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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Mycobacteria have a unique outer membrane (OM) that is thicker than any other known biological membrane. Nutrients cross this permeability barrier by diffusion through porins. MspA is the major porin of Mycobacterium smegmatis. In this study we showed that three paralogues of MspA, namely MspB, MspC and MspD are also porins. However, only the mspA and mspC genes were expressed in the wild-type strain. None of the single deletion mutants displayed a significant OM permeability defect except for the mspA mutant. Deletion of the mspA gene caused activation of transcription of mspB and/or mspD in three independent strains by unknown chromosomal mutations. It is concluded that mspB and mspD provide backup porins for M. smegmatis. This also indicated that a minimal porin-mediated OM permeability is essential for survival of M. smegmatis. Electron microscopy in combination with quantitative image analysis of protein gels revealed that the number of pores per cell dropped from 2400 to 800 and 150 for the ΔmspA and ΔmspA ΔmspC mutant (ML10) respectively. The very low number of pores correlated well with the at least 20-fold lower channel activity of detergent extracts of the ML10 strain and its 15- and 75-fold lower permeability to nutrient molecules such as serine and glucose respectively. The amount of Msp porin and the OM permeability of the triple porin mutant lacking mspA, mspC and mspD was not altered. The growth rate of M. smegmatis dropped drastically with its porin-mediated OM permeability in contrast to porin mutants of Escherichia coli. These results show that porin-mediated influx of nutrients is a major determinant of the growth rate of M. smegmatis.
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 483 (1986), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 126 (1995), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The functional significance of charged amino acids of the anion-selective porin Omp34 from Acidovorax delafieldii was investigated by means of conductance measurements. Chemical modification of Lys and Arg as well as titration of charges by adjusting the pH value revealed that positively charged amino acid residues determine the major functional properties of the porin. Positive charges are involved in creating the protein surface potential, the selectivity filter inside the channels, and the voltage-sensing and/or gating mechanisms.
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Proteinaceous, hair-like appendages known as fimbriae or pili commonly extend from the surface of prokaryotic cells and serve important functions such as cell adhesion, biofilm formation, motility and DNA transfer. Here we show that a novel group of archaea from cold, sulphidic springs has developed cell surface appendages of an unexpectedly high complexity with a well-defined base-to-top organization. It represents a new class of filamentous cell appendages, for which the term ‘hamus’ is proposed. Each archaeal cell is surrounded by a halo of about 100 hami, which mediate strong adhesion of the cells to surfaces of different chemical composition. The hami are mainly composed of 120 kDa subunits and remained stable in a broad temperature and pH range (0–70°C; 0.5–11.5). Electron microscopy and cryo-electron tomography revealed that the hamus filament possesses a helical basic structure. At periodic distances, three prickles emanate from the filament, giving it the character of industrially produced barbwire. At its distal end the hami carry a tripartite, barbed grappling hook (60 nm in diameter). The architecture of this molecular hook is reminiscent of man-made fishhooks, grapples and anchors. It appears that nature has developed a perfect mechanical nano-tool in the course of biological evolution, which also might prove useful in the field of nanobiotechnology.
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water-filled channels in vitro. Immunogold electron microscopy and an enzyme-linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA-specific primers and reverse-transcribed RNA. Detergent extracts of the ΔmspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild-type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the ΔmspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine- and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the ΔmspA mutant. The minimal permeability coefficient of the ΔmspA mutant for glucose was 7.2 × 10−8 cm s−1, which is the lowest value reported so far for bacteria. This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.
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  • 7
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In this study, a flagella-related protein gene cluster is described for Halobacterium salinarum. The fla gene cluster is located upstream of the flagellin genes flgB1–3 and oriented in the opposite direction. It consists of nine open reading frames (ORFs): htpIX, a member of the halobacterial transducer protein gene family, and the genes flaD–K. The genes flaD, E, G, H, I and J share high homologies with genes from other Archaea. Interestingly, flaK shows similarities to bacterial genes involved in the regulation of flagellar synthesis. The ORFs of flaH, flaI and flaK contain sequences coding for nucleotide binding sites. Furthermore, flaI contains a motif called the bacterial type II secretion protein E signature, indicating a functional relation to members of the bacterial pili type IV–type II secretion protein superfamily. Reverse transcription–polymerase chain reaction (RT–PCR) analysis revealed that the genes flaE to flaK are transcribed into one polycistronic message. In frame deletion mutants of flaI were generated by gene replacement. The deletion strain lacks motility and belongs to the fla– mutant class, indicating that it is deficient in flagellar biogenesis. The overall amount of flagellin protein in ΔflaI cells is reduced, although transcription of the flagellin genes is unaffected. Therefore, the flaI gene product is involved in the biosynthesis, transport or assembly of flagella in H. salinarum.
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  • 8
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Selenocysteine synthase of Escherichia coli catalyses the biosynthesis of selenocysteine in the form of the aminoacyl-RNA complex, the reaction intermediate being aminoacrylyl-tRNAsec covalently bound to the prosthetic group of the enzyme. Selenocysteine synthase and the specific aminoacrylyl-tRNA sec-enzyme complex as well as the isolated seryl-tRNAsec were investigated in the electron microscope and analysed by means of image processing to a resolution of 2 nm in projection. The stoichiometric composition of the selenocysteine synthase molecule was elucidated by scanning transmission electron microscopic mass determination. The enzyme has a fivefold symmetric structure and consists of 10 monomers arranged in two rings. The tRNA is bound near the margin of the dimeric subunits. Principal component analysis of the tRNA-enzyme complexes revealed that the selenocysteine synthase appears to bind only one seryl-tRNAsec per dimer, which is consistent with the result of biochemical binding studies.
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  • 9
    ISSN: 1399-0047
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: Omp32, the strongly anion-selective porin from Comamonas acidovorans, has been crystallized. Two crystal forms were observed, both of which belong to space group R3, but exhibit different cell dimensions a = b = 106.7, c = 140.6 Å (crystal form I) and a = b = 87.1, c = 135.3 Å (crystal form II) with one trimer per asymmetric unit. The crystals diffract to 2.2 and 2.3 Å resolution, respectively. Omp32 was chemically modified by introducing negative charges through succinylation. The number and positions of the individual modifications were determined using mass spectrometry and X-ray crystallography. Chemically modified porins yielded crystals of a third form, also of space group R3 but with cell constants of a = b = 109.3 and c = 263.2 Å (crystal form III), showing a virtually doubled c axis. Crystals of form III diffract to 3.5 Å resolution.
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  • 10
    Electronic Resource
    Electronic Resource
    [s.l.] : Macmillan Magazines Ltd.
    Nature 395 (1998), S. 392-395 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Membrane-enclosed organelles, a defining characteristic of eukaryotic cells, are lost during differentiation of specific cell types such as reticulocytes (an intermediate in differentiation of erythrocytes), central fibre cells the eye lens, and keratinocytes. The degradation of these ...
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