Global analysis of sRNA target genes in Mycoplasma hyopneumoniae

Background: Small RNAs (sRNAs) are noncoding molecules that regulate different cellular activities in several bacteria. The role of sRNAs in gene expression regulation is poorly characterized in the etiological agent of porcine enzootic pneumonia Mycoplasma hyopneumoniae. We performed a global analysis of the sRNAs, sRNA target genes and regulatory elements previously identified in their genome and analyzed the expression of some sRNAs and their target genes by quantitative RT-PCR (qPCR) in three different culture conditions. Results: Seven of the 145 sRNA target genes are organized as monocistronic genes (mCs) while the other 138 sRNA target genes are organized into transcriptional units (TU). The identification of transcriptional regulatory elements (promoter motif, DNA repeat sequence or intrinsic terminator) was verified in 116 of the 145 sRNA target genes. Moreover, the 29 sRNA target genes without regulatory elements revealed the presence of at least one regulatory element in the boundaries of the TU or in other internal genes of the TU. We verified that 16 sRNAs showed differential expression, seven in heat shock condition and 14 in oxidative stress condition. Analysis of the differential expression of the sRNA target genes showed that the tested sRNAs possibly regulate gene expression. The sRNA target genes were up- or down-regulated possibly in response to sRNA only under oxidative stress condition. Moreover, the sRNA target genes are involved in diverse processes of the cell, some of which could be linked to transcription processes and cell homeostasis. Conclusion: Our results indicate that bacterial sRNAs could regulate a number of targets with various outcomes, and different correlations between the levels of sRNA transcripts and their target gene mRNAs were found, which suggest that the regulation of gene expression via sRNAs may play an important role in mycoplasma

The Plant Growth-Promoting Bacteria Azospirillum amazonense: Genomic Versatility and Phytohormone Pathway

The rhizosphere bacterium Azospirillum amazonense associates with plant roots to promote plant growth. Variation in replicon numbers and rearrangements is common among Azospirillum strains, and characterization of these naturally occurring differences can improve our understanding of genome evolution. We performed an in silico comparative genomic analysis to understand the genomic plasticity of A. amazonense. The number of A. amazonense-specific coding sequences was similar when compared with the six closely related bacteria regarding belonging or not to the Azospirillum genus. Our results suggest that the versatile gene repertoire found in A. amazonense genome could have been acquired from distantly related bacteria from horizontal transfer. Furthermore, the identification of coding sequence related to phytohormone production, such as flavin-monooxygenase and aldehyde oxidase, is likely to represent the tryptophan-dependent TAM pathway for auxin production in this bacterium. Moreover, the presence of the coding sequence for nitrilase indicates the presence of the alternative route that uses IAN as an intermediate for auxin synthesis, but it remains to be established whether the IAN pathway is the Trp-independent route. Future investigations are necessary to support the hypothesis that its genomic structure has evolved to meet the requirement for adaptation to the rhizosphere and interaction with host plants

Caracterização do gene SRG de Metarhizium anisopliae

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orfatrAb - Caracterização molecular do gene que codifica um ativador de transcrição da família LysR em Azospirillum brasilense

Nitrogen, which is a component of several biomolecules such as nucleic acids and amino acids, is abundant in the atmosphere where, in its gaseous form (N 2 ), it is unavailable for most organisms. However, there are microorganisms, called diazotrophs that are able to convert the atmospheric nitrogen into an assimilable form to other organisms through a process called biological nitrogen fixation. Bacteria of the genus Azospirillum are diazotrophs, free-living or endophytic that are able to associate with grasses like wheat, rice, sugar cane, etc. One of the most used tools for the identification of genes involved in the nitrogen fixation in Azospirillum was the site-direct mutagenesis using the Tn5 transposon. This methodology allowed the isolation of one A. brasilense transconjugant with a capacity of biological nitrogen fixation in vitro that was higher than that of the wild type strain. In the place of transposon insertion into the DNA of this mutant two open reading frames were identified, orfatrAb and orf281, which was interrupted by Tn5. The aim of this work was to determinate the orfatrAb complete nucleotide sequence and the corresponding amino acid sequence which showed great similarity, especially in the amino-terminus, with proteins of the LysR family of transcriptional activators. ORFAtrAb has 297 residues, molecular weight of 33,581.6 kD, isoeletric point of 7.26 and instability index of 33.29. Sequence consensus elements of gene activator proteins involved in the metabolism and in the biological nitrogen fixation were identified in the orfatrAb promoter region.O nitrogênio, elemento-chave na composição de diversas biomoléculas como aminoácidos e ácidos nucléicos, é abundante na atmosfera, onde, sob forma de N 2 , não está disponível para a maioria dos seres vivos. Entretanto, existem microrganismos, chamados de diazotróficos, que são capazes de converter o nitrogênio atmosférico em formas assimiláveis para os outros organismos, através do processo de Fixação Biológica do Nitrogênio. Bactérias do gênero Azospirillum são microrganismos diazotróficos de vida livre ou endofíticos, que se associam com gramíneas como trigo, arroz, cana-de-açúcar, entre outras. Uma das ferramentas mais utilizadas na identificação de genes envolvidos na fixação de nitrogênio em Azospirillum foi a mutagênese sítio-dirigida com o transposon Tn5. Essa metodologia possibilitou o isolamento de um transconjugante de A. brasilense, cuja capacidade de fixação de nitrogênio in vitro foi bastante elevada em relação à da linhagem de tipo selvagem. No local de inserção do transposon no DNA desse transconjugante foram encontradas duas fases abertas de leitura, orfartAb e orf281, tendo sido, essa última, interrompida pelo Tn5. O objetivo deste trabalho foi determinar a seqüência de nucleotídeos de orfatrAb, e, a partir desta, a seqüência de aminoácidos, a qual apresentou grande similaridade, principalmente em sua região amino-terminal, com proteínas da família de ativadores de transcrição LysR. ORFatrAb possui 297 resíduos, peso molecular de 33.581,6 kDa, ponto isoelétrico de 7,26 e índice de instabilidade de 33,29. Na região reguladora de orfatrAb foram iden-tificados elementos de seqüência consensuais para a ligação de proteínas ativadoras de genes envolvidos no metabolismo e na fixação biológica do nitrogênio

