Understanding the role of the lipoprotein DolP in cell envelope Biogenesis

The bacterial cell envelope protects the cell against environmental threats, maintains the cell shape and contributes to metabolism and growth. The Gram-negative bacterial envelope is composed of three-layers, the outer membrane (OM), a peptidoglycan (PG) layer, and the inner membrane. The coordination of proteins involved in cell growth and septation is essential to avoid cell lyses. The aim of this thesis is to study the lipoprotein, DolP (formerly YraP) and its role in the cell envelope biogenesis of the Gram-negative bacterium Escherichia coli. DolP is a dual-BON domain lipoprotein localised in the OM. Studies have suggested that DolP might be a component of the BAM (β-barrel assembly machinery) complex and be a player in cell division. The BAM complex is formed by a β-barrel lipoprotein BamA and four accessory components, BamB, BamC, BamD and BamE. To test the first hypothesis, we deleted the non-essential genes bamB, bamC and bamE in a ΔdolP background. We observed a reduction in cell fitness and increase in the number of lysed cells in ΔbamBΔdolP and ΔbamCΔdolP mutants compared to the single mutants. The results suggest that DolP impacts the OM proteins assembly machinery. The second hypothesis is based on a study that suggested that DolP is an upstream regulator of NlpD. NlpD is the activator of the amidase AmiC. Amidases cleave the shared PG layer of adjunct cells to separate into daughter cells. In E. coli, amidases (AmiA, AmiB and AmiC) are regulated by NlpD, EnvC or ActS. To verify DolP’s link to this process, we first observed that DolP does not regulate amidases activity in vitro and does not interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. In addition, the mutant ΔdolP did not phenocopied ΔnlpD in a range of envelope stresses. Next, we tested the morphology of a panel of double deletion mutants of amidases and amidase regulators with dolP. The analysis showed that ΔamiAΔdolP and ΔenvCΔdolP mutants present longer chain length compared to their parental strains indicating a role for DolP in cell division. In conclusion, we suggest that DolP might not be a NlpD regulator. However, DolP may impact daughter cell separation by interacting with AmiA and AmiC, or by a yet unknown mechanism

Malacofauna Límnica Associada à Macrófitas Aquáticas

This study has as objective to identify the species of freshwatermollusks associated with macrophytes Eichhornia azurea, Sagittariamontevidensis e Myriophyllum aquaticum. The site of study located in the Iguariaçá river in the São Borja city, Rio Grande do Sul. The sample areacharacterized by wetland due the flood of the Iguariaçá river. Quantitativesamples were taken in three epochs: august, october and november of 2005.Totalled 731 specimens mollusks, attributed the six species, five gastropods(n = 727) and one bivalve (n = 4). The gastropods species found werePomacea canaliculata (Lamarck, 1822) (n = 4); Gundlachia moricandi(Orbigny, 1837) (n = 263); Lymnaea columella Say, 1817 (n = 88);Stenophysa marmorata Guilding, 1828 (n = 2) and Biomphalaria tenagophila(Orbigny, 1835) (n = 370), and bivalve Eupera klappenbachi Mansur &Veitenheimer, 1975 (n = 4). The mollusks species registered showpreference between the macrophytes and by position in the plant. Themacrophytes could be used as sampling to obtain species restricted at thishabitat, temporarily or permanently, and its presence associated withsubmerged or floating vegetation.Este estudo teve como objetivo identificar as espécies de moluscoslímnicos associados às macrófitas Eichhornia azurea, Sagittaria montevidensise Myriophyllum aquaticum. O local de estudo situa-se no rio Iguariaçá, nomunicípio de São Borja, Rio Grande do Sul. O local de coleta caracterizasepor uma área alagável devido às inundações pelo rio Iguariaçá. Asamostragens quantitativas foram realizadas em três períodos: agosto, outubroe novembro de 2005. Ao todo 731 espécimes de moluscos foramcoletados, atribuídos a seis espécies, sendo cinco de gastrópodes (n = 727)e uma de bivalve (n = 4). Coletaram-se os gastrópodes Pomacea canaliculata(Lamarck, 1822) (n = 4); Gundlachia moricandi (Orbigny, 1837) (n =263); Lymnaea columella Say, 1817 (n = 88); Stenophysa marmorataGuilding, 1828 (n = 2) e Biomphalaria tenagophila (Orbigny, 1835) (n =370), e o bivalve Eupera klappenbachi Mansur & Veitenheimer, 1975 (n =4). As espécies de moluscos encontradas demonstraram preferências emrelação à macrófita aquática e pela posição no vegetal. A utilização demacrófitas aquáticas como medida amostral possibilita a obtenção de espéciesque estarão restritos a este tipo de hábitat, seja temporário ou permanente,sendo as suas presenças associadas à vegetação submersa ou flutuante

