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R Fontana

Publications and source records attributed to R Fontana.

At least 145 records · Page 8Linked to original sources
PubMed1983

Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium ATCC 9790 has an unusually low affinity for penicillin (50% binding occurred at a penicillin level of 8 micrograms/ml after 60 min of incubation, and the protein only became labeled after 20 min of incubation with high concentrations of radioactive penicillin). PBPs with similar properties are carried by strains of Streptococcus durans, Streptococcus faecalis, and Streptococcus lactis but not by strains of groups A, B, C, and G streptococci or Streptococcus pneumoniae. The strains carrying the slow-reacting PBP demonstrated a sensitivity to penicillin that was several hundred times lower than that of strains not carrying it. Spontaneous mutants with minimal inhibitory concentrations of penicillin of 20, 40, and 80 micrograms/ml were isolated from S. faecium ATCC 9790. They all showed a dramatic increase in the amount of slow-reacting PBP produced. Mutants with increased penicillin resistance were also isolated from wild-type strains of S. durans, S. faecalis, and S. faecium. All of them carried a greater amount of the slow-reacting PBP than that carried by the parent. Finally, it was found that resistant S. faecium ATCC 9790 mutants grew normally in the presence of penicillin concentrations that were far above that saturating all PBPs except PBP 5. Cell growth was, on the contrary, inhibited by a penicillin concentration that saturated the slow-reacting PBP by 90%. This penicillin dose was equal to the minimal inhibitory concentration.

Bacterial Proteins↗
PubMed1983

Division blocks in temperature-sensitive mutants of Streptococcus faecium (S. faecalis ATCC 9790).

Two hundred nine temperature-sensitive growth or division (or both) mutants of Streptococcus faecium ATCC 9790 were isolated. These strains were examined for timing of the division block in the cell division cycle. About 42% of the isolates were blocked at terminal stages of cell division. A second large group appeared to be blocked at various stages of septation. Only five of the temperature-sensitive isolates were blocked at a stage before the completion of chromosome replication. Thirty temperature-sensitive isolates lysed after one or more doublings at the nonpermissive temperature.

Cell Division↗
PubMed1982

Results of Mayo lung project: an interim report.

Screening for lung cancer by chest roentgenography and sputum cytology can be conducted by mail. Detection, localization, and treatment of early presymptomatic lesions are possible. The initial prevalence of lung cancer in men older than age 45 years who were heavy smokers was almost 1%. Rescreening at 4-monthly intervals detected 4.7 new cases of lung cancer per 1,000 subjects per year. Three times as many postsurgical stage I lesions were detected in the screened group as in the control group. Although survival seems to have been improved by screening, mortality from lung cancer is not yet significantly different in the 4-monthly surveillance and control groups. Longer follow-up is necessary for determination of the ultimate usefulness of screening.

Adenocarcinoma↗
PubMed1981

Early initiation of deoxyribonucleic acid replication and shortening of generation time associated with inhibition of lateral wall formation by mecillinam.

The effects of mecillinam on the growth of rods of the pH-conditional morphology mutant MirM7 was studied. It has been found that mecillinam causes, coincident with transition to coccal shape, a balanced rise in the rate of viable count increase and the rate of macromolecular synthesis which lasts either until the cells enter a stationary growth phase or indefinitely, in the case of continuously diluted cultures. When the antibiotic is removed from cells which have already become coccoid, cells continue to grow at a faster rate until they resume the rod shape. No change in the per-cell rate of protein synthesis has been seen in untreated or mecillinam-treated cells before or after the change in growth rate. Studies with synchronously growing cells have shown that the antibiotic causes a shortening in the I period (initiation of deoxyribonucleic acid replication). Evaluation of the residual divisions in nalidixic acid-treated, exponential-phase cells has shown that mecillinam also shortens the D period (cell division). It is proposed that, in strain MirM7, inhibition of lateral wall elongation by the antibiotic allows the initiation of a new septum, though inhibition is still in progress. The initiation of a new septum is, in turn, responsible for both the early inibition of deoxyribonucleic acid replication and accelerated division. In the parental strain, MirA12, as well as in other sensitive gram-negative rods which divide, become cocci, and stop dividing after addition of the antibiotic, inhibition of lateral wall formation activates a feedback mechanism which prevents insertion of new septa (Satta et al., J. Bacteriol. 142:43-51, 1980). Consequently, no early initiation of deoxyribonucleic acid replication is observed, and the last division allowed by the antibiotic occurs in due time. This negative control is missing in MirM7.

