Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Isla Arena está localizada na coordenada 20°70’N - 90°45’W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70N - 90°45W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70'N - 90°45'W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.(AU)

Isla Arena está localizada na coordenada 20°70N - 90°45W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.(AU)

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt

Abstract Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

Resumo As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Abstract Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

Resumo As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.(AU)

As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.(AU)

Effect of Bacillus Subtilis on Immune Function of Hd11 Chicken Macrophages

Poultry is frequently contaminated by Salmonella, a pathogen leading to human health concern worldwide. This study aimed to evaluate the effect of Bacillus subtilis (BS)strain 048 (BS048) on the activation, phagocytosis, sterilization, cytokine secretion, and nitrogen oxide synthesis of HD11 chicken macrophages subjected to Salmonella enteritidis challenge, using lipopolysaccharide treatment as a negative control. The results showed: (1) BS048 had no significant effect on extracellular lactate dehydrogenase activity (p>0.05), while lipopolysaccharide treatment significantly increased extracellular lactate dehydrogenase activity (p >0.05), while lipopolysaccharide treatment significantly increased extracellular lactate dehydrogenase activity (p 0.05);(2)BS048 significantly upregulated the expression levels of pro-inflammatory cytokines (interleukin (IL)- 1â and IL-6), anti-inflammatory cytokines(IL-10 and transforming growth factor-â1), and anti-viral cytokine, interferon-â (p<0.01); ; (3) BS048 significantly upregulated the mRNA expression level of the inducible nitric oxide synthase and its activity as well as extracellular nitrogen oxide level (p <0.01). In conclusion, BS048 could improve antiinflammatory and immune functions of HD11 chicken macrophages, without cytotoxic effects on these cells.(AU)

Saccharomyces cerevisiae OS303 expression of an alkaline protease from a newly isolated Bacillus subtilis D9 / Expressão de Saccharomyces cerevisiae OS303 de uma protease alcalina de um Bacillus subtilis D9 recém-isolado

The aim of this study was to produce high yield of a local bacterial alkaline protease in the yeast system because the scientific involvement of microorganisms in enzyme production is still not given enough attention in Saudi Arabia. Soil samples were collected from the rhizosphere of some desert plants in Saudi Arabia. Ninety-three alkaline protease producing bacterial isolates were recovered on skimmed-milk agar at pH 9.4 and 45°C for 48 hr. Isolate D9 obtained from the rhizosphere of Heliotropium digynum at Dhahran City was the most potent isolate in respect to enzyme productivity (184.6 U/ml). The full gene of alkaline protease was amplified and showed the expected size (1300 bp). Restriction enzymes analysis also verified the integrity of the PCR product. The sequence of the protease gene revealed an open reading frame of 1329 nt correspond to the full length of the protease gene of isolate D9 encoding a 443 aa protein. After ligation of the amplified gene by the TA cloning method, digestion with appropriate restriction enzymes confirmed the integrity of the cloned gene. The insert was prepared by two PCRs that were conducted with a pair of primers specifically designed for this purpose. The digested and purified cloning vector pRS426/GAL1p-207-Glu-MS was ligated with the insert then transformed into various strains of Saccharomyces cerevisiae via the electroporation method. Maximum protease expression was done by recombinant OS303 in galactose containing media (145.5 U/ml) with an approximately 2-fold increase when compared with the wild OS303 strain., this may be due to ability to activate gal operon.

