Biomedical subjects

G Korfmann

Publications and source records attributed to G Korfmann.

4 recordsLinked to original sources

PubMed1991

Altered phenotypes associated with ampD mutations in Enterobacter cloacae.

A study was done to determine the genetic locus responsible for altered expression of AmpC beta-lactamase in Enterobacter cloacae 1194E and several mutants derived from E. cloacae 029. These phenotypes were defined by units of enzyme activity found in sonic extracts of cells before and after induction with cefoxitin and included (units uninduced/units induced) the wild-type (7/219), high-level constitutive (10,911/10,862), temperature-sensitive (at 30 degrees C 82/706 and at 42 degrees C 5,031/6,020), and hyperinducible (19/1,688) phenotypes. When the ampD region of each E. cloacae strain was cloned and introduced into an ampD mutant Escherichia coli strain, the altered phenotypes were found to reside within this locus. Furthermore, transformants containing wild-type ampD were poorly inducible at 42 degrees C while those with high-level constitutive or hyperinducible ampD were unaffected by temperature. Since the source of ampD was the only variable in these E. coli transformants, these results suggested that ampD encodes a protein that is involved in sensing the inducer. To test this possibility, the responses to different inducers of E. coli transformants containing various ampD regions were assessed. In the presence of wild-type ampD, transformants responded equally to cefoxitin and cefotetan, regardless of temperature. In the presence of temperature-sensitive ampD, induction by cefotetan was similar to that by cefoxitin at 30 degrees C but greater than that by cefoxitin at 42 degrees C. These results suggest that ampD encodes a protein involved in induction of AmpC beta-lactamase in E. cloacae.

Anti-Bacterial Agents↗

PubMed1989

ampG is essential for high-level expression of AmpC beta-lactamase in Enterobacter cloacae.

Mutants of Enterobacter cloacae 55 were studied to delineate more completely the genetics of inducible expression of AmpC beta-lactamase. E. cloacae 55M-L, derived by mutagenesis from a mutant with high-level cefotaxime resistance (MIC, greater than 64 micrograms/ml), E. cloacae 55M, demonstrated a novel phenotype by producing only low levels of AmpC constitutively. Neither the parental phenotype of E. cloacae 55M nor the wild-type phenotype of E. cloacae 55 could be restored in E. cloacae 55M-L by the introduction of functional ampR, ampC, or ampD genes. Cloning each of these genes from E. cloacae 55M-L confirmed the same genotype for this mutant as for its parental strain. Mutation of E. cloacae 55M-L to the E. cloacae 55M phenotype was found to occur spontaneously at a frequency of 10(-8). All such revertants demonstrated an inducible wild-type phenotype after introduction of a functional ampD. These results suggested that the E. cloacae 55M-L phenotype was due to a mutation in an as yet unrecognized gene, designated ampG. Verification of this gene was obtained by the restoration of the E. cloacae 55M phenotype in E. cloacae 55M-L by introduction of a cloned 2.9-kilobase BamHI fragment from the E. cloacae 55 chromosome. Transformation of both ampG and ampD into E. cloacae 55M-L reconstituted the inducible wild-type phenotype. These results indicate that ampG is required for the activation of ampC by AmpR. Without ampG, neither induction nor high-level expression of AmpC is possible. It is likely that the ampG gene product and AmpD together modulate the ability of AmpR to activate ampC expression.

Ampicillin↗

PubMedDate not supplied

Genetic control of beta-lactamase production in Enterobacter cloacae.

In Enterobacter cloacae, mutations in favor of overproduction of beta-lactamase--leading to resistance to third-generation cephalosporins--occur at frequencies of 10(-4)-10(-7). Cloning experiments reveal that at least three genes are involved in the regulation of chromosomal beta-lactamase expression. The structural gene, ampC, is located adjacent to the regulatory gene, ampR, coding for a protein that can serve as an activator in the presence of an inducer. An example of an ampR mutant that is independent of an inducer has been studied. More important for the development of cefotaxime resistance in E. cloacae are mutations in the ampD gene and other proposed regulatory genes. Inactivation of the ampD gene leads to elevated beta-lactamase production. Thus, ampD negatively controls ampC expression. Evidence for the existence of a third regulatory gene, ampE, has been found.

Cloning, Molecular↗

PubMedDate not supplied

Impact of the ampD gene and its product on beta-lactamase production in Enterobacter cloacae.

In an investigation of the influence of the ampD gene on beta-lactamase production and induction in Enterobacter cloacae, the ampR-ampC gene region cloned into a plasmid and the ampD gene cloned into another vector were transferred to a strain of Escherichia coli. The genetically manipulated E. coli strains served as a model for study of the inducibility of beta-lactamases in E. cloacae. In addition, beta-lactamase induction in E. cloacae bearing the previously mentioned plasmids was studied. After induction of the beta-lactamase with cefoxitin, the specific hydrolytic activity, the viable cell count, and the degradation of cefoxitin were determined. beta-Lactamase expression decreased with an increasing amount of the ampD gene product. The cefoxitin concentration decreased in proportion to the amount of enzyme, but the induction of beta-lactamase seemed not to be an important factor influencing the viable cell count of E. cloacae as long as cefoxitin concentrations exceeded the MIC. Despite different beta-lactamase concentrations, the decrease in the viable cell count was nearly identical in all experiments.

Cefoxitin↗