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941.
MucR, a Novel Membrane-Associated Regulator of Alginate Biosynthesis in Pseudomonas aeruginosa
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Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3′-5′)-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic ΔmucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44. 相似文献
942.
Stephanie Thoms Klaas E.A. Max Michael Wunderlich Tomas Jacso Bernd Reif Franz X. Schmid 《Journal of molecular biology》2009,391(5):918-2651
In previous work, a strongly stabilized variant of the β1 domain of streptococcal protein G (Gβ1) was obtained by an in vitro selection method. This variant, termed Gβ1-M2, contains the four substitutions E15V, T16L, T18I, and N37L. Here we elucidated the molecular basis of the observed strong stabilizations. The contributions of these four residues were analyzed individually and in various combinations, additional selections with focused Gβ1 gene libraries were performed, and the crystal structure of Gβ1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type Gβ1 by contributions of between 1.6 and 6.0 kJ mol− 1 (at 70 °C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of Gβ1. They also increase the tendency to form dimers, as shown by dependence on the concentration of apparent molecular mass in analytical ultracentrifugation, by concentration-dependent stability, and by a strongly increased van't Hoff enthalpy of unfolding. The 0.88-Å crystal structure of Gβ1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two Gβ1-M2 molecules via six intermolecular hydrogen bonds between the two β strands 2 and 2′ and an adjacent self-complementary hydrophobic surface area, which is created by the T16L and N37L substitutions and a large 120° rotation of the Tyr33 side chain. This removal of hydrophilic groups and the malleability of the created hydrophobic surface provide the basis for the dimer formation of stabilized Gβ1 variants. 相似文献
943.
Fredrik I. Andersson Anders Tryggvesson Michal Sharon Alexander V. Diemand Mirjam Classen Christoph Best Ronny Schmidt Jenny Schelin Tara M. Stanne Bernd Bukau Carol V. Robinson Susanne Witt Axel Mogk Adrian K. Clarke 《The Journal of biological chemistry》2009,284(20):13519-13532
The Clp protease is conserved among eubacteria and most eukaryotes, and
uses ATP to drive protein substrate unfolding and translocation into a chamber
of sequestered proteolytic active sites. The main constitutive Clp protease in
photosynthetic organisms has evolved into a functionally essential and
structurally intricate enzyme. The model Clp protease from the cyanobacterium
Synechococcus consists of the HSP100 molecular chaperone ClpC and a
mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We
have purified the ClpP3/R complex, the first for a Clp proteolytic core
comprised of heterologous subunits. The ClpP3/R complex has unique functional
and structural features, consisting of twin heptameric rings each with an
identical ClpP33ClpR4 configuration. As predicted by its
lack of an obvious catalytic triad, the ClpR subunit is shown to be
proteolytically inactive. Interestingly, extensive modification to ClpR to
restore proteolytic activity to this subunit showed that its presence in the
core complex is not rate-limiting for the overall proteolytic activity of the
ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable
similarities to the 20 S core of the proteasome, revealing a far greater
degree of convergent evolution than previously thought between the development
of the Clp protease in photosynthetic organisms and that of the eukaryotic 26
S proteasome.Proteases perform numerous tasks vital for cellular homeostasis in all
organisms. Much of the selective proteolysis within living cells is performed
by multisubunit chaperone-protease complexes. These proteases all share a
common two-component architecture and mode of action, with one of the best
known examples being the proteasome in archaebacteria, certain eubacteria, and
eukaryotes (1).The 20 S proteasome is a highly conserved cylindrical structure composed of
two distinct types of subunits, α and β. These are organized in
four stacked heptameric rings, with two central β-rings sandwiched
between two outer α-rings. Although the α- and β-protein
sequences are similar, it is only the latter that is proteolytic active, with
a single Thr active site at the N terminus. The barrel-shaped complex is
traversed by a central channel that widens up into three cavities. The
catalytic sites are positioned in the central chamber formed by the
β-rings, adjacent to which are two antechambers conjointly built up by
β- and α-subunits. In general, substrate entry into the core
complex is essentially blocked by the α-rings, and thus relies on the
associating regulatory partner, PAN and 19 S complexes in archaea and
eukaryotes, respectively (1).
