The periplasmic space in Thermus thermophilus: evidence from a regulation-defective S-layer mutant overexpressing an alkaline phosphatase.
The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed in a mutant (HB8(Delta)UTR1) defective in the regulation of its S-layer (surface crystalline layer). This mutant forms round multicellular bodies (MBs) surrounded by a common envelope as the culture approaches the stationary phase. Confocal microscopy revealed that the origin of the MBs is the progressive detachment of the external layers coupled with the accumulation of NH(2)-containing material between the external envelopes and the peptidoglycan. A specific pattern of proteins was found as soluble components of the intercellular space of the MBs by a single fractionation procedure, suggesting that they are periplasmic-like components. To demonstrate this, we cloned a gene ( phoA) from T. thermophilus HB8 encoding a signal peptide-wearing alkaline phosphatase (AP), and engineered it to be overexpressed in the mutant from a shuttle vector. Most of the AP activity (>80%) was found as a soluble component of the MBs' intercellular fraction. All these data indicate that Thermus thermophilus actually has a periplasmic space which is functionally similar to that of Proteobacteria. The potential application of the HB8(Delta)UTR1 mutant for the overexpression of periplasmic thermophilic proteins is discussed.
Castán
Pablo
P
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain.
Zafra
Olga
O
Moreno
Renata
R
de Pedro
Miguel A
MA
Vallés
Cristina
C
Cava
Felipe
F
Caro
Eddy
E
Schwarz
Heinz
H
Berenguer
José
J
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009154:Mutation; D019897:Periplasm / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure
D017931:DNA Primers; D000469:Alkaline Phosphatase