Immunocytochemical analysis of AlgP (Hp1), a histonelike element participating in control of mucoidy in Pseudomonas aeruginosa

Abstract

AlgP, a protein with an unusual carboxy-terminal domain resembling the tails of eukaryotic Hi histones, was detected in whole-cell extracts and within the cells of Pseudomonas aeruginosa by using immunoblotting and immunoelectron microscopy analyses. One known function of AIgP is its participation in the transcriptional activation of the algD gene. This is a pivotal step in the establishment of mucoidy in P. aeruginosa; mucoidy is a critical virulence factor expressed during respiratory infections in patients with cystic fibrosis. Polyclonal and monoclonal antibodies were raised against a synthetic 50-mer peptide containing two sets of six tandem repeats of the motif Lys-Pro-Ala-Ala (and its single-amino-acid substitution variants), based on the sequence of the algP gene from the standard genetic strain PAO. Western immunoblots with these antibodies and total protein extracts from P. aeruginosa revealed two polypeptides that reacted with the antibodies in all of the P. aeruginosa strains tested. The detected polypeptides displayed strain-dependent variability in their electrophoretic mobility, in accordance with the previously noted variability of the algP repeats at the DNA level. In strain PAO, the recognized polypeptides had apparent masses of 46.4

AlgR-binding sites within the algD promoter make up a set of inverted repeats separated by a large intervening segment of DNA

“…The peculiar organization (4) of the algD promoter reported here, in conjunction with the control by multiple signal transduction systems and histone-like elements, involving AlgR (29), AIgB (17,39), AlgP (Hpl) (11,13,22), AlgQ (12,24), and potentially other factors, is a further step toward delineation of the regulation of mucoidy. It is also an example of the elaborate regulatory networks necessary to control such complex phenotypes as the expression of virulence determinants.…”

“…Thus, these putative processes, as well as possible interactions of AlgR bound at RB1 or RB2 with RNA polymerase, may either depend on a protein induced DNA bending or looping, such as in the case of NifA-dependent activation of nifH (20), or NtrC-dependent activation of glnA (37), respectively, or other factors determining local and global nucleoid structure. On the basis of independent genetic studies, it is known that other elements, such as the histone-like protein AlgP (Hpl), can enhance activity of the algD promoter (11,13,22,24). In addition, the algD promoter has several sequences resembling integration host factor (IHF) binding sites (28).…”

Molecular cloning and expression of hctB encoding a strain-variant chlamydial histone-like protein with DNA-binding activity

“…(2) and other prokaryotic proteins termed histone like (reviewed in references 11 and 19). Like Hc2, AlgP (AlgR3) of P. aeruginosa (9,10,20) is a highly basic histone H1 homolog with a lysine-and alanine-rich repetitive protein structure shown to interact with DNA (8,9). As a positive regulator of alginate synthesis, AlgP has been postulated to facilitate the interaction of distally bound transcription factors with RNA polymerase at the algD promoter by introducing or stabilizing DNA bends (10).…”

Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

“…Separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Western blotting was performed as described previously (34) with modifications. Duplicate blots were probed either with an anti-AlgR rabbit serum (Open Biosystems) preabsorbed with a cell extract from an overnight culture of PAO1 ⌬algR or with an anti-OmlA antibody (a gift from Michael Vasil).…”