LI-Detector:a Method for Curating Ordered Gene-Replacement Libraries

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterized genes. To address this gap in functional annotation, many high-throughput screens have been devised to uncover novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered library where a gene has been replaced or disrupted by a uniform linear insertion (LI). We applied our method (LI-detector) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates, with a success rate of 99.7% for identifying the correct kanamycin cassette position. This data set provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of any linear insertion, such as an antibiotic resistance cassette in any ordered library. IMPORTANCE The construction of ordered gene replacement libraries requires significant investment of time and resources to create a valuable community resource. During construction, technical errors may result in a limited number of incorrect mutants being made. Such mutants may confound the output of subsequent experiments. Here, using the remarkable E. coli Keio knockout library, we describe a method to rapidly validate the construction of every mutant.</p

ATLANTIC EPIPHYTES: a data set of vascular and non-vascular epiphyte plants and lichens from the Atlantic Forest

Epiphytes are hyper-diverse and one of the frequently undervalued life forms in plant surveys and biodiversity inventories. Epiphytes of the Atlantic Forest, one of the most endangered ecosystems in the world, have high endemism and radiated recently in the Pliocene. We aimed to (1) compile an extensive Atlantic Forest data set on vascular, non-vascular plants (including hemiepiphytes), and lichen epiphyte species occurrence and abundance; (2) describe the epiphyte distribution in the Atlantic Forest, in order to indicate future sampling efforts. Our work presents the first epiphyte data set with information on abundance and occurrence of epiphyte phorophyte species. All data compiled here come from three main sources provided by the authors: published sources (comprising peer-reviewed articles, books, and theses), unpublished data, and herbarium data. We compiled a data set composed of 2,095 species, from 89,270 holo/hemiepiphyte records, in the Atlantic Forest of Brazil, Argentina, Paraguay, and Uruguay, recorded from 1824 to early 2018. Most of the records were from qualitative data (occurrence only, 88%), well distributed throughout the Atlantic Forest. For quantitative records, the most common sampling method was individual trees (71%), followed by plot sampling (19%), and transect sampling (10%). Angiosperms (81%) were the most frequently registered group, and Bromeliaceae and Orchidaceae were the families with the greatest number of records (27,272 and 21,945, respectively). Ferns and Lycophytes presented fewer records than Angiosperms, and Polypodiaceae were the most recorded family, and more concentrated in the Southern and Southeastern regions. Data on non-vascular plants and lichens were scarce, with a few disjunct records concentrated in the Northeastern region of the Atlantic Forest. For all non-vascular plant records, Lejeuneaceae, a family of liverworts, was the most recorded family. We hope that our effort to organize scattered epiphyte data help advance the knowledge of epiphyte ecology, as well as our understanding of macroecological and biogeographical patterns in the Atlantic Forest. No copyright restrictions are associated with the data set. Please cite this Ecology Data Paper if the data are used in publication and teaching events. © 2019 The Authors. Ecology © 2019 The Ecological Society of Americ

Evaluation of deteriogenic potential of Pseudallescheria boydii and Meyerozyma guilliermondii during simulated storage of fuels (diesel, biodiesel and B10 blend)

