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In some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations are a common route to clinically relevant [beta]-lactam antibiotic resistance. We previously constructed an assay system for studying enterobacterial [beta]-lactam resistance mutations using the well-developed genetics of E. coli by integrating enterobacterial ampRC genes into the E. coli chromosome. Like the cells of other enterobacteria, E. coli cells acquire [beta]-lactam resistance by ampD mutation. Here we show that starvation and stress responses provoke ampD [beta]-lactam resistance mutagenesis. When starved on lactose medium, [Lac.sup.-] strains used in mutagenesis studies accumulate ampD [beta]-lactam resistance mutations independent of Lac reversion. DNA double-strand break repair (DSBR) proteins and the SOS and RpoS stress responses are required for this mutagenesis, in agreement with the results obtained for lac reversion in these cells. Surprisingly, the stress-induced ampD mutations require DinB (DNA polymerase IV) and partially require error-prone DNA polymerase V, unlike lac mutagenesis, which requires only DinB. This assay demonstrates that real-world stressors, such as Starvation, can induce clinically relevant resistance mutations. Finally, we used the ampD system to observe the true forward-mutation sequence spectrum of DSBR-associated stress-induced mutagenesis, for which previously only frameshift reversions were studied. We found that base substitutions outnumber frameshift mutations, as seen in other experimental systems showing stress-induced mutagenesis. The important evolutionary implication is that not only loss-of-function mutations but also change-of-function mutations can be generated by this mechanism. doi: 10.1128/JB.00732-09

Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformabUity of DH10B.

The [gamma]-proteobacterium Francisella tularensis is one of the most infectious human pathogens, and the highly virulent organism F. tularensis subsp, tularensis (type A) and less virulent organism F. tularensis subsp. holarctica (type B) are most commonly associated with significant disease in humans and animals. Here we report the complete genome sequence and annotation for a low-passage type B strain (OSU18) isolated from a dead beaver found near Red Rock, Okla., in 1978. A comparison of the F. tularensis subsp, holarctica sequence with that of F. tularensis subsp, tularensis strain Schu4 (P. Larsson et al., Nat. Genet. 37:153-159, 2005) highlighted genetic differences that may underlie different pathogenicity phenotypes and the evolutionary relationship between type A and type B strains. Despite extensive DNA sequence identity, the most significant difference between type A and type B isolates is the striking amount of genomic rearrangement that exists between the strains. All but two rearrangements can be attributed to homologous recombination occurring between two prominent insertion elements, ISFtu1 and ISFtu2. Numerous pseudogenes have been found in the genomes and are likely contributors to the difference in virulence between the strains. In contrast, no rearrangements have been observed between the OSU18 genome and the genome of the type B live vaccine strain (LVS), and only 448 polymorphisms have been found within non-transposase-coding sequences whose homologs are intact in OSU18. Nonconservative differences between the two strains likely include the LVS attenuating mutation(s).

Eight additional random libraries were generated and screened for specificity-altering mutations to study further how mutations can change the substrate specificity of TEM-1 beta-lactamase in gram-negative bacteria. All were found in the C-terminal half of the active site loop. These mutants seem to be less stable than the wild-type enzyme. Further examination of the 165-YYG-167 triple mutant, chosen for high levels of ceftazidime hydrolytic activity, shows the link between enzyme instability and increased ceftazidime hydrolytic activity in the omega loop mutants.

In some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations in the ampD gene are a common route to beta-lactam antibiotic resistance. We constructed an assay system for studying mechanism(s) of enterobacterial ampD mutation using the well-developed genetics of E. coli. We integrated the Enterobacter ampRC genes into the E. coli chromosome. These cells acquire spontaneous recombination- and SOS response-independent beta-lactam resistance mutations in ampD. This chromosomal system is useful for studying mutation mechanisms that promote antibiotic resistance.