Structure of the HPI-Layer of Micrococcus radiodurans

Kübler, Olaf; Engel, Andréas; Zingsheim, H. P.; Emde, Barbara; Hahn, M.; Heiße, W.; Baumeister, Wolfgang

doi:10.1007/978-3-642-67688-8_2

Cited by 13 publications

(24 citation statements)

References 8 publications

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“…). Saprophytes like M. aurum and M. smegmatis produce carotenoids, which are known scavengers of free radicals (Levy‐Frebault and David ) and enhance the strength of the cell wall due to their lipophilic nature and intercalation into the cell membrane (Kubler and Baumeister ). M. smegmatis mc 2 155 colonies produce pale yellow pigment (carotenoid isorenieratene) when incubated under light for 5–6 days.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Mycobacterium smegmatis sigF mutant and its regulon: overexpression of SigF antagonist (MSMEG_1803) in M. smegmatis mimics sigF mutant phenotype, loss of pigmentation, and sensitivity to oxidative stress

et al. 2015

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In Mycobacterium smegmatis, sigF is widely expressed during different growth stages and plays role in adaptation to stationary phase and oxidative stress. Using a sigF deletion mutant of M. smegmatis mc2155, we demonstrate that SigF is not essential for growth of bacterium. Deletion of sigF results in loss of carotenoid pigmentation which rendered increased susceptibility to H2O2 induced oxidative stress in M. smegmatis. SigF modulates the cell surface architecture and lipid biosynthesis extending the repertoire of SigF function in this species. M. smegmatis SigF regulon included variety of genes expressed during exponential and stationary phases of growth and those responsible for oxidative stress, lipid biosynthesis, energy, and central intermediary metabolism. Furthermore, we report the identification of a SigF antagonist, an anti‐sigma factor (RsbW), which upon overexpression in M. smegmatis wild type strain produced a phenotype similar to M. smegmatis mc2155 ΔsigF strain. The SigF‐anti‐SigF interaction is duly validated using bacterial two‐hybrid and pull down assays. In addition, anti‐sigma factor antagonists, RsfA and RsfB were identified and their interactions with anti‐sigma factor were experimentally validated. Identification of these proteins will help decode regulatory circuit of this alternate sigma factor.

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Section: Resultsmentioning

confidence: 99%

Characterization of Mycobacterium smegmatis sigF mutant and its regulon: overexpression of SigF antagonist (MSMEG_1803) in M. smegmatis mimics sigF mutant phenotype, loss of pigmentation, and sensitivity to oxidative stress

et al. 2015

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“…The fourth layer is the outer membrane, and the fifth layer is a distinct electrolucent zone. The sixth layer consists of regularly packed hexagonal protein subunits (the S-layer, or hexagonally packed intermediate layer), typical of other bacterial S-layers (26,115,206). A few strains of Deinococcus also exhibit a dense carbohydrate coat (25,26,118,187,205,208,221).…”

Section: Cell Structurementioning

confidence: 99%

Genome of the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans Viewed from the Perspective of Comparative Genomics

Makarova

Aravind

Wolf

et al. 2001

Microbiol Mol Biol Rev

646

612

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The bacterium Deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. These characteristics were the impetus for sequencing the genome of D. radiodurans and the ongoing development of its use for bioremediation of radioactive wastes. Although it is known that these multiple resistance phenotypes stem from efficient DNA repair processes, the mechanisms underlying these extraordinary repair capabilities remain poorly understood. In this work we present an extensive comparative sequence analysis of the Deinococcus genome. Deinococcus is the first representative with a completely sequenced genome from a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, supports the hypothesis that it is an ancient group with no clear affinities to any of the other known bacterial lineages. Distinctive features of the Deinococcus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to the collection of clusters of orthologous groups of proteins. Analysis of paralogs in Deinococcus has revealed several unique protein families. In addition, specific expansions of several other families including phosphatases, proteases, acyltransferases, and Nudix family pyrophosphohydrolases were detected. Genes that potentially affect DNA repair and recombination and stress responses were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes and are not present in other bacteria. For example, three proteins homologous to plant desiccation resistance proteins were identified, and these are particularly interesting because of the correlation between desiccation and radiation resistance. Compared to other bacteria, the D. radiodurans genome is enriched in repetitive sequences, namely, IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism

