Regulation of glucosamine utilization in Staphylococcus aureus and Escherichia coli.

Glucosamine- or N-acetylglucosamine-requiring mutants of Staphylococcus aureus 209P and Escherichia coli K12, which lack glucosamine-6-phosphate synthetase [2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring); EC 5.3.1.19], were isolated. Growth of these mutants on glucosamine was inhibited by glucose, but growth on N-acetylglucosamine was not. Addition of glucose to mutant cultures growing exponentially on glucosamine inhibited growth and caused death of bacteria, though chloramphenicol prevented death. Uptake of glucosamine by S. aureus and E. coli mutants was severely inhibited by glucose whereas uptake of N-acetylglucosamine was only slightly inhibited. Uptake of glucose was not inhibited by either glucosamine or N-acetylglucosamine. In glucosamine auxotrophs, glucose causes glucosamine deficiency which interrupts cell wall synthesis and results in some loss of viability in the presence of continued protein synthesis.     

Control of amino sugar metabolism in Escherichia coli and isolation of mutants unable to degrade amino sugars

1. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] and a repression of glucosamine 6-phosphate synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16); glucose abolishes these control effects. 2. Growth of E. coli on N-acetylglucosamine results in an induction of N-acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase, and in a repression of glucosamine 6-phosphate synthetase; glucose diminishes these control effects. 3. The synthesis of amino sugar kinases (EC 2.7.1.8 and 2.7.1.9) is unaffected by growth on amino sugars. 4. Glucosamine 6-phosphate synthetase is inhibited by glucosamine 6-phosphate. 5. Mutants of E. coli that are unable to grow on N-acetylglucosamine have been isolated, and lack either N-acetylglucosamine 6-phosphate deacetylase (deacetylaseless) or glucosamine 6-phosphate deaminase (deaminaseless). Deacetylaseless mutants can grow on glucosamine but deaminaseless mutants cannot. 6. After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamine 6-phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions. In one mutant the acetylamino sugar was shown to be partly as N-acetylglucosamine 6-phosphate. Deaminaseless mutants have no abnormal control effects after growth on glucose. 7. Addition of N-acetylglucosamine or glucosamine to cultures of a deaminaseless mutant caused inhibition of growth. Addition of N-acetylglucosamine to cultures of a deacetylaseless mutant caused lysis, and secondary mutants were isolated that did not lyse; most of these secondary mutants had lost glucosamine 6-phosphate deaminase and an uptake mechanism for N-acetylglucosamine. 8. Similar amounts of (14)C were incorporated from [1-(14)C]-glucosamine by cells of mutants and wild-type growing on broth. Cells of wild-type and a deaminaseless mutant incorporated (14)C from N-acetyl[1-(14)C]glucosamine more efficiently than from N[1-(14)C]-acetylglucosamine, incorporation from the latter being further decreased by acetate; cells of a deacetylaseless mutant showed a poor incorporation of both types of labelled N-acetylglucosamine.     

The site of inhibition of cell wall synthesis by 3-amino-3-deoxy-D-glucose in Staphylococcus aureus.

The inhibition of growth and cell wall synthesis by 3-amino-3-deoxy-D-glucose (3-AG), which is known to be one of the constituents of the kanamycin molecule and a metabolite of Bacillus sp., was almost completely overcome by glucosamine and N-acetylglucosamine in Staphylococcus aureus but scarcely affected by D-glucose and D-fructose. The antibiotic did not inhibit the incorporation of [14C]glucosamine and [3H]N-acetylglucosamine into the acid-insoluble fraction, but rather enhanced the incorporation of [14C]glucosamine. On the other hand, it inhibited the incorporation of D-[14C]fructose into the cell wall fraction but hardly affected the incorporation of D-[14C]fructose into the acid-insoluble fraction in the presence of pencillin G. Based on these results, it is suggested that the site of primary action of 3-AG is the formation of glucosamine-6-phosphate from D-fructose-6-phosphate, which is catalyzed by glucosamine synthetase [EC 2.6.1.16].     

Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine

Glucosamine and N-acetylglucosamine are currently produced by extraction and acid hydrolysis of chitin from shellfish waste. Production could be limited by the amount of raw material available and the product potentially carries the risk of shellfish protein contamination. Escherichia coli was modified by metabolic engineering to develop a fermentation process. Over-expression of glucosamine synthase (GlmS) and inactivation of catabolic genes increased glucosamine production by 15 fold, reaching 60 mg l(-1). Since GlmS is strongly inhibited by glucosamine-6-P, GlmS variants were generated via error-prone PCR and screened. Over-expression of an improved enzyme led to a glucosamine titer of 17 g l(-1). Rapid degradation of glucosamine and inhibitory effects of glucosamine and its degradation products on host cells limited further improvement. An alternative fermentation product, N-acetylglucosamine, is stable, non-inhibitory to the host and readily hydrolyzed to glucosamine under acidic conditions. Therefore, the glucosamine pathway was extended to N-acetylglucosamine by over-expressing a heterologous glucosamine-6-P N-acetyltransferase. Using a simple and low-cost fermentation process developed for this strain, over 110 g l(-1) of N-acetylglucosamine was produced.     