Amdinocillin↗
PubMed1980

Identification of the lethal target of benzylpenicillin in Streptococcus faecalis by in vivo penicillin binding studies.

The mode of bacterial killing by penicillins is still unknown in spite of many studies on the subject. The recent finding of multiple penicillin binding proteins (PBPs) in sensitive bacteria and the possibility of analysing the binding of the antibiotic to exponentially growing cells have provided new directions for investigating this problem. Sensitivity to lethal and other effects of penicillin varies very significantly with the conditions of growth of the cells. If PBPs were the penicillin target, changes in conditions of growth causing variations in penicillin sensitivity should be accompanied by changes in these proteins. Furthermore, if one of PBPs could be identified as the killing target, it could possibly be demonstrated to show changes in cells growing in different conditions. We show here that in Streptococcus faecalis ATCC 9790 changes in conditions of growth are accompanied by changes in PBPs. Furthermore, in the presence of the minimal dose of 14C-benzylpenicillin causing complete inhibition of cell growth, 100% of the total radioactivity is bound to a single protein (PBP 3).

Bacterial Proteins↗
PubMed1980

Stimulation of spreading of trypsinized human fibroblasts by lysozymes from Staphylococcus aureus, hen egg white, and human urine.

The effect of lysozyme from three different sources--Staphylococcus aureus, hen egg white, and human urine--on adhesion to substrate and spreading of trypsinized human fibroblasts was studied. Several fibroblast strains were tested under various conditions. It was found that the different cell strains did not show the same capability of spreading and stably attaching to substrates when resuspended in media not containing serum. Some strains did not spread, whereas others spread even in the absence of serum. Cell spreading in these strains did not occur when the cells were pregrown for 5 weeks in media supplemented with 1% fetal bovine serum. Lysozyme from S. aureus allowed stable adhesion to substrate and spreading of all the fibroblast strains unable to elongate in nonsupplemented minimal essential medium. This enzyme accelerated and augmented spreading of the strains capable of elongating in the absence of serum. S. aureus lysozyme also allowed spreading and stable adhesion to substrates of all these strains when they were pregrown for 5 weeks in the presence of 1% fetal bovine serum. Furthermore, hen egg white lysozyme and the lysozyme purified from human urine were both capable of stimulating anchorage to substrate and spreading of trypsinized fibroblasts although their effect was less pronounced than that of the S. aureus lysozyme. Some tentative hypotheses for the mechanism of cell spreading in the presence of lysozyme are made. The possibility that lysozymes, virtually ubiquitous enzymes, may play a specific role in nature in the regulation of cell differentiation and tissue development is finally raised and discussed in light of several previous observations and findings.

Animals↗
PubMed1980

The penicillin-binding proteins in Streptococcus faecalis ATCC 9790.

Streptococcus faecalis ATCC 9790 possesses seven membrane-bound penicillin-binding proteins. They have been characterized with respect to their apparent molecular weights, relative abundance, specificity profiles for 15 different beta-lactam antibiotics and stability under various conditions. In water and at 37 degrees C, all the native penicillin-binding proteins have half-lives longer than 20 h except protein 3b (half-life of about 600 min) and protein 4 (half-life of about 175 min). The short-lived 80 000-Mr protein 4 is spontaneously converted into a 73 000-Mr water-soluble, penicillin-binding protein 4. Similarly, the short-lived 82 000-Mr protein 3b seems to be the protein from which the 72 000-Mr water-soluble protein X spontaneously originates during incubation of the membranes. Release of both proteins 4 and X from the membrane is maximal under alkaline conditions; it is not inhibited by various protease inhibitors. After exposure to trypsin, the 43 000-Mr membrane-bound penicillin binding protein 6 (a DD-carboxypeptidase) gives to a 30 000-Mr water-soluble protein 6. Like the parent protein, protein 6 exhibits both DD-carboxypeptidase activity and penicillin-binding ability. With proteins 6 and 6, low dose levels of p-chloromercuribenzoate prevent both enzyme activity and combination with penicillin, thus strongly suggesting that a thiol group is involved in the enzyme active center. We have shown previously [Coyette et al. in Eur. J. Biochem. 88, 297--305 (1978) and 75, 231--239 (1977)] that the DD-carboxypeptidase protein 6 fragments the benzylpenicillin molecule with formation of phenylacetylglycine. Breakdown of the complex formed between [14C]benzylpenicillin and 14 000-Mr membrane-bound protein 1 is also 'enzyme-catalysed'. Most likely, however, the released product is penicilloate. With all the other penicillin-binding proteins whose molecular weights are intermediate between those of proteins 1 and 6, breakdown of the complexes formed with [14C]benzylpenicillin results from proteolysis and is not due to the release of the bound metabolite. None of the penicillin-binding proteins behaves, by itself, as a lethal target for beta-lactam antibiotic action on the living cells.

