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Tables
- Table 1.
Oligonucleotides used in site-directed mutagenesis experiments and in DNA sequencing
Procedure and oligonucleotide Sequence (5′-3′)a Codon change and nucleotide positionb Mutagenesis Ile86→Val GGCGTCAACACGGGATAAT ATT→GTT Val184→Ala ATTGCGGCAGGCATCGTGG GTA→GCC Met69→Leu TAAAAGTGCTCAGCATTGGAAAAC ATG→CTG Asn276→Asp TCTGTCTATCTCGTTCATCC AAT→GAT Arg244→Cys ATGATACCGCAAGACCC CGC→TGC Glu104→Lys GTGAGTATTTAACCAAGTC GAG→AAA Gly238→Ser CACGCTCACTGGCTCCAGATTTAT GGT→AGT Arg164→Ser GTTCCCAAGAATCAAGGC CGT→TCT Sequencing f-TEM2F GTATGAGTATTCAACATTTCCGTGTCG 205-231 f-TEM2R ACCAATGCTTAATCAGTGAGGCA 1064-1042 f-TEMi ACTGTCATGCCATCCGTAAGA 556-536 f-TEM2i CTGCGGCCAACTTACTTCTGACAA 598-621 - Table 2.
MICs of β-lactams and β-lactamase inhibitor combinations for E. coli MV1190 producing the TEM-1 and mutant TEM β-lactamases
Strain or amino acid changes from TEM-1a Designation MIC (μg/ml)b AMX AMX + CLAc TIC TIC + CLAc PIP PIP + TZBd CLD CTX CAZ CAZ + CLAc FEP AZM TEM TEM-1e TEM-1 >1,024 256 >1,024 256 128 32 64 0.06 0.5 0.25 0.12 0.25 4 M69L TEM-33 >1,024 >1,024 >1,024 1,024 128 64 16 0.06 0.5 0.25 ≤0.06 0.25 4 M69L, N276D TEM-35 >1,024 >1,024 1,024 512 128 32 16 0.06 0.25 0.25 ≤0.06 0.12 4 R244C TEM-31 1,024 512 64 64 8 4 2 0.06 0.25 0.12 ≤0.06 0.12 4 G238S TEM-19 >1,024 16 1,024 16 32 ≤1 32 1 0.5 0.25 0.12 0.5 4 G238S, M69L >1,024 32 512 16 32 ≤1 32 0.25 0.25 0.25 0.12 0.25 8 G238S, M69L, N276D >1,024 128 512 32 32 ≤1 32 0.12 0.25 0.25 0.25 0.12 4 G238S, R244C 1,024 128 64 16 8 2 4 0.06 0.25 0.25 ≤0.06 0.12 4 G238S, E104K TEM-15 >1,024 16 1,024 32 32 ≤1 32 8 8 0.25 0.25 4 8 G238S, E104K, M69L >1,024 32 1,024 64 32 ≤1 32 4 4 0.25 0.25 1 4 G238S, E104K, M69L, N276D TEM-50 >1,024 128 1,024 128 32 ≤1 32 4 4 0.5 0.5 2 4 G238S, E104K, R244C 1,024 64 256 64 8 ≤1 4 0.06 0.25 0.25 0.25 1 4 R164S TEM-12 >1,024 32 1,024 32 64 2 32 0.12 8 0.25 1 1 8 R164S, M69L >1,024 256 512 64 32 ≤1 8 0.12 8 0.25 0.5 1 4 R164S, M69L, N276D >1,024 1,024 512 128 32 2 8 0.06 8 2 0.5 0.5 4 R164S, R244C 1,024 256 16 8 4 2 2 0.06 0.25 0.25 ≤0.06 0.12 4 R164S, E104K TEM-26 >1,024 16 >1,024 32 64 ≤1 8 1 64 0.5 1 16 8 R164S, E104K, M69L >1,024 64 1,024 64 64 2 8 0.5 64 2 1 16 4 R164S, E104K, M69L, N276D >1,024 256 512 128 32 2 8 0.12 64 8 1 4 4 R164S, E104K, R244C 512 64 128 32 32 8 2 0.06 4 1 0.25 1 8 E. coli MV1190 (recipient strain) 8 4 2 2 ≤1 ≤1 2 0.06 0.12 0.12 ≤0.06 0.12 4 ↵a M69L refers to an amino acid substitution of leucine for methionine at position 69 in the TEM-1 β-lactamase protein sequence. The single amino acid codes for the other substitutions are as follows: N, asparagine; D, aspartate; G, glycine; R, arginine; C, cysteine; S, serine; E, glutamate; and K, lysine.
↵b AMX, amoxicillin; CLA, clavulanate; TIC, ticarcillin; PIP, piperacillin; TZB, tazobactam; CLD, cephaloridine; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; AZM, aztreonam; TEM, temocillin.
↵c Fixed concentration of clavulanate (2 μg/ml).
↵d Fixed concentration of tazobactam (4 μg/ml).
↵e TEM with amino acid modifications engineered to be identical to natural TEM-1.
- Table 3.
β-Lactamase activities and clavulanate and tazobactam IC50s for TEM-1 β-lactamase and TEM mutant enzymes
Amino acid change from TEM-1a Designation β-Lactamase activityb IC50 (μM) Clavulanate Tazobactam TEM-1 TEM-1 5,330 0.08 0.03 M69L TEM-33 586 2 0.5 M69L, N276D TEM-35 1,190 12 0.6 R244C TEM-31 514 10 1.7 G238S TEM-19 2,950 0.001 0.002 G238S, M69L 853 0.03 0.02 G238S, M69L, N276D 2,360 0.38 0.04 G238S, R244C 485 1.5 0.3 G238S, E104K TEM-15 29 0.002 0.004 G238S, E104K, M69L 319 0.07 0.01 G238S, E104K, M69L, N276D TEM-50 952 0.3 0.02 G238S, E104K, R244C 696 1.1 1.2 R164S TEM-12 25 0.02 0.02 R164S, M69L 49 0.25 0.2 R164S, M69L, N276D 1,440 1.8 0.3 R164S, R244C 937 4.5 5 R164S, E104K TEM-26 523 <0.01 0.06 R164S, E104K, M69L 211 0.08 0.3 R164S, E104K, M69L, N276D 458 0.35 0.2 R164S, E104K, R244C 54 3.5 2
