scholarly journals Development of a Markerless Knockout Method for Actinobacillus succinogenes

2014 ◽  
Vol 80 (10) ◽  
pp. 3053-3061 ◽  
Author(s):  
Rajasi V. Joshi ◽  
Bryan D. Schindler ◽  
Nikolas R. McPherson ◽  
Kanupriya Tiwari ◽  
Claire Vieille
Keyword(s):  
High Efficiency ◽  
Direct Selection ◽  
Selection Marker ◽  
Actinobacillus Succinogenes ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Pyruvate Formate Lyase ◽  
Content Type ◽  
Knockout Mutants ◽  
Encoding Genes

ABSTRACTActinobacillus succinogenesis one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method forA. succinogenesusing natural transformation or electroporation. TheEscherichia coliisocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use ofA. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. TheSaccharomyces cerevisiaeflippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressingflpwas cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

scholarly journals Two DHH Subfamily 1 Proteins Contribute to Pneumococcal Virulence and Confer Protection against Pneumococcal Disease

2011 ◽  
Vol 79 (9) ◽  
pp. 3697-3710 ◽  
Author(s):  
L. E. Cron ◽  
K. Stol ◽  
P. Burghout ◽  
S. van Selm ◽  
E. R. Simonetti ◽  
...  
Keyword(s):  
Otitis Media ◽  
Pneumococcal Disease ◽  
Capsular Polysaccharide ◽  
Pneumococcal Infection ◽  
Bacterial Pathogen ◽  
Protein Vaccine ◽  
Double Knockout ◽  
Content Type ◽  
Knockout Mutants ◽  
Different Strains

ABSTRACTStreptococcus pneumoniaeis an important human bacterial pathogen, causing such infections as pneumonia, meningitis, septicemia, and otitis media. Current capsular polysaccharide-based conjugate vaccines protect against a fraction of the over 90 serotypes known, whereas vaccines based on conserved pneumococcal proteins are considered promising broad-range alternatives. The pneumococcal genome encodes two conserved proteins of an as yet unknown function, SP1298 and SP2205, classified as DHH (Asp-His-His) subfamily 1 proteins. Here we examined their contribution to pneumococcal pathogenesis using single and double knockout mutants in three different strains: D39, TIGR4, and BHN100. Mutants lacking both SP1298 and SP2205 were severely impaired in adherence to human epithelial Detroit 562 cells. Importantly, the attenuated phenotypes were restored upon genetic complementation of the deleted genes. Single and mixed mouse models of colonization, otitis media, pneumonia, and bacteremia showed that bacterial loads in the nasopharynx, middle ears, lungs, and blood of mice infected with the mutants were significantly reduced from those of wild-type-infected mice, with an apparent additive effect upon deletion of both genes. Minor strain-specific phenotypes were observed, i.e., deletion of SP1298 affected host-cell adherence in BHN100 only, and deletion of SP2205 significantly attenuated virulence in lungs and blood in D39 and BHN100 but not TIGR4. Finally, subcutaneous vaccination with a combination of both DHH subfamily 1 proteins conferred protection to nasopharynx, lungs, and blood of mice infected with TIGR4. We conclude that SP1298 and SP2205 play a significant role at several stages of pneumococcal infection, and importantly, these proteins are potential candidates for a multicomponent protein vaccine.

scholarly journals Efficient Production of Polymyxin in the Surrogate Host Bacillus subtilis by Introducing a ForeignectBGene and Disrupting theabrBGene

2012 ◽  
Vol 78 (12) ◽  
pp. 4194-4199 ◽  
Author(s):  
Soo-Young Park ◽  
Soo-Keun Choi ◽  
Jihoon Kim ◽  
Tae-Kwang Oh ◽  
Seung-Hwan Park
Keyword(s):  
Bacillus Subtilis ◽  
Paenibacillus Polymyxa ◽  
Biosynthetic Gene Cluster ◽  
Upstream Region ◽  
Efficient Production ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Gene Encoding ◽  
Content Type ◽  
Surrogate Host