Binding, Competitive↗
PubMed1980

Control of cell septation by lateral wall extension in a pH-conditional morphology mutant of Klebsiella pneumoniae.

The pH-conditional morphology mutant of Klebsiella pneumoniae strain MirM7 grows as cocci at pH 7 and as rods at pH 5.8. The mutant has a high-level mecillinam resistance (50% lethal dose greater than 200 micrograms/ml) in both forms. When broth cultures of the rod-shaped mutant were grown with 0.7 microgram of mecillinam per ml, cells assumed a round shape and continued to divided at a higher rate than the untreated control. A MirM7 rod-shaped revertant (MirA12), when treated with the same antibiotic concentration, changed to coccal shape and stopped dividing. The penicillin-binding proteins (PBPs) of strains MirA12 and MirM7 were analyzed. K. pneumoniae had six major PBPs quite similar to those of Escherichia coli. No differences were seen in the PBPs of MirM7 cocci and rods and MirA12 cells. In particular, PBP2 was found to be present and similar in MirM7 rods and cocci and MirA12 cells. We suggest that that in gram-negative rods, a control mechanism exists which prevents further septation in the absence of lateral cell wall elongation. The unique behavior of MirM7 is due to the fact that the control mechanism is not active in this strain. This model allows us to explain the preservation of shape in bacterial rods under various conditions of growth and the mechanism of bacterial killing by mecillinam.

Amdinocillin↗
PubMed1980

Glucagon-, glicentin-, and pancreatic polypeptide-like immunoreativities in rectal carcinoids and related colorectal cells.

Three nonargentaffin rectal carcinoids have been investigated immunohistochemically. In one case most tumor cells reacted with antiglucagon sera as well as with antiglicentin, antibovine pancreatic polypeptide (BPP), and antihuman pancreatic polypeptide (HPP) sera; they were identified ultrastructurally as L cells. Another case showed glucagon-, glicentin-, and BPP-immunoreactive cells but lacked HPP immunoreactivity. In the third case glucagon- and glicentin-immunoreactive cells were well represented, while PP immunoreactivities were scarce. Parallel investigations of human rectal and sigmoid mucosa showed numerous cells reacting with glucagon, glicentin, and BPP antisera, most of which lacked HPP immunoreactivity. Cells reacting with glucagon and glicentin antisera, while lacking PP immunoreactivities, were also found. Thus, both tumor and nontumor cells produce glucagonlike immunoreactive (GLI) peptides--one of which may be glicentin or a related molecule--as well as PP-related sequences, although differing histochemically and ultrastructurally from glucagon or PP cells of the human pancreas. It is concluded that nonargentaffin rectal carcinoids are histogenetically linked to nonargentaffin endocrine cells of the human rectum.

Adult↗
PubMed1979

Peptidoglycan synthesis in cocci and rods of a pH-dependent, morphologically conditional mutant of Klebsiella pneumoniae.

