Assembly of a chemically synthesized peptide of Escherichia coli type 1 fimbriae into fimbria-like antigenic structures.

Escherichia coli type 1 fimbriae are composed of subunits, each of which comprises 158 amino acids. We synthesized a copy of a 13-residue peptide, located near the NH2 terminus of the fimbrial subunit, that assumed some of the properties of type 1 fimbriae. At pH 5.5 the synthetic peptide autoassembled into fibrillar structures that resembled type 1 fimbriae except that they appeared less rigid and rodlike. A quaternary structure-specific monoclonal antibody against type 1 fimbriae recognized the synthetic peptide in the assembled but not the unassembled state. Furthermore, when the synthetic peptide was injected in its fimbrial conformation into rabbits, it evoked antibodies that reacted with type 1 fimbriae isolated from E. coli.     

Cloning and expression in Escherichia coli of Haemophilus influenzae fimbrial genes establishes adherence to oropharyngeal epithelial cells.

In this report the first example of functional expression of a fimbrial gene cluster of a non-enteric human pathogen in Escherichia coli is described. This is shown for Haemophilus influenzae fimbriae which mediate adherence to oropharyngeal epithelial cells. A genomic library of H.influenzae type b, strain 770235f+bo, was constructed using a cosmid vector and screened with a synthetic oligonucleotide probe derived from the N-terminal sequence of the fimbrial subunit of H.influenzae. Four cosmid clones were found which hybridized to this oligonucleotide probe. Escherichia coli strains harbouring these clones expressed the H.influenzae fimbriae at their cell surface, as was demonstrated in a whole-cell ELISA and by immunogold electron microscopy using a monoclonal antibody specific for the H.influenzae fimbriae. Surface expression could be maintained during subcloning until a minimal H.influenzae DNA insert of approximately 8.1 kb was obtained. Escherichia coli strains harbouring the 8.1 kb H. influenzae DNA were able to cause a mannose-resistant adherence to oropharyngeal epithelial cells and a mannose-resistant haemagglutination of human AnWj-positive erythrocytes. The nucleotide sequence of hifA, the gene encoding the major fimbrial subunit, was determined. The predicted amino acid sequence shows a significant homology with a number of E.coli fimbrial subunits.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

A cryptic fimbrial gene in Serratia marcescens.

The gene coding for the mannose-sensitive hemagglutinating fimbriae in Serratia marcescens US5 was cloned into Escherichia coli K4 with a cosmid vector system. One of the transformants, US5-1, expressed two morphologically distinct fimbriae, one that was 5-nm wide and one that was 3-nm wide. The latter fimbria was morphologically and serologically indistinguishable from that of strain US5. Genetic analysis of transformant US5-1 showed that the gene responsible for the 5-nm-wide fimbriae was located more than 10 kilobases away from the gene responsible for the 3-nm-wide fimbriae. The molecular sizes of the subunits of these two fimbriae, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 19 kilodaltons for the 3-nm-wide fimbriae and 20 kilodaltons for the 5-nm-wide fimbriae. Serologically, the 5-nm-wide fimbriae did not cross-react with monoclonal or polyclonal antibodies raised against the mannose-sensitive hemagglutinating fimbriae of strain US5. Strain EL101, which expressed only the 5-nm-wide fimbriae, did not agglutinate chicken or human erythrocytes. These experimental results suggest that the gene for the 5-nm-wide fimbriae is cryptic in strain US5 and is expressed in E. coli K4 only after it is moved by transformation.     

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85.

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.     

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.     

Localization of the neuronal cell adhesion molecule D2-protein in explant cultures of dorsal root ganglia by use of the colloidal-gold immunocytochemical technique

Summary The localization of the neuronal cell adhesion molecule (N-CAM), D2-protein, in explant cultures of rat dorsal root ganglia was investigated at the electron microscope level by the use of 17-nm-diameter colloidal gold particles coated with swine anti-rabbit immunoglobulin molecules. The minimum amount of IgG needed to coat the gold particles and the pH optimal for coating were both determined. Immunocytochemical studies of cultures revealed the binding of gold particles to the neuronal plasma membrane, especially on neuritic processes. Schwann cells were not labeled, and the level of unspecific background staining was very low.     

