Purification and Characterization of a Recombinant Bacteriophytochrome of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pathovar <Emphasis Type="Italic">oryzae</Emphasis>

Phytochrome-like proteins have been recently identified in prokaryotes but their features and functions are not clear. We cloned a gene encoding the phytochrome-like protein (XoBphP) in a pathogenic bacteria, Xanthomonas oryzae pv. oryzae (Xoo) and investigated characteristics of the protein using a recombinant XoBphP. The N-terminal region of XoBphP containing the PAS/GAF/PHY domains is highly similar to most bacteriophytochromes, but Cys4, corresponding to Cys24 of DrBphP, isn’t involved in chromophore attachment. Recombinant XoBphP could bind a bilin molecule and a differential spectrum from Pr/Pfr shows that XoBphP has similar characteristics of known bacteriophytochromes with shifted absorption maxima of 683 and 757 nm for the Pr and Pfr forms. Unlike other bacteriophytochromes, XoBphP has no histidine kinase domain at C-terminus. The domain was predicted from amino-acid 279 to 342 with less significance than the required threshold. This result suggests that XoBphP probably has different signal transduction mechanisms for its intracellular function.     

Production and purification of recombinant human granulocyte–macrophage colony stimulating factor (GM-CSF) from high cell density cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Granulocyte–macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor, which has been used as a therapeutic agent in clinical cases like neutropenia. In this study, we report the production of recombinant human GM-CSF in the methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. Recombinant P. pastoris GS115 cells were grown in fed batch cultures to obtain a biomass density of 55.6 gDCW L⁻¹ and a high volumetric activity of 131 mg L⁻¹ of GM-CSF. The protein migrated as a diffuse band on SDS-PAGE at the range of 28–35 kDa indicating differential glycosylation. The secreted protein was purified to 95% in two steps using cation exchange and size exclusion chromatography.     

In vitro antimicrobial activity of the leaf essential oil of <Emphasis Type="Italic">Spiraea alpina</Emphasis> Pall.

The qualitative and quantitive determination of chemical components of leaf essential oil of Spiraea alpina Pall. with Microwave-assisted Hydrodistillation is carried out by gas chromatography-mass spectrometry. About 69 compounds have been identified from the leaf oil, accounting for 79.39% of the total. The in vitro antifungal activity of S. alpina essential oil was studied against eight test phytopathogenic bacteria and fungi namely Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. citri, Ralstonia solanacearum, Erwinia carotovora subsp. carotovora and Rhizoctonia solani, Fusarium graminerum, Pyricularia oryzea, Exserohilum turcicum by the agar Well Diffusion Method and Poisoned Food Technique, respectively. In the case, R. solanacearum was found to be sensitive to S. alpina oil at a concentration of 10 μl·well⁻¹ and the inhibition zone diameter was found to be 10.7 mm. Concentration- and time-dependent fungitoxicity was recorded from 125 to 1,000 μg·ml⁻¹ concentration. About 125 μg·ml⁻¹ of leaf oil solution partially inhibited the mycelial growth of R. solani to the same extent as 50 μg·ml⁻¹ of miconazole. The oil also affected the mycelial growth of F. graminerum and E. turcicum in a dose-dependent manner but had a weak effect on the growth of P. oryzea.     

<Emphasis Type="Italic">In planta</Emphasis> gene expression analysis of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pathovar <Emphasis Type="Italic">oryzae</Emphasis>, African strain MAI1

Background  

Bacterial leaf blight causes significant yield losses in rice crops throughout Asia and Africa. Although both the Asian and African strains of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), induce similar symptoms, they are nevertheless genetically different, with the African strains being more closely related to the Asian X. oryzae pv. oryzicola (Xoc).     

Xanthomonas oryzae pv. oryzae infection triggers accumulation of phenolics,defense-related enzymes and thaumatin-like proteins in rice leaves

Abstract

The effect of Xanthomonas oryzae pv. oryzae infection on induction of phenylalanine ammonia-lyase (PAL), peroxidase (PO), phenolics and thaumatin-like proteins (TLPs) in rice was studied. PAL activity increased significantly one day after inoculation with X. o. pv. oryzae and the maximum enzyme activity was observed two days after inoculation. The phenolic content in rice leaves increased significantly one day after inoculation and the maximum accumulation of phenols was observed two days after inoculation. Significant increase in peroxidase activity was observed in rice leaves one day after inoculation with X. o. pv. oryzae. Isozyme analysis indicated that three peroxidase isozymes (PO-1, PO-2 and PO-3) were induced after inoculation with X. o. pv. oryzae. Immunoblot analysis of protein extracts from control and pathogen inoculated rice plants revealed the induced accumulation of 16 and 24 kDa TLPs in rice leaves in response to X. o. pv. oryzae infection. TLP mRNA accumulation was induced strongly in rice leaves in response to infection by X. o. pv. oryzae.     

