L-forms of a strain of Pseudomonas aeruginosa were produced by serial subculture of the bacterial form on agar medium containing sucrose as an osmotic stabilizer and carbenicillin. L-forms eventually became stable, i.e., would not revert in the absence of antibiotic, and were adapted to grow well in broth with the osmotic stabilizer. Gross morphology and light microscopic colony morphology were typical of an L-form. L-form cells were approximately spherical and bounded in part by a plasma membrane; they lacked the triple-layer cell wall structure and coarse, electron-dense nucleoidal granules of the parent bacterial form. The L-form, but not the bacterial form, contained cores, organelles previously reported only in group D streptococci. Antibiotic disc-sensitivity studies showed the stable L-form to be as sensitive as, or more sensitive than, the bacterial form to most antibiotics. Exceptions were polymyxin B, colimycin sulfate, and gentamicin, which were more active against the bacterial form. The remainder of the aminoglycosides and cell wall-active antibiotics showed no inhibition of either form. The L-form was more susceptible to cidal activity of normal human serum than the parent form. The L-form exhibited fewer biochemical activities than the parent bacteria or bacterial forms derived by reversion at a time when the L-form was still unstable. L-form colonies appeared colorless, and chemical analysis demonstrated that, if the L-form produces pigment at all, which was not demonstrated, it could not have been more than 3.6% of that produced by the bacterial form.

Anti-Bacterial Agents↗

PubMed1971

Cell wall polysaccharide biosynthesis by membrane fragments from Streptococcus pyogenes and stabilized L-form.

The formation and composition of a cell wall rhamnose-containing polysaccharide by membrane fragments from Streptococcus pyogenes and its stabilized L-form were compared. Also, the effect of prior treatment on the ability of coccal whole-cell and membrane fragments to incorporate radioactivity from thymidine diphosphate-(14)C-rhamnose, and the results of subsequent attempts to remove labeled polysaccharide from such membranes are given. L-form membrane fragments were capable of only 10% uptake of (14)C-rhamnose from this nucleotide as compared with streptococcal membranes. However, once bound, both membrane fragments polymerized rhamnose to the same extent. These findings tend to negate the almost complete lack of polymeric rhamnose within the intact L-form as being due to the absence of membrane enzymes necessary for the transfer of rhamnose from a suitable precursor to membrane acceptor sites or enzymes responsible for rhamnose polymerization. Degradation of labeled rhamnose polysaccharide after isolation from coccal membranes by mild acid hydrolysis showed muramic acid and glucosamine to be attached. This same polysaccharide from L-form membrane fragments was devoid of amino sugars. These data suggest the possible involvement of amino sugars in the attachment of cell wall polymeric rhamnose to the streptococcal cytoplasmic membrane. The absence of attached amino sugars to rhamnose polysaccharide from L-form membrane fragments is discussed in terms of this organism's continued inability for new cell wall formation. The isolation, from streptococcal membrane fragments, of a polysaccharide containing rhamnose and amino sugars common to at least two different streptococcal cell wall-type polymers was demonstrated.

Amino Sugars↗

PubMed1971