Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia.

The cell wall peptodoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined. The petidoglycan of C. botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81. All other types of C. botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type. The peptidoglycans of Clostridium bifermentans and Clostridium histoloyticum contained DAP but they differed from those of C. botulinum in the molar ratio of alanine to glutamic acid. The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90. On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine. In spite of the difference in amino acid composition of peptidoglycans among the clostridia, the quantitative precipitin test demonstrated that antiserum against C. botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other clostridia as well as various types of C. botulinum.     

Mode of Action of Clostridium Phage HM 7-Induced Lytic Enzyme on Clostridium saccharoperbutylacetonicum Cell Wall Peptidoglycan

The action of Clostridium phage HM 7-induced lytic enzyme on the cell wall peptidoglycan of Clostridium saccharoperbutylacetonicum was investigated. The cell wall peptidoglycan of this strain contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00: 2.08: 0.97; 0.92: 0.68. It was strongly digested when incubated with the lytic enzyme. This digestion was accompanied by the release of NH₂-terminal l-alanine without a concomitant release of COOH-terminal amino acids and reducing groups. Chromatography of the lytic enzyme digest resulted in only two fractions, each of which was chromatographically homogeneous. One was a polysaccharide consisting of glucosamine and muramic acid in molar ratios 1.00: 0.78, and other was a peptide composed of glutamic acid, alanine and diaminopimelic acid in molar ratios of 1.00: 2.09: 1.05. These results indicate that phage HM 7-induced lytic enzyme is N-acetylmuramyl-l-alanine amidase, which cleaves the linkage between N-acetylmuramic acid and l-alanine.

A possible structure for the cell wall peptidoglycan was also proposed.     

Peptidoglycan of free-living anaerobic spirochetes

Electron microscope examination of negatively stained or thin-sectioned cells of Spirochaeta stenostrepta treated with penicillin or lysozyme showed that the peptidoglycan was present as a thin, electron-dense layer adjacent and external to the cytoplasmic membrane. The peptidoglycan was isolated from cells of S. stenostrepta and Spirochaeta litoralis by a procedure including treatments with sodium lauryl sulfate and Pronase. Hydrolysates of the isolated S. stenostrepta and S. litoralis peptidoglycans contained glucosamine, muramic acid, glutamic acid, l-ornithine, and alanine in molar ratios of 0.90:0.85:1.00:1.00:1.40 and of 0.63:0.63:0.99:1.00:1.41, respectively. Determination of N-terminal residues suggested that nearly 50% of the ornithine in S. stenostrepta and S. litoralis peptidoglycans was involved in peptide cross-linkage. The peptidoglycan layer of S. stenostrepta was sensitive to lysozyme and myxobacter AL-1 protease.     

Chemical Composition of the Cell Walls of Clostridium botulinum Type A

Cell walls were isolated by sonic disruption of log-phase cells of Clostridium botulinum type A strain 190L and purified by treatment with sodium dodecyl sulfate (SDS) followed by digestion with proteases. Electron microscopy revealed that the cell walls thus obtained were free of both cytoplasmic membrane and cytoplasmic fragments. The purified cell wall contained 8.7% total nitrogen, 15.0% total hexosamines, 22.4% reducing groups, 8.3% carbohydrate, and 3.1% glucose. The content of total phosphorus was very low (0.02%), and therefore it was expected that teichoic acid might be absent in the cell wall. The wall peptidoglycan contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:1.85:0:85:1.06:0.67. A low amount of galactosamine was also present, but no other amino acids were found in significant quantities. The SDS-treated cell walls were not attacked by lysozyme, but after extraction with hot formamide they were completely dissolved by the enzyme and released reducing groups. The lysozyme digest was separated into two constituents, the saccharide moiety and the peptide moiety on Sephadex G-50.     

