scholarly journals Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus

2014 ◽  
Vol 80 (7) ◽  
pp. 2299-2306 ◽  
Author(s):  
Axel Thiel ◽  
Grégoire Michoud ◽  
Yann Moalic ◽  
Didier Flament ◽  
Mohamed Jebbar
Keyword(s):  
Gene Disruption ◽  
Negative Selection ◽  
Deletion Mutants ◽  
Wild Type ◽  
Genetic Studies ◽  
Content Type ◽  
Genetic Manipulations ◽  
Starting Point ◽  
Flanking Regions ◽  
Markerless Deletion

ABSTRACTIn this study, we developed a gene disruption system forThermococcus barophilususing simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-typeT. barophilusMP cells. Initially, apyrFdeletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of thehisBgene was also constructed in the UBOCC-3256 (ΔpyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functionalpyrForhisBallele fromT. barophiluswas inserted into the chromosome of UBOCC-3256 (ΔpyrF) or UBOCC-3260 (ΔpyrFΔhisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

scholarly journals Glutathione Synthesis Contributes to Virulence of Streptococcus agalactiae in a Murine Model of Sepsis

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Elizabeth A. Walker ◽  
Gary C. Port ◽  
Michael G. Caparon ◽  
Blythe E. Janowiak
Keyword(s):  
Streptococcus Agalactiae ◽  
De Novo ◽  
Single Gene ◽  
Neonatal Meningitis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Glutathione Synthesis ◽  
Content Type ◽  
Resistance Pathway

ABSTRACT Streptococcus agalactiae, a leading cause of sepsis and meningitis in neonates, utilizes multiple virulence factors to survive and thrive within the human host during an infection. Unique among the pathogenic streptococci, S. agalactiae uses a bifunctional enzyme encoded by a single gene (gshAB) to synthesize glutathione (GSH), a major antioxidant in most aerobic organisms. Since S. agalactiae can also import GSH, similar to all other pathogenic streptococcal species, the contribution of GSH synthesis to the pathogenesis of S. agalactiae disease is not known. In the present study, gshAB deletion mutants were generated in strains representing three of the most prevalent clinical serotypes of S. agalactiae and were compared against isogenic wild-type and gshAB knock-in strains. When cultured in vitro in a chemically defined medium under nonstress conditions, each mutant and its corresponding wild type had comparable growth rates, generation times, and growth yields. However, gshAB deletion mutants were found to be more sensitive than wild-type or gshAB knock-in strains to killing and growth inhibition by several different reactive oxygen species. Furthermore, deletion of gshAB in S. agalactiae strain COH1 significantly attenuated virulence compared to the wild-type or gshAB knock-in strains in a mouse model of sepsis. Taken together, these data establish that GSH is a virulence factor important for resistance to oxidative stress and that de novo GSH synthesis plays a crucial role in S. agalactiae pathogenesis and further suggest that the inhibition of GSH synthesis may provide an opportunity for the development of novel therapies targeting S. agalactiae disease. IMPORTANCE Approximately 10 to 30% of women are naturally and asymptomatically colonized by Streptococcus agalactiae. However, transmission of S. agalactiae from mother to newborn during vaginal birth is a leading cause of neonatal meningitis. Although colonized mothers who are at risk for transmission to the newborn are treated with antibiotics prior to delivery, S. agalactiae is becoming increasingly resistant to current antibiotic therapies, and new treatments are needed. This research reveals a critical stress resistance pathway, glutathione synthesis, that is utilized by S. agalactiae and contributes to its pathogenesis. Understanding the role of this unique bifunctional glutathione synthesis enzyme in S. agalactiae during sepsis may help elucidate why S. agalactiae produces such an abundance of glutathione compared to other bacteria.

scholarly journals Deciphering Morphological Determinants of the Helix-Shaped Leptospira

2011 ◽  
Vol 193 (22) ◽  
pp. 6266-6275 ◽  
Author(s):  
Leyla Slamti ◽  
Miguel A. de Pedro ◽  
Emilande Guichet ◽  
Mathieu Picardeau
Keyword(s):  
Penicillin G ◽  
Penicillin Binding Protein ◽  
Intact Cells ◽  
Content Type ◽  
Genetic Manipulations ◽  
Content Model ◽  
Starting Point ◽  
Genes Encoding ◽  
Lactam Antibiotic ◽  
Slow Growing

