Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.

O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover.     

A heat-sensitive lysis mutant of Bacillus subtilis 168 with a low activity of pyruvate carboxylase.

A mutant of Bacillus subtilis which grew in complex medium at 30 degrees C but lysed at 45 degrees C has been isolated. It could only grow on minimal medium at 45 degrees C with added aspartate (20 microgram ml-1) but lysed if lysine (20 microgram ml-1) was also present. The requirement for aspartate was due to a low activity of pyruvate carboxylase; the site of the mutation (pyc) was linked (16% cotransducible using phage PBSI) to the pyrD locus, and the order of markers deduced was: pyrD-cysC-pyc. This defect appeared to lead to decreased synthesis of mesodiaminopimelic acid (mesoA2pm), an amino acid unique to peptidoglycan and its precursors. At the restrictive temperature the mutant accumulated uridine-5'-diphosphate N-acetylmuramyl-L-alanyl-D-glutamate, since meso A2pm is the next amino acid to be added to the growing peptide chain of peptidoglycan. This resulted in an inhibition of peptidoglycan synthesis, determined as a reduced incorporation of N-acetyl[14C]glucosamine. Peptidoglycan synthesis was not decreased if the mutant was grown in media containing aspartate but lacking lysine. The sensitivity to lysine may arise because (i) at 45 degrees C the mutant was starved for aspartate and hence mesoA2pm even when aspartate was present, since aspartate utilization, as estimated by the incorporation of [3H]aspartate into trichloroacetic acid precipitable material, was relatively inefficient; and (ii) this diminished level of mesoA2pm synthesis from aspartate was further curtailed since lysine inhibits one of the aspartokinases in B. subtilis. Thus, addition of lysine allowed protein synthesis and hence autolysin production to proceed whilst peptidoglycan synthesis remained inhibited. When autolysis was blocked, either indirectly by stopping protein synthesis through starvation of aspartate and lysine, or directly by introducing a lyt mutation, then shifting the mutant to 45 degrees C did not result in lysis but growth still ceased.     

Defective synthesis of lipid intermediates for peptidoglycan formation in a stabilized L-form of Streptococcus pyogenes.

Membrane preparations obtained from a stabilized L-form of Streptococcus pyogenes are incapable of synthesizing peptidoglycan from uridine-5'-diphospho-N-acetyl-D-muramyl-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala and uridine-5'-diphospho-N-acetyl-D-glucosamine, in contrast with similar preparations from the parental streptococcus. Furthermore, 50-fold higher levels of lipid intermediates which serve as membrane-bound substrates for peptidoglycan synthesis are synthesized in reaction mixtures containing streptococcal membranes than with similar preparations from the L-form. These observations suggest that the inability of this stabilized L-form to form a cell wall in vivo lies, at least in part, in its failure to synthesize significant quantities of the lipid substrates for peptidoglycan synthesis.     

A novel link between the biosynthesis of aromatic amino acids and transfer RNA modification in Escherichia coli

A transposon-induced mutation in Escherichia coli resulted in a lack of two modified nucleosides in the transfer ribonucleic acid. These nucleosides were identified as uridine-5-oxyacetic acid (cmo⁵U)² and its methylester, mcmo⁵U. Both became radioactively labelled using [methyl-¹⁴C]methionine as methyl donor when wild-type cells were grown in a defined rich medium. We believe that both nucleosides have hydroxyuridine as a common precursor, which should be methylated in the first modification step. However, in our system in vitro the tRNA from the mutant was not a methyl group acceptor, indicating that the step affected in the mutant occurs before the methylation step. Thus, the most likely biosynthetic pathway is: formation of (1) hydroxyuridine, (2) methoxyuridine. (3) cmo⁵U and, in some cases, (4) mcmo⁵U. The mutant had also become Aro^?, i.e. it required aromatic amino acids for growth. Genetic analysis revealed that the transposon Tn5 had been integrated close to or within the aroD gene, the gene product of which participates in the synthesis of shikimic acid. The common pathway of the biosynthesis of aromatic amino acids includes the genes aroB, D, E, A and C in that order, and any mutant defective in any of these genes lacked cmo⁵U and mcmo⁵U in their tRNA. When shikimic acid was included in the defined rich medium used, the Tn5-induced mutant regained the normal level of cmo⁵U and mcmo⁵U while an aroC mutant (distal to shikimic acid but prior to chorismic acid) did not. The rich medium used contained, besides the aromatic amino acids, all the precursors for the synthesis of folate, ubiquinone and enterochelin. Thus, chorismic acid itself or a metabolite of it in the synthetic pathway to vitamin K₂ or in an unknown pathway must play a pivotal role in this specific modification of the tRNA. These results reveal a novel link between the biosynthesis of amino acids and modification of tRNA.     

