Tools for genetic manipulation of the plant growth-promoting bacterium Azospirillum amazonense

<p>Abstract</p> <p>Background</p> <p><it>Azospirillum amazonense </it>has potential to be used as agricultural inoculant since it promotes plant growth without causing pollution, unlike industrial fertilizers. Owing to this fact, the study of this species has gained interest. However, a detailed understanding of its genetics and physiology is limited by the absence of appropriate genetic tools for the study of this species.</p> <p>Results</p> <p>Conjugation and electrotransformation methods were established utilizing vectors with broad host-replication origins (pVS1 and pBBR1). Two genes of interest - <it>glnK </it>and <it>glnB</it>, encoding PII regulatory proteins - were isolated. Furthermore, <it>glnK</it>-specific <it>A. amazonense </it>mutants were generated utilizing the pK19MOBSACB vector system. Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium.</p> <p>Conclusion</p> <p>In this work, genetic tools that can support the study of <it>A. amazonense </it>were described. These methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion.</p

Metabolic Investigation of the Mycoplasmas from the Swine Respiratory Tract

International audienceBackgroundThe 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.MethodsIn 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.ResultsEven 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.ConclusionsWe 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

Genomic insights into the versatility of the plant growth-promoting bacterium Azospirillum amazonense

14 páginas.-- 8 figuras.-- 80 referencias.-- 4 ficheros adicionales con 4 tablas en http://www.biomedcentral.com/1471-2164/12/409/additionalBackground: The species Azospirillum amazonense belongs to a well-known genus of plant growth-promoting bacteria. This bacterium is found in association with several crops of economic importance; however, there is a lack of information on its physiology. In this work, we present a comprehensive analysis of the genomic features of this species. Results: Genes of A. amazonense related to nitrogen/carbon metabolism, energy production, phytohormone production, transport, quorum sensing, antibiotic resistance, chemotaxis/motility and bacteriophytochrome biosynthesis were identified. Noteworthy genes were the nitrogen fixation genes and the nitrilase gene, which could be directly implicated in plant growth promotion, and the carbon fixation genes, which had previously been poorly investigated in this genus. One important finding was that some A. amazonense genes, like the nitrogenase genes and RubisCO genes, were closer phylogenetically to Rhizobiales members than to species of its own order. Conclusion: The species A. amazonense presents a versatile repertoire of genes crucial for its plant-associated lifestyle.This work was supported by grants from the Brazilian National Research Council (CNPq) and the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS). FHS, RC, LAR and FMS received scholarships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).Peer reviewe

Molecular analysis of an integrative conjugative element, ICEH, present in the chromosome of different strains of Mycoplasma hyopneumoniae

Diversification of bacterial species and pathotypes is largely caused by lateral gene transfer (LGT) of diverse mobile DNA elements such as plasmids, phages, transposons and genomic islands. Thus, acquisition of new phenotypes by LGT is very important for bacterial evolution and relationship with hosts. This paper reports a 23 kb region containing fourteen CDSs with similarity to the previous described Integrative Conjugal Element of Mycoplasma fermentans (ICEF). This element, named ICEH, is present as one copy at distinct integration sites in the chromosome of 7448 and 232 pathogenic strains and is absent in the type strain J (non-pathogenic). Notable differences in the nucleotide composition of the insertion sites were detected, and could be correlated to a lack of specificity of the ICEH integrase. Although present in strains of the same organism, the ICEH elements are more divergent than the typical similarity between other chromosomal locus of Mycoplasma hyopneunomiae, suggesting an accelerated evolution of these constins or an ongoing process of degeneration, while maintaining conservation of the tra genes. An extrachromosomal form of this element had been detected in the 7448 strain, suggesting a possible involvement in its mobilization and transference of CDSs to new hosts

Análise da contaminação por Salmonella em ovos do tipo colonial através da reação em cadeia da polimerase Analysis of Salmonella in free-range eggs through polymerase chain reaction

A identificação de poedeiras comerciais infectadas por salmonelas tem sido um dos pontos fortes da profilaxia e conseqüente redução de surtos de salmonelose em humanos associados ao consumo de ovos, sendo que a análise dos ovos pode ser mais um dos pontos de detecção da infecção, que, muitas vezes, cursa sem sinais clínicos. A Reação em Cadeia da Polimerase (PCR) parece ser uma estratégia útil para detecção de Salmonella, pois vários autores têm utilizado a PCR para verificar a presença da bactéria em carnes, fezes, tecidos, sangue, leite e ovos, com diferentes metodologias de manipulação das amostras. Foram analisados 360 ovos, procedentes de dez propriedades rurais, produtoras de ovos tipo colonial, no distrito de Camobi, em Santa Maria - RS. Os ovos foram divididos em grupos de seis, totalizando sessenta amostras. O exame bacteriológico foi realizado conforme metodologia preconizada pelas normas técnicas e a metodologia de extração de DNA pelo fenol-clorofórmio. A PCR foi realizada para a amplificação de um fragmento de DNA de 284 pb. A análise dos resultados não demonstrou diferença significativa entre a PCR e o bacteriológico. Todas as amostras positivas ao bacteriológico foram positivas na PCR, sendo que essa última detectou duas amostras a mais, devido a sua alta sensibilidade e especificidade, especialmente quando é sabido que os ovos apresentam uma população microbiana mista que, muitas vezes, impede o isolamento adequado das salmonelas no bacteriológico pela competição com a flora bacteriana normalmente presente.<br>The identification of salmonella infection in commercial poultry has been one of the strong points of prophylaxis and consequent reduction of salmonellosis outbreaks in humans associated to consumption of eggs, considering that the analysis of the eggs can be one more point of detection of infection, which for many times appear without clinical signs. The Polymerase Chain Reaction (PCR) seems to be a useful strategy for Salmonella detection, because various authors have used the PCR to verify the presence of bacteria in meat, feces, tissues, blood, milk and eggs, with different methods of manipulation of samples. We have analyzed 360 eggs from ten farms, producers of free range-eggs, in the district of Camobi, in Santa Maria - RS - Brasil. The eggs were grouped in pools of six, totaling sixty samples. The bacteriological exam was done in compliance with the method preconized by the technical rules and the method for extraction of DNA was by phenol-chloroform. The PCR was performed for the amplification of a 284 bp DNA fragment. The analysis of the results do not show significant difference between the PCR and the bacteriological exam. All positive samples in the bacteriological exam were also positive by PCR, however the PCR detected more two samples due to higher sensitivity and specificity, specially when it is known that the eggs show a mixed population of germs that many times difficult isolation of salmonellas in the bacteriological exam because of the competition with normal flora bacteria