Formation of lipid-linked sugar compounds in Halobacterium salinarium. Presumed intermediates in glycoprotein synthesis.
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The ability of bacitracin to inhibit the growth of Halobacterium salinarium suggested that glycosylation of the major envelope component, a high molecular weight glycoprotein, might occur via a pathway involving lipid intermediates. This report demonstrates that the cells have enzymatic activities for formation of lipid-linked sugar compounds having the expected properties of such intermediates. Whole cell homogenate catalyzed the transfer of sugar from UDP-glucose, GDP-mannose, and UDP-N-acetyglucosamine to endogenous lipid acceptors. Two lipid products were formed from UDP-glucose, two from GDP-mannose, and one from UDP-N-acetylglucosamine. Characterization of the partially purified lipids by ion exchange chromatography, thin layer chromatography, and mild acid and base hydrolysis showed the major product in each case to have the properties expected for polyisoprenyl phosphoglucose, polyisoprenyl phosphomannose, and polyisoprenyl pyrophospho-N-acetylglucosamine. Estimates of chain length by thin layer chromatography indicate that the lipid has 11 to 12 isoprene identity as a C55-60-polyisoprenyl pyrophospho-N-acetylglucosamine. The N-acetylglucosamine transferase, present in cell envelope preparations, was partially characterized. The enzyme was found to be extremely halophilic, specifically requiring a high concentration of KCl. Optimum activity was obtained at 4 m KCl and partial substitution of K+ by Na+ resulted in a decrease in activity. (+info)
Proposal to transfer Halococcus turkmenicus, Halobacterium trapanicum JCM 9743 and strain GSL-11 to Haloterrigena turkmenica gen. nov., comb. nov.
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The 16S rRNA gene sequences of Halococcus saccharolyticus and Halococcus salifodinae were closely related (94.5-94.7% similarity) to that of Halococcus morrhuae, the type species of the genus Halococcus. However, Halococcus turkmenicus was distinct from the other members of this genus, with low 16S rRNA similarities when compared to Halococcus morrhuae (88.7%). On the basis of phylogenetic tree reconstruction, detection of signature bases and DNA-DNA hybridization data, it is proposed to transfer Halococcus turkmenicus to a novel genus, Haloterrigena, as Haloterrigena turkmenica gen. nov., comb. nov., and to accommodate Halobacterium trapanicum JCM 9743 and strain GSL-11 in the same species. On the basis of morphological, cultural and 16S rRNA sequence data, it is also proposed that the culture collection strains of Halobacterium trapanicum NCIMB 767, ATCC 43102 and JCM 8979 should be renamed as Halococcus sp. (+info)
Surface-grafted, environmentally sensitive polymers for biofilm release.
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Controlling bacterial biofouling is desirable for almost every human enterprise in which solid surfaces are introduced into nonsterile aqueous environments. One approach that is used to decrease contamination of manufactured devices by microorganisms is using materials that easily slough off accumulated material (i.e., fouling release surfaces). The compounds currently used for this purpose rely on low surface energy to inhibit strong attachment of organisms. In this study, we examined the possible use of environmentally responsive (or "smart") polymers as a new class of fouling release agents; a surface-grafted thermally responsive polymer, poly(N-isopropylacrylamide) (PNIPAAM), was used as a model compound. PNIPAAM is known to have a lower critical solubility temperature of approximately 32 degrees C (i.e., it is insoluble in water at temperatures above 32 degrees C and is soluble at temperatures below 32 degrees C). Under experimental conditions, >90% of cultured microorganisms (Staphylococcus epidermidis, Halomonas marina) and naturally occurring marine microorganisms that attached to grafted PNIPAAM surfaces during 2-, 18-, 36-, and 72-h incubations were removed when the hydration state of the polymer was changed from a wettability that was favorable for attachment to a wettability that was less favorable. Of particular significance is the observation that an organism known to attach in the greatest numbers to hydrophobic substrata (i.e., H. marina) was removed when transition of PNIPAAM to a more hydrated state occurred, whereas an organism that attaches in the greatest numbers to hydrophilic substrata (i.e., S. epidermidis) was removed when the opposite transition occurred. Neither solvated nor desolvated PNIPAAM exhibited intrinsic fouling release properties, indicating that the phase transition was the important factor in removal of organisms. Based on our observations of the behavior of this model system, we suggest that environmentally responsive polymers represent a new approach for controlling biofouling release. (+info)
Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter.
