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Pleiotropic phenotypes of a Yersinia enterocolitica flhD mutant include reduced lethality in a chicken embryo model.

Townsend MK, Carr NJ, Iyer JG, Horne SM, Gibbs PS, Prüss BM - BMC Microbiol. (2008)

Bottom Line: Compared to the wild-type strain, isogenic flhD and fliA mutants exhibited increased growth on purines and reduced growth on N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source.The flhD mutant caused reduced chicken embryo lethality when compared to wild-type bacteria.Phenotypes of flhD and fliA mutants are related to central metabolism and virulence and correlate with gene regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, USA. megan.townsend@ndsu.edu

ABSTRACT

Background: The Yersinia enterocolitica flagellar master regulator FlhD/FlhC affects the expression levels of non-flagellar genes, including 21 genes that are involved in central metabolism. The sigma factor of the flagellar system, FliA, has a negative effect on the expression levels of seven plasmid-encoded virulence genes in addition to its positive effect on the expression levels of eight of the flagellar operons. This study investigates the phenotypes of flhD and fliA mutants that result from the complex gene regulation.

Results: Phenotypes relating to central metabolism were investigated with Phenotype MicroArrays. Compared to the wild-type strain, isogenic flhD and fliA mutants exhibited increased growth on purines and reduced growth on N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source. Both mutants grew more poorly on pyrimidines and L-histidine as sole nitrogen source. Several intermediates of the tricarboxylic acid and the urea cycle, as well as several dipeptides, provided differential growth conditions for the two mutants. Gene expression was determined for selected genes and correlated with the observed phenotypes. Phenotypes relating to virulence were determined with the chicken embryo lethality assay. The assay that was previously established for Escherichia coli strains was modified for Y. enterocolitica. The flhD mutant caused reduced chicken embryo lethality when compared to wild-type bacteria. In contrast, the fliA mutant caused wild-type lethality. This indicates that the virulence phenotype of the flhD mutant might be due to genes that are regulated by FlhD/FlhC but not FliA, such as those that encode the flagellar type III secretion system.

Conclusion: Phenotypes of flhD and fliA mutants are related to central metabolism and virulence and correlate with gene regulation.

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Related in: MedlinePlus

Lethality of embryos inoculated with Y. enterocolitica strains (flhD and fliA mutants). The ELA was performed with Y. enterocolitica 8081v, as well as with the flhD and fliA mutants in 8081v. Control groups of embryos were either uninoculated or inoculated with PBS. Embryos were inoculated with 108 CFU of the Y. enterocolitica strains. Lethality of the infected embryos was determined for five consecutive days. The experiment was performed three times. Panel A combines the number of dead embryos across the three experiments for each inoculum. Data are expressed as percentage of the total number of embryos for that inoculum. White capital letters in the Y. enterocolitica data bars represent the different groups from the Duncan's multiple group comparison. Panel B shows the bacterial counts in the allantoic fluid for dead (dark bars) and live embryos (light bars). Data are expressed as mean over the three replicate experiments and error bars indicate 1 s.
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Figure 7: Lethality of embryos inoculated with Y. enterocolitica strains (flhD and fliA mutants). The ELA was performed with Y. enterocolitica 8081v, as well as with the flhD and fliA mutants in 8081v. Control groups of embryos were either uninoculated or inoculated with PBS. Embryos were inoculated with 108 CFU of the Y. enterocolitica strains. Lethality of the infected embryos was determined for five consecutive days. The experiment was performed three times. Panel A combines the number of dead embryos across the three experiments for each inoculum. Data are expressed as percentage of the total number of embryos for that inoculum. White capital letters in the Y. enterocolitica data bars represent the different groups from the Duncan's multiple group comparison. Panel B shows the bacterial counts in the allantoic fluid for dead (dark bars) and live embryos (light bars). Data are expressed as mean over the three replicate experiments and error bars indicate 1 s.

Mentions: The pathogenic potential of the Y. enterocolitica 8081v strain and its isogenic flhD and fliA mutants was determined with the ELA. Fig. 7A presents the lethality of the inoculated embryos, calculated over three experiments. This includes the two control groups of embryos (uninoculated and PBS) as a comparison. Lethality of the embryos inoculated with the 8081v strain was highest at 79%, lethality caused by the fliA mutant was similar (76%). The flhD mutant exhibited reduced lethality of 49%. When comparing 8081v to the flhD mutant, the odds ratio resulting from the logistic regression analysis for dead embryos was 0.189. This indicates that the likelihood of death was approximately 5 times higher for embryos that were inoculated with the 8081v strain than with the flhD mutant strain. Comparison of the fliA mutant to 8081v yielded an odds ratio of 0.772, indicating that there was no statistically significant difference between the 8081v strain and the fliA mutant. This result was confirmed with the Duncan's multiple group comparison, which found two significantly different groups. The A group contains the 8081v strain and the fliA mutant, the flhD mutant forms its own group B (Fig. 7A).


