Analyses of ampC gene expression in Serratia marcescens reveal new regulatory properties

Steven D. Mahlen, Stacey S. Morrow, Baha Abdalhamid and Nancy D. Hanson*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Serratia marcescens encodes an inducible, chromosomal ß-lactamase, ampC. Studies addressing the regulation of inducible ampC genes have focused primarily on Enterobacter cloacae and Citrobacter freundii. The purpose of this study was to clone and sequence the ampC, ampR and intergenic region of S. marcescens and examine both inducible and basal level ampC expression. Sequence analysis of the S. marcescens ampC gene identified an extended 5' untranslated region (UTR) of 126 nucleotides, which formed a prominent stem–loop structure. Induction of ampC expression required AmpR, and the start of transcription was determined using primer extension analysis. In vivo half-life analysis revealed that the half-life of the S. marcescens ampC transcript was 7 min. Confirmation of the in vivo half-life and the role of the stem–loop structure in the 5' UTR was demonstrated by comparing transcript half-life and luciferase expression between a wild-type (WT) and a 5' UTR stem–loop deletion mutant. These data demonstrated that the stem–loop structure was involved in transcript stability. Taken together, these findings indicate that constitutive expression of S. marcescens ampC is regulated by both transcriptional initiation and post-transcriptional events.

Keywords: ampC, ß-lactamase, Gram-negative bacteria, regulation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Members of the Enterobacteriaceae that encode an inducible chromosomal ß-lactamase include Citrobacter freundii, Enterobacter cloacae, Yersinia enterocolitica, Morganella morganii, Hafnia alvei and Serratia marcescens among others. Evaluations of ampC expression in C. freundii and E. cloacae have shown that the DNA-binding protein AmpR controls expression of ampC.15 ampC and ampR are transcribed divergently with the AmpR-binding site located within the ampC/ampR intergenic region.4,68 AmpC expression is inducible by some ß-lactam drugs, such as cefoxitin and imipenem, in the presence of AmpR.9,10 Binding of AmpR in the absence of an inducing ß-lactam drug represses ampR expression and results in low constitutive expression of ampC.36,11 In addition to AmpR, two other gene products are required for AmpC induction, a permease (AmpG) and an amidase (AmpD).4,7,12,13 The mechanism of ampC induction has recently been reviewed.14 Perturbations in the induction pathway can lead to constitutive overexpression (derepression) of the ampC gene and an organism resistant to most ß-lactam antibiotics except the fourth-generation cephalosporins and the carbapenems.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and plasmids

All strains and plasmids used in this study are listed in Table 1.


View this table:
[in this window]
[in a new window]
 
Table 1.  Bacterial strains and plasmids used in this study
 
Primers

The primers used in this study are listed in Table 2. Oligonucleotides were synthesized by Genosys (The Woodlands, TX, USA).


View this table:
[in this window]
[in a new window]
 
Table 2.  Primers used in this study
 
Preparation of DNA

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 1–2% 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{Delta} (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{alpha}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.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 1. Cloning scheme of the full-length S. marcescens ampC/ampR. A full description of the cloning strategy is given in Materials and methods. Briefly, two partial clones, pSmC448 and pSmC*, were used to create a full-length inducible clone for S. marcescens ampC. pSmC448 was derived from genomic DNA and encoded the entire ampC, intergenic region and all but 40 nt of the 3' end of ampR. The ampC/ampR region of pSmC* was generated by PCR. This clone contained the entire ampR gene but was missing 14 nt at the 3' end of ampC. The full-length clone was assembled using the unique restriction site (AccI) within the ampC gene and restriction sites found within the vectors, which flanked either the ampC or ampR genes.

 

Generation of UTR plasmid constructions. pSPluc{Delta}RBS 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{Delta}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 pSPluc{Delta}UTR 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{Delta}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{Delta}RBS. This clone was designated pSPlucWTUTR. pSPluc{Delta}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{Delta}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 stem–loop 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 stem–loop 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.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 6. Semi-quantitative RT–PCR depicting half-lives of luciferase construct transcripts. (a) Top panel: RT–PCR of luciferase construct transcripts from the wild-type (WT) clone EcSPlucWTUTR and the mutant (MUT) clone EcSPluc{Delta}UTR from RNA isolated at 0, 2, 5, 10, 15 and 20 min after the addition of 200 mg/L rifampicin using primers SMCMUTC and 5' LUCREV (Table 2). Bottom panel: RT–PCR of transcripts of E. coli 23S rRNA using primers 80 F and 78 R (Table 2) from EcSPlucWTUTR and EcSPluc{Delta}UTR from RNA isolated at 0, 2, 5, 10, 15 and 20 min after the addition of 200 mg/L rifampicin. These bands were used to normalize the data for calculating the % transcript remaining. Lane M is the 100 bp ladder, and the 200 bp band is shown with an arrow. (b) A bar graph representing the % transcript remaining after the addition of rifampicin at the designated time point. WT, black bars; MUT, grey bars. (c) Diagram of the final construction step for the MUT clone, EcSPluc{Delta}UTR, from the WT clone, EcSPlucWTUTR.

