JOURNAL OF BACTERIOLOGY, July 1992, p. 4856-4859
Vol. 174, No. 14
0021-9193/92/144856-04$02.00/0 Copyright © 1992, American Society for Microbiology
Isolation and Characterization of Escherichia coli Mutants Blocked in Production of Membrane-Derived Oligosaccharides AUDREY C. WEISSBORN, MARILYNN K. RUMLEY, AND EUGENE P. KENNEDY* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical Schoo4 Boston, Massachusetts 02115 Received 6 January 1992/Accepted 16 May 1992
We report a new procedure for the facile selection of mutants of Escherichia coli that are blocked in the production of membrane-derived oligosaccharides. Four phenotypic classes were identified, including two with a novel array of characteristics. The mutations mapped to two genetic loci. Mutations in the mdoA region near 23 min are in two distinct genes, only one of which is needed for the membrane-localized glucosyltransferase that catalyzes the synthesis of the beta-1,2-glucan backbone of membrane-derived oligosaccharides. Another set of mutations mapped near 27 min closely linked to osmZ; these appear to be in the galU gene.
Membrane-derived oligosaccharides (MDO) are periplasmic (26), branched glucans of Escherichia coli in which the glucose residues are joined by 13-1,2 and ,B-1,6 linkages (24). They are substituted with sn-1-phosphoglycerol and phosphoethanolamine residues, both derived from membrane phospholipids, and with O-succinyl ester residues (15, 28). The synthesis of MDO is osmotically regulated, with maximum synthesis occurring during growth in medium of low osmolarity (14). MDO may also play a part in the modulation of other osmoregulated systems, including chemotaxis, motility, and production of the colanic acid-containing capsule and of outer membrane porins (8). The enzymology and genetics of MDO biosynthesis are poorly understood. A membrane glucosyltransferase catalyzes the transfer of glucose units from UDP-glucose to a ,B-glucoside "primer" with the formation of P-1,2-glucan chains, thought to represent the backbone of MDO (29). This enzyme requires acyl carrier protein for maximal activity (27) and is stimulated by the addition of polyprenylphosphate (30). Strain TlOGP, constructed by Pluschke et al. (20), was fortuitously discovered by Raetz's laboratory to be defective in MDO production. This strain was found to be missing the membrane glucosyltransferase (29), and the mutation, mdoAl, was mapped to the 23-min region of the E. coli chromosome (2). Subsequently, Bohin and colleagues cloned a 5.5-kb piece of DNA that complements both the mdoAl mutation and also a second mdoA region mutation, mdo-200::TnlO, of identical phenotype (17). Further, they clearly demonstrated (16) that two genes (mdoG and mdoH) are present in the mdoA region by using insertion mutagenesis of an mdoA-containing plasmid and subsequent complementation analysis. The mdoAl and mdoA200::TnlO allele belong to the mdoH complementation group. Fiedler and Rotering (8) recently selected mutants in MDO biosynthesis based on their increased resistance to bacitracin. These mutations also mapped to the 23-min region. They made the important observation that strains lacking MDO also displayed additional phenotypes. Such mutants grew as mucoid colonies on minimal medium, and they no longer exhibited swarming behavior on soft agar plates. Under *
Corresponding author. 4856
certain conditions of culture, MDO mutants also displayed an altered pattern of expression of outer membrane porins OmpF and OmpC. A membrane-localized enzyme, phosphoglycerol transferase I, transfers sn-1-glycerophosphate from phosphatidylglycerol to the glucan backbone of MDO (12). A periplasmic enzyme, phosphoglycerol transferase II (9), cannot use phosphatidylglycerol as a substrate and appears to redistribute sn-1-glycerophosphate residues among periplasmic and membrane-bound MDO species. Mutants in phosphoglycerol transferase I have been isolated by direct selection (11) or a screening method (7, 22), and their mutation was mapped to the mdoB locus, at 99 min on the E. coli chromosome. Interestingly, such mutants exhibit normal swarming behavior on soft agar plates (7). To advance the genetic analysis of MDO biosynthesis, essential to a better understanding of enzymology and function, we have developed a facile, new method for the positive selection of mutants in MDO biosynthesis, and we have characterized a number of the mutants so obtained. Isolation of mutants in MDO biosynthesis. Cells of strain RZ60 dgk-6 (Table 1) lack the enzyme diacylglycerol kinase. The resultant accumulation of diacylglycerol derived principally from the transfer of phospholipid head groups (phosphoglycerol and phosphoethanolamine) to MDO slows the growth of this strain in medium of low osmolarity (21) because the synthesis of MDO and consequently the formation of diacylglycerol are maximal under these conditions. A further mutation that blocks the formation of MDO glucan chains or the transfer of phospholipid head groups to MDO should permit more rapid growth of Dgk- cells in medium of low osmolarity, offering a positive selection for such mutants. A culture of RZ60 dgk-6 was mutagenized with nitrous acid (4). The fraction of viable cells after mutagenesis was about 2 x 10-3. To allow expression of mutations, the culture was incubated in LB medium at 37°C with vigorous aeration. for 90 min (three doubling times). The mutagenized cells were then concentrated by centrifugation and resuspended in a small volume of 1% (wt/vol) Bacto Peptone. From this suspension, 0.05-ml aliquots (about 6 x 106 viable cells) were spread on agar plates containing 1% Bacto Peptone. The plates were examined after 16 h at 37°C and
NOTES
VOL. 174, 1992
TABLE 2. Classification of mutants
TABLE 1. List of bacteria and plasmids Strain or
plasmid Strains JW380
Relevant characteristicsa
Presence (+) or absence (-)
Source or reference
Class
zch-506::TnlO
NFB202 zcd-202::TnlO RZ60 dgk-6 Plasmids pNF239 8-kb fragment including the mdoA region, Cb r pRI40 Low-copy-number vector, Spr
