<Emphasis Type="Italic">Rhizobium</Emphasis> strains in the biological control of the phytopathogenic fungi <Emphasis Type="Italic">Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>) <Emphasis Type="Italic">rolfsii</Emphasis> on the common bean

Aims

To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.

Methods

Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.

Results

Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL^?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.

Conclusions

Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.

     

Synthesis of rosmarinic acid analogues in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To produce rosmarinic acid analogues in the recombinant Escherichia coli BLRA1, harboring a 4-coumarate: CoA ligase from Arabidopsis thaliana (At4CL) and a rosmarinic acid synthase from Coleus blumei (CbRAS).

Results

Incubation of the recombinant E. coli strain BLRA1 with exogenously supplied phenyllactic acid (PL) and analogues as acceptor substrates, and coumaric acid and analogues as donor substrates led to production of 18 compounds, including 13 unnatural RA analogues.

Conclusion

This work demonstrates the viability of synthesizing a broad range of rosmarinic acid analogues in E. coli, and sheds new light on the substrate specificity of CbRAS.

     

Functional analysis of alcohol dehydrogenase (<Emphasis Type="Italic">ADH</Emphasis>) genes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To characterize the genes responsible for ethanol utilization in Pichia pastoris.

Results

ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.

Conclusion

The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.

     

<Emphasis Type="Italic">PiggyBac</Emphasis> transposon-mediated mutagenesis and application in yeast <Emphasis Type="Italic">Komagataella phaffii</Emphasis>

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

     

Development of a novel engineered antibody targeting <Emphasis Type="Italic">Neisseria</Emphasis> species

Objectives

A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv).

Results

Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine.

Conclusions

The recombinant scFv could detect Neisseria strains at 10⁶ CFU/ml.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Niches and routes of transmission of <Emphasis Type="Italic">Xanthomonas citri</Emphasis> pv. <Emphasis Type="Italic">fuscans</Emphasis> to bean seeds

Aims

Seeds are vectors of a diversified microbiota including plant pathogens. To better understand transmission of common bacterial blight (CBB) agents to bean seeds, we analyzed the role of non-pathogenic xanthomonads on seed transmission efficiency and investigated the location of Xanthomonas citri pv. fuscans (Xcf) into seeds and plantlets.

Methods

Competition between CBB and NP strains was initially assessed in vitro and then extended in planta to monitor the impact of co-inoculation on Xcf seed transmission. Moreover, location of Xcf strains in seeds and seedlings was visualized using a combination of gfp-tagged strain and DOPE-FISH/CSLM.

Results

Whereas CBB agent growth was inhibited in vitro by some seed-borne non-pathogenic xanthomonads strains, these strains did not transmit efficiently to seed through floral pathway and did not affect Xcf seed transmission. Xcf cells were observed entering seed through vascular elements and parenchyma of funiculus, but also micropyle and testa. Xcf cells were observed, moreover, among other bacteria on radicle surfaces, especially tip, in cotyledons, and plumules.

Conclusions

CBB agents are more efficient than non-pathogenic xanthomonads in using the floral route to colonize seeds. CBB agents are located within different niches in the seed tissues up to the embryonic axis.

     

Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic <Emphasis Type="Italic">E. coli</Emphasis>

Background

The λ Red recombineering technology has been used extensively in Escherichia coli and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, but less so in pathogenic species of E. coli such as EHEC and EPEC. Our early experiments with the use of λ Red in EHEC and EPEC have led to sporadic results, leading to the present study to identify factors that might improve the efficiency of Red recombineering in these pathogenic strains of E. coli.

Results

In this report, we have identified conditions that optimize the use of λ Red for recombineering in EHEC and EPEC. Using plasmids that contain a P_tac-red-gam operon and a temperature-sensitive origin of replication, we have generated multiple mutations (both marked and unmarked) in known virulence genes. In addition, we have easily deleted five O157-specific islands (O-islands) of EHEC suspected of containing virulence factors. We have examined the use of both PCR-generated substrates (40 bp of flanking homology) and plasmid-derived substrates (~1 kb of flanking homology); both work well and each have their own advantages. The establishment of the hyper-rec phenotype requires only a 20 minute IPTG induction period of red and gam. This recombinogenic window is important as constitutive expression of red and gam induces a 10-fold increase in spontaneous resistance to rifampicin. Other factors such as the orientation of the drug marker in recombination substrates and heat shock effects also play roles in the success of Red-mediated recombination in EHEC and EPEC.