Localization of the neuronal cell adhesion molecule D2-protein in explant cultures of dorsal root ganglia by use of the colloidal-gold immunocytochemical technique

The localization of the neuronal cell adhesion molecule (N-CAM), D2-protein, in explant cultures of rat dorsal root ganglia was investigated at the electron microscope level by the use of 17-nm-diameter colloidal gold particles coated with swine anti-rabbit immunoglobulin molecules. The minimum amount of IgG needed to coat the gold particles and the pH optimal for coating were both determined. Immunocytochemical studies of cultures revealed the binding of gold particles to the neuronal plasma membrane, especially on neuritic processes. Schwann cells were not labeled, and the level of unspecific background staining was very low.     

Regular structures in membranes: the lumenal plasma membrane of the cow urinary bladder.

The ultrastructure of the lumenal plasma membrane of the cow urianry bladder has been studied in thin sections of glutaraldehyde- and glutaraldehyde-H2O2-fixed specimens, by negative staining and freeze fracture. A regular hexagonal array of particles confined to polygonal plaques 0-1-0-4-mum in diameter and separated by 0-02-mum interplaque areas is revealed by all 3 techniques. Cross-sections through particulate areas fixed with glutarayldehyde-H2O2 display a tetralaminar structure consisting of the usual approximately 8-nm-thick trilamellar unit membrane structure, on the external dense leaflet of which is located an additional approximately 4-nm-thick stratum which is occasionally resolved into a row of regulrly spaced approximately 4-nm-diameter particles. Non-particulate areas feature only the approximately 8-nm-thick trilamellar structure. Tangential sections reveal an hexagonal array of particles with a unit cell of approximately 16 nm. Four membrane faces can be revealed by freeze fracture and etching of membranes of the cow urinary bladder; 2 complementary split inner membrane faces (A and B) revealed by the cleaving process and the lumenal and cytoplasmic membrane surfaces exposed by etching. Face B, which belongs to the external membrane leaflet and faces the cytoplasm, displays plaques of particles arranged in a hexagonal lattice with a unit cell of approximately 16 nm. Face A, which belongs to the cytoplasmic membrane leaflet and faces the lumen, displays a complementary array of hexagonally packed pits. The hexagonally arranged particles also protrude into the lumenal membrane surface where they can occasionally be resolved into 6 approximately 5-nm-diameter subunits; the cytoplasmic surface appears smooth. Six approximately 5-nm-diameter subunits are also revealed in negatively stained preparations. The data are consistent with a model for the membrane in which the particles forming the hexagonal structure protrude above the lumenal membrane surface and also bridge most of the thickness of the membrane.     

Purification and characterization of CS2, a sialic acid-specific haemagglutinin of enterotoxigenic Escherichia coli.

CS2 fimbriae of enterotoxigenic Escherichia coli were purified and characterized. The surface haemagglutinins (fimbriae) were detached by sonication from a strain producing only the CS2 fimbriae. Isolation was carried out by gel filtration on a Sepharose 4B column. After depolymerization, the fimbriae subunits were purified on a Sephacryl S-300 column in 8.0 M-guanidinium chloride. From 1 litre of medium, 4-6 mg of purified fimbriae was obtained. We found that CS2 fimbriae were completely dissociated by saturated guanidinium chloride into subunits with a molecular mass of 16.5 kDa. CS2 fimbriae was sialic acid-specific, since sialic acids were the most potent inhibitors, and neuraminidase treatment of erythrocytes abolished haemagglutination. Both fimbriae and fimbrial subunits were found to bind to bovine erythrocytes. The binding of subunits to erythrocytes could be inhibited with low concentrations of sialyl-lactose.     

Oral vaccination of rats with live avirulent Salmonella derivatives expressing adhesive fimbrial antigens of uropathogenic Escherichia coli

Abstract The avirulent Salmonella typhimurium F885 was transformed with a plasmid carrying the cloned S fimbriae genes of a uropathogenic Escherichia coli . The resulting transformant (F885-1) produced efficiently E. coli S fimbriae and was used for live oral vaccination of rats. For comparison rats were immunized subcutaneously with isolated S fimbriae. Both routes of vaccination resulted in a significant IgG antibody response to S fimbriae. In addition live oral vaccination induced a serum IgA response against S fimbriae. After transurethral infection of rats with a S fimbriae producing E. coli a 10-fold reduction of bacterial counts in the kidney was observed in rats orally vaccinated with F885-1 as compared to unvaccinated controls. This study suggests that the avirulent Salmonella F885 may be used as a fimbrial antigen carrier for oral vaccination against renal infections.     