Conforme a previsão do governo brasileiro, até 2019, o óleo diesel (B0) receberá o aumento de 10% de biodiesel, compondo a mistura B10. O biodiesel, devido a sua natureza renovável e sustentável, impulsiona estudos prevendo a estabilidade durante o armazenamento. Neste sentido, o biodiesel utilizado no Brasil é composto por metil ésteres de cadeias longas de ácidos graxos derivados da transesterificação de óleos vegetais ou gorduras animais. Além da possibilidade de degradação química, a natureza das moléculas também o tornam mais suscetível à biodegradação. O objetivo do trabalho foi avaliar a capacidade deteriogênica (crescimento, degradação e detecção de genes) relacionado com a degradação de combustíveis pelo fungo filamentoso Pseudallescheria boydii e pela levedura Meyerozyma guilliermondii em diesel puro (B0); biodiesel puro (B100) e na mistura B10, em meio mineral durante estocagem simulada. Foram incubados por 45 dias a 30°C frascos contendo proporções (1:6, 2:3 e 2:1) de combustível (B0, B10 e B100) em relação ao meio mínimo mineral BH com inóculo de 104 esporos mL-1 de Pseudallescheria boydii. Para a avaliação de Meyerozyma guilliermondii, 102 células mL-1 foram acrescentadas a 5 mL de B10 ou B100 e 30 mL de meio mínimo mineral BH, incubados em agitação (120 rpm) a 28°C por 10 dias. Para o acompanhamento da formação de biomassa foi realizado o método gravimétrico. A curva de crescimento da levedura foi estimada pela contagem de UFC mL-1. A fase aquosa foi analisada pelas metodologias de produção de lipase, medidas de pH e de tensão superficial, IE24 e GC-MS com SPME. A detecção de genes funcionais foi realizada por PCR. A degradação de ésteres e hidrocarbonetos foi avaliada por IV e RMN. Em relação ao fungo filamentoso, as medidas de biomassa produzida ao longo dos 45 dias indicaram maior produção de biomassa no combustível B10. Quanto à levedura, não se obteve diferença significativa na curva de crescimento entre B10 e B100. Em ambos os fungos não houve produção significativa de biossurfactante, mas foi detectada produção de lipase e os genes P450 e de lipase foram amplificados. A emulsificação da fase oleosa foi observada apenas no experimento com a levedura. Os compostos identificados na fase aquosa por cromatografia com os dois fungos foram: álcoois, ésteres, ácidos, sulfurados, cetonas e fenóis. Ambos microrganismos cresceram às expensas de diesel e biodiesel e meio mínimo mineral. Pseudallescheria boydii degradou os hidrocarbonetos e ésteres do combustível e biocombustível, respectivamente.According to the Brazilian government's forecast, until 2019, diesel oil (B0) will receive a 10% increase in biodiesel, composing the B10 blend. Biodiesel due to its renewable and sustainable nature boosts studies predicting stability during storage. In this sense, the biodiesel used in Brazil is composed of methyl esters of long chains of fatty acids derived from transesterification of vegetable oils or animal fats. In addition to the possibility of chemical degradation, the nature of the molecules also make it more susceptible to biodegradation. The aim of this work was to evaluate the deteriogenic potential (growth, degradation and detection of genes) related to the degradation of fuels such as pure diesel (B0), pure biodiesel (B100) and the B10 blend of Pseudallescheria boydii and Meyerozyma guilliermondii in mineral medium during storage. Different ratios (1: 6, 2: 3 and 2: 1) of fuel (B0, B10 and B100) with respect to the minimal mineral medium BH were incubated for 45 days at 30°C with inoculum of 104 spores mL-1. For the evaluation of Meyerozyma guilliermondii, 102 cells mL-1were added to 5 mL of B10 or B100 and 30 mL of minimal BH mineral medium, incubated with shaking (120 rpm) at 28°C for 10 days. We estimates the yeast growth curve by the count of CFU mL-1. We analyzed the aqueous phase by any methodologies as lipase production, pH and surface tension measurements, IE24 and GC-MS with SPME. The detection of functional genes for hydrocarbon degradation was performed by PCR. The degradation of esters and hydrocarbons was evaluated by IR and NMR. In relation to the filamentous fungus, the biomass measurements produced during the 45 days indicated higher biomass production in the B10 fuel. As for yeast, there was no significant difference in the growth curve between B10 and B100. In both fungi, there was no significant production of biosurfactant, but production of lipase and the P450 and lipase gene was detected. Oily emulsification was observed only in the yeast experiment. We identified degradation products by the chromatography in the aqueous phase. The compounds identified were alcohols, esters, acids, sulfur, ketones and phenols. Both microorganisms grew at the expense of diesel, biodiesel and minimal mineral medium. Meyerozyma guilliermondii e Pseudallescheria boydii degradated hydrocarbons and esters of fuel and biofuels, respectivelly

Evaluation of deteriogenic potential of Pseudallescheria boydii and Meyerozyma guilliermondii during simulated storage of fuels (diesel, biodiesel and B10 blend)

