Center for Research in Anti-Infectives & Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178, USA
Received 18 June 2002; returned 10 August 2002; revised 19 December 2002; accepted 24 December 2002
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Abstract |
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Keywords: ampC, ß-lactamase, Gram-negative bacteria, regulation
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Introduction |
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The majority of data on gene expression in prokaryotic organisms have been directed toward control of transcription initiation. ampC gene expression is no exception. The data regarding regulation of ampC gene expression have evaluated the induction of the ampC gene by AmpR.14 This occurs at the level of transcription initiation. However, control of expression for any gene product can occur at several different levels such as mRNA half-life, efficiency of translation and protein stability. Stability of mRNA is an important regulatory mechanism because it can allow a single mRNA to be translated several times, thus increasing the amount of protein product produced by a single mRNA molecule. The half-life of mRNA in Escherichia coli ranges from 40 s to 20 min, but this type of information from other prokaryotic organisms, such as S. marcescens, is extremely limited.15 The longer-lived transcripts are associated with secondary structure in the 5' untranslated region (5' UTR) of the message and this structure is one of the requirements for increased half-life.16 Secondary structure in the 5' UTR has also been associated with a decrease in the efficiency of translation for some transcripts.17
Recently, sequence data revealing the intergenic region and putative 5' UTR of Y. enterocolitica, M. morganii and H. alvei have been published.1820 However, no published data are available describing the intergenic region of S. marcescens or the expression of the S. marcescens ampC gene. Therefore, in this study we have cloned and sequenced the ampC and ampR genes, including the intergenic region of S. marcescens and examined ampC gene expression.
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Materials and methods |
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All strains and plasmids used in this study are listed in Table 1.
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The primers used in this study are listed in Table 2. Oligonucleotides were synthesized by Genosys (The Woodlands, TX, USA).
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DNA template was prepared from an overnight culture of a single colony using 1.5 mL of culture as previously described.21 DNA template from S. marcescens was prepared from a 3 h culture due to the production and secretion of an extracellular nuclease.22 In addition, a final concentration of 0.4 g/L protease K was added to the S. marcescens supernatant before lysis to help protect nucleic acid from nuclease degradation. Genomic DNA from S. marcescens 1 was isolated from 3 h cultures grown in LB broth as previously described.23 Plasmid DNA was isolated from 18 h cultures by alkaline lysis.24
Synthesis of DIG-labelled probe
Gene-specific probes were synthesized by incorporation of digoxigenin-11-dUTP (DIG) into PCR products according to the manufacturer (Roche Molecular Biochemicals, Indianapolis, IN, USA). DNA template was used at a concentration of 1/250 of total template volume (2 µL). An initial 5 min incubation at 95°C was performed to denature the DNA strands, followed by 25 cycles of 96°C for 30 s, 50°C for 15 s and 72°C for 2 min, carried out in a Perkin Elmer thermocycler (PE Applied Biosystems, Foster City, CA, USA). Incorporation of DIG into the PCR product was monitored using 1% agarose gel electrophoresis. The SDMCF/SDMCR primer pair was used to generate the S. marcescens-specific ampC probe (Table 2).
PCR amplification of the S. marcescens DNA
Amplification of the ampC gene was carried out as previously described using 0.5 µM of each primer, and 2 µL (1/250th volume) of template in a final volume of 50 µL.25 PCR was carried out and visualized as described for the DIG-labelled probe using a 12% agarose gel. Amplification of the full-length ampR region of S. marcescens was accomplished using primers SDMCR and AMPR3R (Table 2). PCR was carried out as previously described using Q solution (Qiagen), an annealing temperature of 45°C and a final extension of 10 min at 72°C.
