Thermo-resistant enzyme-producing microorganisms isolated from composting

Abstract Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Resumo Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Thermo-resistant enzyme-producing microorganisms isolated from composting / Microrganismos produtores de enzimas termo-resistentes isolados de compostagem

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Microorganisms from corn stigma with biocontrol potential of Fusarium verticillioides.

The mycotoxigenic fungus Fusarium verticillioides is the primary maize pathogen and causes the maize stalk and ear rot diseases with significant economic losses. Furthermore, the excessive use of fungicides to control F. verticillioides constitutes threats to the environment and human health. Thus, sustainable alternatives such as biological control are needed to minimize the hazards associated with the current method. Although much is known about the vulnerability of the maize silks as a gateway for several fungal pathogens invading the developing grains, studies on the chemical properties of silk extracts and their resident microbiota are scarce. This study isolated and characterized bacteria and fungi that colonize the maize stigma to assess new potential biocontrol agents. The samples were collected from maize fields in the Brazilian localities of Sete Lagoas-MG, Sidrolândia-MS, Sertaneja-PR, and Goiânia-GO. One hundred sixty-seven microorganisms were isolated, 46% endophytic and 54% epiphytic. First, the antagonist activity was evaluated by the agar disc diffusion method performed in triplicate, and 83% of the isolates showed antagonist activity against F. verticillioides. Then, the 42 most efficient isolates were identified based on the partial sequencing of the bacterial 16S rRNA gene and fungi ITS region. The bacteria belong to the genera Bacillus (57.1%), Burkholderia (23.8%), Achromobacter (7.1%), Pseudomonas (2.4%), and Serratia (2.4%), while the fungi are Penicillium (2.4%), Candida (2.4), and Aspergillus (2.4%). The results showed that microorganisms from maize stigma might represent new promising agents for F. verticillioides control.

Thermo-resistant enzyme-producing microorganisms isolated from composting.

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

HydroCalc Proteome: a tool to identify distinct characteristics of effector proteins.

Bacterial pathogenicity is associated with secretion of effector proteins into intra- and extracellular spaces. These proteins interfere with cellular processes such as inhibition of phagosome-lysosome fusion, induction of apoptosis and autophagy, activation and suppression of kinases, regulation of receptor activity, and modulation of transcription factors. Knowledge regarding the characteristics of these proteins would assist in pathogenicity studies, and help to identify possible and novel targets for antibacterial drugs. Amino acid hydropathy is a property that can affect behavior patterns in effector proteins. The HydroCalc Proteome tool analyzes total hydropathy, average hydropathy, C-terminal hydropathy, C-terminal load, and basic polar amino acids at the C-terminus. These five properties could contribute to the identification of proteins with an effector potential. HydroCalc Proteome is a web tool that provides a simple interface for the analysis of hydropathy properties in proteins. This tool permits the analysis of a single protein or even the complete proteome, which cannot be achieved by using other hydropathy tools. The tool displays the result of five properties related to effector proteins in a single table. The HydroCalc Proteome (www.gmb.bio.br/hydrocalc) is a powerful tool for protein analysis, and can contribute to the study of effector proteins.

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

Cytotoxic effects of essential oils from three Lippia gracilis Schauer genotypes on HeLa, B16, and MCF-7 cells and normal human fibroblasts.

This study aimed to evaluate the chemical composition of the essential oils from three genotypes of Lippia gracilis Schauer (Verbenaceae) and investigate the cytotoxic activities of these oils. Essential oils were extracted from the leaves using a Clevenger-type apparatus, and chemical analysis was performed using a gas chromatograph coupled to a mass spectrometer and flame ionization detector. 3T3, MRC5, B16, HeLa, and MCF-7 cell lines were used to study the in vitro cytotoxicity of the essential oils, and the level of cell death was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test with three replicates. The cytotoxic activity was expressed as the concentration that inhibited 50% of cell growth. The main compound in the essential oil of LGRA-106 was thymol (40.52%), while LGRA-109 and LGRA-201 contained 45.84 and 32.60% carvacrol, respectively, as their major compound. The essential oils of L. gracilis showed cytotoxic activity against both normal and tumor cells at concentrations below 100 µg/mL; this demonstrated the antitumor potential of these essential oils, which should be further investigated.

The epimer of kaurenoic acid from Croton antisyphiliticus is cytotoxic toward B-16 and HeLa tumor cells through apoptosis induction.

