Single-gene Deletion Mutants Research Articles

Two citrate chemoreceptors involved in chemotaxis to citrate and/or citrate-metal complexes in Ralstonia pseudosolanacearum

The bacterial wilt pathogen Ralstonia pseudosolanacearum Ps29 exhibited chemotactic responses to citrate. This pathogen expresses 22 putative chemoreceptors. In screening a complete collection of mcp single-gene deletion mutants of Ps29, none showed a significant decrease in response to citrate compared with the wild-type strain. Analysis of a collection of stepwise- and multiple-deletion mutants of Ps29 revealed that the RS_RS07350 homolog (designated McpC) and McpP (chemoreceptor mediating both positive chemotaxis to phosphate and negative chemotaxis to maleate) are chemoreceptors for citrate. Double deletion of mcpC and mcpP markedly reduced the response to citrate, indicating that McpC and McpP are major chemoreceptors for citrate. Wild-type Ps29 was attracted to both free citrate and citrate complexed with divalent metal cations such as magnesium and calcium. The mcpC mcpP double-deletion mutant also showed significant reduction in chemotaxis to Mg2+- and Ca2+-citrate complexes. Introduction of a plasmid harboring the mcpC gene (but not the mcpP gene) restored the ability to respond to these citrate-metal complexes, demonstrating that McpC can sense complexes of citrate and metal ions such as Mg2+ and Ca2+ as well as free citrate. Thus, R.pseudosolanacearum Ps29 expresses two chemoreceptors for citrate. In plant infection assays using tomato seedlings, the mcpC and mcpP single- and double-deletion mutants of the highly virulent R.pseudosolanacearum MAFF106611 strain were as infectious as the wild-type strain, suggesting that citrate chemotaxis does not play an important role in infection of tomato plants in this assay system.

The antimicrobial action of polyaniline involves production of oxidative stress while functionalisation of polyaniline introduces additional mechanisms.

Polyaniline (PANI) and functionalised polyanilines (fPANI) are novel antimicrobial agents whose mechanism of action was investigated. Escherichia coli single gene deletion mutants revealed that the antimicrobial mechanism of PANI likely involves production of hydrogen peroxide while homopolymer poly(3-aminobenzoic acid), P3ABA, used as an example of a fPANI, disrupts metabolic and respiratory machinery, by targeting ATP synthase and causes acid stress. PANI was more active against E. coli in aerobic, compared to anaerobic, conditions, while this was apparent for P3ABA only in rich media. Greater activity in aerobic conditions suggests involvement of reactive oxygen species. P3ABA treatment causes an increase in intracellular free iron, which is linked to perturbation of metabolic enzymes and could promote reactive oxygen species production. Addition of exogenous catalase protected E. coli from PANI antimicrobial action; however, this was not apparent for P3ABA treated cells. The results presented suggest that PANI induces production of hydrogen peroxide, which can promote formation of hydroxyl radicals causing biomolecule damage and potentially cell death. P3ABA is thought to act as an uncoupler by targeting ATP synthase resulting in a futile cycle, which precipitates dysregulation of iron homeostasis, oxidative stress, acid stress, and potentially the fatal loss of proton motive force.

Multiple Applications of a Transient CRISPR-Cas9 Coupled with Electroporation (TRACE) System in the Cryptococcus neoformans Species Complex.

