Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.

We report here the cloning and characterization of a cytoplasmic kinase of Clostridium acetobutylicum, named MurK (for murein sugar kinase). The enzyme has a unique specificity for both amino sugars of the bacterial cell wall, N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), which are phosphorylated at the 6-hydroxyl group. Kinetic analyses revealed K m values of 190 and 127 M for MurNAc and GlcNAc, respectively, and a k cat value (65.0 s ؊1 ) that was 1.5-fold higher for the latter substrate. Neither the non-Nacetylated forms of the cell wall sugars, i.e., glucosamine and/or muramic acid, nor epimeric hexoses or 1,6-anhydro-MurNAc were substrates for the enzyme. MurK displays low overall amino acid sequence identity (24%) with human GlcNAc kinase and is the first characterized bacterial representative of the BcrAD/BadFGlike ATPase family. We propose a role of MurK in the recovery of muropeptides during cell wall rescue in C. acetobutylicum. The kinase was applied for high-sensitive detection of the amino sugars in cell wall preparations by radioactive phosphorylation.Clostridia are anaerobic, spore-forming, and A/T-rich Gram-positive rods that include several toxin-producing pathogens (e.g., Clostridium difficile, C. botulinum, C. tetani, and C. perfringens), as well as nonpathogenic members such as C. acetobutylicum ATCC 824. The latter is a biotechnologically important strain that is used for the production of solvents such as acetone, butanol, and ethanol by fermentation of a broad range of mono-, di-, and polysaccharides (13). Although solvent formation by the conversion of carbohydrates attracted interest in the physiology of carbohydrates in this organism (17) and despite its wide use in metabolic engineering and the recent sequencing of its genome (20), the cell wall (peptidoglycan) catabolism has not been investigated thoroughly in C. acetobutylicum or in any of its pathogenic relatives. For the related members of the firmicutes, Bacillus subtilis, B. megaterium, and B. cereus, it was shown that as much as 50% of the parental peptidoglycan is turned over in one generation (6,16,21). We have recently identified a pathway in B. subtilis for the rescue of peptidoglycan fragments, which involves the sequential processing of N-acetylglucosamine (GlcNAc)-N-acetylmuramic acid (MurNAc) peptides (muropeptides) by the secreted B. subtilis enzymes N-acetylglucosaminidase (NagZ Bs ) and Nacetylmuramyl-L-alanine amidase (AmiE Bs ) (14). Thereby, the amino sugars GlcNAc and MurNAc are generated extracellularly and are presumably further metabolized alike in Escherichia coli. The pathways involve the uptake by specific phosphotransferase system transporters, NagP (25) and MurP (8), respectively, yielding, intracellularly, the respective 6-phosphate sugars, the cytoplasmic recycling enzyme MurNAc-6-phosphate etherase (MurQ), which converts MurNAc-6-phosphate to GlcNAc-6-phosphate (10, 14), and the nag genes (nagA-encoded GlcNAc-6-phosphate deacetylase and nagBencoded glucosamine-6-phosphate deaminase), w...

SUMMARY— The melting point and 1 1% cm‐values of a‐bixin, β‐bixin, and a‐norbixin were determined and the stability of a‐bixin examined under different conditions. The total pigment content of butter colors was determined. a‐Bixin was the principal pigment; in addition, at least eight different pigments were present. The content of a‐ and β‐bixin in butter colors was also determined. Because of complex formation of bixin and norbixin with a yellow pigment, the simple chromatographic method became rather laborious. This complex formation did not occur in the annatto suspensions in oil and fat. These preparations were more concentrated, while the amount of decomposition products of a‐bixin was relatively slight. The stability of the pigments in butter colors appeared to be great. Investigation of annatto cheese colors showed that these extracts contain at least seven color components. The total amount of red pigment and the principal pigment a‐norbixin were determined.