Inhibitory effect of D-glucosamine on glycolysis in bovine retina.

1. The effects of glucosamine concentration on the size of the lactate pool, on the levels of ATP, ADP, AMP and on the radioactivity incorporation from [1-14-C] glucosamine into lactate, N-acetylglucosamine and glucosamine-6-P were studied using whole bovine retinas. 2. The radioactive lactate, evaluated in relation to glucosamine molarity, after a modest initial increase, diminishes significantly. On the contrary the N-acetyl [1-14-C] glucosamine, the [1-14-C] glucosamine-6-P and, consequently, also the [1-14-C] glucosamine-6-P/[-14-C] lactate ratio increase with glucosamine molarity. 3. The retinal content of ATP shows a modest increment after incubation with low concentrations of D-glucosamine (0.5--2.0 mM) and a remarkable fall at higher concentrations. 4. Using retinal homogenates D-glucosamine clearly lowers the lactate production from glucose, glucose-6-P and fructose-1, 6-P2. 5. D-Glucosamine acts as an inhibitor of retinal glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase by decreasing the initial velocity of these reactions. 6. It is concluded that D-glucosamine causes a reduction in the lactate production, by inhibiting two enzymes of the glycolytic pathway: glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase. The fall in the adenine nucleotides content is a consequence of a dephosphorylation of ATP for the phosphorylation of glucosamine without concomitant resynthesis of ATP "via glycolysis".     

An examination of the inhibitory effects of N-iodoacetylglucosamine on Escherichia coli and isolation of resistant mutants

1. After incubation of Escherichia coli with N-iodo[1,2-(14)C(2)]acetylglucosamine, 95-99% of the (14)C taken up by whole cells is located in a cold-trichloroacetic acid-soluble fraction. Two major components of this fraction are S-carboxymethylcysteine and S-carboxymethylglutathione. The same compounds accumulate during incubation with iodo[(14)C]acetate but not with iodo[(14)C]acetamide. The amount of (14)C associated with a cold-trichloroacetic acid-insoluble fraction are comparable for all three alkylating agents. After incubation with iodo[(14)C]acetamide, 50% of the label bound to whole cells is recoverable in a cold-trichloroacetic acid-insoluble fraction. 2. Uptake and incorporation of (14)C from [U-(14)C]glycerol is blocked at an early stage by N-iodoacetylglucosamine. No specific inhibition of macromolecular synthesis could be demonstrated. 3. Mutants selected for resistance to iodoacetate are partially resistant to iodoacetate and N-iodoacetylglucosamine, but show no resistance to iodoacetamide. 4. Mutants selected for resistance to N-iodoacetylglucosamine are not resistant to iodoacetate or iodoacetamide, and are defective in their ability to grow on N-acetylglucosamine. Resistance to N-iodoacetylglucosamine is not absolute, and depends on the presence of glucose or certain other sugars; there is no resistance during growth on maltose, glycerol or succinate. 5. Absolute resistance can be achieved by selecting for a second mutation conferring resistance during growth on maltose; double mutants isolated by this procedure are unable to grow on N-acetylglucosamine and grow poorly on glucosamine. Resistant single mutants have a slightly diminished uptake of N-acetyl[1-(14)C]glucosamine, but in resistant double mutants the uptake of both [1-(14)C]glucosamine and N-acetyl[1-(14)C]glucosamine is severely diminished. 6. These observations are consistent with the presence of two permeases for N-acetylglucosamine, one that also permits uptake of glucosamine and another that allows entry of methyl 2-acetamido-2-deoxy-alpha-d-glucoside. N-Iodoacetylglucosamine can gain entry to the cell by both permeases.     