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“…The structural and chemical characterization of the Slayer of Deinococcus radiodurans, the so-called HPI-layer (23), is particularly advanced. The HPI-layer has been investigated with a variety of electron microscopic techniques (6,7,14), and recently a determination of its three-dimensional structure to a resolution of 1.5 nm has been completed (4).…”

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confidence: 99%

Molecular cloning, expression, and characterization of the gene for the surface (HPI)-layer protein of Deinococcus radiodurans in Escherichia coli

Peters¹,

Baumeister²

1986

J Bacteriol

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The HPI protein of Deinococcus radiodurans belongs to the ciass of surface layer proteins which form crystalline two-dimensional arrays on bacterial cell envelopes. We have cloned and expressed the gene for this protein of Mr about 100,000 by using plasmid pUC8 in Escherichia coli. As judged by immunoreaction with monospecific antibodies, apparent Mr, and limited proteolysis, a single clone contained the gene encoding the complete polypeptide on an 8.9-kilobase (kb) insert. The insert was reduced to a 5.7-kb HindIlI fragment, cloned in pUC18 in both orientations, and subjected to unilateral processive deletion with exonuclease III. The library of deletion derivatives was mapped and, in conjunction with protein immunoblotting of expressed polypeptides, was used to locate the positions of the structural gene and the Deinococcus promoter region that was responsible for expression of the HPI polypeptide. The HPI gene was confined to a stretch 2.95 kb in length.Regular surface layers (S-layers) of protein or glycoprotein are quite common among eubacteria as well as archaebacteria. A variety of functions, all related to interactions between the cell and its environment, has been ascribed to these layers (for recent reviews, see references 9 and 30).The structural and chemical characterization of the Slayer of Deinococcus radiodurans, the so-called HPI-layer (23), is particularly advanced. The HPI-layer has been investigated with a variety of electron microscopic techniques (6,7,14), and recently a determination of its three-dimensional structure to a resolution of 1.5 nm has been completed (4). Measurements of mass, performed with a scanning transmission electron microscope, in conjunction with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis have shown that the unit cell (or the morphological complex) contains six identical polypeptides, each with a molecular weight of about 100,000 (5).Our long-term objective is a high-resolution determination of the structure of the HPI-layer. Another line of investigation aims to elucidate the evolutionary relationships of eubacterial and archaebacterial S-layers. In any case, knowledge of the primary structure is indispensable. A molecular genetic approach to sequence analysis is particularly attractive, as it also creates a perspective for site-directed mutagenesis, which will be an important tool to address questions of function and assembly. Here we report the cloning of the gene for the HPI polypeptide and its characterization and expression in Escherichia coli. Using unilateral deletions generated with exonuclease III, we have mapped the position of the promoter region and the structural gene and constructed an ordered subset of deletion derivatives. MATERIALS AND METHODSMaterials and bacterial strains. Enzymes, nucleotides, M13 reverse sequencing primer, DNA-Mr markers, protein A-Sepharose, and Percoll were bought from Pharmacia P-L, * Corresponding author. D. radiodurans Sark (UWO 298) was kindly provided by R. G. E. Murray, University of Western Ontario, Lo...

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Structure of the HPI-Layer of Micrococcus radiodurans

Cited by 13 publications

References 8 publications

Characterization of Mycobacterium smegmatis sigF mutant and its regulon: overexpression of SigF antagonist (MSMEG_1803) in M. smegmatis mimics sigF mutant phenotype, loss of pigmentation, and sensitivity to oxidative stress

Characterization of Mycobacterium smegmatis sigF mutant and its regulon: overexpression of SigF antagonist (MSMEG_1803) in M. smegmatis mimics sigF mutant phenotype, loss of pigmentation, and sensitivity to oxidative stress

Genome of the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans Viewed from the Perspective of Comparative Genomics

Molecular cloning, expression, and characterization of the gene for the surface (HPI)-layer protein of Deinococcus radiodurans in Escherichia coli

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