In this study, a unique mucA mutation (designated mucA56) was introduced, which was characterized by deletion of bases 166-333, encoding MucA56 protein with the deletion of the trans-membrane region, which then was proved to be cytoplasmic with phoA-mucA fusion method. PAOmucA56 was constructed with homologous recombination; two PAO1 derivatives PAOmucA22 (PDO300) and PAOmucA56 displayed mucoid phenotype on pseudomonas isolation agar (PIA) agar, but PDO300 produced more alginate than PAOmucA56. Scanning confocal laser microscopy was used to observe the biofilm structures of the three strains during various biofilm development stages. PDO300 developed biofilm with low substratum coverage and high structural heterogeneity, while PAOmucA56 and PAO1 formed uniform biofilm with complete substratum coverage. The proteomes of crude protein extracts of biofilm cells revealed that there are 17 candidate proteins differentially expressed between the two kinds of biofilm, which were proteins involved in protein synthesis, MucA degradation, energy metabolism, carbon catabolism and amino acid metabolism and so on. We might conclude that alginate production may affect biofilm architecture, and proteins involved in protein synthesis, MucA degradation, energy metabolism, carbon catabolism and amino acid metabolism might play a role in biofilm development alternatively.