Cryptococcus neoformans is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in C. neoformans drove the investigation of this clinically important pathogen at the molecular level. Although costly and inefficient, biolistic transformation remains the major method for editing the Cryptococcus genome as foreign DNAs introduced by other methods such as electroporation are predominantly not integrated into the genome. Although the majority of DNAs introduced by biolistic transformation are stably inherited, the transformation efficiency and the homologous integration rate (∼1-10%) are low. Here, we developed a Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation (TRACE) system for targeted genetic manipulations in the C. neoformans species complex. This method took advantages of efficient genome integration due to double-strand breaks created at specific sites by the transient CRISPR-Cas9 system and the high transformation efficiency of electroporation. We demonstrated that TRACE can efficiently generate precise single-gene deletion mutants using the ADE2 locus as an example. This system can also effectively delete multiple genes in a single transformation, as evident by the successful generation of quadruple mfα1Δ2Δ3Δ4Δ mutants. In addition to generating gene deletion mutants, we complemented the ade2Δ mutant by integrating a wild-type ADE2 allele at the "safe haven" region (SH2) via homologous recombination using TRACE. Interestingly, introduced DNAs can be inserted at a designated genetic site without any homologous sequences, opening up numerous other applications. We expect that TRACE, an efficient, versatile, and cost-effective gene editing approach, will greatly accelerate research in this field.

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens.

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of <0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full-length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.

Experimental evolution of diverse Escherichia coli metabolic mutants identifies genetic loci for convergent adaptation of growth rate.

Cell growth is determined by substrate availability and the cell’s metabolic capacity to assimilate substrates into building blocks. Metabolic genes that determine growth rate may interact synergistically or antagonistically, and can accelerate or slow growth, depending on genetic background and environmental conditions. We evolved a diverse set of Escherichia coli single-gene deletion mutants with a spectrum of growth rates and identified mutations that generally increase growth rate. Despite the metabolic differences between parent strains, mutations that enhanced growth largely mapped to core transcription machinery, including the β and β’ subunits of RNA polymerase (RNAP) and the transcription elongation factor, NusA. The structural segments of RNAP that determine enhanced growth have been previously implicated in antibiotic resistance and in the control of transcription elongation and pausing. We further developed a computational framework to characterize how the transcriptional changes that occur upon acquisition of these mutations affect growth rate across strains. Our experimental and computational results provide evidence for cases in which RNAP mutations shift the competitive balance between active transcription and gene silencing. This study demonstrates that mutations in specific regions of RNAP are a convergent adaptive solution that can enhance the growth rate of cells from distinct metabolic states.

Identification of Genes Involved in Bacteriostatic Antibiotic-Induced Persister Formation.

Persister cells are metabolically quiescent multi-drug tolerant fraction of a genetically sensitive bacterial population and are thought to be responsible for relapse of many persistent infections. Persisters can be formed naturally in the stationary phase culture, and also can be induced by bacteriostatic antibiotics. However, the molecular basis of bacteriostatic antibiotic induced persister formation is unknown. Here, we established a bacteriostatic antibiotic induced persister model and screened the Escherichia coli single gene deletion mutant library for mutants with defect in rifampin or tetracycline induced persistence to ofloxacin. Thirsty-seven and nine genes were found with defects in rifampin- and tetracycline-induced persister formation, respectively. Six mutants were found to overlap in both rifampin and tetracycline induced persister screens: recA, recC, ruvA, uvrD, fis, and acrB. Interestingly, four of these mutants (recA, recC, ruvA, and uvrD) mapped to DNA repair pathway, one mutant mapped to global transcriptional regulator (fis) and one to efflux (acrB). The stationary phase culture of the identified mutants and parent strain BW25113 were subjected to different antibiotics including ofloxacin, ampicillin, gentamicin, and stress conditions including starvation and acid pH 4.0. All the six mutants showed less tolerance to ofloxacin, but only some of them were more sensitive to other specific stress conditions. Complementation of five of the six common mutants restored the persister level to that of the parent strain in both stationary phase and static antibiotic-induced conditions. In addition to the DNA repair pathways shared by both rifampin and tetracycline induced persisters, genes involved in rifampin-induced persisters map also to transporters, LPS biosynthesis, flagella biosynthesis, metabolism (folate and energy), and translation, etc. These findings suggest that persisters generated by different ways may share common mechanisms of survival, and also shed new insight into the molecular basis of static antibiotic induced antagonism of cidal antibiotics.

Functional Analysis of Mating Type Genes and Transcriptome Analysis during Fruiting Body Development of Botrytis cinerea.

ABSTRACTBotrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (comprising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction.

Sulfolobus acidocaldarius Transports Pentoses via a Carbohydrate Uptake Transporter 2 (CUT2)-Type ABC Transporter and Metabolizes Them through the Aldolase-Independent Weimberg Pathway.

Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources, such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus, only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius MW001 grown on peptides alone and peptides in the presence of d-xylose allowed for the identification of the ABC transporter for d-xylose and l-arabinose transport and the gaining of deeper insights into pentose catabolism under the respective growth conditions. The d-xylose/l-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the carbohydrate uptake transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea Single-gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for d-xylose and l-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and d-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD [also known as KDAD]; Saci_1939) was no longer able to catabolize d-xylose or l-arabinose, suggesting the absence of the aldolase-dependent branch in S. acidocaldariusIMPORTANCE Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications, as their optimal growth conditions resemble conditions used in certain biotechnologies such as industrial plant waste degradation. Because of its high genome stability, Sulfolobus acidocaldarius is especially suited as a platform organism for such applications. For use in (ligno)cellulose degradation, it was important to understand pentose uptake and metabolism in S. acidocaldarius This study revealed that only the aldolase-independent Weimberg pathway is required for growth of S. acidocaldarius MW001 on d-xylose and l-arabinose. Moreover, S. acidocaldarius employs a CUT2 ABC transporter for pentose uptake, which is more similar to bacterial than to archaeal ABC transporters. The identification of pentose-inducible promoters will expedite the metabolic engineering of S. acidocaldarius for its development into a platform organism for (ligno)cellulose degradation.

The Toolbox for Uncovering the Functions of Legionella Dot/Icm Type IVb Secretion System Effectors: Current State and Future Directions

The defective in organelle trafficking/intracellular multiplication (Dot/Icm) Type IVb secretion system (T4SS) is the essential virulence factor for the intracellular life style and pathogenicity of Legionella species. Screens demonstrated that an individual L. pneumophila strain can use the Dot/Icm T4SS to translocate an unprecedented number of more than 300 proteins into host cells, where these, so called Icm/Dot-translocated substrates (IDTS) or effectors, manipulate host cell functions to the benefit of the bacteria. Bioinformatic analysis of the pan-genus genome predicts at least 608 orthologous groups of putative effectors. Deciphering the function of these effectors is key to understanding Legionella pathogenesis; however, the analysis is challenging. Substantial functional redundancy renders classical, phenotypic screening of single gene deletion mutants mostly ineffective. Here, I review experimental approaches that were successfully used to identify, validate and functionally characterize T4SS effectors and highlight new methods, which promise to facilitate unlocking the secrets of Legionella's extraordinary weapons arsenal.

Involvement of many chemotaxis sensors in negative chemotaxis to ethanol in Ralstonia pseudosolanacearum Ps29.

Ralstonia pseudosolanacearum Ps29 showed repellent responses to alcohols including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1,3-propanediol and prenol. R. pseudosolanacearum Ps29 possesses 22 putative chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). To identify a MCP involved in negative chemotaxis to ethanol, we measured ethanol chemotaxis of a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29. However, all the mutants showed repellent responses to ethanol comparable to that of the wild-type strain. We constructed a stepwise- and multiple-mcp gene deletion mutant collection of R. pseudosolanacearum Ps29. Analysis of the collection found that an 18-mcp-knockout mutant (strain POC18) failed to respond to ethanol. Complementation analysis using POC18 as the host strain found that introduction of mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 restored the ability of POC18 to respond to ethanol. However, unexpectedly, strain POC10II, harbouring unmarked deletions in 10 mcp genes including mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 showed repellent responses to ethanol comparable to that of wild-type Ps29. We hypothesised that multiple mcp mutations in POC18 led to a shortage of MCPs required for formation of functional chemoreceptor arrays. When pPS16 (encoding McpP involved in phosphate chemotaxis) was introduced into POC18, POC18(pPS16) did not respond to phosphate. This result supports the hypothesis. But, genetic analysis revealed that MCPs (Mcp07, Mcp13, Mcp20 and Mcp21) are not essential for ethanol chemotaxis. Thus, we conclude that many and unspecified MCPs are involved in negative chemotaxis to ethanol in R. pseudosolanacearum Ps29.