Mir M7 is a spontaneous morphologically conditional mutant of Klebsiella pneumoniae which grows as round cells (cocci) at pH 7 and as normal rods at pH 5.8. We studied the rates of peptidoglycan synthesis of cocci and rods growing at pH values of 7 and 5.8, respectively. It was found that exponentially growing cocci produced a reduced amount of peptidoglycan per cell, compared with rods. Moreover, a shift of cocci to the permissive pH (5.8) caused an increase in the rate of peptidoglycan synthesis, whereas the reverse shift of rods to pH 7 determined a twofold reduction in the rate of [(3)H]diaminopimelic acid incorporation. During synchronous growth at pH 7, the rate of peptidoglycan synthesis after cell division decreased with time and rose before and during the first division. The susceptibilities of rods and cocci to beta-lactam antibiotics were also studied. It was found that cocci were more sensitive both to penicillin G and to cephalexin than were rods, but they showed a high level of resistance to mecillinam. The peculiar behavior of this mutant was interpreted as supporting the existence in bacterial rods of two different sites for peptidoglycan synthesis: one responsible for lateral wall elongation and one responsible for septum formation. In Mir M7, shape damage is described as dependent on the specific inhibition, at the nonpermissive pH, of the site for lateral wall extension.

Amdinocillin↗
PubMed1979

Alterations in peptidoglycan chemical composition associated with rod-to-sphere transition in a conditional mutant of Klebsiella pneumoniae.

Klebsiella pneumoniae Mir M7 is a spontaneous parentless morphology mutant which grows as cocci at pH 7 and as rods at pH 5.8. This strain has been characterized as defective in lateral wall formation (at pH7). Data suggest that the cell wall is mainly made up of poles of the rods (G. Satta, R. Fontana, P. Canepari, and G. Botta, J. Bacteriol. 137:727--734, 1979). In this work the isolation and the biochemical properties of the peptidoglycan of both Mir M7 rods and cocci and a nonconditional rod-shaped Mir M7 revertant (strain Mir A12) are described. The peptidoglycan of Mir M7 (both rods and cocci) and Mir A12 strains carried covalently bound proteins which could be easily removed by pronase treatment in Mir M7 rods and Mir A12 cells, but not in Mir M7 round cells. However, when the sodium dodecyl sulfate-insoluble residues of Mir M7 cocci were pretreated with ethylenediaminetetraacetic acid (EDTA), pronase digestion removed the covalently bound proteins, and pure peptidoglycan was obtained. EDTA treatment of the rigid layer of Mir M7 cocci removed amounts of Mg2+ and Ca2+, which were 10- and 50-fold higher, respectively, than the amount liberated from the rigid layer of Mir M7 rods and Mir A12 cells. Amino acid composition was qualitatively similar in both strains, but Mir M7 cocci contained a higher amount of alanine and glucosamine. Mir M7 cocci contained approximately 50% less peptidoglycan than rods. Under electron microscopy, the rigid layer of the Mir M7 rods and Mir A12 cells appeared to be rod-shaped and their shape remained unchanged after EDTA and pronase treatment. On the contrary, the Mir M7 cocci rigid layer appeared to be round, and after EDTA treatment it collapsed and lost any definite morphology. In spite of these alterations, the peptidoglycan of Mir M7 cocci still appeared able to determine the shape of the cell and protect it from osmotic shock and mechanical damages. The accumluation of divalent cations appeared necessary for the peptidoglycan to acquire sufficient rigidity for shape determination and cell protection. We concluded that the coccal shape in Mir M7 cells is not due to loss of cell wall rigidity but is a consequence of the formation of a round peptidoglycan molecule. The possibility that the alterations found in the Mir M7 cocci rigid layer may reflect natural differences in the biochemical composition of the septa and lateral wall of normally shaped bacteria is discussed.

Amino Acids↗
PubMed1979

Detection of bacterial phosphatase activity by means of an original and simple test.

A new test for the detection of bacterial phosphatase activity has been devised. The test is performed using agar media containing both methyl green (MG) and phenolphthalein diphosphate (PDP); in these media phosphatase-producing strains grow deep-green-stained colonies whereas non-producing strains do not. A total of 739 different strains were tested, including 593 staphylococci, 95 micrococci, 11 streptococci, 10 corynebacteria, 14 enterobacteria, and 16 candidae. All strains found phosphatase-positive according to the conventional phosphatase test displayed deep-green-stained colonies on MG-PDP media, whereas all phosphatase-negative strains showed unstained colonies on the same media. The main advantages of the present phosphatase test as compared with other conventional ones are that it is more simple to perform, it can reveal the phosphatase activity of colonies grown in deep agar, and can be incorporated into commercial multitest kits.

