Cell Architecture of the Giant Sulfur Bacterium Achromatium oxaliferum: Extra-cytoplasmic Localization of Calcium Carbonate Bodies

ABSTRACT Achromatium oxaliferum is a large sulfur bacterium easily recognized by large intracellular calcium carbonate bodies. Although these bodies often fill major parts of the cells’ volume, their role and specific intracellular location are unclear. In this study, we used various microscopy and staining techniques to identify the cell compartment harboring the calcium carbonate bodies. We observed that Achromatium cells often lost their calcium carbonate bodies, either naturally or induced by treatments with diluted acids, ethanol, sodium bicarbonate and UV radiation which did not visibly affect the overall shape and motility of the cells (except for UV radiation). The water-soluble fluorescent dye fluorescein easily diffused into empty cavities remaining after calcium carbonate loss. Membranes (stained with Nile Red) formed a network stretching throughout the cell and surrounding empty or filled calcium carbonate cavities. The cytoplasm (stained with FITC and SYBR Green for nucleic acids) appeared highly condensed and showed spots of dissolved Ca2+ (stained with Fura-2). From our observations, we conclude that the calcium carbonate bodies are located in the periplasm, in extra-cytoplasmic pockets of the cytoplasmic membrane and are thus kept separate from the cell's cytoplasm. This periplasmic localization of the carbonate bodies might explain their dynamic formation and release upon environmental changes.

Exploring Castellaniella defragrans Linalool (De)hydratase-Isomerase for Enzymatic Hydration of Alkenes

Acyclic monoterpenes constitute a large and highly abundant class of secondary plant metabolites and are, therefore, attractive low-cost raw materials for the chemical industry. To date, numerous biocatalysts for their transformation are known, giving access to highly sought-after monoterpenoids. In view of the high selectivity associated with many of these reactions, the demand for enzymes generating commercially important target molecules is unabated. Here, linalool (de)hydratase-isomerase (Ldi, EC 4.2.1.127) from Castellaniella defragrans was examined for the regio- and stereoselective hydration of the acyclic monoterpene β-myrcene to (S)-(+)-linalool. Expression of the native enzyme in Escherichia coli allowed for identification of bottlenecks limiting enzyme activity, which were investigated by mutating selected residues implied in enzyme assembly and function. Combining these analyses with the recently published 3D structures of Ldi highlighted the precisely coordinated reduction–oxidation state of two cysteine pairs in correct oligomeric assembly and the catalytic mechanism, respectively. Subcellular targeting studies upon fusion of Ldi to different signal sequences revealed the significance of periplasmic localization of the mature enzyme in the heterologous expression host. This study provides biochemical and mechanistic insight into the hydration of β-myrcene, a nonfunctionalized terpene, and emphasizes its potential for access to scarcely available but commercially interesting tertiary alcohols.

Discovery and Characterization of Novel Lignocellulose-Degrading Enzymes from the Porcupine Microbiome

Plant cell walls are comprised of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade these components to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with novel lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four genes from the porcupine microbiome encoding putative novel lignocellulose-degrading enzymes, including a β-xylanase, endoxylanase, β-glucosidase, and an ⍺-L-arabinofuranosidase. These genes were identified via conserved catalytic domains associated with cellulose and hemicellulose degradation. We cloned the putative β-xylanase into the pET26b(+) plasmid, enabling inducible gene expression inEscherichia coli(E. coli) and periplasmic localization. We demonstrated IPTG-inducible accumulation of β-xylanase protein but failed to detect xylobiose degrading activity in a reporter assay. Alternative assays may be required to measure activity of this putative β-xylanase. In this report, we describe how a synthetic metagenomic pipeline can be used to identify novel microbial lignocellulose-degrading enzymes and take initial steps to introduce a hemicellulose-degradation pathway intoE. colito enable biofuel production from wood pulp feedstock.

Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans

The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans. Our top-down analysis provided the confident identification of 55 proteins in the periplasm and characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm.

The StkP/PhpP Signaling Couple in Streptococcus pneumoniae: Cellular Organization and Physiological Characterization

ABSTRACT In Streptococcus pneumoniae, stkP and phpP, encoding the eukaryotic-type serine-threonine kinase and PP2C phosphatase, respectively, form an operon. PhpP has the features of a so-called “soluble” protein, whereas StkP protein is membrane associated. Here we provide the first genetic and physiological evidence that PhpP and StkP, with antagonist enzymatic activities, constitute a signaling couple. The StkP-PhpP couple signals competence upstream of the competence-specific histidine kinase ComD, receptor for the oligopeptide pheromone “competence stimulating peptide.” We show that PhpP activity is essential in a stkP + genetic background, suggesting tight control of StkP activity by PhpP. Proteins PhpP and StkP colocalized to the cell membrane subcellular fraction and likely belong to the same complex, as revealed by coimmunoprecipitation in cellular extracts. Specific coimmunoprecipitation of the N-kinase domain of StkP and PhpP recombinant proteins by PhpP-specific antibodies demonstrates direct interaction between these proteins. Consistently, flow cytometry analysis allowed the determination of the cytoplasmic localization of PhpP and of the N-terminal kinase domain of StkP, in contrast to the periplasmic localization of the StkP C-terminal PASTA (penicillin-binding protein and serine-threonine kinase associated) domain. A signaling route involving interplay between serine, threonine, and histidine phosphorylation is thus described for the first time in this human pathogen.

Sulfide-Quinone Reductase from Rhodobacter capsulatus: Requirement for Growth, Periplasmic Localization, and Extension of Gene Sequence Analysis

ABSTRACT The entire sequence of the 3.5-kb fragment of genomic DNA fromRhodobacter capsulatus which contains the sqrgene and a second complete and two further partial open reading frames has been determined. A correction of the previously publishedsqr gene sequence (M. Schütz, Y. Shahak, E. Padan, and G. Hauska, J. Biol. Chem. 272:9890–9894, 1997) which in the deduced primary structure of the sulfide-quinone reductase changes four positive into four negative charges and the number of amino acids from 425 to 427 was necessary. The correction has no further bearing on the former sequence analysis. Deletion and interruption strains document that sulfide-quinone reductase is essential for photoautotrophic growth on sulfide. The sulfide-oxidizing enzyme is involved in energy conversion, not in detoxification. Studies with an alkaline phosphatase fusion protein reveal a periplasmic localization of the enzyme. Exonuclease treatment of the fusion construct demonstrated that the C-terminal 38 amino acids of sulfide-quinone reductase were required for translocation. An N-terminal signal peptide for translocation was not found in the primary structure of the enzyme. The possibility that the neighboring open reading frame, which contains a double arginine motif, may be involved in translocation has been excluded by gene deletion (rather, the product of this gene functions in an ATP-binding cassette transporter system, together with the product of one of the other open reading frames). The results lead to the conclusion that the sulfide-quinone reductase of R. capsulatus functions at the periplasmic surface of the cytoplasmic membrane and that this flavoprotein is translocated by a hitherto-unknown mechanism.