Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Antimicrobial Agents and Chemotherapy
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Mechanisms of Resistance

Promoters P3, Pa/Pb, P4, and P5 Upstream from blaTEM Genes and Their Relationship to β-Lactam Resistance

Marie Frédérique Lartigue, Véronique Leflon-Guibout, Laurent Poirel, Patrice Nordmann, Marie-Hélène Nicolas-Chanoine
Marie Frédérique Lartigue
Service de Microbiologie-Hygiène, Hôpital Ambroise Paré Assistance Publique-Hôpitaux de Paris, BoulogneFaculté Paris Ile-de-France Ouest, Université Versailles Saint-Quentin-en-Yvelines, Saint-Quentin-en-Yvelines
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Véronique Leflon-Guibout
Service de Microbiologie-Hygiène, Hôpital Ambroise Paré Assistance Publique-Hôpitaux de Paris, BoulogneFaculté Paris Ile-de-France Ouest, Université Versailles Saint-Quentin-en-Yvelines, Saint-Quentin-en-Yvelines
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Laurent Poirel
Service de Bactériologie-Virologie, Hôpital BicêtreAssistance Publique-Hôpitaux de Paris
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Patrice Nordmann
Service de Bactériologie-Virologie, Hôpital BicêtreAssistance Publique-Hôpitaux de Paris
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Marie-Hélène Nicolas-Chanoine
Service de Microbiologie-Hygiène, Hôpital Ambroise Paré Assistance Publique-Hôpitaux de Paris, BoulogneFaculté Paris Ile-de-France Ouest, Université Versailles Saint-Quentin-en-Yvelines, Saint-Quentin-en-Yvelines
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: marie-helene.nicolas-chanoine@apr.ap-hop-paris.fr
DOI: 10.1128/AAC.46.12.4035-4037.2002
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Using an isogenic system, we have determined the impact that the four promoters known to control blaTEM gene expression have on β-lactamase activity. For both TEM-1 and TEM-30, this activity gradually increased in relation to the presence of promoters P3, Pa/Pb, and P4 upstream of the corresponding gene. Promoter P5, only found upstream of the blaTEM-1B gene, was related to the highest expression of this gene.

The extensive nucleotide sequence analysis of plasmid-mediated blaTEM genes performed over the last 15 years because of their role in resistance to extended-spectrum cephalosporins and to amoxicillin-clavulanate has allowed us to observe that the expression of these genes is controlled by four different promoters (3, 6). The first promoter, called P3 (Fig. 1,) corresponds to the promoter of the blaTEM-1A gene which is located on a Tn3 transposon and plasmid pBR322 (10). The second promoter, called Pa/Pb and found upstream of the blaTEM-2 gene, is composed, in fact, by two overlapping promoters resulting from the mutation C→T at position 32 according to the Sutcliffe numbering system (10). The −35 and −10 regions of promoter Pa are TTGAAG and TACGCT, respectively, whereas the −35 and −10 regions of promoter Pb are GTGATA and TAATGT, respectively, the first 2 nucleotides of the −10 region of Pa corresponding to the last 2 nucleotides of the −35 region of Pb (Fig. 1). Chen et al. (2), studying the separately cloned promoters P3 and Pa/Pb, showed that the Pb sequence which was present in the fragment containing the promoter P3 did not work as a promoter and that the overlapping promoter P3 were stronger than promoter P3, with the increase in strength resulting from both the strength of Pa and the cooperative activity of Pa and Pb. The third promoter, called P4 by Goussard et al. (3), differs from promoter P3 by the substitution G162T (Sutcliffe numbering system) corresponding to the first nucleotide of the −10 region of the promoter (TACAAT instead of GACAAT). This substitution makes the sequence of the −10 region of promoter P4 closer to the consensus sequence (TATAAT) (Fig. 1). We suggest that the fourth promoter, which we have recently published, be called P5 (7). This promoter has the same sequence as promoter P3 at the −10 region but has a sequence at the −35 region (TTGAAA) closer to the consensus sequence (TTGACA) than that of promoter P3 (TTCAAA) (Fig. 1). To assess and compare the respective impact of the four promoters on β-lactam resistance, we established an isogenic system in which the only difference consisted of the type of promoter upstream from blaTEM genes coding for either a β-lactamase susceptible (TEM-1) or resistant (TEM-30 or IRT-2) to class A β-lactamase inhibitors.

