pan genome in terms of protein content and functions. Moreover, this is the first study identifying antagonistic interactions among mouse-associated B. acidifaciens, B. caecimuris and B. sartorii strains isolated from two host subspecies

Actividade moduladora de extracto de Ginkgo biloba no ciclo celular de Saccharomyces cerevisiae e capacidade de reparação de danos de DNA em células sob stresse replicativo

Dissertação de mestrado em Biotecnologia e Bio-empreendedorismo em Plantas Aromáticas e MedicinaisGinkgo biloba L. possui uma ampla gama de actividades biológicas e o seu extracto padronizado comercial, EGb 761, tem sido um dos produtos medicinais mais vendidos em todo o mundo, devido à sua actividade antioxidante. Efeitos benéficos sobre o sistema nervoso central, incluindo efeitos nos pacientes da doença de Alzheimer, e regulação da expressão genética relacionam-se com os dois principais constituintes do extracto de G. biloba: ginkgolidos e bilobalida. Entre os genes modulados pelo EGb 761, existem vários envolvidos na regulação do ciclo celular, como o gene XPC de mamíferos. A regulação do ciclo celular pode ser crucial para a sobrevivência do organismo, uma vez que, danos de DNA não reparados antes do processo de replicação, podem provocar mutações prejudiciais para as células filhas. O objectivo do presente trabalho foi a investigação dos efeitos de extracto vegetal obtido a partir das folhas de G. biloba, no ciclo celular. Os ensaios envolveram incubações de células de Saccharomyces cerevisiae com o extracto de G. biloba antes da exposição ao choque oxidativo, provocado pelo peróxido de hidrogénio. A estimativa das proporções celulares em cada fase do ciclo celular foi efectuada através da contagem das células ao longo do tempo e da determinação dos índices de gemulação. Foram efectuadas ainda as medições de conteúdo de DNA nas células, através de citometria de fluxo. A capacidade de reparação de danos de DNA nas células com o ciclo celular parado e expostas ao choque oxidativo, também foi investigado. Os ensaios envolveram a incubação das células na presença da hidroxiurea, seguida de exposição a H2O2. Os danos e a reparação de DNA foram determinados através do ensaio cometa. Os resultados demonstraram o efeito protector do extracto de G. biloba contra os danos de DNA provocados pelo H2O2 nas células de levedura, promovendo o progressão do ciclo celular após o choque oxidativo. Além disso, as células sob stress replicativo são menos susceptíveis a danos oxidativos de DNA. No entanto, a hidroxyurea não provoca alterações na dinâmica de reparação de DNA.Ginkgo biloba L. is a plant with a wide range of biological activities and its leaf extract (EGb 761) has been one of the best-selling medicinal products worldwide due to its antioxidant activity. Beneficial effects on the central nervous system, including in patients of Alzheimer's disease, and regulation of gene expression have been related to two main compounds of G. biloba extract: ginkgolides and bilobalide. Among EGb 761 modulated genes, are several involved in cell cycle regulation, like mammalian XPC gene. DNA lesions, which are not repaired before replication, may propagate harmful mutations to the daughter cells, thus, cell cycle control and regulation is crucial to the organism’s survival. DNA damage checkpoints temporarily block the cell cycle progression in G1 or G2 phases in response to genotoxic stress, that allow cells to repair damaged DNA. The aim of the present work was the investigation of G. biloba leaf extract effects on cell cycle. The experiments involved incubation of Saccharomyces cerevisiae cells with G. biloba leaf extract, before the oxidative shock by hydrogen peroxide. Subsequent estimation of cell proportions in each phase of cell cycle by the budding index approach was performed, as well as measurement of cells DNA content by flow cytometry. DNA damage repair ability in yeast cells under replicative stress, exposed to the oxidative shock, was also investigated. Typical experiments included incubation of cells in the presence of hydroxyurea, followed by exposure to hydrogen peroxide. Determination of DNA damage and repair was performed using the comet assay. The results indicate that G. biloba extract protects yeast cells against DNA damage imposed by hydrogen peroxide, promoting progression of the cell cycle after oxidative shock. This protective effect is probably related to a lower degree of DNA damage and higher repair ability. In addition, cells under replicative stress are less susceptible to oxidative DNA damage, however, hydroxyurea does not provoke changes in the dynamics of DNA damage repair ability

