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

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

Properties of Escherichia coli mutants lacking membrane-derived oligosaccharides

Membrane-derived oligosaccharides (MDO) consist of branched substituted beta-glucan chains and are present in the periplasmic space of Escherichia coli and other gram-negative bacteria. A procedure for the isolation of mutants defective in MDO synthesis is described. Their phenotype was compared with a mdoA mutant previously identified, and they are mapped in the mdoA region. Mutants lacking MDO showed imparied chemotaxis on tryptone swarm plates, a reduced number of flagella, and an enhanced expression of the OmpC porin. Revertants able to form swarm rings again had regained the ability to synthesize MDO and showed the wild-type porin pattern. A second group of chemotactic revertants were mutated in the ompB gene region involved in osmoregulation, and they were still devoid of MDO. These findings provide evidence for a link between MDO biosynthesis and other functions of E. coli related to its adaptation to the environment.     

Mapping of a locus (mdoA) that affects the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

Mutants of Escherichia coli defective in the newly discovered mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mutation has now been mapped and found to be located near 23 min on the E. coli chromosome between putA and pyrC. The mdoA mutants are defective in the membrane-localized component of the glucosyl transferase system described by Weissborn and Kennedy (A. C. Weissborn and E. P. Kennedy, Fed. Proc. 42:2122, 1983).     

Membrane-derived oligosaccharides affect porin osmoregulation only in media of low ionic strength.

Gram-negative bacteria grown under conditions of low osmolarity accumulate significant amounts of periplasmic glucans, membrane-derived oligosaccharides (MDO) in Escherichia coli and cyclic glucans in members of the family Rhizobiaceae. It was reported previously (W. Fiedlder and H. Rotering, J. Biol. Chem. 263:14684-14689, 1988) that mdoA mutants unable to synthesize MDO show a number of altered phenotypes, among them a decreased expression of OmpF and an increased expression of OmpC, when grown in a Bacto Peptone medium of low osmolarity and low ionic strength. Although we confirm the findings of Fiedler and Rotering, we find that the regulation of OmpF and OmpC expression in mdoA mutants is normal in cells grown on other low-osmolarity media, eliminating the possibility that MDO itself might control porin expression. Our data suggest that a certain minimal ionic strength in the periplasm is needed for normal porin regulation. In media containing very low levels of salt, this may be contributed by anionic MDO.     

Comparison of the phenotypes of the lpxA and lpxD mutants of Escherichia coli

Abstract We compared the phenotype of two thermosensitive Escherichia coli mutants defective in lipid A biosynthesis i.e. SM101 ( lpxA ) and CDH23-213 ( lpxD ). More than 40% of the periplasmic 27-kDa marker enzyme β-lactamase was released from SM101 at 28°C. At this temperature, the mutant still grew with a generation time (67 min), not much longer than that of the parent control strain (57 min). CDH23-213 released β-lactamase only at higher temperatures. SM101 and CDH23-213 were both unable to grow in hypo-osmotic conditions. Derivatives of SM101 and CDH23-213 with mdoA ::Tn 10 had identical phenotypes (including thermosensitivity and defective outer membrane permeability barrier to hydrophobic probes) to those of SM101 and CDH23-213, indicating that the potential loss of membrane-derived oligosaccharides (MDO) did not explain these phenotypic properties. A method for the estimation of lipid A synthesis rate was developed.     

Lysis induction of Escherichia coli by the cloned lysis protein of the phage MS2 depends on the presence of osmoregulatory membrane-derived oligosaccharides

Expression of the cloned lysis protein of phage MS2, which is sufficient to lyse wild type Escherichia coli, does not cause lysis of mutants lacking the osmoregulatory membrane-derived oligosaccharides (MDO). The lysis gene product normally found in the membrane fraction was not stably inserted into the membranes of a mdoA mutant; rather degradation and release from the membrane occurred. Gentle plasmolysis of the MDO-lacking mutant clearly showed an increased periplasmic space as compared to wild type cells. It is concluded that the MDOs play an important role in maintaining a proper arrangement of inner and outer membrane, a prerequisite for a functional insertion of the MS2 lysis protein.     