ABSTRACTIn our previous study,Bacillus subtilisstrain BSK3S, containing a polymyxin biosynthetic gene cluster fromPaenibacillus polymyxa, could produce polymyxin only in the presence of exogenously addedl-2,4-diaminobutyric acid (Dab). The dependence of polymyxin production on exogenous Dab was removed by introducing anectBgene encoding the diaminobutyrate synthase ofP. polymyxainto BSK3S (resulting in strain BSK4). We found, by observing the complete inhibition of polymyxin synthesis when thespo0Agene was knocked out (strain BSK4-0A), that Spo0A is indispensable for the production of polymyxin. Interestingly, theabrB-spo0Adouble-knockout mutant, BSK4-0A-rB, and the singleabrBmutant, BSK4-rB, showed 1.7- and 2.3-fold increases, respectively, in polymyxin production over that of BSK4. These results coincided with the transcription levels ofpmxAin the strains observed by quantitative real-time PCR (qRT-PCR). The AbrB protein was shown to bind directly to the upstream region ofpmxA, indicating that AbrB directly inhibits the transcription of polymyxin biosynthetic genes. The BSK4-rB strain, producing high levels of polymyxin, will be useful for the development and production of novel polymyxin derivatives.

scholarly journals Strain-Specific Regulatory Role of Eukaryote-Like Serine/Threonine Phosphatase in Pneumococcal Adherence

2012 ◽  
Vol 80 (4) ◽  
pp. 1361-1372 ◽  
Author(s):  
Shivangi Agarwal ◽  
Shivani Agarwal ◽  
Preeti Pancholi ◽  
Vijay Pancholi
Keyword(s):  
High Efficiency ◽  
Regulatory System ◽  
Gene Encoding ◽  
Content Type ◽  
Knockout Mutants ◽  
Catalytically Active

ABSTRACTStreptococcus pneumoniaeexploits a battery of virulence factors to colonize the host. Although the eukaryote-like Ser/Thr kinase ofS. pneumoniae(StkP) has been implicated in physiology and virulence, the role of its cotranscribing phosphatase (PhpP) has remained elusive. The construction of nonpolar markerlessphpPknockout mutants (ΔphpP) in two pathogenic strains, D39 (type 2) and 6A-EF3114 (type 6A), indicated that PhpP is not indispensable for pneumococcal survival. Further, PhpP also participates in the regulation of cell wall biosynthesis/division, adherence, and biofilm formation in a strain-specific manner. Additionally, we provide hitherto-unknownin vitroandin vivoevidence of a physiologically relevant biochemical link between the StkP/PhpP-mediated cognate regulation and the two-component regulatory system TCS06 (RR06/HK06) that regulates the expression of the gene encoding an important pneumococcal surface adhesin, CbpA, which was found to be significantly upregulated in ΔphpPmutants. In particular, StkP (threonine)-phosphorylated RR06 bound to thecbpApromoter with high efficiency even in the absence of the HK06-responsive and catalytically active aspartate 51 residue. Together, our findings unravel the significant contributions of PhpP in pneumococcal physiology and adherence.

scholarly journals High-Level Heterologous Production of d-Cycloserine by Escherichia coli

2015 ◽  
Vol 81 (22) ◽  
pp. 7881-7887 ◽  
Author(s):  
Takanori Kumagai ◽  
Tomoki Ozawa ◽  
Momoko Tanimoto ◽  
Masafumi Noda ◽  
Yasuyuki Matoba ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cells ◽  
Resting Cells ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Biosynthetic Genes ◽  
T7 Promoter ◽  
Α Subunit ◽  
Content Type ◽  
E Coli

