Construction of new β-glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacement

doi:10.1016/0378-1119(93)90691-U

Gene

Volume 129, Issue 1, 15 July 1993, Pages 17-25

https://doi.org/10.1016/0378-1119(93)90691-U Get rights and content

Summary

Five cassettes carrying uidA, encoding β-glucuronidase, were made for the construction of insertion mutants with transcriptional fusions to uidA. Three uidA cassettes contain antibiotic-resistance genes, for chloramphenicol (Cm), for kanamycin (Km) and neomycin (Nm), or for streptomycin (Sm) and spectinomycin (Sp). Some cause polar insertions while others provide a promoter for downstream gene expression. The expression of these uidA cassettes was compared to the expression of lacZ at the same site in phnD, a phosphate-regulated gene for phosphonate use. Several phn::uidA or phn::lacZ insertions were recombined onto the chromosome to test mutational effects and to measure gene expression in single copy. This was done using one of three methods for allele replacement. A new method involved recombination of mutations in M 13 onto the chromosome by infection of an Escherichia coli rep mutant that fails to propagate single-stranded DNA phages. Merodiploid recombinants were selected using a resistance marker carried by the M 13 phage; segregants lacking M 13 sequences were then selected as deoxycholate-resistant (Doc^R) ones. An improved method for recombination of mutations in pir-dependent, ori_R6K vectors involved the use of plasmids containing genes for tetracycline resistance (Tc^R). Merodiploid recombinants were selected by conjugative transfer of such plasmids into a recipient lacking pir (encoding the n protein of the R6K plasmid); segregants lacking vector sequences were subsequently selected as Tc-sensitive ones. Both procedures are efficient and allow for recombining marked as well as unmarked mutations onto the chromosome. In addition, some insertions with an antibiotic-resistance marker were directly recombined onto the chromosome by transformation of a recD mutant with linear DNA. New E. coli mutants deleted for uidA, lacZ and phoA are also described, including ones that allow for the simultaneous use of multiple reporter genes.

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The solute binding proteins (SBPs) of prokaryotes are present in the extracytosolic space. Although their primary function is providing substrates to transporters, SBPs also stimulate different signaling proteins, including chemoreceptors, sensor kinases, diguanylate cyclases/phosphodiesterases and Ser/Thr kinases, thereby causing a wide range of responses. While relatively few such systems have been identified, several pieces of evidence suggest that SBP-mediated receptor activation is a widespread mechanism. (1) These systems have been identified in Gram-positive and Gram-negative bacteria and archaea. (2) There is a structural diversity in the receptor domains that bind SBPs. (3) SBPs belonging to thirteen different families interact with receptor ligand binding domains (LBDs). (4) For the two most abundant receptor LBD families, dCache and four-helix-bundle, there are different modes of interaction with SBPs. (5) SBP-stimulated receptors carry out many different functions. The advantage of SBP-mediated receptor stimulation is attributed to a strict control of SBP levels, which allows a precise adjustment of the systeḿs sensitivity. We have compiled information on the effect of ligands on the transcript/protein levels of their cognate SBPs. In 87 % of the cases analysed, ligands altered SBP expression levels. The nature of the regulatory effect depended on the ligand family. Whereas inorganic ligands typically downregulate SBP expression, an upregulation was observed in response to most sugars and organic acids. A major unknown is the role that SBPs play in signaling and in receptor stimulation. This review attempts to summarize what is known and to present new information to narrow this gap in knowledge.
Labeled Azospirillum brasilense wild type and excretion-ammonium strains in association with barley roots
2017, Plant Physiology and Biochemistry
Citation Excerpt :
Both A. brasilense strains were used to construct mutants which carry a chromosomal PnifH-gusA fusion. The plasmid containing the PnifH-gusA fusion was constructed using the plasmids pSUP202::nifHDK of A. brasilense (Cbr Cmr Tcr; Souza, E. M.) and pWM6 (Metcalf and Wanner, 1993). The plasmid containing the structural genes of nitrogenase has two sites for the enzyme SacI inserted into the nifH gene and pWM6 plasmid releases the promoterless gusA-kanamycin (gusA-Km) cassette when treated with the same enzyme.
Soil bacteria colonization in plants is a complex process, which involves interaction between many bacterial characters and plant responses. In this work, we labeled Azospirillum brasilense FP2 (wild type) and HM053 (excretion-ammonium) strains by insertion of the reporter gene gusA-kanamycin into the dinitrogenase reductase coding gene, nifH, and evaluated bacteria colonization in barley (Hordeum vulgare). In addition, we determined inoculation effect based on growth promotion parameters. We report an uncommon endophytic behavior of A. brasilense Sp7 derivative inside the root hair cells of barley and highlight the promising use of A. brasilense HM053 as plant growth-promoting bacterium.
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We report the construction of a tetracycline inducible expression vector that allows regulated gene expression in the enteric pathogen Vibrio cholerae. The expression vector, named pXB300, contains the tetracycline regulatory elements from Tn10, a multiple cloning site downstream of the tetA promoter and operator sequences, a ColE1 origin of replication, a β-lactamase resistance gene for positive selection, and the hok/sok addiction system for selection in the absence of antibiotic. The function of the tetracycline expression system was demonstrated by cloning lacZ under control of the tetA promoter and quantifying β-galactosidase expression in Escherichia coli and V. cholerae. The utility for pXB300 was documented by complementation of V. cholerae virulence mutants during growth under virulence inducing conditions. The results showed that pXB300 allowed high-level expression of recombinant genes with linear induction in response to the exogenous concentration of the inducer anhydrotetracycline. We further show that pXB300 was reliably maintained in V. cholerae during growth in the absence of antibiotic selection.
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Citation Excerpt :
Chromosomal DNA from P. syringae strains was obtained as previously described (Chen and Kuo, 1993). The PCR-derived amplicon containing the gacA open reading frame was cloned into pUC19 (61) and interrupted at the NaeI site by introduction of the SmaI fragment from pWM6 containing uidA-aph genes (Metcalf and Wanner, 1993). Similarly, gacS was cloned in pCR4.0-TOPO (Invitrogen) and mutated by insertion of a PvuII fragment containing a tetracycline resistance cassette into a unique StuI site located within the gacS coding region.
Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by watersoaked lesions surrounded by a chlorotic halo resulting from the action of a non-host specific toxin known as phaseolotoxin. This toxin inhibits the enzyme ornithine carbamoyltransferase involved in the arginine biosynthesis pathway. It was previously reported that genes within the Pht cluster were involved in the regulation and synthesis of phaseolotoxin. The GacS/GacA two-component signal transduction system controls important pathogenicity and virulence mechanisms in several Gram-negative bacteria. Tox⁻ phenotype gacA⁻ and gacS⁻ mutants were obtained and gacA⁻ transcriptome analysis revealed that this response activator controls expression of genes within the Pht cluster as well as another gene located in a different region in the bacterial chromosome and that has been unambiguously shown to be directly involved in phaseolotoxin biosynthesis. Results presented in this work suggest that phaseolotoxin biosynthesis involve elements within and outside the Pht Cluster, and that the GacS/GacA two-component system exerts control over them.
Suicide vectors for the introduction of genetic markers into Bradyrhizobium strains by site-directed chromosomal integration between repeated sequences (RS-α)
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To achieve stable expression of the heterologous and reporter genes in the Bradyrhizobium genome, we constructed suicide plasmids capable of site-directed genomic integration of the gusA, gfp and nifA genes by homologous recombination into non-essential repeated sequences (RS-α), isolated from B. japonicum strain CPAC7 (SEMIA5080). In this report, we describe the strategies to construct the vectors and their use to obtain mutants with site-specific insertions.
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Summary

