[Show abstract][Hide abstract] ABSTRACT: The bacterial second messenger cyclic di-GMP (c-di-GMP) regulates the transition between sessility and motility. In Salmonella enterica serovar Typhimurium, the expression of CsgD, the regulator of multicellular rdar morphotype behavior, is a major target of
c-di-GMP signaling. CsgD expression is positively regulated by at least two diguanylate cyclases, GGDEF domain proteins, and
negatively regulated by at least four phosphodiesterases, EAL domain proteins. Here, we show that in contrast to EAL domain
proteins acting as phosphodiesterases, the EAL-like protein STM1344 regulated CsgD expression positively and motility negatively.
STM1344, however, did not have a role in c-di-GMP turnover and also did not bind the nucleotide. STM1344 acted upstream of
the phosphodiesterases STM1703 and STM3611, previously identified to participate in CsgD downregulation, where it repressed
their expression. Consequently, although STM1344 has not retained a direct role in c-di-GMP metabolism, it still participates
in the regulation of c-di-GMP turnover and has a role in the transition between sessility and motility.
Journal of bacteriology 05/2009; 191(12):3928-37. DOI:10.1128/JB.00290-09 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The topology of Alg8, the proposed catalytic subunit of the alginate polymerase, was assessed using PhoA and LacZ fusion protein analysis. This analysis suggested that the periplasmic loop comprises only three amino acid residues with the adjacent transmembrane helices at positions 361-387 and 393-416. Accordingly, the extended cytosolic loop could be located at positions 71-361 and was proposed to contain important catalytic residues. Further experimental evidence for this cytosolic domain was obtained by independently demonstrating this protein region as purified soluble protein domain. The soluble protein domain was identified by MALDI-TOF/MS and presumably represents the cytosolic catalytic domain of Alg8. Site-directed mutagenesis of 11 conserved residues in the cytosolic loop showed that D-188/D-190 (DXD motif), D-295/D-296 (acid-base catalysts) and K-297 were each essential for in vivo polymerase activity, whereas D-179/D-181 (DXD motif), C-244, R-263, D-279, and E-282 were not directly involved in the polymerisation reaction. The role of these amino acid residues with respect to the catalysed alginate polymerisation reaction was discussed with the aid of the recently developed structural model of Alg8.
Journal of Biotechnology 04/2009; 140(3-4):176-83. DOI:10.1016/j.jbiotec.2009.02.006 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The physiological response to small molecules (secondary messengers) is the outcome of a delicate equilibrium between biosynthesis and degradation of the signal. Cyclic diguanosine monophosphate (c-di-GMP) is a novel secondary messenger present in many bacteria. It has a complex cellular metabolism whereby usually more than one enzyme synthesizing and degrading c-di-GMP is encoded by a bacterial genome. To assess the in vivo conditions of c-di-GMP signaling, we developed a high-performance liquid chromatography (HPLC)-mass spectrometry-based method to detect c-di-GMP with high sensitivity and to quantify the c-di-GMP concentration in the bacterial cell as described here in detail. We successfully used the methodology to determine and compare the c-di-GMP concentrations in bacterial species such as Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae. We describe the use of the methodology to assess the change in c-di-GMP concentration during the growth phase and the contribution of a point mutation in S. typhimurium to the overall cellular c-di-GMP concentration.
[Show abstract][Hide abstract] ABSTRACT: Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic DeltamucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.
[Show abstract][Hide abstract] ABSTRACT: Alginate is a polysaccharide belonging to the family of linear (unbranched), non-repeating copolymers, consisting of variable amounts of beta-D-mannuronic acid and its C5-epimer alpha- L-guluronic acid linked via beta-1,4-glycosidic bonds. Like DNA, alginate is a negatively charged polymer, imparting material properties ranging from viscous solutions to gel-like structures in the presence of divalent cations. Bacterial alginates are synthesized by only two bacterial genera, Pseudomonas and Azotobacter, and have been extensively studied over the last 40 years. While primarily synthesized in form of polymannuronic acid, alginate undergoes chemical modifications comprising acetylation and epimerization, which occurs during periplasmic transfer and before final export through the outer membrane. Alginate with its unique material properties and characteristics has been increasingly considered as biomaterial for medical applications. The genetic modification of alginate producing microorganisms could enable biotechnological production of new alginates with unique, tailor-made properties, suitable for medical and industrial applications.
[Show abstract][Hide abstract] ABSTRACT: Here the putative alginate biosynthesis gene alg44 of Pseudomonas aeruginosa was functionally assigned. Non-polar isogenic alg44 deletion mutants of P. aeruginosa were generated and did neither produce alginate nor released free uronic acids. No evidence for alginate enrichment in the periplasm was obtained. Alginate production was restored by introducing only the gene alg44. PhoA fusion protein analyses suggested that Alg44 is a soluble protein localized in the periplasm. Hexahistidine-tagged Alg44 was detected by immunoblotting. The corresponding 42.6 kDa protein was purified and identified by MALDI/TOF-MS analysis. Alg44 might be directly involved in alginate polymerization presumably by exerting a regulatory function.
