Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa

Abstract

Transcriptional regulation of alginate biosynthesis by Pseudomonas aeruginosa was studied. A DNA region complementing the alg-5 mutation within the alginate gene cluster was found by RNA-DNA dot blot and Northern hybridization to be transcriptionally activated in mucoid P. aeruginosa. This region was subcloned as a 3.2-kilobase BglII-ClaI DNA fragment on the broad-host-range controlled transcription vector pMMB24, and gene products were analyzed by expression from the tac promoter. A 48-kilodalton polypeptide was detected in extracts of P. aeruginosa and 1'5S-labeled Escherichia coli maxicells. By using the same expression system, GDPmannose dehydrogenase activity was detected in both P. aeruginosa and E. coli. Thus, gene algD coding for this enzyme was found to be present in the transcriptionally active DNA area. Insertion of the xylE gene within the BglI-ClaI fragment disrupted the induction of the 48-kilodalton polypeptide, GDPmannose dehydrogenase activity, and alg-5 complementing ability, With the algD-xyE transcription fusion, activation of algD gene expression was shown to occur in mucoid P. aeruginosa of different origins. In addition, regulation of the algD promoter activity was demonstrated to be mediated by a diffusible factor.

Cloning and Sequencing of the Genes forN-Acetylglucosamine Use That Construct Divergent Operons (nagE-nagAC) fromVibrio choleraeNon-O1

“…Total RNA was isolated from V. eholerae non-O 1 cultivated in medium with or without GlcNAc. 7 ) RNA was separated by electrophoresis on 1 % agarose containing 6% formaldehyde. 8 ) Hybridization probes were synthesized by PCR and labelled with (DIG)-ll-dUTP by PCR DIG Probe Synthesis Kit (Boehringer mannheim).…”

Conversion of Pseudomonas aeruginosa to mucoidy in cystic fibrosis: environmental stress and regulation of bacterial virulence by alternative sigma factors

“…The latter enzyme also has a pyrophosphorylase activity implicated in GDPmannose synthesis (69), The alginate biosynthetic cluster is the major point of convergence for various regulatory factors controlling alginate production. The algD gene is strategically positioned at the beginning of this cluster and undergoes a strong transcriptional activation in mucoid cells (14). The product of this gene, an NAD+-dependent, 4-electron transfer dehydrogenase which converts GDPmannose into GDPmannuronic acid, plays a pivotal role in channeling sugar intermediates into alginate production (14,54).…”

“…The algD gene is strategically positioned at the beginning of this cluster and undergoes a strong transcriptional activation in mucoid cells (14). The product of this gene, an NAD+-dependent, 4-electron transfer dehydrogenase which converts GDPmannose into GDPmannuronic acid, plays a pivotal role in channeling sugar intermediates into alginate production (14,54). Moreover, all characterized genes in the cluster are transcribed in the same direction as algD, and there is now genetic evidence suggesting that the entire cluster may be affected by the algD promoter (9).…”

“…The majority of these and other enzymes modifying alginate are encoded by the large cluster of alginate genes at 34 min (12). This cluster contains in the order of transcription: algD, encoding GDPmannose dehydrogenase, which catalyzes the final step in the precursor biosynthesis (14); algG, encoding the epimerase which converts mannuronic residues into guluronate (8); algL, encoding an enzyme termed alginate lyase, due to its ability to depolymerize alginate (6,67); algF, encoding an acetylase (24,70); and algA, encoding phosphomannose isomerase, which converts fructose 6-phosphate into mannose 6-phosphate (11). The latter enzyme also has a pyrophosphorylase activity implicated in GDPmannose synthesis (69), The alginate biosynthetic cluster is the major point of convergence for various regulatory factors controlling alginate production.…”

Sensor Domain of Histidine Kinase KinB of Pseudomonas

“…Alginate biosynthesis is tightly controlled by a set of genes and a cascade of regulators (2)(3)(4). One of these is AlgB, which was shown to serve as a negative regulator of alginate production (5).…”