Ampicillin

 Ampicillin (D[–]-a-aminobenzylpenicillin; Fig. 1) is a member of a growing family of antimicrobial agents known as the semisynthetic penicillins. The semisynthetic penicillins are derivatives of the natural product, 6-aminopencillanic acid, that have been deliberately modified chemically (1). The chemical modifications are introduced to create new compounds with specific desirable properties.

In the case of ampicillin, the addition of the aminobenzyl side chain results in a product with an increased resistance to acidic pH. Thus, ampicillin is medically significant because it was the first penicillin to be administered orally in chemotherapeutic practice. Furthermore, ampicillin exhibits a broader antibacterial spectrum than do the naturally occurring penicillins and is effective against many Gram-negative bacterial species. The outer membrane component of the Gram-negative bacterial cell wall represents a permeability barrier to many antibiotics, including the natural penicillins (2). The broad activity spectrum and the relative low cost of ampicillin have made it an invaluable tool in molecular biology and genetics for studying Gram-negative model organisms, such as Escherichia coli and Haemophilus influenzae. Applications that employ ampicillin are summarized below.

Figure 1. Structure of ampicillin.

The first application of penicillin in genetics was for the selection of auxotrophic mutants of E. coli (3, 4). This technique, penicillin selection, was based on the fact that penicillin kills only actively growing bacteria, whereas nongrowing bacteria are penicillin-tolerant (see Penicillin). Although benzylpenicillin (penicillin G) was employed in the original studies describing this technique,

ampicillin would be far more effective for this purpose because of its broad spectrum.

Molecular biologists have extensively exploited genetic elements encoding b-lactamase (see Penicillin-Binding Proteins), especially the enzyme designated TEM-1. TEM-1 is a broad spectrum b-lactamase that confers effective high level resistance to penicillins (5). The gene encoding TEM-1 was incorporated into the first plasmid cloning vectors (6) and has been widely used in cloning vectors since. Ampicillin has been used routinely for the selection and maintenance of bacteria carrying recombinant plasmids encoding b-lactamase, and this is undoubtedly its most common application in molecular biology. For this purpose, ampicillin is incorporated into bacteriological media at final concentrations ranging from about 50–100 µg/mL. For E. coli, these levels are about 10–20 times higher than the minimum inhibitory concentration (MIC) of ampicillin. The MIC is defined as the minimum concentration of the antibiotic that is necessary to inhibit bacterial growth.

 When a mixture of ampicillin-sensitive and ampicillin-resistant bacteria are plated on solid media containing ampicillin, such as for selection of transformants carrying a b-lactamase-encoded plasmid, it is not uncommon to find large colonies formed by ampicillin-resistant bacteria surrounded by a zone of smaller colonies. The ampicillin-resistant bacteria in the large central colonies produce and secrete b-lactamase. The activity of the secreted b-lactamase creates a zone of reduced ampicillin concentration around the resistant colonies, and this permits the ampicillin-sensitive bacteria in the vicinity to grow. The subsequent growth of the ampicillin-sensitive bacteria results in the formation of the smaller so-called satellite colonies. The satellite colonies normally do not represent a major hindrance in these procedures, because the desired ampicillin-resistant bacteria can be readily purified by streaking on an ampicillin-containing medium. However, the problem of satellite colony formation can be minimized by substituting carbenicillin, another semisynthetic penicillin (see Fig. 1 in Penicillin) for ampicillin in the selection medium at a concentration of 50100- µg/mL (7). Carbenicillin is less susceptible to hydrolysis by the b-lactamase and is therefore less likely to promote satellite colony formation. It is therefore often used in place of ampicillin.

The b-lactamase gene has also been introduced into plaque-forming and defective derivatives of the E. coli bacteriophage Mu (8). These ampicillin-selectable phages have been used for mutagenesis and for the construction of lac fusions in applications that take advantage of the ability of Mu to transpose randomly.