B. Bachmann, CGSC 6094 J.-P. Bohin (2) C. Raetz (21)
17 Via H. P.
4857
Spaink (11)
of characteristic Map Nof position iNolatef Membrane (min) glucosyl- Mucoidy
Growth on ga-
ing
g
transferase
Ia Ib II (mdoA451) III (mdoX452) IV
27 6 1 1 3
23 23 23 27 27
+ + +
lactose
+ + +
-
+ + + +
-
-
-
a Abbreviations: Cb, carbenicillin; Sp, spectinomycin.
had 6 to 70 well-defined clones of various sizes per plate. An unmutagenized culture of RZ60 gave only 0 to 2 clones per plate. It was important to read the plates after 16 h, because after longer incubation, wild-type clones also appear. The apparently mutant early-appearing clones were purified by streaking for single colonies on 1% Bacto Peptone agar plates. Well-defined, single clones that appeared after 16 h at 37°C were selected and screened for the ability to produce MDO by the following rapid semiquantitative method, which depends on the fact that MDO are readily labeled from [2-3H]glycerol added to the medium. The clones to be tested were inoculated into 1 ml of 1% Bacto Peptone medium supplemented with 50 mM NaCl and 0.1 mM [2-3H]glycerol (about 2 x 106 cpm). The culture was grown overnight at 37°C with aeration. The cells were collected by centrifugation at room temperature and washed twice with growth medium containing unlabeled glycerol. The cells were resuspended in 0.25 ml of 10 mg of bovine serum albumin per ml. A sample (0.025 ml) was removed and counted to measure total cellular radioactivity. The remainder of the cells was treated with 0.025 ml of 33% (wt/vol) trichloroacetic acid. After 30 min, the precipitate was removed by centrifugation. A sample (0.2 ml) of the trichloroacetic acid extract was applied to a column containing exactly 1 ml of Sephadex G-25 medium, suspended in 7% (vol/vol) 1-propanol in a Pasteur pipet. The column was eluted with 0.6 ml of 7% (vol/vol) 1-propanol. The radioactivity in the eluate was then measured. Prior experiments had established that the first 0.8 ml eluted from such a column contains most of the labeled MDO extracted from the culture with only minor non-MDO radiolabeled contaminants. The selection of the first clones to appear on low-osmolarity plates proved to be a very successful strategy for selection of Mdo- mutants. Of 94 clones growing more rapidly than the parent, RZ60, on low osmolarity plates, 46 produced less than 11% of the labeled MDO of the parent strain RZ60. The remaining rapidly growing isolates probably include revertants to Dgk+, but this point was not investigated further. The selection pressure is strong enough so that unmutagenized cultures also yield more rapidly appearing clones, although in lower frequency. Assay of phosphoglycerol transferase I. Of the 46 isolates described above, 38 that were found to have stable phenotypes and little or no production of MDO were characterized in detail. They were first tested for activity of phosphoglycerol transferase I by the method of Bohin and Kennedy (3), which is based on the transfer of labeled phosphoglycerol in vivo to the model substrate arbutin added to the medium. All had activity comparable to that of the wild type and were therefore mdoB+. The failure of the mutants to produce
[2-3H]glycerol-labeled MDO must be attributed to defects in the production of the glucan acceptor for phosphoglycerol transferase I. It may at first seem unexpected that the present selection procedure did not lead to the isolation of mdoB mutants which lack phosphoglycerol transferase I and thus have eliminated an important source of diacylglycerol that accumulates and slows the growth of dgk cells. However, it appears that if phosphoglycerol transferase I is missing (11) in the dgk background, the accumulation of diacylglycerol is not substantially reduced and growth in low-osmolarity medium is still poor, presumably because the transfer of phosphoethanolamine from phosphatidylethanolamine to the glucan backbone is increased in mdoB mutants (7). Only mutations that block the synthesis of the glucan backbone, thus preventing the transfer of both phosphoethanolamine and of phosphoglycerol residues, reduce the diacylglycerol content of dgk mutants sufficiently to increase the growth rate significantly, as shown first by Raetz and Newman (21) with the pgi mutation and here, as will be shown below, by the isolation of a range of different mutants in which the glucan chains are not formed. Assay of glucosyltransferase activity. Membranes were prepared as previously described (30) from the 38 stable isolates, and their glucosyltransferase activity was assayed (30). The majority of the isolates (33 isolates) lacked glucosyltransferase activity (Table 2). The remaining five showed essentially the activity of the parental strain. Two of these isolates that have wild-type glucosyltransferase activity but fail to produce MDO glucan chains represent mutants with a novel phenotypic array (class II and III [Table 2]) as will be described below. Utilization of galactose. Class IV isolates, also with wildtype transglucosylase activity, were the only class of mutants that did not grow on galactose as a carbon source (Table 2). We found that P1 transduction of the Tetr element, zch-506::TnlO (Table 1), which is closely linked to the galU locus near 27 min (18), restored MDO production in the class IV mutants. The phenotype of the class IV mutants suggested that they might be defective in the enzyme glucose1-phosphate uridylyltransferase. This enzyme is needed for the synthesis of UDP-glucose, which is an essential intermediate for galactose metabolism, MDO synthesis (25), and colanic acid synthesis (19). When the glucose-i-phosphate uridylyltransferase activity of the class IV mutants was assayed, activities about 75% of that of the wild type were found. Closer study, however, revealed that this residual activity is that of an enzyme with higher affinity for TTP than for UTP, presumably glucose-i-phosphate thymidylyltransferase (data not shown). Class IV mutations are therefore considered alleles of the galU gene.