O objetivo deste estudo foi produzir alto rendimento de uma protease alcalina bacteriana local no sistema de leveduras, já que o envolvimento científico de microrganismos na produção de enzimas ainda não recebe atenção suficiente na Arábia Saudita. Amostras de solo foram coletadas da rizosfera de algumas plantas do deserto na Arábia Saudita. Noventa e três isolados bacterianos produtores de protease alcalina foram recuperados em ágar de leite desnatado a pH 9,4 e 45°C por 48 horas. O isolado D9 obtido da rizosfera de Heliotropium digynum na cidade de Dhahran foi o mais potente em relação à produtividade da enzima (184,6 U/ml). O gene completo da protease alcalina foi amplificado e apresentou o tamanho esperado (1300 pb). A análise de enzimas de restrição também verificou a integridade do produto de PCR. A sequência do gene da protease revelou uma fase de leitura aberta de 1329 nt, correspondendo ao comprimento total do gene da protease do isolado D9 que codifica uma proteína 443 aa. Após a ligação do gene amplificado pelo método de clonagem TA, a digestão com enzimas de restrição apropriadas confirmou a integridade do gene clonado. A inserção foi preparada por dois PCRs que foram conduzidos com um par de primers projetados especificamente para esta finalidade. O vetor de clonagem digerido e purificado pRS426/GAL1p-207-Glu-MS foi ligado com a inserção e então transformado em várias cepas de Saccharomyces cerevisiae por meio do método de eletroporação. A expressão máxima da protease foi feita por OS303 recombinante em meio contendo galactose (145,5 U/ml) com um aumento de aproximadamente duas vezes quando comparado com a cepa OS303 selvagem, e isso pode ser por causa da capacidade de ativar o operon gal.

Economic feasibility of probiotic use in the diet of Nile tilapia, Oreochromis niloticus, during the reproductive period

This work examines the economic advantages of probiotic use in the diet of Nile tilapia broodstock during the reproductive period. For this purpose, Bacillus subtilis was applied as a feed additive. The experimental design was completely randomized with three treatment groups: the T0 control (without probiotic), the T1 continuous probiotic intake, and the T2 alternate probiotic intake at a dose of 0.50 g kg-1 of feed (1010 CFU g-1 ) with four replicates. For the reproduction assay, 118 females and 48 males of Nile tilapia (proportion 4 males:9 females. hapa-1 ) (weight 527.65 g ± 185.98 g and length 30.16 cm ± 3.57 cm) were distributed into 12 hapas (3.5 × 2.0 × 1.5 m). Reproductive variables (spawning female percentage, egg production, and fry production) were used to calculate the economic feasibility indexes (total cost of nutrition [TcN], gross revenue [GR], and total operational profit [ToP]). The results show increasing values for spawning female number, collected eggs, and surviving fry in the probiotic groups. We recommend continuous intake of probiotic (feed with addition of probiotic) at a dose of 0.5 g kg-1 of feed (1010 CFU g-1 ) during the breeding season of Nile tilapia, due to the suitable reproductive indexes and profitability.(AU)

Economic feasibility of probiotic use in the diet of Nile tilapia, Oreochromis niloticus, during the reproductive period

This work examines the economic advantages of probiotic use in the diet of Nile tilapia broodstock during the reproductive period. For this purpose, Bacillus subtilis was applied as a feed additive. The experimental design was completely randomized with three treatment groups: the T0 control (without probiotic), the T1 continuous probiotic intake, and the T2 alternate probiotic intake at a dose of 0.50 g kg-1 of feed (1010 CFU g-1 ) with four replicates. For the reproduction assay, 118 females and 48 males of Nile tilapia (proportion 4 males:9 females. hapa-1 ) (weight 527.65 g ± 185.98 g and length 30.16 cm ± 3.57 cm) were distributed into 12 hapas (3.5 × 2.0 × 1.5 m). Reproductive variables (spawning female percentage, egg production, and fry production) were used to calculate the economic feasibility indexes (total cost of nutrition [TcN], gross revenue [GR], and total operational profit [ToP]). The results show increasing values for spawning female number, collected eggs, and surviving fry in the probiotic groups. We recommend continuous intake of probiotic (feed with addition of probiotic) at a dose of 0.5 g kg-1 of feed (1010 CFU g-1 ) during the breeding season of Nile tilapia, due to the suitable reproductive indexes and profitability.

