Promoter Sequences Necessary for High-Level Expression of the Plasmid-Associated ampC {beta}-Lactamase Gene blaMIR-1

Little is known about mechanisms involved in high-level expressionof plasmid-associated ampC genes. The sequence for bla_MIR-1has been elucidated, and the gene is not inducible. Althoughthe sequence for the promoter (prA) that drives expression ofEnterobacter cloacae chromosomal ampC is present upstream ofbla_MIR-1, high-level expression from bla_MIR-1 is directed froma hybrid promoter (prB) located further upstream of prA. Thepurpose of this study was to determine the influence of eachpromoter on bla_MIR-1 expression and ß-lactam resistance.RNA expression by deletion clones with both promoters was measuredand compared to that by clones in which –35 and/or –10elements of prA and/or prB were altered. Primer extension revealedtwo start sites for bla_MIR-1 transcription. Expression of bla_MIR-1in clones with both promoters was 171-fold higher than thatin clones carrying only prA. In addition, bla_MIR-1 expressionfrom prA increased 11-fold in the presence of the prB –10element compared to expression driven from prA alone. Ceftazidimeand cefotaxime MICs increased 42- and 64-fold, respectively,for the clone expressing bla_MIR-1 from both promoters comparedto expression from prA alone. The upstream promoter prB of bla_MIR-1is solely responsible for high-level expression required forcefotaxime and ceftazidime resistance. These data suggest thatresistance to extended-spectrum cephalosporins mediated by noninducibleplasmid-associated ampC genes requires the formation of novelpromoter elements that are capable of increasing ampC expression.

INTRODUCTION

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Introduction

The AmpC or group 1, class C ß-lactamase genes arefound on the chromosomes of several gram-negative bacteria andare being characterized with increasing frequency on plasmidsin cephalosporin-resistant clinical isolates. Genetic evidencesuggests that the plasmid-associated ampC genes originated fromchromosomal ampC genes that mobilized onto plasmids via mobilegenetic elements (30). Several plasmid-associated ampC geneswith nucleotide similarity to chromosomal ampC genes from Enterobacterspp., Morganella morganii, Citrobacter freundii, and Hafniaalvei have been identified (1, 8, 9, 17). Recently, sequencingthe chromosomal ampC of Aeromonas caviae revealed greater than96% similarity to the sequences of bla_FOX-1_-₅ (3, 4, 7, 10,19, 25). The remaining plasmid-associated ampC group with sequencessimilar to that of bla_MOX-1 exhibit $~$ 70% identity to the genesof Aeromonas spp. (26).

When AmpC is expressed at high levels, AmpC-producing organismsbecome resistant to almost all ß-lactam antibiotics,with the exception of cefepime, cefpirome, and the carbapenems(30). Most chromosomal ampC genes are inducible in the presenceof certain agents, such as cefoxitin and imipenem (12). InducibleAmpC expression is regulated by AmpR in the presence of twoother gene products, AmpD and AmpG (12, 15, 16, 20). However,AmpR does not regulate expression from the majority of the plasmid-associatedampC genes, and therefore, the mechanisms by which high-levelAmpC expression from noninducible plasmid-associated ampC genesoccurs remain to be elucidated. The plasmid-associated ampCgenes bla_DHA-1 and bla_DHA-2 of M. morganii origin and bla_ACT-1of Enterobacter asburiae origin are inducible, and their inductionis by the general mechanism of inducible expression (2, 6, 27,29). However, a C-to-T transition at the first position of thebla_ACT-1 –10 promoter element is implicated in increasedexpression even in the absence of induction (27, 28).

The other plasmid-carried ampC of Enterobacter origin is bla_MIR-1(14, 22). bla_MIR-1 expression is not inducible, as the genelacks the association of an upstream ampR gene and the bindingsite for AmpR is truncated (14, 28). The 5' flanking sequenceof bla_MIR-1 retains the chromosomal ampC –35 and –10elements (prA) and a portion of the AmpR binding site sequence(Fig. 1B) (14). Upstream of the truncated AmpR binding siteis an insertion element with 96% similarity to a transposasegene nucleotide sequence from Pseudomonas pseudoalcaligenes(14).