Isolation and partial characterization of antibacterial lipopeptide produced by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> HKA-15 against phytopathogen <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. phaseoli M-5

An antibacterial metabolite was isolated from Paenibacillus polymyxa HKA-15, a soybean bacterial endophyte. The purification of the crude metabolite from Paenibacillus polymyxa HKA-15 was done by column chromatography. In TLC, a spot with an R _f value of 0.86 (±0.02) from the purified fraction showed bioactivity against Xanthomonas campestris pv. phaseoli M-5. In SDS-PAGE, the purified antibiotic was separated in the molecular weight range of 3.5 kDa. The exact molecular weight of the active compound was identified as 1,347.7 Da using MS-MS analysis. Infra red spectrum and ¹H NMR analysis showed the presence of amino acids and fatty acids in the active compound. The characterization of the antibacterial compound revealed its lipopeptide nature. In an agar diffusion assay, the crude metabolite showed a broad spectrum of activity, being able to inhibit the growth of the fungal pathogen, Rhizoctonia bataticola, Macrophomina phaseolina and Fusarium udum. A stronger inhibition was observed against bacterial pathogens viz., X. campestris pv.phaseoli M-5, X. campestris pv. phaseoli CP-1-1, Xanthomonas oryzae, Ralstonia solanacearum and Micrococcus luteus.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

1,3-Propandiol production by engineered Hansenula polymorpha expressing dha genes from Klebsiella pneumoniae

Currently, 1,3-propanediol (1,3-PD) is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive chemical synthesis. A methylotrophic yeast Hansenula polymorpha was engineered by expression of dhaB1, dhaB2, dhaB3, dhaB _RA1 and dhaB _RA2 encoding glycerol dehydratase complex and dhaT encoding 1,3-PD oxidoreductase from Klebsiella pneumoniae under direction of promoter of glyceraldehyde-3 phosphate dehydrogenase (GAPDH). The engineered recombinant yeast strain can produce 1,3-PD from glucose (2.4 g L⁻¹) as well as glycerol (0.8 g L⁻¹), which might lead to a safe and cost-effective method for industrial production of 1,3-PD from various biomass resources.     

Activity of a novel bactericide,zinc thiazole against Xanthomonas oryzae pv. oryzae in Anhui Province of China

Bacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv oryzae, is one of the most serious bacterial diseases in China. Presently, bismerthiazol has been the major bactericide for the control of BLB, however, bismerthiazol‐resistant strains of X. oryzae pv. oryzae have appeared in the field in China. Zinc thiazole is a novel bactericide with strong antibacterial activity against Xanthomonas spp. In this study, sensitivity of 109 X. oryzae pv. oryzae strains to zinc thiazole was determined. The EC₅₀ values for zinc thiazole in inhibiting bacterial growth of the 109 X. oryzae pv. oryzae strains were 0.53–9.62 µg mL^?1 with the average EC₅₀ value of 4.82 ± 1.86 µg/ml. The minimum inhibitory concentration (MIC) values of zinc thiazole against the 109 X. oryzae pv. oryzae strains were assessed and the results showed that the MIC values of zinc thiazole for completely inhibiting the growth of these 109 strains ranged from 5.0 to 40.0 µg mL^?1. In the evaluation of protective and curative activity test, zinc thiazole exhibited great activity against BLB and provided over 88% control efficacy (at 300 µg mL^?1) 1 and 3 days before or after inoculations, which was also higher that that of bismerthiazol in the corresponding treatments. Our field trials showed that zinc thiazole at 375 g.a.i ha^?1 provided over 70% control efficacy in 2012 and over 80% control efficacy in 2013 at both sites. Moreover, in all the four field trials, zinc thiazole at 250 g.a.i ha^?1 provided higher control efficacy than that of bismerthiazol at 250 g.a.i ha^?1. Taken together, zinc thiazole is therefore an alternative tool for the management of BLB.     

Detection of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in seeds using a specific TaqMan probe

Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.     

Simple and efficient expression of <Emphasis Type="Italic">Agaricus meleagris</Emphasis> pyranose dehydrogenase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L⁻¹ PDH or 1,330 U L⁻¹ d⁻¹ in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown.     

Individual and combined effects of physicochemical parameters on ellagitannin acyl hydrolase and ellagic acid production from ellagitannin by <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The individual and interactive effects of physicochemical parameters on ellagitannin acyl hydrolase activity and ellagic acid production by Aspergillus oryzae using ellagitannins from acorn fringe of oak as substrate were studied. Ellagitannins concentration, incubation time were identified as important physicochemical parameters influencing the enzyme synthesis and the production accumulation, and the substrate concentration with initial pH was determined to has an interactive effect on the enzyme synthesis, while ellagitannins concentration and initial pH with incubation time were found to have interactions on the production accumulation. Furthermore, the parameters were optimized by quadratic programming. Under optimum condition, the fermentation run lasted 84 h with 4 g L⁻¹ ellagitannins concentration, yielding 17.7% ellagic acid. However, the maximum enzyme activity was obtained in 96 h with 5 g L⁻¹ substrate concentration. The research demonstrated a possible way to develop an efficient approach for recovery of higher added-value product (ellagic acid) from forestry byproduct (acorn fringe of oak).     