Enterobactin : like siderophore gene cluster induction in Cronobacter sakazakii strain

Species belonging to the recently described Cronobacter genus, include several opportunistic foodborne pathogens. These pathogens are capable of causing severe infections in neonates, such as meningitis, septicaemia and necrotizing enterocolitis. Bacterial virulence has been previously correlated with ferric iron acquisition systems. It is known that all plasmid-harbouring Cronobacter species produce an active aerobactin-like siderophore (cronobactin) and the chromosome also contains genes encoding an enterobactin-like siderophore whose production has not been detected so far. Nevertheless it has been determined that, in vivo, enterobactin is upregulated in iron limiting conditions. This work aims to demonstrate that the cluster encoding for the enterobactin-like siderophore synthesis is functional and responsible for the molecule synthesis. To stimulate enterobactin production, the strain used grown in an optimized medium under particular conditions. Overall, siderophore production was detected by using Chrome Azurol S (CAS) indicator solution. A bioinformatic analysis was carried out to identify and annotate the genes in the enterobactin clusters. Genes predicted to be involved in the biosynthesis were mutated by performing Campbell insertions. Enterobactin presence/absence in the wild type and mutant strains, was determined by High Performance Liquid Chromatography. Simultaneously genome analysis showed that some Cronobacter/Enterobacter strains shared iron-siderophore complex receptors, an indicator of the ability to uptake siderophores from unrelated species. This capacity was tested by cross-feeding assays in CAS agar. Results showed that the cluster encoding for the enterobactin-like siderophore is indeed functional and that this molecule might be utilized by other Cronobacter/Enterobacter species

Analysis of DNA damage and repair in saccharomyces cerevisiae using the comet assay in the characterization of antigenotoxicity of plant extracts and phytochemicals

In this work we used the model organism Saccharomyces cerevisiae to characterise the biological activity and the mechanism of action of phytochemicals. One of the goals is to use mutant strains affected in basic mechanisms of oxidative stress response and DNA repair in order to uncover the molecular targets of phytochemicals. We have assessed DNA damage and repair using the comet assay, evaluated as “comet tail length”, which displayed a dose-response relationship with different DNA-damaging agents1. Subsequently, we used this system to assess the antigenotoxic properties of a leaf extract from Ginkgo biloba (GBE). Typical experiments involved incubation of yeast cells, or spheroplasts, with GBE before and during oxidative shock with hydrogen peroxide. Our results obtained with the comet assay show that DNA damage was significantly decreased upon GBE treatment in a dose-dependent manner. In addition, DNA repair kinetics was significantly improved in cells incubated with GBE. However, in the mutant strain affected in CDC9, encoding a DNA ligase involved in the mechanisms of nucleotide excision repair and base excision repair, oxidative DNA damage repair kinetics was unchanged with GBE, suggesting that the activity of this extract involves one of these mechanisms, or both. Hydrogen peroxide-induced cell cycle arrest in G2 was abolished when cells were incubated with GBE after oxidative shock, suggesting that the improved repair kinetics allows progression of the cell cycle and/or GBE can have a direct effect on its regulation. As expected, GBE treatment improved survival of yeast cells when challenged with oxidative shock with H2O2 and intracellular oxidation was considerably decreased upon pre-treatment with GBE as revealed by flow cytometry. Reference(s) 1. Azevedo F., Marques, F., Fokt, H., Oliveira, R. and Johansson, B. (2011) Measuring oxidative DNA damage and DNA repair using the yeast comet assay. Yeast, 28, 55-6

Bacteriocin production by Escherichia coli during biofilm development

Escherichia coli is a highly versatile bacterium ranging from commensal to intestinal pathogen, and is an important foodborne pathogen. E. coli species are able to prosper in multispecies biofilms and secrete bacteriocins that are only toxic to species/strains closely related to the producer strain. In this study, 20 distinct E. coli strains were characterized for several properties that confer competitive advantages against closer microorganisms by assessing the biofilm-forming capacity, the production of antimicrobial molecules, and the production of siderophores. Furthermore, primer sets for E. coli bacteriocins–colicins were designed and genes were amplified, allowing us to observe that colicins were widely distributed among the pathogenic E. coli strains. Their production in the planktonic phase or single-species biofilms was uncommon. Only two E. coli strains out of nine biofilm-forming were able to inhibit the growth of other E. coli strains. There is evidence of larger amounts of colicin being produced in the late stages of E. coli biofilm growth. The decrease in bacterial biomass after 12 h of incubation indicates active type I colicin production, whose release normally requires E. coli cell lysis. Almost all E. coli strains were siderophore-producing, which may be related to the resistance to colicin as these two molecules may use the same transporter system. Moreover, E. coli CECT 504 was able to coexist with Salmonella enterica in dual-species biofilms, but Shigella dysenteriae was selectively excluded, correlating with high expression levels of colicin (E, B, and M) genes observed by real-time PCR.info:eu-repo/semantics/publishedVersio

