A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.     

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.     

pryB mutations as suppressors of arginine auxotrophy in Salmonella typhimurium.

Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation were found to grow more slowly in minimal medium than pyrH+ controls. The addition of arginine or citrulline but not ornithine restored normal growth rates. This requirement for arginine was completely suppressed by pyrB mutations and partially suppressed by pyrC and pyrD mutations. No suppression was observed with mutants at the pyrF locus. Introduction of leaky mutation argI2002 resulted in a more extreme arginine requirement and accentuated suppression by pyrB mutations. Suppression by the pyrC and pyrD mutations was reduced as a result of the incorporation of the leaky argI2002 allele. These results indicate that in pyrH700 strains carbamoyl phosphate is preferentially directed toward the formation of intermediates in the pyrimidine biosynthetic pathway. Arginine auxotrophy results from the reduced availability of carbamoyl phosphate for the biosynthesis of arginine. Suppression of this arginine dependence for growth is used as a convenient positive selection technique for pyrB mutations.     

Isolation of a hybrid F' factor-carrying Escherichia coli lactose region and Salmonella typhimurium histidine region, F42-400 (F' ts114 lac+, his+): its partial characterization and behavior in Salmonella typhimurium.

Episome F' ts114 lac+ (F42-114) was transferred into Salmonella typhimurium carrying an F'his+ (FS400) episome, and fused episome F' ts114 lac+, his+ (F42-400) was obtained. Episome F42-400 could be transferred to S. typhimurium, Escherichia coli and Klebsiella pneumoniae. Identification of the episome was based on: (i) temperature sensitivity of the Lac+ and His+ phenotypes; (ii) the fact that F- segregants, obtained after temperature curing or acridine orange curing, were simultaneously Lac- and His-; and (iii) linkage of lac+ with his+ in episomal transfers to E. coli and S. typhimurium. The frequency of episome transfer was influenced by the genotype of the donor. Plasmid LT2, prevalent in S. typhimurium LT2 strains, was suggested to be responsible for the low fertility of S. typhimurium donors. Episome F42-400 was capable of chromosome mobilization, and the extent of chromosome mobilization was not influenced by the presence or absence of the histidine region on the donor chromosome. Growth in a defined medium with acridine orange was able to cure F42-400. The frequency of curing was increased (the frequency of His+ cells was 0.0001%) if the cells were grown at 40 C in the presence of acridine orange. Selection for temperature-resistant Lac+, His+ derivatives in a strain without histidine deletion yielded Hfr strains. However, similar and stronger selections in strains without the chromosomal histidine region failed to yield Hfr strains. Our inability to obtain Hfr's in strains without the chromosomal histidine region was explained by assuming that the episome F42-400 has lost the F sites involved in integration into the S. typhimurium chromosome.     

Toxicity of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate in Salmonella typhimurium

Growth of Salmonella typhimurium pyrC or pyrD auxotrophs was severely inhibited in media that caused derepressed pyr gene expression. No such inhibition was observed with derepressed pyrA and pyrB auxotrophs. Growth inhibition was not due to the depletion of essential pyrimidine biosynthetic pathway intermediates or substrates. This result and the pattern of inhibition indicated that the accumulation of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate was toxic. This intermediate is synthesized by the sequential action of the first two enzymes of the pathway encoded by pyrA and pyrB and is a substrate for the pyrC gene product. It should accumulate to high levels in pyrC or pyrD mutants when expression of the pyrA and pyrB genes is elevated. The introduction of either a pyrA or pyrB mutation into a pyrC strain eliminated the observed growth inhibition. Additionally, a direct correlation was shown between the severity of growth inhibition of a pyrC auxotroph and the levels of the enzymes that synthesize carbamyl aspartate. The mechanism of carbamyl aspartate toxicity was not identified, but many potential sites of growth inhibition were excluded. Carbamyl aspartate toxicity was shown to be useful as a phenotypic trait for classifying pyrimidine auxotrophs and may also be useful for positive selection of pyrA or pyrB mutants. Finally, we discuss ways of overcoming growth inhibition of pyrC and pyrD mutants under derepressing conditions.     

Salmonella typhimurium metC operator-constitutive mutations

We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (lambda Clac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying lambda Clac mutant phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.     

Salmonella typhimurium metC operator-constitutive mutations

Abstract We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (λClac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying λClac mutant phage exhibit high β-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.     

