Peptidoglycan types of bacterial cell walls and their taxonomic implications.

Processes for fermentative production of xylitol - a sugar substitute

Hemicellulosic sugars, especially d-xylose, are relatively abundant in agricultural and forestry residues. Moreover, they can be recovered from the hemicelluloses by acid hydrolysis more readily and in better yields than can d-glucose from cellulose. These factors favor hemicellulosic sugars as a feedstock for production of ethanol and other chemicals. Unfortunately, d-xylose is not so readily utilized as d-glucose for the production of chemicals by microorganisms. The reason may lie in the biochemical pathways used for pentose and hexose metabolism. Different pathways are employed by prokaryotes and eukaryotes in the initial stages of pentose assimilation. Transport and phosphorylation possibly limit the overall rate of d-xylose utilization. The intermediary steps of pentose metabolism are generally similar for both bacteria and fungi, but substantial variations exist. Phosphoketolase is present in some yeasts and bacteria able to use pentoses. Regulation of the oxidative pentose phosphate pathway occurs at d-glucose-6-phosphate dehydrogenase by the intracellular concentration of NADPH. Regulation of nonoxidative pentose metabolism is not well understood. In some yeasts and fungi, conversion of d-xylose to ethanol takes place under aerobic or anaerobic conditions with rates and yields generally higher in the former than in the latter. Xylitol and acetic acid are major byproducts of such conversions. Many yeasts are capable of utilizing d-xylose for the production of ethanol. Direct conversion of d-xylose to ethanol is compared with two-stage processes employing yeasts and d-xylose isomerase.

Utilization of xylose by bacteria, yeasts, and fungi

Listeria monocytogenes bacteriophages A118, A500 and A511 are members of three distinct phage groups with characteristic host ranges. Their endolysin (ply) genes were cloned and expressed in Escherichia coli as demonstrated by the conferred lytic phenotype when colonies of recombinant cells were overlaid with a lawn of Listeria cells. The nucleotide sequences of the cloned DNA fragments were determined and the individual enzymes (PLY118, 30.8 kDa; PLY500, 33.4 kDa; PLY511, 36.5 kDa) were shown to have varying degrees of homology within their N-terminal or C-terminal domains. Transcriptional analysis revealed them to be 'late' genes with transcription beginning 15-20 min post-infection. The enzymes were overexpressed and partially purified and their individual specificities examined. When applied exogenously, the lysins induced rapid lysis of Listeria strains from all species but generally did not affect other bacteria. Using hydrolysis of purified listerial cell walls, PLY511 was characterized as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and shows homology in its N-terminal domain to other enzymes of this type. In contrast, PLY118 and PLY500 were shown to represent a new class of cell wall lytic enzymes which cleave between the L-alanine and D-glutamate residues of listerial peptidoglycan; these were designated as L-alanoyl-D-glutamate peptidases. These two enzymes share homology in the N-terminal domain which we propose determines hydrolytic specificity. Highly conserved holin (hol) gene sequences are present upstream of ply118 and ply500. They encode proteins of structural similarity to the product of phage lambda gene S, and are predicted to be membrane proteins which form pores to allow access of the lysins to their peptidoglycan substrates. This arrangement of conserved holin genes with downstream lysin genes among the siphoviral lysis cassettes explains why the cytoplasmic endolysins alone are not lethal, since they require a specific transport function across the cell membrane.

Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes

Publisher Summary Polyols (sugar alcohols) are widely distributed in fungi. They could serve several roles in living organisms: (1)act as carbohydrate reserves, (2) act as translocatory compounds, (3) function in osmoregulation, and (4) in coenzyme regulation and storage of reducing power. This chapter describes several enzymes that are concerned with polyol metabolism in fungi. These include Glycerol dehydrogenase (NAD + ), Glycerol-3-phosphate dehydrogenase (NAD + ), Xylitol dehydrogenase (NAD + ), Mannitol-1-phosphate dehydrogenase, Polyol dehydrogenase (NADP + ), Mannitol dehydrogenase (NAD + ), Glyceroldehydrogenase (NADP + ), Ribitol-5-phosphate dehydrogenase (NADP + ), Mannitold ehydrogenase(NADP + ), Glycerophosphate dehydrogenase, Glycerol kinase, Mannitol kinase, Mannitol-1-phosphatase, Sorbitol (glucitol) dehydrogenase, and Glycerol oxidase. A number of enzymes have relatively high K m , values for their substrates and a high pH optimum for the oxidation of polyols, which is likely to be a consequence of the buffer more effectively consuming protons at such pH values. It is conceivable that these particular enzymes are membrane-bound. The groups to which the various fungi belong are identified and designated as—namely, Mastigomycotina (M), Zygomycotina (Z), Ascomycotina (A), Deuteromycotina (D) and Basidiomycotina (B).