Microbiome overview in swine lungs

Mycoplasma hyopneumoniae is the etiologic agent of swine enzootic pneumonia. However other mycoplasma species and secondary bacteria are found as inhabitants of the swine respiratory tract, which can be also related to disease. In the present study we have performed a total DNA metagenomic analysis from the lungs of pigs kept in a field condition, with suggestive signals of enzootic pneumonia and without any infection signals to evaluate the bacteria variability of the lungs microbiota. Libraries from metagenomic DNA were prepared and sequenced using total DNA shotgun metagenomic pyrosequencing. The metagenomic distribution showed a great abundance of bacteria. The most common microbial families identified from pneumonic swine’s lungs were Mycoplasmataceae, Flavobacteriaceae and Pasteurellaceae, whereas in the carrier swine’s lungs the most common families were Mycoplasmataceae, Bradyrhizobiaceae and Flavobacteriaceae. Analysis of community composition in both samples confirmed the high prevalence of M. hyopneumoniae. Moreover, the carrier lungs had more diverse family population, which should be related to the lungs normal flora. In summary, we provide a wide view of the bacterial population from lungs with signals of enzootic pneumonia and lungs without signals of enzootic pneumonia in a field situation. These bacteria patterns provide information that may be important for the establishment of disease control measures and to give insights for further studies

Mycoplasma non-coding RNA: identification of small RNAs and targets

Background: Bacterial non-coding RNAs act by base-pairing as regulatory elements in crucial biological processes. We performed the identification of trans-encoded small RNAs (sRNA) from the genomes of Mycoplama hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis, which are Mycoplasma species that have been identified in the porcine respiratory system. Results: A total of 47, 15 and 11 putative sRNAs were predicted in M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively. A comparative genomic analysis revealed the presence of species or lineage specific sRNA candidates. Furthermore, the expression profile of some M. hyopneumoniae sRNAs was determined by a reverse transcription amplification approach, in three different culture conditions. All tested sRNAs were transcribed in at least one condition. A detailed investigation revealed a differential expression profile for two M. hyopneumoniae sRNAs in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. Moreover, we analyzed sRNA-mRNA hybrids and accessed putative target genes for the novel sRNA candidates. The majority of the sRNAs showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity. Conclusion: This study contributes to our knowledge of Mycoplasma sRNAs and their response to environmental changes. Furthermore, the mRNA target prediction provides a perspective for the characterization and comprehension of the function of the sRNA regulatory mechanisms

Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling

Background: The respiratory tract of swine is colonized by several bacteria among which are three Mycoplasma species: Mycoplasma flocculare, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae is the causative agent of enzootic pneumonia and M. hyorhinis is present in cases of pneumonia, polyserositis and arthritis. The genomic resemblance among these three Mycoplasma species combined with their different levels of pathogenicity is an indication that they have unknown mechanisms of virulence and differential expression, as for most mycoplasmas. Methods: In this work, we performed whole-genome metabolic network reconstructions for these three mycoplasmas. Cultivation tests and metabolomic experiments through nuclear magnetic resonance spectroscopy (NMR) were also performed to acquire experimental data and further refine the models reconstructed in silico. Results: Even though the refined models have similar metabolic capabilities, interesting differences include a wider range of carbohydrate uptake in M. hyorhinis, which in turn may also explain why this species is a widely contaminant in cell cultures. In addition, the myo-inositol catabolism is exclusive to M. hyopneumoniae and may be an important trait for virulence. However, the most important difference seems to be related to glycerol conversion to dihydroxyacetone-phosphate, which produces toxic hydrogen peroxide. This activity, missing only in M. flocculare, may be directly involved in cytotoxicity, as already described for two lung pathogenic mycoplasmas, namely Mycoplasma pneumoniae in human and Mycoplasma mycoides subsp. mycoides in ruminants. Metabolomic data suggest that even though these mycoplasmas are extremely similar in terms of genome and metabolism, distinct products and reaction rates may be the result of differential expression throughout the species. Conclusions: We were able to infer from the reconstructed networks that the lack of pathogenicity of M. flocculare if compared to the highly pathogenic M. hyopneumoniae may be related to its incapacity to produce cytotoxic hydrogen peroxide. Moreover, the ability of M. hyorhinis to grow in diverse sites and even in different hosts may be a reflection of its enhanced and wider carbohydrate uptake. Altogether, the metabolic differences highlighted in silico and in vitro provide important insights to the different levels of pathogenicity observed in each of the studied species

Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador (“gene-cartridge mutagenesis”) foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A combinação entre a disrupção gênica através da técnica de mutagênese utilizada e eletrotransformação foi, provavelmente, a causa principal do rearranjo genômico ocorrido nessas bactérias.In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif - phenotype. Four Nif - mutants were analyzed by Southern blot, using six different probes spanning the nif and Kmr genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance genecartridge mutagenesis combined with electrotransformation