Nature of Lactose-fermenting Salmonella Strains Obtained from Clinical Sources

Six of seven lactose-fermenting (lac(+)) Salmonella strains obtained from clinical sources were found to be capable of transferring the lac(+) property by conjugation to Salmonella typhosa WR4204. All of the six S. typhosa strains which received the lac(+) property transferred it in turn to S. typhimurium WR5000 at the high frequencies typical of extrachromosomal F-merogenotes. These six lac elements were also transmissible from S. typhosa WR4204 to Proteus mirabilis and to some strains of Escherichia coli K-12; moreover, they were capable of promoting low frequency transfer of chromosomal genes from S. typhimurium WR5000 to S. typhosa WR4204. One of these lac elements was shown also to be capable of promoting low frequency chromosome transfer in E. coli K-12. E. coli K-12 strains harboring these lac elements exhibited sensitivity to the male specific phage R-17. Sensitivity to R-17 was not detected in Salmonella strains containing the elements. Examination of the lac elements in P. mirabilis by cesium chloride density gradient centrifugation showed that each element had a guanine plus cytosine content of 50%. The sizes of the elements varied from 0.8 to 3% of the total Proteus deoxyribonucleic acid. The amount of beta-galactosidase produced by induced and uninduced cultures of S. typhimurium WR5000 and S. typhosa WR4204 containing the lac elements was lower than that produced by these strains with the F-lac episome. The heat sensitivity of beta-galactosidase produced by the lac elements in their original Salmonella hosts indicated that the enzyme made by these strains differs from E. coli beta-galactosidase.     

Characterization of a group of anaerobically induced, fnr-dependent genes of Salmonella typhimurium.

We have previously reported the isolation of a group of anaerobically regulated, fnr-dependent lac fusions in Salmonella typhimurium and have grouped these oxd genes into classes based on map position. In order to identify these genes, we have replaced the original Mud-lac fusion in a member of each oxd class with the much smaller Mud-cam element, cloned the fusion, and determined DNA sequence sufficient to define the oxd gene. Several of the fusions correspond to previously known genes from S. typhimurium or Escherichia coli: oxd-4 = cbiA and oxd-11 = cbiK, oxd-5 = hybB, oxd-7 = dcuB, oxd-8 = moaB, oxd-12 = dmsA, and oxd-14 = napB (aeg-46. 5). Two other fusions correspond to previously unknown loci: oxd-2 encodes an acetate/propionate kinase, and oxd-6 encodes a putative ABC transporter present in S. typhimurium but not in E. coli.     

Operon Coordination in Different Bacterial Hosts

Coordination of gene expression in the lac operon was compared in Escherichia coli and Salmonella typhimurium as an approach to detecting possible differences in protein synthesis or membrane structure between organisms. Either a wild-type F' lac pro(AB) episome or the same episome with a polar mutation in one of the lac genes was introduced into pro(-) derivatives of the two strains of bacteria. Activity assays showed that the beta-galactosidase levels were only slightly lower in the S. typhimurium cells than in E. coli cells, whereas the transacetylase levels were significantly higher in S. typhimurium for all of the lac markers tested. Galactoside transport activities were always comparable in the two strains of bacteria; this latter result indicates that the cell envelopes of E. coli and S. typhimurium do not differ sufficiently to affect the membrane-associated lac transport system. It was found, however, that the specific transport activity is very sensitive to culture age in both bacteria, and decreases rapidly in cultures past the mid-exponential phase of growth.     

Use of Staphylococcus aureus 6-P-beta-galactosidase and GFP as fusion partners for lactose-specific IIC domain from Staphylococcus aureus

The hydrophilic part of membrane proteins plays an important role in the formation of 3D crystals. The construction of fusion proteins using well crystallizing proteins as fusion partners is a possibility to increase the hydrophilic part of membrane proteins lacking large hydrophilic domains. These fusion proteins might be easier to crystallize. Two bifunctional fusion proteins containing the membrane-bound, lactose-specific enzyme IIC domain of the lactose transporter (IICB(lac)) from S. aureus as N-terminal fusion partner were constructed by gene fusion. The C-terminal fusion partners were S. aureus 6-P-beta-Galactosidase and GFP, respectively. Both proteins were overexpressed in E. coli, purified to homogeneity and kinetically characterized: In the presence of the components of the lactose phosphotransferase system of S. aureus, the hybrid proteins phosphorylated their substrates, indicating that the fusion partners are sufficiently flexibly linked to allow the interaction of the IIC(lac) domain with the IIB(lac) domain of the lactose transporter. The activity of the 6-P-beta-Galactosidase as well as the fluorescence of GFP were preserved in the fusion proteins. The Vmax values determined for the IIC domain in the fusion proteins were dramatically reduced compared with the values determined for the separate IIC(lac) domain and the complete lactose transporter (IICB(lac)). The Km values were only slightly increased indicating that the Vmax values are much more influenced by the fusion than the substrate affinities. The substrate affinity and the Vmax value determined for the GFP-fused IIC(lac) domain are higher than for the 6-P-beta-Galactosidase-fused IIC(lac). The results suggest that the fusion with GFP enables a better interaction with the IIB(lac) domain than the fusion with 6-P-beta-Galactosidase. Moreover, the GFP-fused IIC(lac) domain proved to be more stable than the 6-P-beta-Galactosidase fusion protein.     

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.     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.