Plasmid Integration Research Articles

Emergence of carbapenem-resistant Klebsiella pneumoniae harbouring bla OXA-48-like genes in China.

Klebsiella pneumoniae strains carrying OXA-48-like carbapenemases are increasingly prevalent across the globe. There is thus an urgent need to better understand the mechanisms that underpin the dissemination of bla OXA-48-like carbapenemases. To this end, four ertapenem-resistant K. pneumoniae isolates producing OXA-48-like carbapenemases were isolated from two patients. Genome sequencing revealed that one sequence type (ST) 17 isolate carried bla OXA-181, whilst three isolates from a single patient, two ST76 and one ST15, carried bla OXA-232. The 50514 bp bla OXA-181-harbouring plasmid, pOXA-181_YML0508, was X3-type with a conjugation frequency to Escherichia coli of 1.94×10−4 transconjugants per donor. The bla OXA-232 gene was located on a 6141 bp ColKP3-type plasmid, pOXA-232_WSD, that was identical in the ST76 and ST15 K. pneumoniae isolates. This plasmid could be transferred from K. pneumoniae to E. coli at low frequency, 8.13×10−6 transconjugants per donor. Comparative analysis revealed that the X3 plasmid acquired the bla OXA-48-like gene via IS3000-mediated co-integration of the ColKP3-type plasmid. Our study highlights how plasmid integration and rearrangements can contribute to the spread of bla OXA-48-like genes, which provides important clues for clinical prevention of the dissemination of K. pneumoniae strains carrying bla OXA-48-like carbapenemases.

Development and Optimization of Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis Reporter Constructs.

Mycobacterium tuberculosis resides in the lungs in various lesion types with unique microenvironmental conditions. This diversity is in line with heterogeneous disease progression and divergent drug efficiency. Fluorescent reporter strains can be used to decipher the micromilieu and to guide future treatment regimens. Current reporters using replicating plasmids, however, are not suitable for long-term mouse infections or studies in non-human primates. Using a combination of recombinant DNA and protein optimization techniques, we have developed reporter strains based on integrative plasmids, which exhibit stimulus-response characteristics and fluorescence intensities comparable to those based on replicating plasmids. We successfully applied the concepts by constructing a multi-color reporter strain able to detect simultaneous changes in environmental pH, Mg2+ concentrations, and protein expression levels.

Hybrid genome assembly of Shigella sonnei reveals the novel finding of chromosomal integration of an IncFII plasmid carrying a mphA gene.

Azithromycin is increasingly being used for the treatment of shigellosis despite a lack of interpretative guidelines and with limited clinical evidence. The present study determined azithromycin susceptibility and correlated this with macrolide-resistance genes in Shigella spp. isolated from stool specimens in Vellore, India. The susceptibility of 332 Shigella isolates to azithromycin was determined using the disc diffusion method. Of these, 31 isolates were found to be azithromycin resistant. The azithromycin minimum inhibitory concentration (MIC) was determined using the broth microdilution method. In addition, isolates were screened for mphA and ermB genes using conventional PCR. Furthermore, an isolate that was positive for resistance genes was subjected to complete genome analysis, and was analysed for mobile genetic elements. The azithromycin MIC for the 31 resistant Shigella isolates ranged between 2 and 16 mg l−1. PCR results showed that a single isolate of Shigella sonnei carried a mphA gene. Complete genome analysis revealed integration of an IncFII plasmid into the chromosome of S. sonnei , which was also found to carry the following resistance genes: sul1, bla DHA1, qnrB4, mphA, tetR. Mutations in the quinolone-resistance-determining region (QRDR) were also observed. Additionally, prophages, insertion sequences and integrons were identified. The novel finding of IncFII plasmid integration into the chromosome of S. sonnei highlights the potential risk of Shigella spp. becoming resistance to azithromycin in the future. These suggests that it is imperative to monitor Shigella susceptibility and to study the resistance mechanism of Shigella to azithromycin considering the limited treatment choices for shigellosis.

