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1.
Fig 7

Fig 7. From: Study of the Response Regulator Rrp1 Reveals Its Regulatory Role in Chitobiose Utilization and Virulence of Borrelia burgdorferi.

Rrp1 regulates RpoS expression through BosR. (a) Detection of BosR, Pta, and Rrp2 via immunoblot analyses was performed as described for . (b) qRT-PCR analysis of bosR, rpoN, rpoS, and eno transcripts was performed as described for . Data are expressed as expression of the rrp1mut transcripts relative to that of the corresponding wild-type transcripts. Asterisks indicate that the differences between WT and rrp1mut transcript levels were statistically significant at a P value of <0.01 (two-way ANOVA).

Ching Wooen Sze, et al. Infect Immun. 2013 May;81(5):1775-1787.
2.
Fig 5

Fig 5. From: Study of the Response Regulator Rrp1 Reveals Its Regulatory Role in Chitobiose Utilization and Virulence of Borrelia burgdorferi.

Detecting N-acetylglucosamine (GlcNAc) in B. burgdorferi culture media and cells. The intracellular level of GlcNAc and the level of GlcNAc in culture media were measured using LC-MS/MS. (a) The intracellular concentration of GlcNAc was determined at various stages of the growth curve for BSK-II(−GlcNAc)+chitobiose medium as indicated and at the late stationary phase for cultures in BSK-II medium. Samples were measured in triplicate, and data are represented as means ± SEM in μM/108 cells. (b) Concentration of GlcNAc in normal BSK-II, GlcNAc-depleted BSK-II BSK-II(−GlcNAc) and BSK-II(−GlcNAc)+chitobiose. Asterisks indicate no GlcNAc peak detected.

Ching Wooen Sze, et al. Infect Immun. 2013 May;81(5):1775-1787.
3.
Fig 6

Fig 6. From: Study of the Response Regulator Rrp1 Reveals Its Regulatory Role in Chitobiose Utilization and Virulence of Borrelia burgdorferi.

The rrp1mut mutant has decreased RpoS expression. A total of 105 cells/ml of the WT, rrp1mut, and rrp1com strains were inoculated into BSK-II and cultivated at 34°C and pH 7.6. (a) Cells were harvested after the stationary phase (108 cells/ml) at the indicated time points (D1 to D3). Similar amounts of whole-cell lysates were analyzed by SDS-PAGE. (b) Detection of RpoS. (c) Detection of OspA, OspC, DbpA, and DbpB. DnaK was used as an internal control as previously described (). W = WT strain, M = rrp1mut mutant, C = rrp1com strain.

Ching Wooen Sze, et al. Infect Immun. 2013 May;81(5):1775-1787.
4.
Fig 2

Fig 2. From: Study of the Response Regulator Rrp1 Reveals Its Regulatory Role in Chitobiose Utilization and Virulence of Borrelia burgdorferi.

The growth curves of WT, rrp1mut, and rrp1com strains. Growth curves were measured under the following conditions: (a) cells were grown in BSK-II medium at 23°C and pH 7.6; (b) cells were grown in BSK-II(−GlcNAc)+chitobiose medium at 23°C and pH 7.6; (c) cells were grown in BSK-II medium at 34°C and pH 7.6; and (d) cells were grown in BSK-II(−GlcNAc)+chitobiose medium at 34°C and pH 7.6. Asterisks indicate that the difference in cell densities between the rrp1mut mutant and the WT was statistically significant at a P value of <0.001. Cell counting was repeated in triplicate with at least two independent samples, and the results are expressed as means ± SEM.

Ching Wooen Sze, et al. Infect Immun. 2013 May;81(5):1775-1787.
5.
Fig 3

Fig 3. From: Study of the Response Regulator Rrp1 Reveals Its Regulatory Role in Chitobiose Utilization and Virulence of Borrelia burgdorferi.

The rrp1mut mutant forms membrane blebs in chitobiose-supplemented medium. (a) Dark-field microscopic analysis of B. burgdorferi strains grown in BSK-II(−GlcNAc)+chitobiose medium at 34°C and pH 7.6. The cultures were collected at day 4 and day 6 and B. burgdorferi cells observed under dark-field illumination at ×200 magnification using a Zeiss Axiostar Plus microscope. Scale bars represent 10 μm. (b) Scanning electron microscope analysis of the rrp1mut membrane bleb formation. A total of 105 cells/ml of the rrp1mut mutant were inoculated into 5 ml BSK-II(−GlcNAc)+chitobiose medium, and cells were collected for scanning electron microscope analysis at the different time points indicated to observe the formation of membrane blebs. Arrowheads point to membrane blebs observed.

Ching Wooen Sze, et al. Infect Immun. 2013 May;81(5):1775-1787.
6.
Fig 1

Fig 1. From: Study of the Response Regulator Rrp1 Reveals Its Regulatory Role in Chitobiose Utilization and Virulence of Borrelia burgdorferi.

