Multiple genes involved in chitin degradation from the marine bacterium Pseudoalteromonas sp. strain S91

Abstract

A cluster of three closely linked chitinase genes organized in the order chiA, chiB and chic, with the same transcriptional direction, and two unlinked genes, chip and chiQ, involved in chitin degradation in Pseudoalteromnas sp. strain S91 were cloned, sequenced and characterized. The deduced amino acid sequences revealed that ChiA, Chis and Chic exhibited similarities to chitinases belonging to family 18 of the glycosyl hydrolases while Chip and ChiQ belonged to family 20. Chip and ChiQ showed different enzymic activities against fluorescent chitin analogues, but neither was able to degrade colloidal chitin. ChiA possessed chitinase activity but did not bind chitin; Chis bound chitin but had no chitinase activity; Chic possessed strong chitinase activity and also bound chitin. Production of Chic in 591 appeared to be controlled by chiA expression, since insertion of a transposon into the ORF of chiA resulted in the loss of chitinase activity as well as loss of Chic proteins in a chitinasenegative mutant. In Escherichia coli, Chic appeared to be expressed from its own promoter.

Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster

“…The amino acid positions correspond to those recorded Sections of the S91 chiABC sequence highlighting the potential promoter region upstream of each putative TSP with homology to: (a) the À10 and À35 sites of the E. coli s 70 -dependent promoter consensus sequence (bold and underlined; Hawley and McClure 1983); (b) the À12 and À24 sites of the E. coli s 54 -dependent promoter consensus sequence (underlined; Barrios et al, 1999) (the most conserved residues in the À12 and À24 positions are bold and underlined). Numbers, before and after each sequence, indicate the base number in each GenBank sequence (chiA, AF007894; chiB, AF007895; chiC, AF007896; Techkarnjanaruk and Goodman, 1999). Each base in bold and double underlined is that at which transcription initiates and the subscript numeral beside it indicates its base number, as found in GenBank.…”

“…Each base in bold and double underlined is that at which transcription initiates and the subscript numeral beside it indicates its base number, as found in GenBank. N with bracketed numeral indicates the number of bases to the putative ribsome binding site (italics) located upstream of a putative in-frame translation initiation codon (ATG; underlined (Techkarnjanaruk and Goodman, 1999), there was a chitinase C (ChiC) domain, involved in chitin-binding (Pfam 06483; Watanabe et al, 1994), of 128 residues, Glu 793 -Gly 980 (Table 5 and Figure 1). Also, ChiA was found to have a chitin-binding type 3 domain (ChtBD3; smart 00495) at its N-terminal end of 52 residues, Cys 31 -Cys 82 (Table 5 and Figure 1).…”

“…The sequence was also analyzed to see if domains in addition to those reported previously (Techkarnjanaruk and Goodman, 1999) could be identified (Table 5 and Figure 1). The amino acid positions correspond to those recorded Sections of the S91 chiABC sequence highlighting the potential promoter region upstream of each putative TSP with homology to: (a) the À10 and À35 sites of the E. coli s 70 -dependent promoter consensus sequence (bold and underlined; Hawley and McClure 1983); (b) the À12 and À24 sites of the E. coli s 54 -dependent promoter consensus sequence (underlined; Barrios et al, 1999) (the most conserved residues in the À12 and À24 positions are bold and underlined).…”

“…Strain S91CX, a chiA interrupted transposon mutant, has no ChiA or ChiC chitinolytic activity, indicating that the gene cluster may be an operon, with transcription initiating at chiA (Techkarnjanaruk et al, 1997). Sequencing of chiABC, however, revealed a potential promoter region for each gene, and when cloned into Escherichia coli, chiC was expressed from its own promoter (Techkarnjanaruk, 1998;Techkarnjanaruk and Goodman, 1999). Techkarnjanaruk et al (1997) found that the chiA promoter was active at a basal level when S91CX was grown in marine minimal medium plus glutamate (MMM glt) and was induced three-fold and ten-fold on the addition chitin and GlcNAc, respectively.…”

“…Pseudoalteromonas S91 is a non-pathogenic Gram-negative marine bacterium in which three adjacent genes, chiABC, in the same transcriptional direction on the chromosome, encode two chitinases with catalytic activity (ChiA and ChiC) and a chitin-binding protein (ChiB) (Techkarnjanaruk et al, 1997;Techkarnjanaruk and Goodman, 1999). The transcriptional organization of this cluster is not known.…”