New Insights into Beta-Lactam Resistance of Streptococcus pneumoniae: Serine Protease HtrA Degrades Altered Penicillin-Binding Protein 2x

Reduced amounts of the essential penicillin-binding protein 2x (PBP2x) were detected in two cefotaxime-resistant Streptococcus pneumoniae laboratory mutants C405 and C606. These mutants contain two or four mutations in the penicillin-binding domain of PBP2x, respectively. The transcription of the pbp2x gene was not affected in both mutants; thus, the reduced PBP2x amounts were likely due to post-transcriptional regulation. The mutants carry a mutation in the histidine protein kinase gene ciaH, resulting in enhanced gene expression mediated by the cognate response regulator CiaR. Deletion of htrA, encoding a serine protease regulated by CiaR, or inactivation of HtrA proteolytic activity showed that HtrA is indeed responsible for PBP2x degradation in both mutants, and that this affects β-lactam resistance. Depletion of the PBP2xC405 in different genetic backgrounds confirmed that HtrA degrades PBP2xC405. A GFP-PBP2xC405 fusion protein still localized at the septum in the absence of HtrA. The complementation studies in HtrA deletion strains showed that HtrA can be overexpressed in pneumococcal cells to specific levels, depending on the genetic background. Quantitative Western blotting revealed that the PBP2x amount in C405 strain was less than 20% compared to parental strain, suggesting that PBP2x is an abundant protein in S. pneumoniae R6 strain.

Serine–Arginine Protein Kinase-like Protein, SrpkF, Stimulates Both Cellobiose-responsive and D-Xylose-Responsive Signaling Pathways in Aspergillus Aculeatus

To elucidate the regulatory mechanisms of various cellulolytic enzyme genes in Aspergillus aculeatus , we identified one mutant that reduced the expression of FIII-avicelase ( chbI ) in response to cellulose from 12,000 A . aculeatus T-DNA-inserted mutants. The T-DNA inserted into a putative protein kinase gene similar to AN10082 in A . nidulans , the serine–arginine protein kinase F, SrpkF. The fold increase in srpkF gene expression in response to various carbon sources was 2.3 (D-xylose), 44 (Avicel®), 59 (Bacto™ Tryptone), and 98 (no carbon) compared with D-glucose. The deletion of srpkF in A . aculeatus resulted in a significant reduction in the cellulose-responsive expression of chbI , hydrocellulase ( cel7b ), and FIb-xylanase ( xynIb ) genes at an early induction phase. However, the srpkF deletion did not affect the expression of xynIb in response to D-xylose. Furthermore, the srpkF -overexpressing strain that expresses the srpkF gene at levels from four- to nine-fold higher than the control strain stimulated the expression of cbhI and cel7b in response to cellobiose and the FI-carboxymethyl cellulase gene ( cmc1 ) and xynIb in response to xylose. The expression of cbhI and cel7b is regulated by a transcriptional activator, ManR, and the expression of cmc1 and xynIb is regulated by XlnR. Our data demonstrate that SrpkF can stimulate both the ManR- and XlnR-dependent signaling pathways in response to cellobiose and D-xylose in A . aculeatus .