Improvement on the genetic engineering of an invasive agricultural pest insect, the cherry vinegar fly, Drosophila suzukii

BackgroundThe invasive fly Drosophila suzukii has become an established fruit pest in Europe, the USA, and South America with no effective and safe pest management. Genetic engineering enables the development of transgene-based novel genetic control strategies against insect pests and disease vectors. This, however, requires the establishment of reliable germline transformation techniques. Previous studies have shown that D. suzukii is amenable to transgenesis using the transposon-based vectors piggyBac and Minos, site-specific recombination (lox/Cre), and CRISPR/Cas9 genome editing.ResultsWe experienced differences in the usability of piggyBac-based germline transformation in different strains of D. suzukii: we obtained no transgenic lines in a US strain, a single rare transgenic line in an Italian strain, but observed a reliable transformation rate of 2.5 to 11% in a strain from the French Alps. This difference in efficiency was confirmed by comparative examination of these three strains. In addition, we used an attP landing site line to successfully established φC31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with two attP sequences to effectively perform φC31-Recombinase Mediated Cassette Exchange (φC31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions of Ds nanos to express φC31 integrase maternally to generate self-docking lines for φC31-RMCE. Besides, we isolated the promoter/enhancer of Ds serendipity α to drive the heterologous tetracycline-controlled transactivator (tTA) during early embryonic development and generated a testes-specific tTA driver line using the endogenous beta-2-tubulin (β2t) promoter/enhancer.ConclusionOur results provide evidence that the D. suzukii strain AM derived from the French Alps is more suitable for piggyBac germline transformation than other strains. We demonstrated the feasibility of using φC31-RMCE in the cherry vinegar fly and generated a set of lines that can be used for highly efficient integration of larger constructs. The φC31-based integration will facilitate modification and stabilization of previously generated transgenic lines that carry at least one attP site in the transgene construction. An early embryo-specific and a spermatogenesis-specific driver line were generated for future use of the binary expression system tet-off to engineer tissue- and stage-specific effector gene expression for genetic pest control strategies.

High-risk ST772-MRSA lineage in South Asia: potential for global dissemination

Background: Methicillin resistant Staphylococcus aureus (MRSA) is a clinically important pathogen with an abundance of virulence factors and multi-drug resistance, that are essential for survival within host. In recent decades, a community-acquired lineage, ST772-MRSA are being frequently reported cause of hospital-onset infections and outbreak. ST772-MRSA is currently circulating in South Asia, Australia and Europe. We hypothesis that the presence of multiple resistance determinants in ST772-MRSA, is the driver for potential adaptation and evolution. Genome-wide association study was conducted to understand the evolutionary events within ST772-MRSA and propensity for dissemination. Methods and materials: Whole genome sequencing was performed for ST772-MRSA (n = 33), isolated from blood culture during 2013-2018. Global collection ST772 S. aureus genome (n = 120) were retrieved from National Center for Biotechnology Information (NCBI) and European Nucleotide Archive (ENA) databases. Core genome alignment and variant calling analysis were carried out using parsnp and snippy pipeline respectively. Divergence and evolution of ST772 S. aureus were estimated using phylogenetic BEAST analysis. Results: ST772-MRSA was emerged in the early 1970s. In this lineage, mecA gene was identified on a short variant (5C2) or composite (5C2&5) SCCmec V element. Among the investigated genome, 90% carried Panton Valentine leukocicidin (PVL) toxin, which is encoded by the prophage Φ-IND772PVL. vSaα, vSaβ and vSaγ pathogenicity islands carrying exotoxins, enterotoxin gene cluster and exfoliative toxin were identified on all ST772-MRSA genome. Mutation in parC (S80Y) and gyrA (S82L), contributed for quinolone resistance in 72% of the genome. A copy of dfrA gene, encoding for trimethoprim resistance was identified on all the core genome. An integrated resistance plasmid (IRP) containing resistant determinants for beta-lactam (blaZ), aminoglycosides (aphA3-sat-aadE), macrolide (msrA, mphC), and bacitracin (bacA, bacB) was found in all MRSA genome. Another landmark event, is the acquisition of IRP, in the early 1990s, resulted in a highly distinct uniform clade comprised of multi-drug resistant variant. Most of this variant mediated intercontinental transmission is typically associated with travel from South Asia. Conclusion: Acquisition of IRP contributed for the emergence of ST772-MRSA lineage, as a multi-drug resistant variant. Genetic changes associated with the adaptation and increasing multi-drug resistance in ST772-MRSA, represents the potential threat for global dissemination.

Whole-genome characterisation of TEM-1 and CMY-2 β-lactamase-producing Salmonella Kentucky ST198 in Lebanese broiler chain