Osmotic regulation of biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

The osmotic regulation of the biosynthesis of membrane-derived oligosaccharides (MDO) in strains UB1005 and DC2 of Escherichia coli K-12 was examined; this regulation was previously reported by Clark (J. Bacteriol. 161:1049-1053, 1985) to be different from that observed by Kennedy for other strains of E. coli (Proc. Natl. Acad. Sci. USA 79:1092-1095, 1982). Osmotic regulation of the synthesis of MDO in UB1005 and DC2 is in fact indistinguishable from that previously reported for other strains of E. coli, with maximum production of MDO occurring in the medium of lowest osmolarity. The report of Clark to the contrary was apparently based on the inadequate methods for the measurement of MDO employed in that study. MDO are localized in the periplasm of wild-type E. coli cells. However, strain DC2, selected for hypersensitivity to a range of antibiotics, released most of its MDO into the medium, apparently as a result of greater outer membrane permeability.     

Mutant of Escherichia coli deficient in osmoregulation of periplasmic oligosaccharide synthesis.

A mutant of Escherichia coli (mdoR) has been isolated which is defective in synthesis of the membrane-derived oligosaccharides (MDO) normally found in the periplasmic space. In media of high osmotic pressure this defect is suppressed and MDO levels approaching those of the wild type are produced. The mdoR mutant also fails to accumulate glycogen; however, genetic analysis showed that mdoR was not cotransducible with the known glg (glycogen) locus. A further relationship between MDO and glycogen metabolism was suggested by two observations that (i) certain glg mutants affect MDO accumulation and (ii) elevated osmotic pressure inhibits glycogen accumulation, in both wild-type and mdoR cells.     

Transfer of phosphoethanolamine residues from phosphatidylethanolamine to the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents composed of glucose residues linked by beta-1,2 and beta-1,6 glycosidic bonds. MDO are substituted with phosphoglycerol, phosphoethanolamine, and succinic acid moieties. The phosphoglycerol residues present on MDO are derived from phosphatidylglycerol (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983), but evidence as to the source of the phosphoethanolamine residues has been lacking. We now report that phosphatidylethanolamine, exogenously added to intact cells of E. coli, provides a source of phosphoethanolamine residues that are transferred to MDO. The biosynthesis of phosphoethanolamine-labeled MDO is osmotically regulated, with maximum synthesis occurring during growth in medium of low osmolarity.     

Membrane-derived oligosaccharides (MDOs) are essential for sodium dodecyl sulfate resistance in Escherichia coli

We studied the role of membrane-derived oligosaccharides (MDOs) in sodium dodecyl sulfate (SDS) resistance by Escherichia coli. MDOs are also known as osmoregulated periplasmic glucans. Wild-type E. coli MC4100 grew in the presence of 10% SDS whereas isogenic mdoA and mdoB mutants could not grow above 0.5% SDS. Similarly, E. coli DF214, a mutant (pgi, zwf) unable to grow on glucose, exhibited conditional sensitivity to SDS in that it grew in gluconate and glucose or galactose but not in gluconate and mannose or sorbose. DF214 requires both gluconate and glucose/galactose because the gluconate is used for energy production, while glucose/galactose is used for MDO synthesis. Finally, the fate of E. coli cells subjected to SDS shock either during growth or when used as an inoculum is dependent on the presence or absence of sufficient MDOs. In both cases, cells grown under high-osmolarity (low-MDO) conditions were rapidly lysed by 5% SDS. Based on findings from a wild-type E. coli (MC4100), two mdo mutants and strain DF214 we conclude that MDOs are required for SDS resistance.     

Characterization of an Escherichia coli mdoB mutant strain unable to transfer sn-1-phosphoglycerol to membrane-derived oligosaccharides

A procedure for the isolation of mutants affected in components containing glycerol derived from phospholipids yielded two mutant strains that contain membrane-derived oligosaccharides (MDO) devoid of glycerol (Rotering, H., Fiedler, W., Rollinger, W., and Braun, V. (1984) FEMS Microbiol. Lett. 22, 61-68). MDO are found in the periplasmic space of Escherichia coli and other Gram-negative bacteria, and they may comprise up to 7% of the cells dry weight. The biosynthesis of MDO is osmoregulated (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095) and linked to the metabolism of phospholipids (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368-1372). This leads to substitution of MDO with sn-1-phosphoglycerol and phosphoethanolamine (Kennedy, E. P., Rumley, M. K., Schulman, P., and van Golde, L. M. G. (1976) J. Biol. Chem. 251, 4208-4213). MDO also contain succinate in O-ester linkage. We now report that one mutant strain lacks phosphoglycerol transferase I activity and thus is unable to transfer sn-1-phosphoglycerol residues from phosphatidylglycerol to MDO. The mdoB gene affected in this mutant has been located at 99.2 min on the E. coli chromosome. The ethanolamine content of MDO isolated from the mutant strain is elevated, whereas the number of succinate residues is not affected. The only phenotype of mdoB mutants we found is a dramatic reduction of the diglyceride content observed in dgk mdoB double mutants when the beta-glucoside arbutin is present in the growth medium.     

