Fusion Strains Research Articles

Engineering a Sinorhizobium meliloti Chassis with Monopartite, Single Replicon Genome Configuration.

Multipartite bacterial genomes pose challenges for genome engineering and the establishment of additional replicons. We simplified the tripartite genome structure (3.65 Mbp chromosome, 1.35 Mbp megaplasmid pSymA, 1.68 Mbp chromid pSymB) of the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Strains with bi- and monopartite genome configurations were generated by targeted replicon fusions. Our design preserved key genomic features such as replichore ratios, GC skew, KOPS, and coding sequence distribution. Under standard culture conditions, the growth rates of these strains and the wild type were nearly comparable, and the ability for symbiotic nitrogen fixation was maintained. Spatiotemporal replicon organization and segregation were maintained in the triple replicon fusion strain. Deletion of the replication initiator-encoding genes, including the oriVs of pSymA and pSymB from this strain, resulted in a monopartite genome with oriC as the sole origin of replication, a strongly unbalanced replichore ratio, slow growth, aberrant cellular localization of oriC, and deficiency in symbiosis. Suppressor mutation R436H in the cell cycle histidine kinase CckA and a 3.2 Mbp inversion, both individually, largely restored growth, but only the genomic rearrangement recovered the symbiotic capacity. These strains will facilitate the integration of secondary replicons in S. meliloti and thus be useful for genome engineering applications, such as generating hybrid genomes.

Development of a Robust Saccharomyces cerevisiae Strain for Efficient Co-Fermentation of Mixed Sugars and Enhanced Inhibitor Tolerance through Protoplast Fusion.

The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior S. cerevisiae strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals.

The galactose metabolism genes UGE1 and UGM1 are novel virulence factors of the maize anthracnose fungus Colletotrichum graminicola.

Fungal cell walls represent the frontline contact with the host and play a prime role in pathogenesis. While the roles of the cell wall polymers like chitin and branched β-glucan are well understood in vegetative and pathogenic development, that of the most prominent galactose-containing polymers galactosaminogalactan and fungal-type galactomannan is unknown in plant pathogenic fungi. Mining the genome of the maize pathogen Colletotrichum graminicola identified the single-copy key galactose metabolism genes UGE1 and UGM1, encoding a UDP-glucose-4-epimerase and UDP-galactopyranose mutase, respectively. UGE1 is thought to be required for biosynthesis of both polymers, whereas UGM1 is specifically required for fungal-type galactomannan formation. Promoter:eGFP fusion strains revealed that both genes are expressed in vegetative and in pathogenic hyphae at all stages of pathogenesis. Targeted deletion of UGE1 and UGM1, and fluorescence-labeling of galactosaminogalactan and fungal-type galactomannan confirmed that Δuge1 mutants were unable to synthesize either of these polymers, and Δugm1 mutants did not exhibit fungal-type galactomannan. Appressoria of Δuge1, but not of Δugm1 mutants, were defective in adhesion, highlighting a function of galactosaminogalactan in the establishment of these infection cells on hydrophobic surfaces. Both Δuge1 and Δugm1 mutants showed cell wall defects in older vegetative hyphae and severely reduced appressorial penetration competence. On intact leaves of Zea mays, both mutants showed strongly reduced disease symptom severity, indicating that UGE1 and UGM1 represent novel virulence factors of C. graminicola.

Discovery of N-Benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine as a Potential Antifungal Agent against Sporidia Growth and Teliospore Germination of Sporisorium scitamineum.

The cultivation of sugar cane using perennial roots is the primary planting method, which is one of the reasons for the serious occurrence of sugar cane smut disease caused by the basidiomycetous fungus Sporisorium scitamineum in the sugar cane perennial root planting area. Consequently, it is crucial to eliminate pathogens from perennial sugar cane buds. In this study, we found that MAP kinase Hog1 is necessary for heat stress resistance. Subsequent investigations revealed a significant reduction in the expression of the heat shock protein 104-encoding gene, SsHSP104, in the ss1hog1Δ mutant. Additionally, the overexpression of SsHSP104 partially restored colony growth in the ss1hog1Δ strain following heat stress treatment, demonstrating the crucial role of SsHsp104 in SsHog1-mediated heat stress tolerance. Hence, we constructed the ss1hsp104:eGFP fusion strain in the wild type of S. scitamineum to identify small-molecule compounds that could inhibit the heat stress response, leading to the discovery of N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine as a potential compound that targets the SsHog1 mediation SsHsp104 pathway during heat treatment. Furthermore, the combination of N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine and warm water treatment (45 °C for 15 min) inhibits the growth of S. scitamineum and teliospore germination, thereby reducing the occurrence of sugar cane smut diseases and indicating its potential for eliminating pathogens from perennial sugar cane buds. In conclusion, these findings suggest that N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine is promising as a targeted compound for the SsHog1-mediated SsHsp104 pathway and may enable the reduction of hot water treatment duration and/or temperature, thereby limiting the occurrence of sugar cane smut diseases caused by S. scitamineum.

