Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (K m , 8.6 ؎ 0.4 M) with high maximum velocity (110 nmol ⅐ min). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na ؉ ions. It is thus driven both by the membrane potential and the Na ؉ gradient. An extremely high accumulation (internal/external) ratio of up to 4 ؋ 10 6 was measured, which represents the highest accumulation ratio observed for any transport system.
SummaryAmtR, the master regulator of nitrogen control in Corynebacterium glutamicum , represses transcription of a number of genes during nitrogen surplus. Repression is released by an interaction of AmtR with signal transduction protein GlnK. As shown by pulldown assays and gel retardation experiments, only adenylylated GlnK, which is present in the cells during nitrogen limitation, is able to bind to AmtR.The AmtR regulon was characterized in this study by a combination of bioinformatics, transcriptome and proteome analyses. At least 33 genes are directly controlled by the repressor protein including those encoding transporters and enzymes for ammonium assimilation ( amtA , amtB , glnA , gltBD ), urea and creatinine metabolism ( urtABCDE , ureABCEFGD , crnT , codA ), a number of biochemically uncharacterized enzymes and transport systems (NCgl1099, NCgl1100, NCgl 1915-1918) as well as signal transduction proteins ( glnD , glnK ). For the AmtR regulon, an AmtR box has been defined which comprises the sequence tttCTATN 6 AtAGat/aA. Furthermore, the transcriptional organization of AmtR-regulated genes and operons was characterized.
Lipids are important constituents of the human epidermis. Either free and organized into broad lipid bilayers in the intercorneocytes spaces, or covalently bound to the corneocyte envelope, they play a crucial role in permeability barrier function and are major contributors to cutaneous anti-microbial defense. Free sphingoid bases are a recent addition to this family of active lipids, which emerged from studies of breakdown products from ceramides. Phytosphingosine (PS) is a lipid occurring naturally in the stratum corneum, both in its free form and as a part of the major fraction of ceramides. The biotechnological production of PS patented by Degussa yields to PS with the correct configuration present in the skin. So, application of a PS containing formulation leads to its integration into the natural lipid structures of the skin. In acne, different pathogenetic factors contribute to the inflammation process, defect in keratinization, increased sebaceous gland activity and increased colonization of Propionibacterium acnes. The results of in vitro and in vivo studies confirm the previous reports on strong anti-microbial effectiveness of skin-identical PS produced by Degussa in vitro and in vivo. In addition, PS shows excellent clinical results in the context of skin care in acne, based on both anti-inflammatory and anti-microbial activity. These results demonstrate the potential of PS to enhance or complement existing acne therapies acting as an active cosmetic ingredient.
In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using [(33)P]-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies. After two-dimensional gel electrophoresis (2-DE), around 60 [(33)P]-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.
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