Table 1.
Bacterial Strains and Plasmids.
Fig 1.
Y. pestis crp-deficient Mutant 26°C Growth Kinetics.
(A) Growth curves of CO92, CO92Δcrp, and plasmid complemented CO92Δcrp: pBluescript/crp when grown in BCS medium supplemented with 0.2% glucose or 0.2% K-gluconate. Growth of the CO92 crp-deficient mutant when cultured in medium supplemented with 0.2% K-gluconate was significantly impaired relative to all other bacterial strain and media conditions (Tukey’s HSD P < 0.05). (B) Growth curves of KIM6+ and KIM6+Δcrp when cultured in BCS medium supplemented with either 0.2% glucose, 0.2% glycerol, or a combination of both 0.2% glucose and 0.2% glycerol. Growth of the KIM6+ crp-deficient mutant when cultured in medium solely supplemented with 0.2% glycerol was significantly impaired relative to all other bacterial strain and media conditions (Tukey’s HSD P < 0.05). (C) Growth kinetics of the CO92 and KIM6+ crp-deficient mutants and respective isogenic controls when grown in HIB medium at 26°C.
Fig 2.
Y. pestis csrA-deficient Mutant 26°C Growth Kinetics.
(A) Growth of CO92, CO92ΔcsrA, and the chromosomal restoration mutant CO92ΔcsrA csrA’ in BCS medium supplemented with either 0.2% glucose or 0.2% K-gluconate during incubation at 26°C. (B) Growth kinetics of the CO92 and KIM6+ csrA-deficient mutants and respective controls when cultured in HIB medium at 26°C. No significant alteration in growth kinetics as determined by repeated measures ANOVA was calculated among the csrA-deficient mutants and the respective controls, regardless of media type or available carbon source.
Fig 3.
Carbon Catabolite Regulation of Y. pestis Biofilm Production.
Crystal violet biofilm quantification assay of KIM6+ and CO92 when cultured in BCS medium supplemented with primary (glucose) and/or alternate (glycerol or K-gluconate) carbon sources. Error bars reflect standard deviation from the mean derived from two independent experiments, each consisting of 6 technical replicates. * P-value <0.005 determined by Tukey’s HSD post-hoc analysis.
Fig 4.
CRP Enables Robust Y. pestis Biofilm Production.
Relative biofilm production of CO92, CO92Δcrp, and plasmid complemented CO92Δcrp: pBluescript/crp when cultured in HIB medium or BCS medium supplemented with either 0.2% K-gluconate or 0.2% glucose. (A) Following 24 hours post-inoculation. (B) After 72 hours post-inoculation. Error bars reflect standard deviation from the mean derived from two independent experiments, each consisting of 6 technical replicates. * P-value <0.005 determined by Tukey’s HSD post-hoc analysis.
Fig 5.
Y. pestis Biofilm Production is not Influenced by Exogenous cAMP.
(A) Relative biofilm production of KIM6+ and KIM6+Δcrp when grown in BCS medium supplemented with 0.2% glucose containing either 3 mM cAMP prepared in K-phosphate buffer or an equal volume of plain K-phosphate buffer. (B) Relative biofilm production of CO92 and CO92Δcrp when grown in BCS medium supplemented with 0.2% glucose containing either 3 mM cAMP prepared in K-phosphate buffer or an equal volume of plain K-phosphate buffer. Identical source inoculum per strain was utilized for each media type. Error bars reflect standard deviation from the mean derived from two independent experiments, each consisting of 6 technical replicates. No statistically significant change in crystal violet absorption as determined by one-way ANOVA was calculated for both the crp-deficient mutants and the respective parental controls when cultured in media containing 3mM cAMP or an equal volume of K-phosphate buffer.
Fig 6.
Semi-quantitative RT-PCR of the Y. pestis Hms System.
Depiction of semi-quantitative RT-PCR fold changes in expression of the Y. pestis Hms biofilm formation/regulation system following normalization to the gyrB gene. “OM” reflects the bacterial outer membrane; whereas, “IM” indicates the inner membrane. Factors highlighted in green represent characterized pro-biofilm factors. Factors colored in red have been shown to impair biofilm production. I. Fold changes in mRNA abundance amongst CO92 grown in BCS medium supplemented with 0.2% glucose to CO92 cultured in BCS supplemented with 0.2% K-gluconate. II. Fold changes in mRNA abundance of CO92 to CO92Δcrp when grown in HIB medium.
Fig 7.
CsrA is a Positive Regulator of Y. pestis Biofilm Production.
Relative biofilm production of the csrA-deficient mutants after 24 hours inoculation of BCS medium supplemented with 0.2% K-gluconate, HIB medium, or BCS medium supplemented with 0.2% glucose. (A) CO92, CO92ΔcsrA, and the chromosomal restoration mutant CO92ΔcsrA csrA’. (B) KIM6+, KIM6+ΔcsrA, and the chromosomal restoration mutant KIM6+ΔcsrA csrA’. Results reflect the average of two independently derived csrA-deficient mutants (CO92 clones 5a and 3b, and KIM6+ clones 2:14 and 4:12). Two biological replicates were assessed per mutant, each consisting of 6 technical replicates. Error bars reflect standard deviation from the mean among biological replicates. * P-value < 0.005 as determined by Tukey’s HSD post-hoc analysis. (C) Impaired Congo red assimilation of the Y. pestis csrA-deficient mutants. Phenotypic evaluation of Congo red binding of the CO92 and KIM6+ csrA-deficient mutants, chromosomal csrA restoration mutants, parental isogenic controls, and pigmentation locus negative (biofilm-deficient) mutants after 48 hours post-inoculation of Congo red plates supplemented with 0.2% K-gluconate.
Table 2.
Relative Crystal Violet Absorption of the CO92 Glycogen-deficient Mutants.
Fig 8.
CsrA does not alter Y. pestis hmsH, hmsP, and hmsT Transcript Levels.
Relative mRNA abundance of hmsH, hmsP, and hmsT among the csrA-deficient mutants and the respective parental strains calculated via the ΔΔCT method in reference to the gyrB gene. Dashed lines reflect two-fold thresholds for alterations in relative transcript levels. No significant change in relative mRNA abundance among the deletion mutants and the respective controls were determined by one-way ANOVA comparing the average of 2 independent cDNA samples per strain, each consisting of 3 technical replicates per target.
Fig 9.
Deletion of hmsP Enables Excessive Biofilm Production in the csrA-deficient Mutant.
Relative biofilm production of CO92, CO92ΔcsrA, CO92ΔhmsP, and CO92ΔcsrAΔhmsP following 24 hours post-inoculation in HIB medium. Error bars reflect standard deviation from the mean of 2 independent experiments, each consisting of 6 technical replicates. * P-value < 0.005 as determined by Tukey’s HSD post-hoc analysis.