Biosynthesis of membrane-derived oligosaccharides. Membrane-bound glucosyltransferase system from Escherichia coli requires polyprenyl phosphate.

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) of Escherichia coli and the cyclic glucans of the Rhizobiaceae, are now recognized to be a family of closely related substances with important functions in osmotic adaptation and cell signaling. The synthesis of the beta-1,2-glucan backbone of MDO is catalyzed by a membrane-bound glucosyltransferase system previously shown to require UDP-glucose and (surprisingly) acyl carrier protein (Therisod, H., Weissborn, A. C., and Kennedy, E. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7236-7240). In the present study, no glucan intermediates bound to acyl carrier protein or to UDP could detected. The enzyme system, however, was found to be strongly inhibited by bacitracin and by amphomycin. Because the two antibiotics function by forming specific complexes with polyprenyl phosphates, their inhibitory effect suggests a prenol requirement for MDO biosynthesis. Furthermore, the activity of the glucosyltransferase was greatly stimulated by the addition of polyprenyl phosphates such as decaprenyl-P and dihydroheptaprenyl-P, but not by farnesyl-P. The same membrane preparations carry out the synthesis of polyprenyl-P-glucose, which is also stimulated by added polyprenyl-P, including farnesyl-P, the most active of those tested. Pulse chase experiments, however, indicate that the endogenous pool of polyprenyl-P-glucose cannot be an obligate intermediate in the MDO glucosyltransferase system.     

Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti.

Rhizobium species produce an inducible acyl carrier protein (ACP), encoded by the nodF gene, that somehow functions in an exchange of cell signals between bacteria and specific plant hosts, leading to nodulation of plant roots and symbiotic nitrogen fixation, as well as a constitutive ACP needed for the synthesis of essential cell lipids. The periplasmic cyclic glucans of Rhizobium spp. are also involved in specific rhizobium-plant interaction. These glucans are strongly similar to the periplasmic membrane-derived oligosaccharides (MDO) of Escherichia coli. E. coli ACP is an essential component of a membrane-bound transglucosylase needed for the biosynthesis of MDO, raising the possibility that either or both of the rhizobial ACPs might have a similar function. We have now isolated the constitutive ACP of R. meliloti and determined its primary structure. We have also examined its function, together with those of ACPs from E. coli, Rhodobacter sphaeroides, and spinach, in the MDO transglucosylase system and as substrate for the E. coli ACP acylase enzyme. All four ACPs act as acceptors of acyl residues, but only the E. coli ACP functions in the transglucosylase system.     

Interstrain Variation of the Polysaccharide B Biosynthesis Locus of Bacteroides fragilis: Characterization of the Region from Strain 638R

The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of Bacteroides fragilis 638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of B. fragilis NCTC 9343. Two genes of the region, wcgD and wcgC, are shown by complementation to encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.     

Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites

Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K_d = 0.15 μM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1″ phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.     

Isolation and characterization of Escherichia coli mutants blocked in production of membrane-derived oligosaccharides.

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.     

Gene-Enzyme Relationships in Histidine Biosynthesis in Bacillus subtilis

Mutants for 9 of the 10 steps in histidine biosynthesis have been isolated and identified by enzyme assay. Each locus has been mapped in relation to the aro cluster and to other histidine loci by deoxyribonucleic acid-mediated transformation. The genes which code for enzymes 3, 6, and 8 of the pathway are linked to the aro cluster. A major histidine linkage group is composed of the genes which specify enzymes 1, 2, 5, 7, and 10. The locus which codes for step 9 of the pathway is unlinked to any other identified his loci. The major histidine cluster is loosely linked to cysB and is unlinked to any of the loci concerned with aromatic amino acid biosynthesis.     

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)

Abstract A mutant screening procedure is described which allows the identification of mutants carrying lesions in lipoprotein, membrane-derived oligosac-charides (MDO), and other compounds of the E. coli cell envelope containing glycerol derived from phospholipid metabolism. Two mutants lacking glycerol in MDO and one mutant devoid of lipoprotein demonstrate the usefulness of the procedure.     

Genetic analysis of Proteus mirabilis mutants defective in swarmer cell elongation.

Swarmer cell differentiation is a complex process involving the activity of many gene products. In this report, we characterized the genetic locus of Tn5 insertion in each of 12 mutants defective in swarmer cell elongation. The mutations fell into four categories affecting either flagellar biosynthesis or energetics, lipopolysaccharide and cell wall biosynthesis, cellular division, or proteolysis of peptides.