 The concept of transposon mutagenesis has been applied to transposable genetic elements encoding b-lactamases for the generation of random gene fusions that are directly selectable with ampicillin (or carbenicillin). For example, a derivative of Tn3 designated Tn3-HoHo1 (9) is a lacZ-containing transposon capable of producing both transcriptional and translational beta-galactosidase fusions. Although it was originally developed for studies on Agrobacterium tumefaciens, it has been adapted for use in other bacterial genera.

b-Lactamase is a periplasmic enzyme. It has served as an important model for studying protein export to the bacterial periplasm. Urbain et al. (10) have recently developed a technique for quantifying b-lactamase activity in cultures of E. coli carrying recombinant plasmids that confer ampicillin resistance. Their assay involved determining the conversion of ampicillin to aminobenzylpenicilloic acid in periplasmic extracts of cells by quantitative high performance liquid chromatography (HPLC). The procedure may be useful for investigating the mechanism of b-lactamase translocation. It may also prove useful in studies on protein expression. For example, since b-lactamase is expressed constitutively from ampicillin-selectable recombinant plasmids, its activity could serve as a useful internal standard in protein coexpression studies.

 b-lactamase has also been used as a genetic tool for studying membrane proteins, and these applications are based on the fact that b-lactamase is an exported protein (11, 12). A plasmid vector that permits the in vitro construction of translational fusions between a gene of interest, encoding either a membrane protein or an exported protein, and the mature form (ie, the exported form) of the TEM b-lactamase has been described (13). Transformants carrying recombinant plasmids with in-frame fusions are ampicillin-selectable. This technique may be used for the analysis of protein export signals or for determining the topological organization of membrane proteins. A strategy for maximizing yields of membrane and exported proteins based on this vector has also been described (14). It is notable that alkaline phosphatase has been used widely for studying protein export signals and for topological mapping of membrane proteins (15). The b-lactamase system is an attractive alternative to alkaline phosphatase for these purposes (13, 14). For example, b-lactamase fusions are directly selectable (with ampicillin), whereas the identification of alkaline phosphatase fusions is based on phenotypic screening. Moreover, only the periplasmic form of alkaline phosphatase is enzymatically active, whereas both the cytoplasmic and periplasmic forms of b-lactamase are active. Consequently, the cellular location of the b-lactamase fusions can be determined on the basis of the levels of ampicillin resistance; cytoplasmic b-lactamase confers ampicillin resistance only at high cell density, whereas periplasmic b-lactamase confers ampicillin resistance at low cell density. As an extension of these studies, Broome-Smith et al. (16) have constructed a transposable b-lactamase element, designated TnblaM, that is equivalent to the transposable alkaline phosphatase element, TnphoA. The b-lactamase fusions constructed with TnblaM are directly selectable with ampicillin, and this is a major advantage over alkaline phosphatase system, which as already noted is based on phenotypic screening.

References

1. J. H. C. Nayler (1991) in 50 Years of Penicillin Application. History and Trends, Public Ltd, Czech Republic, 64–74. 

2. H. Nikaido and M. Vaara (1985) Microbiol. Rev. 49, 1–32. 

3. J. Lederberg and N. Zinder (1948) J. Am. Chem Soc. 70, 467–468. 

4. B. D. Davis (1949) Proc. Natl. Acad. Sci. USA 35, 1–10. 

5. N. Datta and M. D. Richmond (1966) Biochem. J. 98, 204–210. 

6. F. Bolivar, R. L. Rodriguez, M. C. Betlach, and H. W. Boyer (1977) Gene 2, 75–93. 

7. F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, S. D. Seidman, J. A. Smith, and K. Struhl (1994) Current Protocols in Molecular Biology, Wiley, New York, p.1.8.7. 

8. E. A. Groisman (1991) Meth. Enzymol. 204, 180–212. 

9. S. E. Stachel, G. An, C. Flores, and E. W. Nester (1985) EMBO J. 4, 891–898. 

10. J. L. Urbain, C. M. Wittich, and S. R. Campion (1998) Anal. Biochem. 260, 160–165. 

11. J. K. Broome-Smith, M. Tadayyon, and Y. Zhang (1990) 4, 1637–1644. 

12. M. Tadayyon, Y. Zhang, S. Gnaneshan, L. Hunt, F. Mehraein-Ghomi, and J. K. Broome-Smith (1992) Biochem. Soc. Trans. 20, 598–601. 

13. J. K. Broome-Smith and B. G. Spratt (1986) Gene 49, 341–349. 

14. J. K. Broome-Smith, L. D. Bowler, and B. G. Spratt (1989) Mol. Microbiol. 3, 1813–1817. 

15. C. Manoil, J. J. Mekalanos, and J. Beckwith (1989) J. Bacteriol. 172, 515–518. 

16. M. Tadayyon and J. K. Broome-Smith (1992) Gene 111, 21–26.