Isolation and characterization of the peptidoglycans from selected gram-positive and gram-negative periodontal pathogens

The peptidoglycans from several Gram-negative and Gram-positive periodontal pathogens were isolated, purified, and characterized both morphologically and chemically. In addition, the effects of the mureolytic enzymes, lysozyme, M-1 N-acetyl-muramidase, and the AM-3 endopeptidase, on the peptidoglycans were examined. These enzymes were found to be highly effective in the degradation of the purified peptidoglycans; however, a Bacteroides capillus peptidoglycan-protein complex exhibited a greater resistance to these enzymes. Morphologically, the peptidoglycans consisted of large saccular sheets which, when viewed by scanning electron microscopy, contained numerous holes and tears. Chemically, the peptidoglycans consisted of muramic acid, glucosamine, alanine, glutamic acid, and meso-diaminopimelic acid (DAP). One Bacteroides species, Bacteroides gingivalis strain W, contained glycine and LL-DAP, suggestive of an indirectly cross-linked A3 gamma peptidoglycan.     

Novel peptidoglycans in Caulobacter and Asticcacaulis spp.

Peptidoglycan sacculi free of poly-beta-hydroxybutyric acid were prepared from whole cells of four species of Caulobacter and two species of Asticcacaluis and from morphological mutants of Caulobacter crescentus and Caulobacter leidyi. Acid hydrolysates of the sacculi were analyzed quantitatively, and each of the hydrolysates was found to contain significant amounts of only five ninhydrin-reactive compounds: alanine, glutamic acid, alpha , omega-diaminopimelic acid, muramic acid, and glucosamine. Four types of peptidoglycans were distinguishable on the basis of the molar ratios among these five compounds. The respective ratios were as follows: in C. leidyi, 2:1:1:1:0.8; in Asticcacaulis biprosthecum, 1.7:1.6:1.1:0.7; in the cells of the remaining species, 2:1:1:1.2:0.8; and in stalks shed by the abscission mutant 2NY66, 2:1:1:1:1.67. Thus, in addition to some species differences among these caulobacters, it was found that the peptidoglycan sacculus of the stalked C. crescentus cell is chemically differentiated; the cellular peptidoglycan is richer in muramic acid than is the peptidoglycan of typical gram-negative bacteria, and the peptidoglycan of the stalk is correspondingly rich in glucosamine. Empirical formulas for the repeating units of the peptidoglycans have been inferred on the basis of the molar ratios of their amino components.     

Amino acid composition of peptidoglycan in Caulobacter crescentus.

Peptidoglycan of a gram-negative stalked bacterium, Caulobacter crescentus CB13, contained alanine, diaminopimelic acid, and glutamic acid, in molar ratios of 2 : 1 : 1. The amino acid compositions of peptidoglycans isolated from cultures enriched in swarmer and stalked cells, and from a stalk-less mutant were similar. This finding conflicts with a previous observation that swarmer peptidoglycan does not contain diaminopimelic acid (Goodwin and Shedlarski (1975) Arch. Biochem. Biophys. 170, 23-36). It appears that, despite the morphological differences, the Caulobacter cells all contain a similar peptidoglycan in the cell wall.     

Cell walls from Actinopolyspora halophila,an extremely halophilic actinomycete

Cell wall components were prepared from Actinopolyspora halophila (strain wt), an extremely halophilic actinomycete requiring a minimum 12% NaCl concentration for growth, and from an erythromycin-resistant strain of A. halophila (strain ER) that required only 6% NaCl for growth. Both cell wall preparations contained glutamic acid, alanine, and diaminopimelic acid in a 1:2:1 molar ratio. On the basis of muramic acid content, peptidoglycans from the wt and ER strains contained 255 and 245 disaccharide units per mg dry weight respectively. In addition, both cell wall preparations contained from 10 to 20% more glucosamine than muramic acid, and equimolar amounts of d-galactose and d-arabinose. Analysis of cell walls before and after digestion with Myxobacter AL-1 protease indicated that nearly all glycan disaccharide units were peptide-substituted and that peptide cross-bridging was facilitated by direct peptide linkages between N-diaminopimelic acid and C-terminal alanine. While the peptidoglycan of A. halophila wt was 50% peptide cross-linked, that from A. halophila ER was approximately 67% peptide cross-linked. Chemical modifications involving substitution of non-N-acetylated hexosamines of the cell walls greatly enhanced their sensitivity to lysozyme. Although differences in peptidoglycan structure between the two strains of A. halophila were observed, these probably do not account for the reduced salt requirement for growth of the erythromycin-resistant strain.Issued as NRCC 25165     

Analysis of the peptidoglycan of Rickettsia prowazekii.

