CSS motif at the N-terminal region, whereas the C-terminal contains a conserved EAL domain of the PDE enzyme [155]. In addition, the encoding gene yjcC is cluster-located with soxRS genes, suggesting that it plays a role in the oxidative stress response. This study investigates whether YjcC plays a role in oxidative stress defenses and if YjcC uses PDE activity to execute its regulation.
2.3. Results
2.3.1The YjcC expression is paraquat inducible, and SoxRS and RpoS dependent
To confirm the previously reported paraquat-induced expression phenotype [115],
the IVE DNA containing the 5’ non-coding region and part of the coding sequence of
yjcC was isolated from K. pneumoniae CG43S3 and cloned in front of the
promoterless lacZ gene of pLacZ15 [152]. The resulting plasmid was called pPyjcC1. The sequence analysis of PyjcC1 shows a conserved Fnr box TGTGA-N6-TCACA [156]
centered approximately 400-bp upstream of the yjcC start codon. This process also generated recombinant plasmids pPyjcC2 and pPyjcC0 carrying truncated forms of PyjcC1. These plasmids respectively removed the putative Fnr box and the small stem-loop sequence of the 33-bp coding region shown in Fig.2.1 (A). As Fig. 2.1 (B) shows, the bacteria containing pPyjcC1 exhibited the highest level of
-galactosidase activity,
whereas CG43S3[pPyjcC2] had the lowest activity. In addition, the activity of PyjcC1, but24
not PyjcC2 nor PyjcC0, increased after adding 10 μM of paraquat to the culture medium.
This paraquat-induced characteristic also appeared when the concentration increased to 30 μM, further enhancing the activity of PyjcC1.
As Fig. 2.1 (C) shows, the addition 30 μM paraquat to the bacterial culture significantly increased the yjcC mRNA level. Compared to the expression of the well-characterized stress response regulators SoxS, SoxR, RpoS, and Fnr, the yjcC gene expression was more responsive to paraquat than to hydrogen peroxide exposure.
This study also investigates whether yjcC is subjected to regulation by SoxRS or RpoS. As Fig.2.1 (D) shows, the deletion of soxR, soxS, or rpoS reduces the yjcC expression, implying that SoxRS and RpoS play a positive role in yjcC expression.
2.3.2 YjcC plays a positive role in the oxidative stress response
Paraquat is a superoxide anion generator. Thus, the paraquat-inducible expression suggests that YjcC plays a role in the oxidative stress response. To investigate this possibility, an yjcC deletion mutant was generated through an allelic exchange strategy. As Fig. 2.2 (A) shows, the yjcC deletion mutant was more sensitive to paraquat and hydrogen peroxide when compared to the wild type bacteria K.
pneumoniae CG43S3. The deletion effect could be complemented by transforming the
yjcC expression plasmid pJR1 into the mutant. However, introducing the mutant pJR2,
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which expresses the mutant form of YjcC with the conserved E residue of the EAL domain replaced by A or pJR3 (which carries the coding region of the YjcC EAL domain), had no complementation effect. Neither of the two EAL-domain protein encoding plasmids pmrkJ and pfimK, which carry PDE activity, could complement the yjcC deletion effect. These results suggest that the stress response is YjcC dependent
and both the N-terminal signaling receiving region and the EAL domain of YjcC are required and specific for an oxidative stress response.
To determine if the YjcC-EAL domain exhibits PDE activity, the recombinant expression plasmid containing the DNA coding for the EAL domain of YjcC or the AAL coding region of pJR2 was constructed and overexpresed in E. coli, and the recombinant proteins were purified. Figure 2.2 (B) shows that the purified EAL domain protein exhibits PDE activity towards pNpp. This activity is lower than the level of the recombinant MrkJ [157], but considerably higher than the activity of the
recombinant protein AALyjcC. As Fig.2.2 (C) shows, the c-di-GMP level of CG43S3ΔyjcC[pJR1] was significantly lower than those of CG43S3[pRK415], CG43S3ΔyjcC[pJR2], or CG43S3ΔyjcC[pJR3]. This suggests that YjcC in vivo
functions as a PDE enzyme capable of reducing the intracellular c-di-GMP levels. The deletion of yjcC gene from CG43S3 increased the c-di-GMP amounts and the difference between the levels was much more apparent after the bacteria exposure to
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30 µM paraquat Fig. 2.2(D). This also suggests that YjcC is able to degrade c-di-GMP and the catalytic activity could be enhanced by oxidative stress.