ABSTRACTPreviously, we successfully cloned ad-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designateddcsAtodcsJ) fromd-CS-producingStreptomyces lavendulaeATCC 11924. In this study, we put fourd-CS biosynthetic genes (dcsC,dcsD,dcsE, anddcsG) in tandem under the control of the T7 promoter in anEscherichia colihost. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. Whenl-serine and hydroxyurea (HU), the precursors ofd-CS, were incubated together with theE. coliresting cell suspension, the cells produced significant amounts ofd-CS (350 ± 20 μM). To increase the productivity ofd-CS, thedcsJgene, which might be responsible for thed-CS excretion, was connected downstream of the four genes. TheE. coliresting cells harboring the five genes producedd-CS at 660 ± 31 μM. ThedcsDgene product, DcsD, formsO-ureido-l-serine fromO-acetyl-l-serine (OAS) and HU, which are intermediates ind-CS biosynthesis. DcsD also catalyzes the formation ofl-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, theE. colichromosomalcysJandcysKgenes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the fourd-CS biosynthetic genes, together withdcsJ, were incubated withl-serine and HU, thed-CS production was 980 ± 57 μM, which is comparable to that ofd-CS-producingS. lavendulaeATCC 11924 (930 ± 36 μM).

scholarly journals A Helicobacter pylori TolC Efflux Pump Confers Resistance to Metronidazole

2005 ◽  
Vol 49 (4) ◽  
pp. 1477-1482 ◽  
Author(s):  
Karin van Amsterdam ◽  
Aldert Bart ◽  
Arie van der Ende
Keyword(s):  
Helicobacter Pylori ◽  
Efflux Pump ◽  
Sodium Deoxycholate ◽  
Loss Of Function ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Active Efflux ◽  
Knockout Mutants ◽  
H Pylori ◽  
Susceptibility Profiles

ABSTRACT In Helicobacter pylori, the contribution of efflux proteins to antibiotic resistance is not well established. As translocases that act in parallel may have overlapping substrate specificities, the loss of function of one such translocase may be compensated for by that of another translocase with no effect on susceptibilities to antibiotics. The genome of H. pylori 26695 was assessed for the presence of putative translocases and outer membrane efflux or TolC-like proteins which could interact to form efflux systems involved in drug resistance. Twenty-seven translocases were identified, of which HP1184 was the sole representative of the multidrug and toxic compound extrusion family of translocases and which could thus have a unique substrate specificity. In addition, four TolC-like proteins (HP0605, HP0971, HP1327, and HP1489) were identified. Thus, it is feasible that inactivation of a TolC-like protein would affect the functions of multiple translocases. We aimed to determine whether efflux systems contribute to antimicrobial susceptibility by evaluation of the susceptibility profiles of an HP1184-knockout mutant, four mutants in which one of the four TolC homologs was inactivated, as well as a mutant in which both HP0605 and HP0971 were inactivated. The HP1184- and HP1489-knockout mutants both showed increased susceptibilities to ethidium bromide, while the HP0605-knockout mutant exhibited increased susceptibilities to novobiocin and sodium deoxycholate. The HP0605 and HP0971 double-knockout mutant was also more susceptible to metronidazole, in addition to being susceptible to novobiocin and sodium deoxycholate. Thus, active efflux is an eminent means of resistance to antimicrobials in H. pylori and resembles the situation in other bacteria.

scholarly journals New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Valmik K. Vyas ◽  
G. Guy Bushkin ◽  
Douglas A. Bernstein ◽  
Matthew A. Getz ◽  
Magdalena Sewastianik ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Genome Engineering ◽  
High Efficiency ◽  
Selection Marker ◽  
Important Species ◽  
Content Type ◽  
Guide Rna ◽  
Wide Range ◽  
Repair Mechanisms ◽  
The Creation

ABSTRACT We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans , Saccharomyces cerevisiae , Candida glabrata , and Naumovozyma castellii . These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms. IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata , as well as in the related yeasts S. cerevisiae and N. castellii . The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

scholarly journals PafR, a Novel Transcription Regulator, Is Important for Pathogenesis in Uropathogenic Escherichia coli

2014 ◽  
Vol 82 (10) ◽  
pp. 4241-4252 ◽  
Author(s):  
Mordechai Baum ◽  
Mobarak Watad ◽  
Sara N. Smith ◽  
Christopher J. Alteri ◽  
Noa Gordon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Gene Knockout ◽  
Sensory System ◽  
Phosphotransferase System ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Content Type