4858
NOTES
Strains bearing the class III mdoX452 allele are Gal' and thus distinctly different from the galU mutants of class IV. Genetic mapping by conjugation and P1 transduction (data not shown) revealed, however, that mdoX, like galU, is closely linked to but distinct from the osmZ locus, at 27 min. Growth on galactose provides an alternative supply of glucose-i-phosphate via galactose-1-phosphate. If the mdoX452 mutation is an allele of galU encoding an enzyme with reduced affinity for glucose-i-phosphate, growth on galactose might sufficiently increase levels of glucose-iphosphate to permit the synthesis of UDP-glucose, needed both for growth on galactose and for MDO production. To test this idea, the mdoX452 mutant was examined for MDO production in medium supplemented with galactose. Supplementation with galactose restored MDO production to about one-third the level of the wild-type parent. It seems likely that the mdoX452 mutation and the class IV mutations are alleles of the galU gene, but this will be further examined as part of a comprehensive study of the galU locus presently under way. Swarming behavior. When the swarming behavior of the 38 isolates was tested on tryptone soft agar plates as described by Adler (1), all failed to produce the clearly delineated 2 to 3 swarm rings of the parental strain. This corroborates the important finding of Fiedler and Rotering (8) that strains lacking in MDO are impaired in motility or chemotaxis. Although all were clearly defective in swarming behavior as compared to the wild type, about one-fifth of the isolates appeared to have swarming ability intermediate between that of the wild type and the completely defective swarming of the majority of the mutants. This intermediate swarming phenotype was not due to an incomplete block in MDO production and is presently not understood. Colanic acid production. Fiedler and Rotering (8) also reported that their mutants lacking MDO produced mucoid colonies when grown at 27°C on minimal medium plates. The 38 mutants were examined for this phenotype both by examination of colony morphology on minimal medium plates and by direct determination of the amount of capsular polysaccharide produced after growth for 4 days at 30°C in the low-nitrogen medium of Duguid and Wilkinson (6). For the latter determination, the colanic acid was prepared as described by Kang and Markovitz (13) and the quantitation of its component sugar, fucose, was done by the method of Dische and Shettles (5). Twenty-eight mutants were found to have the mucoid phenotype. However, 10 (class Ib, III, or IV) were found to produce little or no capsular polysaccharide. When the mdoX452 allele listed in class III and a representative class IV mutation (Table 2) were moved into other genetic backgrounds as described below, the nonmucoid phenotype always accompanied the Mdo- phenotype, consistent with the interpretation that these are galU mutations. In contrast, the failure of the isolates of class Ib to produce capsule is more complex. When certain of these mutations were moved into other backgrounds by P1 transduction, the Mdo- transductants were in fact mucoid, suggesting either that the original isolates contained two distinct mutations, one affecting MDO production and the other mucoidy, or that the nonmucoid phenotype of these class Tb mutants is expressed only in certain genetic backgrounds. Localization of mutations to the mdoA region. Each of the class I and class II isolates was transduced with a Plvir lysate prepared on NFB202, which contains the zcd202::TnlO insertion closely linked to the mdoA region (2). After selection for tetracycline resistance, the transductants were screened for their mucoid phenotype (class Ia and II) or
J.
