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Distinct patterns of mutational sensitivity for ? resistance and maltodextrin transport in Escherichia coli LamB.

ABSTRACT: Bacteria can evade cohabiting phages through mutations in phage receptors, but these mutations may come at a cost if they disrupt the receptor's native cellular function. To investigate the relationship between these two conflicting activities, we generated sequence-function maps of Escherichia coli LamB with respect to sensitivity to phage ? and transport of maltodextrin. By comparing 413 missense mutations whose effect on both traits could be analysed, we find that these two phenotypes were correlated, implying that most mutations affect these phenotypes through a common mechanism such as loss of protein stability. However, individual mutations could be found that specifically disrupt ?-sensitivity without affecting maltodextrin transport. We identify and individually assay nine such mutations, whose spatial positions implicate loop L6 of LamB in ? binding. Although missense mutations that lead to ?-resistance are rare, they were approximately as likely to be maltodextrin-utilizing (Mal+) as not (Mal-), implying that E. coli can adapt to ? while conserving the receptor's native function. We propose that in order for E. coli and ? to stably cohabitate, selection for ?-resistance and maltose transport must be spatially or temporally separated.

SUBMITTER: Andrews B

PROVIDER: S-EPMC7276705 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Distinct patterns of mutational sensitivity for λ resistance and maltodextrin transport in Escherichia coli LamB.

Andrews Bryan B Fields Stanley S

Microbial genomics 20200402 4

Bacteria can evade cohabiting phages through mutations in phage receptors, but these mutations may come at a cost if they disrupt the receptor's native cellular function. To investigate the relationship between these two conflicting activities, we generated sequence-function maps of Escherichia coli LamB with respect to sensitivity to phage λ and transport of maltodextrin. By comparing 413 missense mutations whose effect on both traits could be analysed, we find that these two phen ...[more]

PMID: 32238226

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Project description:The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination.

Dataset Information

Distinct patterns of mutational sensitivity for ? resistance and maltodextrin transport in Escherichia coli LamB.

Publications

Distinct patterns of mutational sensitivity for <i>λ</i> resistance and maltodextrin transport in <i>Escherichia coli</i> LamB.

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