Ganoderma Fusions with High Yield of Ergothioneine and Comparative Analysis of Its Genomics.

Ergothioneine (EGT), an exceptional antioxidant found ubiquitously across diverse living organisms, plays a pivotal role in various vital physiological regulatory functions. Its principal natural sources are mushrooms and animal liver tissues. Ganoderma spp., a traditional Chinese food and medicinal mushroom, boasts high concentrations of EGT. To advance the development of novel Ganoderma spp. strains with enhanced EGT yields, we employed an efficient Ganoderma spp. protoplasmic fusion system. Through molecular and biological characterization, we successfully generated seven novel fusion strains. Notably, fusion strain RS7 demonstrated a remarkable increase in mycelial EGT production (12.70 ± 1.85 mg/L), surpassing the parental strains FQ16 and FQ23 by 34.23% and 39.10%, respectively. Furthermore, in the context of the fruiting body, fusion strain RS11 displayed a notable 53.58% enhancement in EGT production (11.24 ± 1.96 mg/L) compared to its parental strains. Genomic analysis of the RS7, the strain with the highest levels of mycelial EGT production, revealed mutations in the gene EVM0005141 associated with EGT metabolism. These mutations led to a reduction in non-productive shunts, subsequently redirecting more substrate towards the EGT synthesis pathway. This redirection significantly boosted EGT production in the RS7 strain. The insights gained from this study provide valuable guidance for the commercial-scale production of EGT and the selective breeding of Ganoderma spp. strains.

Improving the regeneration rate of deep lethal mutant protoplasts by fusion to promote efficient L-lysine fermentation

BackgroundL-lysine is widely used for feed and special diet products. The transformation of fermentation strains plays a decisive role in the development of these industries. Based on the mutation breeding theory and metabolic engineering methods, this study aimed to improve the regeneration rate of high-lethality protoplasts by combining multiple mutagenesis and homologous cell fusion techniques to efficiently concentrate multiple dominant mutations and optimize the L-lysine production strain Escherichia coli QDW.ResultsIn order to obtain the best protoplasts, the optimal enzymolysis time was selected as 4 h. The optimal lysozyme concentration was estimated at 0.8 mg/mL, because the protoplast formation rate and regeneration rate reached 90% and 30%, respectively, and their product reached the maximum. In this study, it was necessary that UV mutagenesis be excessive to obtain an expanded mutation library. For high lethality protoplasts, under the premise of minimal influence on its recovery, the optimal time for UV mutagenesis of protoplasts was 7 min, and the optimal time for thermal inactivation of protoplasts at 85 ℃ was 30 min. After homologous fusion, four fusion strains of E. coli were obtained, and their stability was analyzed by flow cytometry. The L-lysine yield of QDW-UH3 increased by 7.2% compared with that of QDW in a fermentation experiment, which promoted the expression of key enzymes in L-lysine synthesis, indicating that the combination of ultraviolet mutagenic breeding and protoplast fusion technology improved the acid-production level of the fusion strain.ConclusionThis method provides a novel approach for the targeted construction of microbial cell factories.

Metabolic flux enhancement from the translational fusion of terpene synthases is linked to terpene synthase accumulation

The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable >110-fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), corresponding with increases of a similar magnitude in terpene synthase levels. Our data demonstrate that increased in vivo enzyme levels – resulting from improved expression and/or improved protein stability – is a major driver of catalytic enhancement from enzyme fusion.

