galE Mutant Research Articles

UDP-glucose is a potential intracellular signal molecule in the control of expression of sigma S and sigma S-dependent genes in Escherichia coli

The sigma S subunit of RNA polymerase is the master regulator of a regulatory network that controls stationary-phase induction as well as osmotic regulation of many genes in Escherichia coli. In an attempt to identify additional regulatory components in this network, we have isolated Tn10 insertion mutations that in trans alter the expression of osmY and other sigma S-dependent genes. One of these mutations conferred glucose sensitivity and was localized in pgi (encoding phosphoglucose isomerase). pgi::Tn10 strains exhibit increased basal levels of expression of osmY and otsBA in exponentially growing cells and reduced osmotic inducibility of these genes. A similar phenotype was also observed for pgm and galU mutants, which are deficient in phosphoglucomutase and UDP-glucose pyrophosphorylase, respectively. This indicates that the observed effects on gene expression are related to the lack of UDP-glucose (or a derivative thereof), which is common to all three mutants. Mutants deficient in UDP-galactose epimerase (galE mutants) and trehalose-6-phosphate synthase (otsA mutants) do not exhibit such an effect on gene expression, and an mdoA mutant that is deficient in the first step of the synthesis of membrane-derived oligosaccharides, shows only a partial increase in the expression of osmY. We therefore propose that the cellular content of UDP-glucose serves as an internal signal that controls expression of osmY and other sigma S-dependent genes. In addition, we demonstrate that pgi, pgm, and galU mutants contain increased levels of sigma S during steady-state growth, indicating that UDP-glucose interferes with the expression of sigma S itself.

Gonococcal rfaF mutants express Rd2 chemotype LPS and do not enter epithelial host cells

We have investigated the function of the Isi-1 gene of Neisseria gonorrhoeae previously implicated in lipopolysaccharide (LPS)-inner-core biosynthesis (Petricoin et al., 1991). Disruption of the gene in gonococcal strain MS11 resulted in the production of LPS that migrated faster than that from an isogenic galE mutant, typical for a mutation that influences the inner-core region. Complementation of a panel of Salmonella typhimurium mutants with defined defects in rfa loci demonstrated conclusively that the Isi-1 gene of MS11 is functionally homologous to the rfaF gene, which encodes heptosyltransferase II in both E. coli and S. typhimurium. Comparison of deduced amino acid sequences of the gonococcal and the Salmonella RfaF demonstrated 70% similarity, including 47% identical amino acid residues. Immunochemical analysis of the LPS using monoclonal antibodies directed against chemically defined inner-core glycoconjugates revealed that the gonococcal and Salmonella Rd2-chemotypes were antigenically similar, further extending the genetic and functional homology. Infection experiments in vitro demonstrated that the Isi-1 mutant could not invade human Chang epithelial cells despite expression of a genetically defined invasion-promoting gonococcal opacity protein. These data imply that the LPS phenotype is a critical factor for gonococcal invasiveness.

A bacteriocidal effect of 2-deoxy-D-galactose on Escherichia coli due to inhibition of amino sugar metabolism.

A bacteriocidal effect of 2-deoxy-D-galactose (dGal) in addition to the well known static effect was newly demonstrated on the basis of various physiological and biochemical events in Escherichia coli. Unlike D- galactose-induced lysis of a E. coli galE mutant via the process of uridine 5′-diphosphate galactose accumulation, dGal-induced growth inhibition was characterized by cell burst without swelling and elongation of the original rod shaped cells. 2-Deoxy-D-galactose 1-phosphate (dGal1P) was the most probable effector for a bacteriocidal effect of dGal owing to the relationship between dGal-sensitive phenotype and plasmid-dependent galK gene expression. The synthesis of cell wall polysaccharides was functional in dGal-grown cells with the added D-glucosamine and N-acetyl-D-glucosamine, which were protective against dGal-induced growth inhibition. The present study proposes inhibition of fructose 1, 6- bisphosphatase related to de novo synthesis of amino sugar with accumulation of dGal1P as a cause of overall growth inhibition by dGal.

Cloning and molecular analysis of the galE gene of Neisseria meningitidls and its role in lipopolysaccharide biosynthesis.