[Show abstract][Hide abstract] ABSTRACT: AlgX was found to be an essential protein for alginate biosynthesis, but its function is unknown. In this study, an isogenic, marker-free algX-knock out mutant was generated. In-frame fusions of algX with phoA and lacZ were analysed, respectively. No LacZ-activity was detected, but the PhoA fusion showed alkaline phosphatase activity. These data indicated that the C-terminus of AlgX is located in the periplasm, but is not required for protein function. Accordingly, AlgX with C-terminal fusion of strep tag II restored alginate production in the algX-negative mutant and was purified under native conditions from periplasmic and crude cell extracts, respectively. AlgX was identified by MALDI/TOF-MS analysis of tryptic peptides. TritonX-100 mediated solubilisation of cytoplasmic membrane and subsequent strep tag II affinity chromatography led to purification of an AlgX-MucD (AlgY) protein complex as identified by MALDI/TOF-MS analysis. This data suggested a protein-protein interaction between AlgX and MucD (AlgY) with a 1:1 stoichiometry. Thus AlgX might exert its function via interaction with MucD (AlgY). Immunoelectron microscopic localisation of AlgX-strep tag II suggested a localisation close to the cytoplasmic membrane.
[Show abstract][Hide abstract] ABSTRACT: An enzymatic in vitro alginate polymerization assay was developed by using 14C-labeled GDP-mannuronic acid as a substrate and subcellular fractions of alginate overproducing Pseudomonas aeruginosa FRD1 as a polymerase source. The highest specific alginate polymerase activity was detected in the envelope fraction, suggesting
that cytoplasmic and outer membrane proteins constitute the functional alginate polymerase complex. Accordingly, no alginate
polymerase activity was detected using cytoplasmic membrane or outer membrane proteins, respectively. To determine the requirement
of Alg8, which has been proposed as catalytic subunit of alginate polymerase, nonpolar isogenic alg8 knockout mutants of alginate-overproducing P. aeruginosa FRD1 and P. aeruginosa PDO300 were constructed, respectively. These mutants were deficient in alginate biosynthesis, and alginate production was
restored by introducing only the alg8 gene. Surprisingly, this resulted in significant alginate overproduction of the complemented P. aeruginosa Δalg8 mutants compared to nonmutated strains, suggesting that Alg8 is the bottleneck in alginate biosynthesis. 1H-NMR analysis of alginate isolated from these complemented mutants showed that the degree of acetylation increased from 4.7
to 9.3% and the guluronic acid content was reduced from 38 to 19%. Protein topology prediction indicated that Alg8 is a membrane
protein. Fusion protein analysis provided evidence that Alg8 is located in the cytoplasmic membrane with a periplasmic C terminus.
Subcellular fractionation suggested that the highest specific PhoA activity of Alg8-PhoA is present in the cytoplasmic membrane.
A structural model of Alg8 based on the structure of SpsA from Bacillus subtilis was developed.
[Show abstract][Hide abstract] ABSTRACT: The development of non-petrochemical sources for the plastics industry continues to progress as large multinationals focus on renewable resources to replace fossil carbon. Many bacteria are known to accumulate polyoxoesters as water-insoluble granules in the cytoplasm. The thermoplastic and/or elastomeric behaviour of these biodegradable polymers holds promise for the development of various technological applications. Here, we report the synthesis and characterization of microbial polythioesters (PTEs), a novel class of biopolymers of general technological relevance. Biosynthesis of PTE homopolymers was achieved using a recombinant strain of Escherichia coli that expressed a non-natural pathway consisting of a butyrate kinase, a phosphotransbutyrylase, and a PHA synthase. Different homopolymers were produced, consisting of either 3-mercaptopropionate, 3-mercaptobutyrate, or 3-mercaptovalerate repeating units, if the respective mercaptoalkanoic acids were provided as precursor substrates to the fermentative process. The PTEs contributed up to 30% (w/w) of the cellular dry weight and were identified as hydrophobic inclusions in the cytoplasm. The chemical and stereochemical homogeneity of the purified PTEs were identified by different methods, and the estimated physical properties were compared to the oxypolyester equivalents, revealing low crystalline order and, for the poly(3-mercaptopropionate) improved thermal stability. The ability to produce PTEs through a biosynthetic route opens up new avenues in the field of biomaterials.
Nature Material 01/2003; 1(4):236-40. DOI:10.1038/nmat773 · 36.50 Impact Factor