DNA sequence mapping using electron microscopy

DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe followed by detection with a two-step antibody reaction employing a colloidal gold tag. Hybridization probes can be modified with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP, or N-acetoxy-2-acetylaminofluorene (AAF). The availability of different sizes of colloidal gold particles permits the simultaneous detection of several sequences. In addition, low signals can be amplified either with an antibody sandwich scheme or by silver intensification. This technology is applicable both to TEM and SEM preparations of chromosomes, and we have used it to map a number of highly and moderately repeated sequences on whole mount metaphase chromosomes.     

A rapid method for assessing the distribution of gold labeling on thin sections.

Particulate gold labeling on ultrathin sections is in widespread use for antigen localization at the EM level. To extend the usefulness of gold labeling technology, we are evaluating different methods for sampling and estimating quantities of gold labeling. Here we present a simple, rapid, and unbiased method for assessing the relative pool sizes of immunogold labeling distributed over different cell compartments. The method uses a sampling approach developed for stereology in which a regular array of microscopic fields or linear scans is positioned randomly on labeled sections. From these readouts, gold particles are counted and assigned to identifiable cell structures to construct a gold labeling frequency distribution of those labeled compartments. Here we use ultrathin cryosections labeled for a range of different proteins and for a signaling lipid. We show by scanning labeled sections at the electron microscope that counting 100-200 particles on each of two grids is sufficient to obtain a reproducible and rapid assessment of the pattern of labeling proportions over 10-16 compartments. If more precise estimates of labeling proportions over individual compartments are required (e.g., to achieve coefficients of error of 10-20%), then 100-200 particles need to be counted over each compartment of interest.     

Oral vaccination of rats with live avirulent Salmonella derivatives expressing adhesive fimbrial antigens of uropathogenic Escherichia coli

The avirulent Salmonella typhimurium F885 was transformed with a plasmid carrying the cloned S fimbriae genes of a uropathogenic Escherichia coli. The resulting transformant (F885-1) produced efficiently E. coli S fimbriae and was used for live oral vaccination of rats. For comparison rats were immunized subcutaneously with isolated S fimbriae. Both routes of vaccination resulted in a significant IgG antibody response to S fimbriae. In addition live oral vaccination induced a serum IgA response against S fimbriae. After transurethral infection of rats with a S fimbriae producing E. coli a 10-fold reduction of bacterial counts in the kidney was observed in rats orally vaccinated with F885-1 as compared to unvaccinated controls. This study suggests that the avirulent Salmonella F885 may be used as a fimbrial antigen carrier for oral vaccination against renal infections.     

Appearance of newly formed mRNA and rRNA as ribonucleoprotein-particles in the cytoplasmic subribosomal fraction of pea embryos

Incorporation studies with ³H-uridine or ³H-adenosine showedthat germinating pea embryos synthesize all types of poly A(+)RNA, rRNA and 4–5S RNA at the early stage of germination.After the pulse labeling for 30 min, only heterodisperse RNAand 4–5S RNA appeared in the cytoplasm as labeled RNAspecies. At this time the radioactivity was associated withcytoplasmic structures heavier than 80S and RNP particles of68–70S, 52–55S, 36–38S and 20–22S whichare presumed to be free mRNP particles in plants. When the pulse-labeledembryos were incubated for a further 60 min in an isotope-freemedium, the labeled 17S and 25S rRNA emerged in the cytoplasm,together with labeled heterodisperse and 4–5S RNAs. Moreradioactivity accumulated in the regions of the polysome, 62–65Sand 38–42S particles. The results of analysis of RNAsextracted from the whole cytoplasm, polysome or subribosomalfractions indicated that small subunits of newly formed ribosomesappear more rapidly in the cytoplasm than new large subunits,which accumulate for a while as free particles in the cytoplasmthen are incorporated into polysomes. The actino-mycin treatmentwhich caused preferential inhibition of rRNA synthesis reducedthe accumulation of free, newly formed ribosome subunits andpartially permitted detection of the presumed mRNP particlesin the subribosomal region even after the chase treatment. (Received June 28, 1976; )     