The galE gene from Haemophilus influenzae was used as a hybridization probe for the galE gene of Neisseria meningitidis Group B, identifying two different homologous loci. Each of the loci was cloned and nucleotide sequence analysis revealed that both loci contained sequences similar to galE. One contained a functional galE gene and mapped to the capsule biosynthetic locus. The second contained only a partial galE-coding sequence, which did not express a functional gene product. A galE mutant meningococcal strain was constructed by transformation with an inactivated galE gene. Analysis of the LPS from the galE mutant strain revealed an apparent reduction in molecular weight and a loss of reactivity with monoclonal antibodies specific for structures known to contain galactose. These results are consistent with an essential role for galE in the incorporation of galactose into meningococcal lipopolysaccharide.

Effect of Antibiotic Class and Concentration on the Release of Lipopolysaccharide from Escherichia coli

The ability of six antibiotics from different classes to release radiolabeled lipopolysaccharide (LPS) from a phenotypically smooth galE mutant of Escherichia coli O111:B4 was examined. Antibiotic concentrations were 0.0625-512 micrograms/mL. LPS release increased as a function of the antibiotic concentration, reaching a limit at or near the concentration that killed the majority of bacteria. The maximum amount of LPS released by polymyxin B was 40.6% +/- 0.9%, by gentamicin 58.2% +/- 2.5%, by ciprofloxacin 65.8% +/- 2.5%, by ceftazidime 73.1% +/- 0.9%, by tetracycline 75.3% +/- 10.0%, and by imipenem 79.7% +/- 2.3%. In timed experiments, ceftazidime released 61.9% +/- 1.2%, imipenem 51.1% +/- 8.8%, and tetracycline 39.7% +/- 4.4% of the LPS within the first hour of incubation, whereas polymyxin B released 13.5% +/- 1.9%, gentamicin 9.8% +/- 3.6%, and ciprofloxacin 12.7% +/- 2.6% of the LPS (P < .05). Fluoro-radiography and immunoblot analyses revealed similar migration patterns for antibiotic-released and cell-bound LPS on SDS-PAGE gels, suggesting similar O-polysaccharide content in the two LPS fractions. The amount and rate of LPS release from an E. coli strain was dependent upon antibiotic class and concentration.

The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide.

Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and an important virulence factor of many pathogens, such as Neisseria gonorrhoeae. We have cloned the gonococcal galE gene which was found to be located in the gonococcal homologue of the meningococcal capsule gene complex region D. Sequence alignment indicated extensive homology with the Escherichia coli and Salmonella GalE proteins. Mutants with insertions in the galE gene were used as a tool to characterize the structure and function of gonococcal lipopolysaccharide. They displayed deep rough phenotypes, and chemical analysis confirmed the loss of galactose from the mutant lipopolysaccharide. Functional analysis indicated that the terminal oligosaccharides contain galactose and that these are lost in galE mutants. The importance of these oligosaccharides in gonococcal biology is clear from the fact that they contain the epitopes that are the targets for killing by normal human serum, and the acceptor site for sialic acid, which acts to protect the gonococcus from this killing. Furthermore, infection experiments in vitro indicate that the galE mutants exhibit unaltered intergonococcal adhesion as well as adhesion to, and invasion of, epithelial cells.

Stability, immunogenicity and expression of foreign antigens in bacterial vaccine vectors

The use of attenuated strains of Salmonella as vaccine vectors frequently involves the introduction of heterologous antigens on recombinant plasmids. To overcome the problem of plasmid instability, we have integrated the gene that codes for a potential immunogen into the chromosome of a galE mutant of Salmonella typhimurium. Comparative in vitro and in vivo studies were conducted between the strain carrying the gene chromosomally integrated and an isogenic strain carrying the same gene on a multicopy plasmid. Levels of expression of the foreign antigen were significantly lower when the antigen was expressed from the chromosome than when it was expressed from the plasmid. The in vivo maintenance of the genes coding for antigen expression was determined on organisms recovered from spleen, liver and Peyer's patches of orally inoculated mice. By 24 h postinoculation, the majority of tissue isolates from the plasmid-containing strain had lost the plasmid and the ability to synthesize the antigen. By contrast, 100% of the recovered cointegrate isolates retained the ability to express the antigen throughout the 21 days of the experiment. Significantly, humoral and mucosal antibody levels against the antigen were greater when the antigen was expressed from the plasmid stabilized by the presence of the antibiotic than when the antigen was expressed from the chromosome. These observations indicate that the most important event for the development of an immune response against a foreign antigen delivered by these vectors may be the initial amount of antigen that primes the gut-associated lymphoid tissue and not persistence of the vector in tissues.

