2-keto-3-deoxygluconate transport system in Erwinia chrysanthemi.

In Erwinia chrysanthemi, the gene kdgT encodes a transport system responsible for the uptake of ketodeoxyuronates. We studied the biochemical properties of this transport system. The bacteria could grow on 2,5-diketo-3-deoxygluconate but not on 2-keto-3-deoxygluconate. The 2-keto-3-deoxygluconate entry reaction displayed saturation kinetics, with an apparent Km of 0.52 mM (at 30 degrees C and pH 7). 5-Keto-4-deoxyuronate and 2,5-diketo-3-deoxygluconate appeared to be competitive inhibitors, with Kis of 0.11 and 0.06 mM, respectively. The 2-keto-3-deoxygluconate permease could mediate the uptake of glucuronate with a low affinity. kdgT was cloned on an R-prime plasmid formed by in vivo complementation of a kdgT mutation of Escherichia coli. After being subcloned, it was mutagenized with a mini-Mu-lac transposable element able to form fusions with the lacZ gene. We introduced a kdgT-lac fusion into the E. chrysanthemi chromosome by marker exchange recombination and studied its regulation. kdgT product synthesis was not induced by external 2-keto-3-deoxygluconate in the wild-type strain but was induced by galacturonate and polygalacturonate. Two types of regulatory mutants able to grow on 2-keto-3-deoxygluconate as the sole carbon source were studied. Mutants of one group had a mutation in the operator region of kdgT; mutants of the other group had a mutation in kdgR, a regulatory gene controlling kdgT expression.     

Anaerobiosis induces expression of ant, a new Escherichia coli locus with a role in anaerobic electron transport

Escherichia coli has a formate hydrogenlyase system which allows it to maintain an electron balance during anaerobic growth by passing electrons from formate to H+ ions, thus generating H2. The Mu d1(Ap lac) bacteriophage was used to generate mutants that were defective in passing electrons from formate to benzyl viologen, an artificial electron acceptor. A subset of these mutants was studied in which beta-galactosidase was expressed at much higher levels under anaerobic conditions than under aerobic conditions. If nitrate was present during anaerobic growth, the same levels of beta-galactosidase were seen in these fusion strains as were seen under aerobic conditions. The Mu d1(Ap lac) insertions in these mutants were genetically mapped between mutS and srl and thus define a new locus we have termed ant (anaerobic electron transport). Recombinant lambda derivatives were isolated which complemented the deficiency of the ant mutants in anaerobic electron transport and also carried a trans-acting region of DNA which reduced expression of the ant-lac fusions under anaerobic conditions; a probe to the ant region was generated from one of these recombinant lambda derivatives. Southern hybridization analysis revealed that the four independent ant::Mu d1(Ap lac) fusions we isolated spanned an approximately 5-kilobase region and that all were transcribed in the same direction, counterclockwise on the E. coli genetic map.     

Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions.

Fusions of the lac genes to the promoters of four structural genes in the methionine biosynthetic pathway, metA, metB, metE, and metF, were obtained by the use of the Mu d(Ap lac) bacteriophage. The levels of beta-galactosidase in these strains could be derepressed by growth under methionine-limiting conditions. Furthermore, growth in the presence of vitamin B12 repressed the synthesis of beta-galactosidase in strains containing a fusion of lacZ to the metE promoter, phi(metE'-lacZ+). Mutations affecting the regulation of met-lac fusions were generated by the insertion of Tn5. Tn5 insertions were obtained at the known regulatory loci metJ and metK. Interestingly, a significant amount of methionine adenosyltransferase activity remained in the metK mutant despite the fact that the mutation was generated by an insertion. Several Tn5-induced regulatory mutations were isolated by screening for high-level beta-galactosidase expression in a phi(metE'-lacZ+) strain in the presence of vitamin B12. Tn5 insertions mapping at the btuB (B12 uptake), metH (B12 dependent tetrahydropteroylglutamate methyltransferase), and metF (5,10-methylenetetrahydrofolate reductase) loci were obtained. The isolation of the metH mutant was consistent with previous suggestions that the metH gene product is required for the repression of metE by vitamin B12. The metF::Tn5 insertion was of particular interest since it suggested that a functional metf gene product was also needed for repression of metE by vitamin B12.     

Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature.

We describe defective Mu phage Mu dX (Mu d1 Bx::Tn9 [lac Apr Cmr]) which is useful for insertion mutagenesis and for construction of lac operon fusions in vivo. Mu dX retains the insertion properties of Mu d1 but produces temperature-resistant lysogens and transposes at a reduced frequency. A method is described to convert existing Mu d1 insertions to Mu dX.     

Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant

We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease.     

Effects of glnL and other regulatory loci on regulation of transcription of glnA-lacZ fusions in Klebsiella aerogenes

Mutants of Klebsiella aerogenes containing genetic fusions of glnA to lacZ were isolated by using Mu dl (lac, bla) bacteriophage and a Mu Kmr helper phage with the host range of bacteriophage P1. Synthesis of beta-galactosidase in these strains is regulated in response to nitrogen metabolites and regulatory gln loci and is rendered constitutive by a mutation in the linked glnL gene. Complementation studies indicated that glnL is a separate locus from glnA and glnG and that insertions in glnA are partially polar on glnL expression. These results support the hypothesis that glnA, glnL, and glnG are organized in an operon with multiple promoters.     

Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions.

A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.     

Transposition of lambda placMu is mediated by the A protein altered at its carboxy-terminal end

Lambda placMu phages are derivatives of bacteriophage lambda that use the transposition machinery of phage Mu to insert into chromosomal and cloned genes. When inserted in the proper fashion, these phages yield stable fusions to the Escherichia coli lac operon in a single step. We have determined the amount of DNA from the c end of phage Mu present in one of these phages, lambda placMu3, and have shown that this phage carries a 3137-bp fragment of Mu DNA. This DNA segment carries the Mu c-end attachment site and encodes the Mu genes cts62, ner+, and gene A lacking 179 bp at its 3' end (A'). The product of this truncated gene A' retains transposase activity and is sufficient for the transposition of lambda placMu. This was demonstrated by showing that lambda placMu derivatives carrying the A am1093 mutation in the A' gene are unable to transpose by themselves in a Su- strain, but their transposition can be triggered by coinfection with lambda pMu507(A+ B+). We have constructed several new lambda placMu phages that carry the A' am1093 gene and the kan gene, which confers resistance to kanamycin. Chromosomal insertions of these new phages are even more stable than those of the previously reported lambda placMu phages, which makes them useful tools for genetic analysis.     

Bacteriophage P22 as a vector for Mu mutagenesis in Salmonella typhimurium: isolation of nad-lac and pnc-lac gene fusions.

Salmonella phage P22 was utilized as a vector for phage Mu cts d1(Apr lac) mutagenesis in Salmonella typhimurium. Efficient transposition of phage Mu d1 and the construction of gene fusions were readily accomplished with this procedure. Mutants blocked in the biosynthesis of NAD+ and in pyridine nucleotide cycle metabolism were isolated by this method, resulting in nadB-lac, nadC-lac, and pncB-lac gene fusions.     

Identification and characterization of a relA-dependent starvation-inducible locus (sin) in Salmonella typhimurium

By use of Mu cts d1(Ap lac) phage, a strain of Salmonella typhimurium was isolated containing a Mu d insertion in a locus (sinA) which is induced during nicotinate, thiamine, purine, amino acid, phosphate, and carbon starvation conditions. Depending on the starvation condition, a 2- to 10-fold increase in beta-galactosidase activity was demonstrated. The sinA locus, which mapped at 32 U, became induced after a decline in growth rate due to starvation. The introduction of relA into the sinA-lac strain prevented induction by nicotinate starvation and partially prevented induction by phosphate starvation. The data suggest that sinA responds to changes in growth rate due to various nutrient starvation conditions and probably responds in part to changes in guanosine tetraphosphate levels.     

Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp. atroseptica: identification of kduD gene

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-mini-Tn5xylE transposon into the chromosome of phytopathogenic bacteria Erwinia carotovora subsp. atroseptica. The inability of mutant cells to utilize these substrates was caused by a failure to accomplish the catabolism of unsaturated digalacturonic acid (UDA). Study of enzymatic activities has established that mutant bacteria lost the ability to produce 2,5-diketo-3-deoxygluconate dehydrogenase, an enzyme of intracellular UDA utilization. Molecular cloning of the mutant gene was conducted, and its nucleotide sequence was determined. It was shown that the nucleotide sequence of this gene had an 82% homology with the sequence of Erwinia chrysanthemi EC3937 kduD gene encoding 2,5-diketo-3-deoxygluconate dehydrogenase. The intergene kdul-kduD region in bacteria Erwinia carotovora subsp. atroseptica is shorter in length by 98 nucleotides than the corresponding region of Erwinia chrysanthemi and does not contain promoter sequences. The kduD gene was located at 126.8 min of the Erwinia carotovora subsp. atroseptica genetic map.     

Unmasking of bacteriophage Mu lipopolysaccharide receptors in Salmonella enteritidis confers sensitivity to Mu and permits Mu mutagenesis.

The human pathogen Salmonella enteritidis 3b was found to be highly resistant to phage P22 and Mu derivatives. The Mu sensitivity (musA1) allele from Salmonella typhimurium could be transferred to S. enteritidis 3b at low frequency by cotransduction with hisG::Tn10. Sensitivity to Mu resulted in a large reduction in the number of lipopolysaccharide core-region oligosaccharides that were substituted with O-antigen polysaccharide. The residual high-molecular-weight lipopolysaccharide appeared to be a hybrid displaying O antigens which were immunologically related to those of S. typhimurium and not to those of S. enteritidis. Consequently, Mu d1(Ap lac) could then be transduced into Mus strains forming stable lysogens. On temperature induction, Mu transposition could easily be used to generate mutations in genes coding for cell surface antigens including fimbriae, lipopolysaccharide, and flagella.