The chromosomal ampC beta-lactamase in Citrobacter freundii and Enterobacter cloacae is inducible by beta-lactam antibiotics. When an inducible ampC gene is introduced on a plasmid into Escherichia coli together with its transcriptional regulator ampR, the plasmid-borne beta-lactamase is still inducible. We have isolated mutants, containing alterations in a novel E. coli gene, ampG, in which a cloned C. freundii ampC gene is unable to respond to beta-lactam inducers. The ampG gene was cloned, sequenced and mapped to minute 9.6 on the E. coli chromosome. The deduced amino acid sequence predicted AmpG to be a 53 kDa, transmembrane protein, which we propose acts as a signal transducer or permease in the beta-lactamase induction system. Immediately upstream of ampG there is another 579-base-pair-long open reading frame (ORF) encoding a putative lipoprotein shown to be non-essential for beta-lactamase induction. We have found that ampG and this ORF form an operon, whose promoter is located in front of the ORF. Located closely upstream of the putative promoter is the morphogene bolA, which is transcribed in the opposite orientation. However, using transcription fusions, we have found that the ampG transcription is not regulated by bolA. In addition, we show that transcription is probably not regulated by either the starvation specific sigma factor RpoS, which controls bolA, or by AmpD the negative regulator for ampC transcription.
Composition of cell wall peptidoglycan was studied in the gram‐negative bacterium Proteus mirabilis and in different wall‐defective growth forms of this organism which survive and multiply in the presence of penicillins. The results show that action of the classical benzylpenicillin does not proceed by a uniform mechanism of inhibition of peptide crosslinkage in peptidoglycan.
Peptidoglycan of the unstable spheroplast L‐form of P. mirabilis grown in medium with 120 μg/ml benzylpenicillin contained monomers, and peptide‐crosslinked dimers and trimers of disaccharide‐peptide building blocks in a similar ratio as peptidoglycan of the normal bacterial form. Moreover, peptide side‐chains in peptidoglycan from penicillin‐grown L‐form spheroplasts were predominantly tetrapeptides l‐alanyl‐d‐γ‐glutamyl‐meso‐diaminopimelyl‐d‐alanine. Thus, specific penicillin‐sensitive enzymes of peptidoglycan synthesis, peptidoglycan transpeptidase and dd‐carboxypeptidase, must have remained uninhibited by the antibiotic in the L‐form. L‐form peptidoglycan, however, differed characteristically from peptidoglycan of normal Proteus bacteria by having a much lower content of O‐acetyl groups linked to muramic acid residues of the polymer.
P. mirabilis grew actively in the form of osmotically stable spheres in medium with up to 100 μg/ml of the amidino penicillin mecillinam. Neither the peptide crosslinkage nor the O‐acetyl content of peptidoglycan were influenced to any great extent by mecillinam.
Only the combined action of benzylpenicillin and mecillinam caused complete inhibition of growth in P. mirabilis, concomitant with deformation of cells to very large spheres. It is hypothesized that benzylpenicillin and mecillinam each inhibit different parts of a multiple, isofunctional enzyme system whose total inhibition requires the presence of both antibiotics.
SUMMARYContinuous, cell-shaped mucopolymer layers were isolated from cell walls of some unstable and stable L-forms of Proteus mirabilis. The chemical compositions of these non-rigid mucopolymers and of normal rigid mucopolymer basal-structures of rod-shaped Proteus bacteria were very similar. Electron microscopy showed that the protein and mucopolymer of the complex 'rigid layers' were organized differently in cell walls of rods and L-form cells. The results suggest that the rigid-layer protein plays a role in the morphogenesis of the mucopolymer.
Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine with cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes.
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