Investigations into Multiple Acyl‐CoA Assimilation Pathways in a Single Organism

By 2050 there could be around 12 billion tons of plastic in landfills and the natural environment, which could be avoided by using a more environmentally friendly alternative: biologically produced plastic, or bioplastic. Polyhydroxybutyrate (PHB) is a bioplastic whose precursor is an intermediate in the ethylmalonyl‐CoA pathway (EMCP), a pathway for acetyl‐CoA assimilation in bacteria. The genes that encode the enzymes for two different acetyl‐CoA assimilation pathways, the glyoxylate bypass (GB) and the EMCP, have been observed in two PHB‐producing bacteria: Rhodobacter capsulatus and Paracoccus denitrificans. It was previously observed that P. denitrificans asynchronously uses both pathways when grown with acetate. We produced R. capsulatus fusion strains by fusing the gene for a fluorescent protein, mCherry, to the gene of a key enzyme of the EMCP or GB and observed the fluorescence during growth with acetate. Tracking fluorescence allowed us to track the accumulation of each key enzyme, which indirectly indicates the activity of the pathway. We observed that unlike P. denitrificans, the GB in R. capsulatus is not essential to growth with acetate. Understanding the use/occurrence of the EMCP in bacteria is a step to being able to manipulate the EMCP to produce commercially relevant levels of bioplastic and related products.

EppR, a new LysR-family transcription regulator, positively influences phenazine biosynthesis in the plant growth-promoting rhizobacterium Pseudomonas chlororaphis G05

Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds, including phenazines and pyrrolnitrin. Although some regulatory genes have been identified to be involved in antifungal metabolite production, the regulatory mechanism and pathway of phenazine-1-carboxylic acid biosynthesis remain poorly defined. To identify more new regulatory genes, we applied transposon mutagenesis with the chromosomal lacZ fusion strain G05Δphz::lacZ as an acceptor. In the white conjugant colony G05W05, a novel transcriptional regulator gene, eppR, was verified to be interrupted by the transposon mini-Tn5Kan. To evaluate the specific function of eppR, we created a set of eppR-deletion mutants, including G05ΔeppR, G05Δphz::lacZΔeppR and G05Δprn::lacZΔeppR. By quantifying the production of antifungal compounds and β-galactosidase expression, we found that the expression of the phenazine biosynthetic gene cluster (phz) and the production of phenazine-1-carboxylic acid were markedly reduced in the absence of EppR. Moreover, the pathogen suppression test verified that the yield of phenazine-1-carboxylic acid was significantly decreased when eppR was deleted in frame. At the same time, no changes in the expression of the phzI/phzR quorum-sensing (QS) system and the production of N-acyl homoserine lactones (AHLs) and pyrrolnitrin were found in the EppR-deficient mutant. In addition, chromosomal fusion analyses and quantitative real-time polymerase chain reaction (qRT–PCR) results also showed that EppR could positively mediate the expression of the phz cluster at the posttranscriptional level. In summary, EppR is specifically essential for phenazine biosynthesis but not for pyrrolnitrin biosynthesis in P. chlororaphis.

467 Post-transcriptional regulation of the MiaA prenyl transferase by the small RNA CsrB in Escherichia coli (E. coli)

OBJECTIVES/GOALS: MiaA is a highly conserved prenyl transferase that catalyzes synthesis of the i6A37 tRNA modification in E. coli. While transcriptional regulation of MiaA is well characterized, there is no information on the MiaA post-transcriptional regulation. The aim of this study is to characterize the post-transcriptional regulation of the MiaA gene in E. coli. METHODS/STUDY POPULATION: To characterize the post-transcriptional regulation of miaA, we executed a targeted genetic screen of an E. coli small RNA library on a miaA-lacZ translational reporter fusion strain to identify small RNAs (sRNAs) that modulate MiaA translation or transcription termination. We also measured MiaA mRNA levels and miaA-lacZ activity in the absence or over-expression of candidate sRNA regulators of MiaA. We also measured MiaA mRNA levels in the absence of RNaseE and PNPase, two enzymes involved in mRNA turnover. Finally, we measured the ability of purified recombinant CsrA to bind to the MiaA mRNA transcript in vitro. RESULTS/ANTICIPATED RESULTS: We identified the carbon sensing sRNA CsrB and its cognate protein interaction partner CsrA, as potential post-transcriptional regulators of MiaA. Over-expression of CsrB fully repressed miaA-lacZ activity and MiaA mRNA levels. The absence of CsrA resulted in a defective miaA-lacZ activity and a 10-fold decrease in MiaA mRNA levels. We also identified an increase in the MiaA mRNA half-life particularly in the absence of RNaseE. Our results demonstrate an additional layer of regulation for the miaA operon by the CsrA/CsrB protein-sRNA system. DISCUSSION/SIGNIFICANCE: MiaA is a highly conserved bacterial protein. Our data may represent phenomena in an array of bacteria that could be targeted by novel antibiotics. The human MiaA homologue, TRIT1, plays a role in mitochondrial disorders. We anticipate that information garnered from MiaA studies will elucidate TRIT1 function and its role in mitochondrial disorders.