Studies on Cellular Response of Escherichia coli to UDP-Galactose Accumulation.

Cellular responses to intracellular accumulation of UDP-galactose (UDP-Gal) were investigated for D-galactose-induced lysis of an Escherichia coli galE mutant. The mutant, ara-207, showed dramatic lysis in a nutrient broth supplemented with D-galactose after 3h of cultivation when transformation of normal rod cells into bulged- and spheroplast-like cells was clearly observed under electron microscope. UDP-Gal had been already detected with the optimal level at 2 h of cultivation, followed by a drastic decrease in its level as well as a significant increase in the level of active (inhibitor-free) UDP-sugar hydrolase (USH) together with the bacterial lysis. Significant loss of UTP, UDP, and UDP-N-acetylglucosamine agreed with the in vivo activity of USH thus produced. Addition of D-sorbitol did not restrict UDP-Gal accumulation but did mostly protect ara-207 against the above morphological changes of the cells in which overproduction of USH was repressed over 3 to 4 h of cultivation. These results suggested that accumulation of UDP-Gal could have some growth inhibitory effect on E. coli cells but the compound was not directly involved with the following lytic process via spheroplast formation. A possibility was taken into account, that the accumulation of UDP-Gal caused overproduction of USH, which may provoke inhibition of bacterial peptidoglycan synthesis.

Colonizing ability and immunogenicity of a galE mutant of an enteroadherent Escherichia coli

Colonizing ability and immunogenicity of a galE mutant of an enteroadherent Escherichia coli

Efficient incorporation of galactose into lipopolysaccharide by Escherichia coli K-12 strains with polar galE mutations

Efficient incorporation of exogenous galactose into lipopolysaccharide was observed in a strain with a galE::Tn10 insertion and a strain with a deletion of galOPE. No incorporation was observed in a strain with a longer (galETK) deletion, indicating that incorporation into the galE mutants was due to a low level of expression of galTK from internal or fortuitous promoters. These galE mutants provided a convenient and specific way to radiolabel lipopolysaccharide.

RfaP mutants of Salmonella typhimurium.

Salmonella typhimurium rfaP mutants were isolated and characterised with respect to their sensitivity towards hydrophobic antibiotics and detergents, and their lipopolysaccharides were chemically analysed. The rfaP mutants were selected after diethylsulfate mutagenesis or as spontaneous mutants. The mutation in two independent mutants SH7770 (line LT2) and SH8551 (line TML) was mapped by cotransduction with cysE to the rfa locus. The mutants were sensitive to hydrophobic antibiotics (clindamycin, erythromycin and novobiocin) and detergents (benzalkoniumchloride and sodium dodecyl sulfate). Analysis of their lipopolysaccharides by chemical methods and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that their saccharide portion was, to a large extent, of chemotype Rc with small proportions of material containing a more complete core oligosaccharide and O-specific chains. Only 2.5 mol phosphate/mol lipopolysaccharide was found whereas the phosphate content of the lipopolysaccharide of a galE mutant strain was 4.8 mol. Thus the rfaP mutant lipopolysaccharides lacked more than two phosphate residues. Assessment of the location of phosphate groups in rfaP lipopolysaccharides revealed the presence of at least 2 mol phosphate in lipid A, indicating that the core oligosaccharide was almost devoid of phosphate. The chemical, physiological and genetic data obtained for these mutants are in full agreement with those reported earlier for rfaP mutants of Salmonella minnesota.

