Complementation Vector Research Articles

Genetic Manipulation of Klebsiella pneumoniae.

Klebsiella pneumoniae is a Gram-negative, rod-shaped bacterium commonly found in the human intestine. Although it typically exists as part of the normal flora, it can also cause healthcare-associated infections with severe consequences. Understanding the specific genes responsible for its virulence through genetic manipulation is crucial for potential therapeutic interventions. However, manipulating K. pneumoniae presents challenges due to its exopolysaccharide capsule. This article presents a comprehensive collection of protocols designed to facilitate the genetic manipulation of K. pneumoniae. By following these protocols, researchers will acquire the necessary skills to prepare electrocompetent cells, utilize electroporation for efficient plasmid DNA introduction, construct isogenic mutants using the λ Red recombinase system, and generate a complementation vector for restoring the phenotypic traits of knockout strains. These protocols provide valuable tools and techniques to navigate the intricacies associated with studying and modifying K. pneumoniae. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing electrocompetent K. pneumoniae cells Alternate Protocol 1: Preparing electrocompetent K. pneumoniae cells for recombineering Basic Protocol 2: Transforming K. pneumoniae using electroporation Basic Protocol 3: Constructing isogenic mutants in K. pneumoniae using the λ Red recombinase system Support Protocol 1: Confirming a knockout via colony PCR Support Protocol 2: Verifying absence of secondary mutations Basic Protocol 4: Generating unmarked knockout mutants in K. pneumoniae using the pFLP plasmid Basic Protocol 5: Constructing a complementation vector for K. pneumoniae.

Swnk plays an important role in the biosynthesis of swainsonine in Metarhizium anisopliae.

Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by multiple fungi. A key enzyme in the SW synthesis pathway is a hybrid swnk/nrps. To analyze the role of swnk in the SW biosynthesis pathway of Metarhizium anisopliae. The concentration of SW and the swnk expression in M. anisopliae fermentation from 1st to 7th day were determined using LC-MS and RT-qPCR, respectively. M. anisopliae had the highest SW content and swnk expression on the 5th day of fermentation; Mutant strain (MT) were obtained by PEG-mediated homologous recombination (HR) which knocked out swnk in the wild-type (WT) strain. Complemented-type (CT) strain were obtained by transforming a modified PUC19 complementation vector containing the geneticin (G418) resistance gene and swnK. SW was not detected in the MT strain and reverted to its original level in the CT strain; A Psilent-1 plasmid with Benomyl (ben)-resistant that was used interfered with swnk of WT strain. The level of SW was markedly diminished in the RNAi strain. RNAi of swnk affects the formation of the cell wall in M. anisopliae. These results indicate that swnk plays a crucial role in the SW biosynthesis of M. anisopliae.

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase–related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid–containing peptides and other periplasmic biomolecules in manifold pathways.

SCL, Encoding a Chloroplast Signal Recognition Particle Receptor, Affects Chlorophyll Synthesis and Chloroplast Development in Rice

Crop yield is largely determined by the solar energy utilization efficiency of photosynthesis; plants with long stay-green periods have greater total photosynthetic production levels and crop yields. Here, a novel seedling chlorosis and lethality (scl) mutant exhibiting a yellow leaf and seedling-lethal phenotype was identified in rice (Oryza sativa L.). The mutant had deformed chloroplasts and almost no protein complexes in thylakoid membranes. The expression levels of photosynthesis-associated genes were significantly down-regulated in scl compared with the wild-type (WT). Positive transgenic lines generated by Agrobacterium tumefaciens-mediated transformation of the scl mutant with a complementation vector harboring SCL cDNA exhibited the normal green leaf phenotype, whereas the scl seedling harboring the empty vector displayed the yellow leaf phenotype, indicating that SCL is LOC_Os01g72800. A fusion protein expressing SCL with green fluorescent protein revealed the fluorescence signal localized to chloroplasts. The expression patterns of chloroplast development and chlorophyll biosynthesis and degradation-related genes were disordered in scl mutant, possibly resulting in the yellow leaf phenotype. These results indicated that the SCL loss of function impaired chloroplast development, chlorophyll biosynthesis, and light-harvesting chlorophyll-binding protein transportation in rice.

The β-galactosidase LacLM plays the major role in lactose utilization of Lactiplantibacillus plantarum

Lactiplantibacillus plantarum has often been used for fermentation of lactose-rich substrates. However, its growth in lactose has not been studied at the molecular level and the role of different β-galactosidases is also unknown. In this study, transcriptome sequencing was performed to reveal the difference of gene expression when Lp. plantarum LP01 was cultivated in glucose and lactose medium. Two genetic loci involved in lactose metabolism were highly transcripted by lactose induction, in which β-galactosidase coding gene lacA, lacL and lacM were up-regulated by 9.86, 9.12 and 9.53-fold, respectively. To explore the detail role of different β-galactosidase, mutant strains Lp. plantarum ΔlacA, Lp. plantarum ΔlacLM and Lp. Plantarum ΔlacAΔlacLM was constructed by gene knockout. Results showed that the growth of Lp. plantarum ΔlacA was slightly weakened in lactose medium, while the growth of Lp. plantarum ΔlacLM and Lp. plantarum ΔlacAΔlacLM was completely inhibited. Furthermore, the growth of Lp. plantarum ΔlacLM could be restored after transformed into an complementation vector pNZ8148-PlacLM-lacLM. Therefore, the β-galactosidase LacLM plays the major role in lactose utilization of Lp. plantarum, providing a theoretical foundation for the growth and metabolism of Lp. plantarum in lactose medium.