Leptospiraspp. are thin, highly motile, slow-growing spirochetes that can be distinguished from other bacteria on the basis of their unique helical shape. Defining the mechanisms by which these bacteria generate and maintain this atypical morphology should greatly enhance our understanding of the fundamental physiology of these pathogens. In this study, we showed that peptidoglycan sacculi fromLeptospiraspp. retain the helical shape of intact cells. Interestingly, the distribution of muropeptides was different from that in theEscherichia colimodel, indicating that specific enzymes might be active on the peptidoglycan macromolecule. We could alter the shape ofLeptospira biflexawith the broad-spectrum β-lactam antibiotic penicillin G and with amdinocillin and aztreonam, which are β-lactams that preferentially target penicillin-binding protein 2 (PBP2) and PBP3, respectively, in some species. Although genetic manipulations ofLeptospiraspp. are scarce, we were able to obtain mutants with alterations in genes encoding PBPs, including PBP3. Loss of this protein resulted in cell elongation. We also generated anL. biflexastrain that conditionally expresses MreB. Loss of the MreB function was correlated with morphological abnormalities such as a localized increased diameter and heterogeneous length. A prolonged depletion of MreB resulted in cell lysis, suggesting that this protein is essential. These findings indicate that important aspects of leptospiral cell morphology are determined by the cytoskeleton and the murein layer, thus providing a starting point for a better understanding of the morphogenesis in these atypical bacteria.

scholarly journals Utilization of Host Iron Sources by Corynebacterium diphtheriae: Multiple Hemoglobin-Binding Proteins Are Essential for the Use of Iron from the Hemoglobin-Haptoglobin Complex

2014 ◽  
Vol 197 (3) ◽  
pp. 553-562 ◽  
Author(s):  
Courtni E. Allen ◽  
Michael P. Schmitt
Keyword(s):  
Double Mutant ◽  
Biological Function ◽  
Corynebacterium Diphtheriae ◽  
Enzyme Linked Immunosorbent Assay ◽  
Deletion Mutants ◽  
Wild Type ◽  
Content Type ◽  
Iron Source ◽  
Hemoglobin Binding ◽  
Single Deletion

The use of hemin iron byCorynebacterium diphtheriaerequires the DtxR- and iron-regulated ABC hemin transporter HmuTUV and the secreted Hb-binding protein HtaA. We recently described two surface anchored proteins, ChtA and ChtC, which also bind hemin and Hb. ChtA and ChtC share structural similarities to HtaA; however, a function for ChtA and ChtC was not determined. In this study, we identified additional host iron sources that are utilized byC. diphtheriae. We show that severalC. diphtheriaestrains use the hemoglobin-haptoglobin (Hb-Hp) complex as an iron source. We report that anhtaAdeletion mutant ofC. diphtheriaestrain 1737 is unable to use the Hb-Hp complex as an iron source, and we further demonstrate that achtA-chtCdouble mutant is also unable to use Hb-Hp iron. Single-deletion mutants ofchtAorchtCuse Hb-Hp iron in a manner similar to that of the wild type. These findings suggest that both HtaA and either ChtA or ChtC are essential for the use of Hb-Hp iron. Enzyme-linked immunosorbent assay (ELISA) studies show that HtaA binds the Hb-Hp complex, and the substitution of a conserved tyrosine (Y361) for alanine in HtaA results in significantly reduced binding.C. diphtheriaewas also able to use human serum albumin (HSA) and myoglobin (Mb) but not hemopexin as iron sources. These studies identify a biological function for the ChtA and ChtC proteins and demonstrate that the use of the Hb-Hp complex as an iron source byC. diphtheriaerequires multiple iron-regulated surface components.

scholarly journals Supplementation of Intracellular XylR Leads to Coutilization of Hemicellulose Sugars

2012 ◽  
Vol 78 (7) ◽  
pp. 2221-2229 ◽  
Author(s):  
Dan Groff ◽  
Peter I. Benke ◽  
Tanveer S. Batth ◽  
Gregory Bokinsky ◽  
Christopher J. Petzold ◽  
...  
Keyword(s):  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Gene Insertion ◽  
Starting Point ◽  
Different Types ◽  
Important Challenge ◽  
Biomass Sugars ◽  
Hemicellulosic Sugars ◽  
Type Strains

ABSTRACTEscherichia colihas the potential to be a powerful biocatalyst for the conversion of lignocellulosic biomass into useful materials such as biofuels and polymers. One important challenge in usingE. colifor the transformation of biomass sugars is diauxie, or sequential utilization of different types of sugars. We demonstrate that, by increasing the intracellular levels of the transcription factor XylR, the preferential consumption of arabinose before xylose can be eliminated. In addition, XylR augmentation must be finely tuned for robust coutilization of these two hemicellulosic sugars. Using a novel technique for scarless gene insertion, an additional copy ofxylRwas inserted into thearaBADoperon. The resulting strain was superior at cometabolizing mixtures of arabinose and xylose and was able to produce at least 36% more ethanol than wild-type strains. This strain is a useful starting point for the development of anE. colibiocatalyst that can simultaneously convert all biomass sugars.