Characterization of two catalase-peroxidase-encoding genes in Fusarium verticillioides reveals differential responses to invitro versus inplanta oxidative challenges.

Catalase-peroxidases (KatGs) are a superfamily of reactive oxygen species (ROS)-degrading enzymes believed to have been horizontally acquired by ancient Ascomycota from bacteria. Subsequent gene duplication resulted in two KatG paralogues in ascomycetes: the widely distributed intracellular KatG1 group and the phytopathogen-dominated extracellular KatG2 group. To functionally characterize FvCP01 (KatG1) and FvCP02 (KatG2) in the maize pathogen Fusarium verticillioides, single and double gene deletion mutants were examined in response to hydrogen peroxide (H2 O2 ). Both ΔFvCP01 and ΔFvCP02 were more sensitive to H2 O2 than the wild-type invitro, although their sensitivity differed depending on the type of inoculum. Inoculations using mycelial agar plugs demonstrated an additive effect of the mutants, with the ΔFvCP01/ΔFvCP02 double deletion being the most sensitive to H2 O2 . In general, conidia were much more sensitive than agar plugs to H2 O2 , and conidial inoculations indicated that FvCP01 conferred more H2 O2 tolerance than FvCP02. Transcriptional analysis showed the induction of FvCP01, but decreased expression of FvCP02, in both mycelia and spores in the wild-type after H2 O2 exposure, but this trend was reversed when the fungus was grown on germinating maize seeds. This interaction with the plant increased the expression of FvCP02, but not FvCP01, indicating that FvCP02 may be responsive to plant-derived H2 O2 . Yet, FvCP01 was induced more than three-fold in the ΔFvCP02 mutant grown on germlings, suggesting that FvCP01 can compensate for the loss of FvCP02. Given the differential responses of these two F.verticillioides genes to invitro versus inplanta challenges, a model is proposed to illustrate the differing roles of FvCP01 and FvCP02 in protective responses against H2 O2 -derived oxidative stress.

Clade II nitrous oxide respiration of Wolinella succinogenes depends on the NosG, -C1, -C2, -H electron transport module, NosB and a Rieske/cytochrome bc complex.

Microbial reduction of nitrous oxide (N2 O) is an environmentally significant process in the biogeochemical nitrogen cycle. However, it has been recognized only recently that the gene encoding N2 O reductase (nosZ) is organized in varying genetic contexts, thereby defining clade I (or 'typical') and clade II (or 'atypical') N2 O reductases and nos gene clusters. This study addresses the enzymology of the clade II Nos system from Wolinella succinogenes, a nitrate-ammonifying and N2 O-respiring Epsilonproteobacterium that contains a cytochrome c N2 O reductase (cNosZ). The characterization of single non-polar nos gene deletion mutants demonstrated that the NosG, -C1, -C2, -H and -B proteins were essential for N2 O respiration. Moreover, cells of a W. succinogenes mutant lacking a putative menaquinol-oxidizing Rieske/cytochrome bc complex (QcrABC) were found to be incapable of N2 O (and also nitrate) respiration. Proton motive menaquinol oxidation by N2 O is suggested, supported by the finding that the molar yield for W. succinogenes cells grown by N2 O respiration using formate as electron donor exceeded that of fumarate respiration by about 30%. The results demand revision of the electron transport chain model of clade II N2 O respiration and challenge the assumption that NosGH(NapGH)-type iron-sulfur proteins are menaquinol-reactive.

Characterization of the Burkholderia cenocepacia TonB Mutant as a Potential Live Attenuated Vaccine.

