SHV extended-spectrum ␤-lactamases (ESBLs) arise through single amino acid substitutions in the parental enzyme, SHV-1. In order to evaluate the effect of genetic dissimilarities around the structural gene on MICs, we had previously devised an isogenic system of strains. Here, we present an extended version of the system that now allows assessment of all major types of SHV ␤-lactamases as well as of two types of promoters of various strengths. Moreover, we devised a novel vector, pCCR9, to eliminate interference of the selection marker. A substitution within the signal sequence, I8F found in SHV-7, slightly increased MICs, suggesting more efficient transfer of enzyme precursor into the periplasmic space. We also noted that combination of G238S and E240K yielded higher resistance than G238S alone. However, the influence of the additional E240K change was more pronounced with ceftazidime and aztreonam than with cefotaxime and ceftriaxone. The SHV enzymes characterized by the single change, D179N, such as SHV-8, turned out to be the weakest SHV ESBLs. Only resistance to ceftazidime was moderately increased compared to SHV-1. Since 1983 (15, 16), clinical isolates resistant to expandedspectrum cephalosporins have increasingly been reported. They were derived through single amino acid substitutions from one of three parental enzymes, TEM-1, TEM-2, or SHV-1. The resulting structures were designated extendedspectrum ␤-lactamases (ESBLs) (13, 29), and they were classified in a new subgroup, 2be (4). Since the responsible genes are easily transferable due to frequent localization on plasmids (34) the situation has recently been called a "plague of plasmids" (6).Phenotypic differences due to the variably mutated ␤-lactamases were noted early in vitro, and, although ESBL production appears to frequently lead to treatment failure, MICs for ESBL producers may be barely significantly increased compared to those for fully susceptible variants (18). Therefore, it is crucial to be able (i) to detect ESBLs or bla ESBL genes easily and reliably (19) and (ii) to judge the clinical significance of given ESBLs by studying the structure-function relationships of the various ESBLs. The present study is a contribution to the second aim.In order to examine the influence of amino acid substitutions in known SHV ␤-lactamases as well as of various promoter strengths on the level of resistance, we exploited a previously developed system of strains (26) which allows direct phenotypic comparison of such derivatives under isogenic conditions. To some extent, the effect of the plasmid copy number could also be estimated through introduction of a novel lowcopy-number vector system. MATERIALS AND METHODSBacterial strains and plasmids. Escherichia coli DH5␣ (10) was used as a recipient for transformation with cloned bla SHV genes based on a novel plasmid vector, pCCR9 (this study). Relevant information on recombinant strains and plasmids are given in Tables 1 and 2.Antibiotics. Ampicillin was obtained from SmithKline Beecham Pharmaceuticals (Surrey...