Typically, the archaeal core structure is assembled from only one type of
α- and β-subunit, so that the central proteolytic chamber contains
14 catalytic active sites (2).
In contrast, each ring of the eukaryotic 20 S complex has seven distinct
α- and β-subunits. Moreover, only three of the seven
β-subunits in each ring are proteolytically active
(3). Having a strictly
conserved architecture, the main difference between the 20 S proteasomes is
one of complexity. In mammalian cells, the three constitutive active subunits
can even be replaced with related subunits upon induction by
γ-interferon to generate antigenic peptides presented by the class 1
major histocompatibility complex
(4).Two chambered proteases architecturally similar to the proteasome also
exist in eubacteria, HslV and ClpP. HslV is commonly thought to be the
prokaryotic counterpart to the 20 S proteasome mainly because both are Thr
proteases. A single type of HslV protein, however, forms a proteolytic chamber
consisting of twin hexameric rather than heptameric rings
(5). Also displaying structural
similarities to the proteasome is the unrelated ClpP protease. The model Clp
protease from Escherichia coli consists of a proteolytic ClpP core
flanked on one or both sides by the ATP-dependent chaperones ClpA or ClpX
(6). The ClpP proteolytic
chamber is comprised of two opposing homo-heptameric rings with the catalytic
sites harbored within (7). ClpP
alone displays only limited peptidase activity toward short unstructured
peptides (8). Larger native
protein substrates need to be recognized by ClpA or ClpX and then translocated
in an unfolded state into the ClpP proteolytic chamber
(9,
10). Inside, the unfolded
substrate is bound in an extended manner to the catalytic triads (Ser-97,
His-122, and Asp-171) and degraded into small peptide fragments that can
readily diffuse out (11).
Several adaptor proteins broaden the array of substrates degraded by a Clp
protease by binding to the associated HSP100 partner and modifying its protein
substrate specificity (12,
13). One example is the
adaptor ClpS that interacts with ClpA (EcClpA) and targets N-end rule
substrates for degradation by the ClpAP protease
(14).Like the proteasome, the Clp protease is found in a wide variety of
organisms. Besides in all eubacteria, the Clp protease also exist in mammalian
and plant mitochondria, as well as in various plastids of algae and plants. It
also occurs in the unusual plastid in Apicomplexan protozoan
(15), a family of parasites
responsible for many important medical and veterinary diseases such as
malaria. Of all these organisms, photobionts have by far the most diverse
array of Clp proteins. This was first apparent in cyanobacteria, with the
model species Synechococcus elongatus having 10 distinct Clp
proteins, four HSP100 chaperones (ClpB1–2, ClpC, and ClpX), three ClpP
proteins (ClpP1–3), a ClpP-like protein termed ClpR, and two adaptor
proteins (ClpS1–2) (16).
Of particular interest is the ClpR variant, which has protein sequence
similarity to ClpP but appears to lack the catalytic triad of Ser-type
proteases (17). This diversity
of Clp proteins is even more extreme in photosynthetic eukaryotes, with at
least 23 different Clp proteins in the higher plant Arabidopsis
thaliana, most of which are plastid-localized
(18).We have recently shown that two distinct Clp proteases exist in
Synechococcus, both of which contain mixed proteolytic cores. The
first consists of ClpP1 and ClpP2 subunits, and associates with ClpX, whereas
the other has a proteolytic core consisting of ClpP3 and ClpR that binds to
ClpC, as do the two ClpS adaptors
(19). Of these proteases, it
is the more constitutively abundant ClpCP3/R that is essential for cell
viability and growth (20,
21). It is also the ClpP3/R
complex that is homologous to the single type in eukaryotic plastids, all of
which also have ClpC as the chaperone partner
(16). In algae and plants,
however, the complexity of the plastidic Clp proteolytic core has evolved
dramatically. In Arabidopsis, the core complex consists of five ClpP
and four ClpR paralogs, along with two unrelated Clp proteins unique to higher
plants (22). Like ClpP3/R, the
plastid Clp protease in Arabidopsis is essential for normal growth
and development, and appears to function primarily as a housekeeping protease
(23,
24).One of the most striking developments in the Clp protease in photosynthetic
organisms and Apicomplexan parasites is the inclusion of ClpR within the
central proteolytic core. Although this type of Clp protease has evolved into
a vital enzyme, little is known about its activity or the exact role of ClpR
within the core complex. To address these points we have purified the intact
Synechococcus ClpP3/R proteolytic core by co-expression in E.