Arabinan-deficient mutants of Corynebacterium glutamicum and the consequent flux in decaprenylmonophosphoryl-D-arabinose metabolism

The arabinogalactan (AG) of Corynebacterianeae is a critical macromolecule that tethers mycolic acids to peptidoglycan, thus forming a highly impermeable cell wall matrix termed the mycolyl-arabinogalactan peptidoglycan complex (mAGP). The front line anti-tuberculosis drug, ethambutol (Emb), targets the Mycobacterium tuberculosis and Corynebacterium glutamicum arabinofuranosyltransferase Mt-EmbA, Mt-EmbB and Cg-Emb enzymes, respectively, which are responsible for the biosynthesis of the arabinan domain of AG. The substrate utilized by these important glycosyltransferases, decaprenylmonophosphoryl-D-arabinose (DPA), is synthesized via a decaprenylphosphoryl-5-phosphoribose (DPPR) synthase (UbiA), which catalyzes the transfer of 5-phospho-ribofuranose-pyrophosphate (pRpp) to decaprenol phosphate to form DPPR. Glycosyl compositional analysis of cell walls extracted from a C. glutamicum::ubiA mutant revealed a galactan core consisting of alternating beta(1-->5)-Galf and beta(1-->6)-Galf residues, completely devoid of arabinan and a concomitant loss of cell-wall-bound mycolic acids. In addition, in vitro assays demonstrated a complete loss of arabinofuranosyltransferase activity and DPA biosynthesis in the C. glutamicum::ubiA mutant when supplemented with p[14C]Rpp, the precursor of DPA. Interestingly, in vitro arabinofuranosyltransferase activity was restored in the C. glutamicum::ubiA mutant when supplemented with exogenous DP[14C]A substrate, and C. glutamicum strains deficient in ubiA, emb, and aftA all exhibited different levels of DPA biosynthesis.     

The synthesis of beta-galactosidase by constitutive and other regulatory mutants of Escherichia coli in chemostat culture.

The synthesis of beta-galactosidase by an E. coli constitutive mutant was examined in a chemostat using glucose-, glycerol-, succinate- or N-limited growth media. Except for glucose-grown bacteria, the steady-state intracellular level of beta-galactosidase was maximal at dilution rates between 0-2 and 0-3 h-1. At higher dilution rates enzyme synthesis was reduced by catabolite repression, which could be relieved by the addition of cyclic AMP. With a catabolite-resistant mutant (UV5c), no decrease in enzyme level at high dilution rates were observed. All mutants examined were constitutive and gave decreased enzyme levels at low dilution rates, with the exception of lac-/F'lac UV5c mutants where the enzyme levels rose at low dilution rates. Hyper-producing mutants were isolated but were unstable. A constitutive mutant growing on glycerol-limited media was considered the most suitable for large-scale production of beta-galactosidase in a chemostat.     

Molecular characterization of an atl null mutant of Staphylococcus aureus

atl is a gene encoding a bifunctional peptidoglycan hydrolase of Staphylococcus aureus. The gene product of atl is a 138 kDa protein that has an amidase domain and a glucosaminidase domain, and undergoes processing to generate two major peptidoglycan hydrolases, a 51 kDa glucosaminidase and a 62 kDa amidase in culture supernatant. An atl null mutant was isolated by allelic replacement and characterized. The mutant grew in clusters and sedimented when grown in broth culture. Analysis of peptidoglycan prepared from the wild type and the mutant revealed that there were no differences in muropeptide composition or in glycan chain length distribution. On the other hand, the atl mutation resulted in pleiotropic effects on cell surface nature. The mutant cells showed complete inhibition of metabolic turnover of cell wall peptidoglycan and revealed a rough outer cell wall surface. The mutation also decreased the amount of protein non-covalently bound to the cell surface and altered the protein profile, but did not affect proteins covalently associated with the cell wall. Lysis of growing cells treated with otherwise lytic concentration of penicillin G was completely inhibited in the mutant, but that of non-growing cells was not affected by the mutation. The atl mutation did not significantly affect the ability of S. aureus to provoke an acute infection when inoculated intraperitoneally in a mouse sepsis model. These results further support the supposition that atl gene products are involved in cell separation, cell wall turnover and penicillin-induced lysis of the cells.     

