oniae CpxR very likely binds to DNA, given its sequence homology to other effector domains that have this function. A multiple sequence alignment of the proteins clearly showed that they were highly conserved throughout bacterial kingdom. Overall, in silico analysis suggests that KP1_0078/KP1_0079 is indeed a CpxA/CpxR signaling system worthy to be characterized. Construction and morphological analysis of cpxAR deletion mutant The nucleotide sequence deduced from the 2073 bp DNA fragment obtained from K. pneumoniae NTUH-K2044 shared.75% identity with the CpxA/CpxR regulatory system of Gram CpxAR Confers b-Lactam Resistance negative pathogens. To determine the role of cpxAR, a cpxAR mutant was created by conjugation in the wild-type K. pneumoniae NTUH-K2044. This strain was selected due to its high virulence in a murine model of pneumonia. We used insertionduplication mutagenesis to interrupt cpxAR, required for the synthesis of a functional signaling system. PCR followed by DNA sequencing was done to confirm the disruption of the operon in K. pneumoniae. RT-PCR analysis confirmed that the mutations abolished the transcription of cpxA and cpxR. The cpxAR mutant appeared as smaller colonies compared to wild-type,. The average lengths of strings generated by NTUH-K2044 and NTUH-K2044DcpxAR mutant as per hypermucoviscosity string test were 5.12, 5.08 cm respectively. The precipitation test was carried out on the 12 h grown culture in LB broth at 37uC. Both NTUH-K2044 and NTUHK2044DcpxAR did not form a dense pellet after centrifuging at 10,000 g for 10 min. Visualization of cultures under microscope using 20% CuSO4 by Anthony’s capsule staining methodology revealed no difference in the exopolysaccharide production around NTUH-K2044 and NTUH-K2044DcpxAR. Quantification of glucuronic acid content reconfirmed the same observation. The effect of deleting cpxAR on the colony morphology of K. pneumoniae was JW 55 cost evaluated by scanning electron microscopy. The results indicated no significant difference in cell size of NTUH-K2044 and NTUH-K2044DcpxAR . Role of cpxAR in bacterial growth To decipher the involvement of Cpx signal transduction system in inducing a general or global response, the growth kinetics of CpxAR Confers b-Lactam Resistance DcpxAR strain was compared with that of the wild type strain. Experimentally the growth characteristics of NTUH-K2044 and NTUH-K2044DcpxAR were determined over a period of,10 h in LB medium with different pH and analysis revealed unique patterns. We tested the growth kinetics at pH 3.0, 6.0, 8.0 and 12.0 respectively. It was interesting to note that ” mutant exhibited 1.21.3 fold reduced growth compared to the wild type strain in LB at pH 6.0 . The NTUH-K2044DcpxAR exhibited 5.6 fold stunted growth compared to NTUH-K2044 after 4 h, and displayed a 1.2 fold difference thereafter in LB at pH 8.0 . The differences observed in growth profiles at pH6.0 and pH8.0 were not statistically different. The other tested pH 3.0 and 12.0 was toxic to both the cultures. The ” apparent density of the DcpxAR culture was 1.3 fold lower compared to wild-type parent strain after 8 h. were exposed to different concentrations of bile. The ability of NTUH-K2044 to grow in the presence of 0.75% bile was 2.6 fold, 1% was 3.5 fold and 2% was 3.2 fold 
higher when compared to NTUH-K2044DcpxAR, while transcomplemented NTUH-K2044DcpxARVcpxAR strain restored the ability to tolerate stress . The ability of NTUH-K2044 to grow in the presence of NaCl at
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