Bioinformatic and functional analysis of a PHB polymerase (PhbC) from Azospirillum baldaniorum.

BACKGROUND: Azospirillum baldaniorum Sp245 produces poly-ß-hydroxybutyrate, a biodegradable polymer with characteristics similar to synthetic thermoplastics, including polypropylene. In the synthesis pathway, the poly-ß-hydroxybutyrate synthase enzyme uses thioesters of 3-hydroxy butyryl-CoA as a substrate and catalyzes their polymerization with HS-CoA release. METHODS: A study was conducted using in silico analysis of the two phbC genes of A. baldaniorum Sp245. One was selected for amplification and cloning into the pEXP5- CT/TOPO® vector, which was analysed by restriction pattern, polymerase chain reaction, and sequencing. SDS-PAGE analysis determined the molecular weight of the PhbC1 protein from Azospirillum baldaniorum (AbPhbC1). The presence of the protein was confirmed by Western blotting using anti-polyhistidine monoclonal antibodies. The enzymatic activity in the crude extract of AbPhbC1 was determined by measuring the concentration of sulfhydryl groups using the Ellman method. A UV-Vis assay was performed. To confirm the presence of the poly-ß-hydroxybutyrate product, an NMR assay was performed. RESULTS: In silico analyses, it is revealed that AbPhbC1 and the PhbC2 protein from Azospirillum baldaniorum (AbPhbC2) retain the poly-ß-hydroxybutyrate polymerase and α/ß hydrolase domain. The Cys-His-Asp catalytic triad is highly conserved in all four polyß-hydroxyalkanoate synthases in the central subdomain, structurally similar to the reported crystallized proteins. The dimerization subdomain is different; in AbPhbC1, it is in the closed form; in AbPhbC2, it is in the open form; and in AbPhbC2, it lacks the EC region as class III and IV poly-ß-hydroxyalcanoate synthases. In vitro, the molecular weight of AbPhbC1 was 68 kDa. The polymerization of PHB by AbPhbC1 was detected by the release of HS-CoA from the quantification of SH. The UV-Vis scan showed a characteristic peak at 264 nm. A comparison of the NMR spectra of the bacterial and commercial poly-ß-hydroxybutyrate samples suggested their presence. CONCLUSION: In silico analyses suggested that AbPhbC1 and AbPhbC2 are structurally functional, except that AbPhbC2 might require the PhaR subunit for its activity; this strongly suggests that it could be a class IV poly-ß-hydroxyalcanoate synthase. UV-Vis scanning and NMR spectroscopy revealed the synthesis of poly-ß-hydroxybutyrate by the A. baldaniorum enzyme AbPhbC1, indicating that the enzyme is functional.

Computational Analysis of the Tripartite Interaction of Phasins (PhaP4 and 5)-Sigma Factor (σ24)-DNA of Azospirillum brasilense Sp7.

Azospirillum brasilense Sp7 produces PHB, which is covered by granule-associated proteins (GAPs). Phasins are the main GAPs. Previous studies have shown phasins can regulate PHB synthesis. When A. brasilense grows under stress conditions, it uses sigma factors to transcribe genes for survival. One of these factors is the σ24 factor. This study determined the possible interaction between phasins and the σ24 factor or phasin-σ24 factor complex and DNA. Three-dimensional structures of phasins and σ24 factor structures were predicted using the I-TASSER and SWISS-Model servers, respectively. Subsequently, a molecular docking between phasins and the σ24 factor was performed using the ClusPro 2.0 server, followed by molecular docking between protein complexes and DNA using the HDOCK server. Evaluation of the types of ligand-receptor interactions was performed using the BIOVIA Discovery Visualizer for three-dimensional diagrams, as well as the LigPlot server to obtain bi-dimensional diagrams. The results showed the phasins (Pha4Abs7 or Pha5Abs7)-σ24 factor complex was bound near the -35 box of the promoter region of the phaC gene. However, in the individual interaction of PhaP5Abs7 and the σ24 factor, with DNA, both proteins were bound to the -35 box. This did not occur with PhaP4Abs7, which was bound to the -10 box. This change could affect the transcription level of the phaC gene and possibly affect PHB synthesis.

Identification of residues for chaperone-like activity of OppA protein in Yersinia pseudotuberculosis.