Construction of the rrp1mut mutant and its complemented rrp1com strain. (a) To construct the rrp1::kan plasmid, the entire ORF of bb0419 (rrp1) was PCR amplified. A kan cassette was inserted into the XbaI and BalI cut sites that are present within the rrp1 ORF, resulting in a 151-bp deletion, generating the rrp1::kan plasmid. (b) Pbb0420, 218 bp from the upstream region of bb0420 that contains the native promoter of the bb0419-bb0420 operon, was PCR amplified and fused to the 5′ end of bb0419. The obtained fragment was then inserted into the pCisCom construct at the BamHI cut site, yielding Rrp1/cis com. (c) Immunoblot analysis of the whole-cell lysate of the wild-type (WT), rrp1mut, and rrp1com strains probed with a specific antibody against Rrp1. DnaK was used as an internal control, as previously described (). α, anti-.

Ching Wooen Sze, et al. Infect Immun. 2013 May;81(5):1775-1787.
7.
Fig 4

Fig 4. From: Study of the Response Regulator Rrp1 Reveals Its Regulatory Role in Chitobiose Utilization and Virulence of Borrelia burgdorferi.

Constitutive expression of chbC restored the growth defects of the rrp1mut mutant. (a) The rrp1 mutant has decreased chbC transcript. The WT rrp1mut, and rrp1com strains were cultured at 34°C and pH 7.6 and harvested at the early stationary phase (∼108 cells/ml). Total RNA was extracted for qRT-PCR. Data are expressed as expression of the rrp1mut or rrp1com transcripts relative to the wild-type level. Asterisks indicate that the difference between WT and rrp1mut transcript levels was statistically significant at a P value of <0.01 (two-way ANOVA). (b) Constitutive expression of chbC rescues the growth defects of the rrp1mut mutant. Growth curve experiments similar to those described for were repeated using BSK-II and BSK-II(−GlcNAc)+chitobiose media at 34°C and pH 7.6. Cell counting was repeated in triplicate with at least two independent samples, and the results are expressed as means ± SEM. Asterisks indicate that the difference between the rrp1mut and rrp1mut chbC+ strains in cell densities was statistically significant at a P value of <0.001.

Ching Wooen Sze, et al. Infect Immun. 2013 May;81(5):1775-1787.
8.
Fig 9

Fig 9. From: Study of the Response Regulator Rrp1 Reveals Its Regulatory Role in Chitobiose Utilization and Virulence of Borrelia burgdorferi.

Model for Rrp1 regulation of chitobiose metabolism in B. burgdorferi. During the feeding process, certain host factors or chitobiose or both serve as a ligand(s) to activate the histidine kinase, Hk1, leading to phosphorylation of the response regulator, Rrp1. Phosphorylated Rrp1 activates BosR (the oxidative stress regulator) and RpoN, which then activate the transcription of rpoS. The expression of RpoS is further enhanced via the signaling by Rrp2-RpoN as well as other regulators such as DsrABb. An increase in the level of RpoS leads to repression of glycerol metabolism genes and activation of the components of the chitobiose metabolic pathway, especially the chitobiose transporter gene, chbC. Expression of the chitobiose utilization pathway allows B. burgdorferi to utilize chitobiose shed from the peritrophic membrane which is formed after a blood meal as a source for GlcNAc, a precursor for cell wall synthesis. A switch from glycerol to chitobiose for utilization as the main carbon source allows the spirochete to multiply in numbers and completes the transmission cycle.

Ching Wooen Sze, et al. Infect Immun. 2013 May;81(5):1775-1787.
9.
Fig 8

Fig 8. From: Study of the Response Regulator Rrp1 Reveals Its Regulatory Role in Chitobiose Utilization and Virulence of Borrelia burgdorferi.

Supplementation of GlcNAc fails to rescue the rrp1mut bacterial load in fed ticks but enables transmission via tick bite. (a) RNA samples were extracted from whole fed ticks (after repletion; 5 to 7 days) and subjected to qRT-PCR analysis. The bacterial burdens in ticks were measured by the number of copies of flaB mRNA compared to the number of copies of tick-actin transcript as previously described (). The data are presented as the means of relative levels of flaB transcript ± SEM for three groups (each group contained 4 replete ticks) for each strain (WT, rrp1mut mutant, and rrp1com strain). Asterisks indicate that the differences in bacterial load between the WT strain, the rrp1mut mutant, and the rrp1mut mutant supplemented with GlcNAc were statistically significant at a P value of <0.05. (b) Detection of spirochete burdens in C3H mice infected via tick bite using qRT-PCR. At day 14 after tick feeding, mice were sacrificed. Tissues (skin, heart, joint, and bladder) were collected and total RNA from heart and bladder tissues subjected to qRT-PCR analysis as described in Materials and Methods. Statistical analysis showed no significant difference between the WT, the rrp1com strain, and the rrp1mut mutant supplemented with GlcNAc at P = 0.05.

Ching Wooen Sze, et al. Infect Immun. 2013 May;81(5):1775-1787.

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