Fabrizio Giannotta

Subdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies

Haydon and Guest (Haydon, D. J, and Guest, J. R. (1991) FEMS Microbiol. Lett. 63, 291–295) first described the helix-turn-helix GntR family of bacterial regulators. They presented them as transcription factors sharing a similar N-terminal DNA-binding (d-b) domain, but they observed near-maximal divergence in the C-terminal effector-binding and oligomerization (E-b/O) domain. To elucidate this C-terminal heterogeneity, structural, phylogenetic, and functional analyses were performed on a family that now comprises about 270 members. Our comparative study first focused on the C-terminal E-b/O domains and next on DNA-binding domains and palindromic operator sequences, has classified the GntR members into four subfamilies that we called FadR, HutC, MocR, and YtrA. Among these subfamilies a degree of similarity of about 55% was observed throughout the entire sequence. Structure/function associations were highlighted although they were not absolutely stringent. The consensus sequences deduced for the DNA-binding domain were slightly different for each subfamily, suggesting that fusion between the D-b and E-b/O domains have occurred separately, with each subfamily having its own D-b domain ancestor. Moreover, the compilation of the known or predicted palindromiccis-acting elements has highlighted different operator sequences according to our subfamily subdivision. The observed C-terminal E-b/O domain heterogeneity was therefore reflected on the DNA-binding domain and on the cis-acting elements, suggesting the existence of a tight link between the three regions involved in the regulating process.

Purification and properties of three endo-β-1,4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of kraft pulps.

Abstract In the presence of xylan, Streptomyces sp. strain S38 secretes three xylanases (Xyl1, Xyl2, and Xyl3) that were purified to protein homogeneity and characterized. When used in bleach boosting tests on kraft hardwood and softwood, Xyl1, a family-11 enzyme, was more effective than Xyl2 and Xyl3 that belonged to family-10. Xyl1 was fully responsible for the biodelignification potential of the culture supernatants with a minimal effective amount of 10 IU per gram of dry pulp for both softwood and hardwood pulp. Complete conventional CEDED bleaching sequences showed that enzymatic pretreatment (20 IU/g dry pulp) could result in active chlorine savings of 8.6 and 4.9 kg/ton of dry pulp with hardwood and softwood, respectively. The purified enzymes were totally devoid of cellulase activity on CM-cellulose and their activities were optimal at about 60°C and pH 6. Moreover, the V max value of Xyl1 at 50°C measured on birchwood xylan (5,700 μmoles/min/mg prot.) was significantly higher than those of Xyl2 and Xyl3 whereas their K m values were similar. Their half-lives at 50°C were larger than 16 h but sharply decreased at 60°C where the family-11 Xyl1 was less stable (t 1/2 60°C = 10 min) than both family-10 enzymes Xyl2 (t 1/2 60°C = 30 min) and Xyl3 (t 1/2 60°C = 70 min).

SEQUENCE, OVERPRODUCTION AND PURIFICATION OF THE FAMILY 11 ENDO-BETA -1,4-XYLANASE ENCODED BY THE XYL1 GENE OF STREPTOMYCES SP. S38

The xyl1 gene encoding the Xyl1 xylanase of Streptomyces sp. strain S38 was cloned by screening an enriched DNA library with a specific DNA probe and sequenced. Three short 5 bp -CGAAA- sequences are located upstream of the Streptomyces sp. S38 xyl1 gene 105, 115 and 250 bp before the start codon. These sequences, named boxes 1, 2 and 3, are conserved upstream of the Actinomycetales xylanase genes and are specifically recognized by a DNA-binding protein (Giannotta et al., 1994. FEMS Microbiol. Lett. 142, 91-97) and could be probably involved in the regulation of xylanase production. The Xyl1 ORF encodes a 228 residue polypeptide and the Xyl1 preprotein contains a 38 residue signal peptide whose cleavage yields a 190 residue mature protein of calculated M(r) = 20,585 and basic pI value of 9.12. The molecular mass of the produced and purified mature protein determined by mass spectrometry (20,586 +/- 1 Da) and its pI (9.8) agree with these calculated values. Its N-terminal amino-acid sequence confirmed the proposed cleavage site between the signal peptide and the mature protein. Comparisons between Xyl1 and the 62 other xylanases belonging to family 11 allowed the construction of a phylogenetic tree and revealed its close relationship with Actinomycetales enzymes. Moreover, nine residues were found to be strictly conserved among the 63 xylanases.

BlaB, a protein involved in the regulation of Streptomyces cacaoi β-lactamases, is a penicillin-binding protein

AbstractStreptomyces cacaoi β-lactamase genes are controlled by two regulators named blaA and blaB. Whereas BlaA has been identified as a LysR-type activator, the function of BlaB is still unknown. Its primary structure is similar to that of the serine penicillin-recognizing enzymes (PREs). Indeed, the SXXK and KTG motifs are perfectly conserved in BlaB, whereas the common SXN element found in PREs is replaced by a SDG motif. Site-directed mutations were introduced in these motifs and they all disturb β-lactamase regulation. A water-soluble form of BlaB was also overexpressed in the Streptomyces lividans TK24 cytoplasm and purified. To elucidate the activity of BlaB, several compounds recognized by PREs were tested. BlaB could be acylated by some of them, and it can therefore be considered as a penicillin-binding protein. BlaB is devoid of β-lactamase, D-aminopeptidase, DD-carboxypeptidase or thiolesterase activity.