BACT1EPRIOL.
swarming behavior (class Tb). All members of classes I and II (Table 2) could be transduced to the corresponding wild-type phenotype, with an average cotransduction frequency of 93% between tetracycline resistance and the wild-type phenotype. Subsequent analysis of a tetracycline-resistant and nonmucoid or wild-type swarming transductant from each member of class I and II for the production of [2-3H]glycerollabeled MDO confirmed that MDO was produced in these clones. The swarming phenotype of the Mdo+ transductants from a representative number of class I mutants that were initially identified by their nonmucoid character was checked, and in each case it had returned to the wild-type behavior. That the mutations of classes I and II are in the mdoA region was confirmed by the finding that they can be complemented by the plasmid pNF239 (17) containing an 8.0-kb piece of DNA that includes the known mdoA region. The 8.0-kb insert of pNF239 was subcloned by standard procedures (23) into a low-copy-number plasmid, pRI40 (10), because the RZ60 dgk-6 background did not appear to tolerate the original multicopy plasmid which allows an elevated level of MDO synthesis. The mutants that contained the plasmid now produced MDO as measured by the [2-3H]glycerol-labeling test for MDO synthesis and were nonmucoid. Some of the transformants were also tested for their swarming phenotype; all showed the wild-type pattern. The mdoA locus has been recently reported (16) to contain at least two genes, designated mdoG and mdoH. Lesions in mdoH cause defects in the membrane glucosyltransferase. Class I mutants may therefore be assigned to the mdoH gene. The class II mutant (mdoA451 allele) although it is MDO- and has the mucoid and defective swarming phenotype, retains glucosyltransferase activity. It is probably allelic to the mutation mdoG202::neo of Lacroix et al. (16), which, like the class II mutation, blocks MDO production without affecting the membrane glucosyltransferase. Summary. We describe a new method for the positive selection of mutants in the synthesis of MDO that is based on the fact that MDO production slows the growth of Dgkcells in low-osmolarity medium. The utility of this method is shown by the isolation of four different phenotypic classes of mutants whose mutations map to two different loci in the chromosome. We thank J.-P. Bohin for generously providing strains and the
plasmid pNF239 and Barbara Bachmann for the many strains used in the genetic mapping. We thank Herman P. Spaink for his invaluable help in the subcloning of the mdoA fragment. This work was supported by grants GM19822 and GM22057 from the National Institute of General Medical Sciences.
REFERENCES 1. Adler, J. 1966. Chemotaxis in bacteria. Science 153:708-716. 2. Bohin, J.-P., and E. P. Kennedy. 1984. Mapping of a locus (mdoA) that affects the biosynthesis of membrane-derived oli-
gosaccharides in Escherichia coli. J. Bacteriol. 157:956-957.
3. Bohin, J.-P., and E. P. Kennedy. 1984. Regulation of the synthesis of membrane-derived oligosaccharides in Escherichia coli. Assay of phosphoglycerol transferase I in vivo. J. Biol. Chem. 259:8388-8393. 4. Carlton, B. C., and B. J. Brown. 1981. Gene mutation, p. 227. In P. Gerhardt (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. 5. Dische, Z., and L. B. Shettles. 1948. A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J. Biol. Chem. 175:595-603. 6. Duguid, J. P., and J. F. Wdikinson. 1953. The influence of cultural conditions on polysaccharide production byAerobacter
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aerogenes. J. Gen. Microbiol. 9:174-189. 7. Fiedler, W., and H. Rotering. 1985. Characterization of an Eschenichia coli mdoB mutant strain unable to transfer sn-1phosphoglycerol to membrane-derived oligosaccharides. J. Biol. Chem. 260:4799-4806. 8. Fiedler, W., and H. Rotering. 1988. Properties of Escherichia coli mutants lacking membrane-derived oligosaccharides. J. Biol. Chem. 263:14684-14689. 9. Goldberg, D. E., M. K. Rumley, and E. P. Kennedy. 1981. Biosynthesis of membrane-derived oligosaccharides: a periplasmic phosphoglyceroltransferase. Proc. Natl. Acad. Sci. USA 78:5513-5517. 10. Innes, R. W., M. A. Hirose, and P. L. Kuempel. 1988. Induction of nitrogen-fixing nodules on clover requires only 32 kilobases of DNA from the Rhizobium tnifolii symbiosis plasmid. J. Bacteriol. 170:3793-3802. 11. Jackson, B. J., J.-P. Bohin, and E. P. Kennedy. 1984. Biosynthesis of membrane-derived oligosaccharides: characterization of mdoB mutants defective in phosphoglycerol transferase I activity. J. Bacteriol. 160:976-981. 12. Jackson, B. J., and E. P. Kennedy. 1983. The biosynthesis of membrane-derived oligosaccharides. A membrane-bound phosphoglycerol transferase. J. Biol. Chem. 258:2394-2398. 13. Kang, S., and A. Markovitz. 1967. Induction of capsular polysaccharide synthesis by p-fluorophenylalanine in Escherichia coli wild type and strains with altered phenylalanyl soluble ribonucleic acid synthetase. J. Bacteriol. 