Construction and Characterization of an Intergeneric Fusant That Degrades the Fungicides Chlorothalonil and Carbendazim.

Bordetella sp. CTN-16 (GenBank FJ598326) can degrade chlorothalonil (CTN) but not carbendazim (MBC), and Microbacterium sp. MBC-3 (GenBank OK667229) can degrade MBC but not CTN. A functional strain BD2 was obtained by protoplast fusion of CTN-16 and MBC-3 to generate a fusant with improved degradation efficiency of CTN and MBC. Fusant-BD2 with eighth transfer on a medium containing CTN and two antibiotics was obtained. To identify and confirm the genetic relationship between parental strains and fusion strain BD2, scanning electron microscopy (SEM), random amplified polymorphic DNA (RAPD), and 16S ribosomal RNA (rRNA) gene sequences analysis were carried out. SEM analysis illustrated BD2 and its parents had some slight differences in the cell morphology. Fusant-BD2 not only possessed the same bands as parental strains but also had its specific bands analyzed through RAPD. The genetic similarity indices for BD2 and its parental strains CTN-16 and MBC-3 are 0.571 and 0.428, respectively. The degradation rates of CTN and MBC were 79.8% and 65.2% in the inorganic salt solution containing 50 mg·L−1 CTN and 50 mg·L−1 MBC, respectively, and the degradation efficiencies were better than the parental strains CTN-16 and MBC-3. This study provides a prospect for the application of fusion strain BD2 in bioremediation of CTN and MBC contaminated sites.

SpoVG is an important regulator of sporulation and affects biofilm formation by regulating Spo0A transcription in Bacillus cereus 0\u20139

BackgroundBacillus cereus 0–9, a Gram-positive, endospore-forming bacterium isolated from healthy wheat roots in our previous research, is considered to be an effective biocontrol strain against several soil-borne plant diseases. SpoVG, a regulator that is broadly conserved among many Gram-positive bacteria, may help this organism coordinate environmental growth and virulence to survive. This study aimed to explore the multiple functions of SpoVG in B. cereus 0–9.MethodsThe gene knockout strains were constructed by homologous recombination, and the sporulation process of B. cereus 0–9 and its mutants were observed by fluorescence staining method. We further determined the spore yields and biofilm formation abilities of test strains. Transcriptional fusion strains were constructed by overlapping PCR technique, and the promoter activity of the target gene was detected by measuring its fluorescence intensity. The biofilm production and colonial morphology of B. cereus 0–9 and its mutants were determined to study the functions of the target genes, and the transcription level of the target gene was determined by qRT-PCR.ResultsAccording to observation of the sporulation process of B. cereus 0–9 in germination medium, SpoVG is crucial for regulating sporulation stage V of B. cereus 0–9, which is identical to that of Bacillus subtilis but differs from that of Bacillus anthracis. In addition, SpoVG could influence biofilm formation of B. cereus 0–9. The transcription levels of two genes closely related to biofilm-formation, sipW and calY, were downregulated in a ΔspoVG mutant. The role of SpoVG in regulating biofilm formation was further explored by deleting the genes abrB and sinR in the ΔspoVG mutant, respectively, generating the double mutant strains ΔspoVGΔabrB and ΔspoVGΔsinR. The phenotypes of these double mutants were congruent with those of the single abrB and sinR deletion strains, respectively, which showed increased biofilm formation. This indicated that spoVG was located upstream of abrB and sinR in the regulatory pathway of B. cereus biofilm formation. Further, the results of qRT-PCR and the luminescence intensity of transcriptional fusion strains indicated that spoVG gene deletion could inhibit the transcription of Spo0A.ConclusionsSpoVG, an important regulator in the sporulation of B. cereus, is located upstream of Spo0A and participates in regulation of biofilm formation of B. cereus 0–9 through regulating the transcription level of spo0A. Sporulation and biofilm formation are crucial mechanisms by which bacteria respond to adverse conditions. SpoVG is therefore an important regulator of Spo0A and is crucial for both sporulation and biofilm formation of B. cereus 0–9. This study provides a new insight into the regulatory mechanism of environmental adaptation in bacteria and a foundation for future studies on biofilm formation of B. cereus.