Expression,characterization and 2,4,6-trichlorophenol degradation of laccase from <Emphasis Type="Italic">Monilinia fructigena</Emphasis>

A novel laccase gene from Monilinia fructigena was synthesized chemically according to the yeast bias codon and integrated into the genome of Pichia pastoris GS115 by electroporation. The expressed enzyme was recovered from the culture supernatant and purified. The result of enzyme activity assay and SDS-PAGE demonstrated that the recombinant laccase was induced and extracellularly expressed in P. pastoris. Main biochemical properties of this laccase, such as thermodependence and thermostability, optimal pH and pH stability, and the effect of metal ions and inhibitors, were characterized. With 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) as the substrate, MfLcc had its optimal pH at 3.5 and optimal temperature at 45°C. The Km values of the ABTS, guaiacol were 0.012 and 0.016 Mm, respectively, and the corresponding V _max values are 243.9 and 10.55 Um min⁻¹ mg⁻¹, respectively. The recombinant laccase degraded 80% 2,4,6-trichlorophenol after 8 h under the optimal conditions. The recombinant strain and its laccase can be considered as candidate for treating waste water polluted with trichlorophenols.     

Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

Zinc Uptake Regulator (<Emphasis Type="Italic">zur</Emphasis>) Gene Involved in Zinc Homeostasis and Virulence of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in Rice

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. In order to understand the gene of zinc uptake regulator (zur) involved in virulence of the pathogen in rice, we generated a mutant OSZRM by homologous suicide plasmid integration. The mutant failed to grow in NYGB medium supplemented with Zn²⁺ or Fe³⁺ at a concentration of 500 μM or 6 mM, whereas the wild-type strain grew well at the same conditions. The zur mutant was hypersensitive to hydrogen peroxide and exhibited reduction catalase activity and the production of extracellular polysaccharide (EPS). Interestingly, the mutant showed a reduction in virulence on rice but still kept triggering hypersensitive response (HR) in tobacco. When the mutant was complemented with the zur gene, the response was recovered to wild-type. These results suggested that zur gene is a functional member of the Zur regulator family that controls zinc and iron homeostasis, oxidative stress, and EPS production, which is necessary for virulence in X. oryzae pv. oryzae. Wanfeng Yang and Yan Liu contributed equally to this work     

Compositional Difference of the Exopolysaccharides Produced by the Virulent and Virulence-Deficient Strains of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, is known to produce phytotoxic polysaccharides. The extracellular polysaccharide (EPS) was isolated from virulent (BXO1) and virulence-deficient gum G mutant (BXO1002) strains of X. oryzae pv. oryzae and characterized using fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR). Data from the FT-IR suggested that the aldehyde (R-CHO) group and C=O of acid anhydride are present in BXO1 but absent in BXO1002. The ¹H-NMR spectra showed the presence of an acetyl amine of hexose or pentose, free amines of glucose, an β-anomeric carbon of hexose and pentose, hydrogen next to hydroxyl group, an acetyl amine of hexose and pentose in the polysaccharides of both BXO1 and BXO1002, and the absence of α-anomeric carbon of hexose or pentose and the glucuronic acid in the polysaccharides produced by BXO1002. The test for glucuronic acid also confirmed the absence of glucuronic acid in the polysaccharides of BXO1002 and the presence glucuronic acid (32 μg/mg) in the polysaccharides produced by BXO1. Received: 14 May 2002 / Accepted: 21 June 2002     

Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae

Aims: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co²⁺-containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. Conclusions: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co²⁺-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. Significance and Impact of the Study: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.     

Suppression of XopQ–XopX-induced immune responses of rice by the type III effector XopG

Effectors that suppress effector-triggered immunity (ETI) are an essential part of the arms race in the co-evolution of bacterial pathogens and their host plants. Xanthomonas oryzae pv. oryzae uses multiple type III secretion system (T3SS) secreted effectors such as XopU, XopV, XopP, XopG, and AvrBs2 to suppress rice immune responses that are induced by the interaction of two other effectors, XopQ and XopX. Here we show that each of these five suppressors can interact individually with both XopQ and XopX. One of the suppressors, XopG, is a predicted metallopeptidase that appears to have been introduced into X. oryzae pv. oryzae by horizontal gene transfer. XopQ and XopX interact with each other in the nucleus while interaction with XopG sequesters them in the cytoplasm. The XopG E76A and XopG E85A mutants are defective in interaction with XopQ and XopX, and are also defective in suppression of XopQ–XopX-mediated immune responses. Both mutations individually affect the virulence-promoting ability of XopG. These results indicate that XopG is important for X. oryzae pv. oryzae virulence and provide insights into the mechanisms by which this protein suppresses ETI in rice.