93:584-591. 14. Kennedy, E. P. 1982. Osmotic regulation and the biosynthesis of membrane-derived oligosaccharides in Escherichia coli. Proc. Natl. Acad. Sci. USA 79:1092-1095. 15. Kennedy, E. P., M. K. Rumley, H. Schulman, and L. M. G. van Golde. 1976. Identification of sn-glycero-1-phosphate and phosphoethanolamine residues linked to the membrane-derived oligosaccharides of Escherichia coli. J. Biol. Chem. 251:42084213. 16. Lacroix, J. M., I. Loubens, M. Tempete, B. Menichi, and J.-P. Bohin. 1991. The mdoA locus of Escherichia coli consists of an operon under osmotic control. Mol. Microbiol. 5:1745-1753. 17. Lacroix, J. M., M. Tempete, B. Menichi, and J.-P. Bohin. 1989. Molecular cloning and expression of a locus (mdoA) implicated in the biosynthesis of membrane-derived oligosaccharides in Escherichia coli. Mol. Microbiol. 3:1173-1182. 18. Lejeune, P., P. Bertin, C. Walon, K. Willemot, C. Colson, and A. Danchin. 1989. A locus involved in kanamycin, chloramphenicol and L-serine resistance is located in the bglY-galU region of the Escherichia coli K12 chromosome. Mol. Gen. Genet. 218:361363. 19. Markovitz, A. 1977. Genetics and regulation of bacterial capsu-
20. 21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
4859
lar polysaccharide biosynthesis and radiation sensitivity, p. 415-462. In I. Sutherland (ed.), Surface carbohydrates of the prokaryotic cell. Academic Press, New York. Pluschke, G., Y. Hirota, and P. Overath. 1978. Function of phospholipids in Escherichia coli. Characterization of a mutant deficient in cardiolipin synthesis. J. Biol. Chem. 253:5048-5055. Raetz, C. R. H., and K. F. Newman. 1979. Diglyceride kinase mutants of Escherichia coli: inner membrane association of 1,2-diglyceride and its relation to synthesis of membrane-derived oligosaccharides. J. Bacteriol. 137:860-868. Rotering, H., W. Fiedler, W. Rollinger, and V. Braun. 1984. Procedure for the identification of Escherichia coli mutants affected in components containing glycerol derived from phospholipid turnover: isolation of mutants lacking glycerol in membrane-derived oligosaccharides (MDO). FEMS Microbiol. Lett. 22:61-68. Sambrook, J., E. F. Fritsch, and T. Maniatis (ed.). 1989. Molecular cloning: a laboratory manual, 2nd ed., vol. 1 and 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Schneider, J. E., V. Reinhold, M. K. Rumley, and E. P. Kennedy. 1979. Structural studies of the membrane-derived oligosaccharides of Eschenichia coli. J. Biol. Chem. 254:1013510138. Schulman, H., and E. P. Kennedy. 1977. Identification of UDPglucose as an intermediate in the biosynthesis of the membranederived oligosaccharides of Escherichia coli. J. Biol. Chem. 252:6299-6303. Schulman, H., and E. P. Kennedy. 1979. Localization of membrane-derived oligosaccharides in the outer envelope of Eschenichia coli and their occurrence in other gram-negative bacteria. J. Bacteriol. 137:686-688. Therisod, H., A. C. Weissborn, and E. P. Kennedy. 1986. An essential function for acyl carrier protein in the biosynthesis of membrane-derived oligosaccharides of Escherichia coli. Proc. Natl. Acad. Sci. USA 83:7236-7240. Van Golde, L. M. G., H. Schulman, and E. P. Kennedy. 1973. Metabolism of membrane phospholipids and its relation to a novel class of oligosaccharides in Escherichia coli. Proc. Natl. Acad. Sci. USA 70:1368-1372. Weissborn, A. C., and E. P. Kennedy. 1984. Biosynthesis of membrane-derived oligosaccharides. Novel glucosyltransferase system from Escherichia coli for the elongation of P1-2 linked polyglucose chains. J. Biol. Chem. 259:12644 12651. Weissborn, A. C., M. K. Rumley, and E. P. Kennedy. 1991. Biosynthesis of membrane-derived oligosaccharides: membrane-bound glucosyl transferase system from Escherichia coli requires polyprenyl phosphate. J. Biol. Chem. 266:8062-8067.
Vol. 174, No. 14
0021-9193/92/144856-04$02.00/0 Copyright © 1992, American Society for Microbiology
Isolation and Characterization of Escherichia coli Mutants Blocked in Production of Membrane-Derived Oligosaccharides AUDREY C. WEISSBORN, MARILYNN K. RUMLEY, AND EUGENE P. KENNEDY* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical Schoo4 Boston, Massachusetts 02115 Received 6 January 1992/Accepted 16 May 1992
We report a new procedure for the facile selection of mutants of Escherichia coli that are blocked in the production of membrane-derived oligosaccharides. Four phenotypic classes were identified, including two with a novel array of characteristics. The mutations mapped to two genetic loci. Mutations in the mdoA region near 23 min are in two distinct genes, only one of which is needed for the membrane-localized glucosyltransferase that catalyzes the synthesis of the beta-1,2-glucan backbone of membrane-derived oligosaccharides. Another set of mutations mapped near 27 min closely linked to osmZ; these appear to be in the galU gene.