Evaluation of the Effect of Bacillus Subtilis and Pediococcus Acidilactici Mix on Serum Biochemistry, Growth Promotation of Body and Visceral Organs in Lohmann Brown Chicks

Recently the use of antibiotic growth promoter (AGPs) in poultry feed is banned in China, leading to the increasing demand for AGPs substitutes. Probiotics have been considered as a potential substitute. The purpose of this study was to evaluate the effects of probiotic on serum biochemistry, and growth promotion of body weight and visceral organs and in Lohmann Brown chicks aged 0 to 9 weeks. Five hundred and forty 1-day-old male chicks were randomly divided into three groups, with six replicates and 30 chicks in each replicate. The experiment was carried out over 70 days. Dietary treatments were: (1) basal diet alone (control group); (2) basal diet containing 0.1% probiotics (probiotic group); and (3) basal diet containing 0.01% zinc bacitracin in the feed (antibiotic group). The body weight (BW) of probiotic group was significantly higher than that of the control group during the whole trial period (p 0.05). ADG (0~70 d) in the probiotic group and the antibiotic group was higher than that in the control group (p 0.05). The Oliver weight and serum total protein (TP) in the probiotic group was higher than that in the control group on the 63rd day (p 0.05). The length of the duodenum, the weight of the spleen, duodenum and Jejunum in both the probiotic and antibiotic groups were higher than those in the control group (p 0.05). The ileum weight in the probiotic group was significantly higher than those in the control group (p 0.05). The ileum length in the probiotic group was significantly longer than that in both the antibiotic and control groups (p 0.05). Probiotic mix with Bacillus subtilis and Pediococcus acidilactici play a similar role in promoting growth of body and visceral organs, and it is a promising growth promoter for Lohmann Brown chicks (0 to 9 weeks).(AU)

Evaluation of the Effect of Bacillus Subtilis and Pediococcus Acidilactici Mix on Serum Biochemistry, Growth Promotation of Body and Visceral Organs in Lohmann Brown Chicks

Recently the use of antibiotic growth promoter (AGPs) in poultry feed is banned in China, leading to the increasing demand for AGPs substitutes. Probiotics have been considered as a potential substitute. The purpose of this study was to evaluate the effects of probiotic on serum biochemistry, and growth promotion of body weight and visceral organs and in Lohmann Brown chicks aged 0 to 9 weeks. Five hundred and forty 1-day-old male chicks were randomly divided into three groups, with six replicates and 30 chicks in each replicate. The experiment was carried out over 70 days. Dietary treatments were: (1) basal diet alone (control group); (2) basal diet containing 0.1% probiotics (probiotic group); and (3) basal diet containing 0.01% zinc bacitracin in the feed (antibiotic group). The body weight (BW) of probiotic group was significantly higher than that of the control group during the whole trial period (p 0.05). ADG (0~70 d) in the probiotic group and the antibiotic group was higher than that in the control group (p 0.05). The Oliver weight and serum total protein (TP) in the probiotic group was higher than that in the control group on the 63rd day (p 0.05). The length of the duodenum, the weight of the spleen, duodenum and Jejunum in both the probiotic and antibiotic groups were higher than those in the control group (p 0.05). The ileum weight in the probiotic group was significantly higher than those in the control group (p 0.05). The ileum length in the probiotic group was significantly longer than that in both the antibiotic and control groups (p 0.05). Probiotic mix with Bacillus subtilis and Pediococcus acidilactici play a similar role in promoting growth of body and visceral organs, and it is a promising growth promoter for Lohmann Brown chicks (0 to 9 weeks).

A novel expression vector for the secretion of abaecin in Bacillus subtilis

ABSTRACT This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5 µM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5 µM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics.(AU)

Fusarium culmorum affects expression of biofilm formation key genes in Bacillus subtilis