A chromosomal integration system for stabilization of heterologous genes in Salmonella based vaccine strains

We have developed a system whereby heterologous DNA encoding an antigen from an enteropathogen may be recombined into the chromosome of an attenuated Salmonella carrier strain. The system involves two steps; (i) integration of a hisOG deletion mutation into the chromosome; (ii) replacement of the hisOG deletion by the complete hisOG region and the segment of heterologous DNA which encodes the antigen of interest. Recombinants may be selected ( his +). The system was used to integrate the genes encoding K88 fimbriae from enterotoxigenic Escherichia coli into the chromosome of a galE mutant of Salmonella typhimurium (LT2H1). Recombinants were detected at a frequency of between 1.0 × 10 −3 and 1.5 × 10 −3. A variety of tests confirmed that the K88 genes were integrated into the chromosome of LT2H1 and were expressed. The stability of the recombinant was tested both in vivo and in vitro. When administered orally to mice, the recombinant elicited a serum antibody response to K88, and retained the Salmonella vaccine potential of the vector strain.

Paradoxes of immunity and immunosuppression in Salmonella infection.

There is continuing interest in mechanisms of immunity to systemic Salmonella infection, as concepts regarding the relative contributions of humoral and cellular immunity to host defense against these organisms form the theoretical basis for development of improved vaccines against typhoid fever (1,2). (Experimentally, investigations are carried out using a mouse model of systemic infection with Salmonella typhimurium as S. typhi is not virulent for mice.) Although the current typhoid vaccine, consisting of acetone-killed cells, has been shown to be protective in humans (3), it has an undesirable level of toxicity (1). Recent efforts have focused on live, attenuated strains of S. typhi which can be given orally, as an alternative vaccine strategy. A galE mutant, Ty21a, has been shown to have efficacy in a field trial in Egypt (4) although results have been less promising in field trials in Chile (5). One drawback of the Ty2la strain is that the exact nature of the genetic lesions which confer loss of virulence are not known (6). Stocker and colleagues have developed a series of mutants in various species of Salmonella which are blocked in aromatic synthesis due to transposon-mediated gene deletions or inversions (7,8). Based on high levels of protection induced in mouse models of typhoid fever using these aroAstrains of S. typhimurium Stocker has developed attenuated S. typhi with similar types of lesions for potential use in humans against typhoid fever (9). In addition, there is also considerable interest in using these aro-strains as vectors for cloned genes of other organisms such as E. coli enterotoxins (10,11).

Gene organization and structure of the Streptomyces lividans gal operon.

We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes. Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK. Comparison of the inferred protein sequences for the S. lividans gal gene products to the corresponding E. coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes. Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism.

Construction of defined galE mutants of Salmonella for use as vaccines.

We describe the molecular cloning of the gal operon of Salmonella typhimurium LT2 and the localization of the gal promoter and the genes galE, galT, and galK. The order of the genes and the direction of transcription was the same as in Escherichia coli K12. A restriction enzyme map of the operon was obtained, and the approximate termini of the gal genes were located by using transposon insertion mutagenesis and minicell analysis. We constructed a plasmid that contained a defined 0.4-kilobase deletion in the galE but still expressed galT and galK activities from the gal promoter. This galE deletion was recombined into the chromosomal gal operons of S. typhimurium and Salmonella typhi Ty2. The resulting strains were vigorous, nonreverting galE mutants that were sensitive to galactose-induced lysis at 0.2 mM galactose. The S. typhimurium galE derivatives were avirulent and protective in mice.