ObjectivesSalmonella enterica subsp. enterica serovar Kentucky has been associated with the worldwide ciprofloxacin-resistant (CIPR) Salmonella Kentucky sequence type 198 (ST198) epidemic clone, mostly recovered from poultry farms and products. The aim of this study was to examine whether this expanding clone exists in the Lebanese broiler chain. MethodsEight CIPR and extended-spectrum cephalosporin-resistant Salmonella Kentucky isolates previously recovered from Lebanese broilers were genetically characterised by whole-genome sequencing. ResultsSeven of the eight isolates belonged to ST198 and were phylogenetically closely related. They all harboured mutations in the chromosomal quinolone resistance genesgyrA and parC with double and single substitutions, respectively. The blaTEM-1B and blaCMY-2 genes were both detected in six isolates. Insertion sequence ISEcp1 was located upstream of blaCMY-2, harboured by IncI1 plasmids in four strains. An IS10 transposition coupled to homologous recombination at transposition sites mediated CMY-2 plasmid integration into the chromosome of one strain. Resistance genes to aminoglycosides [aadA7 and aac(3)-Id], tetracyclines [tet(A)] and sulfonamides (sul1) were detected in five strains, among which four were positive for the presence of Salmonella genomic island 1 (SGI1) variant SGI1-K. All studied isolates harboured a variety of Salmonella pathogenicity islands (SPIs) as well as common regulatory and virulence genes. ConclusionHere we report for the first time in Lebanon the detection and dissemination of the emerging highly drug-resistantSalmonella Kentucky ST198. Our findings shed new light on this clone as a potential public-health threat.

First isolation of an IMP-1 metallo-β-lactamase-producing Kluyvera ascorbata in Japan

ObjectivesThe emergence of carbapenemase-producing Enterobacterales has been increasing globally, causing growing concerns. Although Kluyvera ascorbata is not known as a metallo-β-lactamase (MBL)-producer, in the present study we isolated a K. ascorbata strain producing IMP-1 MBL from catheter-associated urine of a paediatric patient and performed whole-genome analysis to elucidate the features of this strain and the origin of IMP-1. MethodsCarbapenemase production was confirmed by a modified carbapenemase inactivation method. Whole-genome sequencing was performed using NovaSeq 6000 and GridION. Conjugation ability was evaluated using Escherichia coli ML1410 by a broth mating assay. ResultsTheblaIMP-1 gene was located on a 149 316-bp transferable plasmid (pKATP2) and formed a class 1 integron structure. In addition, this plasmid had two types of repA genes as well as astA encoding a putative heat-stable enterotoxin. Comparison with other plasmids from Enterobacterales and Pseudomonas aeruginosa suggested that this plasmid might have originated by the integration of multiple plasmids. In addition, pKATP2 harboured conjugation-associated genes and was transferable. ConclusionThis is the first report of MBL-producing K. ascorbata. Therefore, our findings suggest that species which do not typically produce MBL could acquire the corresponding genes, attracting attention as potential MBL-producing pathogens.

Overexpression of the celA1 gene in BCG modifies surface pellicle, glucosamine content in biofilms, and affects in vivo replication

Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.

Integration of the pSLT Plasmid into the Salmonella Chromosome Results in a Temperature-Sensitive Growth Defect Due to Aberrant DNA Replication.

A mutant of Salmonella enterica serovar Typhimurium was isolated that simultaneously affected two metabolic pathways as follows: NAD metabolism and DNA repair. The mutant was isolated as resistant to a nicotinamide analog and as temperature-sensitive for growth on minimal glucose medium. In this mutant, Salmonella's 94-kb virulence plasmid pSLT had recombined into the chromosome upstream of the NAD salvage pathway gene pncA This insertion blocked most transcription of pncA, which reduced uptake of the nicotinamide analog. The pSLT insertion mutant also exhibited phenotypes associated with induction of the SOS DNA repair system, including an increase in filamentous cells, higher exonuclease III and catalase activities, and derepression of SOS gene expression. Genome sequencing revealed increased read coverage extending out from the site of pSLT insertion. The two pSLT replication origins are likely initiating replication of the chromosome near the normal replication terminus. Too much replication initiation at the wrong site is probably causing the observed growth defects. Accordingly, deletion of both pSLT replication origins restored growth at higher temperatures.IMPORTANCE In studies that insert a second replication origin into the chromosome, both origins are typically active at the same time. In contrast, the integrated pSLT plasmid initiated replication in stationary phase after normal chromosomal replication had finished. The gradient in read coverage extending out from a single site could be a simple but powerful tool for studying replication and detecting chromosomal rearrangements. This technique may be of particular value when a genome has been sequenced for the first time to verify correct assembly.

