DNA Uptake during Genetic Transformation and the Growing Zone of the Cell Envelope

Abstract

Continuous cellular incorporation of choline molecules is essential for DNA binding to competent pneumococci. The choline molecules are incorporated into the cell surface at the equatorial region of the cocci. As a working hypothesis, it is proposed that DNA molecules enter these bacteria at the growing zone(s) of the cell envelope.The presence of choline residues in the teichoic acid portion of the pneumococcal cell envelope (1) is essential for several physiological functions of this bacterium. Not only is choline required for growth (2), but biosynthetic replacement of choline in the cell surface by structural analogs (such as ethanolamine) causes several striking physiological changes in the bacteria (2-5); one such change is a defect in cell division that prevents the physical separation of daughter cells from one another. The incorporation of new choline molecules into the cell surface takes place at a distinct growing zone, localized at the equatorial region of pneumococci (6). We now describe evidence which suggests that the cell-surface changes that make pneumococci capable of absorbing and integrating extracellular DNA are in some way coupled to the functioning of the growing zone of the cell envelope. MATERIALS AND METHODSCultures of the R36A strain of Diplococcus pneumoniae were used in all experiments at a concentration of 107-108 cells/ml. The composition of a chemically defined (Cd) (7) and a semisynthetic (C) (8) medium was described earlier. An enriched defined medium (Cden) was prepared by supplementing Cd medium with all the amino acids present in acid-hydrolyzed casein, at the same concentrations as in the C medium. "Choline-phase" and "ethanolamine-phase" bacteria were grown in Cden medium supplemented with 5 MLg/ml of choline or 40 Mg/ml of ethanolamine, respectively. Bacterial growth was monitored with a Coleman nephelometer. Cells were transferred from one medium to another by filtration (Millipore filters, pore size 0.45 Mm). Radioactive tracers were used at 5 Mg/ml and 1 MCi/ml concentrations, unless otherwise noted. The incorporation of radioactive tracers into cold trichloroacetic acid (TCA)-insoluble material was measured by collecting the samples on glass-fiber disks (Schleicher and Schuell); after drying, the disks were counted in a toluene-based scintillator in a scintillation spectrometer (Unilux, Nuclear-Chicago). The preparation and assay of competence factor (CF) (9) and transforming DNA (10), determination of CF-receptor activity (11), and the tests for the competence-specific agglutinin (12) were all performed by published procedures. The concentration of CF during the "activation" assay was usually 100-500 Units (13) per ml. Lysis sensitivity of pneumococci was determined as follows: 0.9 ml of the culture was pipetted into tubes prepared with 0.1 ml of phosphate buffer (1 M, pH 8) and 50 Ml of a 5% solution of deoxycholate. The preparation of Bacillus subtilis DNA labeled with [3H]thymidine and radioautography of cell-bound DNA were described (14). Pneumococcal DNA ...

Bacterial Cell Wall Synthesis: New Insights from Localization Studies

“…In a different study, Tomasz and colleagues took advantage of the fact that choline-containing cell wall is sensitive to autolysin whereas ethanolamine-containing cell wall is not. Autolysin treatment of choline-pulsed ethanolamine-grown cells confirmed that new cell wall is inserted in the central portion of dividing cells (187,188). These results were recently supported by Van-FL staining of S. pneumoniae, which clearly indicated PG synthesis activity at the septal and (stained less brightly) the equatorial rings (31).…”

The CiaRH System ofStreptococcus pneumoniaePrevents Lysis during Stress Induced by Treatment with Cell Wall Inhibitors and by Mutations inpbp2xInvolved in β-Lactam Resistance

“…It is possible that the CiaRH system is also involved in licD2 expression under choline starvation, but further experiments are required to reveal the relation between CiaRH, the licD2 product, and cellular lysis. Interestingly, cells also respond to choline deprivation with a rapid decline of cellular competence (52), another indication that the cia system might be activated and indeed required under these conditions.…”

Pneumococcal Virulence Factors: Structure and Function

“…The other domain, located at the N terminus, is probably directly responsible for the lytic activity against pneumococcal peptidoglycan structures. Attachment of the enzyme to the choline of the S. pneumoniae cell wall teichoic acid is essential for the lytic activity of the enzyme (155). The carboxy-terminal attachment module probably influences the activity of the enzyme by stabilizing or inducing its active conformation (17).…”

“…Examples of other pneumococcal CBPs are the major cell wall hydrolase LytA (54) and an adhesin choline binding protein A (CbpA) (129). LytA is an amidase functioning in the separation of daughter cells during cell division (127) and is required for cell lysis (155), whereas CbpA appears to be the first known protein adhesin on the pneumococcal surface (129). The CBR motif has also been found among the surface proteins of other bacteria (164) like Clostridium acetobutylicum, Clostridium difficile, Streptococcus mutans, and Streptococcus downei.…”