Expression, crystallization and preliminary X-ray analysis of an outer membrane protein from<i>Thermus thermophilus</i>HB27

Brosig, Alexander; Nesper, Jutta; Welte, Wolfram; Diederichs, Kay

doi:10.1107/s1744309108013602

Cited by 4 publications

(5 citation statements)

References 14 publications

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“…TtOmp85 with an N-terminal Strep tag and TtoA with an N-terminal His tag were expressed in T. thermophilus HB27 cells transformed with derivatives of plasmid pMK18 as described previously. , Growth of transformed bacteria was performed according to the method described in ref . In brief, T. thermophilus was grown at 70 °C in autoclaved medium containing 8 g/L Trypticase, 4 g/L yeast extract, and 3 g/L NaCl dissolved in distilled water (pH 7.5) supplemented with 100 μg/mL kanamycin.…”

Section: Methodsmentioning

confidence: 99%

TtOmp85, a β-Barrel Assembly Protein, Functions by Barrel Augmentation

et al. 2015

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Outer membrane proteins are vital for Gram-negative bacteria and organisms that inherited organelles from them. Proteins from the Omp85/BamA family conduct the insertion of membrane proteins into the outer membrane. We show that an eight-stranded outer membrane β-barrel protein, TtoA, is inserted and folded into liposomes by an Omp85 homologue. Furthermore, we recorded the channel conductance of this Omp85 protein in black lipid membranes, alone and in the presence of peptides comprising the sequence of the two N-terminal and the two C-terminal β-strands of TtoA. Only with the latter could a long-living compound channel that exhibits conductance levels higher than those of the Omp85 protein alone be observed. These data support a model in which unfolded outer membrane protein after docking with its C-terminus penetrates into the transmembrane β-barrel of the Omp85 protein and augments its β-sheet at the first strand. Augmentation with successive β-strands leads to a compound, dilated barrel of both proteins.

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Section: Methodsmentioning

confidence: 99%

TtOmp85, a β-Barrel Assembly Protein, Functions by Barrel Augmentation

et al. 2015

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“…The proteins from thermophiles exhibit unusually high stabilities and have a greater ability to crystallize compared with their mesophilic counterparts, which makes evident the relevance of the T. thermophilus DNA translocator as a model for further studies at the structural level. The crystallization potential of T. thermophilus proteins is supported by numerous reports of successful crystallization of T. thermophilus proteins and protein complexes, including V‐ATPase, isocitrate dehydrogenase, uroporphyrinogen II synthase, an outer membrane protein and 360 proteins of T. thermophilus HB8; the latter were successfully crystallized in a whole cell‐project (Toei et al , 2007; Brosig et al , 2008; Iino et al , 2008; Ishii et al , 2008; Schubert et al , 2008).…”

Section: Introductionmentioning

confidence: 99%

Shuffling genes around in hot environments: the unique DNA transporter ofThermus thermophilus

Averhoff

2009

FEMS Microbiol Rev

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Natural transformation permits the transport of DNA through bacterial membranes and represents a dominant mode for the transfer of genetic information between bacteria and between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal, or lateral, gene transfer, has been a major force for genome plasticity over evolutionary history, and is largely responsible for the spread of fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Here, we present a survey of the natural transformation machinery of the thermophile Thermus thermophilus HB27. A tentative model of the transformation machinery comprising of components similar to proteins of type IV pili and type II secretion systems is presented. A comparative discussion of the subunits and the structure of the DNA translocator and the underlying mechanism of transfer of free DNA in T. thermophilus highlights conserved and unique features of the DNA translocator in T. thermophilus. We hypothesize that the extraordinary broad substrate specificity and the high efficiency of the T. thermophilus DNA uptake system is of major importance for thermoadaptation and interdomain DNA transfer in hot environments.

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“…2A). As has been done to investigate various types of OM proteins, including other iron transporters (22,23,50,89,106,122,127,130), we introduced a His 6 -tagged version of LbtU into 130b and then used anti-His antibodies and immunoblot analysis to track the location of LbtU in cellular fractions. LbtU-His 6 did not diminish the growth of the wild type and complemented an iron-related growth defect of an lbtU mutant (see below, Fig.…”

Section: Vol 193 2011mentioning

confidence: 99%

Legionella pneumophila LbtU Acts as a Novel, TonB-Independent Receptor for the Legiobactin Siderophore

et al. 2011

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Gram-negative Legionella pneumophila produces a siderophore (legiobactin) that promotes lung infection. We previously determined that lbtA and lbtB are required for the synthesis and secretion of legiobactin. DNA sequence and reverse transcription-PCR (RT-PCR) analyses now reveal the presence of an iron-repressed gene (lbtU) directly upstream of the lbtAB-containing operon. In silico analysis predicted that LbtU is an outer membrane protein consisting of a 16-stranded transmembrane ␤-barrel, multiple extracellular domains, and short periplasmic tails. Immunoblot analysis of cell fractions confirmed an outer membrane location for LbtU. Although replicating normally in standard media, lbtU mutants, like lbtA mutants, were impaired for growth on iron-depleted agar media. While producing typical levels of legiobactin, lbtU mutants were unable to use supplied legiobactin to stimulate growth on iron-depleted media and displayed an inability to take up iron. Complemented lbtU mutants behaved as the wild type did. The lbtU mutants were also impaired for infection in a legiobactin-dependent manner. Together, these data indicate that LbtU is involved in the uptake of legiobactin and, based upon its location, is most likely the Legionella siderophore receptor. The sequence and predicted two-dimensional (2D) and 3D structures of LbtU were distinct from those of all known siderophore receptors, which generally contain a 22-stranded ␤-barrel and an extended N terminus that binds TonB in order to transduce energy from the inner membrane. This observation coupled with the fact that L. pneumophila does not encode TonB suggests that LbtU is a new type of receptor that participates in a form of iron uptake that is mechanistically distinct from the existing paradigm.

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Expression, crystallization and preliminary X-ray analysis of an outer membrane protein fromThermus thermophilusHB27

Cited by 4 publications

References 14 publications

TtOmp85, a β-Barrel Assembly Protein, Functions by Barrel Augmentation

TtOmp85, a β-Barrel Assembly Protein, Functions by Barrel Augmentation

Shuffling genes around in hot environments: the unique DNA transporter ofThermus thermophilus

Legionella pneumophila LbtU Acts as a Novel, TonB-Independent Receptor for the Legiobactin Siderophore

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