Some galE mutants of Salmonella choleraesuis retain virulence

galE mutants were isolated from three mouse-virulent strains of Salmonella choleraesuis (of group C1, O antigen 6,7) by selection for resistance to 2-deoxygalactose. The galE derivative of strain 381 comprised two components: galactose sensitive, thought to be the original mutant; and galactose resistant, presumably by a second mutation reducing galK or galT function or both. The galactose-sensitive component had an intraperitoneal 50% lethal dose for BALB/c mice of ca. 4 X 10(6) CFU, whereas the galactose-resistant component was about as virulent as its gal+ parent, with a 50% lethal dose of ca. 100 CFU. The galE mutant of strain 110 was somewhat sensitive to galactose, as shown by retardation of growth; its 50% lethal dose, ca. 500 CFU, was not much greater than the ca. 200 CFU value for its parent. The galE mutant of strain 117 showed the same partial sensitivity to galactose as strain 110 galE, but was nonvirulent (50% lethal dose of ca. 10(6) CFU versus ca. 400 CFU for its parent). Growth on galactose-supplemented medium restored the smooth phenotype, as indicated by phage sensitivity to three of the four galE strains, but only partially so for the strain 117 galE mutant. The retention of parental virulence by galE mutants of S. choleraesuis which are galactose resistant or somewhat galactose sensitive contrasts with the greatly reduced virulence of galactose-resistant galE mutants of Salmonella typhimurium and Salmonella typhi; this difference may result from the absence of galactose from the O repeat unit in the lipopolysaccharide of group C1 salmonellae.

Magnesium-dependent plaque formation by bacteriophage P1 cinC(-) on Escherichia coli C and Shigella sonnei

Phage P1C(−), in a state of the phage not infective to Escherichia coli K12, was able to form plaques on a wild-type strain of E. coli C and on Shigella sonnei in the presence of Mg 2+. Citrobacter freundii, Enterobacter aerogenes, and a Salmonella typhimurium galE mutant were not lysed by, but were lysogenized with P1 cinC(−), whereas Klebsiella pneumoniae, Proteus rettgeri, and S. typhimurium LT2 were not susceptible to either P1 cinC(−) or P1 cinC(+). The lipopolysaccharide structure of E. coli C and Sh sonnei is discussed with reference to receptors for P1 cinC(−) and P1 cinC(+).

Control of lipopolysaccharide biosynthesis and release by Escherichia coli and Salmonella typhimurium.

The influence of the relA gene on lipopolysaccharide (LPS) biosynthesis and release by Escherichia coli and Salmonella typhimurium was investigated. Similar results were obtained with both species. The incorporation of [3H]galactose into LPS by galE mutants was inhibited by at least 50% (as compared with normal growing controls) during amino acid deprivation of relA+ strains. This inhibition could be prevented by the treatment of the amino acid-deprived relA+ bacteria with chloramphenicol, a known antagonist of the stringent control mechanism. Furthermore, LPS biosynthesis was not inhibited during amino acid deprivation of isogenic relA mutant strains. These results indicate that LPS synthesis is regulated by the stringent control mechanism. Normal growing cells of both relA+ and relA strains released LPS into the culture fluid at low rates. Amino acid deprivation stimulated the rate of LPS release by relA mutants but not by relA+ bacteria. Chloramphenicol treatment markedly stimulated the release of cell-bound LPS by amino acid-deprived relA+ cells. Thus, a low rate of LPS release was characteristic of normal growth and could be increased in nongrowing cells by relaxing the control of LPS synthesis.

Construction of a potential live oral bivalent vaccine for typhoid fever and cholera-Escherichia coli-related diarrheas.

We used the Salmonella typhi galactose epimerase (galE) mutant strain Ty21a, shown to be a safe, effective, living, attenuated oral typhoid vaccine, as a recipient for a recombinant plasmid containing the gene for production of the nontoxic B subunit of the heat-labile enterotoxin of Escherichia coli. The S. typhi derivative, strain SE12, produced heat-labile enterotoxin subunit B that was structurally and immunologically indistinguishable from heat-labile enterotoxin subunit B produced by strains of E. coli harboring the same plasmid. Tests in mice and guinea pigs showed that strain SE12 was safe when given orally and was capable of inducing a significant antitoxic antibody response when injected parenterally. Moreover, it retained the galactose sensitivity of the parent strain, preserving its utility as a typhoid vaccine. This strain may prove to be a useful live oral bivalent vaccine strain for typhoid fever and cholera-E. coli-related diarrheas.

Transformability of galE variants derived from uropathogenic Escherichia coli strains.

Transformation of uropathogenic Escherichia coli strains with plasmid DNA was in general unsuccessful or very inefficient. Transformation was much more efficient when galE mutants of such strains, in which the lipopolysaccharide chains appeared shorter, were used as recipients.