Surrogate Genetics: The Use of Bacterial Hybrids as a Genetic Tool

Maloy, Stanley; Zahrt, Thomas C.

doi:10.1006/meth.1999.0907

Cited by 7 publications

(4 citation statements)

References 35 publications

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“…These results also suggest the Pullorum biotype is diversifying faster than biotype Gallinarum due to an increased accumulation of point mutations and pseudogenes. Hypermutable strains of pathogenic bacteria have been hypothesized to provide an advantage during host adaptation and colonization (reviewed in [ 84 , 85 ]) and mismatch deficient mutator strains are more susceptible to homeologous recombination (reviewed in [ 86 ]). The processes driving the evolution of these Salmonella serovars will be better understood as more genomes of strains belonging to these serovars are sequenced and analyzed.…”

Section: Discussionmentioning

confidence: 99%

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum

et al. 2015

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The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.

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Section: Discussionmentioning

confidence: 99%

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum

et al. 2015

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“…Surrogate genetics exploits a well-characterized host to study gene function from a related but experimentally lesstractable species (37). The close phylogenetic affiliation between E. coli and H. influenzae (14) makes the former a suitable surrogate for the latter.…”

Section: Discussionmentioning

confidence: 99%

Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

Stewart

Bledsoe

2005

J Bacteriol

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Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions ؊64.5 and ؊44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position ؊60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions ؊81.5 and ؊42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.Facultative aerobes such as Escherichia coli can respire with a variety of terminal electron acceptors, including oxygen, nitrate, dimethyl sulfoxide (DMSO), and fumarate. Synthesis of the corresponding respiratory enzymes is regulated in response to the preferred acceptors, oxygen and nitrate. During anaerobic growth, the Fnr protein (fumarate and nitrate reductases) activates transcription initiation at many operons, including the narGHJI and dmsABC operons encoding the respiratory enzymes cytochrome b-linked nitrate reductase and DMSO reductase, respectively (17,21,59).The Fnr protein is homologous to the well-studied Crp protein (cyclic AMP receptor protein; also known as Cap, catabolite gene activator protein). The Crp and Fnr proteins bind as homodimers to DNA sites of dyad symmetry upstream of regulated promoters, from whence they stimulate transcription initiation. The Crp protein is activated upon binding its allosteric effector, cyclic AMP, whereas the Fnr protein is activated upon assembly of its oxygen-labile iron-sulfur cluster (23, 28).Two types of simple Crp-and Fnr-dependent transcription control regions are defined (6, 8). Class I control regions have a Crp or Fnr binding site located 60 or more nucleotides (nt) upstream of the transcription initiation point (8,65). From these distal locations, Crp and Fnr make specific contacts with the RNA polymerase ␣-subunit carboxyl-term...

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“…typhi and S. typhimurium, allowing potential virulence genes from S. typhi to be directly studied in vivo in a surrogate S. typhimurium host (15). However, certain genes involved in DNA repair and recombination are required for growth or survival of Salmonella in animals.…”

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confidence: 99%

Effect of mutS and recD Mutations on Salmonella Virulence

1999

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Hybrid derivatives of closely related bacteria may be used to dissect strain-specific functions that contribute to virulence within a host. However, mismatches between DNA sequences are a potent barrier to recombination. Recipients with mutS and recDmutations overcome this barrier, allowing construction of genetic hybrids. To determine whether Salmonella hybrids constructed in a mutS recD host can be used to study virulence, we assayed the effect of mutS andrecD mutations on the virulence of Salmonella typhimurium 14028s in mice. Mutants defective in eithermutS or recD do not affect the time course or the 50% lethal dose (LD50) of the infection. In contrast, the inactivation of both mutS and recD results in a synthetic phenotype which substantially increases the time required to cause a lethal infection without changing the LD50. This phenotype results from an inability ofmutS recD double mutants to rapidly adapt to purine-limiting conditions present within macrophages. Although the disease progression is slower, S. typhimurium mutS recDmutants retain the ability to cause lethal infections, and, thus, hybrids constructed in mutS recD hosts may permit the analysis of virulence factors in a surrogate animal model.

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Surrogate Genetics: The Use of Bacterial Hybrids as a Genetic Tool

Cited by 7 publications

References 35 publications

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum

Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

Effect of mutS and recD Mutations on Salmonella Virulence

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