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FIG. 1. Genetic organization of the chromosomal ampC gene of E. cloacae (A) and the plasmid-carried ampC gene bla_MIR-1 (B). The E. cloacae ampC is inducible and is expressed from prA. bla_MIR-1 is not inducible; however, prA remains intact, upstream of the structural gene.

A previous study reported that bla_MIR-1 is expressed from aputative hybrid promoter (prB) likely formed during mobilizationof bla_MIR-1 from the chromosome to the plasmid (28). The joiningof the upstream insertion element from P. pseudoalcaligeneswith the truncated AmpR binding site created prB with the –35element located in the insertion sequence and the –10element located 17 bp downstream, within the remnant of theAmpR binding site (Fig. 1B) (28). In this study, the role ofexpression from the bla_MIR-1 promoter prB and the vestigialpromoter prA were investigated in relation to ß-lactamantibiotic susceptibility.

(A preliminary account of this work has been presented previously[M. D. Reisbig and N. D. Hanson, Abstr. 43rd Intersci. Conf.Antimicrob. Agents Chemother., abstr. C1-676, 2003].)

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listedin Tables 1 to 3.

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TABLE 1. Clinical isolates used in this study

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TABLE 3. Plasmids used in this study

Cloning of bla_MIR-1 and deletion clones. The primers used to amplify by PCR each bla_MIR-1 deletion fragmentare listed in Table 4. PCR was carried out as previously describedto create amplicons containing the entire bla_MIR-1 structuralgene and various lengths of the 5' upstream region includingdifferent components of prB and prA (Fig. 2). Table 4 identifiesthe PCR-amplified fragment lengths and the nucleotide positionspresent in each fragment and the resulting promoter changes.These changes are illustrated in Fig. 2. The seven cDNA ampliconswere cloned by using the TOPO-XL cloning kit (Invitrogen). Eachfragment was subcloned as an EcoRI fragment into plasmid pMDR009,a pACYC184 derivative that allows for EcoRI insertion withoutexpression interference from the chloramphenicol acetyltransferasepromoter (5). Plasmid constructs were transformed into Escherichiacoli Top10 (Invitrogen) (11). The promoter inserts were manuallysequenced from the plasmid with the Pfu polymerase sequencingkit as described by the manufacturer (Stratagene). The bla_MIR-1structural gene from each clone was amplified by PCR as previouslydescribed with Platinum Taq Plus DNA polymerase (28). Theseamplicons were analyzed by automated sequencing as previouslydescribed (23). The constructed plasmids are listed in Tables2 and 3.

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TABLE 4. Primers used to amplify bla_MIR-1 deletion fragments

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FIG. 2. Diagram of the 5' flanking regions of each bla_MIR-1 construct. Solid lines, wild-type sequence; hatched lines, vector sequence. prA is the vestigial promoter, a remnant of the chromosomal ampC gene of Enterobacter origin. prB is the hybrid promoter, composed of the insertion sequence (IS) and remnant portions of the 5' flanking region of the chromosomal ampC gene. Deletions of both prA and prB components were generated to assess the contributory effects of these elements on transcription and the susceptibility phenotype. EcM1 ${Delta}$ 05 and EcM1 ${Delta}$ 06 harbored constructs with mutations that destroyed the –35 and –10 prB elements but kept the original nucleotide spacing. These clones were created to ensure that any changes in the observed in bla_MIR-1 transcript levels were not a result of differences in promoter topography. For the specific mutations generated for these clones see Table 4.

