Identification and Characterization of a New Type of Holin-Endolysin Lysis Cassette in Acidovorax oryzae Phage AP1.

Phages utilize lysis systems to allow the release of newly assembled viral particles that kill the bacterial host. This is also the case for phage AP1, which infects the rice pathogen Acidovorax oryzae. However, how lysis occurs on a molecular level is currently unknown. We performed in silico bioinformatics analyses, which indicated that the lysis cassette contains a holin (HolAP) and endolysin (LysAP), which are encoded by two adjacent genes. Recombinant expression of LysAP caused Escherichia coli lysis, while HolAP arrested growth. Co-expression of both proteins resulted in enhanced lysis activity compared to the individual proteins alone. Interestingly, LysAP contains a C-terminal region transmembrane domain, which is different from most known endolysins where a N-terminal hydrophobic region is found, with the potential to insert into the membrane. We show that the C-terminal transmembrane domain is crucial for protein localization and bacterial lysis in phage AP1. Our study characterizes the new phage lysis cassette and the mechanism to induce cell disruption, giving new insight in the understanding of phage life cycles.

Genome re-seqeunce and analysis of Burkholderia glumae strain AU6208 and evidence of toxoflavin: A potential bacterial toxin.

Burkholderia glumae, the primary causative agent of bacterial panicle blight in rice, has been reported as an opportunistic pathogen in patients with chronic infections. This study aimed to re-sequence the clinical isolate B. glumae strain AU6208 and comparatively analyze its genome using B. glumae strain BGR1 from rice plant as the reference. Re-sequencing results revealed that the genome of strain AU6208 comprised 96 contigs corresponding to a 6.1 Mbp genome of the strain AU6208, with 5322 coding sequences and 68.2 % GC content; this is much larger compared to the genome previously sequenced by us and described by Seo et al (2015), which was reported to be 4.1 Mbp comprising >1200 contigs, 4361 coding sequences, and 67.31 % GC content. Moreover, this updated genome shares >80 % identity to the 7.2 Mbp genome of BGR1, which encodes 6491 coding sequences and has 68.3 % GC content. Further computational analysis revealed that the strain AU6208 encodes several bacteriocin biosynthesis genes, antibiotic, as well as virulent genes such as toxoflavin genes, which included 425 specialty genes and 12 toxoflavin genes. Upon further characterization, 12 toxoflavins (ToxA, B, C, D, E, F, G, H, I, J, TofI, and TofR) were found in AU6208 with 70-100 % sequence, family, and domain similarity with that of BGR1. Upon comparison with BGR1, the structural characterizations of selected toxoflavin genes (ToxB, ToxC, ToxG, H, and TofI) revealed variations in 2D and 3D structures such as differences in α-helix, ß-sheets, loops, physiological properties of proteins, RMSD values, etc. These variations may play significant role in different mode of action in different hosts thereby indicating that in addition to their respective hosts, toxoflavins could also contribute to exploit other hosts across the kingdom. In addition to understanding the epidemiology of strain AU6208, this updated genomics data will also unfold the pathogenicity of bacteria in diversity of various hosts and anti-virulence.

Green Synthesis of Silver Nanoparticles with Culture Supernatant of a Bacterium Pseudomonas rhodesiae and Their Antibacterial Activity against Soft Rot Pathogen Dickeya dadantii.

Bacterial stem and root rot disease of sweet potato caused by Dickeya dadantii recently broke out in major sweet potato planting areas in China and calls for effective approaches to control the pathogen and disease. Here, we developed a simple method for green synthesis of silver nanoparticles (AgNPs) using bacterial culture supernatants. AgNPs synthesized with the cell-free culture supernatant of a bacterium Pseudomonas rhodesiae displayed the characteristic surface plasmon resonance peak at 420-430 nm and as nanocrystallites in diameters of 20-100 nm determined by transmission electron microscopy, scanning electron microscopy, and X-ray diffraction spectroscopy. Functional groups associated with proteins in the culture supernatant may reduce silver ions and stabilize AgNPs. The AgNPs showed antibacterial activities against D. dadantii growth, swimming motility, biofilm formation, and maceration of sweet potato tubers whereas the culture supernatant of P. rhodesiae did not. AgNPs (12 µg∙ml-1) and AgNO3 (50 µg∙ml-1) showed close antibacterial activities. The antibacterial activities increased with the increase of AgNP concentrations. The green-synthesized AgNPs can be used to control the soft rot disease by control of pathogen contamination of sweet potato seed tubers.

The Green Synthesis of MgO Nano-Flowers Using Rosmarinus officinalis L. (Rosemary) and the Antibacterial Activities against Xanthomonas oryzae pv. oryzae.

Recently, the use of herbs in the agriculture and food industry has increased significantly. In particular, Rosmarinus officinalis L. extracts have been reported to have strong antibacterial properties, which depend on their chemical composition. The present study displayed a biological method for synthesis of magnesium oxide (MgO) nano-flowers. The nano-flowers are developed without using any catalyst agent. Aqueous Rosemary extract was used to synthesize MgO nano-flowers (MgONFs) in stirring conditions and temperature at 70°C for 4 h. The mixture solution was checked by UV-Vis spectrum to confirm the presence of nanoparticles. The MgO nano-flowers powder was further characterized in this study by the X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy. In addition, bacteriological tests indicated that MgO nano-flowers significantly inhibited bacterial growth, biofilm formation, and motility of Xanthomonas oryzae pv. oryzae, which is the causal agent of bacterial blight disease in rice. The electronic microscopic observation showed that bacterial cell death may be mainly due to destroy of cell integrity, resulting in leakage of intracellular content. As recommended, the use of Rosemary extract is an effective and green way to produce the MgO nano-flowers, which can be widely used in agricultural fields to suppress bacterial infection.