Cancer has become the leading cause of death in developing countries due to increased life expectancy of the population and changes in lifestyle. Studies on active principles of plant have motivated researchers to develop new antitumor agents that are specific and effective for treatment of neoplasms. Kaurane diterpenes are considered important compounds in the development of new and highly effective anticancer chemotherapeutic agents due to their cytotoxic properties in the induction of apoptosis. We evaluated the cytotoxic and apoptotic activity of the epimer of kaurenoic acid (EKA) isolated from the medicinal plant Croton antisyphiliticus (Euphorbiaceae) toward tumor cell lines HeLa and B-16 and normal fibroblasts 3T3. Based on analyses with the MTT test, EKA showed cytotoxic activity, with half maximal inhibitory concentration values of 59.41, 68.18 and 60.30 µg/mL for the B-16, HeLa and 3T3 cell lines, respectively. The assay for necrotic or apoptotic cells by differential staining showed induction of apoptosis in all three cell lines. We conclude that EKA is not selective between tumor and normal cell lines; the mechanism of action of EKA is induction of apoptosis, which is part of the innate mechanism of cell defense against neoplasia.

In silico characterization of three two-component systems of Ehrlichia canis and evaluation of a natural plant-derived inhibitor.

Two-component signal transduction systems (TCS) are important elements in the interaction of endobacteria with host cells. They are basically composed of two proteins, an environmental signal sensor and a response regulator, which activate genes involved in a wide range of bacterial responses to their environment. We analyzed three sets of genes corresponding to TCS of Ehrlichia canis, a common tick-borne canine pathogen and the etiologic agent of canine monocytic ehrlichiosis, in order to identify the characteristic domains of the sensor and response regulator components. Analysis of sequence alignments of the corresponding proteins indicated a high degree of similarity to other members of the Anaplasmataceae TCS proteins, demonstrating that they could be useful as universal targets for development of new drugs against these bacteria. We also evaluated by quantitative PCR inhibition of E. canis by (2H)-1,4-benzoxazin-3(4H)-one (BOA), the core compound of the plant phenolic compound DIMBOA, which shows inhibitory action against TCS of the phytopathogen Agrobacterium tumefasciens. This bacterium exerts its pathogenicity by transferring oncogenic DNA (T-DNA) into plant cells; this transfer is mediated through a type-IV secretion system, which is regulated by the VirA/VirG TCS. The process of infection and pathogenesis of E. canis is associated with the secretion of effector proteins into the host cell cytoplasm through a T4SS system, which blocks the cell defense response. We suggest that BOA, and possibly other plant phenolic compounds that are TCS inhibitors, can be exploited in the search for new antiehrlichial drugs to be used alone or as complements in the treatment of canine monocytic ehrlichiosis.

A glass bead protocol for recovery of host cell free Ehrlichia canis and quantification by Sybr-green real-time PCR.

E. canis infection of the canine cell line DH82 is a routine in studies with this bacteria. A protocol for isolation of host cell free bacteria was developed based on the use of glass beads. Improvement of infection with E. canis isolated by this method was detected by real-time PCR.

A glass bead protocol for recovery of host cell free Ehrlichia canis and quantification by Sybr-green real-time PCR

E. canis infection of the canine cell line DH82 is a routine in studies with this bacteria. A protocol for isolation of host cell free bacteria was developed based on the use of glass beads. Improvement of infection with E. canis isolated by this method was detected by real-time PCR.

Molecular analysis of the rRNA genes of Babesia spp and Ehrlichia canis detected in dogs from Ribeirão Preto, Brazil / Análise dos genes rRNA de Babesia spp e Ehrlichia canis detectados em cães de Ribeirão Preto, Brasil

The partial DNA sequences of the 18S rRNA gene of Babesia canis and the 16S rRNA gene of Ehrlichia canis detected in dogs from Ribeirão Preto, Brazil, were compared to sequences from other strains deposited in GenBank. The E. canis strain circulating in Ribeirão Preto is identical to other strains previously detected in the region, whereas the subspecies Babesia canis vogeli is the main Babesia strain circulating in dogs from Ribeirão Preto.

As sequências parciais dos genes RNAr 18S de Babesia canis e RNAr 16S e Ehrlichiacanis detectados em cães de Ribeirão Preto, Brasil, foram comparadas à sequências de outras linhagens depositadas no GeneBank. A linhagem de E. canis circulando em Ribeirão Preto é idêntica a outras detectadas previamente na região, enquanto a sub-espécie B. canis vogeli é a principal linhagem de Babesia circulando em cães de Ribeirão Preto.