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TABLE 2. Other strains used in this study^a

Primer extension analysis. RNA isolation and primer extension analysis were carried outas previously described using 20 µg of RNA for experimentalsamples and 1 µg for the 16S rRNA control lanes (27).Primers used for primer extension are listed in Table 4. Extensionproducts were visualized, normalized, and quantified as previouslydescribed (27).

MICs. Cefoxitin, cephalothin, ampicillin, cefotaxime, and ceftazidimeMICs for each strain were determined by Etest (AB Biodisk NorthAmerica, Piscataway, N.J.) with a 0.5 McFarland's standard inoculumon 56-ml Mueller-Hinton agar plates incubated in ambient airfor 18 h at 37°C.

RESULTS

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Results

Expression levels of promoter clones were compared to the uninducedwild-type ampC expression from Enterobacter cloacae 55. ampCexpression from clone EcM1 ${Delta}$ 01 is driven by prA, which includes–10 and –35 elements identical to those found inthe wild-type E. cloacae 55 promoter. As shown in Fig. 3, overallexpression levels for these two strains were comparable. Inaddition, the start sites for ampC transcription mapped to position+36 (Fig. 3B) in each clone. As the upstream sequence was extendedto include nucleotides which represented the –10 and –35elements of prA and the –10 element of prB (EcM1 ${Delta}$ 04), ampCexpression from the prA promoter increased 11-fold comparedto expression from EcM1 ${Delta}$ 01 and E. cloacae 55 (Fig. 3). In addition,expression from the prB –10 element was seen, as determinedby analysis of two ampC transcriptional start sites locatedat positions +1 for prB and +36 for prA (Fig. 3B). However,the increases in expression from each promoter compared to thatfrom EcM1 ${Delta}$ 01 and E. cloacae 55 were almost equivalent (11-foldfrom prA and 7-fold from prB).

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FIG. 3. (A) Transcription levels expressed relative to the expression observed from prA in EcM1 ${Delta}$ 06, as measured by primer extension analysis. Relative expression was calculated by setting the lowest detectable expression level to 1 (prA, EcM1 ${Delta}$ 06) after normalization and comparing all expression levels to this value. A value of zero, indicated by the absence of a bar, was given when bands were below the level of detection by primer extension analysis. Solid black bars, expression from prA; striped bars, expression from prB. The key below indicates the presence (+), deletion ( ${Delta}$ ), or mutation (MUT) of the promoter elements listed at the left. The insertion sequence (IS) and prB are not present (np) in the E. cloacae 55 clinical isolate. (B) Partial sequence of the upstream region of bla_MIR-1. The start sites of transcription are shown in the sequence map below, indicated by G for prA and by C for prB. prB is boldface, and prA is italicized and underlined.

By including the –35 element in prB, represented by cloneEcM1 ${Delta}$ 03, full bla_MIR-1 expression was restored, as expressionlevels from this clone were comparable to expression levelsfrom the clinical isolate Klebsiella pneumoniae 96D and thefull-length clone EcM1wt (Fig. 3). Interestingly, sequence elementsother than the –35 promoter element of prB, upstream withinthe insertion sequence (Fig. 3), did not influence bla_MIR-1expression. To ensure that positional effects due to deletionsof sequence within the promoter did not influence the observedlevels of expression from clones EcM1 ${Delta}$ 01 and EcM1 ${Delta}$ 04, clones EcM1 ${Delta}$ 06and EcM1 ${Delta}$ 05, which included nonsense sequences in place of thedeleted –10 and –35 elements of prB, respectively,were constructed. No difference between levels of bla_MIR-1 expressionfrom EcM1 ${Delta}$ 01 and EcM1 ${Delta}$ 06 (Fig. 3) or EcM1 ${Delta}$ 04 and EcM1 ${Delta}$ 05 (Fig. 3)was observed. Furthermore, no expression from EcM1 ${Delta}$ 02, whichcontained only the –10 element for prA, was detected (Fig.3).