By using primers 5′-ATA AAA TTC TTG AAG AC-3′ and 5′-TTA CCA ATG CTT AAT CA-3′, four blaTEM-1B genes, each preceded by one of the four promoters, were amplified and cloned from previously published Escherichia coli isolates (6). The blaTEM-30 gene coding for the inhibitor-resistant TEM-30 or IRT-2 enzyme was also amplified and cloned from previously published E. coli isolates but only with promoter P3, Pa/Pb, or P4 upstream from it, as a blaTEM-30 gene controlled by promoter P5 has not been discovered thus far (6). Plasmid pPCR Script (Camr) (Stratagene, La Jolla, Calif.) and E. coli strain Epicurian Coli XL10-Gold (Stratagene) were used in the first stage of the cloning experiments. After the sequences of inserted fragments (promoter and coding region) were checked (automated cycle sequencing system on a Perkin-Elmer R377 sequencer), the fragments were then cloned into the BamHI-restricted and dephosphorylated plasmid pACYC184 (Tetr and Camr) and expressed in E. coli strain NM554 (8). The size of the inserts and their insertion in the opposite orientation of the promoter of the pACYC184 tetracycline resistance-encoding gene were assessed by restriction analyses with BamHI and AseI enzymes.

The E. coli NM554 transformants containing the different recombinant plasmids were then tested in three independent experiments for β-lactam susceptibility by using the agar dilution method on Mueller-Hinton agar with a Steers multiple inoculator and 104 CFU per spot. The β-lactamase activity of each transformant was also tested from the crude extracts of three independent cultures, as previously described (5). Briefly, β-lactamase was extracted from 100 ml of an exponential-growth-phase culture at 37°C in Trypticase soy broth containing amoxicillin (100 μg/ml) combined with chloramphenicol (30 μg/ml). Bacterial suspensions were disrupted by sonication, and the β-lactamase activity was measured from the crude extracts with a Pharmacia UV2000 spectrophotometer, using benzylpenicillin (100 μM) as the substrate. One unit of β-lactamase activity was defined as the amount of enzyme which hydrolyzed 1 μM benzylpenicillin per min, and the specific activity was defined as the β-lactamase activity per milligram of protein.

As indicated in Table 1, β-lactamase activity as well as MICs of amoxicillin-clavulanate, ticarcillin-clavulanate, piperacillin, and cephalothin gradually increased from the E. coli transformant expressing the blaTEM-1B gene controlled by the P3 promoter to that expressing the blaTEM-1B gene controlled by the P5 promoter, with higher values for the two types of parameters when blaTEM-1B was controlled by the P4 promoter than when it was controlled by the Pa/Pb promoters. Concerning ticarcillin, the concentrations used in this study did not allow us to observe a gradual increase in MICs in relation to the promoter type. For piperacillin-tazobactam, we observed different levels of MICs: low MICs, from 1 to 2 μg/ml for the transformants whose blaTEM-1B gene was controlled by promoters P3 and Pa/Pb, respectively, and high MICs, from 128 to 512 μg/ml for those whose blaTEM-1B gene was under the control of promoters P4 and P5, respectively. The first two transformants were categorized as susceptible to piperacillin-tazobactam, whereas the last two were categorized as resistant following the recommendations of the French Antibiogram Committee (9). These results indicate that the C→G substitution, which occurred in the −35 region of promoter P5, is more efficient in terms of promoter strength than the G→T substitution, which occurred in the −10 region of promoter P4. In fact, promoter P5 possesses the trimer TTG in the −35 region that was considered by Kobayashi et al. (4) to be the most essential sequence for determining promoter strength. However, although the overlapping promoters Pa/Pb possess the trimer TTG in the −35 region of promoter Pa, they were related to a weaker β-lactamase activity than that related to promoter P5. This difference suggests that other nucleotide motifs than TTG in the promoter region might be involved in gene expression.

