Abstract
Serratia marcescens encodes an inducible, chromosomal beta-lactamase, ampC. ampC is inducible in S. marcescens and several other Gram-negative bacteria in the presence of different p- lactam antibiotics, and induction is dependent on the products of ampK, ampl.), and ampG. Sequence analysis of S. marcescens ampC revealed that the 5’-untranslated region (5’-UTR) is 126 nucleotides long, which is at least 74 bases longer than the 5’-UTR from related organisms with inducible ampC genes such as Enierobacter cloacae, Citrobacter freundii, Morganella morganii, and Yersinia enterocolitica. In addition, the GCG program MFold predicted extensive secondary structure in the 5’- UTR of 5. marcescens ampC, while secondary structure is not evident in the ampC 5’-UTRs of E. cloacae, C. freundii, M. morganii, and Y. enterocolitica. It was hypothesized that the role of the secondary structure in S. marcescens ampC was to increase transcript stability and/or influence post- transcriptional regulation. The purpose of this research was to examine the regulation of the ampC gene of S. marcescens and determine the role of the stem-loop present in the 5’-UTR of S. marcescens ampC. Primer extension analysis revealed the transcriptional start site of S. marcescens ampC 126 bases upstream of the ATG start codon, and Northern blot analysis showed that the S. marcescens ampC transcript was longer than the ampC Uanscripts from E. cloacae and C. freundii, indicating that the difference in size was due to the longer 5’-UTR of S. marcescens ampC. S. marcescens ampC was induced with different P-lactam antibiotics, and primer extension analysis showed a 3.5-fold, 2.3-fold, and 3.0-fold increase in transcript level over enzymatic activity with cefoxitin, imipenem, and cefamandole, respectively. Half-life analysis showed that the S. marcescens ampC transcript had a half-life of 7 minutes, compared to 8 and 15 minutes for the E. cloacae and C. freundii ampC transcripts, respectively. Thus, the S. marcescens ampC transcript does not have a longer half-life than either the E. cloacae or C. freundii ampC transcript. In addition, constitutive expression of 5. marcescens ampC transcript was repeatedly 2-fold less than the constitutive expression of the E. cloacae and C. freundii ampC transcripts. To examine the role of the stem-loop structure identified in the 5’-UTR of the S. marcescens ampC transcript a deletion mutant was constructed. Comparisons between the wild-type and mutant were made using the luciferase reporter gene assay. The production of luciferase in the clone containing the deleted stem-loop in the 5’-(JTR was 2.9-fold and 2.3-fold lower than the wild-type clone in E. coli and S. marcescens, respectively. These data indicate a possible role for the secondary structure in post-transcriptional regulation and/or translational efficiency. The transcript half-life between the wild-type and deletion mutant 5’-UTR clones was compared using RT-PCR. The transcript half-lives were 7 minutes and 2 minutes, respectively, for the wild-type and deletion mutant clones. Thus, the half-life studies confirmed that the stem-loop structure was involved in transcript stability. Finally, a novel factor was observed to bind to a m-acting element downstream of the S. marcescens ampC transcriptional start site in a growth phase dependent manner. Late in log phase, the intensity of binding increased and was concomitant with a decrease in steady state levels of ampC transcript. Taken together, these data indicate at least two different levels of regulation for S. marcescens ampC: 1) initiation of transcription, both constitutive and inducible, and 2) post-transcriptional regulation as it relates to transcript haif-life. These data indicate that the regulation of S. marcescens ampC occurs at multiple levels which has not been indicated in other bacteria with inducible ampC genes.