2.3.3 Deletion of yjcC places bacteria in an oxidative stress state
As Figs. 2.3 (A) and (B) show, the deletion of yjcC after treatment of H2O2 or paraquat significantly raised the levels of the fluorescent probe H2DCFDA (used to
monitor the formation of ROS) and carbonyl proteins. The introduction of pJR1 into CG43S3ΔyjcC mutant appeared to reduce the levels of ROS and the carbonyl proteins,
showing that YjcC is involved in the removal of ROS or damaged molecules. Thus, this study also investigates the anti-oxidant activity of YjcC. As Fig. 3C shows, the deletion of yjcC reduced the oxidant scavenging activity, as assessed by the absorbance change at 517 nm for the decolorization degree of the purple color, supporting the possibility that YjcC modulates anti-oxidant activity in a certain manner. Numerous studies have shown that Fur and RpoS affect and regulate numerous SODs and catalases [139,158-161]. Figure 2.3 (D) shows that zymogel analysis and total activity measurement exhibit significant changes in the SOD or catalase activity after the deletion of fur or rpoS. However, the deletion of yjcC has no apparent influence on SOD or catalase activity, suggesting that the YjcC-dependent anti-oxidant enzyme remains to be identified.
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2.3.4 YjcC plays a regulatory role in the virulence, CPS production, biofilm
formation, and type 3 fimbriae expression
YjcC, previously identified as an IVE gene product, is likely involved in infection [115]. To investigate whether YjcC is a virulence factor for the bacteria to establish infection, a mouse peritonitis model was employed. As Table 1 shows, the LD50 to Balb/c mice increased approximately 10-fold after yjcC deletion; introducing ΔyjcC with pJR1, but not pJR2 or pJR3, could restore the LD50. This indicates that YjcC expression at a certain stage is required for mouse infection. It is interesting to note that the ΔyjcC colony is smaller and less mucoid, as determined by a string test [39], than its parental strain on LB agar plate. Therefore, sedimentation analysis and glucuronic acid content measurement are carried out to determine the CPS production.
As Fig. 2.4 (A) shows the deletion of yjcC reduces CPS production. The CPS
deficient phenotype can be fully complemented with the transformation of pJR1 into the ΔyjcC mutant. However, transforming the mutant with pJR2 or pJR3 partially
restores glucuronic acid production.
The second messenger c-di-GMP plays an important role in bacterial biofilm formation [14-16]. Figure 2.4 (B) shows that the biofilm formation activity of ΔyjcC appears to increase compared to the parental strain, whereas that transformed with pJR1 decreases biofilm formation. This can be attributed to the level changes of
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c-di-GMP (Fig. 2.2 C), which indicates that the YjcC expression of pJR1 significantly reduces the c-di-GMP level, thus reducing biofilm forming activity. Moreover, type 3 fimbriae is a major determinant of biofilm formation in K. pneumoniae [64].
Therefore, this study also investigates the deletion effect on type 3 fimbriae expression. As Fig. 2.4 (C) shows, the western blot hybridization with anti-MrkA antibody shows that the yjcC deletion also increased the major pilin MrkA production of type 3 fimbriae.
2.3.5 Effects of YjcC overexpression assessed using a transcriptome study
This study uses comparative transcriptome analysis between CG43S3[pRK415] and CG43S3[pJR1] to gain further insights into how YjcC executes its regulation.
Analysis of the genome annotation of liver abscess isolate K. pneumoniae NTHU-K2044 [112] shows that the increased expression of yjcC significantly enhances the expression of 34 genes. As Table 2.2 shows, the YjcC-activated genes can be categorized into 12 functional groups. These include the oxidative stress response genes grxA, ybbN, dinI, priB, and stpA, which are involved in anti-oxidation [162,163] or DNA repair [163], the heat shock chaperone protein encoding genes ibpB, ibpA, htpG, and dnaK, which are generally induced in stress conditions [164],
and the genes coding for chaperone ClpB and PspB to protect protein from
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aggregation and help maintain proton motive force (PMF) to counteract stress conditions [165,166]. Increasing the expression of yjcC also enhanced the expression of PilZ domain protein MrkH and the LuxR-type transcription factor MrkI.
Conversely, 29 genes whose expressions were significantly repressed by the increase of YjcC expression include fumB, which is regulated by fnr under limited oxygen and anaerobic conditions [124]. Other YjcC negatively affected genes are metabolite transporter genes and genes coding for permease and energy metabolism involved in the synthesis of amino acids (Table 2.3).
As assessed by qRT-PCR analysis, Fig. 2.5 shows that the mRNA level of mrkH and mrkI, respectively increased 2.87- and 3.24-fold in CG43S3[pJR1] compared to that of CG43S3[pRK415]. In contrast, the mrkA transcript levels dropped to approximately 1/3 of that of CG43S3[pRK415].