Hybrid assembly of multi-drug resistant, highly virulent methicillin resistant Staphylococcus aureus ST772-SCCmec V lineage: Maximising its potential for dissemination similar to USA300 clone

• ST772-SCC mec V is a multi-drug resistant and highly virulent MRSA lineage. • The complete genome of VB9352 (ST772-SCC mec V) S. aureus genome was generated using hybrid assembly of ion Torrent reads overlayed with the long reads of minIon. • The complete genome analysis showed the presence of mosaic integrated resistance plasmid (IRP) element contained loci for beta-lactam resistance operon ( bla Z, bla I, bla R), aminoglycosides resistance ( aph A3- sat - aad E), macrolide resistance ( msrA , mphC ), and bacitracin resistant genes ( bcr A/B). • A prophage Φ-IND772PVL co-carrying PVL and sea was also identified. • In the isolate VB9352, three distinct pathogenicity islands were identified. This includes vSaα carrying both toxins and superantigens, vSaβ carrying enterotoxin gene cluster ( egc ) and vSaγ containing super-antigen like protein, alpha hemolysin and exfoliative ( eta ) toxin.

Molecular characteristics of optrA-carrying Enterococcus faecalis from chicken meat in South Korea

The purpose of this study was to identify the genetic environment of optrA gene in linezolid (LZD)-resistant Enterococcus faecalis from chicken meat and to describe the probable mechanism of dissemination of the optrA gene through plasmid or chromosomal integration. Whole genome sequencing and analysis revealed that all 3 E. faecalis isolates confirmed as LZD- and chloramphenicol-resistant carried fexA adjacent to the optrA gene as well as a variety of resistance genes for macrolides, tetracyclines, and aminoglycosides, simultaneously. But, the other genes conferring LZD resistance, cfr and poxtA, were not detected in those strains. Two isolates harboring the optrA gene in their chromosomal DNA showed >99% similarity in arrangement to the transposon Tn6674 and the transposase genes, tnpA, tnpB, and tnpC and were located in the first open reading frame for transposase. One isolate harboring an optrA-carrying plasmid also showed >99% similarity with the previously reported pE439 plasmid but had 2 amino acid changes (Thr96Lys and Tyr160Asp) and a higher minimum inhibitory concentration against LZD of 16 mg/L than that of pE439 (8 mg/L). Mobile genetic elements such as transposons or plasmids flanking the optrA gene conduct a crucial role in the dissemination of antimicrobial resistance genes. Further investigations are required to identify the way by which optrA is integrated into chromosomal DNA and plasmids.

The effect of copy number on α-synuclein's toxicity and its protective role in Bax-induced apoptosis, in yeast.

Apoptosis is a form of programmed cell death which is essential for the growth of dividing human cells whereas, in contrast, it is deleterious for post-mitotic cells such as neurons. Bax and α-synuclein are two human proteins which play a role in the induction of neuronal apoptosis in neurodegenerative diseases like Alzheimer’s and Parkinson’s. Human Bax and α-synuclein also induce cell death when expressed in baker’s yeast, Saccharomyces cerevisiae. Quite unexpectedly, the human α-synuclein gene had been identified as an inhibitor of pro-apoptotic Bax using a yeast-based screen of a human hippocampal cDNA library. Plasmids were constructed with different promoters, which allow expression of wildtype and Parkinson’s disease (PD)-related mutant α-synuclein genes, from (i) multi-copy 2µ (episomal) plasmids and (ii) integrative plasmids that compel expression of genes from chromosomal sites in varying copy numbers (1–3). All α-synuclein-containing plasmids were introduced, through transformation, into a yeast strain which already contained a chromosomally integrated copy of Bax. It is for the first time that it was observed that, depending on gene dosage, only wildtype α-synuclein is anti-apoptotic while mutant α-synuclein is not. The results also indicate that wildtype α-synuclein has a remarkable ability to manifest two contrasting effects depending on its level of expression: (i) normally, it would negate apoptosis but (ii) when overexpressed, it tends to induce apoptosis which is probably what happens in PD.

Generation of a human iPSC line (IMEDEAi008-A) derived from natural homozygous CCR5-Δ32 PBMCs enriched in the pro-erythroblast population.

A 32 base pair deletion in the C-C chemokine receptor type gene (CCR5-Δ32), the main Human Immunodeficiency Virus (HIV) co-receptor results in a non-functional protein. Individuals homozygous for the CCR5-Δ32 mutation are resistant to HIV infection. Here we report the generation, from pro-erythroblast enriched Peripheral Blood Mononuclear Cells (PBMCs) from a naturally occurring CCR5-Δ32/Δ32 individual, of the fully characterized iPSC line IMEDEAi008-A. The new line has normal karyotype, carry the Δ32 mutation in homozygosity, is free of plasmid integrations, express high levels of pluripotency markers and can differentiate into all three germ layers.

Construction and characterization of centromeric plasmids for Komagataella phaffii using a color-based plasmid stability assay.