Membrane-derived oligosaccharides (MDO) are periplasmic (26), branched glucans of Escherichia coli in which the glucose residues are joined by 13-1,2 and ,B-1,6 linkages (24). They are substituted with sn-1-phosphoglycerol and phosphoethanolamine residues, both derived from membrane phospholipids, and with O-succinyl ester residues (15, 28). The synthesis of MDO is osmotically regulated, with maximum synthesis occurring during growth in medium of low osmolarity (14). MDO may also play a part in the modulation of other osmoregulated systems, including chemotaxis, motility, and production of the colanic acid-containing capsule and of outer membrane porins (8). The enzymology and genetics of MDO biosynthesis are poorly understood. A membrane glucosyltransferase catalyzes the transfer of glucose units from UDP-glucose to a ,B-glucoside "primer" with the formation of P-1,2-glucan chains, thought to represent the backbone of MDO (29). This enzyme requires acyl carrier protein for maximal activity (27) and is stimulated by the addition of polyprenylphosphate (30). Strain TlOGP, constructed by Pluschke et al. (20), was fortuitously discovered by Raetz's laboratory to be defective in MDO production. This strain was found to be missing the membrane glucosyltransferase (29), and the mutation, mdoAl, was mapped to the 23-min region of the E. coli chromosome (2). Subsequently, Bohin and colleagues cloned a 5.5-kb piece of DNA that complements both the mdoAl mutation and also a second mdoA region mutation, mdo-200::TnlO, of identical phenotype (17). Further, they clearly demonstrated (16) that two genes (mdoG and mdoH) are present in the mdoA region by using insertion mutagenesis of an mdoA-containing plasmid and subsequent complementation analysis. The mdoAl and mdoA200::TnlO allele belong to the mdoH complementation group. Fiedler and Rotering (8) recently selected mutants in MDO biosynthesis based on their increased resistance to bacitracin. These mutations also mapped to the 23-min region. They made the important observation that strains lacking MDO also displayed additional phenotypes. Such mutants grew as mucoid colonies on minimal medium, and they no longer exhibited swarming behavior on soft agar plates. Under *
Corresponding author. 4856
certain conditions of culture, MDO mutants also displayed an altered pattern of expression of outer membrane porins OmpF and OmpC. A membrane-localized enzyme, phosphoglycerol transferase I, transfers sn-1-glycerophosphate from phosphatidylglycerol to the glucan backbone of MDO (12). A periplasmic enzyme, phosphoglycerol transferase II (9), cannot use phosphatidylglycerol as a substrate and appears to redistribute sn-1-glycerophosphate residues among periplasmic and membrane-bound MDO species. Mutants in phosphoglycerol transferase I have been isolated by direct selection (11) or a screening method (7, 22), and their mutation was mapped to the mdoB locus, at 99 min on the E. coli chromosome. Interestingly, such mutants exhibit normal swarming behavior on soft agar plates (7). To advance the genetic analysis of MDO biosynthesis, essential to a better understanding of enzymology and function, we have developed a facile, new method for the positive selection of mutants in MDO biosynthesis, and we have characterized a number of the mutants so obtained. Isolation of mutants in MDO biosynthesis. Cells of strain RZ60 dgk-6 (Table 1) lack the enzyme diacylglycerol kinase. The resultant accumulation of diacylglycerol derived principally from the transfer of phospholipid head groups (phosphoglycerol and phosphoethanolamine) to MDO slows the growth of this strain in medium of low osmolarity (21) because the synthesis of MDO and consequently the formation of diacylglycerol are maximal under these conditions. A further mutation that blocks the formation of MDO glucan chains or the transfer of phospholipid head groups to MDO should permit more rapid growth of Dgk- cells in medium of low osmolarity, offering a positive selection for such mutants. A culture of RZ60 dgk-6 was mutagenized with nitrous acid (4). The fraction of viable cells after mutagenesis was about 2 x 10-3. To allow expression of mutations, the culture was incubated in LB medium at 37°C with vigorous aeration. for 90 min (three doubling times). The mutagenized cells were then concentrated by centrifugation and resuspended in a small volume of 1% (wt/vol) Bacto Peptone. From this suspension, 0.05-ml aliquots (about 6 x 106 viable cells) were spread on agar plates containing 1% Bacto Peptone. The plates were examined after 16 h at 37°C and
NOTES
VOL. 174, 1992
TABLE 2. Classification of mutants
TABLE 1. List of bacteria and plasmids Strain or
plasmid Strains JW380
Relevant characteristicsa
Presence (+) or absence (-)
Source or reference
Class
zch-506::TnlO
NFB202 zcd-202::TnlO RZ60 dgk-6 Plasmids pNF239 8-kb fragment including the mdoA region, Cb r pRI40 Low-copy-number vector, Spr
B. Bachmann, CGSC 6094 J.-P. Bohin (2) C. Raetz (21)
17 Via H. P.
4857
Spaink (11)
of characteristic Map Nof position iNolatef Membrane (min) glucosyl- Mucoidy
Growth on ga-
ing
g
transferase
Ia Ib II (mdoA451) III (mdoX452) IV
27 6 1 1 3
23 23 23 27 27
+ + +
lactose
+ + +
-
+ + + +
-
-
-
a Abbreviations: Cb, carbenicillin; Sp, spectinomycin.
