Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA

Summary The omega locus controls the polarity of recombination and transmission of genetic markers in the 21S ribosomal RNA region in yeast mtDNA. Polarity is observed in crosses between omega⁺ and omega^- strains. These two strains differ by the presence of an intervening sequence in the 21S ribosomal RNA gene of omega⁺ strains. Mutations of the omega^- allele, omega neutral (omegaⁿ), can eliminate the polarity effect. We have made DNA:RNA hybrids containing ribosomal RNA from an omegaⁿ strain and mtDNA from Saccharomyces carlsbergensis (identical to omega^- in the nucleotide sequence of the omega region). These hybrids contain no mismatch at the omega region detectable by digestion with S₁ nuclease. We conclude that omegaⁿ differs from omega^- only in a point mutation or analogous small alteration and that the omegaⁿ mutation can result either m a C^r phenotype (omegaⁿC^r) or in the phenotypic suppression of pre-existing C^r mutations (omegaⁿC^s). All results can be explained by a model which postulates interaction in the ribosome between the C^r and omegaⁿ regions of the ribosomal RNA and interference of the omegaⁿ mutation with splicing of the precursor ribosomal RNA in omega⁺ strains. The mechanism of omega-directed polarity is discussed.Abbreviations rRNA ribosomal RNA - bp base pair(s) - kb kilo base pair(s)     

Characterization and quantification of the peroxidase in human monocytes.

1. The lifetime of thiamine pyrophosphate-Sepharose 2B affinity matrices synthesized according to Matsuura et al. (Matsuura, A., Iwashina, A. and Nose, Y. (1973) Biochem. Biophys. Res. Commun. 51, 241-246) has been improved. The matrix interacts with bacterial pyruvate dehydrogenase complexes. 2. The synthesis of a stable thiochrome-Sepharose 2B matrix is described. 3. Both matrices bind the pyruvate dehydrogenase complex of Escherichia coli in a 50 mM phosphate buffer, pH 7.0. Elution is possibly by an increase in ionic strength but not by the cofactor or metal-cofactor complexes. 4. The presence of Mg2+, reduces the capacity of the affinity matrices but leads to higher specificity for the multienzyme complex. 5. The pyruvate dehydrogenase complex of E. coli has been successfully purified by combining a classical purification step with these affinity chromatography systems. The method is less suitable for large scale operation.     

Characterization of Aspergillus nidulans mutants in carbon metabolism isolated after D-galacturonate enrichment

A selective method for the isolation of Aspergillus nidulans mutants defective in the pyruvate dehydrogenase complex was devised. The essential steps in the procedure were a mutagenic treatment of conidia with X-rays to about 50% survival, followed by filtration enrichment in minimal medium with D-galacturonate as sole carbon source, and rescue on complete medium with acetate. The mutants thus isolated were phenotypically characterized on the basis of growth tests, and different genotypes were assigned on the basis of complementation tests. The majority of the mutants that were unable to utilize galacturonate were defective in one of the components of the pyruvate dehydrogenase complex. In addition, mutants defective in pyruvate carboxylase, mutants defective in glycerol catabolism and some novel mutants which were only unable to use D-galacturonate as carbon source were found. At least two genes were shown to be involved in D-galacturonate metabolism.     

Rap1 Can Bypass the FAK-Src-Paxillin Cascade to Induce Cell Spreading and Focal Adhesion Formation

We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.     

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Netherlands Heart Journal -     

Zinc Piracy as a Mechanism of Neisseria meningitidis for Evasion of Nutritional Immunity

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.     

Species-Specificity of the BamA Component of the Bacterial Outer Membrane Protein-Assembly Machinery

The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP) in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner in vitro. In this work, we further studied species specificity in vivo by testing the functioning of BamA homologs of the proteobacteria Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Burkholderia mallei, and Escherichia coli in E. coli and in N. meningitidis. We found that no BamA functioned in another species than the authentic one, except for N. gonorrhoeae BamA, which fully complemented a N. meningitidis bamA mutant. E. coli BamA was not assembled into the N. meningitidis outer membrane. In contrast, the N. meningitidis BamA protein was assembled into the outer membrane of E. coli to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of N. meningitidis and E. coli BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded β-barrel conformation of the membrane-embedded domain of BamA.