Concerning the three transformants containing the blaTEM-30gene, they differed from each other with regard to the β-lactamase activity and the β-lactam MICs with the exception of cephalothin. The lowest MICs, regardless of the penicillins tested, were found in the transformant harboring the blaTEM-30B gene preceded by promoter P3, whereas the highest MICs were found in the transformant harboring the blaTEM-30B gene controlled by promoter P4; intermediate MICs were found in the transformant harboring the blaTEM-30 gene preceded by promoters Pa/Pb (Table 1). For cephalothin, all of the transformants were susceptible. For piperacillin-tazobactam, only those transformants with promoters P3 and Pa/Pb upstream from the blaTEM-30B gene were susceptible. For piperacillin, only the transformant with promoter P3 was susceptible. For the transformants with blaTEM-30B, the lowest β-lactamase activity was observed in the transformant harboring the blaTEM-30B gene with promoter P3, whereas the highest activity was observed in the transformant harboring the blaTEM-30B gene with promoter P4.

Overall, the increase in β-lactamase activity observed in relation to the presence of promoters P3, Pa/Pb, and P4 was demonstrated by using either the blaTEM-1B gene coding for TEM-1 or the blaTEM-30B gene coding for TEM-30 or IRT-2. Thus, we were able to compare not only the expression of a given blaTEM gene in relation to different promoters but also the expression of different blaTEM genes in relation to a given promoter. In this study, it has been unambiguously demonstrated that, for any one promoter, the β-lactamase activity measured by using benzylpenicillin as the substrate was higher for TEM-30 or IRT-2 than for TEM-1 from which the IRT enzyme is derived (1). However, despite this higher β-lactamase activity, the MICs of the penicillins tested were lower, with the exception of amoxicillin-clavulanate, for the transformants producing TEM-30 or IRT-2 than for those producing TEM-1 (Table 1). This result emphasizes that MICs are linked to both the type of promoters upstream of the TEM-encoding gene and to the biochemical properties of the TEM enzyme.

In conclusion, although our isogenic system has probably slightly overestimated MICs and β-lactamase activity for all of the transformants, as the vector used (pACYC184) is a low-number but not a single-copy plasmid, it has allowed us to compare the roles that the four promoters that have been described thus far and that are upstream from the blaTEM genes have on β-lactam resistance according to the type of TEM enzyme involved.

FIG. 1.
  • Open in new tab
  • Download powerpoint
FIG. 1.

Nucleotide sequences of promoters P3, Pa/Pb, P4, and P5 of blaTEM genes. The sequences of the −35 and −10 regions of promoters P3, P4, and P5 are boxed by solid lines, whereas these of the overlapping promoters Pa/Pb are boxed by broken lines. Boldface letters indicate the mutations which led to the creation of promoters Pa/Pb, P4, and P5. The numbers under sequences indicate Sutcliffe numbering positions.

View this table:
  • View inline
  • View popup
TABLE 1.

β-Lactam susceptibility and β-lactamase activity of TEM-1- and TEM-30 (IRT-2)-producing E. coli transformants according to the type of promoter upstream from the blaTEM-1B and blaTEM-30B genes

FOOTNOTES

    • Received 23 May 2002.
    • Returned for modification 24 July 2002.
    • Accepted 25 August 2002.
  • American Society for Microbiology