DNA damage repair ability in cell cycle arrested saccharomyces cerevisiae cells

Cells are exposed permanently to different types of environmental stress and to stress caused by oxidative metabolism in mitochondria that target macromolecules like DNA. Although cells dispose a range of stress response mechanisms, balance between DNA damage-causing stress and DNA repair capacity is crucial. Cells have evolved molecular repair mechanisms in case of DNA damage so that genome stability is maintained. Once DNA damage occurs, cell cycle is arrested, allowing the focus of all resources needed for the repair process. Hydroxyurea (HU), a compound that blocks the synthesis of deoxynucleotides, inhibiting DNA synthesis, is able to induce cells to arrest in S phase of the cell cycle. In the present study we aim to investigate the effect of cell cycle arrest on the DNA repair capacity of Saccharomyces cerevisiae cells treated with hydrogen peroxide. We used the comet assay, previously optimized for yeast in our laboratory, in cells grown in medium containing HU and then submitted to oxidative shock. In this test, cells are embedded on agarose, lysed and a short electrophoresis is applied in order to move de genomic DNA out of the nucleoid. The length of the "DNA tail" correlates with DNA damage. Determination of DNA damage and repair was performed through the measurement of the DNA tail length immediately after the oxidative shock and after incubation at 30ºC for different time points, respectively. Also, we have tested HU on cells pre-treated with Ginkgo biloba L. leaves extract (GBE), suggested by previous studies by our group to have an antigenotoxic effect on cells challenged with oxidative shock. Preliminary results obtained show that cells grown in medium containing HU are less susceptible to oxidative stress provoked by different concentrations of H2O2, compared to the control situation. In addition, pre-incubation with GBE improved DNA damage repair. Thus, these results suggest that cells with arrested cell cycle are more efficient in DNA damage repair and less susceptible to oxidative stress. Furthermore, we show evidence suggesting that GBE improves this repair process in cell cycle-arrested cells

The archaeome in metaorganism research, with a focus on marine models and their bacteria–archaea interactions

Metaorganism research contributes substantially to our understanding of the interaction between microbes and their hosts, as well as their co-evolution. Most research is currently focused on the bacterial community, while archaea often remain at the sidelines of metaorganism-related research. Here, we describe the archaeome of a total of eleven classical and emerging multicellular model organisms across the phylogenetic tree of life. To determine the microbial community composition of each host, we utilized a combination of archaea and bacteria-specific 16S rRNA gene amplicons. Members of the two prokaryotic domains were described regarding their community composition, diversity, and richness in each multicellular host. Moreover, association with specific hosts and possible interaction partners between the bacterial and archaeal communities were determined for the marine models. Our data show that the archaeome in marine hosts predominantly consists of Nitrosopumilaceae and Nanoarchaeota, which represent keystone taxa among the porifera. The presence of an archaeome in the terrestrial hosts varies substantially. With respect to abundant archaeal taxa, they harbor a higher proportion of methanoarchaea over the aquatic environment. We find that the archaeal community is much less diverse than its bacterial counterpart. Archaeal amplicon sequence variants are usually host-specific, suggesting adaptation through co-evolution with the host. While bacterial richness was higher in the aquatic than the terrestrial hosts, a significant difference in diversity and richness between these groups could not be observed in the archaeal dataset. Our data show a large proportion of unclassifiable archaeal taxa, highlighting the need for improved cultivation efforts and expanded databases

Data_Sheet_1_The archaeome in metaorganism research, with a focus on marine models and their bacteria–archaea interactions.docx