The influence of these promoter mutations on the susceptibilityphenotype was examined by using MICs of cefotaxime, ceftazidime,ampicillin, cefoxitin, and cephalothin determined by Etest analysis.MICs of the extended-spectrum cephalosporins for all of theclones that had mutations or deletions in any of the prA orprB promoter elements remained in the susceptible category.Etest analysis revealed ceftazidime MICs of 16 and 24 µg/mlfor clones EcM1 ${Delta}$ 03 and EcM1wt, respectively, which both expressedbla_MIR-1 from prB. A significantly lower ceftazidime MIC of0.75 µg/ml was observed for clones EcM1 ${Delta}$ 01 and EcM1 ${Delta}$ 06,both of which expressed bla_MIR-1 from only the prA promoter.Cefotaxime MICs of 8 µg/ml, observed for clones EcM1 ${Delta}$ 03and EcM1wt, were also significantly higher than MICs for clonesEcM1 ${Delta}$ 01 and EcM1 ${Delta}$ 06, which were 0.125 and 0.094 µg/ml, respectively(Table 5). Even though bla_MIR-1 prA expression from clones EcM1 ${Delta}$ 04and EcM1 ${Delta}$ 05 showed an $~$ 11-fold increase over that from EcM1 ${Delta}$ 01and E. cloacae 55, no significant increases in cefotaxime andceftazidime MICs were observed. However, the cefoxitin MIC forclones EcM1 ${Delta}$ 04 and EcM1 ${Delta}$ 05 was 24 µg/ml, while ampicillinMICs were 32 and 12 µg/ml, respectively, and cephalothinMICs were >256 and 192 µg/ml, respectively. Cefoxitin,cephalothin, and ampicillin MICs for clones EcM1 ${Delta}$ 03 and EcM1wt,both containing the intact prB promoter, were >256 µg/ml.Cephalothin MICs for all clones were above the resistance breakpoint.However, regardless of which ß-lactam was tested,the highest MICs correlated to the clones expressing bla_MIR-1at the highest level (Table 5).

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TABLE 5. ß-Lactam MICs determined by Etest for clinical isolates and bacterial transformants^a

DISCUSSION

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Discussion

The events that mobilized bla_MIR-1 from the chromosome to theplasmid resulted in the formation of hybrid promoter prB, whichis responsible for high-level MIR-1 expression and resistanceto all the ß-lactam antibiotics tested. In the process,the chromosomal promoter prA was rendered ineffective, as theupstream regulatory elements were not mobilized with the ampCstructural gene onto the plasmid. Therefore, prA representsa vestigial promoter associated with bla_MIR-1 as a remnant ofthe chromosomal ampC expression system. Although the level ofexpression from prA resulted in resistance to cephalothin, thislevel of expression was not enough to confer resistance to theother ß-lactams tested in this study. These data indicatethat prB is responsible for high-level expression required forresistance to the extended-spectrum cephalosporins, ceftazidime,and cefotaxime, as well as cefoxitin and ampicillin. Removalof prB from the upstream flanking region of bla_MIR-1 did notaid in the ability of prA to express levels of bla_MIR-1 sufficientto confer resistance to these drugs. These data suggest that,even in the presence of prA, resistance to the extended-spectrumß-lactams requires the formation of a novel promoter,such as prB, to drive high-level expression.

In the absence of the AmpR regulatory protein, model systemshave shown that constitutive ampC expression increased 2.5-to 5.8-fold. However, the data presented from this study showthat, in the presence of a stronger promoter, levels of expressionof the –35 and –10 elements of the vestigial ampCpromoter do not increase above the constitutive level observedfrom a wild-type strain of E. cloacae. This observation wasnot predicted by data from promoter gene constructs comparingexpression among cloning vectors expressing chromosomal ampCgenes with or without the ampR gene (18, 24).

