Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt.
In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.
Velasco-García
Roberto
R
Laboratorio de Osmorregulación, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, México, Mexico.
Villalobos
Miguel Angel
MA
Ramírez-Romero
Miguel A
MA
Mújica-Jiménez
Carlos
C
Iturriaga
Gabriel
G
Muñoz-Clares
Rosario A
RA
D001483:Base Sequence; D050728:Betaine-Aldehyde Dehydrogenase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002794:Choline / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005947:Glucose / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012965:Sodium Chloride / Q000378:metabolism*
D011994:Recombinant Proteins; D012965:Sodium Chloride; D050728:Betaine-Aldehyde Dehydrogenase; D005947:Glucose; D002794:Choline