It is known that there is correlation between biofilm formation and antagonistic activities of Bacillus subtilis strains; but, the mechanism of this correlation is not clear. So, the effect of the plant pathogen (Fusarium culmorum) on the biofilm formation in a B. subtilis strain with high antagonistic and biofilm formation activities was studied. The expression of sinR and tasA genes involved in the biofilm formation was studied in both single culture of bacterium (B) and co-culture with F. culmorum (FB) using real-time PCR. The results revealed that the expression of the sinR gene in both B and FB conditions was continuously decreased during the biofilm formation period and, after 24 h (B4 and FB4), it reached 1% and 0.3% at the planktonic phase (B1), respectively, whereas the expression of the tasA was continuously increased and was 5.27 and 30 times more than that at the planktonic phase (B1) after 24 h, respectively. So, the expression reduction rate for sinR (3 times) and the expression increasing rate for tasA (6 times) were significantly higher in FB conditions than the B ones. The relative expression of sinR in FB1 (planktonic phase), FB2 (8 h), FB3(12 h), and FB4 (24 h) times was 0.65, 0.44, 0.35, and 0.29, whereas the tasA gene expression was 2.98, 3.44, 4.37, and 5.63-fold of the one at coordinate time points in B conditions, respectively. The significant expression reduction of sinR and increase of tasA confirmed that the presence of pathogen could stimulate biofilm formation in the antagonistic bacterium. (AU)

Simultaneous production of amylases and proteases by Bacillus subtilis in brewery wastes

The simultaneous production of amylase (AA) and protease (PA) activity by Bacillus subtilis UO-01 in brewery wastes was studied by combining the response surface methodology with the kinetic study of the process. The optimum conditions (T = 36.0 °C and pH = 6.8) for high biomass production (0.92 g/L) were similar to the conditions (T = 36.8 °C and pH = 6.6) for high AA synthesis (9.26 EU/mL). However, the maximum PA level (9.77 EU/mL) was obtained at pH 7.1 and 37.8 °C. Under these conditions, a considerably high reduction (between 69.9 and 77.8%) of the initial chemical oxygen demand of the waste was achieved. In verification experiments under the optimized conditions for production of each enzyme, the AA and PA obtained after 15 h of incubation were, respectively, 9.35 and 9.87 EU/mL. By using the Luedeking and Piret model, both enzymes were classified as growth-associated metabolites. Protease production delay seemed to be related to the consumption of non-protein and protein nitrogen. These results indicate that the brewery waste could be successfully used for a high scale production of amylases and proteases at a low cost.(AU)

Prebiótico, probiótico e simbiótico para larvas de Trichogaster leeri (Bleeker, 1852, Perciformes, Osphronemidae) / Prebiotic, probiotic and symbiotic for Trichogaster leeri larvae (Bleeker, 1852, Perciformes, Osphronemidae)

Avaliou-se a suplementação de prebiótico (mananoligossacarídeo - 2g/kg), probiótico (Bacillus subtilis, Cohn, 1872, Bacillales, Bacillaceae - 2g/kg) e simbiótico (mananoligossacarídeo - 2g/kg + B. subtilis - 2g/kg), em rações para larvas de Trichogaster leeri (Bleeker, 1852, Perciformes, Osphronemidae), durante a transição alimentar, sobre o crescimento, a sobrevivência, a morfometria intestinal e a resistência ao estresse por exposição aérea. Para isso, 420 larvas (0,45±0,05mg) foram distribuídas em 20 aquários (3,5L), em delineamento inteiramente ao acaso, com cinco tratamentos e quatro repetições, durante 28 dias, a saber: 12 dias de alimento vivo (AV) + oito dias de coalimentação (AV + ração controle) + oito dias de ração controle; 12 dias de AV + oito dias de coalimentação (AV + ração prebiótico) + oito dias de ração prebiótico; 12 dias de AV + oito dias de coalimentação (AV + ração probiótico) + oito dias de ração probiótico; 12 dias de AV + oito dias de coalimentação (AV + ração simbiótico) + oito dias de ração simbiótico; 28 dias de AV. Larvas que receberam apenas AV apresentaram resultados de desempenho zootécnico significativamente superiores às larvas dos demais tratamentos, exceto para taxa de crescimento específico em comprimento e sobrevivência, que foram semelhantes aos resultados obtidos por larvas que receberam rações suplementadas com simbiótico. Larvas que receberam a ração controle apresentaram resultados significativamente inferiores para altura das vilosidades intestinais e taxa de resistência ao estresse comparando-se aos demais tratamentos. Os resultados deste estudo indicam que a suplementação com prebiótico, probiótico e simbiótico exerce efeito positivo sobre a sobrevivência, a morfometria intestinal e a resistência ao estresse em larvas de T. leeri, no entanto somente a suplementação com simbiótico resulta em melhora no crescimento.(AU)