Generation of the S. marcescens ampC/ampR and ampR deletion clones
The S. marcescens ampC genomic fragment was identified by Southern analysis by restricting S. marcescens 1 with BamHI (Gibco Life-Technologies, Rockville, MD, USA) and hybridizing with the DIG-labelled probe described above as suggested by the manufacturer (Roche Molecular Biochemicals). Probe hybridization conditions were carried out, with the following modifications: 5 x SSC, 1% blocking reagent, 0.1% sarcosine and 2% SDS at 37°C for 18 h and washed three times at 65°C with 2 x SSC and 1% SDS. The membrane was exposed to Lumi-Film Chemiluminescent Detection Film (Roche Molecular Biochemicals) for 20 min at room temperature. The BamHI restricted vector, pCRII (Table 1), and a 3.7 kb fragment containing S. marcescens 1 ampC DNA, were excised from a 1% agarose gel and purified using the GeneClean II kit (Bio101, Inc.). The vector and genomic fragments were ligated and transformed into One Shot INV
F' chemically competent E. coli (Invitrogen), as recommended by the manufacturer, and selected on LB agar containing 50 mg/L kanamycin (Sigma Chemical Co., St Louis, MO, USA). The plasmid from the ampC-positive genomic clone, pSmC448, was transformed into E. coli strain HB101 using a modified Hanahan method.26 In addition, a 2.1 kb PCR product was generated using the primers AMPR3R and SDMCR identified in Table 2. This amplification product contained the full-length ampR gene of S. marcescens and was purified after gel electrophoresis in 1% agarose using an S.N.A.P. gel purification kit (Invitrogen), cloned into the vector pCR-XL-Topo (Invitrogen) and sequenced. Figure 1 shows the steps used to obtain the full-length ampC/ampR plasmid. The plasmid derived from genomic DNA (pSmC448) was restricted with the endonuclease XhoI, whereas the PCR-derived plasmid (pSmC*) was restricted with HindIII. The respective fragments were treated with Klenow and subsequently treated by the restriction endonuclease AccI, which cleaves the fragments within the ampC gene. The two fragments were ligated together to generate the functional S. marcescens ampC/ampR plasmid (pSmCR) in a pCR-XL-Topo vector background cloned into One Shot TOP10 E. coli (Invitrogen). The full-length clone was subsequently subcloned into the EcoRI site of a modified pACYC184 vector (pACdpr1) in which the chloramphenicol promoter had been deleted using PvuII restriction sites resulting in the final clone, pSm1. The ampR deletion clone (pSm1dR) was generated by restricting pSm1 with BglII and DraIII restriction endonucleases, treated with Klenow, and religated, deleting a 528 bp region from the ampR gene.
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Generation of UTR plasmid constructions. pSPlucRBS construction. The plasmid vector pSPluc+ was purchased from Promega (Madison, WI, USA). This vector contains the ribosomal binding site (RBS) of E. coli.27 In order to construct promoter/UTR regions, which are indicative of the organism from which it originated, the RBS site from pSPluc+ was removed. pSPluc
RBS was constructed by restricting pSPluc+ with endonucleases AvrII and HindIII, treating the restricted plasmid with T4 DNA polymerase and religating the vector.24
pSPlucWTUTR and pSPlucUTR constructions. pSPlucWTUTR was constructed by generating a 215 bp fragment representing the entire ampC/ampR intergenic region, including the 5' UTR of ampC from S. marcescens by PCR using the primers SMCP1F and SMCP1R (Table 2). pSPluc
UTR represents the mutant constructed by insertion of a 159 bp fragment representing a deletion of the UTR from nucleotides +51 to +106 of the S. marcescens ampC/ampR 5' UTR. The 215 bp fragment was initially cloned into pCR2.1 and excised from pCR2.1 using EcoRI endonuclease. The resulting fragment was restricted with KpnI and BglII, and subcloned into vector pSP73 and finally subcloned from pSP73 as a KpnI/BglII fragment upstream of the luciferase gene of pSPluc
RBS. This clone was designated pSPlucWTUTR. pSPluc
UTR was constructed using the internal deletion PCR method (see Figure 6).28 In this method, two primer sets are used that contain overlapping sequences and flank the region of DNA to be deleted. To construct pSPluc
UTR from pSPlucWTUTR, primers SMCP1F and SMCMUTB were used to generate the PCR product containing the region of the 5' UTR up to position +50, whereas SMCMUTC and SP73MUTD were used to generate the PCR product corresponding to the region downstream of the prominent stemloop structure from position +107 (Table 2). Primer SP73MUTD was specific for a region of vector pSP73 represented in pSPlucWTUTR. Primer SMCMUTB has 10 bases on its 5' end, which were complimentary to the 10 bases on the 5' end of primer SMCMUTC (Table 2). These two PCR fragments were gel purified, and used as template in a subsequent PCR using primers SMCP1F and SMCP1R to synthesize a new fragment that lacked the predicted stemloop secondary structure from S. marcescens ampC. The initial PCR products and excised fragments were purified by agarose gel electrophoresis using 1 x TAE.24 All enzymes were purchased from Gibco-BRL/Life Technologies and used according to the manufacturer. The S. marcescens ampC/ampR intergenic region insert was confirmed in vector pCR2.1 by DNA sequencing.
