Penicillin-binding protein 2x of Streptococcus pneumoniae: the mutation Ala707Asp within the C-terminal PASTA2 domain leads to destabilization.
Streptococcus pneumoniae penicillin-binding protein 2x (PBP2x) is an enzyme involved in the last stages of peptidoglycan assembly and essential for bacterial growth and survival. PBP2x localizes to the division site, a process that depends on its Penicillin-Binding Protein And Serine-Threonine-kinase Associated (PASTA) domains, which was previously demonstrated via GFP-PBP2x in living cells. During this study a mutant strain was isolated in which the GFP-PBP2x fusion protein did not localize at division sites and it contained reduced amounts of the full-length GFP-PBP2x. We now show that this defect is due to a point mutation within the C-terminal PASTA2 domain of PBP2x. The mutant protein was analyzed in detail in terms of beta-lactam binding, functionality, and localization in live cells. We demonstrate that the mutation affects the GFP-tagged PBP2x variant severely and renders it susceptible to the protease/chaperone HtrA.
Department of Microbiology, University of Kaiserslautern , Kaiserslautern, Germany .
Inga
I
Schweizer
Katharina
K
Peters
Christoph
C
Stahlmann
Regine
R
Hakenbeck
Dalia
D
Denapaite
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010457:Peptidoglycan / Q000096:biosynthesis; D033903:Periplasmic Proteins / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D047090:beta-Lactams / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D047090:beta-Lactams; C072223:PBP 2x protein, Streptococcus; D049452:Green Fluorescent Proteins; C061682:DegP protease; D012697:Serine Endopeptidases