Inhibition of peptidoglycan biosynthesis at a postcytoplasmic reaction in a stable L-phase variant of Streptococcus faecium.

Cultures of a stable L-phase variant of Streptococcus faecium F24 produced and retained peptidoglycan precursors intracellularly over the entire growth cycle in a chemically defined medium. The identity of the most abundant precursor, UDP N-acetylmuramyl-L-alanyl-D-glutamyl-L-lysyl-D-alanyl-D-alanine (UDP-MurNAc-pentapeptide), was confirmed by demonstrating in vitro the presence of enzymes required for the cytoplasmic stage of peptidoglycan biosynthesis. The initial membrane-bound reaction in peptidoglycan biosynthesis involving phospho-MurNAc-pentapeptide translocase and undecaprenyl-phosphate membrane carrier was catalyzed by protoplast membrane preparations but not by L-phase membrane preparations. However, both protoplast and L-phase membranes incorporated radioactivity from dTDP-L-[14C]rhamnose, the presumed precursor to a non-peptidoglycan cell surface component, into high-molecular-weight material. dTDP-L-rhamnose did not accumulate in growing cultures but was synthesized from D-glucose-1-phosphate and dTTP by cell-free extracts of the streptococcus and L-phase variant. Neither rhamnose- nor muramic acid-containing compounds were detected in culture fluids. It is suggested that continued inhibition of cell wall biosynthesis in this stable L-phase variant is the result of a defect expressed at the membrane stage of peptidoglycan biosynthesis specifically involving the translocation step.     

Galactose repression of beta-galactosidase induction in Escherichia coli

Beggs, William H. (University of Minnesota, Minneapolis), and Palmer Rogers. Galactose repression of beta-galactosidase induction in Escherichia coli. J. Bacteriol. 91:1869-1874. 1966.-Galactose repression of beta-galactosidase induction in Escherichia coli was investigated to determine whether the galactose molecule itself is the catabolite repressor of this enzyme system. Without exception, beta-galactosidase induction by cells grown in a synthetic salts medium with lactate or glycerol as the carbon source was more strongly repressed by glucose than by galactose. This relationship existed even when the organism was previously grown in the synthetic medium containing galactose as the source of carbon. Two observations suggested that the ability of galactose to repress beta-galactosidase formation by Escherichia coli depends directly upon the cells' capacity to catabolize galactose. First, galactose repression of beta-galactosidase synthesis was markedly enhanced in bacteria tested subsequent to gratuitous induction of the galactose-degrading enzymes with d-fucose. Second, galactose failed to exert a repressive effect on beta-galactosidase in a galactose-negative mutant lacking the first two enzymes involved in galactose catabolism. Glucose completely repressed enzyme formation in this mutant. This same mutant, into which the genes for inducible galactose utilization had been introduced previously by transduction, again exhibited galactose repression. Pyruvate was found to be at least as effective as galactose in repressing beta-galactosidase induction by cells grown in synthetic salts medium plus glycerol. It is concluded that the galactose molecule itself is not the catabolite repressor of beta-galactosidase, but that repression is exerted through some intermediate in galactose catabolism.     

Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli.

In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source. The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate. These cells were devoid of alkaline phosphatase activity. However, the phoA promoter was fully active in a glpT mutant. These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium.     