Isolation and characterization of a glucosamine-requiring mutant of Escherichia coli K-12 defective in glucosamine-6-phosphate synthetase

A mutant was isolated from Escherichia coli K-12 which requires glucosamine or N-acetylglucosamine for growth. Depriving the mutant of glucosamine resulted in a rapid loss of viability of the cells, followed by a decrease in the turbidity of the culture. When the mutant cells were resuspended in broth media containing 10% sucrose, the rod-shaped cells became spheroplasts. However, the presence of sucrose in the media did not prevent the cells from losing their viability. This mutant was shown to be deficient in the activity of l-glutamine:d-fructose-6-phosphate aminotransferase (EC 2.6.1.16). The activity of the deaminating enzyme, 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (EC 5.3.1.10), appeared to be normal in this mutant. The position of the mutation has been determined to be at the 74th min of the Taylor and Trotter map, as shown by cotransduction with phoS (90%) and ilv (25%) by using bacteriophage P1.     

Directed evolution and characterization of Escherichia coli glucosamine synthase

Glucosamine synthase (GlmS) converts fructose-6-phosphate to glucosamine-6-phosphate. Overexpression of GlmS in Escherichia coli increased synthesis of glucosamine-6-P, which was dephosphorylated and secreted as glucosamine into the growth medium. The E. coli glmS gene was improved through error-prone polymerase chain reaction (PCR) in order to develop microbial strains for fermentation production of glucosamine. Mutants producing higher levels of glucosamine were identified by a plate cross-feeding assay and confirmed in shake flask cultures. Over 10 mutants were characterized and all showed significantly reduced sensitivity to inhibition by glucosamine-6-phosphate. Ki of mutants ranged from 1.4 to 4.0 mM as compared to 0.56 mM for the wild type enzyme. Product resistance resulted from single mutations (L468P, G471S) and/or combinations of mutations in the sugar isomerase domain. Most overexpressed GlmS protein was found in the form of inclusion bodies. Cell lysate from mutant 2123-72 contained twice as much soluble GlmS protein and enzyme activity as the strain overexpressing the wild type gene. Using the product-resistant mutant, glucosamine production was increased 60-fold.     

Bacteriolytic effect of cessation of glucosamine supply, induced by specific inhibition of glucosamine-6-phosphate synthetase

The antibiotic tetaine (bacilysin) and its C-terminal epoxyaminoacid--anticapsin--are powerful inhibitors of glucosamine-6-phosphate synthetase (EC 5.3.1.19.) in cell-free extracts of Escherichia coli K-12. Tetaine acts on growing cells as a bactericidal agent. This bactericidal action, measured from 10 to 160 muM concentration, is a consequence of the induction of lysis of growing cells. The induction of lysis by tetaine is compared with the lytic action of some beta-lactams. Hypertonic medium, destruction of the antibiotic, presence of chloramphenicol or the addition of N-acetylglucosamine protect E. coli K-12 cells against lysis induced by tetaine. These effects are compared with those observed in the presence of penicillin G. The results indicate that inhibition of early or late stages of peptidoglycan synthesis all result in more or less the same consequence, i.e. death via cell lysis.     

Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and Essentiality of M. smegmatis MSMEG_1556

The normal growth of mycobacteria attributes to the integrity of cell wall core which consists of peptidoglycan (PG), arabinogalactan (AG) and mycolic acids. N-acetyl glucosamine (GlcNAc) is an essential component in both PG and AG of mycobacterial cell wall. The biosynthetic pathway for UDP-N-acetylglucosamine (UDP-GlcNAc), as a sugar donor of GlcNAc, is different in prokaryotes and eukaryotes. The conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is catalyzed by phosphoglucosamine mutase (GlmM), is unique to prokaryotes. Bioinformatic analysis showed that Msm MSMEG_1556 and Mtb Rv3441c are homologous to Ec GlmM. In this study, soluble Msm MSMEG_1556 protein and Mtb Rv3441c protein were expressed in E. coli BL21(DE3) and their phosphoglucosamine mutase activity were detected. In order to further investigate the essentiality of MSMEG_1556 for the growth of M. smegmatis, we generated a conditional MSMEG_1556 knockout mutant, which harbored thermo-sensitive rescue plasmid carrying Mtb Rv3441c. As the rescue plasmid was unable to complement MSMEG_1556 deficiency at 42°C, MSMEG_1556 knockout mutant did not grow. The dramatic morphological changes of MSMEG_1556 knockout mutant after temperature shift from 30°C to 42°C have been observed by scanning electron microscope. These results demonstrated that MSMEG_1556 is essential for growth of M. smegmatis. This study provided evidence that GlmM enzyme could be as a potential target for developing anti-tuberculosis drugs.     