Conclusions

The λ Red recombineering technology has been optimized for use in pathogenic species of E. coli, namely EHEC and EPEC. As demonstration of this technology, five O-islands of EHEC were easily and precisely deleted from the chromosome by electroporation with PCR-generated substrates containing drug markers flanked with 40 bp of target DNA. These results should encourage the use of λ Red recombineering in these and other strains of pathogenic bacteria for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development.

     

Inactivation of virginiamycin by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Objective

To test the inactivation of the antibiotic, virginiamycin, by laccase-induced culture supernatants of Aureobasidium pullulans.

Results

Fourteen strains of A. pullulans from phylogenetic clade 7 were tested for laccase production. Three laccase-producing strains from this group and three previously identified strains from clade 5 were compared for inactivation of virginiamycin. Laccase-induced culture supernatants from clade 7 strains were more effective at inactivation of virginiamycin, particularly at 50 °C. Clade 7 strain NRRL Y-2567 inactivated 6 µg virginiamycin/ml within 24 h. HPLC analyses indicated that virginiamycin was degraded by A. pullulans.

Conclusions

A. pullulans has the potential for the bioremediation of virginiamycin-contaminated materials, such as distiller’s dry grains with solubles (DDGS) animal feed produced from corn-based fuel ethanol production.

     

Characterization of a novel <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> xanthine dehydrogenase expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To characterize a novel xanthine dehydrogenase (XDH) from Acinetobacter baumannii by recombinant expression in Escherichia coli and to assess its potential for industrial applications.

Results

The XDH gene cluster was cloned from A. baumannii CICC 10254, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant XDH consisted of two subunits with the respective molecular weights of 87 kDa and 56 kDa according to SDS-PAGE. XDH catalysis was optimum at pH 8.5 and 40–45 °C, was stable under alkaline conditions (pH 7–11) and the half-inactivation temperature was 60 °C. The K _m, turnover number and catalytic efficiency for xanthine were 25 μM, 69 s^?1 and 2.7 μM^?1 s^?1, respectively, which is an improvement over XDHs characterized previously. A. baumannii XDH is less than 50 % identical to previously identified XDH orthologs from other species, and is the first from the Acinetobacter genus to be characterized.

Conclusion

The novel A. baumannii enzyme was found to be among the most active, thermostable and alkaline-tolerant XDH enzymes reported to date and has potential for use in industrial applications.

     

Isolation and identification of <Emphasis Type="Italic">Salmonella</Emphasis> spp. from red foxes (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) and badgers (<Emphasis Type="Italic">Meles meles</Emphasis>) in northern Italy

Background

Salmonella spp. have been isolated from a wide range of wild animals. Opportunistic wild carnivores such as red foxes (Vulpes vulpes) and badgers (Meles meles) may act as environmental indicators or as potential sources of salmonellosis in humans. The present study characterizes Salmonella spp. isolated from the intestinal contents of hunted or dead red foxes (n?=?509) and badgers (n?=?17) in northern Italy.

Findings

Thirty-one strains of Salmonella belonging to 3 Salmonella enterica subspecies were isolated. Fourteen different serovars of S. enterica subsp. enterica were identified, among which were serovars often associated with human illness.

Conclusions

Wild opportunistic predators can influence the probability of infection of both domestic animals and humans through active shedding of the pathogen to the environment. The epidemiological role of wild carnivores in the spread of salmonellosis needs to be further studied.

     

Production of caffeoylmalic acid from glucose in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.

Results

We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli–E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.

Conclusions

Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

     

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To engineer Escherichia coli for the heterologous production of di-rhamnolipids, which are important biosurfactants but mainly produced by opportunistic pathogen Pseudomonas aeruginosa.

Results

The codon-optimized rhlAB and rhlC genes originating from P. aeruginosa and Burkholderia pseudomallei were combinatorially expressed in E. coli to produce di-rhamnolipids with varied congeners compositions. Genes involved in endogenous upstream pathways (rhamnose and fatty acids synthesis) were co-overexpressed with rhlAB–rhlC, resulting in variations of rhamnolipids production and congeners compositions. Under the shake-flask condition, co-overexpression of rfbD with rhlAB–rhlC increased rhamnolipids production (0.64 ± 0.02 g l^?1) than that in strain only expressing rhlAB–rhlC (0.446 ± 0.009 g l^?1), which was mainly composed of di-rhamnolipids congeners Rha–Rha–C₁₀–C₁₀.

Conclusion

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically engineered E. coli strains were achieved via combiniations of mono-/di-rhamnolipids synthesis modules and endogenous upstream modules.

     

Enhanced <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> immune responses caused by heterologous plant genes from <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Background

Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F₁ hybrid was proposed.

Results

By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F₁ hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance.

Conclusions

Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F₁ hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.

     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.