 
Transformations of E. coli were carried out using E. coli INV{alpha}F competent cells (Stratagene, La Jolla, CA, USA) or TSS competent E. coli HB101.26 S. marcescens cells were made competent for electroporation by chilling 1.5 mL overnight cultures on ice for 15 min and washing the cell pellets in 1 mL of sterile, ice-cold nanopure dH2O a total of three times.23 Gel-purified plasmid DNA (0.5 µg) was added to the cells, and incubated on ice for 10 min before electroporation. The cell suspension was placed in a chilled electroporation cuvette (0.2 cm gap), and pulsed using the parameters 2.5 kV, 25 µF and 400 {Omega}. All transformation mixtures were plated onto LB agar containing the appropriate antibiotic for the vector used and incubated at 37°C overnight. Colonies were screened for inserts by PCR using primers SMCP1F and SMCP1R.

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.

RT–PCR

The half-life of transcripts expressed from the luciferase constructs was determined by semi-quantitative RT–PCR. Total RNA samples used for RT–PCR were isolated from strains EcSPlucWTUTR and EcSPluc{Delta}UTR 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. RT–PCR was carried out using the Qiagen OneStep RT–PCR 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 RT–PCR. The cycling parameters for all RT–PCR 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 RT–PCR 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, EcSPluc{Delta}UTR, SmSPlucWTUTR and SmSPluc{Delta}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 Mueller–Hinton 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cloning and sequence analysis of the ampC/ampR intergenic region of S. marcescens

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.



View larger version (47K):
[in this window]
[in a new window]
 
Figure 2. Nucleotide sequence alignments. The overlapping ampR/ampC promoter region of S. marcescens (Sm) and its alignment with analogous regions of C. freundii (Cf), E. cloacae (Ec), Y. enterocolitica (Ye), M. morganii (Mm), P. stuartii (Ps) and H. alvei (Hf). The start codons for both AmpC and AmpR are in bold and indicated by a directional arrow. The –10 and –35 sequences are in bold. The S. marcescens ampC transcriptional start site, in bold and underlined, is denoted by +1. The ampC and ampR start sites for Ec and the putative start sites for Cf, Mm, Ye, Ps and Hf are also in bold and underlined. The LysR binding motif is italicized and underlined with the TN11A motif noted. Nucleotides corresponding to the predominant stem–loop structure in S. marcescens predicted by the DNASIS program are noted, underlined and in bold. The putative ribosomal binding site for S. marcescens is underlined.

 
AmpR is required for induction

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 stem–loop 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.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 3. Primer extension analysis of S. marcescens ampC. The primary start site of S. marcescens ampC transcription is indicated by the labelled arrow. Premature stop sites by reverse transcriptase are designated by the unlabelled arrows and correspond to the secondary structure within the 5' UTR (Figure 4). Sequence ladder was obtained using the same reverse primer used for primer extension (SMPE2). U, uninduced; I, induced with one quarter the MIC of the ß-lactam antibiotic cefoxitin. The bands corresponding to S. marcescens 16S rRNA indicate the RNA from the uninduced cells was not under-represented.

 
Computer analysis of the 5' UTR

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 stem–loop 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.



View larger version (17K):
[in this window]
[in a new window]
 
Figure 4. Predicted RNA secondary structure of the S. marcescens ampC 5' UTR. The predicted secondary structure was determined by the DNASIS program. The {Delta}G is predicted to be 57 kcal/mol. The ribosomal binding site is shaded and the start codon for AmpC is indicated by the arrow.

 
Evaluation of 5' UTR function

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 6–7 min of incubation with rifampicin (Figure 5).



View larger version (34K):
[in this window]
[in a new window]
 
Figure 5. Determination of ampC half-lives. (a) ampC RNA half-life of S. marcescens was determined using northern analysis. Time points, in minutes, are noted above each lane. (b) Decay curve of S. marcescens ampC RNA half-life. Bands were quantified by densitometry and plotted semi-logarithmically as a percentage of transcript remaining compared with zero time (which equals 100% transcript remaining). All blots were stained with Methylene Blue to ensure equal loading of total RNA in each lane.31 DIG-labelled RNA markers are indicated in lane M and represent transcripts of 1.0 and 1.6 kb in size.