Characterization of the pathways for phosphatidylethanolamine biosynthesis in Chinese hamster ovary mutant and parental cell lines

A tritium suicide procedure was devised to facilitate the isolation of Chinese hamster ovary cell mutants defective in phosphatidylethanolamine biosynthesis. One mutant with a 20-50% reduction in [3H]ethanolamine incorporation was chosen for further analysis and was shown to have reduced activity of CTP: phosphoethanolamine cytidylyltransferase. Levels of phosphatidylethanolamine and rates of its biosynthesis were compared in the mutant and parent cell lines. Despite the reduced activity of the CDP-ethanolamine pathway in the mutant, levels of phosphatidylethanolamine were the same in mutant and parent cells. Rates of phosphatidylethanolamine synthesis de novo, as measured by incorporation of 32PO4 into phosphatidylethanolamine, were also the same in mutant and parent cells, as was the rate of incorporation of [3H]serine into both phosphatidylserine and phosphatidylethanolamine. After a long term labeling with [3H]serine, the specific radioactivity of phosphatidylserine was the same as that of phosphatidylethanolamine, and there was no difference in the specific radioactivities of the two lipids between mutant and parent cells. These results implicate decarboxylation of phosphatidylserine as the sole route for synthesis of phosphatidylethanolamine under normal culture conditions.     

Formation of cell wall polymers by reverting protoplasts of Bacillus licheniformis.

The biosynthesis of peptidoglycan and teichoic acid by reverting protoplasts of Bacillus licheniformis 6346 His-, in cubated at 35 C on medium containing 2.5% agar, is detectable after 40 min. The amount of N-acetyl-[1-14C]glucosamine incorporated into peptidoglycan and teichoic acid on continued incubation doubles at the same rate as the incorporation of [3H]tryptophan into protein. At the early stages of reversion the average glycan chain length, measured by the ratio of free reducing groups of muramic acid and glucosamine to total muramic acid present, is very short. As reversion proceeds, the average chain length increases to a value similar to the found in the wall of the parent bacillus. The extent of cross-linkage found in the peptide side chains of the peptidoglycan also increases as reversion proceeds. At the completion of reversion the wall material synthesized has similar characteristics to those of the walls of the parent bacilli, containing peptidoglycan and teichoic and teichuronic acids in about the same proportions. Soluble peptidoglycan can be isolated from the reversion medium, amounting to 30% of the total formed after 3 h of incubation and 8% after 12 h. This amount was reduced by the presence in the medium of the walls of an autolysin-deficient mutant; they were not formed at all by reverting protoplasts of the autolysin-deficient mutant itself. Analysis of the soluble material provided additional evidence for their being autolytic products rather than small unchanged molecules. When protoplasts were incubated on medium containing only 0.8% agar, 53 to 67% of the peptidoglycan formed after 3 h of incubation was soluble, and 21% after 12 h. Fibers that appeared to be sheared from the protoplasts at intermediate stages of reversion on medium containing 2.5% agar were similar in composition to the bacillary walls.     

Temperature-dependent variation in the synthesis of streptococcal group-specific carbohydrate. II. Biosynthetic studies in group A and variant strains.

The biosynthesis of the cell wall polysaccharide and peptidoglycan of group A and A-486-Var streptococci was studied with N-acetyl-[14C]glucosamine, UDP-N-acetyl-[14C]glucosamine, and [14C]glucose. The incorporation of N-acetyl-[14C]-glucosamine into the cell wall four times greater in the A-486-Var cells than in the group A cells. However, the percentage of the total label incorporated into the cell wall polysaccharide at 37 degrees C by the A-486-Var strain was 12%, compared with 66% for the group A cells. When the A-486-Var was grown at 22 degrees C, the proportion of the label incorporated into the cell wall polysaccharide increased to 41%. At 37 degrees C, N-acetyl-[14C]glucosamine was incorporated preferentially into the peptidoglycan of the A-486-Var; almost three times as much of the label was incorporated into the peptidoglycan at 37 degrees C as was incorporated at 22 degrees C. Studies with protoplast membranes of these organisms showed similar differences, with a fourfold greater uptake of UDP-N-acetyl-[14C]glucosamine by the A-486-Var membranes at both incubation temperatures. These studies suggest that a defect in the incorporation of N-acetylglucosamine into the side chain of the polysaccharide is present in the A-486-Var strain at a step following the synthesis of UDP-N-acetylglucosamine. This defect, which may involve the UDP-N-acetylglucosamine transferase, is temperature dependent in the A-486-Var strain.     

Negative control of cell division by mreB, a gene that functions in determining the rod shape of Escherichia coli cells.

