Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant

doi:10.1016/0378-1119(95)00193-A

Gene

Volume 158, Issue 1, 1995, Pages 9-14

https://doi.org/10.1016/0378-1119(95)00193-A Get rights and content

Abstract

Two cassettes with tetracycline-resistance (Tc^R) and kanamycin-resistance (Km^R) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli. In both cassettes, the resistance determinants are flanked by the short direct repeats (FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase. In addition, a plasmid with temperature-sensitive replication for temporal production of the Flp enzyme in E. coli has been constructed. After a gene disruption or deletion mutation is constructed in vitro by insertion of one of the cassettes into a given gene, the mutated gene is transferred to the E. coli chromosome by homologous recombination and selection for the antibiotic resistance provided by the cassette. If desired, the resistance determinant can subsequently be removed from the chromosome in vivo by Flp action, leaving behind a short nucleotide sequence with one FRT site and with no polar effect on downstream genes. This system was applied in the construction of an E. coli endA deletion mutation which can be transduced by P1 to the genetic background of interest using Tc^R as a marker. The transductant can then be freed of the Tc^R if required.

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Gene transfer with subsequent removal of the selection gene from the host genome

Cited by (1493)

COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells
2024, Molecular Cell
Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role but also via the oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis and shed light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.
ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis
2024, mSphere
Gastrointestinal disease caused by Salmonella enterica is associated with the pathogen’s ability to replicate within epithelial cells and macrophages. Upon host cell entry, the bacteria express a type-three secretion system encoded within Salmonella pathogenicity island 2, through which host-manipulating effector proteins are secreted to establish a stable intracellular niche. Transcription of this intracellular virulence program is activated by the PhoPQ two-component system that senses the low pH and the reduced magnesium concentration of host cell vacuoles. In addition to transcriptional control, Salmonella commonly employ RNA-binding proteins (RBPs) and small regulatory RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. ProQ is a globally acting RBP in Salmonella that promotes expression of the intracellular virulence program, but its RNA repertoire has previously been characterized only under standard laboratory growth conditions. Here, we provide a high-resolution ProQ interactome during conditions mimicking the environment of the Salmonella-containing vacuole (SCV), revealing hundreds of previously unknown ProQ binding sites in sRNAs and mRNA 3′UTRs. ProQ positively affected both the levels and the stability of many sRNA ligands, some of which were previously shown to associate with the well-studied and infection-relevant RBP Hfq. We further show that ProQ activates the expression of PhoP at the post-transcriptional level, which, in turn, leads to upregulation of the intracellular virulence program.
Salmonella enterica is a major pathogen responsible for foodborne gastroenteritis, and a leading model organism for genetic and molecular studies of bacterial virulence mechanisms. One key trait of this pathogen is the ability to survive within infected host cells. During infection, the bacteria employ a type three secretion system that deliver effector proteins to target and manipulate host cell processes. The transcriptional regulation of this virulence program is well understood. By contrast, the factors and mechanisms operating at the post-transcriptional level to control virulence gene expression are less clear. In this study, we have charted the global RNA ligand repertoire of the RNA-binding protein ProQ during in vitro conditions mimicking the host cell environment. This identified hundreds of binding sites and revealed ProQ-dependent stabilization of intracellular-specific small RNAs. Importantly, we show that ProQ post-transcriptionally activates the expression of PhoP, a master transcriptional activator of intracellular virulence in Salmonella.
Salmonella enterica is a major pathogen responsible for foodborne gastroenteritis, and a leading model organism for genetic and molecular studies of bacterial virulence mechanisms. One key trait of this pathogen is the ability to survive within infected host cells. During infection, the bacteria employ a type three secretion system that deliver effector proteins to target and manipulate host cell processes. The transcriptional regulation of this virulence program is well understood. By contrast, the factors and mechanisms operating at the post-transcriptional level to control virulence gene expression are less clear. In this study, we have charted the global RNA ligand repertoire of the RNA-binding protein ProQ during in vitro conditions mimicking the host cell environment. This identified hundreds of binding sites and revealed ProQ-dependent stabilization of intracellular-specific small RNAs. Importantly, we show that ProQ post-transcriptionally activates the expression of PhoP, a master transcriptional activator of intracellular virulence in Salmonella.
Efficient production of isobutanol from glycerol in Klebsiella pneumoniae: Regulation of acetohydroxyacid synthase, a rate-limiting enzyme in isobutanol biosynthesis
2024, Renewable Energy
Glycerol is an abundant and inexpensive resource that can be used to produce valuable industrial products. Isobutanol is an important industrial chemical that has been studied for biosynthesis from various carbon sources and microorganisms. So far, isobutanol production by Klebsiella pneumoniae has mainly been studied using glucose. In this study, we produced isobutanol from glycerol based on the K. pneumoniae ΔldhAΔbudA mutant harboring pBR-iBO, which was used in a previous study with K. pneumoniae. We investigated the effect of different acetohydroxyacid synthase (AHAS) isoenzymes (rate-limiting enzymes in isobutanol biosynthesis), plasmid copy number, and different promoters as a method to increase isobutanol production through regulation at gene expression level. The K. pneumoniae Cu ΔldhAΔbudA, pUC-tac-BN-ISO strain produced 2.56-fold more isobutanol than reported previously for glycerol-derived isobutanol production. Additionally, the in vitro enzyme activity of AHAS I (ilvBN) was greater than that of the other two isoenzymes (ilvIH and ilvGM). The evaluation of process factors indicated that an agitation speed of 200 rpm with the culture maintained at pH 6.0 was a favorable condition for isobutanol production (1.02 g/L). Our results demonstrated enhanced production of isobutanol from glycerol and provide insights for future research on isobutanol production from renewable feedstock.
The virulence regulator AbsR in avian pathogenic Escherichia coli has pleiotropic effects on bacterial physiology
2024, Journal of Integrative Agriculture
Avian pathogenic Escherichia coli (APEC) belonging to extraintestinal pathogenic E. coli (ExPEC) can cause severe infections in extraintestinal tissues in birds and humans, such as the lungs and blood. MprA (microcin production regulation, locus A, herein renamed AbsR, a blood survival regulator), a member of the MarR (multiple antibiotic resistance regulator) transcriptional regulator family, governs the expression of capsule biosynthetic genes in human ExPEC and represents a promising druggable target for antimicrobials. However, a deep understanding of the AbsR regulatory mechanism as well as its regulon is lacking. In this study, we present a systems-level analysis of the APEC AbsR regulon using ChIP-Seq (chromatin immunoprecipitation sequencing) and RNA-Seq (RNA sequencing) methods. We found that AbsR directly regulates 99 genes and indirectly regulates 667 genes. Furthermore, we showed that: 1) AbsR contributes to antiphagocytotic effects by macrophages and virulence in a mouse model for systemic infection by directly activating the capsular gene cluster; 2) AbsR positively impacts biofilm formation via direct regulation of the T2SS (type II secretion system) but plays a marginal role in virulence; and 3) AbsR directly upregulates the acid tolerance signaling system EvgAS to withstand acid stress but is dispensable in ExPEC virulence. Finally, our data indicate that the role of AbsR in virulence gene regulation is relatively conserved in ExPEC strains. Altogether, this study provides a comprehensive analysis of the AbsR regulon and regulatory mechanism, and our data suggest that AbsR likely influences virulence primarily through the control of capsule production. Interestingly, we found that AbsR severely represses the expression of the type I-F CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) systems, which could have implications in CRISPR biology and application.
Salmonella Gallinarum mgtC mutant shows a delayed fowl typhoid progression in chicken
2024, Gene
Salmonella Gallinarum (SG) provokes fowl typhoid, an infectious disease of acute clinical course that affects gallinaceous of any age and leads to high mortality rates. During the typhoid-like systemic infection of S. Typhimurium (STM) in mice, the bacterium expresses the mgtC gene, which is encoded in the Salmonella Pathogenecity Island – 3 (SPI-3). In this serovar, the function is linked to bacterial replication within macrophages, and its absence attenuates the pathogen. We hypothesized that deleting mgtC from SG genome would alter the microorganism pathogenicity in susceptible commercial poultry in a similar manner. Thus, the present study sought to elucidate the importance of mgtC on SG pathogenicity. For this, a mgtC-mutant lacking S. Gallinarum mutant was constructed (SG ΔmgtC). Its ability to replicate in medium that mimicries the mgtC-related intracellular environment of macrophages as well as in primary macrophages from chicken was evaluated. Moreover, the infection of susceptible chickens was performed to elucidate its pathogenicity and the elicited immune responses by measuring key interleukins by qRT-PCR and the population of macrophages and lymphocytes T CD4⁺ and CD8⁺ by means of immunohistochemistry. It was observed that mgtC was required for S. Gallinarum replication in acidified low-Mg²⁺ media and survival within macrophages. However, unlike its requirement for initial phase of STM infection in mice, lower bacterial counts were only observed at the late stage of macrophage infection without affecting the citotoxicity. Experiments showed that knocking-out the mgtC gene neither altered bacterial uptake by macrophages nor affects bacterial counts in liver and spleen and total chicken mortality. However, plotting a survival curve and analyzing the clinical-pathologic conditions, it was observed a slower progression of the disease in chickens infected by SG ΔmgtC compared to those challenged by the wild-type strain. Furthermore, the mRNA expression of IFN-γ and LITAF were similar between the infected chickens, but higher than in the uninfected group. The same was observed in macrophages and lymphocytes T CD4⁺ populations. On the other hand, the presence of lymphocytes T CD8⁺ was increased in the initial phase of the disease provoked by the wild-type strain over the mutant strain. We concluded that the role of mgtC in Fowl Typhoid in susceptible chickens differs from the role in typhoid-like infections in mammals. Thus, the deletion of mgtC gene from S. Gallinarum genome does not affect the overall pathogenicity, but slightly alters the pathogenesis.
Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG
2024, Journal of Biological Chemistry
Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a β-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin β-lactone warheads.

