An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-,I-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude P-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing I-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the I-lactamase II of Bacillus cereus and the CfiA I-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-,I-lactamases.In recent classification schemes for ,B-lactamases, a distinct grouping has been reserved for those enzymes requiring a metal cofactor for activity (metallo-1-lactamases) (1, 4, 5). The prototype for this group of enzymes is the 1-lactamase II of Bacillus cereus, which has long been studied as a model of metallo-13-lactamases and for which molecular sequence data, along with enzymologic and crystallographic data, are available (3,12,17,27). In the molecular classification of 1-lactamases, a class B was devised for this enzyme (1).Interest in this group of enzymes has grown recently since an increasing number of metallo-1-lactamases have been described in several gram-negative species which share the important characteristic of being able to hydrolyze carbapenem compounds (2,7,9,22,25,26,31). Carbapenems are new P-lactam antibiotics of great therapeutic potential, since they are not hydrolyzed by most bacterial 3-lactamases and show a broad spectrum of activity.Information currently available on carbapenem-hydrolyzing (CH) metallo-1-lactamases is limited, in most cases, to a few biochemical features and suggests the existence of different molecular species within this group of enzymes (5). Molecular sequence data are presently available only for the CfiA P-lactamase of Bacteroides fragilis TAL2480 (28) and for the ,B-lactamase II of B. cereus 569/H (12) and 5/B/6 (17). A strong sequence similarity exists between these two enzymes (28), suggesting that the CfiA enzyme also belongs to molecular class B...

Daptomycin at the MIC allowed the cell mass increase of enterococcal strains and Bacilus subtilis to continue for 2 to 3 h at rates comparable to those of the controls. During this time the cell shape of the former changed to a rod configuration and that of the latter changed to long rods. In these bacteria, in which cell mass continued to increase, the MIC of daptomycin inhibited peptidoglycan synthesis by no more than 20% after 20 min of incubation and by roughly 50% after 2 h of incubation. Other macromolecules, such as DNA, RNA, and proteins, were only slightly affected. In contrast, incorporation of [14C]acetate into lipids was reduced by about 50% in the various strains after 20 min of treatment with daptomycin at the MIC. When the effect of the major lipid-containing polymers on synthesis was evaluated in detail, it was found that under conditions in which peptidoglycan and the other macromolecules mentioned above were inhibited only slightly (20%) and total lipid synthesis was inhibited by 50%, synthesis of teichoic and lipoteichoic acids was inhibited by 50 and 93%, respectively. Daptomycin was not found to enter the cytoplasm of either bacterial or mammalian cells. It bound, in the presence of calcium ions only, to whole bacterial cells, cell walls (both those that contained and those that did not contain membranes), and isolated membranes of bacterial and mammalian cells. Washing with EDTA removed daptomycin from all cells mentioned above and cell fractions except the bacterial membrane. It is concluded that lipoteichoic acid is most likely the primary target of daptomycin.Daptomycin (LY146032) is an acidic lipopeptide antibiotic that is active against gram-positive bacteria (F. T. Counter,

The two-competing-sites model for peptidoglycan assembly for bacterial cell shape regulation suggests that in rods, bacterial cell shape depends on the balance between two reactions (sites), one responsible for lateral wall elongation and the other responsible for septum formation. The two reactions compete with each other so that no lateral wall can be formed during septum formation and vice versa. When the site for lateral wall elongation overcomes that for septum formation, long rods or filaments are formed and cell division may be blocked. When the reaction leading to septum formation is hyperactive compared with the other, coccobacilli or cocci are formed. Other bacteria carry only one site for peptidoglycan assembly and can grow only as cocci. The two-competing-sites model predicts that two different types of cocci exist (among both morphology mutants and wild-type strains); one carries only the site for septum formation, whereas the other also carries the site for lateral wall elongation, the former site predominating over the latter. As a consequence of the inhibition (by antibiotics or by mutations) of septum formation in wild-type cocci of various species and in coccoid morphology mutants, some cocci are expected to undergo transition to rod shape and others are not. We have evaluated these predictions and show that they are in agreement. In fact, we found that among wild-type cocci belonging to 13 species, those of 6 species formed rods, whereas the remaining organisms maintained their coccal shape when septa were inhibited by antibiotics. Some coccoid morphology mutants of rod-shaped bacteria underwent coccus-to-rod transition after septum inhibition by antibiotics, whereas others maintained their coccal shape. When a mutation that causes septum inhibition was expressed in a morphology mutant of Klebsielia pneumoniae grown as a coccus, transition to rod shape was observed. A total of 914 mutants unable to form colonies at 42TC were isolated from the coccoid species mentioned above. Between 75 and 95% of the mutants isolated from the species that formed rods when septum formation was inhibited by antibiotics but none of those isolated from the others underwent coccus-to-rod transition upon incubation at the nonpermissive temperature.In previous papers, we proposed a model for shape regulation in bacteria and presented a large body of experimental data in support of it (33,34,37). In this study, we have extensively tested predictions concerning the effects on cell shape of septum inhibition by antibiotics and mutations in various wild-type and mutant coccoid bacteria (see predictions 6 and 7 of Table 1, which have previously been evaluated only preliminarily). We show that all predictions are fulfilled and demonstrate that some coccoid species but not others undergo transition to rod shape when septa are specifically blocked by antibiotics and that mutants undergoing coccus-to-rod transition can be isolated in some coccoid species but not in others.The two-competing-sites (TCS) model for peptidoglycan ...