 
Determination of stem–loop function

It is possible that the stem–loop 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 stem–loop 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 stem–loop structure deleted (nucleotides +51 to +106) (pSPluc{Delta}UTR). 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 stem–loop 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 stem–loop 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 RT–PCR 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).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The data provided in this paper are the first genetic evidence of inducible ampC regulation in S. marcescens and indicate that regulation of the S. marcescens ampC ß-lactamase gene occurs at multiple levels: RNA initiation with induction through AmpR and events downstream of transcriptional initiation including regulation of RNA half-life. In this study, the S. marcescens ampC/ampR intergenic region was identified and compared with intergenic regions of other genera. All sequence motifs describing the –35 and –10 boxes are putative sites, with only the S. marcescens and E. cloacae start sites of the ampC transcripts mapped. Both similarities and differences were observed for the putative –35 and –10 boxes of ampC and ampR when compared with these same regions in C. freundii, E. cloacae, Y. enterocolitica, M. morganii, H. alvei and P. stuartii (Figure 2).

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 stem–loops has been predicted in the 5' UTRs of the transcripts listed above. Stem–loop 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 stem–loop 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 stem–loop 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 1–2 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.


    Acknowledgements
 
We would like to thank William Tapprich and John Mullican for the use of the 23S rRNA primers. We would like to thank Mark Reisbig for constructing pACdpr1 and we thank Alika Maunakea for technical support. Funding for the research in this study was provided by the Center for Research in Anti-Infectives & Biotechnology. DNA sequencing in this study was supported in part by UNMC/Eppley Cancer Center grant P30CA36727 and the DNA core facility at Creighton University.


    Footnotes
 
* Corresponding author. Tel: +1-402-280-5837; Fax: +1-402-280-1875; E-mail:ndhanson@creighton.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Bartowsky, E. & Normark, S. (1991). Purification and mutant analysis of Citrobacter freundii AmpR, the regulator for chromosomal AmpC beta-lactamase. Molecular Microbiology 5, 1715–25.[ISI][Medline]

2 . Henikoff, S., Haughn, G. W., Calvo, J. M. & Wallace, J. C. (1988). A large family of bacterial activator proteins. Proceedings of the National Academy of Sciences, USA 85, 6602–6.[Abstract]

3 . Jacobs, C., Frere, J. M. & Normark, S. (1997). Cytosolic intermediates for cell wall biosynthesis and degradation control inducible beta-lactam resistance in Gram-negative bacteria. Cell 88, 823–32.[ISI][Medline]

4 . Lindberg, F., Westman, L. & Normark, S. (1985). Regulatory components in Citrobacter freundii AmpC beta-lactamase induction. Proceedings of the National Academy of Sciences, USA 82, 4620–4.[Abstract]

5 . Lindquist, S., Lindberg, F. & Normark, S. (1989). Binding of the Citrobacter freundii AmpR regulator to a single DNA site provides both autoregulation and activation of the inducible ampC beta-lactamase gene. Journal of Bacteriology 171, 3746–53.[ISI][Medline]

6 . Honore, N., Nicolas, M. H. & Cole, S. T. (1986). Inducible cephalosporinase production in clinical isolates of Enterobacter cloacae is controlled by a regulatory gene that has been deleted from Escherichia coli. EMBO Journal 5, 3709–14.[Abstract]

7 . Lindberg, F., Lindquist, S. & Normark, S. (1987). Inactivation of the ampD gene causes semiconstitutive overproduction of the inducible Citrobacter freundii beta-lactamase. Journal of Bacteriology 169, 1923–8.[ISI][Medline]

8 . Lindquist, S., Weston-Hafer, K., Schmidt, H., Pul, C., Korfmann, G., Erickson, J. et al. (1993). AmpG, a signal transducer in chromosomal beta-lactamase induction. Molecular Microbiology 9, 703–15.[ISI][Medline]

9 . Gatus, B. J., Bell, S. M. & Jimenez, A. S. (1988). Comparison of glycine enhancement with cefoxitin induction of class 1 beta-lactamase production in Enterobacter cloacae ATCC 13047. Journal of Antimicrobial Chemotherapy 21, 163–70.[Abstract]