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Gene

Abstract

References (24)

Precise deletions in large bacterial genomes by vector-mediated excision (VEX)

J. Mol. Biol.

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

Gene

Endonuclease I-deficient and ribonuclease-deficient Escherichia coli

J. Mol. Biol.

The transposon Tn5 carries a bleomycin-resistance determinant

Gene

Specific-purpose plasmid cloning vectors, I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors

Gene

The periplasmic endonuclease I of Escherichia coli has amino acid sequence homology to the extracellular DNases of Vibrio cholerae and Aeromonas hydrophila

Gene

Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activated att-nutL-p-att-N module

Gene

A multifunctional gene (tetR) controls Tn10-encoded tetracycline resistance

J. Bacteriol.

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the pA15 cryptic miniplasmid

J. Bacteriol.

The FLP protein of the yeast 2-μ plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli

FLP site-specific recombination system of Saccharomyces cerevisiae

Gene transfer with subsequent removal of the selection gene from the host genome

Cited by (1493)

COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells

ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis

Efficient production of isobutanol from glycerol in Klebsiella pneumoniae: Regulation of acetohydroxyacid synthase, a rate-limiting enzyme in isobutanol biosynthesis

The virulence regulator AbsR in avian pathogenic Escherichia coli has pleiotropic effects on bacterial physiology

Salmonella Gallinarum mgtC mutant shows a delayed fowl typhoid progression in chicken

Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG