Bacteriophage Mu as a genetic tool to study Erwinia amylovora pathogenicity and hypersensitive reaction on tobacco

Erwinia amylovora 1430 was shown to be sensitive to Mu G(-) particles. Infection resulted either in lytic development or in lysogenic derivatives with insertion of the Mu genome at many sites in the bacterial chromosome. We used the Mu d1Bx::Tn9 (lac Apr Cmr) derivative, called Mu dX, to identify mutants affected in pathogenicity and in their ability to induce a hypersensitive reaction (HR) on tobacco plants. Inoculation of 1,400 lysogenic derivatives on apple root calli led to the identification of 12 mutants in three classes: (i) class 1 mutants were nonpathogenic and unable to induce an HR on tobacco plants; (ii) class 2 mutants were nonpathogenic but retained the ability to induce an HR; and (iii) class 3 mutants showed attenuated virulence. Of the 12 mutants, 8 had a single insertion of the Mu dX prophage. For class 1 and 2 mutants, reversion to pathogenicity was concomitant with the loss of the Mu dX prophage. Furthermore, revertants from the class 1 mutants also recovered the ability to induce an HR on tobacco plants. Five of the six class 3 mutants were impaired in exopolysaccharide production. No changes of the envelope structure (lipopolysaccharide and outer membrane proteins) were correlated with differences in pathogenicity. One class 3 mutant did not produce any functional siderophore, suggesting that iron uptake could be involved in pathogenicity.     

Thiol-sensitive promoters of Escherichia coli.

Mu dX(lac) insertion mutants of Escherichia coli CSH50 in which the expression of the lacZ gene was sensitive to the presence of exogenous 1-thioglycerol or dithiothreitol were isolated. Both stimulatory and inhibitory mutants were found. The existence of several thiol-sensitive promoters suggests that exogenous thiols may provoke global stress responses in E. coli.     

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.     

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.     

Mu insertion duplicates a 5 base pair sequence at the host inserted site.

Nucleotide sequences were analyzed across the two ends of lysogenic Mu DNA. These ends were cloned separately in lambdapMu hybrid particles that derived from a single Mu lysogen in the lac Z part of lambdaplac5. The obtained data imply that Mu lysogenization was associated with the duplication of 5 base pairs present in lac DNA at the Mu insertion site. As a result of this duplication, Mu DNA is flanked by two copies of five identical base pairs oriented as direct repeats. A similar conclusion has been obtained independently by other investigators with the use of a different Mu lysogen (D. Kamp and R. Kahmann, personal communication). Thus Mu insertion seems to have a striking similarity to typical IS-mediated insertions that were found to be associated with a short DNA duplication at the target site.     

A new insertion sequence, IS121, is found on the Mu dI1 (Ap lac) bacteriophage and the Escherichia coli K-12 chromosome

We have discovered a new insertion sequence, now designated IS121, as a component of the Mu dI1 (Ap lac) phage. This sequence is 1.2 kilobases long and contains single recognition sites for the HincII, Bg1II, and HindIII restriction endonucleases. IS121 is present in at least three copies in the chromosome of several Escherichia coli K-12 strains. When present in the nonconjugative plasmid pBR322, IS121 can mediate cointegrate formation with an F' lac plasmid and transfer of pBR322 sequences to suitable recipients. IS121 is also capable of precise or nearly precise excision. As part of the study of IS121, we have determined the physical structure of the Mu dI1 (Ap lac) phage and established an extensive restriction endonuclease map of this phage. A revised schema for the formation of the Mu dI1 (Ap lac) phage is presented.     

DNA intermediates in transposition of phage Mu

Transposable genetic elements can insert into DNA sites that have no homology to themselves. Evidence that there is a physical linkage between a transposable element and its target DNA sequence during transposition comes from studies on bacteriophage Mu DNA transposition in which plasmids containing Mu DNA have been shown to attach to host DNA. We report the isolation of key structures, seen after induction of Mu DNA replication, after cloning lac operator into Mu DNA and using the lac repressor-operator interaction to trap Mu DNA on nitrocellulose filters. We have localized Mu sequences within these structures in the electron microscope by visualizing the lac operator-repressor interaction after binding with ferritin-conjugated antibody. This analysis shows that key structures contain replicating Mu DNA linked to non-Mu DNA and that replication can begin at either end of Mu.     