Exponentially growing Escherichia coli cells containing additional copies of the shape-determining gene mreB were found to be elongated, whereas mreB mutant cells were spherical and overproduced penicillin-binding protein 3, a septum peptidoglycan synthetase. The effect of the mreB gene on expression of ftsI, the structural gene for penicillin-binding protein 3, was examined by using an ftsI-lacZ fusion gene on a plasmid. Formation of beta-galactosidase from the fusion gene was significantly increased in mreB129 mutant cells, and its overproduction was suppressed to a normal level by the presence of a plasmid containing the mreB gene. These results indicate a negative mechanism of control of cell division by this morphology gene and suggest that the gene functions in determining whether division or elongation of the cells occurs.     

Properties of a D-glutamic acid-requiring mutant of Escherichia coli

Some properties of a d-glutamic acid auxotroph of Escherichia coli B were studied. The mutant cells lysed in the absence of d-glutamic acid. Murein synthesis was impaired, accompanied by accumulation of uridine-5'-diphosphate-N-acetyl-muramyl-l-alanine (UDP-MurNac-l-Ala), as was shown by incubation of the mutant cells in a cell wall medium containing l-[(14)C]alanine. After incubation of the parental strain in a cell wall medium containing l-[(14)C]glutamic acid, the acid-precipitable radioactivity was lysozyme degradable to a large extent. Radioactive UDP-MurNac-pentapeptide was isolated from the l-[(14)C]glutamic acid-labeled parental cells. After hydrolysis, the label was exclusively present in glutamic acid, the majority of which had the stereo-isomeric d-configuration. Compared to the parent the mutant incorporated less l-[(14)C]glutamic acid from the wall medium into acid-precipitable material. Lysozyme degraded a smaller percentage of the acid-precipitable material of the mutant than of that of the parent. No radioactive uridine nucleotide precursors could be isolated from the mutant under these conditions. Attempts to identify the enzymatic defect in this mutant were not successful. The activity of UDP-MurNac-l-Ala:d-glutamic acid ligase (ADP; EC 6.3.2.9) (d-glutamic acid adding enzyme) is not affected by the mutation. Possible pathways for d-glutamic acid biosynthesis in E. coli B are discussed.     

In vitro metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal protozoa and bacteria.

Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid [( 3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. These radiolabeled bacterial mutants were incubated alone (control) and with mixed ruminal bacteria or protozoa, and the metabolic processes, rates, and patterns of radiolabeled products released from them were studied. Control incubations revealed an inherent difference between the two substrates; gram-positive supernatants consistently contained 5% radioactivity, whereas even at 0 h, those from the gram-negative mutant released 22%. Incubations with ruminal microorganisms showed that the two mutants were metabolized differently and that protozoa were the major effectors of their metabolism. Protozoa exhibited differential rates of engulfment (150 B. megaterium GW1 and 4,290 E. coli W7-M5 organisms per protozoan per h), and they extensively degraded [3H]DAP-labeled B. megaterium GW1 at rates up to nine times greater than those of ruminal bacteria. By contrast, [3H]DAP-labeled E. coli W7-M5 degradation by either ruminal bacteria or ruminal protozoa was more limited. These fundamental differences in the metabolism of the two mutants, especially by ruminal protozoa, were reflected in the patterns and rates of radiolabeled metabolites produced; many were rapidly released from [3H]DAP-labeled B. megaterium GW1, whereas few were slowly released from [3H]DAP-labeled E. coli W7-M5. Most radiolabeled products derived from [3H]DAP-labeled B. megaterium GW1 were peptides of bacterial peptidoglycan origin. The ruminal metabolism of DAP-containing gram-positive and gram-negative bacteria, even with the same peptidoglycan chemotype, is thus likely to be profoundly different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship of macromolecular synthesis to competence induction in a group H streptococcus.

Group H streptococcus strain Wicky, which was induced to competence for genetic transformation with competence factor (CF) derived from a related strain, displayed reduced rates of ribonucleic acid (RNA) and peptidoglycan synthesis. Pulse-labeling studies revealed that the inhibition of both RNA and peptidoglycan synthesis was maximal at the peak of competence and decreased as competence declined. These studies indicated that competence induction had only a slight effect on the rate of protein synthesis. Trypsin inactivation of CF prevented the reductions in synthesis normally elicited by CF preparations. If the addition of trypsin was delayed until 5 min after the addition of CF, competence induction and decreased synthesis of RNA and peptidoglycan were again apparent. Thus, the alterations in the synthesis of these macromolecules appeared to be related to the induction of competence. Further studies indicated that the apparent reductions in biosynthesis were not caused by decreased uptake of the labeled precursors by intact Wicky cells. In addition, these effects were probably not the result of turnover of macromolecules induced by CF. The lack of turnover of labeled peptidoglycan suggested that competence induction may not involve an autolysin.     