REFERENCES

  1. ↵
    Chaibi, E. B., D. Sirot, G. Paul, and R. Labia. 1999. Inhibitor-resistant TEM β-lactamases: phenotypic, genetic and biochemical characteristics. J. Antimicrob. Chemother. 43:447-458.
    OpenUrlCrossRefPubMedWeb of Science
  2. ↵
    Chen, C. 1984. Two improved promoter sequences for the β-lactamase expression arising from a single base pair substitution. Nucleic Acids Res. 12:3219-3234.
    OpenUrlCrossRefPubMedWeb of Science
  3. ↵
    Goussard, S., and P. Courvalin. 1999. Updated sequence information for TEM beta-lactamase genes. Antimicrob. Agents Chemother. 43:367-370.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Kobayashi, M., K. Nagata, and A. Ishihama. 1990. Promoter selectivity of Escherichia coli RNA polymerase: effect of base substitutions in the promoter −35 region on promoter strength. Nucleic Acids Res. 18:7367-7372.
    OpenUrlCrossRefPubMedWeb of Science
  5. ↵
    Laurent, F., L. Poirel, T. Naas, E. B. Chaibi, R. Labia, P. Boiron, and P. Nordmann. 1999. Biochemical-genetic analysis and distribution of FAR-1, a class A beta-lactamase from Nocardia farcinica. Antimicrob. Agents Chemother. 43:1644-1650.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Leflon-Guibout, V., B. Heym, and M. H. Nicolas-Chanoine. 2000. Updated sequence information and proposed nomenclature for blaTEM genes and their promoters. Antimicrob. Agents Chemother. 44:3232-3234.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Leflon-Guibout, V., V. Speldooren, B. Heym, and M.-H. Nicolas-Chanoine. 2000. Epidemiological survey of amoxicillin-clavulanate resistance and corresponding molecular mechanisms in Escherichia coli isolates in France: new genetic features of blaTEM genes. Antimicrob. Agents Chemother. 44:2709-2714.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Raleigh, E. A., N. E. Murray, H. Revel, R. M. Blumenthal, D. Westaway, A. D. Reith, P. W. Rigby, J. Elhai, and D. Hanahan. 1988. McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning. Nucleic Acids Res. 16:1563-1575.
    OpenUrlCrossRefPubMedWeb of Science
  9. ↵
    Soussy, C. J., G. Carret, J. D. Cavallo, H. Chardon, C. Chidiac, P. Choutet, P. Courvalin, H. Dabernat, H. Drugeon, L. Dubreuil, F. Goldstein, V. Jarlier, R. Leclercq, M. H. Nicolas-Chanoine, A. Philippon, C. Quentin, B. Rouveix, and J. Sirot. 2000. Antibiogram Committee of the French Microbiology Society. Report 2000-2001. Pathol. Biol. 48:832-871.
    OpenUrlPubMed
  10. ↵
    Sutcliffe, J. G. 1978. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc. Natl. Acad. Sci. USA 75:3737-3741.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Promoters P3, Pa/Pb, P4, and P5 Upstream from blaTEM Genes and Their Relationship to β-Lactam Resistance
Marie Frédérique Lartigue, Véronique Leflon-Guibout, Laurent Poirel, Patrice Nordmann, Marie-Hélène Nicolas-Chanoine
Antimicrobial Agents and Chemotherapy Dec 2002, 46 (12) 4035-4037; DOI: 10.1128/AAC.46.12.4035-4037.2002

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Antimicrobial Agents and Chemotherapy article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Promoters P3, Pa/Pb, P4, and P5 Upstream from blaTEM Genes and Their Relationship to β-Lactam Resistance
(Your Name) has forwarded a page to you from Antimicrobial Agents and Chemotherapy
(Your Name) thought you would be interested in this article in Antimicrobial Agents and Chemotherapy.
Share
Promoters P3, Pa/Pb, P4, and P5 Upstream from blaTEM Genes and Their Relationship to β-Lactam Resistance
Marie Frédérique Lartigue, Véronique Leflon-Guibout, Laurent Poirel, Patrice Nordmann, Marie-Hélène Nicolas-Chanoine
Antimicrobial Agents and Chemotherapy Dec 2002, 46 (12) 4035-4037; DOI: 10.1128/AAC.46.12.4035-4037.2002
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

About

  • About AAC
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AACJournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

Copyright © 2019 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0066-4804; Online ISSN: 1098-6596