ABSTRACTThemetVgenomic island in the chromosome of uropathogenicEscherichia coli(UPEC) encodes a putative transcription factor and a sugar permease of the phosphotransferase system (PTS), which are predicted to compose a Bgl-like sensory system. The presence of these two genes, hereby termedpafRandpafP, respectively, has been previously shown to correlate with isolates causing clinical syndromes. We show here that deletion of both genes impairs the ability of the resulting mutant to infect the CBA/J mouse model of ascending urinary tract infection compared to that of the parent strain, CFT073. Expressing the two genes intransin the two-gene knockout mutant complemented full virulence. Deletion of either gene individually generated the same phenotype as the double knockout, indicating that bothpafRandpafPare important to pathogenesis. We screened numerous environmental conditions but failed to detect expression from the promoter that precedes thepafgenesin vitro, suggesting that they arein vivoinduced (ivi). Although PafR is shown here to be capable of functioning as a transcriptional antiterminator, its targets in the UPEC genome are not known. Using microarray analysis, we have shown that expression of PafR from a heterologous promoter in CFT073 affects expression of genes related to bacterial virulence, biofilm formation, and metabolism. Expression of PafR also inhibits biofilm formation and motility. Taken together, our results suggest that thepafgenes are implicated in pathogenesis and that PafR controls virulence genes, in particular biofilm formation genes.

scholarly journals Elimination of Manganese(II,III) Oxidation in Pseudomonas putida GB-1 by a Double Knockout of Two Putative Multicopper Oxidase Genes

2012 ◽  
Vol 79 (1) ◽  
pp. 357-366 ◽  
Author(s):  
Kati Geszvain ◽  
James K. McCarthy ◽  
Bradley M. Tebo
Keyword(s):  
Pseudomonas Putida ◽  
Multicopper Oxidase ◽  
Genome Comparison ◽  
Double Knockout ◽  
Oxidase Gene ◽  
Content Type ◽  
Heme Peroxidase ◽  
A Genome ◽  
Oxidation Phenotype ◽  
Encoding Genes

ABSTRACTBacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase inPseudomonas putidaGB-1 has long remained unknown. To identify theP. putidaGB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (mnxG,mofA,moxA, andmopA). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11Pseudomonasspecies. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1_2447, which encodes an MCO homologous to MnxG, and PputGB1_2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1_2447 and PputGB1_2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes.

scholarly journals Dictyostelium discoideum Dgat2 Can Substitute for the Essential Function of Dgat1 in Triglyceride Production but Not in Ether Lipid Synthesis

2014 ◽  
Vol 13 (4) ◽  
pp. 517-526 ◽  
Author(s):  
Xiaoli Du ◽  
Cornelia Herrfurth ◽  
Thomas Gottlieb ◽  
Steffen Kawelke ◽  
Kristin Feussner ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Mammalian Cells ◽  
Lipid Droplets ◽  
Lipid Synthesis ◽  
Ether Lipid ◽  
Natural Food ◽  
Ether Lipids ◽  
Double Knockout ◽  
Content Type ◽  
Knockout Mutants

ABSTRACT Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate.

scholarly journals Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

2015 ◽  
Vol 81 (24) ◽  
pp. 8500-8506 ◽  
Author(s):  
Bo Wang ◽  
Jianping Yu ◽  
Weiwen Zhang ◽  
Deirdre R. Meldrum
Keyword(s):  
High Efficiency ◽  
Transformation Efficiency ◽  
Content Type ◽  
E Coli ◽  
Integrative Transformation ◽  
High Transformation ◽  
Host Genetic ◽  
Foreign Dna ◽  
Encoding Genes ◽  
Pcc 6803

ABSTRACTRestriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium,Synechocystissp. strain PCC 6803 (Synechocystis6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e.,sll0729(geneM) andslr0214(geneC), were cloned from the chromosome ofSynechocystis6803 and expressed inEscherichia coliharboring an integration plasmid. After premethylation treatment inE. coli, the integration plasmid was extracted and used for transformation ofSynechocystis6803. The results showed that although expression of methyltransferase M had little impact on the transformation ofSynechocystis6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation ofSynechocystis6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency inSynechocystis6803. Since premethylating foreign DNA prior to transformingSynechocystisavoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation ofSynechocystis6803.

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