had 6 to 70 well-defined clones of various sizes per plate. An unmutagenized culture of RZ60 gave only 0 to 2 clones per plate. It was important to read the plates after 16 h, because after longer incubation, wild-type clones also appear. The apparently mutant early-appearing clones were purified by streaking for single colonies on 1% Bacto Peptone agar plates. Well-defined, single clones that appeared after 16 h at 37°C were selected and screened for the ability to produce MDO by the following rapid semiquantitative method, which depends on the fact that MDO are readily labeled from [2-3H]glycerol added to the medium. The clones to be tested were inoculated into 1 ml of 1% Bacto Peptone medium supplemented with 50 mM NaCl and 0.1 mM [2-3H]glycerol (about 2 x 106 cpm). The culture was grown overnight at 37°C with aeration. The cells were collected by centrifugation at room temperature and washed twice with growth medium containing unlabeled glycerol. The cells were resuspended in 0.25 ml of 10 mg of bovine serum albumin per ml. A sample (0.025 ml) was removed and counted to measure total cellular radioactivity. The remainder of the cells was treated with 0.025 ml of 33% (wt/vol) trichloroacetic acid. After 30 min, the precipitate was removed by centrifugation. A sample (0.2 ml) of the trichloroacetic acid extract was applied to a column containing exactly 1 ml of Sephadex G-25 medium, suspended in 7% (vol/vol) 1-propanol in a Pasteur pipet. The column was eluted with 0.6 ml of 7% (vol/vol) 1-propanol. The radioactivity in the eluate was then measured. Prior experiments had established that the first 0.8 ml eluted from such a column contains most of the labeled MDO extracted from the culture with only minor non-MDO radiolabeled contaminants. The selection of the first clones to appear on low-osmolarity plates proved to be a very successful strategy for selection of Mdo- mutants. Of 94 clones growing more rapidly than the parent, RZ60, on low osmolarity plates, 46 produced less than 11% of the labeled MDO of the parent strain RZ60. The remaining rapidly growing isolates probably include revertants to Dgk+, but this point was not investigated further. The selection pressure is strong enough so that unmutagenized cultures also yield more rapidly appearing clones, although in lower frequency. Assay of phosphoglycerol transferase I. Of the 46 isolates described above, 38 that were found to have stable phenotypes and little or no production of MDO were characterized in detail. They were first tested for activity of phosphoglycerol transferase I by the method of Bohin and Kennedy (3), which is based on the transfer of labeled phosphoglycerol in vivo to the model substrate arbutin added to the medium. All had activity comparable to that of the wild type and were therefore mdoB+. The failure of the mutants to produce
[2-3H]glycerol-labeled MDO must be attributed to defects in the production of the glucan acceptor for phosphoglycerol transferase I. It may at first seem unexpected that the present selection procedure did not lead to the isolation of mdoB mutants which lack phosphoglycerol transferase I and thus have eliminated an important source of diacylglycerol that accumulates and slows the growth of dgk cells. However, it appears that if phosphoglycerol transferase I is missing (11) in the dgk background, the accumulation of diacylglycerol is not substantially reduced and growth in low-osmolarity medium is still poor, presumably because the transfer of phosphoethanolamine from phosphatidylethanolamine to the glucan backbone is increased in mdoB mutants (7). Only mutations that block the synthesis of the glucan backbone, thus preventing the transfer of both phosphoethanolamine and of phosphoglycerol residues, reduce the diacylglycerol content of dgk mutants sufficiently to increase the growth rate significantly, as shown first by Raetz and Newman (21) with the pgi mutation and here, as will be shown below, by the isolation of a range of different mutants in which the glucan chains are not formed. Assay of glucosyltransferase activity. Membranes were prepared as previously described (30) from the 38 stable isolates, and their glucosyltransferase activity was assayed (30). The majority of the isolates (33 isolates) lacked glucosyltransferase activity (Table 2). The remaining five showed essentially the activity of the parental strain. Two of these isolates that have wild-type glucosyltransferase activity but fail to produce MDO glucan chains represent mutants with a novel phenotypic array (class II and III [Table 2]) as will be described below. Utilization of galactose. Class IV isolates, also with wildtype transglucosylase activity, were the only class of mutants that did not grow on galactose as a carbon source (Table 2). We found that P1 transduction of the Tetr element, zch-506::TnlO (Table 1), which is closely linked to the galU locus near 27 min (18), restored MDO production in the class IV mutants. The phenotype of the class IV mutants suggested that they might be defective in the enzyme glucose1-phosphate uridylyltransferase. This enzyme is needed for the synthesis of UDP-glucose, which is an essential intermediate for galactose metabolism, MDO synthesis (25), and colanic acid synthesis (19). When the glucose-i-phosphate uridylyltransferase activity of the class IV mutants was assayed, activities about 75% of that of the wild type were found. Closer study, however, revealed that this residual activity is that of an enzyme with higher affinity for TTP than for UTP, presumably glucose-i-phosphate thymidylyltransferase (data not shown). Class IV mutations are therefore considered alleles of the galU gene.