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DNA sequencing of the S. marcescens ampC/ampR
Sequencing was carried out either manually using the Exo Pfu DNA cycle sequencing kit from Stratagene or by automated sequencing using a DNA stretch sequencer and dye terminator technology. All primers used for sequence analysis are listed in Table 2.
DNA sequence analysis
Secondary structure analysis was carried out using the DNASIS for Windows analysis program (Hatachi Software Engineering Co., Ltd, San Francisco, CA, USA) based on the method of Zuker & Stiegler.29 Sequence alignments and analyses were carried out online using the BLAST program of the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov).30
RNA extraction
Total RNA was extracted using the hot-phenol extraction method of Chen et al.22 The final RNA pellet was resuspended in 200 µL of 1 x TE, and stored at 80°C. Normalization of RNA expression between cultures of different genera and/or clones required that RNA be isolated from an equivalent cell number, 8 x 108, as determined by growth curve analysis.
Primer extension analysis
Primers were labelled and primer extension was carried out as described previously.25 Primer extension products were visualized by exposing the gel to X-OMAT AR Scientific Imaging Film (Eastman Kodak Co., Rochester, NY, USA) for 2 days.
Northern blotting
Northern analysis was carried out as described previously, with the following modifications.23 Thirty micrograms of total RNA was electrophoresed in a 2% agarose/formaldehyde gel for 16 h at 30 V. Methylene Blue staining of the blots was carried out to ensure equal loading of RNA in all lanes.31 The membrane was pre-hybridized for 1 h at 50°C in pre-hybridization solution (50% deionized formamide, 5 x SSC, 50 mM sodium phosphate, pH 7.0, 2% blocking reagent, 0.1% N-laurylsarcosine) and hybridized in fresh pre-hybridization solution with the addition of saturating amounts of denatured DIG-labelled probe (20 µg) for 18 h at 50°C. The membrane was washed twice for 15 min each at 50°C with 2 x SSC/0.1% SDS, then washed twice for 15 min each at 68°C with 0.5 x SSC/0.1% SDS. Chemiluminescent detection of the hybridized probe was as directed by the manufacturer (Roche Molecular Biochemicals). The membrane was exposed to Lumi-Film Chemiluminescent Detection Film for 20 min at room temperature.
Transcript half-life studies
Overnight cultures were diluted 1:100 and incubated to an A600 of 0.45, at which time rifampicin (Sigma) was added to a final concentration of 200 mg/L.32 Total RNA was extracted at indicated time points after addition of rifampicin and evaluated by northern analysis or reverse transcriptase (RT)PCR as indicated. Band intensities were quantified by densitometry.