The prebiotic (mannan oligosaccharide - 2g/kg), probiotic (Bacillus subtilis Cohn, 1872, Bacillales, Bacillaceae - 2g/kg) and symbiotic (mannan oligosaccharide - 2g/kg + Bacillus subtilis - 2g/kg) supplementation in diets during food transition in Trichogaster leeri (Bleeker, 1852, Perciformes, Osphronemidae) larvae on growth, survival, intestinal morphology and stress resistance to air exposure was evaluated. For this, 420 larvae (0.45±0.05mg) were distributed in 20 aquaria (3.5L) in a completely randomized design with five treatments and four replications for 28 days, namely: 12 days of live food (LF) + eight days of co-feeding (LF + control diet) + eight days of control diet; 12 days of LF + eight days of co-feeding (LF + prebiotic feed) + eight days of prebiotic feed; 12 days of LF + eight days of co-feeding (LF + probiotic feed) + eight days of probiotic feed; 12 days of LF + eight days of co-feeding (LF + symbiotic feed) + eight days symbiotic feed; 28 days of LF. Larvae fed only LF showed growth performance significantly higher than the larvae of other treatments, except for specific growth rate in length and survival that were similar to the results obtained by larvae fed diets symbiotically supplemented. Larvae fed the control diet showed significantly lower results for height of intestinal villi and stress resistance rate comparing to other treatments. The results of this study indicate that prebiotic, probiotic and symbiotic supplementation has a positive effect on survival, intestinal morphology and resistance to stress in larvae of T. leeri, however, only symbiotic supplementation results in improved growth.(AU)

Prebiótico, probiótico e simbiótico para larvas de Trichogaster leeri (Bleeker, 1852, Perciformes, Osphronemidae) / Prebiotic, probiotic and symbiotic for Trichogaster leeri larvae (Bleeker, 1852, Perciformes, Osphronemidae)

Avaliou-se a suplementação de prebiótico (mananoligossacarídeo - 2g/kg), probiótico (Bacillus subtilis, Cohn, 1872, Bacillales, Bacillaceae - 2g/kg) e simbiótico (mananoligossacarídeo - 2g/kg + B. subtilis - 2g/kg), em rações para larvas de Trichogaster leeri (Bleeker, 1852, Perciformes, Osphronemidae), durante a transição alimentar, sobre o crescimento, a sobrevivência, a morfometria intestinal e a resistência ao estresse por exposição aérea. Para isso, 420 larvas (0,45±0,05mg) foram distribuídas em 20 aquários (3,5L), em delineamento inteiramente ao acaso, com cinco tratamentos e quatro repetições, durante 28 dias, a saber: 12 dias de alimento vivo (AV) + oito dias de coalimentação (AV + ração controle) + oito dias de ração controle; 12 dias de AV + oito dias de coalimentação (AV + ração prebiótico) + oito dias de ração prebiótico; 12 dias de AV + oito dias de coalimentação (AV + ração probiótico) + oito dias de ração probiótico; 12 dias de AV + oito dias de coalimentação (AV + ração simbiótico) + oito dias de ração simbiótico; 28 dias de AV. Larvas que receberam apenas AV apresentaram resultados de desempenho zootécnico significativamente superiores às larvas dos demais tratamentos, exceto para taxa de crescimento específico em comprimento e sobrevivência, que foram semelhantes aos resultados obtidos por larvas que receberam rações suplementadas com simbiótico. Larvas que receberam a ração controle apresentaram resultados significativamente inferiores para altura das vilosidades intestinais e taxa de resistência ao estresse comparando-se aos demais tratamentos. Os resultados deste estudo indicam que a suplementação com prebiótico, probiótico e simbiótico exerce efeito positivo sobre a sobrevivência, a morfometria intestinal e a resistência ao estresse em larvas de T. leeri, no entanto somente a suplementação com simbiótico resulta em melhora no crescimento.(AU)