Binding of S21 to the 50S subunit and the effect of the 50S subunit on nonradiative energy transfer between the 3' end of 16S RNA and S21

Escherichia coli ribosomal protein S21 was labeled at its single cysteine group with a fluorescent probe. Labeled S21 showed full activity in supporting MS2 RNA-dependent binding of formylmethionyl-tRNAf to 30S ribosomal subunits. Fluorescence anisotropy measurements and direct analysis on glycerol gradients demonstrate conclusively that labeled S21 binds to 50S ribosomal subunits as well as to 30S and 70S particles. The relative binding affinities are in the order 70S greater than 30S greater than 50S. Other results presented appear to indicate that S21 is bound in the same position on either 50S subunits or 30S subunits as in 70S ribosomes, suggesting that the protein is bound simultaneously to both subunits in the latter. Addition of 50S subunits to 30S particles containing probes on S21 and at the 3' end of 16S RNA caused a decrease in the energy transfer between these points. The results correspond to an apparent change in distance from 51 to 61 A.     

Fimbrial adhesins: similarities and variations in structure and biogenesis

Abstract Fimbriae are wiry (2 to 4 nm diam.) or rod-shaped (6 to 8 nm diam.), fibre-like structures on the surfaces of bacteria which mediate attachment to host cells. Much has been learned in recent years about the biogenesis, structure and regulation of expression of these adhesive organelles in Gram-negative bacteria. Analyses of the genetic determinants encoding the biogenesis of fimbriae has revealed that the adhesive interaction of fimbriae can be mediated by major subunits (CFA/I and CS1 fimbriae) or minor subunits (P, S, and type 1 fimbriae), with the adhesin being located either at the tip of the fimbria or along the length of the fimbrial shaft. Minor subunits can also act as adapters, anchors, initiators or elongators. Post-translational glycosylation of the type 4 pilins of Neisseria gonorrhoeae, Neisseria meningitidis and Pseudomonas aeruginosa has been demonstrated. The structures of the PapD chaperone of Escherichia coli and of N. gonorrhoeae type 4 fimbrin have been resolved at 2.0–2.6 Å. Rod-shaped fimbriae should not be thought of as being rigid inflexible structures but rather as dynamic structures which can undergo transition from a helicoidal to a fibrillar conformation to provide a degree of elasticity and plasticity to the fimbriae so that they can resist shear forces, rather like a bungee cord. At least four mechanisms have been identified in the assembly of fimbriae from fimbrin subunits, namely the chaperone-usher pathway (e.g., P-fimbriae of uropathogenic E. coli ), the general secretion assembly pathway (e.g., type 4 fimbriae or N -methylphenylalanine fimbriae of P. aeruginosa , the extracellular nucleation-precipitation pathway (e.g., curli of E. coli ) and the CFA/I, CS1 and CS2 fimbrial pathway.     

大肠杆菌K88ac菌毛操纵子fae全基因的克隆、表达及生物学活性初步研究

目的：在体外克隆和表达猪肠产毒性大肠杆菌（ETEC）K88ae菌毛操纵子,触结构基因,并检测重组菌毛的相关生物学活性。方法：利用长PCR技术以猪ETECK88ae株C83902基因组DNA为模板扩增编码K88菌毛操纵子触基因,克隆入表达质粒载体pBR322,构建和筛选重组质粒pBR322-fae,转化至不含任何菌毛的大肠杆菌EP株;电镜观察重组菌表面菌毛表达情况;用热抽提法提纯表达的重组菌毛;用纯化菌毛免疫小鼠制备高效价抗血清;用SDS-PAGE和Western blot检测重组菌毛的抗原性,用细胞黏附和黏附抑制试验检测其生物学活性。结果和结论：在电镜下观察到重组菌表面大量表达K88ae菌毛,该重组菌与兔抗K88ae菌毛单因子阳性血清、鼠抗K88ac菌毛单克隆抗体均产生凝集反应;纯化菌毛经SDS-PAGE,结构单位菌毛呈单一的相对分子质量约26×10^3的蛋白条带;纯化菌毛免疫小鼠后可制备出高效价的鼠抗血清,玻板凝集试验和Western blot结果表明体外表达的K88ae菌毛具有与K88ae野生菌毛相同的抗原性;猪小肠上皮细胞系黏附和黏附抑制实验结果表明重组EP菌和野生菌株一样具有较强的黏附猪小肠上皮细胞系的能力,而且提纯重组菌毛制备出的鼠抗血清能有效抑制上述重组菌或野生菌株对猪小肠上皮细胞系的黏附结合。     