References (33)

Molecular biology of carbon-phosphorus bond cleavage: cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli B

J. Biol. Chem.

Evidence for a fourteen-gene, phnC to phnP, locus for phosphonate metabolism Escherichia coli

Gene

Plasmid vectors for selecting IS1-promoted deletions in cloned DNA: sequence analysis of the omega interposon

Gene

Phosphate-controlled gene expression in Escherichia coli using Mu dl-directed lacZ fusions

J. Mol. Biol.

'uidA-antibiotic-resistance cassettes for insertion mutagenesis, gene fusions and genetic constructions

FEMS Microbiol. Lett.

Gene replacement and retrieval with recombinant M13mp bacterio-phages

J. Bacteriol.

Positive selection for loss of tetracycline resistance

J. Bacteriol.

Cloning, mapping, and expression of the fumarase gene of Escherichia coli K-12

J. Bacteriol.

The fumarase genes of Escherichia coli: location of the fumB gene and discovery of a new gene (fumC)

J. Gen. Microbiol.

Replacement and amplification of bacterial genes with sequences altered in vitro

Beta-glucuronidase from Escherichia coli as a gene-fusion marker

Location on the chromosome of Escherichia coli of genes governing purine metabolism

Mol. Gen. Genet.

Hybrid pUC vectors for addition of new restriction enzyme sites to the ends of DNA fragments

Nucleic Acids Res.

Genetic evidence for a repressor of synthesis of cytosine deaminase and purine biosynthesis enzymes in Escherichia coli

J. Bacteriol.

Evidence for two phosphonate degradative pathways in Enterobacter aerogenes

J. Bacteriol.

Cited by (79)

The role of solute binding proteins in signal transduction

Labeled Azospirillum brasilense wild type and excretion-ammonium strains in association with barley roots

Construction of a tetracycline inducible expression vector and characterization of its use in Vibrio cholerae

Gene expression of Pht cluster genes and a putative non-ribosomal peptide synthetase required for phaseolotoxin production is regulated by GacS/GacA in Pseudomonas syringae pv. phaseolicola

Suicide vectors for the introduction of genetic markers into Bradyrhizobium strains by site-directed chromosomal integration between repeated sequences (RS-α)

Rotation of the Fla2 flagella of Cereibacter sphaeroides requires the periplasmic proteins MotK and MotE that interact with the flagellar stator protein MotB2