In the present study, peptidoglycan from Rickettsia prowazekii, an obligate intracellular bacterium, was purified. The rickettsial peptidoglycan is like that of gram-negative bacteria; that is, it is sodium dodecyl sulfate insoluble, lysozyme sensitive, and composed of glutamic acid, alanine, and diaminopimelic acid in a molar ratio of 1.0:2.3:1.0. The small amount of lysine found in the peptidoglycan preparation suggests that a peptidoglycan-linked lipoprotein(s) may be present in the rickettsiae. D-Cycloserine, a D-alanine analog which inhibits the biosynthesis of bacterial cell walls, prevented rickettsial growth in mouse L929 cells at a high concentration and altered the morphology of the rickettsiae at a low concentration. These effects were prevented by the addition of D-alanine. This suggests that R. prowazekii contains D-alanine in the peptidoglycan and has D-Ala-D-Ala ligase and alanine racemase activities.     

Amino acid utilization patterns in clostridial taxonomy

The polyamide layer technique for the chromatographic separation of dimethylaminonaphthalene sulphonyl amino acids has been adapted to the qualitative analysis of amino acids in media before and after the growth of micro-organisms. The method has been used to study the amino acids metabolized by cultures of proteolytic clostridia growing in a medium consisting of an acid hydrolysate of casein as a source of amino acids and small amounts of yeast extract and trypticase as sources of growth factors. The chromatograms of the media after growth showed which amino acids were used and which new amino acids were produced. Clostridium botulinum type F (proteolytic), C. ghoni, C. mangenoti and C. putrificum were found to reduce proline to 5-aminovaleric acid and to produce 2-aminobutyric acid, properties they shared with C. sporogenes and C. sticklandii. C. botulinum type G and C. subterminale used glycine, lysine, serine, and arginine but in contrast to C. sticklandii they neither reduced proline to 5-aminovaleric acid nor produced 2-aminobutyric acid. Both organisms oxidized phenylalanine, tyrosine and tryptophan to phenylacetic acid, p-hydroxyphenyl acetic acid and indole acetic acid respectively. C. lituseburense and C. scatologenes used serine, threonine and arginine and produced 2-amino butyric acid and ornithine. C. lentoputrescens, C. limosum and C. malenomenatum resembled C. tetanomorphum by using glutamic acid and tyrosine. The chromatograms always showed the physiological group to which an organism belonged and in some cases were characteristic of the species.Abbreviations Abu 2-aminobutyric acid - Ava 5-aminovaleric acid - DNS 1-dimethyaminonaphthalene-5-sulphonyl - DNS-Cl the sulphonyl chloride - DNS-NH₂ the sulphonamide - DNS-OH the sulphonic acid - VFA steam volatile fatty acid - u unknown     

Recovery of Clostridium botulinum from mud samples incubated at different temperatures

Summary In mud samples naturally contaminated with Clostridium botulinum type C, other types cannot be recovered when incubated at 37°C. At incubation temperatures equal to or lower than 30°C and in the presence of type E, Clostridium botulinum type C cannot always be detected. The use of two incubation temperatures to increase the probability of detecting all types of Clostridium botulinum can therefore be recommended.     

Isolation of lipid activated pseudomurein precursors from Methanobacterium thermoautotrophicum

From cell extracts of the pseudomurein possessing methanogen Methanobacterium thermoautotrophicum two putative pseudomurein precursors were isolated and characterized: (1) an undecaprenyl pyrophosphate activated disaccharide pentapeptide composed of N-acetylglucosamine, N-acetyltalosaminuronic acid, alanine, glutamic acid and lysine in a molar ratio of 1:1:2:2:1 and (2) the corresponding undecaprenyl pyrophosphate activated tetrapeptide lacking one alanine residue. The isolation of these precursors show that the biosynthesis of the eubacterial murein and the methano-bacterial pseudomurein differs not only in the cytoplasmic step, as recently described, but also in the lipid stage.Abbreviations GlcNitol glucosaminitol - NAcTalNUA N-acetyltalosaminuronic acid - Udp undecaprenol - TLC thin layer chromatography     

Autolytic Enzyme System of Clostridium botulinum

The mode of action of the autolytic enzymes of Clostridium botulinum type A strain 190L was investigated using a partially purified autolysin. The autolysin completely solubilized SDS-treated cell walls of the organism, liberating 1.2 moles of NH₂-terminal-L-alanine and 0.6 moles of reducing groups per mole of glutamic acid. Neither the NH₂-termini of other amino acids nor COOH-termini of any amino acids were released. These results show that the autolysin contains an N-acetylmuramyl-L-alanine amidase and a hexosaminidase. A disaccharide and peptides were isolated from the wall lysate in a chromatographically homogeneous state. The reducing end of the disaccharide was elucidated to be N-acetylglucosamine by borohydride reduction. This fact indicates that the hexosaminidase is likely to be an endo-β-N-acetylglucosaminidase. A possible structure of the cell wall peptidoglycan is proposed.     

Structure of rigid-layer of Rhizobium cell wall. I. Purification on the peptidoglycan from the cellulose microfibrils

The bag shaped peptidoglycan layer of Rhizobium cell wall was isolated from intact cells after treatment with sodium dodecylsulfate and trypsin, chymotrypsin or pepsin digestion. Results of chemical analysis of acid hydrolyzed peptidoglycan revealed beside two amino sugars: glucosamine and muramic acid, three major amino acids; alanine, glutamic acid and 2,6-diaminopimelic acid and also significant amount of glucose. Evidence were provided that the polyglucose found in peptidoglycan preparations of three strains of Rhizobium trifolii, one of Rhizobium leguminosarum and one of Rhizobium meliloti consist of cellulose microfibrils. The content of cellulose present in Rhizobium peptidoglycans ranged from 60 to 80%. Methods of peptidoglycan purification from the cellulose microfibrils are described.     

Structure of the peptidoglycans of Moraxella glucidolytica and Moraxella lwoffi grown on hydrocarbons

The peptidoglycans of Moraxella glucidolytica and Moraxella lwoffi grown on aliphatic hydrocarbons were isolated. They contained muramic acid. glucosamine, alanine, d-glutamic acid and meso-diaminopimelic acid in a molar ratio of about 0.5:0.5:1.6:1.0:1.0 (M. glucidolytica) and 0.8:0.7:1.3:1.0:1.0 (M. lwoffi).The peptidoglycans were lysozyme-resistant. However, when treated with formamide, they could be partially degraded by lysozyme. The fragments were purified and their structure determined. In both strains, the peptide subunits consisted mainly of tripeptides (l-Ala-d-Glu-meso-DAP) and tetrapeptides (l-Ala-d-Glu-meso-DAP-d-Ala), most of them being directly cross-linked. It is concluded that in both strains the primary structures of the peptidoglycans are closely related.     

Analysis of the Botulinum Neurotoxin Type F Gene Clusters in Proteolytic and Nonproteolytic Clostridium botulinum and Clostridium barati

Comparison of genes encoding type F botulinum neurotoxin progenitor complex in strains of proteolytic Clostridium botulinum strain Langeland, nonproteolytic Clostridium botulinum strain 202F, and Clostridium barati strain ATCC 43256 reveals an identical organization of genes encoding a protein of molecular mass of approx. 47 kDa (P-47), nontoxic-nonhemagglutinin (NTNH) and botulinum toxin (BoNT). Although homology between the protein components of the complexes encoded by these different species all producing botulinum neurotoxin type F is considerable (approx. 69–88% identity), exceptionally high homology is observed between the C-termini of the P-47s (approx. 96% identity) and the NTNHs (approx. 94% identity) encoded by Clostridium botulinum type F strain Langeland and Clostridium botulinum type A strain Kyoto. Such a region of extremely high sequence identity is strongly indicative of recombination in these strains synthesizing botulinum neurotoxins of different antigenic types. Received: 13 April 1998 / Accepted: 9 May 1998     

Evidence for peptidoglycan-associated protein(s) in Neisseria gonorrhoeae.