Pleiotropic phenotypes of a Yersinia enterocolitica flhD mutant include reduced lethality in a chicken embryo model.

Townsend MK, Carr NJ, Iyer JG, Horne SM, Gibbs PS, Prüss BM - BMC Microbiol. (2008)

Lethality of embryos inoculated with Y. enterocolitica strains (flhD and fliA mutants). The ELA was performed with Y. enterocolitica 8081v, as well as with the flhD and fliA mutants in 8081v. Control groups of embryos were either uninoculated or inoculated with PBS. Embryos were inoculated with 108 CFU of the Y. enterocolitica strains. Lethality of the infected embryos was determined for five consecutive days. The experiment was performed three times. Panel A combines the number of dead embryos across the three experiments for each inoculum. Data are expressed as percentage of the total number of embryos for that inoculum. White capital letters in the Y. enterocolitica data bars represent the different groups from the Duncan's multiple group comparison. Panel B shows the bacterial counts in the allantoic fluid for dead (dark bars) and live embryos (light bars). Data are expressed as mean over the three replicate experiments and error bars indicate 1 s.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262085&req=5

Figure 7: Lethality of embryos inoculated with Y. enterocolitica strains (flhD and fliA mutants). The ELA was performed with Y. enterocolitica 8081v, as well as with the flhD and fliA mutants in 8081v. Control groups of embryos were either uninoculated or inoculated with PBS. Embryos were inoculated with 108 CFU of the Y. enterocolitica strains. Lethality of the infected embryos was determined for five consecutive days. The experiment was performed three times. Panel A combines the number of dead embryos across the three experiments for each inoculum. Data are expressed as percentage of the total number of embryos for that inoculum. White capital letters in the Y. enterocolitica data bars represent the different groups from the Duncan's multiple group comparison. Panel B shows the bacterial counts in the allantoic fluid for dead (dark bars) and live embryos (light bars). Data are expressed as mean over the three replicate experiments and error bars indicate 1 s.
Mentions: The pathogenic potential of the Y. enterocolitica 8081v strain and its isogenic flhD and fliA mutants was determined with the ELA. Fig. 7A presents the lethality of the inoculated embryos, calculated over three experiments. This includes the two control groups of embryos (uninoculated and PBS) as a comparison. Lethality of the embryos inoculated with the 8081v strain was highest at 79%, lethality caused by the fliA mutant was similar (76%). The flhD mutant exhibited reduced lethality of 49%. When comparing 8081v to the flhD mutant, the odds ratio resulting from the logistic regression analysis for dead embryos was 0.189. This indicates that the likelihood of death was approximately 5 times higher for embryos that were inoculated with the 8081v strain than with the flhD mutant strain. Comparison of the fliA mutant to 8081v yielded an odds ratio of 0.772, indicating that there was no statistically significant difference between the 8081v strain and the fliA mutant. This result was confirmed with the Duncan's multiple group comparison, which found two significantly different groups. The A group contains the 8081v strain and the fliA mutant, the flhD mutant forms its own group B (Fig. 7A).

Bottom Line: Compared to the wild-type strain, isogenic flhD and fliA mutants exhibited increased growth on purines and reduced growth on N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source.The flhD mutant caused reduced chicken embryo lethality when compared to wild-type bacteria.Phenotypes of flhD and fliA mutants are related to central metabolism and virulence and correlate with gene regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, USA. megan.townsend@ndsu.edu

ABSTRACT

Background: The Yersinia enterocolitica flagellar master regulator FlhD/FlhC affects the expression levels of non-flagellar genes, including 21 genes that are involved in central metabolism. The sigma factor of the flagellar system, FliA, has a negative effect on the expression levels of seven plasmid-encoded virulence genes in addition to its positive effect on the expression levels of eight of the flagellar operons. This study investigates the phenotypes of flhD and fliA mutants that result from the complex gene regulation.

Results: Phenotypes relating to central metabolism were investigated with Phenotype MicroArrays. Compared to the wild-type strain, isogenic flhD and fliA mutants exhibited increased growth on purines and reduced growth on N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source. Both mutants grew more poorly on pyrimidines and L-histidine as sole nitrogen source. Several intermediates of the tricarboxylic acid and the urea cycle, as well as several dipeptides, provided differential growth conditions for the two mutants. Gene expression was determined for selected genes and correlated with the observed phenotypes. Phenotypes relating to virulence were determined with the chicken embryo lethality assay. The assay that was previously established for Escherichia coli strains was modified for Y. enterocolitica. The flhD mutant caused reduced chicken embryo lethality when compared to wild-type bacteria. In contrast, the fliA mutant caused wild-type lethality. This indicates that the virulence phenotype of the flhD mutant might be due to genes that are regulated by FlhD/FlhC but not FliA, such as those that encode the flagellar type III secretion system.

Conclusion: Phenotypes of flhD and fliA mutants are related to central metabolism and virulence and correlate with gene regulation.

Show MeSH
Related in: MedlinePlus