scholarly journals Identification of themcpAandmcpMGenes, Encoding Methyl-Accepting Proteins Involved in Amino Acid and l-Malate Chemotaxis, and Involvement of McpM-Mediated Chemotaxis in Plant Infection by Ralstonia pseudosolanacearum (Formerly Ralstonia solanacearum Phylotypes I and III)

2015 ◽  
Vol 81 (21) ◽  
pp. 7420-7430 ◽  
Author(s):  
Akiko Hida ◽  
Shota Oku ◽  
Takeru Kawasaki ◽  
Yutaka Nakashimada ◽  
Takahisa Tajima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Gene Deletion ◽  
Pcr Analysis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Complete Collection ◽  
Content Type ◽  
Plant Infection ◽  
Tomato Roots ◽  
Ralstonia Pseudosolanacearum

ABSTRACTSequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in theRalstonia pseudosolanacearumGMI1000 genome. PCR analysis and DNA sequencing showed that the highly motileR. pseudosolanacearumstrain Ps29 possesses homologs of all 22R. pseudosolanacearumGMI1000mcpgenes. We constructed a complete collection of singlemcpgene deletion mutants ofR. pseudosolanacearumPs29 by unmarked gene deletion. Screening of the mutant collection revealed thatR. pseudosolanacearumPs29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis tol-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designatedmcpMandmcpA. While wild-typeR. pseudosolanacearumstrain Ps29 displayed attraction to 16 amino acids, themcpAmutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructedmcpAandmcpMdeletion mutants of highly virulentR. pseudosolanacearumstrain MAFF106611 to investigate the contribution of chemotaxis tol-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, themcpMmutant (but not themcpAmutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis tol-malate, facilitatesR. pseudosolanacearummotility to tomato roots in sand.

scholarly journals PFP1, a Gene Encoding an Epc-N Domain-Containing Protein, Is Essential for Pathogenicity of the Barley Pathogen Rhynchosporium commune

2014 ◽  
Vol 13 (8) ◽  
pp. 1026-1035 ◽  
Author(s):  
Sylvia Siersleben ◽  
Daniel Penselin ◽  
Claudia Wenzel ◽  
Sylvie Albert ◽  
Wolfgang Knogge
Keyword(s):  
Upstream Region ◽  
Insertion Mutagenesis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Homologous Proteins ◽  
Gene Encoding ◽  
Fungal Virulence ◽  
Content Type ◽  
Wild Type Level ◽  
Type Gene

ABSTRACTScald caused byRhynchosporium communeis an important foliar disease of barley. Insertion mutagenesis ofR. communegenerated a nonpathogenic fungal mutant which carries the inserted plasmid in the upstream region of a gene namedPFP1. The characteristic feature of the gene product is an Epc-N domain. This motif is also found in homologous proteins shown to be components of histone acetyltransferase (HAT) complexes of fungi and animals. Therefore, PFP1 is suggested to be the subunit of a HAT complex inR. communewith an essential role in the epigenetic control of fungal pathogenicity. TargetedPFP1disruption also yielded nonpathogenic mutants which showed wild-type-like growthex planta, except for the occurrence of hyphal swellings. Complementation of the deletion mutants with the wild-type gene reestablished pathogenicity and suppressed the hyphal swellings. However, despite wild-type-levelPFP1expression, the complementation mutants did not reach wild-type-level virulence. This indicates that the function of the protein complex and, thus, fungal virulence are influenced by a position-affected long-range control ofPFP1expression.

scholarly journals Marker Removal in Staphylococci via Cre Recombinase and Different lox Sites

2007 ◽  
Vol 74 (5) ◽  
pp. 1316-1323 ◽  
Author(s):  
Martina Leibig ◽  
Bernhard Krismer ◽  
Martina Kolb ◽  
Alexandra Friede ◽  
Friedrich Götz ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Deletion Mutants ◽  
Origin Of Replication ◽  
Wild Type ◽  
Efficient Removal ◽  
Expression Plasmid ◽  
Staphylococcus Carnosus ◽  
Allelic Replacement ◽  
Resistance Markers ◽  
Markerless Deletion

ABSTRACT Allelic replacement in staphylococci is frequently aided by antibiotic resistance markers that replace the gene(s) of interest. In multiply modified strains, the number of mutated genes usually correlates with the number of selection markers in the strain's chromosome. Site-specific recombination systems are capable of eliminating such markers, if they are flanked by recombinase recognition sites. In this study, a Cre-lox setting was established that allowed the efficient removal of resistance genes from the genomes of Staphylococcus carnosus and S. aureus. Two cassettes conferring resistance to erythromycin or kanamycin were flanked with wild-type or mutant lox sites, respectively, and used to delete single genes and an entire operon. After transformation of the cells with a newly constructed cre expression plasmid (pRAB1), genomic eviction of the resistance genes was observed in approximately one out of ten candidates analyzed and subsequently verified by PCR. Due to its thermosensitive origin of replication, the plasmid was then easily eliminated at nonpermissive temperatures. We anticipate that the system presented here will prove useful for generating markerless deletion mutants in staphylococci.