Anaerobiosis, formate, nitrate, and pyrA are involved in the regulation of formate hydrogenlyase in Salmonella typhimurium.

Three groups of mutants defective in the fermentative production of gas were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). One group consisted of strains which lacked hydrogenase. The mutation site for this group was located in the vicinity of the known hyd gene. A second group consisted of mutants which lacked the formate dehyrogenase associated with hydrogenase. The mutation site was located in four of them. It was not in the vicinity of the previously described fhlD gene but was instead located at 93 min on the Salmonella map. The third mutant group, which consisted of strains that produced gas in triple sugar iron agar but not in nutrient agar supplemented with glucose, appeared to be pyrA mutants. The insertion site was located in the vicinity of pyrA , and they required arginine and pyrimidines for growth. Expression of the lac operon in the hyd mutants was induced by anaerobiosis. It was only slightly increased by the addition of formate under anaerobic conditions and slightly decreased by the addition of nitrate. Nitrate had no effect in an hyd ::Mu d strain that also carried a chlC::Tn10 insertion. Full expression of the lac operon in the fhl mutants required both formate and anaerobic conditions. The presence of nitrate in addition to formate resulted in activities about half those obtained in its absence, even in the fhl ::Mu d chlC::Tn10 double mutant. In the absence of formate, nitrate reduced expression only in the fhl ::Mu d single mutants. Expression of the lac operon among the pyrA mutants was repressed by arginine and cytosine and also by anaerobiosis. An explanation for the involvement of pyrA in aerobic and anaerobic energy metabolism is proposed.     

Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac.

Bacteriophage Mu d1 (lac Aprr) was used to obtain strains of Escherichia coli K-12 in which the lac genes are expressed from the promoter of pheA, the structural gene for the enzyme chorismate mutase P-prephenate-dehydratase. A derivative of bacteriophage lambda which carries the pheA-lac fusion was prepared; the method used is generally applicable for the construction, from Mu dl lysogens, of specialized transducing lambda phage carrying the promoter-lac fusions. A restriction enzyme cleavage map of lambda ppheA-lac for the enzymes HindIII and PstI is presented.     

Conditionally transposition-defective derivative of Mu d1(Amp Lac).

A Mu d1 derivative is described which is useful for genetic manipulation of Mu-lac fusion insertions. A double mutant of the specialized transducing phage Mu d1(Amp Lac c62ts) was isolated which is conditionally defective in transposition ability. The Mu d1 derivative, designated Mu d1-8(Tpn[Am] Amp Lac c62ts), carries mutations which virtually eliminate transposition in strains lacking an amber suppressor. In such strains, the Mu d1-8 prophage behaves like a standard transposon. It can be moved from one strain of Salmonella typhimurium to another by the general transducing phage P22 with almost 100% inheritance of the donor insertion mutation. When introduced into a recipient carrying supD, supE, or supF, 89 to 94% of the Ampr transductants were transpositions of the donor Mu d1-8, from the transduced fragment into new sites. The stability of Mu d1-8 in a wild-type, suppressor-free background was sufficient to permit use of the fusion to select constitutive mutations without prior isolation of deletions to stabilize the fusion. Fusion strains could be grown at elevated temperature without induction of the Mu d prophage. The transposition defect of Mu d1-8 was corrected by a plasmid carrying the Mu A and B genes.     

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.     

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.     

Mutagenesis of Erwinia carotovora subsp. carotovora with bacteriophage Mu d1 (Apr lac cts62): construction of his-lac gene fusions.

The bacteriophage Mu d1(Apr lac cts62 ) obtained from an Escherichia coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid phage was utilized as a vector for phage mutagenesis in Erwinia carotovora subsp. carotovora. Among ampicillin-resistant transductants. 1.4% were auxotrophs. The synthesis of beta-galactosidase was derepressed upon starvation for histidine in two different his-lac fusion strains.     

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis.     

Precise excision of bacteriophage Mu DNA

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.