RTPCR
The half-life of transcripts expressed from the luciferase constructs was determined by semi-quantitative RTPCR. Total RNA samples used for RTPCR were isolated from strains EcSPlucWTUTR and EcSPlucUTR at 0, 2, 5, 10, 15 and 20 min after the addition of 200 mg/L rifampicin as described above. RNA (5 µg) was treated with 5 U of RQ1 RNase-free DNase (Promega) as recommended by the manufacturer. RNA was resuspended in 10 µL of DEPC-dH2O (where DEPC stands for diethyl pyrocarbonate) and concentration was determined by UV spectrophotometry. RTPCR was carried out using the Qiagen OneStep RTPCR kit (Qiagen, Valencia, CA, USA) as suggested by the manufacturer using 250 ng of total RNA. A linearity curve was carried out with RNA isolated from EcSPlucWTUTR from the 0 min sample to optimize the number of cycles required for RTPCR. The cycling parameters for all RTPCR protocols consisted of 20 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 1 min. The samples were incubated at 72°C for 10 min after the last cycle for a final extension reaction. Products were separated using a 1.5% agarose gel at 70 V for 2 h. Primers SMCMUTC and 5' LUCREV were used to generate a 157 bp fragment specific for the luciferase constructs, whereas primers 80 F and 78 R were used to generate a 196 bp fragment specific for E. coli 23S rRNA as an internal control. In addition, PCR using only Taq DNA polymerase and the same RNA template used for RTPCR was carried out using 30 cycles to ensure that contaminating DNA was not present in the RNA samples. To ensure that Taq DNA polymerase was functional, PCR amplification was carried out using primers SMCP1F and SMCP1R (Table 2) and plasmid DNA template from strain pSPlucWTUTR(E. coli).
Luciferase assays
Single colonies of the clones to be tested, EcSPluc, SmSPluc, EcSPlucWTUTR, EcSPlucUTR, SmSPlucWTUTR and SmSPluc
UTR were inoculated into 5 mL of LB broth with the appropriate antibiotic and incubated overnight. A 1:20 dilution of the overnight culture was inoculated into LB broth with antibiotic and incubated to an A600 of 2.0, at which time 90 µL samples were taken from each culture. Luciferase assays were carried out as suggested by the manufacturer (Promega). The luminometer was programmed for each sample as follows: 2 s time delay, 10 s integrate time and injection of 100 µL of luciferin substrate. The assay was carried out at least three times for each strain.
ß-Lactamase induction assays
Induction of S. marcescens cultures was carried out as described previously, except that all cultures were induced with 1/4 x MIC of cefoxitin (16 mg/L) at an A600 of 0.45.10
Disc induction
Disc induction was investigated using the disc approximation test and commercially available discs of cefoxitin (30 µg) and cefotaxime (30 µg) placed 13 mm apart (edge to edge) on MuellerHinton agar plates inoculated with the E. coli clones containing the full-length or ampR deletion constructs.33 The plates were examined after overnight incubation at 37°C.
Nucleotide accession number
The GenBank accession numbers for the S. marcescens strain 1 ampR/ampC genes are AY125470 and AF384203.
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Results |
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A 3.7 kb BamHI fragment isolated from the genomic DNA of S. marcescens 1 containing the ampC/ampR region was cloned (Table 1). Unfortunately, the clone was missing 40 nt of the 3' end of the ampR gene, due to the position of a BamHI site, and was not inducible. To obtain a clone containing the full-length genes of ampC and ampR, including the intergenic region from S. marcescens, a series of steps involving the genomic clone (3.7 kb) (pSmC448) and a PCR-generated clone (pSmC*) were used (Figure 1). The final clone (pSm1) contained ampC, the intergenic region, ampR and a 1.2 kb segment of genomic DNA of unknown identity downstream of the ampC gene (Table 1). Sequence data indicated that the organization of the S. marcescens ampC/ampR genes and intergenic region would result in divergent transcription of the two genes as seen for other genera expressing inducible ampC genes.5,6,1820 Both similarities and differences were observed for the intergenic region of ampC and ampR for S. marcescens when compared with intergenic regions of C. freundii, E. cloacae, Y. enterocolitica, M. morganii, H. alvei and Providencia stuartii (Figure 2). Interestingly, the sequence for the putative AmpR-binding sites was variable between these organisms.
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Demonstration that the S. marcescens AmpC ß-lactamase was inducible and required AmpR was achieved using the disc approximation test and E. coli clones possessing either the full-length construct (pSm1) or an ampR deletion mutant (pSm1dR). A characteristic flattening of the cefotaxime zone of inhibition was observed on a lawn of E. coli expressing the full-length clone. However, no induction (zone flattening) was observed when this test was used to examine the ampR deletion mutant (data not shown).