Distribution of newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus of an Escherichia coli lac-lpp strain.

The insertion of newly synthesized lipoprotein molecules into the cell wall of Escherichia coli was studied topographically by immunoelectron microscopy. Lipoprotein was briefly induced with isopropyl-beta-D-thiogalactopyranoside in cells carrying lac-lpp on a low-copy-number plasmid in an E. coli lpp host. Specific antibodies bound to the newly inserted lipoprotein molecules, which were exposed at the cell surface after treatment of the cells with Tris-EDTA, were detected with a protein A-gold probe. The average distribution of the gold particles over the cell surface of noninduced cells was determined for cells induced for 5 and 10 min. Analysis of 250 to 350 cells showed that the distribution of newly synthesized lipoprotein over the cell surface was homogeneous in both cases. The binding of lipoprotein to the peptidoglycan layer was studied by the same technique, and visual inspection again revealed a homogeneous distribution of bound lipoprotein over the entire sacculus surface. It is therefore concluded that free lipoprotein is inserted equally over the entire cell wall of E. coli, while binding to peptidoglycan also occurs over the entire cell surface. The rate of lipoprotein synthesis increased with cell length in nondividing cells, whereas it was constant in cells which had initiated constriction. Analysis of cells having different amounts of lipoprotein in their cell wall revealed that the cell shape depended on the total lipoprotein content of the cell. Cells having no or only a small amount of lipoprotein grew as spheres, whereas cells with increasing numbers of lipoprotein molecules gradually changed their shape to short rods.     

Attachment of lipoprotein to murein (peptidoglycan) of Escherichia coli in the presence and absence of penicillin FL 1060.

In vivo studies on the attachment of lipoprotein to the murein (peptidoglycan) of Escherichia coli showed that it takes several generations of growth until the amount of lipoprotein on newly made murein is equilibrated. The technique used involves degradation of the sodium dodecyl sulfate-insoluble murein-lipoprotein complex (sacculus, rigid layer) with lysozyme and separation of the labeled products on paper. No lipoprotein was found on murein subunits incorporated during a pulse of [3H]diaminopimelate for 1 min in logarithmically growing cells at 37 C. Even after one doubling of the cell mass, only 4 to 8% of the labeled murein was isolated as bound to lipoprotein. With uniformly labeled murein, 30% remains bound to lipoprotein after lysozyme treatment, corresponding to three murein subunits. Therefore it can be concluded that during pulse labeling either no lipoprotein is incorporated into the newly synthesized murein or no murein subunits are inserted into existing murein around lipoprotein attachment sites. Longer pulse and pulse-chase experiments argue for the latter interpretation. It is therefore concluded that incorporation of murein subunits into the growing murein polymer is not at all a random process. Instead, quite large areas of murein, on which lipoprotein is situated, seem to be preserved. Under the influence of penicillin FL 1060 murein synthesis is 50% inhibited. The rate of lipoprotein attachment is less affected so that increasing amounts of lipoprotein become attached during spheroplast formation. By the time the stationary growth phase has been reached, the lipoprotein content of the murein has doubled. Diaminopimelate auxotrophic mutants require, in the presence of penicillin FL 1060, more diaminopimelate for full growth than in the absence of penicillin FL 1060. This finding and the fact that murein synthesis is always inhibited by 50% over a wide range of penicillin concentration (1 to 1,000 mug/ml) point to the inhibition of an enzymatic step of murein synthesis which can be partially bypassed by a second enzyme, less efficient but resistant to penicillin FL 1060.