10 . Sanders, C. C. & Sanders, W. E. (1986). Type I beta-lactamases of Gram-negative bacteria: interactions with beta-lactam antibiotics. Journal of Infectious Diseases 154, 792–800.[ISI][Medline]

11 . Bishop, R. E. & Weiner, J. H. (1993). Overproduction, solubilization, purification and DNA-binding properties of AmpR from Citrobacter freundii. European Journal of Biochemistry 213, 405–12.[Abstract]

12 . Korfmann, G. & Sanders, C. C. (1989). AmpG is essential for high-level expression of AmpC beta-lactamase in Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 33, 1946–51.[ISI][Medline]

13 . Nicolas, M. H., Honore, N., Jarlier, V., Philippon, A. & Cole, S. T. (1987). Molecular genetic analysis of cephalosporinase production and its role in beta-lactam resistance in clinical isolates of Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 31, 295–9.[ISI][Medline]

14 . Hanson, N. D. & Sanders, C. C. (1999). Regulation of inducible AmpC beta-lactamase expression among Enterobacteriaceae. Current Pharmaceutical Design 5, 881–94.[ISI][Medline]

15 . Pedersen, S. & Reeh, S. (1978). Functional mRNA half lives in E. coli. Molecular and General Genetics 166, 329–36.[Medline]

16 . Rosenbaum, V., Klahn, T., Lundberg, U., Holmgren, E., Von Gabain, A. & Riesner, D. (1993). Co-existing structures of an mRNA stability determinant. The 5' region of the Escherichia coli and Serratia marcescens ompA mRNA. Journal of Molecular Biology 229, 656–70.[CrossRef][ISI][Medline]

17 . Yamanaka, K., Mitta, M. & Inouye, M. (1999). Mutation analysis of the 5' untranslated region of the cold shock cspA mRNA of Escherichia coli. Journal of Bacteriology 181, 6284–91.[Abstract/Free Full Text]

18 . Girlich, D., Karim, A., Spicq, C. & Nordmann, P. (2000). Plasmid-mediated cephalosporinase ACC-1 in clinical isolates of Proteus mirabilis and Escherichia coli. European Journal of Clinical Microbiology and Infectious Diseases 19, 893–5.[CrossRef][ISI][Medline]

19 . Poirel, L., Guibert, M., Girlich, D., Naas, T. & Nordmann, P. (1999). Cloning, sequence analyses, expression, and distribution of ampCampR from Morganella morganii clinical isolates. Antimicrobial Agents and Chemotherapy 43, 769–76.[Abstract/Free Full Text]

20 . Seoane, A., Francia, M. V. & Garcia Lobo, J. M. (1992). Nucleotide sequence of the ampCampR region from the chromosome of Yersinia enterocolitica. Antimicrobial Agents and Chemotherapy 36, 1049–52.[Abstract]

21 . Pitout, J. D., Thomson, K. S., Hanson, N. D., Ehrhardt, A. F., Moland, E. S. & Sanders, C. C. (1998). Beta-lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa. Antimicrobial Agents and Chemotherapy 42, 1350–4.[Abstract/Free Full Text]

22 . Chen, Y. C., Shipley, G. L., Ball, T. K. & Benedik, M. J. (1992). Regulatory mutants and transcriptional control of the Serratia marcescens extracellular nuclease gene. Molecular Microbiology 6, 643–51.[ISI][Medline]

23 . Ausebel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. et al. (1989). Short Protocols in Molecular Biology, vol. 1. John Wiley & Sons, New York, NY, USA.

24 . Manniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.

25 . Reisbig, M. D. & Hanson, N. D. (2002). The ACT-1 plasmid-encoded AmpC ß-lactamase is inducible: detection in a complex ß-lactamase background. Journal of Antimicrobial Chemotherapy 49, 557–60.[Abstract/Free Full Text]

26 . Hanson, N. D., Thomson, K. S., Moland, E. S., Sanders, C. C., Berthold, G. & Penn, R. G. (1999). Molecular characterization of a multiply resistant Klebsiella pneumoniae encoding ESBLs and a plasmid-mediated AmpC. Journal of Antimicrobial Chemotherapy 44, 377–80.[Abstract/Free Full Text]

27 . Snyder, L. & Champness, W. (1997). Introduction to macromolecular synthesis: gene expression. In Molecular Genetics of Bacteria, pp. 43–72. ASM Press, Washington, DC, USA.

28 . White, R., Butler, A. & Parker, M. (1995). Directed mutagenesis and mutant analysis. In Gene Probes 2 (Hames, B. D. & Higgins, S. J., Eds), pp. 329–55. Oxford University Press, New York, NY, USA.

