Regulatory components in Citrobacter freundii ampC beta-lactamase induction.

Abstract

Citrobacter freundU encodes an inducible chromosomal P-lactamase similar to the constitutively expressed ampC .8-lactamase of Escherichia coli. In the latter species the ampC gene is located next to the fumarate reductase (frd) operon, whereas in C. freundii the ampC gene is known to be separated from frd by 1100 base pairs. P3-lactamase (8, 9). This enzyme acts by either hydrolyzing or by simply binding the ,B-lactam that reaches the periplasmic space (10-12).The ampC gene of Citrobacter freundii has been cloned and was shown to be closely linked to the frd operon, which codes for the fumarate reductase complex (4), as is the case in Escherichia coli (13). In C. freundii, however, there is an 1100-base-pair (bp) DNA segment, not present in E. coli, that separates ampC from frd (4). We show that this segment codes for a trans-acting 31-kilodalton protein, AmpR, which is responsible for the inducibility of,-lactamase synthesis from the C. freundii ampC gene, and that it can have both a positive and a negative effect on ampC expression. We further show that mutations leading to high constitutive levels of ampC f3-lactamase production map outside the ampR-ampC region. MATERIALS AND METHODSMedia and Growth Conditions. L medium (14) was used for routine purposes. M9 medium (15) containing 0.2% glucose, 0.2% casamino acids, uracil at 50 ,ug/ml, and thiamin at 1 ,ug/ml (M9CA) was used in the determination of ampicillin resistance and .-lactamase expression. When necessary, the media were supplemented with ampicillin (concentration as indicated), tetracycline (10 ,ug/ml), or chloramphenicol (10 Ag/ml).Determination of Ampicillin Resistance and Relative ,-Lactamase Production. Ampicillin resistance on M9CA medium was determined as the LD50-i.e., the concentration of ampicillin required to inhibit 50% of the single cells from forming colonies (16). To determine the relative production of ,B-lactamase, cells were washed twice (50 mM potassium phosphate buffer, pH 7.4) and sonicated (17). (3-Lactamase activity was determined spectrophotometrically (18) at 255 nm, using 100 uM cephalosporin C as substrate in 10 mM MgCl2/10 mM potassium phosphate buffer, pH 7.4. Specific activity was expressed as ,mol of substrate hydrolyzed per min at 22°C per mg of total protein (19).Induction of f-Lactamase Expression in C. freundu and E. coli. Bacteria were grown exponentially in M9CA medium for at least eight generations to an OD420 of 0.8. Subsequently, they were diluted into an equal volume of prewarmed medium containing twice the final inducer concentration. Samples, 20 or 40 ml, were withdrawn at various times into centrifuge tubes on ice, containing chloramphenicol (200 ,g/ml) to inhibit protein synthesis. Relative 83-lactamase production was then determined.Induction of ,B-Lactamase Expression in Minicells. Strain AA10, obtained from P. Orndorff (Stanford University), is a recA derivative of P678-54 (20) and was used for the minicell experiments. Minicells from AA10 harboring the approprate plasmids were prepared (21) a...

A Common System Controls the Induction of Very Different Genes

“…Escherichia coli strains were DH5 a (Gibco BRL) as host for cloning vectors, S17-1 (Simon, 1989) as mobilizing donor for plasmid transconjugation, and SN03/pNU305 (Lindberg et al, 1985) and SN0301/pNU413 (Lindquist et al, 1989b), for expression of AmpR protein.…”

“…Genes ampR and ampC in Ent. cloacae (Honor6 et al, 1986), Citrobacter freundii, (Lindberg et al, 1985;Lindquist et al, 1989a), Yersinia enterocolitica (Seoane et al, 1992) and Pseudomonas aeruginosa (Lodge el al., 1990) and also the regulator and structural genes of the class-A P-lactamase in Rhodopseudomonas capsulata (Campbell et al, 1989) are equally located in adjacent chromosomal positions. They are also separated by short intercistronic regulatory regions from which they are transcribed in opposite directions.…”

“…Induction of p-lactamase was carried out as described by Lindberg et al (1985Lindberg et al ( , 1987. P-lactamase activities and values of K, and k,, were determined by computerized spectrophotometry, using the wavelengths and absorbance variations described by Matagne et al (1990) for different /3-lactam substrates.…”

“…Detailed studies of the AmpC induction mechanism have revealed the essential participation of additional genes, ampR, ampG and the ampDE operon. These encode a transcriptional regulator (Lindberg et al, 1985;HonorC et al, 1986;Lindquist et al, 1989a), a transducer of the external induction signal (Korfmann and Sanders, 1989;Lindquist et al, 1993) and a negative modulator of AmpD (Lindberg et al, 1987) Enzymes. /I-Lactamase (EC 3.5.2.6); DNA-ligase (EC 6.5.1.1); RNdse A (EC 3.1.27.5); alkaline phosphatase (EC 3.1.3.1); DNA polymerase (EC 2.7.7.7); lysozyme (3.2.1.17); fumarate reductase (EC 1.3.99.1).…”

Nucleotide Sequence of the Chromosomal ampC Gene of Enterobacter aerogenes

“…In the uninduced state, transcription of the structural ␤-lactamase gene, ampC, is repressed by the product of the linked ampR gene (6,12,16). Chromosomal ampC-ampR systems have been described for Citrobacter freundii (11, 13), Enterobacter cloacae (4, 6), Morganella morganii (2, 16), and Yersinia enterocolitica (21).…”