Bacteria↗
PubMed1978

Solubilization and isolation of the membrane-bound DD-carboxypeptidase of Streptococcus faecalis ATCC9790. Properties of the purified enzyme.

Streptococcus faecalis ATCC 9790 possesses six membrane-bound, penicillin-binding proteins. That numbered 6 (Mr 43000) is the most abundant one and is the DD-carboxypeptidase studied previously. The enzyme has been solubilized and purified to the stage where one single protein band can be detected by gel electrophoresis. The purification procedure does not alter the properties that the enzyme exhibits when it is membrane-bound. The DD-carboxypeptidase itself may be a killing target for penicillin in S. faecalis.

Carboxypeptidases↗
PubMed1978

Close association between shape alteration and loss of immunity to superinfection in a wild-type Klebsiella pneumoniae stable lysogen which can be both immune and nonimmune to superinfection.

Klebsiella pneumoniae MirM7 is a wild-type strain which grows as cocci at pH 7 and above and as rods at pH 6.5 and below. Cultures of this strain and an auxotrophic derivative, MirM7b, have been found to undergo spontaneous lysis after purification from possible contaminating viruses. Lysates always contained two phages, FR2 and AP3, most often at high titers. FR2 and AP3 plated with the same efficiency on both MirM7b and K59 (another K. pneumoniae strain sensitive to FR2 and AP3) and lysogenized 45 and 54% of the K59-infected cells, respectively. These findings raise the possibility that MirM7b is lysogenic for FR2 and AP3, although nonimmune to their superinfection. The fact that mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine can induce phages FR2 and AP3 from MirM7b confirmed this possibility. When MirM7b was infected with FR2 several strains immune to FR2 and AP3, which were all rod shaped, were obtained. Furthermore, 19 derivatives, rod shaped at all pH's have been isolated from MirM7b. They were all immune to both FR2 and AP3. From mating experiments between the MirM7b donor derivative, strain M720, and either K59 or MirCV5, a rod-shaped MirM7b derivative cured from the prophages, cysteine recombinants were obtained which were most often (80%) immune to FR2 and AP3. Nonimmune and still lysogenic recombinants were obtained by mating M720 with a rod-shaped immune MirM7b derivative; the majority of the non-immune strains maintained the rod shape. Five coccus-shaped recombinants were also isolated; they were nonimmune to superinfection. Several physiological properties of strain MirM7b and the other nonimmune coccal recombinants have been studied in comparison with those of the rod-shaped immune derivatives. All of the coccal strains have shown several alterations with respect to the rods. The role of possible derepressed prophage genes in the various physiological alterations of MirM7 is discussed, and the analogies between this system and those of vertebrate cells transformed by proviruses are stressed.

Bacteriophages↗
PubMed1977

Penicillins activate autolysins extracted from both Escherichia coli and Klebsiella pneumoniae envelopes.

Ampicillin at a 50-mug/ml concentration effects a doubling of the autolysis rate of cell envelopes isolated from Klebsiella pneumoniae Mir A12. This antibiotic increases also by two- to threefold the depolymerizing activity of both K. pneumoniae and Escherichia coli crude extracts on a labeled peptidoglycan-lipoprotein complex. Several other penicillins also activate autolysins.

Bacteriolysis↗
PubMed1977

Bacteriolytic activity in staphylococci.

The bacteriolytic activity of Staphylococcus aureus and Staphylococcus albus strains was tested with various media. Whereas S. aureus strains were found to be active under all conditions, the percentage of active S. albus strains was significantly influenced by the composition of the medium. Ionic strength and concentration of the organic nitrogen source were found to be the main factors affecting the expression of bacteriolytic activity of straphylococci. Virtually all of 318 S. aureus and 603 S. albus strains were active on a medium containing 3% peptone, 0.3% glucose, 0.2% yeast extract, 0.1% disodium phosphate, 2.2% sodium chloride and 0.9% agar. The optimal conditions for the bacteriolytic activity of S. aureus strains were different from those of most S. albus strains. Within S. albus, optimal conditions differed also from strain to strain. It is suggested that further studies on this subject may prove useful for the identification and taxonomy of staphylococci. A possible relationship between the production of extracellular bacteriolytic enzymes and pathogenic properties of staphylococci is also considered.

Bacteriolysis↗