Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable.

The lon gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in the intracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, such as UV sensitivity and overproduction of capsular polysaccharide. We have isolated lambda transducing phage carrying the lon gene and used the lon phage as a target for insertional mutagenesis by a defective transposon Tn10 to produce lon::delta 16 delta 17Tn10 derivatives. The delta 16 delta 17Tn10 (hereafter called delta Tn10) elements were inserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distance from the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different lambda lon::delta Tn10 phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. The lon::delta Tn10 mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis of intact Lon protein, as assayed by antibody on Western blots. An analysis of the protein-degradative ability of lon::delta Tn10 cells suggests that although the insertions in lon caused a reduction in ATP-dependent protein degradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::delta Tn10 mutations and a lon deletion retaining only the amino-terminal 25% of the gene did not affect the energy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in the ATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provide clear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli.     

The mdoA locus of Escherichia coli consists of an operon under osmotic control

In Escherichia coli, the 5 kb mdoA locus is involved in the osmotically controlled biosynthesis of periplasmic membrane-derived oligosaccharides (MDOs). The structure of this locus was analysed by in vitro cassette insertion, transposon mutagenesis, and gene-fusion analysis. A 'neo' cassette, derived from the neomycin phosphotransferase II region of transposon Tn5, was inserted into mdoA, borne by a multicopy plasmid. This plasmid was shown to complement two previously described mdoA mutations, depending on the orientation of the exogenous gene. Thus, the gene altered by these mutations could be expressed under the control of the exogenous promoter. Moreover, the 'neo' cassette inactivated another, uncharacterized, mdo gene, because when this insertion was transferred into the chromosome MDO synthesis was abolished. The existence of a second gene was confirmed by complementation analysis with a collection of Tn1000 insertions into mdoA. Two groups were defined, and the two genes are organized into an operon (mdoGH). This conclusion was reached because Tn1000 insertions in the first gene displayed a polar effect on the expression of the second gene. An active gene fusion was obtained on a multicopy plasmid between the beginning of mdoH and lacZ. The hybrid beta-galactosidase activity followed the same osmotically controlled response as that described for of MDO synthesis. This regulation was unaffected by the presence, or absence, of MDOs in the periplasm. Finally, the amount of mdoA-specific mRNAs, determined by dot blot hybridization, decreased when the osmolarity of the growth medium increased.     

Identification of a second RcsA protein, a positive regulator of colanic acid capsular polysaccharide genes, in Escherichia coli.

A second form of RcsA, a positive activator of the capsular polysaccharide genes (cps), has been identified in Escherichia coli. Ferguson plot analysis suggests that the two RcsA proteins differ by size rather than by charge. Both RcsA proteins are expressed from a single rcsA gene. Detection of both RcsA proteins in delta lon cells is RcsB dependent.     

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.     

A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli.

We present a collection of 182 isogenic strains containing genetically linked antibiotic resistance elements located at approximately 1-min intervals around the Escherichia coli chromosome. At most positions both Tn10 (Tetr) and TN10kan (Kanr) elements are available, so that the collection contains a linked set of alternating antibiotic resistance markers. The map position of each insertion has been aligned to the E. coli genetic map as well as to the Kohara ordered clone bank. These strains are designed to be used in a rapid two-step mapping system in E. coli. In the first step, the mutation is localized to a 5- to 15-min region of the chromosome by Hfr mapping with a set of Hfr strains containing either Tn10 or Tn10kan elements located 20 min from their respective origins of transfer. In the second step, the mutation is localized to a 1-min region by P1 transduction, with a collection of isogenic insertion strains as donors. We discuss the uses of this collection of strains to map and eventually to clone a variety of mutations in E. coli.     

Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance.

The proU locus in Escherichia coli encodes an important osmoregulatory function which mediates the growth-promoting effect of L-proline and glycine betaine in high-osmolarity media. This locus was cloned, in contiguity with a closely linked Tn10 insertion, onto a multicopy plasmid directly from the E. coli chromosome. For a given level of osmotic stress, the magnitude of osmoresponsive induction of a single-copy proU::lac fusion was reduced in strains with multiple copies of the proU+ genes; in comparison with haploid proU+ strains, strains with the multicopy proU+ plasmids also exhibited enhanced osmotolerance in media supplemented with 1 mM L-proline or glycine betaine. Experiments involving subcloning, Tn1000 mutagenesis, and interplasmid complementation in a deletion mutant provided evidence for the presence at this locus of two cistrons, both of which are necessary for the expression of ProU function. We propose the designations proU for the gene originally identified by the proU224::Mu d1(lac Ap) insertion and proV for the gene upstream (that is, counterclockwise) of proU.     