Transcript analysis of the S. marcescens ampC
Primer extension was carried out to map the start of ampC transcription and demonstrate that the extra length of the transcript observed by sequence analysis was due to an extended 5' UTR. The start site of transcription was mapped to the cytosine residue 126 bases upstream of the ATG codon (Figure 3, lanes U and I, and Figure 1). Treatment of S. marcescens cells with the inducing agent cefoxitin had no effect on the start of transcription. In addition to mapping the transcriptional start site for the ampC transcript, primer extension analysis of the induced ampC transcripts also revealed numerous premature stops (Figure 3, lane I). These stops map to GC-rich regions within the 5' UTR (Figure 3) and correspond to the major stemloop structure. These data indicated physical hindrance for the reverse transcriptase and suggested the presence of secondary structure within the 5' UTR of the S. marcescens ampC transcript.
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In addition to sequence variations within the intergenic region of these Gram-negative organisms, the 5' UTR of S. marcescens was unusually long in comparison. The ampC 5' UTRs in Enterobacteriaceae range from 28 bases in M. morganii, to 50 bases in organisms such as C. freundii (48 bases), E. cloacae (49 bases) and Y. enterocolitica (54 bases).5,6,19,20 The putative UTRs of P. stuartii and H. alvei were 81 and 118 bp, respectively18 (www.ncbi.nlm.nih.gov/entrez). Unlike these previously identified ampC 5' UTR sequences, the S. marcescens ampC 5' UTR was found to contain 126 nucleotides (Figure 2). Extended 5' UTRs are indicative of post-transcriptional regulation due to the potential for secondary structure formation. Considerable secondary RNA structure was observed when the sequence of the 5' UTR of the S. marcescens ampC transcript was analysed using the sequence analysis software program DNASIS (Figure 4).29 The predominant stemloop structure was composed of 56 bases and corresponded to nucleotides +51 to +106 of the 5' UTR (Figures 2 and 4). The entire secondary structure had a predicted free energy of 57 kcal/mol and forms upstream of the putative ribosome binding site (AAGAGCT) and initiation codon. No secondary structure was predicted for E. cloacae, C. freundii, Y. entercolitica or M. morganii. However, secondary structure was predicted for both the 5' UTRs of P. stuartii and H. alvei, with predicted free energies of 15.9 and 37.2 kcal/mol, respectively (data not shown). However, these structures were not similar to the predicted secondary structure for the S. marcescens 5' UTR.
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Secondary structure in the 5' UTR of transcripts has been associated with prolonged RNA half-life. The half-life of S. marcescens ampC transcripts in vivo was determined using northern analysis and the densitometry values plotted semi-logarithmically (Figure 5). As indicated in Figure 5, the half-life of S. marcescens ampC mRNA was 7 min. Transcript half-life studies usually reveal gradual decay patterns;32,34 however, the level of ampC transcripts for S. marcescens decayed very sharply after 67 min of incubation with rifampicin (Figure 5).
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It is possible that the stemloop structure played a role in the half-life of the ampC transcript. However, data on the average half-life of RNA in S. marcescens are not available for comparison with that of the half-life of the ampC transcript. Therefore, in order to determine the role, if any, that the stemloop structure played in the expression of the AmpC ß-lactamase transcript, two clones were constructed and tested using the reporter gene luciferase (see Materials and methods and Figure 6). These clones represented the wild-type S. marcescens intergenic region with the full-length 5' UTR (pSPlucWTUTR) and a secondary structure mutant with the prominent stemloop structure deleted (nucleotides +51 to +106) (pSPlucUTR). Luciferase expression by these clones was examined in both E. coli and S. marcescens. A 2.1-fold decrease in luciferase activity in S. marcescens was observed for the deletion mutant compared with the wild-type 5' UTR, whereas a 2.9-fold decrease in luciferase activity was observed in E. coli between mutant and wild-type.
The luciferase data indicated that the stemloop structure within the 5' UTR played some role in post-transcriptional regulation of AmpC expression. Because luciferase activity is a measurement of a post-translational product, the role of the 5' UTR stemloop could be related to RNA stability or translational efficiency of the message. Therefore, ampC transcript half-life determinations were carried out using RNA isolated from either the wild-type or deletion mutant 5' UTR clones expressed in E. coli (Figure 6). Semi-quantitative RTPCR analysis indicated that the half-life of the wild-type and mutant 5' UTR clones expressed in E. coli was 7 and 2 min, respectively (Figure 6a and b). The levels of E. coli 23S rRNA did not vary between samples and were used to normalize the data (Figure 6a). Taq polymerase PCR was used to evaluate contamination of RNA samples by DNA. No amplification product was observed using the RNA as template (data not shown).