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NOTES
Strains bearing the class III mdoX452 allele are Gal' and thus distinctly different from the galU mutants of class IV. Genetic mapping by conjugation and P1 transduction (data not shown) revealed, however, that mdoX, like galU, is closely linked to but distinct from the osmZ locus, at 27 min. Growth on galactose provides an alternative supply of glucose-i-phosphate via galactose-1-phosphate. If the mdoX452 mutation is an allele of galU encoding an enzyme with reduced affinity for glucose-i-phosphate, growth on galactose might sufficiently increase levels of glucose-iphosphate to permit the synthesis of UDP-glucose, needed both for growth on galactose and for MDO production. To test this idea, the mdoX452 mutant was examined for MDO production in medium supplemented with galactose. Supplementation with galactose restored MDO production to about one-third the level of the wild-type parent. It seems likely that the mdoX452 mutation and the class IV mutations are alleles of the galU gene, but this will be further examined as part of a comprehensive study of the galU locus presently under way. Swarming behavior. When the swarming behavior of the 38 isolates was tested on tryptone soft agar plates as described by Adler (1), all failed to produce the clearly delineated 2 to 3 swarm rings of the parental strain. This corroborates the important finding of Fiedler and Rotering (8) that strains lacking in MDO are impaired in motility or chemotaxis. Although all were clearly defective in swarming behavior as compared to the wild type, about one-fifth of the isolates appeared to have swarming ability intermediate between that of the wild type and the completely defective swarming of the majority of the mutants. This intermediate swarming phenotype was not due to an incomplete block in MDO production and is presently not understood. Colanic acid production. Fiedler and Rotering (8) also reported that their mutants lacking MDO produced mucoid colonies when grown at 27°C on minimal medium plates. The 38 mutants were examined for this phenotype both by examination of colony morphology on minimal medium plates and by direct determination of the amount of capsular polysaccharide produced after growth for 4 days at 30°C in the low-nitrogen medium of Duguid and Wilkinson (6). For the latter determination, the colanic acid was prepared as described by Kang and Markovitz (13) and the quantitation of its component sugar, fucose, was done by the method of Dische and Shettles (5). Twenty-eight mutants were found to have the mucoid phenotype. However, 10 (class Ib, III, or IV) were found to produce little or no capsular polysaccharide. When the mdoX452 allele listed in class III and a representative class IV mutation (Table 2) were moved into other genetic backgrounds as described below, the nonmucoid phenotype always accompanied the Mdo- phenotype, consistent with the interpretation that these are galU mutations. In contrast, the failure of the isolates of class Ib to produce capsule is more complex. When certain of these mutations were moved into other backgrounds by P1 transduction, the Mdo- transductants were in fact mucoid, suggesting either that the original isolates contained two distinct mutations, one affecting MDO production and the other mucoidy, or that the nonmucoid phenotype of these class Tb mutants is expressed only in certain genetic backgrounds. Localization of mutations to the mdoA region. Each of the class I and class II isolates was transduced with a Plvir lysate prepared on NFB202, which contains the zcd202::TnlO insertion closely linked to the mdoA region (2). After selection for tetracycline resistance, the transductants were screened for their mucoid phenotype (class Ia and II) or
J.
BACT1EPRIOL.
swarming behavior (class Tb). All members of classes I and II (Table 2) could be transduced to the corresponding wild-type phenotype, with an average cotransduction frequency of 93% between tetracycline resistance and the wild-type phenotype. Subsequent analysis of a tetracycline-resistant and nonmucoid or wild-type swarming transductant from each member of class I and II for the production of [2-3H]glycerollabeled MDO confirmed that MDO was produced in these clones. The swarming phenotype of the Mdo+ transductants from a representative number of class I mutants that were initially identified by their nonmucoid character was checked, and in each case it had returned to the wild-type behavior. That the mutations of classes I and II are in the mdoA region was confirmed by the finding that they can be complemented by the plasmid pNF239 (17) containing an 8.0-kb piece of DNA that includes the known mdoA region. The 8.0-kb insert of pNF239 was subcloned by standard procedures (23) into a low-copy-number plasmid, pRI40 (10), because the RZ60 dgk-6 background did not appear to tolerate the original multicopy plasmid which allows an elevated level of MDO synthesis. The mutants that contained the plasmid now produced MDO as measured by the [2-3H]glycerol-labeling test for MDO synthesis and were nonmucoid. Some of the transformants were also tested for their swarming phenotype; all showed the wild-type pattern. The mdoA locus has been recently reported (16) to contain at least two genes, designated mdoG and mdoH. Lesions in mdoH cause defects in the membrane glucosyltransferase. Class I mutants may therefore be assigned to the mdoH gene. The class II mutant (mdoA451 allele) although it is MDO- and has the mucoid and defective swarming phenotype, retains glucosyltransferase activity. It is probably allelic to the mutation mdoG202::neo of Lacroix et al. (16), which, like the class II mutation, blocks MDO production without affecting the membrane glucosyltransferase. Summary. We describe a new method for the positive selection of mutants in the synthesis of MDO that is based on the fact that MDO production slows the growth of Dgkcells in low-osmolarity medium. The utility of this method is shown by the isolation of four different phenotypic classes of mutants whose mutations map to two different loci in the chromosome. We thank J.-P. Bohin for generously providing strains and the
plasmid pNF239 and Barbara Bachmann for the many strains used in the genetic mapping. We thank Herman P. Spaink for his invaluable help in the subcloning of the mdoA fragment. This work was supported by grants GM19822 and GM22057 from the National Institute of General Medical Sciences.
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