Gram-negative Azospirillum brasilense accumulates approximately 80% of polyhydroxybutyrate (PHB) as dry cell weight. For this reason, this bacterium has been characterized as one of the main microorganisms that produce PHB. PHB is synthesized inside bacteria by the polymerization of 3-hydroxybutyrate monomers. In this review, we are focusing on the analysis of the PHB production by A. brasilense in order to understand the metabolism during PHB accumulation. First, the carbon and nitrogen sources used to improve PHB accumulation are discussed. A. brasilense accumulates more PHB when it is grown on a minimal medium containing a high C/N ratio, mainly from malate and ammonia chloride, respectively. The metabolic pathways to accumulate and mobilize PHB in A. brasilense are mentioned and compared with those of other microorganisms. Next, we summarize the available information to understand the role of the genes involved in the regulation of PHB metabolism as well as the role of PHB in the physiology of Azospirillum. Finally, we made a comparison between the properties of PHB and polypropylene, and we discussed some applications of PHB in biomedical and commercial areas.

Bactericidal efficiency of Au and Ag nanoparticles (NPs) is reported with and without photoactivation by white light. Au and Ag NPs were synthesized with an average size of $14\pm 1.2\mathrm{nm}$ and of $4.6\pm 0.5\mathrm{nm}$ , respectively. The size distribution of the Ag colloid was relatively wide. Less than 4$\mathrm{\%}$ of these NPs were largely decahedral, which, based on numerical calculations, determined the position of the optical band. In contrast, the Au colloid had a narrow optical band; a concentration of $1.3\mu \mathrm{g}/\mathrm{ml}$ was determined by theoretical and experimental spectra. Ag and Au NPs showed a superficial charge of $-35\mathrm{mV}$ and $+57\mathrm{mV}$ due to the presence of the citrate ions and cetyltrimethylammonium bromide on their surface, respectively. The effect of the NPs concentration on the viability of Escherichia coli and Staphylococcus aureus strains was investigated. It was found that Ag NPs were more effective against E. coli than Au NPs, whereas Au NPs were more effective against S. aureus than Ag NPs. The induced damage to the bacteria by the NPs was evaluated by AFM. The images show that the bacterial cell wall was changed in shape and in surface roughness, being more noticeable in S. aureus than in E. coli. The bactericidal activity of the photoactivated Ag NPs was almost doubled for both bacteria, whereas for the Au NPs, no bactericidal enhancement was observed for either strain. This can be explained by the high efficiency of Ag NPs to absorb white light and the consequent creation of hot spots that contribute to kill the bacteria.

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 (PhaP1_Abs) 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 PhaP1_Abs 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.

This work presents the effect of Polyhydroxybutyrate nanospheres (PHB-NSs) on the bacterial activity of plasmonic nanoparticles (NPs). The PHB-NSs were used as a substrate for the metal-NPs. Silver and gold NPs in colloidal solution were synthesized by chemical reduction, while PHB-NSs were synthesized by a physical method. A normal size distribution around 27 and 208 nm characterizes gold and PHB colloids, respectively. The Ag-NP colloid has a size distribution with a positive skewness and a mean size of ~19.6 nm. Ag-NP surface plasmon resonance is 400 nm, and Au-NP is 526 nm. A resonance shift is observed when the metal-NPs are on the PHB-NSs. Numerical calculations based on the discrete dipole approximation theory provide insight into the resonance position of the composite as a function of the nanoparticle concentration. The bactericidal effect on the viability of Escherichia coli and Staphylococcus aureus strains was evaluated. Ag-NPs were more effective against E. coli than Au-NPs, while the latter were more effective against S. aureus than the former. Interestingly, the PHB-NSs caused a delay in the bacterial activity of both metal-NPs. We proposed a model to explain this delay and the increase in contact time.

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 (Pha4_Abs7 or Pha5_Abs7)-σ²⁴ factor complex was bound near the −35 box of the promoter region of the phaC gene. However, in the individual interaction of PhaP5_Abs7 and the σ²⁴ factor, with DNA, both proteins were bound to the −35 box. This did not occur with PhaP4_Abs7, which was bound to the −10 box. This change could affect the transcription level of the phaC gene and possibly affect PHB synthesis.

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.