A dnaT mutant with phenotypes similar to those of a priA2::kan mutant in Escherichia coli K-12

The ability to repair damaged replication forks and restart them is important for cell survival. DnaT is essential for replication restart in vitro and yet no definite genetic analysis has been done in Escherichia coli K-12. To begin, dnaT822, an in-frame six-codon (87-92) deletion was constructed. DnaT822 mutants show colony size, cell morphology, inability to properly partition nucleoids, UV sensitivity, and basal SOS expression similar to priA2::kan mutants. DnaT822 priA2::kan double mutants had phenotypes similar to those of the single mutants. DnaT822 and dnaT822 priA2::kan mutant phenotypes were fully suppressed by dnaC809. Previously, a dominant temperature-sensitive lethal mutation, dnaT1, had been isolated in E. coli 15T(-). DnaT1 was found to have a base-pair change relative to the E. coli 15T(-) and E. coli K-12 dnaT genes that led to a single amino acid change: R152C. A plasmid-encoded E. coli K-12 mutant dnaT gene with the R152C amino acid substitution did not display a dominant temperature-sensitive lethal phenotype in a dnaT(+) strain of E. coli K-12. Instead, this mutant dnaT gene was found to complement the E. coli K-12 dnaT822 mutant phenotypes. The significance of these results is discussed in terms of models for replication restart.     

Growth of a mutant of Escherichia coli K-12 on xylitol by recruiting enzymes for D-xylose and L1,2-propanediol metabolism.

Wild type Escherichia coli K-12 cannot grow on xylitol and we have been unsuccessful in isolating a mutant directly which had acquired this new growth ability. However, a mutant had been selected previously for growth on L-1,2-propanediol as the sole source of carbon and energy. This mutant constitutively synthesized a propanediol dehydrogenase. Recently, we have found that this dehydrogenase fortuitously converted xylitol to D-xylose which could normally be metabolized by E. coli K-12. In addition, it was also discovered that the D-xylose permease fortuitously transported xylitol into the cell. A second mutant was thus isolated from the L-1,2-propanediol-growing mutant that was constitutive for the enzymes of the D-xylose pathway. This mutant could indeed grow on xylitol as the sole source of carbon and energy, by utilizing the enzymes normally involved in D-xylose and L-1,2-propanediol metabolism.     

Alternative route for biosynthesis of amino sugars in Escherichia coli K-12 mutants by means of a catabolic isomerase.

By inserting a lambda placMu bacteriophage into gene glmS encoding glucosamine 6-phosphate synthetase (GlmS), the key enzyme of amino sugar biosynthesis, a nonreverting mutant of Escherichia coli K-12 that was strictly dependent on exogenous N-acetyl-D-glucosamine or D-glucosamine was generated. Analysis of suppressor mutations rendering the mutant independent of amino sugar supply revealed that the catabolic enzyme D-glucosamine-6-phosphate isomerase (deaminase), encoded by gene nagB of the nag operon, was able to fulfill anabolic functions in amino sugar biosynthesis. The suppressor mutants invariably expressed the isomerase constitutively as a result of mutations in nagR, the locus for the repressor of the nag regulon. Suppression was also possible by transformation of glmS mutants with high-copy-number plasmids expressing the gene nagB. Efficient suppression of the glmS lesion, however, required mutations in a second locus, termed glmX, which has been localized to 26.8 min on the standard E. coli K-12 map. Its possible function in nitrogen or cell wall metabolism is discussed.     

Rhizobium nodM and nodN genes are common nod genes: nodM encodes functions for efficiency of nod signal production and bacteroid maturation.

Earlier, we showed that Rhizobium meliloti nodM codes for glucosamine synthase and that nodM and nodN mutants produce strongly reduced root hair deformation activity and display delayed nodulation of Medicago sativa (Baev et al., Mol. Gen. Genet. 228:113-124, 1991). Here, we demonstrate that nodM and nodN genes from Rhizobium leguminosarum biovar viciae restore the root hair deformation activity of exudates of the corresponding R. meliloti mutant strains. Partial restoration of the nodulation phenotypes of these two strains was also observed. In nodulation assays, galactosamine and N-acetylglucosamine could substitute for glucosamine in the suppression of the R. meliloti nodM mutation, although N-acetylglucosamine was less efficient. We observed that in nodules induced by nodM mutants, the bacteroids did not show complete development or were deteriorated, resulting in decreased nitrogen fixation and, consequently, lower dry weights of the plants. This mutant phenotype could also be suppressed by exogenously supplied glucosamine, N-acetylglucosamine, and galactosamine and to a lesser extent by glucosamine-6-phosphate, indicating that the nodM mutant bacteroids are limited for glucosamine. In addition, by using derivatives of the wild type and a nodM mutant in which the nod genes are expressed at a high constitutive level, it was shown that the nodM mutant produces significantly fewer Nod factors than the wild-type strain but that their chemical structures are unchanged. However, the relative amounts of analogs of the cognate Nod signals were elevated, and this may explain the observed host range effects of the nodM mutation. Our data indicate that both the nodM and nodN genes of the two species have common functions and confirm that NodM is a glucosamine synthase with the biochemical role of providing sufficient amounts of the sugar moiety for the synthesis of the glucosamine oligosaccharide signal molecules.     