The plasmid-associated ampC genes of Enterobacter origin asa group are distinctive in that the mechanisms driving expressionare unique to each gene, bla_ACT-1 or bla_MIR-1. The genetic contextof the bla_ACT-1 promoter is similar to what is observed forthe promoter prA of the Enterobacter sp. chromosomal ampC gene,whereas bla_MIR-1 is expressed constitutively from the novelhybrid promoter prB. Although prB and prA both exhibit 67% percentidentity to the overall E. coli consensus promoter sequence,the observed expression from prB is higher than that observedfrom prA (13). The difference between prB and prA is in thepositions at which these promoters match the consensus sequence.The sequence of prB is more similar than the sequence of prAat positions that have been shown to dramatically increase expressionaccording to studies of the E. coli promoter consensus sequence(13). The prB promoter sequence matched the consensus at position1 of the –10 element and position 4 of the –35 elementcompared to that of prA, which did not match the consensus sequenceat these positions. Such positional differences would allowthe RNA polymerase initiation complex to recognize the prB promoterbetter than the prA promoter.

It is possible that, because prB expression is 170-fold higherthan prA expression, quenching could play a role in promoterusage. Quenching of promoter usage occurs when a strong promoteroutcompetes a weak promoter for RNA polymerase recruitment (21).The lack of bla_MIR-1 expression in the absence of the upstreampromoter, prB, indicated that quenching was not responsiblefor decreased expression from the vestigial promoter, prA.

Although the formation of new promoters that constitutivelyexpress high levels of ampC transcripts is the means by whichbla_MIR-1 is expressed and is probable in other plasmid-associatedampC promoters, other mechanisms for increased promoter expressioncannot be ruled out. The $~$ 11-fold increase in expression fromprA in EcM1 ${Delta}$ 04 and EcM1 ${Delta}$ 05 compared to expression from prA alonein EcM1 ${Delta}$ 06 was likely the result of the prB –10 elementattracting the RNA polymerase to the region. Although this increasein expression did not result in a significant change to theobserved cefotaxime and ceftazidime MICs, an element with agreater ability to recruit RNA polymerase may enhance the expressionof a weak promoter. The expression of bla_MIR-1 in EcM1 ${Delta}$ 04 andEcM1 ${Delta}$ 05 increased cefoxitin and ampicillin MICs to nonsusceptiblelevels according to breakpoints for resistance set by the NationalCommittee for Clinical Laboratory Standards.

The MICs for EcM1wt and K. pneumoniae 96D were comparable becausethe copy number of the vector did not result in a differencein relative copy number between the two plasmid-associated systems.bla_MIR-1 is carried on plasmids with relative copy numbers of12 and 11 in K. pneumoniae 96D and EcM1wt, respectively (28).

This study of the bla_MIR-1 promoter has provided insight asto how noninducible plasmid-associated ampC genes are expressed.An ampC gene that mobilizes from the chromosome to a plasmidand leaves behind the genetic elements that control inducibleexpression must evolve some other means of high-level, constitutiveexpression. As indicated by this study, it is inappropriateto select putative –10 and –35 elements of a sequencedplasmid-associated ampC gene based on the location of the wild-typepromoter in the gene of origin. It is likely that the formationof new promoters or the acquisition of strong promoters locatedwithin upstream insertion elements (V. L. Herrera and N. D.Hanson, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother.,abstr. 1488, 2001; M. D. Reisbig and N. D. Hanson, Abstr. 13thEur. Congr. Clin. Microbiol. Infect. Dis., abstr. P574, 2003)is necessary in order for ß-lactam resistance to beobserved in an organism that expresses a plasmid-associatedampC gene in the absence of AmpR.

FOOTNOTES

* Corresponding author. Mailing address: Center for Research in Anti-Infectives and Biotechnology, Department of Microbiology and Immunology, Creighton University School of Medicine, 2500 California Pl., Omaha, NE 68178. Phone: (402) 280-5837. Fax: (402) 280-1875. E-mail: ndhanson{at}creighton.edu.

REFERENCES

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