Growth pH markedly influenced the composition of the cell envelope of $\text{Neisseria gonorrhoeae}$. The composition of the peptidoglycan from cells grown at pH 7.2 and 8.0 consisted primarily (91%) of muramic acid, glutamic acid, alanine, $\text{meso}$-diaminopimelic acid, and glucosamine in approximate molar ratios of 1:1:2:1:1. The peptidoglycan from cells grown at pH 6.0 contained an accessory protein(s) which accounted for 42% of the weight of the isolated complex.     

Volatile acid production from threonine,valine, leucine and isoleucine by clostridia

The amounts of the volatile acids produced from thereonine, valine, leucine and isoleucine by growing cultures of clostridia have been measured. The species used were Clostridium sporogenes; C. caloritolerans; C. botulinum proteolytic type A; C. botulinum proteolytic type B; C. botulinum proteolytic type F; C. botulinum proteolytic type G; C. putrificum; C. difficile; C. ghoni; C. bifermentans; C. sordellii; C. mangenoti; C. cadaveris; C. lituseburense; C. propionicum; C. sticklandii; C. scatologenes; C. subterminale; C. putrefaciens; C. histolyticum; C. tetanomorphum; C. limosum; C. lentoputrescens; C. tetani; C. melanomenatum; C. cochlearium; C. sporospheroides. Most of the species tested gave increased yields of propionic acid when grown in the threonine medium; in addition, some species resembled C. propionicum and produced n-butyric acid when grown in this medium. C. histolyticum produced only acetic acid in the basal medium; all seven strains of this species produced more acetic acid when grown in the threonine medium than in the basal medium. Species which oxidize valine to iso-butyric acid also oxidize leucine to 3-methyl butyric acid and isoleucine to 2-methylbutyric acid. The iso-caproic fraction produced by some species is shown to be derived from leucine. The identitity of the branched-chain acids produced by C. sporogenes has been confirmed by gas liquid chromatography/mass spectrometry.Abbreviations GLC gas liquid chromatography - RCM reinforced clostridial medium - VFA volatile fatty acid     

Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus

The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod‐shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme‐resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD‐crosslinks between meso‐diaminopimelic acid and D‐alanine residues, whereas stalk peptidoglycan had more LD‐transpeptidation (meso‐diaminopimelic acid‐meso‐diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD‐transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD‐crosslinking. Despite no obvious selection pressure for LD‐crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP‐DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.     

Streptomyces species with madurose (3-O-methyl-D-galactose) as a whole-cell sugar

Madurose, an actinomycete whole-cell sugar, was found in the strains of the genus Streptomyces: three strains of S. platensis, one strain each of S. platensis subsp. malvinus, and S. albus subsp. albus. The sugar was isolated from the hydrolysate of S. platensis IFO 14008 cells, and was identified as madurose (3-O-methyl-D-galactose) by chromatographic analyses, ¹H-NMR spectrometry, mass spectrometry as its alditol acetate, and demethylation with boron trichloride. Cell walls of the strain contained peptidoglycan and teichoic acids. ll-Diaminopimelic acid, glycine, glutamic acid, and alanine were present in the peptidoglycan fraction in molar ratios of 1.0:1.3:1.2:2.3. Madurose was detected in the teichoic acid fraction, which was composed of phosphorus, glycerol, galactose, and madurose in molar ratios of 9.3:8.5:2.9:1.0. Thus, madurose was found in the glycerol teichoic acid moiety of the cell walls of this strain.Abbreviations PC paper chromatography - TLC thin-layer chromatography - HPLC high performance liquid chromatography - GLC gas-liquid chromatography - TCA trichloroacetic acid