No abstract

S-adenosylmethionine:S-adenosylhomocysteine (SAM/SAH) ratio, 5-methylcytosine (5mC) DNA content, and methylation and expression of c-myc, c-Ha-ras and c-Ki-ras have been studied in liver nodules, induced by diethylnitrosamine according to the 'resistant hepatocyte' model, and in regenerating liver (RL) between 0.5 and 72 h after partial hepatectomy (PH). Nodules, 11, 13 and 21 weeks after initiation, grew actively, showed a low tendency to remodel (persistent nodules), and did not exhibit carcinomatous changes. They underwent extensive remodeling after a 1-week SAM treatment (64 mumol/kg/day), and decreased in size and number after a 3-11-week treatment. A low SAM/SAH ratio was coupled, in nodules, with a high labeling index (LI), 2-fold fall in 5mC DNA content, increase in c-myc, c-Ha-ras and c-Ki-ras expression and hypomethylation of CCGG sequences in the DNA hybridizing with the three protooncogenes. In RL a low SAM/SAH ratio, overall DNA hypomethylation and enhanced c-myc expression were first observed 0.5 h after PH, reached a peak at 5 h and progressively returned to pre-PH levels later on. Maximum expression of c-Ha-ras and c-Ki-ras occurred 24-30 h after PH, roughly coincident with the LI peak. However, no great modifications of the methylation pattern of protooncogene CCGG sequence occurred at any time after PH, indicating the presence of hypomethylated genes and/or DNA sequences different from those investigated in this paper. SAM injection to nodule-bearing rats, for 1-11 weeks before killing, and to hepatectomized rats, 2 days before PH and then up to killing, largely prevented decrease in the SAM/SAH ratio and overall DNA methylation and inhibited LI and protooncogene expression. In nodules these effects were proportional to the treatment length and coupled with methylation of CpG residues in the CCGG sequence of the three protooncogenes studied. SAM treatment left the methylation pattern of these genes unchanged in RL. Kinetics of increase in protooncogene expression suggest a role in the regulation of cell cycle in RL. However, decrease in the SAM/SAH ratio, protooncogene hypomethylation and enhanced expression are apparently stable in nodules 11-21 weeks after initiation and could be implicated in continuous nodule growth and progression. Control of DNA methylation and gene expression by exogenous SAM could be a mechanism of the SAM anti-progression effect.

Five ampicillin-resistant clinical isolates of Enterococcus faecium were analyzed for a correlation between overproduction of the low-affinity penicillin-binding protein (PBP 5) and the level of ampicillin resistance.Comparison was made with one susceptible clinical isolate and its ampicillin-resistant derivative obtained in the laboratory by selection with increasing concentrations of penicillin. Overproduction of the low-affinity PBP relative to the susceptible isolate was noted in moderately resistant strains (MIC, 32 ,ug/ml) but not in highly resistant strains (MIC, 128 ,ug/ml). Polyclonal antibodies specifically reacting with the low-affinity PBP of Enterococcus hirae, Enterococcusfaecalis, and Enterococcusfaecium (M. Ligozzi, M. Aldegheri, S. C. Predari, and R. Fontana, FEMS Microbiol. Lett. 83:335-340, 1991) were used to determine the amount of this PBP in the E. faecium isolates. In all strains, the antibody preparation reacted with a membrane protein of the same molecular mass as PBP 5. The amount of this protein was very small in the susceptible strain but large in all of the resistant strains. These results suggest that the highly resistant strains also overproduced the low-affinity PBP, which, compared with PBP 5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability.Despite several reports of enterococcal strains producing a P-lactamase (16,17,19), the main pathway of penicillin resistance development in these microorganisms still remains modification of penicillin-binding proteins (PBPs), in particular, overproduction of a low-affinity PBP which is a normal component of the PBP pattern of these bacteria (9, 10). The mechanism by which overproduction of PBP 5 confers resistance has been ascribed to the ability of this PBP to substitute the functions of the other PBPs (3,4,7,8,10). This proposal stems from the finding that ATCC 9790 mutants overproducing the low-affinity PBP grow normally in the presence of penicillin concentrations which saturate all PBPs but not PBP 5, which, in these conditions, is apparently the only active PBP. In addition, it has been shown that, in ATCC 9790 derivatives which carry a temperature-sensitive defect in the essential PBPs 1, 2, and 3 associated with a temperature-* Corresponding author. sensitive cell division, a second mutation causing overproduction of PBP 5 allows normal growth at the nonpermissive temperature (4).The recent cloning of the E. hirae PBP 5 gene (pbp5) (5) has provided insights into the genetic mechanism of penicillin resistance development (14). It has been shown that, in resistant mutants, PBP 5 overproduction is associated with a deletion in a genetic element, located 1 kb upstream of pbp5, which negatively controls PBP 5 synthesis (14). Hypersusceptibility to penicillin is associated with a point mutation inpbp5, which causes premature termination of translation. E. hirae pbpS does not hybridize with DNA of other enterococcal species, suggesting that the genes which code for low-affinity PBPs are not str...

