Analysis ofN-acetylglucosamine metabolism in the marine bacteriumPirellulasp. strain 1 by a proteomic approach

Abstract

Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.

Proteomic analysis of carbohydrate catabolism and regulation in the marine bacteriumRhodopirellula baltica

“…Thus, a correlation between experimentally determined protein abundance and codon usage features as it already has been shown for fast-growing bacteria [43] could also be observed for R. baltica, a slowly growing environmental bacterium (doubling times between 10-14 h, [32]). However, there are some exceptions where the genes of proteins appearing as highly abundant on 2-DE gels are not PHX; this applies mainly for proteins that were, e.g., specifically induced during growth with ribose (see accompanying publication) or proteins affiliated with lipid metabolism.…”

“…Cells of R. baltica (DSM 10527) were grown in mineral medium with ribose (10 mM), glucose (10 mM) or N-acetylglucosamine (10 mM) as sole source of organic carbon [32].…”

“…Harvesting of cells was essentially performed as previously described [32]. Cells were harvested in the exponential growth phase by centrifugation (10 0006g, 15 min, 47C).…”

“…2-DE was essentially performed as described before [19,32,34]. In brief, IEF was performed using the IPGphor  system and 24 cm long IPG strips (linear pH gradient from 4 to 7; Amersham Biosciences), followed by equilibration of the gels with DTT and iodoacetamide.…”

Taxonomic heterogeneity within the Planctomycetales as derived by DNA–DNA hybridization, description of Rhodopirellula baltica gen. nov., sp. nov., transfer of Pirellula marina to the genus Blastopirellula gen. nov. as Blastopirellula marina comb. nov. and emended description of the genus Pirellula

“…It could be speculated that growth with chondroitin sulfate, as found here, may require the activity of a specific sulfatase that liberates the carbohydrate moiety. The first insights into the regulation of carbohydrate metabolism were recently obtained by means of a proteomic approach (Rabus et al, 2002).…”