Inducible high level expression of a variant ΔD19A,D58A-ferredoxin-hydrogenase fusion increases photohydrogen production efficiency in the green alga Chlamydomonas reinhardtii

Oxygenic photosynthetic organisms use solar energy to split water into protons, electrons, and oxygen. Unicellular green algae like Chlamydomonas reinhardtii have evolved the additional ability to reduce protons to produce hydrogen (H2) via chloroplast hydrogenases (HYDA1 and HYDA2) under light-driven anaerobic conditions. In wild type cells, H2 production is limited by proton and electron supply to HYDA; most electrons are diverted to ferredoxin-NADP+-reductase. To enhance electron supply to HYDA and improve H2 production, variant ferredoxins with reduced ferredoxin-NADP+-reductase affinity have been demonstrated in vitro. Ferredoxin-hydrogenase fusions have also been tested, but achieving robust expression in vivo has been challenging. To improve efficiency, we here combine (1) a ferredoxin-HYDA fusion containing (2) a mutant FDX1 ferredoxin (D19A, D58A) with lowered affinity for ferredoxin-NADP+-reductase, utilising (3) a strongly inducible lhcbm9 promoter for controllable expression, and (4) a transgene construct containing introns for higher nuclear expression levels. Fusion constructs with 15 (pDK6-15) or 25 (pDK6-25) amino acid linkers were expressed in the nucleus of a HYDA1/HYDA2 knockout strain. Under illuminated, sulfur-deprived conditions, the variant ferredoxin-HYDA fusion strains yielded 3.0 and 4.6-fold more H2 than the wild type background strain over a 6-day period (typical purity range attained; 92–97%), confirming that the variant ferredoxin fused to HYDA is functional in vivo, strongly inducible, expresses at a similar level to the native HYDA gene, and significantly increases light-driven H2 production over wild type levels, likely by reducing losses to other electron acceptors such as ferredoxin-NADP+-reductase.

31547 Regulation and function of the i6A37 tRNA modification

ABSTRACT IMPACT: MiaA has a human homolog known as TRIT1. Mutations in TRIT1 have been associated with rare diseases such as MELAS and MERRF syndromes. These diseases are associated with mitochondrial disfunction.Understanding the mechanisms of bacterial sRNAs, and the miRNAs associated with these diseases could potentially afford the insight into effective cures. OBJECTIVES/GOALS: The aim is to investigate the regulation and function of tRNA isopentyladenine transferase enzyme in Escherichia coli. We aimed to execute screens for the identification of small RNA regulators of MiaA. The study will also investigate if i6A tRNA modification is necessary for the expression of major heat shock and mitochondrial proteins. METHODS/STUDY POPULATION: We constructed a chromosomal miaA-lacZ translational fusion driven by the arabinose responsive PBAD promoter and used it to screen against an Escherichia coli small RNA library. Using CsrB, one of our candidate sRNA regulators from our genetic screen, we measured the steady state levels of MiaA by Northern Blot in a PBAD-miaA2(P2HS)-lacZ translational fusion strain whereby pBR-pLac-csrB, pBR-pLac-csrA and the pBR-pLac vector are over-expressed, and under the control of an IPTG inducible promoter. Additionally, and in the same PBAD-miaA2(P2HS)-lacZ translational fusion strain background, we measured the steady state levels of MiaA in the wild type, csrA:zeo mutant strain, and csrA:zeo pBR-pLac-csrA complementation strain to determine if a combination of the pair would restore the wild-type genotype. RESULTS/ANTICIPATED RESULTS: Upon measuring the effect of small RNAs on miaA expression using quantitative b-galactosidase assays, we saw a 5-fold decrease in the expression of MiaA in the miaA-lacZ translational fusion containing sRNA CsrB, suggesting that this sRNA may play a role in the regulation of post-transcriptional expression of MiaA.From our northern blotting analysis, we observed a 6-fold decrease in MiaA expression in the absence of csrA, suggesting that csrA is essential for MiaA expression. DISCUSSION/SIGNIFICANCE OF FINDINGS: Identifying, mapping and characterizing how MiaA is regulated post-transcriptionally will give us an increased understanding in the maintenance and regulation of the normal function of E.coli to conserve homeostasis and translation fidelity.