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.     

Glucosamine as carbon source for amino acid-producing Corynebacterium glutamicum

Corynebacterium glutamicum grows with a variety of carbohydrates and carbohydrate derivatives as sole carbon sources; however, growth with glucosamine has not yet been reported. We isolated a spontaneous mutant (M4) which is able to grow as fast with glucosamine as with glucose as sole carbon source. Glucosamine also served as a combined source of carbon, energy and nitrogen for the mutant strain. Characterisation of the M4 mutant revealed a significantly increased expression of the nagB gene encoding the glucosamine-6P deaminase NagB involved in degradation of glucosamine, as a consequence of a single mutation in the promoter region of the nagAB-scrB operon. Ectopic nagB overexpression verified that the activity of the NagB enzyme is in fact the growth limiting factor under these conditions. In addition, glucosamine uptake was studied, which proved to be unchanged in the wild-type and M4 mutant strains. Using specific deletion strains, we identified the PTS^Glc transport system to be responsible for glucosamine uptake in C. glutamicum. The affinity of this uptake system for glucosamine was about 40-fold lower than that for its major substrate glucose. Because of this difference in affinity, glucosamine is efficiently taken up only if external glucose is absent or present at low concentrations. C. glutamicum was also examined for its suitability to use glucosamine as substrate for biotechnological purposes. Upon overexpression of the nagB gene in suitable C. glutamicum producer strains, efficient production of both the amino acid l-lysine and the diamine putrescine from glucosamine was demonstrated.     

Enzymatic deacetylation of N-acetylglucosamine residues in cell wall peptidoglycan

An enzyme which catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine residues in cell wall peptidoglycan was found in the supernatant and 20,000 X g pellet fractions of Bacillus cereus. Autolysis of the latter fraction resulted in solubilization and activation of the deacetylase. Among various bacteria, strains of B. cereus which contain high proportions of N-unsubstituted glucosamine residues in their cell wall peptidoglycan components are particularly rich in the deacetylase. The peptidoglycan deacetylase is distinguishable from N-acetylglucosamine-6-phosphate deacetylase [EC 3.5.1.25] on the basis of their cellular distribution and chromatographic behavior. The rate of reaction of the deacetylase with (N-acetylglucosaminyl-N-acetylmuramic acid)3 [abbreviated as (GlcNAc-MurNAc)3] is less than 1/100 of that with peptidoglycan, while the enzyme is inactive towards (GlcNAc-MurNAc)2, GlcNAc-MurNAc, and monomeric N-acetylglucosamine derivatives. The enzyme also deacetylates partially O-hydroxyethylated chitin. The concentrations of peptidoglycan and partially O-hydroxyethylated chitin required for half-maximum activities were found to be 0.29 and 6.9 mg per ml (or 0.17 and 20 mM with respect to N-acetylglucosamine residues), respectively. The occurrence of this enzyme accounts for the formation of cell wall peptidoglycan N-unsubstituted at the glucosamine residues.     

Metabolism of glucosamine in the liver of Scyliorhinus canicula (selachian)]

(1) The metabolism of the glucosamine was studied in the liver of S. canicula, after injection of D-(1-14C) glucosamine into the animal. (2) The labelled acid-soluble derivatives were separated by ion exchange columns and characterized by chromatography and electrophoresis, and were identified as glucosamine, glucosamine 6-P, N-acetylglucosamine, N-acetylmannosamine, N-acetylneuraminic acid, N-acetylglucosamine 6-P, N-acetylglucosamine 1-P, N-acetylmannosamine 6-P, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine. (3) The variation with time after glucosamine injection of the radioactivity of the fractions separated on Dowex 1-X4 was investigated. This study showed a decrease of the radioactivity in the fraction 1 (glucosamine and glucosamine 6-P), an increase in the fraction II (N-acetylneuraminic acid) and the fraction IV (UDP-N-acetylhexosamines), and a stability in the fraction III (phosphorylated N-acetylhexosamines). (4) The absence of label in neutral hexoses and their phosphorylated derivatives was interpretated as due to the weak activity of the glucosamine 6-P isomerase, which is positively modulated by the N-acetylglucosamine 6-P.     

Analysis of N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1 by a proteomic approach

Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.