A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum

The recent availability of genome editing tools like TALEN (Transcription activator-like effector nuclease) and CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) for the diatom Phaeodactylum tricornutum has dramatically increased the options to explore diatom biology via reverse genetics. In order to verify that an observed phenotype indeed is directly related to a specific gene knockout and not due to a secondary effect, complementation of the inactivated gene with the wildtype gene and restoration of the wild type phenotype is an essential tool in molecular biology. So far, no strategy for a complementation method has been published for P. tricornutum. Here we demonstrate, as a proof-of principle, the complementation of P. tricornutum AUREO1a knockout strains previously created by TALEN technology. These strains are deficient in the PtAureo1a gene, which is encoding a blue-light dependent transcription factor. pPTbsr, a modified pPha-T1 vector with an antibiotic resistance cassette against Blasticidin served as a complementation vector. In order to avoid the modification of the complementing gene via the potentially still active TALEN nucleases, we have modified the TALEN binding sites of the complementing PtAureo1a gene using synonymous codons. The altered PtAureo1a gene along with its native promoter and terminator was transformed by particle gun bombardment into PtAUREO1a TALEN knockout strains of P. tricornutum. The integration and the expression of PtAUREO1a was confirmed by PCR and western blotting. Due to random integration within the genome, the expression level of the complemented gene may be variable in different lines. Physiological parameters indicated the successful rescue of the wild type phenotype in several lines that showed a similar PtAUREO1a protein content as wild type cells. Our method provides a rapid and efficient tool to complement knockout lines generated by genome editing approaches in P. tricornutum.

Lighting up my life: a LOV-based fluorescent reporter for Campylobacter jejuni

In this study, a LOV-based fluorescent reporter (light, oxygen, or voltage-sensing domains of phototropin), termed iLOV, was adapted for Campylobacter jejuni and used to investigate promoter activity via monitoring fluorescence intensity and to study the localisation of two chemotaxis proteins. The pC46 complementation vector contains coding sequence from cj0046, a C. jejuni NCTC11168 pseudo-gene and is used to integrate cloned genes onto the C. jejuni chromosome. The pC46 vector was used to construct plasmids containing iLOV, driven by three different C. jejuni constitutive promoters and plasmids containing transcriptional fusions of the iLOV reporter and two chemoreceptors, tlp5 and tlp8. Expression from the porA promoter, pporA, produced the highest fluorescence signals compared to pfdxA (intermediate level) and pmetK (lowest level). The cellular localisation pattern of transducer-like protein (Tlp) clusters, containing Tlp5 and Tlp8, was predominately polar, with Tlp5 positioned only at one and Tlp8 at both poles. Here, we demonstrate that a iLOV fluorescent reporter can be used as a promoter probe or as a gene fusion reporter in Campylobacter spp. This is a new system uniquely placed for studying Campylobacter spp., as it combines resistance to photobleaching and functionality under microaerobic conditions.

Disruption of the pdhB pyruvate dehydrogenase [corrected] gene affects colony morphology, in vitro growth and cell invasiveness of Mycoplasma agalactiae.

The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae.

A Protoplast Transformation System for Gene Deletion and Complementation in Sclerotinia sclerotiorum

AbstractProtoplast transformation is an important technique for establishing a mutation library and determining gene function for Sclerotinia sclerotiorum and other plant pathogenic fungi. In this study, we determined the effect of various conditions on preparation of protoplasts for transformation. These conditions included the age of the culture providing the hyphae to be digested; enzyme composition, buffer solution and concentration; and digestion time and temperature. The optimum conditions for preparing protoplasts were as follows: 10 ml of enzyme solution (1.5% lysing enzyme in 0.8 m mannitol and citric acid‐sodium citrate buffer) reacting with 0.1 g of hyphae (cultured for 36 h) at 30°C for 2.5 h. The transformation efficiency was 60–85 transformants per microgram of DNA. In addition, an expression vector for gene complementation was constructed, and an additional dominant selectable marker (neomycin) was demonstrated. To verify the reliability of the expression vector, we constructed and transformed the complementation vector of Shk1 for gene complementation based on the Shk1 deletion mutant △Shk1. The results showed that the expression level and biological phenotypes of Shk1 were restored in the complementary strain △Shk1+Shk1. The techniques and procedures described will improve our ability to study gene function in S. sclerotiorum and are likely applicable to other plant pathogens.

ALK, The Key Gene for Gelatinization Temperature, is a Modifier Gene for Gel Consistency in Rice (Oryza sativa L.)