Green synthesis of zinc oxide nanoparticles using different plant extracts and their antibacterial activity against Xanthomonas oryzae pv. oryzae.

The synthesis of metal oxide nanoparticles with the use of plant extract is a promising alternative to the conventional chemical method. This work aimed to synthesize zinc oxide nanoparticles (ZnONPs) using plant extract of chamomile flower (Matricaria chamomilla L.), olive leave (Olea europaea) and red tomato fruit (Lycopersicon esculentum M.). The synthesized ZnONPs were characterized by UV-Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) with EDS profile. The XRD studies confirmed the presence of pure crystalline shapes of ZnO nanoparticles. The ZnONPs synthesized by Olea europaea had the least size range of 40.5 to 124.0 nm as revealed by the SEM observation while XRD revealed a dominant average size of 48.2 nm in the sample which is similar to the size distribution analysis obtained from TEM. The antibacterial effect of ZnONPs synthesized by Olea europaea on Xanthomonas oryzae pv. oryzae (Xoo) strain GZ 0003 had an inhibition zone of 2.2 cm at 16.0 µg/ml which was significantly different from ZnONPs synthesized by Matricaria chamomilla and Lycopersicon esculentum. Also, the bacterial growth, biofilm formation, swimming motility and bacterial cell membrane of Xoo strain GZ 0003 were significantly affected by ZnO nanoparticle. Overall, zinc oxide nanoparticles are promising biocontrol agents that can be used to combat bacterial leaf blight diseases of rice.

Role of type IV secretion system genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae strain RS-2.

Type IV secretion system (T4SS) is a specialized nanomachine that is utilized for the pathogenicity of gram-negative bacteria. However, the role of T4SS genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae (Ao) strain RS-2 is not clear, which contains T4SS gene cluster based on genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type strain RS-2 and nine T4SS mutants, which were constructed in this study. Results indicated that mutation of pilT, pilM, pilQ, or pilZ3 genes not only significantly reduced bacterial virulence, but also caused a reduction of 20.4-62.0% in biofilm formation and 37.7-47.7% reduction in motility, but had no effect on exopolysaccharide (EPS) production or extracellular enzymatic activities when compared to the wild type. The four T4SS genes had a differential effect on bacterial growth after 24 h post-incubation. The complemented strains of the four T4SS mutants restored similar virulence symptom as the wild type. In addition, no change was observed in bacterial virulence by mutation of the other five T4SS genes. Totally, these results demonstrated that T4SS played vital roles in bacterial virulence, motility and biofilm formation in plant pathogen Ao strain RS-2.

Flowerlike Mg(OH)2 Cross-Nanosheets for Controlling Cry1Ac Protein Loss: Evaluation of Insecticidal Activity and Biosecurity.

Bacillus thuringiensis (Bt) can produce Cry proteins during the sporulation phase, and Cry protein is effective against lepidopteran, coleopteran, and dipteran insects and nematodes. However, Cry protein tends to be discharged into soil and nontarget plants through rainwater runoff, leading to reduced effective period toward target insects. In the present study, nano-Mg(OH)2 (magnesium hydroxide nanoparticles, MHNPs) were synthesized to control the loss of Cry1Ac protein and deliver protein to Helicoverpa armigera (Lepidoptera: Noctuidae). The results showed that Cry1Ac protein could be loaded onto MHNPs through electrostatic adsorption, and both MHNPs and Cry protein were stable during the adsorption process. Meanwhile, the Cry1Ac-loaded MHNPs could remain on the surface of cotton leaves, resulting in enhanced adhesion of Cry1Ac protein by 59.50% and increased pest mortality by 75.00%. Additionally, MHNPs could be slowly decomposed by acid medium and MHNPs showed no obvious influence on cotton, Bt, Escherichia coli, and H. armigera. Therefore, MHNPs could serve as an efficient nanocarrier for delivery of Cry1Ac protein and be used as a potential adjuvant for biopesticide in agricultural applications.

The adsorption features between insecticidal crystal protein and nano-Mg(OH)2.

Nano-Mg(OH)2, with low biological toxicity, is an ideal nano-carrier for insecticidal protein to improve the bioactivity. In this work, the adsorption features of insecticidal protein by nano-Mg(OH)2 have been studied. The adsorption capacity could reach as high as 136 mg g-1, and the adsorption isotherm had been fitted with Langmuir and Freundlich models. Moreover, the adsorption kinetics followed a pseudo-first or -second order rate model, and the adsorption was spontaneous and an exothermic process. However, high temperatures are not suitable for adsorption, which implies that the temperature would be a critical factor during the adsorption process. In addition, FT-IR confirmed that the protein was adsorbed on the nano-Mg(OH)2, zeta potential analysis suggested that insecticidal protein was loaded onto the nano-Mg(OH)2 not by electrostatic adsorption but maybe by intermolecular forces, and circular dichroism spectroscopy of Cry11Aa protein before and after loading with nano-Mg(OH)2 was changed. The study applied the adsorption information between Cry11Aa and nano-Mg(OH)2, which would be useful in the practical application of nano-Mg(OH)2 as a nano-carrier.