The prebiotic (mannan oligosaccharide - 2g/kg), probiotic (Bacillus subtilis Cohn, 1872, Bacillales, Bacillaceae - 2g/kg) and symbiotic (mannan oligosaccharide - 2g/kg + Bacillus subtilis - 2g/kg) supplementation in diets during food transition in Trichogaster leeri (Bleeker, 1852, Perciformes, Osphronemidae) larvae on growth, survival, intestinal morphology and stress resistance to air exposure was evaluated. For this, 420 larvae (0.45±0.05mg) were distributed in 20 aquaria (3.5L) in a completely randomized design with five treatments and four replications for 28 days, namely: 12 days of live food (LF) + eight days of co-feeding (LF + control diet) + eight days of control diet; 12 days of LF + eight days of co-feeding (LF + prebiotic feed) + eight days of prebiotic feed; 12 days of LF + eight days of co-feeding (LF + probiotic feed) + eight days of probiotic feed; 12 days of LF + eight days of co-feeding (LF + symbiotic feed) + eight days symbiotic feed; 28 days of LF. Larvae fed only LF showed growth performance significantly higher than the larvae of other treatments, except for specific growth rate in length and survival that were similar to the results obtained by larvae fed diets symbiotically supplemented. Larvae fed the control diet showed significantly lower results for height of intestinal villi and stress resistance rate comparing to other treatments. The results of this study indicate that prebiotic, probiotic and symbiotic supplementation has a positive effect on survival, intestinal morphology and resistance to stress in larvae of T. leeri, however, only symbiotic supplementation results in improved growth.(AU)

Análise morfométrica da parede intestinal e dinâmica de mucinas secretadas no jejuno de frangos suplementados com probiótico Bacillus subtilis cepa C3102 / Morphometric analysis of the intestinal wall and the dynamic of mucins secreted in the jejunum of broilers supplemented with Bacillus subtilis strain C3102

O objetivo deste estudo foi analisar a morfometria da parede intestinal e a dinâmica de mucinas secretadas no jejuno de frangos de corte suplementadas com probiótico Bacillus subtilis cepa C3102. Foram utilizados 720 pintinhos com um dia de vida, divididos em dois grupos: GA (Grupo controle) - aves alimentadas com ração sem suplementação de Bacillus subtilis cepa C3102 (1º ao 35º dia) e GB (Grupo tratado) - aves alimentadas com ração com suplementação de Bacillus subtilis cepa C3102 (1º ao 35º dia). Aos 7 e 35 dias 2cm do jejuno foram coletados e submetidos à rotina de processamento histológico. Cortes transversais de 1μm foram corados pela técnica do Alcian Blue (AB) para detecção de sialomucinas (pH 0,5) e sulfomucinas (pH 2,5), como também para contagem de células caliciformes presentes. A análise histoquímica revelou uma maior quantidade de sialomucinas presentes no jejuno dos animais que foram suplementados com probiótico Bacillus subtilis cepa C3102 (GB), quando comparados com os animais do grupo controle (GA). O número de células caliciformes presentes no grupo tratado (GB) foi superior ao grupo controle (GA).(AU)

The aim of this study was to analyze the morphology of the intestinal wall and the dynamics of mucins secreted in the jejunum of broilers supplemented with probiotic Bacillus subtilis strain C3102. We used 720 chicks with a day of life, divided into two groups: GA (control group) - birds fed diet without supplementation of Bacillus subtilis strain C3102 (1 to 35 days) and GB (treated group) - birds fed with supplementation of Bacillus subtilis strain C3102 (1 to 35 days). On the 7th and the 35th day, 2cm of jejunum were collected and submitted to histological processing. 1μm cross sections were stained by Alcian blue (AB) for detecting of sialomucins (pH 0.5) and sulphomucins (pH 2.5) as well as to count goblet cells. Histochemical analysis revealed a greater amount of sialomucins in the jejunum of animals that were supplemented with probiotic Bacillus subtilis strain C3102 (GB), when compared with the control group (GA). The number of goblet cells in the treated group (GB) was higher than the control group (GA).(AU)