29 . Zuker, M. & Stiegler, P. (1981). Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Research 9, 133–48.[Abstract]

30 . Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology 215, 403–10.[CrossRef][ISI][Medline]

31 . Wade, M. F. & O’Conner, J. L. (1992). Using a cationic carbocyanine dye to assess RNA loading in northern gel analysis. Biotechniques 12, 794–6.[ISI][Medline]

32 . Wang, N., Yamanaka, K. & Inouye, M. (1999). CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock. Journal of Bacteriology 181, 1603–9.[Abstract/Free Full Text]

33 . Sanders, C. C. & Sanders, W. E. (1979). Emergence of resistance to cefamandole: possible role of cefoxitin-inducible beta-lactamases. Antimicrobial Agents and Chemotherapy 15, 792–7.[ISI][Medline]

34 . Bricker, A. L. & Belasco, J. G. (1999). Importance of a 5' stem–loop for longevity of papA mRNA in Escherichia coli. Journal of Bacteriology 181, 3587–90.[Abstract/Free Full Text]

35 . Etchegaray, J. P., Jones, P. G. & Inouye, M. (1996). Differential thermoregulation of two highly homologous cold-shock genes, cspA and cspB, of Escherichia coli. Genes to Cells 1, 171–8.[Abstract/Free Full Text]

36 . Tanabe, H., Goldstein, J., Yang, M. & Inouye, M. (1992). Identification of the promoter region of the Escherichia coli major cold shock gene, cspA. Journal of Bacteriology 174, 3867–73.[Abstract]

37 . Chen, L. H., Emory, S. A., Bricker, A. L., Bouvet, P. & Belasco, J. G. (1991). Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA. Journal of Bacteriology 173, 4578–86.[ISI][Medline]

38 . Brumlik, M. J. & Storey, D. G. (1998). Post-transcriptional control of Pseudomonas aeruginosa lasB expression involves the 5' untranslated region of the mRNA. FEMS Microbiology Letters 159, 233–9.[CrossRef][ISI][Medline]

39 . Hansen, M. J., Chen, L. H., Fejzo, M. L. & Belasco, J. G. (1994). The ompA 5' untranslated region impedes a major pathway for mRNA degradation in Escherichia coli. Molecular Microbiology 12, 707–16.[ISI][Medline]

40 . Jiang, W., Fang, L. & Inouye, M. (1996). The role of the 5'-end untranslated region of the mRNA for CspA, the major cold-shock protein of Escherichia coli, in cold-shock adaptation. Journal of Bacteriology 178, 4919–25.[Abstract]

41 . Matsumura, N., Minami, S. & Mitsuhashi, S. (1998). Sequences of homologous beta-lactamases from clinical isolates of Serratia marcescens with different substrate specificities. Antimicrobial Agents and Chemotherapy 42, 176–9.[Abstract/Free Full Text]

42 . Sanders, C. C. (1989). Beta-lactamase stability and in vitro activity of oral cephalosporins against strains possessing well-characterized mechanisms of resistance. Antimicrobial Agents and Chemotherapy 33, 1313–7.[ISI][Medline]

43 . Okonogi, K., Sugiura, A., Kuno, M., Higashide, E., Kondo, M. & Imada, A. (1985). Effect of beta-lactamase induction on susceptibility to cephalosporins in Enterobacter cloacae and Serratia marcescens. Journal of Antimicrobial Chemotherapy 16, 31–42.[Abstract]

44 . Perez-Perez, F. J. & Hanson, N. D. (2002). Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. Journal of Clinical Microbiology 40, 2153–62.[Abstract/Free Full Text]

45 . Philippon, A., Arlet, G. & Jacoby, G. A. (2002). Plasmid-determined AmpC-type beta-lactamases. Antimicrobial Agents and Chemotherapy 46, 1–11.[Free Full Text]

46 . Everett, M., Walsh, T., Guay, G. & Bennett, P. (1995). GcvA, a LysR-type transcriptional regulator protein, activates expression of the cloned Citrobacter freundii ampC beta-lactamase gene in Escherichia coli: cross-talk between DNA-binding proteins. Microbiology 141, 419–30.[Abstract]

47 . Henderson, T. A., Young, K. D., Denome, S. A. & Elf, P. K. (1997). AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli. Journal of Bacteriology 179, 6112–21.[Abstract]

48 . Nomura, K. & Yoshida, T. (1990). Nucleotide sequence of the Serratia marcescens SR50 chromosomal ampC beta-lactamase gene. FEMS Microbiology Letters 58, 295–9.[Medline]