Molecular analysis of the rRNA genes of Babesia spp and Ehrlichia canis detected in dogs from RibeirÃo Preto, Brazil.

The partial DNA sequences of the 18S rRNA gene of Babesia canis and the 16S rRNA gene of Ehrlichia canis detected in dogs from Ribeirão Preto, Brazil, were compared to sequences from other strains deposited in GenBank. The E. canis strain circulating in Ribeirão Preto is identical to other strains previously detected in the region, whereas the subspecies Babesia canis vogeli is the main Babesia strain circulating in dogs from Ribeirão Preto.

Analysis of the 16S rRNA gene of Anaplasma platys detected in dogs from Brazil

Comparison of the partial DNA sequence of the 16S rRNA gene of Anaplasma platys detected in dogs from Ribeirão Preto, Brazil, to sequences of other strains previously deposited in GenBank showed that there are at least three A. platys strains circulating in South America.

A comparação de sequências parciais do gene 16S RNAr de Anaplasma platys detectadas em cães de Ribeirão Preto, Brasil, com sequências de outras linhagens previamente depositadas no GenBank indicam que existem pelo menos três linhagens de A. platys circulando na América do Sul.

Cell migration in the postnatal subventricular zone

New neurons are constantly added to the olfactory bulb of rodents from birth to adulthood. This accretion is not only dependent on sustained neurogenesis, but also on the migration of neuroblasts and immature neurons from the cortical and striatal subventricular zone (SVZ) to the olfactory bulb. Migration along this long tangential pathway, known as the rostral migratory stream (RMS), is in many ways opposite to the classical radial migration of immature neurons: it is faster, spans a longer distance, does not require radial glial guidance, and is not limited to postmitotic neurons. In recent years many molecules have been found to be expressed specifically in this pathway and to directly affect this migration. Soluble factors with inhibitory, attractive and inductive roles in migration have been described, as well as molecules mediating cell-to-cell and cell-substrate interactions. However, it is still unclear how the various molecules and cells interact to account for the special migratory behavior in the RMS. Here we will propose some candidate mechanisms for roles in initiating and stopping SVZ/RMS migration

Cell migration in the postnatal subventricular zone.

New neurons are constantly added to the olfactory bulb of rodents from birth to adulthood. This accretion is not only dependent on sustained neurogenesis, but also on the migration of neuroblasts and immature neurons from the cortical and striatal subventricular zone (SVZ) to the olfactory bulb. Migration along this long tangential pathway, known as the rostral migratory stream (RMS), is in many ways opposite to the classical radial migration of immature neurons: it is faster, spans a longer distance, does not require radial glial guidance, and is not limited to postmitotic neurons. In recent years many molecules have been found to be expressed specifically in this pathway and to directly affect this migration. Soluble factors with inhibitory, attractive and inductive roles in migration have been described, as well as molecules mediating cell-to-cell and cell-substrate interactions. However, it is still unclear how the various molecules and cells interact to account for the special migratory behavior in the RMS. Here we will propose some candidate mechanisms for roles in initiating and stopping SVZ/RMS migration.

The effect of abiotic factors on the hatching of Moina micrura Kurz, 1874 (Crustacea: Cladocera) ephippial eggs.

The roles of some abiotic factors in controlling the hatching rates of Moina micrura ephippial eggs were investigated. Determination of optimum hatching conditions would be important in developing the use of this species as a food for fish larvae in aquaculture. Ephippia were exposed to different treatments in the laboratory, and monitored for hatching over a period of seven days. Optimum hatching conditions were: pH 5-9, temperature 25 degrees C, photoperiod eight or more hours light per day and light intensity equal to or greater than 850 lux. Differences in water ionic concentrations (from deionized water to 880 mg.L-1 of selected salts) had no effect.

The effect of abiotic factors on the hatching of Moina micrura Kurz, 1874 (Crustacea: Cladocera) ephippial eggs

The roles of some abiotic factors in controlling the hatching rates of Moina micrura ephippial eggs were investigated. Determination of optimum hatching conditions would be important in developing the use of this species as a food for fish larvae in aquaculture. Ephippia were exposed to different treatments in the laboratory, and monitored for hatching over a period of seven days. Optimum hatching conditions were: pH 5-9, temperature 25ºC, photoperiod eight or more hours light per day and light intensity equal to or greater than 850 lux. Differences in water ionic concentrations (from deionized water to 880 mg.L-1 of selected salts) had no effect