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Discussion |
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Most prokaryotic mRNA half-life studies have been carried out using E. coli. Little is known about the half-life of transcripts from other Gram-negative organisms. An extended 5' UTR in prokaryotes such as the one identified for S. marcescens ampC (126 bases) is unusual, but not unique. The average length of 5' UTRs in prokaryotic organisms is 50 bases; however, examples of prokaryotic transcripts with extended 5' UTRs include cspA (159 bases), cspB (161 bases) and cspI (145 bases) from E. coli, and ompA from E. coli, E. aerogenes and S. marcescens (133 bases each).32,3537 As in the 5' UTR of S. marcescens ampC, extensive secondary structure with stemloops has been predicted in the 5' UTRs of the transcripts listed above. Stemloop and 5' end hairpin formation in extended 5' UTRs have been shown to influence both RNA stability and translation initiation.17,34,3740
The longer leader length reported here is not unique to this strain of S. marcescens but was also present in the ampC sequence data of S. marcescens strains SRT-1, SST-1 and SLS7341 (www.ncbi.nlm.nih.gov/entrez/). In this study, DNASIS analysis revealed a secondary structure with a free energy of 57 kcal/mol (Figure 4). The free energy associated with this secondary structure together with the experimental data presented indicate that the stemloop structure plays a role in S. marcescens ampC RNA stability. In addition, these data also indicate that the half-life of the S. marcescens ampC transcript relies on the stemloop structure and that the RNases required for degradation are not S. marcescens specific. The uncharacteristic decay rate of the ampC transcript could reflect a physical hindrance for RNases by the secondary structure present in the 5' UTR of the transcript. Further studies comparing decay rates of S. marcescens with other Enterobacteriaceae with inducible ß-lactamases are warranted.
Expression of the ampC gene of S. marcescens deviates from what has been described in the literature for other members of the Enterobacteriaceae. The clinical significance for understanding the regulation of the S. marcescens ampC gene is reflected by studies comparing the induction potential of inducing ß-lactam drugs between different genera with inducible AmpC ß-lactamases.10,42 Differences in inducer potential have been suggested to be due, in part, to variability among species.10 Semi-continuous cultures were used to examine changes in ß-lactamase activity and susceptibility to cephalosporins in E. cloacae and S. marcescens.43 These studies revealed that upon induction with cefoxitin, the ß-lactamase activity remained high in cultures of S. marcescens, 1.5 h longer than ß-lactamase activity found in E. cloacae. Because early studies relied on ß-lactamase activity or MIC data to differentiate the differences between induction potential and specific drug effects on MICs, it was impossible to determine the mechanism responsible for these differences. Analysis of induction studies using ß-lactamase activity is generally carried out 12 h post-induction to allow for maximum ß-lactamase activity.10,42,43 Therefore, a stabilized transcript could explain variations in response to induction between genera by a particular drug, which could reflect differences in the mechanisms used to control ampC ß-lactamase gene expression. In addition to induction potential differences, there is increasing concern for the generation and spread of plasmid-encoded ampC ß-lactamases from different genera of Enterobacteriaceae that encode inducible ampC genes. It is of interest to note that to date, no plasmid-encoded ampC gene has been identified from S. marcescens origin.44,45 It is possible that the innate characteristic of the S. marcescens ampC gene itself plays a role in the lack of plasmid-encoded ampC genes of Serratia origin.
Data from other laboratories indicate that expression of the ampC gene may play a role in Gram-negative cells other than simply protecting the cell from antibiotic threat.46,47 The identification of multiple levels of regulation for ampC expression in S. marcescens strengthens and supports these observations. Understanding the mechanism of ampC gene expression and the requirements of this expression during cell growth is important in understanding AmpC-mediated ß-lactam resistance.
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Acknowledgements |
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Footnotes |
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References |
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