Burkholderia cenocepacia is an opportunistic pathogen prevalent in cystic fibrosis patients, which is particularly difficult to treat, causing chronic and eventually fatal infections. The lack of effective treatment options makes evident the need to develop alternative therapeutic or prophylactic approaches. Vaccines, and live attenuated vaccines, are an unexplored avenue to treat B. cenocepacia infections. Here we constructed and characterized a B. cenocepacia tonB mutant strain, which was unable to actively transport iron, to test whether this single gene deletion mutant (strain renamed GAP001) protected against an acute respiratory B. cenocepacia lethal infection. Here we show that the mutant strain GAP001 is attenuated, and effective at protecting against B. cenocepacia challenge. Intranasal administration of GAP001 to BALB/c mice resulted in almost complete survival with high degree of bacterial clearance.

Elimination of the last reactions in ergosterol biosynthesis alters the resistance of Saccharomyces cerevisiae to multiple stresses.

The sterol composition of membranes is known to influence many phenotypes of yeast. However, a systematic study of the relationship between sterol composition and stress resistances has not been conducted. Here, we therefore constructed single or double gene deletion mutants of the last four enzymes in ergosterol biosynthesis in a prototrophic genetic background of Saccharomyces cerevisiae. Identification of the sterol composition of these mutants revealed a high flexibility of the sterol-processing steps instead of the previously proposed sequential conversion. Compared with the wild type, the mutants showed altered resistances to different exogenous stresses regarding the specific growth rate and duration of lag phase. The erg5 deletion mutant whose sterol has a saturated side chain exhibited overall robust growth under the tested stress conditions. The thermotolerant phenotype of erg5 deletion mutant was reproduced in filamentous fungus Penicillium oxalicum. These results highlight the important role of sterols in the response of yeast cells to environmental stresses, and suggest the possibility of improving the robustness of industrial yeast strains by engineering their sterol composition.

Establishment of a markerless gene deletion system in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow over a wide range of salinity, which makes it as an excellent model organism for understanding the mechanism of prokaryotic osmoregulation. Functional analysis of C. salexigens genes is an essential way to reveal their roles in cellular osmoregulation. However, the lack of an effective markerless gene deletion system has prevented construction of multiple gene deletion mutants for the members in the genus. Here, we report the development of a markerless gene deletion system in C. salexigens using allelic exchange method. In this system, the in vitro mutant allele of target gene was inserted into a pK18mobsacB-based integrative vector pMDC21, which contained a chloramphenicol resistance cassette as the positive selection marker and a sacB gene from Bacillus subtilis as the counterselectable marker. To validate this system, two single-gene deletion mutants and a double-gene deletion mutant were constructed. In addition, our results showed that growth of the merodiploids and sucrose screening at 25°C were more effective to decrease the occurrence of spontaneous sucrose resistance colonies than at higher temperature (30 or 37°C), and growth of the merodiploids in mineral salt medium instead of the complex medium was critical to increase the recovery rate of deletion mutants.

Genomic and transcriptomic analyses reveal distinct biological functions for cold shock proteins (VpaCspA and VpaCspD) in Vibrio parahaemolyticus CHN25 during low-temperature survival