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Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site. A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity. (+info)
Bioenergetic aspects of halophilism.
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Examination of microbial diversity in environments of increasing salt concentrations indicates that certain types of dissimilatory metabolism do not occur at the highest salinities. Examples are methanogenesis for H2 + CO2 or from acetate, dissimilatory sulfate reduction with oxidation of acetate, and autotrophic nitrification. Occurrence of the different metabolic types is correlated with the free-energy change associated with the dissimilatory reactions. Life at high salt concentrations is energetically expensive. Most bacteria and also the methanogenic Archaea produce high intracellular concentrations of organic osmotic solutes at a high energetic cost. All halophilic microorganisms expend large amounts of energy to maintain steep gradients of NA+ and K+ concentrations across their cytoplasmic membrane. The energetic cost of salt adaptation probably dictates what types of metabolism can support life at the highest salt concentrations. Use of KCl as an intracellular solute, while requiring far-reaching adaptations of the intracellular machinery, is energetically more favorable than production of organic-compatible solutes. This may explain why the anaerobic halophilic fermentative bacteria (order Haloanaerobiales) use this strategy and also why halophilic homoacetogenic bacteria that produce acetate from H2 + CO2 exist whereas methanogens that use the same substrates in a reaction with a similar free-energy yield do not. (+info)
Membrane insertion kinetics of a protein domain in vivo. The bacterioopsin n terminus inserts co-translationally.
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The pathway by which segments of a polytopic membrane protein are inserted into the membrane has not been resolved in vivo. We have developed an in vivo kinetic assay to examine the insertion pathway of the polytopic protein bacterioopsin, the apoprotein of Halobacterium salinarum bacteriorhodopsin. Strains were constructed that express the bacteriorhodopsin mutants I4C:H(6) and T5C:H(6), which carry a unique Cys in the N-terminal extracellular domain and a polyhistidine tag at the C terminus. Translocation of the N-terminal domain was detected using a membrane-impermeant gel shift reagent to derivatize the Cys residue of nascent radiolabeled molecules. Derivatization was assessed by gel electrophoresis of the fully elongated radiolabeled population. The time required to translocate and fully derivatize the Cys residues of I4C:H(6) and T5C:H(6) is 46 +/- 9 and 61 +/- 6 s, respectively. This is significantly shorter than the elongation times of the proteins, which are 114 +/- 26 and 169 +/- 16 s, respectively. These results establish that translocation of the bacterioopsin N terminus and insertion of the first transmembrane segment occur co-translationally and confirm the use of the assay to monitor the kinetics of polytopic membrane protein insertion in vivo. (+info)
Detergent-free membrane protein crystallization.
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A comprehensive understanding of structure-function relationships of proteins requires their structures to be elucidated to high resolution. With most membrane proteins this has not been accomplished so far, mainly because of their notoriously poor crystallizability. Here we present a completely detergent-free procedure for the incorporation of a native purple membrane into a monoolein-based lipidic cubic phase, and subsequent crystallization of three-dimensional bacteriorhodopsin crystals therein. These crystals exhibit comparable X-ray diffraction quality and mosaicity, and identical crystal habit and space group to those of bacteriorhodopsin crystals that are grown from detergent-solubilized protein in cubic phase. (+info)
Structural (shape-maintaining) role of the cell surface glycoprotein of Halobacterium salinarium.
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The obligate halophile, Halobacterium salinarium, maintains a rod-shaped morphology under normal growth conditions. Lactoperoxidase(EC 1.11.1.7;donor:hydrogen-peroxide oxidoreductase)-catalyzed iodination and treatment with proteolytic enzymes were used to demonstrate that the recently described envelope glycoprotein (Mescher, M.F. & Strominger, J.L. (1976) J. Biol. Chem. 251, 2005-2014) is the only major cell surface component of this organism. The morphological changes that accompany alteration of the structure of the glycoprotein by growth in the presence of bacitracin or its removal with proteolytic enzymes strongly suggest that it forms a rigid matrix at the cell surface and is responsible for maintenance of the characteristic rod shape. (+info)