scholarly journals Identification and Characterization of theNeisseria gonorrhoeaeMscS-Like Mechanosensitive Channel

2018 ◽  
Vol 86 (6) ◽  
Author(s):  
Zhemin Wang ◽  
Xiaomin Wang ◽  
Ping Lu ◽  
Chunshan Ni ◽  
Yuezhou Li ◽  
...  
Keyword(s):  
Turgor Pressure ◽  
Bacterial Pathogen ◽  
Mechanosensitive Channel ◽  
Infection Model ◽  
Deletion Mutants ◽  
Mechanosensitive Channels ◽  
Wild Type ◽  
Content Type ◽  
Type Strains

ABSTRACTMechanosensitive channels are ubiquitous in bacteria and provide an essential mechanism to survive sudden exposure to a hypo-osmotic environment by the sensing and release of increased turgor pressure. No mechanosensitive channels have thus far been identified and characterized for the human-specific bacterial pathogenNeisseria gonorrhoeae. In this study, we identified and characterized theN. gonorrhoeaeMscS-like mechanosensitive channel (Ng-MscS). Electrophysiological analyses by the patch clamp method showed that Ng-MscS is stretch activated and contains pressure-dependent gating properties. Further mutagenesis studies of critical residues forming the hydrophobic vapor lock showed that gain-of-function mutations in Ng-MscS inhibited bacterial growth. Subsequent analysis of the function of Ng-MscS inN. gonorrhoeaeby osmotic down-shock assays revealed that the survival of Ng-mscSdeletion mutants was significantly reduced compared with that of wild-type strains, while down-shock survival was restored upon the ectopic complementation ofmscS. Finally, to investigate whether Ng-MscS is important forN. gonorrhoeaeduring infections, competition assays were performed by using a murine vaginal tract infection model. Ng-mscSdeletion mutants were outcompeted byN. gonorrhoeaewild-type strains for colonization and survival in this infection model, highlighting that Ng-MscS contributes toin vivocolonization and survival. Therefore, Ng-MscS might be a promising target for the future development of novel antimicrobials.

scholarly journals Precise Null Deletion Mutations of the Mycothiol Synthesis Genes Reveal Their Role in Isoniazid and Ethionamide Resistance in Mycobacterium smegmatis

2011 ◽  
Vol 55 (7) ◽  
pp. 3133-3139 ◽  
Author(s):  
Xia Xu ◽  
Catherine Vilchèze ◽  
Yossef Av-Gay ◽  
Anaximandro Gómez-Velasco ◽  
William R. Jacobs
Keyword(s):  
Mycobacterium Smegmatis ◽  
Deletion Mutant ◽  
Drug Sensitivity ◽  
Deletion Mutants ◽  
Null Mutant ◽  
Gene Encoding ◽  
Genetic Studies ◽  
Content Type ◽  
Deletion Mutations ◽  
Single Deletion

ABSTRACTMycothiol (MSH; AcCys-GlcN-Ins) is the glutathione analogue for mycobacteria. Mutations in MSH biosynthetic genes have been associated with resistance to isoniazid (INH) and ethionamide (ETH) in mycobacteria, but rigorous genetic studies are lacking, and those that have been conducted have yielded different results. In this study, we constructed independent null deletion mutants for all four genes involved in the MSH biosynthesis pathway (mshA,mshB,mshC, andmshD) inMycobacterium smegmatisand made complementing constructs in integrating plasmids. The resulting set of strains was analyzed for levels of MSH, INH resistance, and ETH resistance. ThemshAandmshCsingle deletion mutants were devoid of MSH production and resistant to INH, whereas themshBdeletion mutant produced decreased levels of MSH yet was sensitive to INH, suggesting that MSH biosynthesis is essential for INH susceptibility inM. smegmatis. Further evidence supporting this conclusion was generated by deleting the gene encoding the MSH S-conjugate amidase (mca) from the ΔmshBnull mutant. This double mutant, ΔmshBΔmca, completely abolished MSH production and was resistant to INH. ThemshA,mshC, andmshBsingle deletion mutants were also resistant to ETH, indicating that ETH resistance is modulated by the level of MSH inM. smegmatis. Surprisingly, themshDdeletion mutant lacked MSH production but was sensitive to both INH and ETH. The drug sensitivity was likely mediated by the compensated synthesis ofN-formyl-Cys-GlcN-Ins, previously demonstrated to substitute for MSH in anmshDmutant ofM. smegmatis. We conclude that MSH orN-formyl-Cys-GlcN-Ins is required for susceptibility to INH or ETH inM. smegmatis.

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