The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work, we designed a color-based genetic assay to investigate plasmid stability in K. phaffii and constructed vectors bearing K. phaffii centromeres and the ADE3 marker. These genetic tools were evaluated in terms of mitotic stability by transforming an ade2/ade3 auxotrophic strain and regarding plasmid copy number by quantitative PCR (qPCR). Our results confirmed that the centromeric plasmids were maintained at low copy numbers as a result of typical chromosome-like segregation during cell division. These features, combined with in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.

The Role of Integrative and Conjugative Elements in Antibiotic Resistance Evolution.

Mobile genetic elements (MGEs), such as plasmids and integrative and conjugative elements (ICEs), are main drivers for the spread of antibiotic resistance (AR). Coevolution between bacteria and plasmids shapes the transfer and stability of plasmids across bacteria. Although ICEs outnumber conjugative plasmids, the dynamics of ICE-bacterium coevolution, ICE transfer rates, and fitness costs are as yet largely unexplored. Conjugative plasmids and ICEs are both transferred by type IV secretion systems, but ICEs are typically immune to segregational loss, suggesting that the evolution of ICE-bacterium associations varies from that of plasmid-bacterium associations. Considering the high abundance of ICEs among bacteria, ICE-bacterium dynamics represent a promising challenge for future research that will enhance our understanding of AR spread in human pathogens.

Response to Comment on “RNA-guided DNA insertion with CRISPR-associated transposases”

Rice et al suggest that the CRISPR-associated transposase ShCAST system could lead to additional insertion products beyond simple integration of the donor. We clarify the outcomes of ShCAST-mediated insertions in Escherichia coli, which consist of both simple insertions and integration of the donor plasmid. This latter outcome can be avoided by use of a 5' nicked DNA donor.

Genomics of Atlantic Forest Mycobacteriaceae strains unravels a mobilome diversity with a novel integrative conjugative element and plasmids harbouring T7SS.

Mobile genetic elements (MGEs) are agents of bacterial evolution and adaptation. Genome sequencing provides an unbiased approach that has revealed an abundance of MGEs in prokaryotes, mainly plasmids and integrative conjugative elements. Nevertheless, many mobilomes, particularly those from environmental bacteria, remain underexplored despite their representing a reservoir of genes that can later emerge in the clinic. Here, we explored the mobilome of the Mycobacteriaceae family, focusing on strains from Brazilian Atlantic Forest soil. Novel Mycolicibacterium and Mycobacteroides strains were identified, with the former ones harbouring linear and circular plasmids encoding the specialized type-VII secretion system (T7SS) and mobility-associated genes. In addition, we also identified a T4SS-mediated integrative conjugative element (ICEMyc226) encoding two T7SSs and a number of xenobiotic degrading genes. Our study uncovers the diversity of the Mycobacteriaceae mobilome, providing the evidence of an ICE in this bacterial family. Moreover, the presence of T7SS genes in an ICE, as well as plasmids, highlights the role of these mobile genetic elements in the dispersion of T7SS.

Multi-Stress Induction of the Mycobacterium tuberculosis MbcTA Bactericidal Toxin-Antitoxin System.

MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD+ and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (PmbcA) in a variety of stress conditions, including in vivo in mice and in macrophages.

Overexpression of wysR gene enhances wuyiencin production in ΔwysR3 mutant strain of Streptomyces albulus var. wuyiensis strain CK-15.

The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK-15 based on the ΔwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin. We developed a method to create a wysR overexpression strain based on the ΔwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into ΔwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin-resistant overexpression strain was confirmed by PCR using the Apra-F and Apra-R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in ΔwysR3 mutant. We can conclude that overexpression of wysR gene in ΔwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real-time RT-PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development. Overexpression of wysR gene in the ΔwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes.

Optimised red- and green-fluorescent proteins for live cell imaging in the industrial enzyme-producing fungus Trichoderma reesei

The filamentous fungus Trichoderma reesei is a major source of cellulolytic enzymes in biofuel production. Despite its economic relevance, our understanding of its secretory pathways is fragmentary. A major challenge is to visualise the dynamic behaviour of secretory vesicles in living cells. To this end, we establish a location juxtaposing the succinate dehydrogenase locus as a “soft-landing” site for controlled expression of 4 green-fluorescent and 5 red-fluorescent protein-encoding genes (GFPs, RFPs). Quantitative and comparative analysis of their fluorescent signals in living cells demonstrates that codon-optimised monomeric superfolder GFP (TrmsGFP) and codon-optimised mCherry (TrmCherry) combine highest signal intensity with significantly improved signal-to-noise ratios. Finally, we show that integration of plasmid near the sdi1 locus does not affect secretion of cellulase activity in RUT-C30. The molecular and live cell imaging tools generated in this study will help our understanding the secretory pathway in the industrial fungus T. reesei.