Genes encoding proteins of unknown function 
Several membrane proteins such as 05SSU0677 and 05SSU1912 were also repressed in DeltasalKR.
It is well documented that surface proteins or cell wall-anchored proteins are usually thought to contribute to the immunogenicity [33], [34], invasiveness [35]-[37], and even pathogenesis [38], [39] of pathogens.
Moreover, it should be noted that numerous proteins of unknown function are also depressed in DeltasalKR, which may in part lead to the virulence loss of the mutant via some thus-far-unknown mechanisms.
Remarkably, no gene was found to be up-regulated (more than twice) in DeltasalKR.
To examine the validity of the microarray data, we performed further quantitative real-time PCR analysis on 42 genes using RNA from three independent cultures of each strain (WT, the DeltasalKR mutant and the complemented strain CDeltasalKR).
These genes include the total 26 ones that are down-regulated in the DeltasalKR mutant, four virulence factors (CPS, MRP, EF, SLY) coding genes [40]-[44], genes immediately flanking the salKR locus (05SSU0945 and 05SSU0942), and 10 genes that selected from the chromosome of 05ZYH33 every 200 ORFs.
As shown in Table S1, the quantitative results of RT-PCR were highly compatible to the microarray data, in the DeltasalKR mutant, 41 out of 42 selected genes (97.6%) were confirmed to have similar transcript levels using these two different techniques, thus independently confirming the microarray results.
The discrepancies of target genes expression between microarray data and real-time quantitative PCR results probably best reflects differences in the relative sensitivity and specificity of the two methods.
Among the 26 down-regulated genes in the DeltasalKR mutant, five did not recover to expression level of the wild-type strain, indicating that the complemented strain does not imitate completely the exact tuning of gene expression as in the wild-type strain.