Chromosomal inversion accompanied by an enhancement of uridine phosphorylase gene expression in Escherichia coli K-12

In thymine requiring auxotrophs of Escherichia coli the uridine phosphorylase enzyme (udp gene) can catalyze nonspecifically conversion of thymine to thymidine. By selection for effective utilization of exogenous thymine, it is possible to isolate forms with increased expression of the udp gene. Mutants with increased gene expression were isolated from the strain with transposon Tn10 within the metE gene closely linked to udp. Some mutants (designated udpPf) losing Tn10 but retaining the Met- phenotype are characterized by disturbance of recombination in the metE-udp region: they do not form Met+ transductants in P1 transduction with the wild-type donor strain. However, recovery of homology in the chromosomal metE-udp region takes place with low frequency in P1 transduction using the strain with Tn10 insertion in metE as a donor. Data obtained in transductional and conjugational experiments demonstrate that the udpPf1 mutant studied is an inversion extending about 3 min of the E. coli chromosome and including the region of chromosomal replication origin (oriC).     

Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase.

A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.     

A mutation defining ultrainduction of the Escherichia coli gal operon.

Tn10 insertion in the galS (ultrainduction factor) gene of Escherichia coli allows the gal operon to be constitutively expressed at a very high level, equal to that seen in a delta galR strain in the presence of an inducer. The insertion has been mapped by criss-cross Hfr matings and by marker rescue into Kohara phages at 46 min on the E. coli chromosome.     

Identification of genes and gene products whose expression is activated during nitrogen-limited growth in Bacillus subtilis.

The levels of urease and asparaginase were elevated 25- and 20-fold, respectively, in extracts of Bacillus subtilis cells grown in medium containing nitrogen sources that are poor sources of ammonium (NH4+) compared with the levels seen in extracts of cells grown in medium containing nitrogen sources that are good sources of NH4+. To determine whether a collection of genes whose expression responds to nitrogen availability could be isolated, a library of Tn917-lacZ insertions was screened for nitrogen-regulated beta-galactosidase expression. Two fusion strains were identified. beta-Galactosidase expression was 26- and 4,000-fold higher, respectively, in the nrg-21::Tn917-lacZ and the nrg-29::Tn917-lacZ insertion strains during NH4(+)-restricted growth than during growth on nitrogen sources that are good sources of NH4+. PBS1 transduction analysis showed that the nrg-21::Tn917-lacZ insertion mapped between gutB and purB and that the nrg-29::Tn917-lacZ insertion mapped between degSU and spoIID. The repression of expression of these four gene products during growth on good sources of NH4+ required the wild-type glutamine synthetase protein but not the glutamine synthetase regulatory protein, GlnR.     

Activation of the lac genes of Tn951 by insertion sequences from Pseudomonas cepacia.

We have identified three transposable gene-activating elements from Pseudomonas cepacia on the basis of their abilities to increase expression of the lac genes of the broad-host-range plasmid pGC91.14 (pRP1::Tn951). When introduced into auxotrophic derivatives of P. cepacia 249 (ATCC 17616), this plasmid failed to confer the ability to utilize lactose. The lac genes of Tn951 were poorly expressed in P. cepacia and were not induced by isopropyl-beta-D-thiogalactopyranoside. Lac+ variants of the pGC91.14-containing strains which formed beta-galactosidase at high constitutive levels as a consequence of transposition of insertion sequences from the P. cepacia genome to sites upstream of the lacZ gene of Tn951 were isolated. Certain of the elements also increased gene expression in other bacteria. For example, IS407 strongly activated the lacZ gene of Tn951 in Pseudomonas aeruginosa and Escherichia coli, and IS406 (but not IS407) did so in Zymomonas mobilis. The results indicate that IS elements from P. cepacia have potential for turning on the expression of foreign genes in a variety of gram-negative bacteria.     

In vitro construction of gshB::kan in Escherichia coli and use of gshB::kan in mapping the gshB locus.

The Escherichia coli structural gene for glutathione synthetase, gshB, was cloned into pBR322. Plasmids containing gshB were able to complement the glutathione requirement of a trxA gshB double mutant, and cells containing the plasmids were found to have elevated levels of glutathione synthetase. A mutant gshB allele was constructed by inserting the kan gene from pUC4K into a unique HpaI site located within gshB. The resulting plasmid-encoded allele was used to replace a genomic gshB+ by homologous recombination. The resulting strain had no detectable glutathione synthetase activity. The gshB allele containing the kan insertion was used to map gshB on the E. coli chromosome by P1 transduction. The results indicated that gshB is located at 63.4 min, between metK and speC. The allele was further localized to a region of 3,100 to 3,120 kilobase pairs on the physical map (restriction map) of E. coli by DNA-DNA hybridization to a series of lambda bacteriophages (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987).