Gelatinization temperature (GT) is an important parameter in evaluating the cooking and eating quality of rice. Indeed, the phenotype, biochemistry and inheritance of GT have been widely studied in recent times. Previous map-based cloning revealed that GT was controlled by ALK gene, which encodes a putative soluble starch synthase II-3. Complementation vector and RNAi vector were constructed and transformed into Nipponbare mediated by Agrobacterium. Phenotypic and molecular analyses of transgenic lines provided direct evidence for ALK as a key gene for GT. Meanwhile, amylose content, gel consistency and pasting properties were also affected in transgenic lines. Two of four nonsynonymous single nucleotide polymorphisms in coding sequence of ALK were identified as essential for GT. Based on the single nucleotide polymorphisms (SNPs), two new sets of SNP markers combined with one cleaved amplified polymorphic sequence marker were developed for application in rice quality breeding.

Static Test Data Volume Reduction Using Complementation or Modulo-$M$ Addition

Both test compaction and test data compression methods provide an opportunity for a tester to apply modified versions of each test, in addition to the original test. We take advantage of this opportunity to achieve additional test data volume reductions. One way to modify a test is to complement some or all of its bits. We represent the way in which modified tests will be obtained by a complementation vector. Experimental results demonstrate that, even when a test set has minimum or close-to-minimum size, the use of a complementation vector allows us to reduce the size of the stored test set further, and almost always below the known lower bound on the size of a test set. The use of a complementation vector is equivalent to a modulo-2 addition operation. We generalize it to modulo- <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">M</i> addition, for a constant <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">M</i> ≥ 2. With modulo- <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">M</i> addition, each stored test yields up to <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">M</i> tests. It is thus possible to reduce the size of the stored test set even further.

Construction of an unmarked recombinant BCG expressing a pertussis antigen by auxotrophic complementation: Protection against Bordetella pertussis challenge in neonates

Mycobacterium bovis BCG has long been investigated as a candidate for heterologous antigen presentation. We have previously described an rBCG-Pertussis that confers protection against challenge with Bordetella pertussis in neonate and adult mice. In order to obtain stable expression in vivo, we constructed an unmarked BCG lysine auxotrophic and a complementation vector containing the lysine and the genetically detoxified S1 pertussis toxin genes, both under control of the same promoter. Complemented BCG-Δlysine growth and expression of the pertussis antigen were stable, without the use of an antibiotic marker. Our results show that the complemented rBCG-ΔlysA-S1PT-lysA +(kan −), which is now suitable to be evaluated in clinical trials, maintains similar characteristics of the original rBCG-pNL71S1PT strain, such as the antigen expression level, cellular immune response and protection against the same model challenge in neonatal-immunized mice.

Expanding the Helicobacter pylori Genetic Toolbox: Modification of an Endogenous Plasmid for Use as a Transcriptional Reporter and Complementation Vector

Helicobacter pylori is an important human pathogen. However, the study of this organism is often limited by a relative shortage of genetic tools. In an effort to expand the methods available for genetic study, an endogenous H. pylori plasmid was modified for use as a transcriptional reporter and as a complementation vector. This was accomplished by addition of an Escherichia coli origin of replication, a kanamycin resistance cassette, a promoterless gfpmut3 gene, and a functional multiple cloning site to form pTM117. The promoters of amiE and pfr, two well-characterized Fur-regulated promoters, were fused to the promoterless gfpmut3, and green fluorescent protein (GFP) expression of the fusions in wild-type and delta fur strains was analyzed by flow cytometry under iron-replete and iron-depleted conditions. GFP expression was altered as expected based on current knowledge of Fur regulation of these promoters. RNase protection assays were used to determine the ability of this plasmid to serve as a complementation vector by analyzing amiE, pfr, and fur expression in wild-type and delta fur strains carrying a wild-type copy of fur on the plasmid. Proper regulation of these genes was restored in the delta fur background under high- and low-iron conditions, signifying complementation of both iron-bound and apo Fur regulation. These studies show the potential of pTM117 as a molecular tool for genetic analysis of H. pylori.

In vivo characterization of Escherichia coli ftsZ mutants: effects on Z-ring structure and function.

We have characterized the in vivo phenotypes of 17 mutations of Escherichia coli ftsZ. In particular, we determined whether these mutations can complement a null ftsZ phenotype, and we demonstrated that two noncomplementing mutations show partial dominant-negative behavior. We performed immunofluorescence microscopy to determine whether these mutants could assemble into normal or abnormal structures in vivo. The mutants separated into four classes-those that complemented the null and formed normal FtsZ rings, those that complemented the null but formed aberrant FtsZ structures, those that formed aberrant FtsZ structures and did not complement, and those that were unable to form any FtsZ structures. We did not find any mutations that produced nonfunctional Z rings of normal appearance. Surprisingly, some mutants that produced extensively spiraled Z-ring structures divided and grew with a normal doubling time. The analysis was carried out using a complementation system based on an ftsZ deletion strain, a temperature-sensitive rescue plasmid, and a complementation vector that placed mutated ftsZ alleles under the control of the pBAD promoter, which offered several advantages over previous systems.