BackgroundVibrio parahaemolyticus causes serious seafood-borne gastroenteritis and death in humans. Raw seafood is often subjected to post-harvest processing and low-temperature storage. To date, very little information is available regarding the biological functions of cold shock proteins (CSPs) in the low-temperature survival of the bacterium. In this study, we determined the complete genome sequence of V. parahaemolyticus CHN25 (serotype: O5:KUT). The two main CSP-encoding genes (VpacspA and VpacspD) were deleted from the bacterial genome, and comparative transcriptomic analysis between the mutant and wild-type strains was performed to dissect the possible molecular mechanisms that underlie low-temperature adaptation by V. parahaemolyticus.ResultsThe 5,443,401-bp V. parahaemolyticus CHN25 genome (45.2% G + C) consisted of two circular chromosomes and three plasmids with 4,724 predicted protein-encoding genes. One dual-gene and two single-gene deletion mutants were generated for VpacspA and VpacspD by homologous recombination. The growth of the ΔVpacspA mutant was strongly inhibited at 10 °C, whereas the VpacspD gene deletion strongly stimulated bacterial growth at this low temperature compared with the wild-type strain. The complementary phenotypes were observed in the reverse mutants (ΔVpacspA-com, and ΔVpacspD-com). The transcriptome data revealed that 12.4% of the expressed genes in V. parahaemolyticus CHN25 were significantly altered in the ΔVpacspA mutant when it was grown at 10 °C. These included genes that were involved in amino acid degradation, secretion systems, sulphur metabolism and glycerophospholipid metabolism along with ATP-binding cassette transporters. However, a low temperature elicited significant expression changes for 10.0% of the genes in the ΔVpacspD mutant, including those involved in the phosphotransferase system and in the metabolism of nitrogen and amino acids. The major metabolic pathways that were altered by the dual-gene deletion mutant (ΔVpacspAD) radically differed from those that were altered by single-gene mutants. Comparison of the transcriptome profiles further revealed numerous differentially expressed genes that were shared among the three mutants and regulators that were specifically, coordinately or antagonistically modulated by VpaCspA and VpaCspD. Our data also revealed several possible molecular coping strategies for low-temperature adaptation by the bacterium.ConclusionsThis study is the first to describe the complete genome sequence of V. parahaemolyticus (serotype: O5:KUT). The gene deletions, complementary insertions, and comparative transcriptomics demonstrate that VpaCspA is a primary CSP in the bacterium, while VpaCspD functions as a growth inhibitor at 10 °C. These results have improved our understanding of the genetic basis for low-temperature survival by the most common seafood-borne pathogen worldwide.

Deletion of mazF increases Staphylococcus aureus biofilm formation in an ica-dependent manner.

Toxin-antitoxin (TA) systems are composed of a toxin that inhibits an essential cellular process (e.g. DNA replication, transcription, membrane integrity) and its cognate antitoxin that neutralizes the effect of the toxin. Staphylococcus aureus harbors two types of chromosomally encoded TA systems, namely mazEFsa encoding a UACAU-specific mRNA interferase and two paralogous genes of yefM-yoeBsa encoding a ribosome-dependent endoribonuclease system. However, little is known about the physiological role of MazEFsa and YefM-YoeBsa in S. aureus. Upon characterizing the phenotypes of single, double and triple gene deletion mutants, we found that mazFsa deletion led to increased biofilm formation. Subsequently, transcriptional analysis revealed that expression of intercellular adhesin (ica) gene, icaADBC, increased in a mazFsa deletion mutant. mazFsa/icaADBC double gene deletion and genetic complementation approaches provided convincing evidence that increased biofilm formation was caused by an increase in polysaccharide intercellular adhesin synthesized by icaADBC-encoded proteins. Furthermore, through the use of alanine substitutions at the conserved active residues of MazFsa, our results suggested that ica-mediated biofilm formation depended on the mRNA interferase activity of MazFsa. These findings give new insights not only into the physiological role of MazEFsa in S. aureus, but also into the regulatory mechanism of ica-dependent biofilm formation.

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

Calpains are involved in asexual and sexual development, cell wall integrity and pathogenicity of the rice blast fungus.

Calpains are ubiquitous and well-conserved proteins that belong to the calcium-dependent, non-lysosomal cysteine protease family. In this study, 8 putative calpains were identified using Pfam domain analysis and BlastP searches in M. oryzae. Three single gene deletion mutants (ΔMocapn7, ΔMocapn9 and ΔMocapn14) and two double gene deletion mutants (ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7) were obtained using the high-throughput gene knockout system. The calpain disruption mutants showed defects in colony characteristics, conidiation, sexual reproduction and cell wall integrity. The mycelia of the ΔMocapn7, ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7 mutants showed reduced pathogenicity on rice and barley.