Penicillin-binding protein (PBP) 5 of Streptococcusfaecium has been shown to have a very low affinity for penicillin, and this PBP was suggested to be responsible for both the natural low susceptibility and high resistance to the antibiotic in this species (R. Fontana, R. Cerini, P. Longoni, A. Grossato, and P. Canepari, J. Bacteriol. 155:1343Bacteriol. 155: -1350Bacteriol. 155: , 1983. In this study, an S. faecium mutant (Revl4) hypersusceptible to penicillin was derived from the highly resistant S. faecium R40 treated with novobiocin, and its properties were compared with those of the parent and S. faecium PS, a relatively susceptible strain from which R40 was isolated. The hypersusceptible strain did not synthesize PBP 5, but it did resemble the parent in cell morphology, growth rate, and autolytic activity. In addition, it was highly susceptible to other beta-lactams but remained as susceptible as R40 and PS to antibiotics of a different mechanism of action. The affinity of individual PBPs for the beta-lactams tested was the same in all the strains. This finding suggested that Revl4 hypersusceptibility was due to the lack of PBP 5 and strongly supported the role of this protein in the mechanism of both natural low susceptibility and high-level resistance to beta-lactams in S. faecium.The synthesis of penicillin-binding proteins (PBPs) that show a very low affinity for beta-lactams (low-affinity PBPs) appears to be a newly recognized mechanism of intrinsic resistance to these antibiotics found in several gram-positive species (1, 9, 11).Described for the first time in a methicillin-resistant Staphylococcus aureus strain by Brown and Reynolds (1), lowaffinity PBPs are also produced by Streptococcus faecium and other species belonging to the enterococcus group (6, 9). S. faecium strains overproducing this protein (PBP 5) show very high penicillin MICs, can grow in the presence of a penicillin concentration saturating all PBPs but the lowaffinity PBP, and stop growing in the presence of the minimal concentration of antibiotic saturating this protein (9). These findings have led to the conclusion that low-affinity PBPs may be responsible for the characteristic low susceptibility of enterococci to beta-lactams and, when overproduced, for the capability of these microorganisms to become highly resistant to these antibiotics, a property not shared with other streptococci (9, 12). If this suggestion is correct, S. faecium strains unable to synthesize the low-affinity PBP should demonstrate a susceptibility for beta-lactams similar to that of other streptococci.In this report we describe a mutant of S. faecuium hypersusceptible to penicillin and lacking PBP 5. The comparison of the properties of this mutant with those of strains producing a different amount of PBP S provides fuirther strong evidence of the role of the low-affinity PBP in the mechanism of resistance to beta-lactams in this species.(This work was presented, in part, at the FEMS Symposium: Bacterial morphogenesis, Marseilles, France, 5 to 7 September 198...

The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-1-lactamase highly active against carbapenem antibiotics. This enzyme was induced in strain AE036 to the same extent by both benzylpenicillin and imipenem. When the cphA gene was inserted into plasmid pACYC184, used to transform Escherichia coli DH5a, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DHSa(pAA2OR), expressing the Aeromonas metallo-13-lactamase, were significantly increased, but those of penicillins and cephalosporins were not. When the metallo-13-lactamase purified from E. coli DH5a(pAA20R) was assayed with several 13-lactam substrates, it hydrolyzed carbapenems but not penicillins or cephalosporins efficiently. These results demonstrate that this metallo-13-lactamase possesses an unusual spectrum of activity compared with all the other class B enzymes identified so far, being active on penems and carbapenems only.This enzyme may thus contribute to the development of resistance to penems and carbapenems but not other -lactams.