Periplasmic oligopeptide binding protein (OppA) is part of a multimeric cytoplasmic membrane protein complex, whose function is known as peptide transporters found in Gram-negative bacteria. A chaperone-like activity has been found for the OppA from Yersinia pseudotuberculosis, using biochemical experiments. Through computational analysis, we selected two amino acid residues (R41 and D42) that probably are involved in the chaperone-like activity. Our results to corroborate how OppA assists refolding and renaturation of lactate dehydrogenase and alpha-glucosidase denatured enzymes.

Phasin PhaP1 is involved in polyhydroxybutyrate granules morphology and in controlling early biopolymer accumulation in Azospirillum brasilense Sp7.

Phasins are amphiphilic proteins involved in the regulation of the number and size of polyhydroxybutyrate (PHB) granules. The plant growth promoting bacterium Azospirillum brasilense Sp7 accumulates high quantities of bioplastic PHB as carbon and energy source. By analyzing the genome, we identified six genes that code for proteins with a Phasin_2 domain. To understand the role of A. brasilense Sp7 PhaP1 (PhaP1Abs) on PHB synthesis, the phaP1 gene (AMK58_RS17065) was deleted. The morphology of the PHB granules was analyzed by transmission electron microscopy (TEM) and the PHB produced was quantified under three different C:N ratios in cultures subjected to null or low-oxygen transfer. The results showed that PhaP1Abs is involved in PHB granules morphology and in controlling early biopolymer accumulation. Using RT-PCR it was found that phasin genes, except phaP4, are transcribed in accordance with the C:N ratio used for the growth of A. brasilense. phaP1, phaP2 and phaP3 genes were able to respond to the growth conditions tested. This study reports the first analysis of a phasin protein in A. brasilense Sp7.

The ipdC, hisC1 and hisC2 genes involved in indole-3-acetic production used as alternative phylogenetic markers in Azospirillum brasilense.

Plant growth-promoting bacteria of the genus Azospirillum are present in the rhizosphere and as endophytes of many crops. In this research we studied 40 Azospirillum strains isolated from different plants and geographic regions. They were first characterized by 16S rDNA restriction analysis, and their phylogenetic position was established by sequencing the genes 16S rDNA, ipdC, hisC1, and hisC2. The latter three genes are involved in the indole-3-pyruvic acid (IPyA) biosynthesis pathway of indole-3-acetic acid (IAA). Furthermore, the suitability of the 16S-23S rDNA intergenic spacer sequence (IGS) for the differentiation of closely related Azospirillum taxa and development of PCR protocols allows for specific detection of strains. The IGS-RFLP analysis enabled intraspecies differentiation, particularly of Azospirillum brasilense and Azospirillum lipoferum strains. Results demonstrated that the ipdC, hisC1, and hisC2 genes are highly conserved in all the assessed A. brasilense isolates, suggesting that these genes can be used as an alternative phylogenetic marker. In addition, IAA production determined by HPLC ranged from 0.17 to 98.2 µg mg(-1) protein. Southern hybridization with the A. brasilense ipdC gene probe did not show, a hybridization signal with A. lipoferum, Azospirillum amazonense, Azospirillum halopreferans and Azospirillum irakense genomic DNA. This suggests that these species produce IAA by other pathways. Because IAA is mainly synthesized via the IPyA pathway in A. brasilense strains, a species that is used worldwide in agriculture, the identification of ipdC, hisC1, and hisC2 genes by PCR may be suitable for selecting exploitable strains.

Indole-3-butyric acid (IBA) production in culture medium by wild strain Azospirillum brasilense.

Some microorganisms found in the soil are able to produce substances which regulate plant growth. In this study, we show the presence of a substance associated with auxin activity, identified as indole-3-butyric acid (IBA), in Azospirillum brasilense UAP 154 growth medium. A. brasilense was grown and indolic compounds were extracted from the supernatant. These were then analyzed by high performance liquid chromatography (HPLC), gas chromatography and gas chromatography mass spectrometry. The retention time was similar to those of the authentic IBA standard. The compound obtained from HPLC was collected and applied to maize seedlings (Zea mays), inducing biological activity along the roots, similar to that induced by an authentic IBA standard.