Differential expression of nifH and anfH genes in Paenibacillus durus analysed by reverse transcriptase-PCR and denaturing gradient gel electrophoresis.

AIMS: The aim of this study was to develop an approach based on a reverse transcriptase (RT)-PCR/denaturing gradient gel electrophoresis (DGGE) for the detection of the functional genes nifH and anfH in Paenibacillus durus. METHODS AND RESULTS: Two sets of primers were employed to study the expression of the nitrogen fixation genes in a pure-culture system of P. durus grown in media with increasing concentrations of ammonium (NH(4)(+)), tungsten (W) or molybdenum (Mo). The results obtained indicate that the expression of nitrogenase genes from P. durus can take place in the presence of relatively high levels of fixed nitrogen. It was also observed that the addition of 20 micromol l(-1) molybdenum and 2 mmol l(-1) tungstate did not interfere in the mRNA levels of nifH and anfH genes. CONCLUSIONS: Our results demonstrate the presence and transcription of nifH and anfH in P. durus under a variety of growth conditions. A specific set of primers was designed for the detection of the alternative system for nitrogen fixation in P. durus. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-PCR/DGGE system enables the rapid gathering of incremental data about the regulation of conventional and alternative nitrogenase genes in P. durus strains.

Diversity of Paenibacillus polymyxa strains isolated from the rhizosphere of maize planted in Cerrado soil.

Paenibacillus polymyxa populations present in the rhizosphere of maize (cultivar BR-201) planted in Cerrado soil were investigated in order to assess their diversity at four stages of plant growth. A total of 67 strains were isolated and all strains were identified as P. polymyxa by classical biochemical tests, API 50CH tests and a set of species-specific primers based on the 23S rDNA sequence. To compare the isolated strains, phenotypic characteristics (utilization of different carbohydrates, resistance to antibiotics and production of antimicrobial substances) and genetic approaches (hybridization with a Klebsiella pneumoniae nifKDH probe and BOX-PCR) were used. Fermentation of glycerol, arabinose, xylose and rhamnose varied among the isolates and these data divided the strains into five groups. Fifty strains (75%) showed homology to plasmid pSA30 (containing the nifKDH genes) resulting in five different hybridization patterns. Using BOX-PCR, 18 groups were observed. Phenetic analyses were applied based on the unweighted pair group method with arithmetic means using the phenotypic and genetic data, separately. All P. polymyxa isolates could be divided into two main clusters at approximately 52% and into 18 groups at approximately 89% of similarity, when phenotypic data were used. Also, two main clusters were formed at 65% of similarity when genetic data were used. In this dendrogram, clusters were further split into 10 and 22 groups, at about 88 and 97% of similarity, respectively. Finally, all phenotypic and genetic data, or just the genetic data, were used in a multivariate analysis of variance (MANOVA) in order to address the heterogeneity among P. polymyxa populations during the different stages of maize growth. The resulting data showed that strains isolated 10, 30, 60 and 90 days after maize sowing were statistically different.

Influence of growth conditions on the production of extracellular proteolytic enzymes in Paenibacillus peoriae NRRL BD-62 and Paenibacillus polymyxa SCE2.

AIMS: To analyse the extracellular protease profile of two Paenibacillus species, Paenibacillus peoriae and Paenibacillus polymyxa, as well as how different growth media influenced its expression. METHODS AND RESULTS: Both bacteria were cultured in five media [Luria-Bertani broth, glucose broth, thiamine/biotin/nitrogen broth (TBN), trypticase soy broth and a defined medium] for 48 h at 32 degrees C. Our results showed a heterogeneous protease secretion pattern whose expression was dependent on medium composition. However, TBN induced the most quantitative and qualitative protease production on both Paenibacillus. The proteases were detected in neutral-alkaline pH range, being totally inhibited by 1,10-phenanthroline, a zinc-metalloprotease inhibitor. We also analysed the protease expression during the growth and, at least to P. peoriae, the most elevated protease activity was measured at 96 h, in which the highest number of spores and a low concentration of viable cells were observed. CONCLUSIONS: The results presented add P. peoriae and P. polymyxa to the list of neutral-alkaline extracellular protease producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Paenibacillus species are ubiquitous in nature, are capable to form resistant spores and to produce several hydrolytic enzymes, including proteases. However, only few data concerning the production of these enzymes are available. Proteases produced by Paenibacillus strains may represent new sources for biotechnological use.

Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi.

AIMS: To investigate the potential antagonistic activity of Paenibacillus peoriae strain NRRL BD-62 against phytopathogenic micro-organisms and to determine the physiological and biochemical characteristics of the antimicrobial compound produced by this strain. METHODS AND RESULTS: Strain NRRL BD-62 showed a broad inhibition spectrum with activity against various phytopathogenic bacteria and fungi. Physico-chemical characterization of the antimicrobial activity showed that it was stable during heat treatment and was retained even after autoclave at 121 degrees C for 10 min. The compound was also stable after the treatment with organic solvents, hydrolytic enzymes and its activity was preserved at a wide range of pH. The partial purification carried out by Sephadex G25 gel filtration showed two profiles of inhibition against the indicator strains tested, suggesting at least two different substances with distinct molecular weight. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the production of antimicrobial substances in P. peoriae. Besides the antimicrobial inhibition capability, the strain NRRL BD-62 is also able to effectively fix molecular nitrogen, and produce chitinases and proteases as well, suggesting that further studies should be addressed to use P. peoriae strain NRRL BD-62 as a plant growth promoter and/or as a biocontrol agent in field experiments.