Many bacteria, in particular Gram-positive bacteria, contain high proportions of non-N-acetylated amino sugars, i.e., glucosamine (GlcN) and/or muramic acid, in the peptidoglycan of their cell wall, thereby acquiring resistance to lysozyme. However, muramidases with specificity for non-N-acetylated peptidoglycan have been characterized as part of autolytic systems such as of Clostridium acetobutylicum. We aim to elucidate the recovery pathway for non-N-acetylated peptidoglycan fragments and present here the identification and characterization of an acetyltransferase of novel specificity from C. acetobutylicum, named GlmA (for glucosamine/glucosaminide N-acetyltransferase). The enzyme catalyzes the specific transfer of an acetyl group from acetyl coenzyme A to the primary amino group of GlcN, thereby generating N-acetylglucosamine. GlmA is also able to N-acetylate GlcN residues at the nonreducing end of glycosides such as (partially) non-Nacetylated peptidoglycan fragments and ␤-1,4-glycosidically linked chitosan oligomers. K m values of 114, 64, and 39 M were determined for GlcN, (GlcN) 2 , and (GlcN) 3 , respectively, and a 3-to 4-fold higher catalytic efficiency was determined for the di-and trisaccharides. GlmA is the first cloned and biochemically characterized glucosamine/glucosaminide N-acetyltransferase and a member of the large GCN5-related N-acetyltransferases (GNAT) superfamily of acetyltransferases. We suggest that GlmA is required for the recovery of non-N-acetylated muropeptides during cell wall rescue in C. acetobutylicum.The glycan chains of the peptidoglycan of the bacterial cell wall are composed of alternating, ␤-1,4-glycosidically linked amino sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) (30). The two glycosidic bonds of these glycans are targeted by muramidases, such as lysozymes that hydrolyze the MurNAc-GlcNAc linkages, and by (endo-)Nacetylglucosaminidases that cleave the GlcNAc-MurNAc bonds. Many bacteria, in particular Gram-positive pathogens, have been reported to acquire resistance against cell lysis due to the action of lysozyme and N-acetylglucosaminidases by Ndeacetylation of a great portion of the amino sugars of the peptidoglycan of their cell wall (29). Araki et al. showed that the majority of glucosamine residues of the peptidoglycans of Bacillus cereus, B. subtilis, and B. megaterium have free amino groups and that non-N-acetylated glucosamine residues accounted for the resistance of these strains to lysozyme (1, 2, 12). The peptidoglycan of Streptococcus pneumoniae, which contains 40 to 80% glucosamine (GlcN) and up to 10% muramic acid (19), is N-deacetylated at the GlcNAc residues in the peptidoglycan by the GlcNAc deacetylase PgdA, the first characterized peptidoglycan deacetylase (31). Clostridium acetobutylicum, which is an endospore-forming, anaerobic firmicute that is closely related to bacilli, presumably also contains N-deacetylated peptidoglycan since orthologs of pgdA are present on its chromosome. Moreover, an autolytic muramidase has been i...

It is possible to determine 2.5 to 250 y of mercury photometrically by extracting the mercury salt solution with an excess of dithizon solution in chloroform and measuring the extinctions of the chloroform layer a t 500 and 610 mp. T h e amount of mercury is calculated from the volume of dithizon solution used and the extinctions E, and E,,, measured, thus standardization of the dithizon solution is unnecessary.%he pure solution of the orange coloured mercury dithizonate in chloroform turns a dirty greenish orange in daylight, in direct sunlight it becomes bluish purple in a few minutes. T h e original colour is quickly recovered again in the dark. This reversible photochemical process is completely inhibited by acetic acid.For the quantitative determination of small quantities of mercury the use of dithizon ( D z ) is at present preferred. W i t h this substance the mercury can be determined according to two principles 1 ) . by titration and by so-called mixedcolour colorimetry. Photometric methods do not seem to be in use, which is surprizing when it is kept in mind that f o r the determination of lead, copper and zinc with dithizon photometric methods are much preferred. During the years 1935-1939 W i n k 1 e r did attempt to determine mercury quantitatively by means of the extinctions of mercury dithizonate (HgDz,), but he later declared his own method to be useless,), due to the instability of the compounds when exposed to light. A photometric method was proposed by F i s c h e r and L e o p o 1 d i 3 ) ; no experience of this method by other investigators is however known, and it seems to us to be more laborious than the new method described below.Our method can be explained as follows. W h e n a solution of a mercuric salt in dilute sulphuric acid is shaken with the green solution of Dz in chloroform, HgDz, is formed and dissolves in the chloroform forming an orange solution. (In this mineral acid medium only-gold and platinum may constitute a hindrance). W i t h a slight excess (5-10 percent) of Dz, which is manifested by a dirty greenish orange colour of the chloroform layer, the complete extraction of the mercury is insured. If it is permissible to apply the law of L a m b e r t -B e e r to this solution, the concentrations c,)~ and clIF of the two coloured substances Dz and HgDz, can be calculated from the extinctions at two wave *lengths chosen as suitably as possible, namely that wave length at which the light absorption by the Dz is relatively the greatest and that wave length at which the light absorption of the Dz is relatively the smallest compared with that of the HgDz,.In order to work out this principle we determined the extinction curves of