Identification of the chromosome region responsible for pathogenicity of Verticillium dahliae on tomato using genetic recombination through protoplast fusion

The host range of Verticillium dahliae, causal agent of verticillium wilt in various dicot plants, differs among strains, but the mechanism responsible for the host-specific pathogenicity of the strains remains unclear. In this study, protoplast fusion of a tomato-pathogenic and a nonpathogenic strain of V. dahliae was used for genetic recombination of the isolates to localize the fungal genomic region involved in the pathogenicity on tomato. Twenty fusion strains resistant to two antibiotics, hygromycin B and geneticin, were obtained by protoplast fusion between two parental strains resistant to one of these antibiotics. Genomic Southern hybridization probed with telomere sequences revealed that these fusion strains were haploid and inherited chromosomes from both parental strains. Eight fusion strains were pathogenic on tomato. In PCR analysis of the fusion strains using DNA markers specific to a parental strain TV103, two DNA markers (T12 and VDA787) were amplified only in strains pathogenic on tomato. The genomic region mapped around these two DNA markers for a parental strain pathogenic on tomato was similar to that on the map for chromosome 3 of a reference strain (JR2). The analysis of 35 fusion strains with additional DNA markers revealed that one of the markers was completely accorded with pathogenicity of the strains on tomato. Therefore, the genomic region around this DNA marker is possibly involved in pathogenicity of V. dahliae on tomato.

Molecular Characterization of the Purine Degradation Pathway Genes ALA1 and URE1 of the Maize Anthracnose Fungus Colletotrichum graminicola Identified Urease as a Novel Target for Plant Disease Control.

Fungal pathogenicity is governed by environmental factors, with nitrogen playing a key role in triggering pathogenic development. Spores germinating on the plant cuticle are exposed to a nitrogen-free environment, and reprograming of nitrogen metabolism is required for bridging the time needed to gain access to the nitrogen sources of the host. Although degradation of endogenous purine bases efficiently generates ammonium and may allow the fungus to bridge the preinvasion nitrogen gap, the roles of the purine degradation pathway and of the key genes encoding allantoicase and urease are largely unknown in plant pathogenic fungi. To investigate the roles of the allantoicase and urease genes ALA1 and URE1 of the maize anthracnose fungus Colletotrichum graminicola in pathogenic development, we generated ALA1:eGFP and URE1:eGFP fusion strains as well as allantoicase- and urease-deficient mutants. Virulence assays, live cell, and differential interference contrast imaging, chemical complementation and employment of a urease inhibitor showed that the purine degradation genes ALA1 and URE1 are required for bridging nitrogen deficiency at early phases of the infection process and for full virulence. Application of the urease inhibitor acetohydroxamic acid did not only protect maize from C. graminicola infection, but also interfered with the infection process of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici, the maize and broad bean rusts Puccinia sorghi and Uromyces viciae-fabae, and the potato late blight pathogen Phytophthora infestans. Our data strongly suggest that inhibition of the purine degradation pathway might represent a novel approach to control plant pathogenic fungi and oomycetes.

Genetic Analysis of Four Sexual Differentiation Process Proteins (isp4/SDPs) in Chaetomium thermophilum and Thermomyces lanuginosus Reveals Their Distinct Roles in Development