Polyol metabolism in fungi

Aldose reductase, NADPH and NADP+ in normal, galactose-fed and diabetic rat lens.

Purification and properties of D-glycerate 3-kinase from Escherichia coli.

Doughty, C. C. (University of Illinois College of Medicine, Chicago) and James A. Hayashi. Enzymatic properties of a phage-induced lysin affecting group A streptococci. J. Bacteriol. 83:1058-1068. 1962.-Phage-induced lysis of group C streptococci releases into the medium a lysin which completely lyses group A streptococci. Partial purification of the lytic activity yields 47% of the original activity with a 17-fold purification. The activity was assayed by observing lysis of group A streptococci under standard conditions. The optimal pH range for lysis is from 6.0 to 6.7. A monovalent cation requirement satisfied by Na(+), K(+), or Li(+) is shown by the lysin. Lysis is stimulated by ethylenedi-aminetetraacetic acid (EDTA), chlortetracycline, streptomycin, and penicillin. It is inhibited by p-hydroxymercuribenzoate (pHMB), and the inhibition is reversed by cysteine. Other inhibitors include ristocetin A and specific antisera against the lysin. Isolated group A streptococcal cell walls are partially lysed by massive amounts of lysin. This partial lysis is not affected by EDTA, pHMB, chlortetracycline, streptomycin, or ristocetin A. It is concluded that the enzymatic process of lysis of isolated cell walls is not identical to the more complex process resulting in lysis of intact cells.

Enzymatic properties of a phage-induced lysin affecting group A streptococci.

A phage-induced cell wall solubilizing enzyme isolated from phage-infected Staphylococcus aureus phage type 80 was purified 588-fold. The pH optimal activity was 6.8 to 7.3, and pH optimal stability, 6.5 to 7.5. It was inhibited by p-hydroxymercuribenzoate, ethylenediaminetetraacetic acid, and specific rabbit antisera. The cell wall lytic reaction is a peptidase resulting in cleavage of the cell wall peptide at N-terminal alanine, glutamic acid, and glycine. Electron micrographs are shown of cell wall “ghosts” remaining after the enzymatic digestion of cell walls.

Purification and Properties of a Bacteriophage-induced Cell Wall Peptidase from Staphylococcus aureus

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-aldehyde reductase was isolated in 24% yield and 66-fold purification from a dl-glyceraldehyde-grown Rhodotorula species. This enzyme was specific for NADPH, and d-, l-, or dl-glyceraldehyde were equally good substrates. Other substrates had activities as follows: methylglyoxal, 50%; fructose, 33%; d- and l-arabinose, 12%; d-xylose, 8%; d-glucose, 5%; d-erythrose and d-threose, 0 to 5%. The product from the reduction of dl-glyceraldehyde was glycerol, as shown by high voltage electrophoresis, paper chromatography, and direct enzymatic analysis. Kinetic studies gave K m values of 0.89 mm and 0.013 mm for dl-glyceraldehyde and NADPH, respectively. An optimal p H range of 6.3 to 6.7 was found for maximal activity. Reduction of NADP + by glycerol was not demonstrable. This Rhodotorula NADPH-aldehyde reductase activity was compared to similar enzymes from other sources.

C. C. Doughty

Papers

Aldose reductase, NADPH and NADP+ in normal, galactose-fed and diabetic rat lens.

Purification and properties of D-glycerate 3-kinase from Escherichia coli.

Enzymatic properties of a phage-induced lysin affecting group A streptococci.

Purification and Properties of a Bacteriophage-induced Cell Wall Peptidase from Staphylococcus aureus

Identification of Reduced Nicotinamide Adenine Dinucleotide Phosphate-Dependent Aldehyde Reductase in A Rhodotorula Strain