Comparative analysis of the K. pneumoniae cps gene clusters. In a previous study, K. pneumoniae CPS has been shown to be made from a similar biosynthetic pathway to that of E. coli group 1 CPS (Whitfield and Roberts, 1999). The sequence of K2 cps gene cluster of K. pneumoniae Chedid has been determined, which is composed of 19 open reading frames organized into 3 transcriptional units identified (Arakawa et al., 1995). K. pneumoniae CG43, as well as K. pneumoniae Chedid, produce CPS of K2 serotype (Chang et al., 1996). Comparative analysis of the cps gene cluster of K. pneumoniae Chedid with that of E. coli K30 revealed several conserved genes involved in regulation of high-level polymerization and translocation of CPS, and hence they are called as core elements. These proteins, Wzi, Wza, Yor5, Yco6, Wzx and Wzy, of K. pneumoniae Chedid exhibit striking sequence homologies with the proteins, Wzi, Wza, Wzb, Wzc, Wzx and Wzy, of E. coli K30 (identity and similarity: 97.6% and 98.4%, 91.0% and 95.5%, 53.1% and 64.6%, 66.5% and 78.3%, 14.1% and 31.4%, 15.9% and 30.3%, respectively). Besides, the genes, galF, orf2, gnd and manCB, are also conserved in the two genomes (Fig. 1).
Characterizations of the deletion mutants. In addition to yor5－ and yoc6－ mutants, which have been constructed previously (白平輝, 2004), deletion mutations of wza and wzx were also constructed by using allelic exchange strategy. As shown in Fig. 3 and Fig. 4, Southern blotting analysis using the probes amplified by specific
primer pairs, wzad3 and wzad4 for wza－ mutation and wzxM3 and wzxM4 for wzx－ mutation, demonstrated that the specific deletions have been introduced. As shown in Fig. 3, 1688-bp signal was detected in the wild type strain and 1238-bp signal was detected in the wza－ mutant. Although 2210-bp and 1653-bp signal were detected respectively in wild type strain and wzx－ mutant, additional unexpected band was also observed (Fig. 4). The additional unexpected band was about 1800-bp and might be de to incomplete digestion of chromosome or additional deletion of wzx homolog. In addition, analysis of Western blotting and RT-PCR indicated that Yco6 protein was not detected in the yor5－ mutant and yco6 mRNA was apparently less than wild type strain (data not shown), suggesting a polar effect of the yor5 deletion.
Phenotypes of the mutants. Colony morphology of all four mutants appeared to be smaller compared to that of the wild type strain CG43-S3 and the mucoidy was dramatically reduced as determined by string formation (data not shown). As shown in Fig. 5, all the mutants showed higher growth rates than wild type strain in LB broth.
In the sedimentation test using a galU deletion mutant (U9451) as a negative control, the mutants appeared to precipitate readily, particularly the wza－ mutant (Fig. 6).
Mucoidy of the wza complementing strain appeared to be recovered and the defect in the sedimentation test was also partially restored.
Capsular analysis of the deletion mutants. Since the core elements were assumed to play critical roles in CPS biosynthesis, the defects of these mutants ought to result from modification of CPS biosynthesis. As shown in Fig. 7, the glucuronic acid contents, which serve as indicator of K2 CPS amount (Vodonik and Gray, 1988), of all the mutants were significantly reduced. Furthermore, the CPS purified and analyzed in Fig. 8 showed that all, except the wza－ mutant, lost their polymeric CPS, and in turn increased the corresponding oligomeric CPS. In the periplasmic fraction
of the wza－ mutant, some polymeric CPS were detected indicating that Wza is involved in translocation of polymeric CPS but not in CPS polymerization. Block of the polymeric CPS translocation could feedback-inhibit the upstream CPS biosynthesis and hence resulted in the reduction of total CPS amount. In addition, some polymeric CPS was found in wzx－ mutant suggesting the existence of an alternative system with low efficiency for the translocation of the CPS oligomer. In the yor5－ and yco6－ mutants, disappearance of the high-molecular-weight CPS demonstrated a regulatory role of the tyrosine phosphorylation executed by Yco6 and Yor5 in polymerization of CPS.
Visualization of K. pneumoniae CPS by transmission electron microscopy (TEM). The surface-expressed CPS of each of the mutants was labeled with cationized ferritin (Sigma F-7879) as shown in the electron micrograph (Fig. 9), K.
pneumoniae wild-type strain CG43-S3 appeared to carry an intact thick capsule but the mutants exhibited different levels of unorganized capsules. The disorderly electron-dense materials seen on the surfaces of mutants in micrographs were labeled CPS which might result form dehydration of cells, the treatment for TEM. Notably, the wza－ mutant had the most smooth surface, and the wzx－ mutant appeared to carry more of the disrupted CPS on the surface than the other mutants.
Reduction of CPS affected the bacterial susceptibility to polymyxin B and ability of biofilm formation. According to the above mentioned results, deletions of the genes encoding the core elements indeed affected normal CPS expression, but the influences of these defects on bacterial physiology are still not clear. It has been demonstrated in K. pneumoniae that increasing amount of CPS and upregulated cps transcription were induced by polymyxin B thereby led to the phenotype of polymyxin B resistance (Campos et al., 2004). As shown in Fig. 10, yor5－ and wzx－
mutants appeared to be more sensitive to polymyxin B. Capsular expression has been shown to exert significant effect on K. pneumoniae biofilm formation (Schembri et al., 2005). Increasing capability of biofilm formation was also observed in either the yor5
－ or the wzx－ mutant (Fig. 11).
Part 2: Autophosphorylation of protein-tyrosine kinase Yco6 regulates tyrosine