Fungal sexual development requires the involvement of a large number of functional genes. Fungal genes encoding sexual differentiation process proteins (SDPs), isps, have been known for decades. isp4/SDP and its homologs function as oligopeptide transporters (OPTs), yet their roles in reproduction are unknown. Here, we genetically analyzed all four isp4/SDP homologs in the sexual species Chaetomium thermophilum and asexual species Thermomyces lanuginosus. Using single gene deletion mutants, we found that T. lanuginosus SDP (TlSDP) participated in asexual sporulation, whereas the other homologs participated in sexual morphogenesis. In complementary tests, C. thermophilum SDPs (CtSDP1-3) restored sporulation defects in TlSDP deletion strains (ΔTlSDP), and their translated proteins, which were localized onto the cytomembrane, possessed OPT activity. Interestingly, CtSDP2 accumulated at the top of the hyphae played a distinct role in determining the sexual cycle, glutathione transport, and lifespan shortening. A unique 72nt-insertion fragment (72INS) was discovered in CtSDP2. Biological analysis of the 72INS deletion and DsRED-tagged fusion strains implied the involvement of 72INS in fungal growth and development. In contrast to TlSDP, which only contributes to conidial production, the three CtSDPs play important roles in sexual and asexual reproduction, and CtSDP2 harbors a unique functional 72INS that initiates sexual morphogenesis.

Open-Source High-Throughput Phenomics of Bacterial Promoter-Reporter Strains.

Open-source electronics are becoming more prevalent in biological sciences, enabling novel and unique means of data acquisition. Here, we present 3D-printed, open-source tools to acquire fluorescence phenotypes with high temporal resolution. Printed fluorescence imaging boxes (PFIboxes) cost approximately 200US dollars to assemble, can be placed in incubators or hypoxic chambers, and accurately read high-density colony arrays of microorganisms. We demonstrate the utility of PFIboxes using a timecourse gene expression approach, examining globalEscherichia coli promoter activity using a fluorescent reporter library across a diverse panel of 15 antibiotics, each at several concentrations. Many secondary and indirect effects were observed when E.coli was challenged with various drugs, including increased gene expression in carbon metabolism processes. Further, kinetic data acquisition enabled non-destructive time course gene expression, clustering of which revealed patterns of co-expression. In all, PFIboxes provide an open solution to gene expression, for about 2US dollars per treatment condition, including technical replicates.

Intracellular localization of sirtuin and cell length analysis of Lactobacillus paracasei suggest possible role of sirtuin in cell division and cell shape regulation.

Sirtuin has been associated in prolonging lifespan of different model organisms. It has been shown to have an enzymatic activity of NAD+-dependent protein deacetylation targeting acetylated proteins. To determine targets and possible roles of sirtuin (LpSirA) in the Lactobacillus paracasei BL23, deletion (ΔsirA), sirtuin overexpressor (highsirA) and GFP fusion (highsirA-Venus) strains were generated, and microscopic localization and cell length analysis were done. Microscopic analysis revealed localization of LpSirA at cell division plates, at cell poles and all throughout the cell length in a spiral manner. Cell length analysis revealed that 46.9% of the ΔsirA cells were observed to be shorter (<2μm), whereas 12.6% of the highsirA cells were observed to be longer (>4μm) in comparison with the wild-type with only 17.1% short cells and 5.3% long cells. Our results suggest that sirtuin may have an essential role in cell division and cell shape regulation.

Heterologous expression of predicted promoter site for paraquat-inducible genes of the bacterium Chromobacterium violaceum is increased by plumbagin

http://dx.doi.org/10.5007/2175-7925.2017v30n2p1O objetivo deste estudo foi avaliar funcionalmente a influência do composto plumbagin sobre a indução heteróloga de uma região promotora predita de genes paraquat-induzíveis revelada durante as análises de anotação do genoma da bactéria Chromobacterium violaceum. A região promotora de interesse de C. violaceum foi amplificada a partir de sequências oligonucleotídicas específicas e clonada em vetor conjugativo, sendo acoplada à região codificante do gene lacZ de Escherichia coli. Em seguida, a indução heteróloga desse segmento regulatório foi estimada em cepa de E. coli na presença do plumbagin em uma concentração final de 50 µg/mL por mensurações dos níveis de expressão da enzima β-galactosidase. Diferenças significativas nos níveis da β-galactosidase foram observadas como resultado da ativação da região promotora de interesse pelo plumbagin na concentração testada em comparação às condições controle. Por outro lado, nenhum crescimento da cepa selvagem de C. violaceum foi observado durante a incubação dessas células em meio nutritivo contendo diferentes concentrações do plumbagin. As descobertas descritas aqui sugerem que uma região promotora predita para genes paraquat-induzíveis da bactéria C. violaceum sofra indução heteróloga pelo plumbagin, reforçando evidências acerca da caracterização funcional de motivos regulatórios intrínsecos a essa bactéria.