A Bacillus subtilis gene encoding a protein similar to nucleotide sugar transferases influences cell shape and viability.

Bacillus subtilis gene ypfP, which is located at 196 degrees on the genetic map, shows similarity to both the monogalactosyldiacylglycerol synthase gene of Cucumis sativus, which encodes a galactosyltransferase, and the murG genes of B. subtilis, Escherichia coli, Haemophilus influenzae, and Synechocystis sp. strain PCC6803, which encode N-acetylglucosaminyltransferases involved in peptidoglycan biosynthesis. Cells containing a null mutation of ypfP are shorter and rounder than wild-type cells during growth in Luria-Bertani medium and glucose minimal medium. In addition, the mutant cells preferentially undergo lysis when grown on solid Luria-Bertani medium.     

Turnover of beta-galactosidase in fibroblasts from patients with genetically different types of beta-galactosidase deficiency.

The turnover of lysosomal beta-galactosidase was studied in fibroblast cultures from patients with Gm1-gangliosidosis and combined beta-galactosidase and neuraminidase deficiency, which had 5-10% residual beta-galactosidase activity. beta-Galactosidase was specifically inactivated with the suicide substrate beta-D-galactopyranosylmethyl-p-nitro-phenyltriazene (beta-Gal-MNT) and from the subsequent restoration of enzyme activity in cell cultures turnover times were calculated. By using [3H]beta-Gal-MNT, the hydrolytic activity per molecule of beta-galactosidase was determined. 3H-labelled beta-D-galactopyranosylmethylamine, the precursor of [3H]beta-gal-MNT, was obtained by Raney-nickel-catalysed exchange with 3H2O. The rate of synthesis of beta-galactosidase in normal and all mutant cells tested was found to be 0.4-0.5 pmol/day per mg of cellular protein. The GM1-gangliosidosis cells tested contain the normal amount of 0.5 pmol of beta-galactosidase/mg of protein with a normal turnover time of about 10 days, but only 10% of beta-galactosidase activity per enzyme molecule. Cells with combined beta-galactosidase and neuraminidase deficiency contain only 0.3 pmol of beta-galactosidase/mg of protein with a decreased turnover time of 1 day and normal hydrolytic properties (200 nmol of 4-methylumbelliferyl galactoside/h pmol of beta-galactosidase).     

In vitro metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal protozoa and bacteria

Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid [( 3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. These radiolabeled bacterial mutants were incubated alone (control) and with mixed ruminal bacteria or protozoa, and the metabolic processes, rates, and patterns of radiolabeled products released from them were studied. Control incubations revealed an inherent difference between the two substrates; gram-positive supernatants consistently contained 5% radioactivity, whereas even at 0 h, those from the gram-negative mutant released 22%. Incubations with ruminal microorganisms showed that the two mutants were metabolized differently and that protozoa were the major effectors of their metabolism. Protozoa exhibited differential rates of engulfment (150 B. megaterium GW1 and 4,290 E. coli W7-M5 organisms per protozoan per h), and they extensively degraded [3H]DAP-labeled B. megaterium GW1 at rates up to nine times greater than those of ruminal bacteria. By contrast, [3H]DAP-labeled E. coli W7-M5 degradation by either ruminal bacteria or ruminal protozoa was more limited. These fundamental differences in the metabolism of the two mutants, especially by ruminal protozoa, were reflected in the patterns and rates of radiolabeled metabolites produced; many were rapidly released from [3H]DAP-labeled B. megaterium GW1, whereas few were slowly released from [3H]DAP-labeled E. coli W7-M5. Most radiolabeled products derived from [3H]DAP-labeled B. megaterium GW1 were peptides of bacterial peptidoglycan origin. The ruminal metabolism of DAP-containing gram-positive and gram-negative bacteria, even with the same peptidoglycan chemotype, is thus likely to be profoundly different.(ABSTRACT TRUNCATED AT 250 WORDS)