Amine and carboxyl groups of the cell wall of Bacillus subtilis were chemically modified individually to neutralize their electrochemical charge for determination of their contribution to the metal uptake process. Mild alkali treatment removed ca. 94% of the constituent teichoic acid (expressed as inorganic phosphorus) and allowed estimation of metal interaction with phosphodiester bonds. Chemical modifications of amine functions did not reduce the metal uptake values as compared to native walls, whereas extraction of teichoic acid caused a stoichiometric reduction in levels. In contrast, alteration of carboxyl groups severely limited metal deposition of most of the metals tested. X-ray diffraction and electron microscopy suggested, in this case, that the form and structure of the metal deposit could be different from that found in native walls. The observations suggest that carboxyl groups provide the major site of metal deposition in the B. subtilis wall.

Sites of metal deposition in the cell wall of Bacillus subtilis.

Covalent lipoprotein from the outer membrane of escherichia coli

Isolated walls of Bacillus subtilis Marburg, prepared in a manner which avoided metal contamination other than by the growth medium, were incubated in dilute metal solutions, separated by membrane filtration (0.22 mum), and monitored by atomic absorption to give uptake data for 18 metals. Substantial amounts of Mg2+, Fe3+, Cu2+, Na+, and K+ (amounts which were often visible as Au3+, and Ni2+ (the higher atomic-numbered elements also visible as electron scattering), and small amounts of Hg2+, Sr2+, Pb2+, and Ag+ were taken into the wall. Some (Li+, Ba2+, Co2+, and Al3+) were not absorbed. Most metals which had atomic numbers greater than 11 and which could be detected by electron microscopy appeared to diffusely stain thin sections of the wall. Magnesium, on the other hand, partitioned into the central region, and these sections of walls resisted ruthenium red staining, which was not true for the other metals. Areas of the walls also acted as nucleation sites for the growth of microscopic elemental gold crystals when incubated in solutions of auric chloride. Retention or displacement of the metals was estimated by a "chromatographic" method using the walls cross-linked by the carbodiimide reaction to adipic hydrazide agarose beads (which did not take up metal but reduced the metal binding capacity of the walls by ca. 1%) packed in a column. When a series of 12 metal solutions was passed through the column, it became evident that Mg2+, Ca2+, Fe3+, and Ni2+ were strongly bound to the walls and could be detected by both atomic absorption and by their electron-scattering power in thin sections, qhereas the other metals were fisplaced or replaced. Partial lysozyme digestion of the walls (causing a 28% loss of a [3H]diaminopimelic acid label) greatly diminished the Mg2+ retention but not that of Ca2+, Fe3+, or Ni2+, indicating that there are select sites for various cations.

Uptake and retention of metals by cell walls of Bacillus subtilis.

Publisher Summary This chapter describes the ultrastructure, chemistry and function of the bacterial wall. Cell walls are dynamic and change to fulfill functions dictated by the cell in response to the environment. Most bacteria respond unequivocally to the Gram reaction; that is, some retain the large crystal violet-iodine complex (Gram-positive), whereas others are decolorized by the alcohol treatment (Gram-negative) and can be counterstained. Cell age, autolysin levels, and growth conditions can affect the Gram reaction. Unlike Gram-positive bacteria, freeze-cleaved and -etched Gram-negative cells present a number of cleavage sites within the wall that is an indication of multilayering. This wall is chemically and structurally more complex than its Gram-positive counterpart. Each of the layers of capsule, slime layers, and surface arrays reside above the wall and may be singular or in combination with one another. Each presents unique problems for preservation and visualization by electron microscopy. The chapter also discusses the functional aspects of walls that include (1) interaction with metals; (2) β-lactam drugs and low-level antibiotic resistance; and (3) functional discontinuities in the wall fabric.

Ultrastructure, Chemistry, and Function of the Bacterial Wall

The 3D structure of the bacterial peptidoglycan, the major constituent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmuramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacterium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its complexity and the lack of pure samples. Herein we report the 3D solution structure as determined by NMR of the 2-kDa NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) synthetic fragment of the cell wall. The glycan backbone of this peptidoglycan forms a right-handed helix with a periodicity of three for the NAG-NAM repeat (per turn of the helix). The first two amino acids of the pentapeptide adopt a limited number of conformations. Based on this structure a model for the bacterial cell wall is proposed.

https://www.pnas.org/content/pnas/103/12/4404.full.pdf

Three-dimensional structure of the bacterial cell wall peptidoglycan

X-Ray diffraction, infrared spectra and density of murein, the rigid component of almost all bacterial cell walls, were measured using gram-negative (Spirillum serpens) and gram-positive (Lactobacillus plantarum) strains.



X-Ray diffraction of dried foils of murein showed Debye-Scherrer rings, indicating periodicities of 0.93 to 1.00 and 0.44 nm. The comparison of the infrared spectra of chitin and murein showed that their amide I and amide II bands have the same frequencies. With isopycnic density-gradient centrifugation of murein a density of ϱ= 1.46 g × cm−3 was observed.



Based on these data, on theoretical considerations and on model building the following structure of murein is proposed: the structure of the glycan chains in murein is similar to that of chitin and cellulose. The peptides consisting of alternating d- and l-amino acids have the 2.27 helical conformation. They are linked to the carbohydrate chains through their N-terminus and form two hydrogen bonds with the sugar residues. This linkage causes an angle of about 150° between the carbohydrate and peptide chains.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03620.x

Sibylle Formanek

Papers

A three-dimensional atomic model of the murein layer of bacteria.