The enzymic digest of cell wall peptidoglycan from Clostridium saccharoperbutylacetonicum by phage HM 7 endolysin (N-acetylmuramyl-L-alanine amidase) was separated into two constituents on ion-exchange chromatography. One was a polysaccharide, which contained N-acetylglucosamine and N-acetylmuramic acid in the molar ratio of 1.00:0.78. This polysaccharide was digested by phage HM 3 endolysin (N-acetylmuramidase), and the digested product was a saccharide composed of N-acetylglucosamine and N-acetylmuramic acid. The other was a peptide composed of glutamic acid, alanine, and diaminopimelic acid in the molar ratio of 1.00:2.09:1.05. Amino acid sequence of the isolated peptide was determined by Edman degradation method, and optical configuration of the component amino acids was confirmed by gas chromatography using their N-trifiuoroacetyl menthyl esters. These analyses indicated that the isolated peptide was composed of a tetrapeptide subunits of NH2-terminal-L-Ala-D-Glu-Dpm-D-Ala. A reasonable structure for the cell wall peptidoglycan was also proposed.
The effect of inoculum size and nutrients on bacterial growth was examined using a Biophotometer (Jouan, Paris). The inoculum size greatly affected the length of lag time but not the growth rate. There were two types of reduction of lag time. In one the lag time was reduced regularly in parallel with an increase in inoculum size, and in the other the lag time was reduced irregularly. Composition of the media and supplementation of nutrients influenced the reduction of lag time, but the effect of compounds used were varied from strain to strain. Among the compounds employed, no common substance was found that would change an irregular reduction of lag time to a regular reduction of lag time with an increase of inoculum size. Reduction of lag time when inoculum size was increased ten times was termed L10, and a simple calculation of doubling time is discussed on the basis of cell number in the inoculum and L10.
The effects of various long-chain fatty acids supplied in the culture medium on fatty acid composition in a strain of Staphylococcus aureus (SA-36) and its stable L-form variant (SL-36) are described. L-form variant SL-36 did not grow on a medium supplemented with oleic acid or linoleic acid. Neither L-form nor bacterial parent grew in media supplemented with palmitoleic acid. Gas-liquid chromatography profiles were obtained on isolated cell fractions by thin-layer chromatography after the microorganisms had been cultivated in each experimental medium. Analysis of fatty acids derived from bacterial strain SA-36 showed that the major fatty acid peaks reflected the particular fatty acids used as supplements. SA-36 incorporated heptadecanoic, oleic, and linoleic acids although none of these is normally encountered in this microorganism after cultivation in conventional media. In contrast to the bacterial parent, fatty acids appearing in isolates of L-form SL-36 after growth in each experimental medium were found to be semi-independent of fatty acid supplementation. Additionally, SL-36 incorporated heptadecanoic acid, although this fatty acid was not detectable when SL-36 was grown in unsupplemented media. Tuberculostearic acid was detected uniquely in the L-form following cultivation in diverse experimental media.
Phospholipid compositions were radiochemically studied on spherical bacteria belonging to the genera of Micrococcus, Planococcus, Staphylococcus, and Sporosarcina, and two unidentified motile cocci. The strains of the genera of Planococcus and Sporosarcina exhibited the presence of phosphatidylethanolamine (PE), cardiolipin (CL), phosphatidylglycerol (PG), and an unidentified phospholipid b. The genus Micrococcus showed the presence of CL, PG, and unidentified phospholipids a and b, and the genus Staphylococcus, the presence of CL, PG, and the unidentified phospholipid b. The phospholipid composition indicated a close relation to other taxonomic characteristics in this group of bacteria, and was not affected qualitatively by cultural conditions. From these findings, the phospholipid composition is considered to be useful for differentiation of aerobic gram-positive cocci.
Large amounts of alkaline phosphatase and 2, 3-cyclic phosphodiesterase were released into the culture medium during the active growth of heptose-deficient mutants of Escherichia coli NS1 to NS3, but no periplasmic enzymes were released from cells of the wild type strain JE. The NS mutants were hyper-sensitive to lysozyme, ethylenedia-minetetraacetate (EDTA), sodium deoxycholate (DOC), sodium dodecyl sulfate (SDS), and Triton X100; the sensitivity to detergent was increased several fold when the treatment was carried out in the presence of potassium cyanide. The wild type strain was not sensitive to lysozyme or detergents, even in the presence of KCN. Both the wild type and mutant strains were sensitive to colicins, E1, E2, E3, G, H, and K, and to phages T2, T3, T6, and BF23, but they were resistant to phages P22, FO, Ffm, 6SR, Br2, Br60, C21, and T5. Unlike the wild type strain, the NS mutants were resistant to phages T4, T7, and Br10. A comparison of the protein composition of the outer membrane of wild type and NS1 strains indicated that one major protein species of molecular weight (mol. wt.) 50, 000 was absent while two other proteins of mol. wt. 40, 000 and 33, 400 were present only in reduced amounts in the outer membrane of the NS1 mutant. These observations suggest that both the polysaccharide and protein components of the outer membrane of E. coli participate in the normal functioning of the outer membrane as a ‘permeability barrier.’