Metaorganism research contributes substantially to our understanding of the interaction between microbes and their hosts, as well as their co-evolution. Most research is currently focused on the bacterial community, while archaea often remain at the sidelines of metaorganism-related research. Here, we describe the archaeome of a total of eleven classical and emerging multicellular model organisms across the phylogenetic tree of life. To determine the microbial community composition of each host, we utilized a combination of archaea and bacteria-specific 16S rRNA gene amplicons. Members of the two prokaryotic domains were described regarding their community composition, diversity, and richness in each multicellular host. Moreover, association with specific hosts and possible interaction partners between the bacterial and archaeal communities were determined for the marine models. Our data show that the archaeome in marine hosts predominantly consists of Nitrosopumilaceae and Nanoarchaeota, which represent keystone taxa among the porifera. The presence of an archaeome in the terrestrial hosts varies substantially. With respect to abundant archaeal taxa, they harbor a higher proportion of methanoarchaea over the aquatic environment. We find that the archaeal community is much less diverse than its bacterial counterpart. Archaeal amplicon sequence variants are usually host-specific, suggesting adaptation through co-evolution with the host. While bacterial richness was higher in the aquatic than the terrestrial hosts, a significant difference in diversity and richness between these groups could not be observed in the archaeal dataset. Our data show a large proportion of unclassifiable archaeal taxa, highlighting the need for improved cultivation efforts and expanded databases.</p

Table_3_The archaeome in metaorganism research, with a focus on marine models and their bacteria–archaea interactions.xlsx

Metaorganism research contributes substantially to our understanding of the interaction between microbes and their hosts, as well as their co-evolution. Most research is currently focused on the bacterial community, while archaea often remain at the sidelines of metaorganism-related research. Here, we describe the archaeome of a total of eleven classical and emerging multicellular model organisms across the phylogenetic tree of life. To determine the microbial community composition of each host, we utilized a combination of archaea and bacteria-specific 16S rRNA gene amplicons. Members of the two prokaryotic domains were described regarding their community composition, diversity, and richness in each multicellular host. Moreover, association with specific hosts and possible interaction partners between the bacterial and archaeal communities were determined for the marine models. Our data show that the archaeome in marine hosts predominantly consists of Nitrosopumilaceae and Nanoarchaeota, which represent keystone taxa among the porifera. The presence of an archaeome in the terrestrial hosts varies substantially. With respect to abundant archaeal taxa, they harbor a higher proportion of methanoarchaea over the aquatic environment. We find that the archaeal community is much less diverse than its bacterial counterpart. Archaeal amplicon sequence variants are usually host-specific, suggesting adaptation through co-evolution with the host. While bacterial richness was higher in the aquatic than the terrestrial hosts, a significant difference in diversity and richness between these groups could not be observed in the archaeal dataset. Our data show a large proportion of unclassifiable archaeal taxa, highlighting the need for improved cultivation efforts and expanded databases.</p

Table_4_The archaeome in metaorganism research, with a focus on marine models and their bacteria–archaea interactions.xls

Metaorganism research contributes substantially to our understanding of the interaction between microbes and their hosts, as well as their co-evolution. Most research is currently focused on the bacterial community, while archaea often remain at the sidelines of metaorganism-related research. Here, we describe the archaeome of a total of eleven classical and emerging multicellular model organisms across the phylogenetic tree of life. To determine the microbial community composition of each host, we utilized a combination of archaea and bacteria-specific 16S rRNA gene amplicons. Members of the two prokaryotic domains were described regarding their community composition, diversity, and richness in each multicellular host. Moreover, association with specific hosts and possible interaction partners between the bacterial and archaeal communities were determined for the marine models. Our data show that the archaeome in marine hosts predominantly consists of Nitrosopumilaceae and Nanoarchaeota, which represent keystone taxa among the porifera. The presence of an archaeome in the terrestrial hosts varies substantially. With respect to abundant archaeal taxa, they harbor a higher proportion of methanoarchaea over the aquatic environment. We find that the archaeal community is much less diverse than its bacterial counterpart. Archaeal amplicon sequence variants are usually host-specific, suggesting adaptation through co-evolution with the host. While bacterial richness was higher in the aquatic than the terrestrial hosts, a significant difference in diversity and richness between these groups could not be observed in the archaeal dataset. Our data show a large proportion of unclassifiable archaeal taxa, highlighting the need for improved cultivation efforts and expanded databases.</p