Conforme a previsão do governo brasileiro, até 2019, o óleo diesel (B0) receberá o aumento de 10% de biodiesel, compondo a mistura B10. O biodiesel, devido a sua natureza renovável e sustentável, impulsiona estudos prevendo a estabilidade durante o armazenamento. Neste sentido, o biodiesel utilizado no Brasil é composto por metil ésteres de cadeias longas de ácidos graxos derivados da transesterificação de óleos vegetais ou gorduras animais. Além da possibilidade de degradação química, a natureza das moléculas também o tornam mais suscetível à biodegradação. O objetivo do trabalho foi avaliar a capacidade deteriogênica (crescimento, degradação e detecção de genes) relacionado com a degradação de combustíveis pelo fungo filamentoso Pseudallescheria boydii e pela levedura Meyerozyma guilliermondii em diesel puro (B0); biodiesel puro (B100) e na mistura B10, em meio mineral durante estocagem simulada. Foram incubados por 45 dias a 30°C frascos contendo proporções (1:6, 2:3 e 2:1) de combustível (B0, B10 e B100) em relação ao meio mínimo mineral BH com inóculo de 104 esporos mL-1 de Pseudallescheria boydii. Para a avaliação de Meyerozyma guilliermondii, 102 células mL-1 foram acrescentadas a 5 mL de B10 ou B100 e 30 mL de meio mínimo mineral BH, incubados em agitação (120 rpm) a 28°C por 10 dias. Para o acompanhamento da formação de biomassa foi realizado o método gravimétrico. A curva de crescimento da levedura foi estimada pela contagem de UFC mL-1. A fase aquosa foi analisada pelas metodologias de produção de lipase, medidas de pH e de tensão superficial, IE24 e GC-MS com SPME. A detecção de genes funcionais foi realizada por PCR. A degradação de ésteres e hidrocarbonetos foi avaliada por IV e RMN. Em relação ao fungo filamentoso, as medidas de biomassa produzida ao longo dos 45 dias indicaram maior produção de biomassa no combustível B10. Quanto à levedura, não se obteve diferença significativa na curva de crescimento entre B10 e B100. Em ambos os fungos não houve produção significativa de biossurfactante, mas foi detectada produção de lipase e os genes P450 e de lipase foram amplificados. A emulsificação da fase oleosa foi observada apenas no experimento com a levedura. Os compostos identificados na fase aquosa por cromatografia com os dois fungos foram: álcoois, ésteres, ácidos, sulfurados, cetonas e fenóis. Ambos microrganismos cresceram às expensas de diesel e biodiesel e meio mínimo mineral. Pseudallescheria boydii degradou os hidrocarbonetos e ésteres do combustível e biocombustível, respectivamente.According to the Brazilian government's forecast, until 2019, diesel oil (B0) will receive a 10% increase in biodiesel, composing the B10 blend. Biodiesel due to its renewable and sustainable nature boosts studies predicting stability during storage. In this sense, the biodiesel used in Brazil is composed of methyl esters of long chains of fatty acids derived from transesterification of vegetable oils or animal fats. In addition to the possibility of chemical degradation, the nature of the molecules also make it more susceptible to biodegradation. The aim of this work was to evaluate the deteriogenic potential (growth, degradation and detection of genes) related to the degradation of fuels such as pure diesel (B0), pure biodiesel (B100) and the B10 blend of Pseudallescheria boydii and Meyerozyma guilliermondii in mineral medium during storage. Different ratios (1: 6, 2: 3 and 2: 1) of fuel (B0, B10 and B100) with respect to the minimal mineral medium BH were incubated for 45 days at 30°C with inoculum of 104 spores mL-1. For the evaluation of Meyerozyma guilliermondii, 102 cells mL-1were added to 5 mL of B10 or B100 and 30 mL of minimal BH mineral medium, incubated with shaking (120 rpm) at 28°C for 10 days. We estimates the yeast growth curve by the count of CFU mL-1. We analyzed the aqueous phase by any methodologies as lipase production, pH and surface tension measurements, IE24 and GC-MS with SPME. The detection of functional genes for hydrocarbon degradation was performed by PCR. The degradation of esters and hydrocarbons was evaluated by IR and NMR. In relation to the filamentous fungus, the biomass measurements produced during the 45 days indicated higher biomass production in the B10 fuel. As for yeast, there was no significant difference in the growth curve between B10 and B100. In both fungi, there was no significant production of biosurfactant, but production of lipase and the P450 and lipase gene was detected. Oily emulsification was observed only in the yeast experiment. We identified degradation products by the chromatography in the aqueous phase. The compounds identified were alcohols, esters, acids, sulfur, ketones and phenols. Both microorganisms grew at the expense of diesel, biodiesel and minimal mineral medium. Meyerozyma guilliermondii e Pseudallescheria boydii degradated hydrocarbons and esters of fuel and biofuels, respectivelly