coli. The recombinant ClpP3/R forms a double heptameric ring complex,
with each ring having a specific ClpP3/R stoichiometry and arrangement.
Together with ClpC, the ClpP3/R complex degrades several polypeptide
substrates, but at a rate considerably slower than that by the E.
coli ClpAP protease. Interestingly, although ClpR is shown to be
proteolytically inactive, its inclusion in the core complex is not
rate-limiting to the overall activity of the ClpCP3/R protease. In general,
the results reveal remarkable similarities between the evolutionary
development of the Clp protease in photosynthetic organisms and the eukaryotic
proteasome relative to their simpler prokaryotic counterparts. 相似文献
944.
Friederike Hoffmann Regina Radax Dagmar Woebken Moritz Holtappels Gaute Lavik Hans Tore Rapp Marie-Lise Schläppy Christa Schleper Marcel M. M. Kuypers 《Environmental microbiology》2009,11(9):2228-2243
Marine sponges constitute major parts of coral reefs and deep‐water communities. They often harbour high amounts of phylogenetically and physiologically diverse microbes, which are so far poorly characterized. Many of these sponges regulate their internal oxygen concentration by modulating their ventilation behaviour providing a suitable habitat for both aerobic and anaerobic microbes. In the present study, both aerobic (nitrification) and anaerobic (denitrification, anammox) microbial processes of the nitrogen cycle were quantified in the sponge Geodia barretti and possible involved microbes were identified by molecular techniques. Nitrification rates of 566 nmol N cm?3 sponge day?1 were obtained when monitoring the production of nitrite and nitrate. In support of this finding, ammonia‐oxidizing Archaea (crenarchaeotes) were found by amplification of the amoA gene, and nitrite‐oxidizing bacteria of the genus Nitrospira were detected based on rRNA gene analyses. Incubation experiments with stable isotopes (15NO3‐ and 15NH4+) revealed denitrification and anaerobic ammonium oxidation (anammox) rates of 92 nmol N cm?3 sponge day?1 and 3 nmol N cm?3 sponge day?1 respectively. Accordingly, sequences closely related to ‘Candidatus Scalindua sorokinii’ and ‘Candidatus Scalindua brodae’ were detected in 16S rRNA gene libraries. The amplification of the nirS gene revealed the presence of denitrifiers, likely belonging to the Betaproteobacteria. This is the first proof of anammox and denitrification in the same animal host, and the first proof of anammox and denitrification in sponges. The close and complex interactions of aerobic, anaerobic, autotrophic and heterotrophic microbial processes are fuelled by metabolic waste products of the sponge host, and enable efficient utilization and recirculation of nutrients within the sponge–microbe system. Since denitrification and anammox remove inorganic nitrogen from the environment, sponges may function as so far unrecognized nitrogen sinks in the ocean. In certain marine environments with high sponge cover, sponge‐mediated nitrogen mineralization processes might even be more important than sediment processes. 相似文献
945.
Stefan Kirsch Juanjo Pasantes Andreas Wolf Nadia Bogdanova Claudia Münch Arseni Markoff Petra Pennekamp Michael Krawczak Bernd Dworniczak Werner Schempp 《BMC evolutionary biology》2009,9(1):14-1
Correction to Kirsch S, Pasantes J, Wolf A, Bogdanova N, Münch C, Pennekamp P, Krawczak M, Dworniczak B, Schempp W: Chromosomal evolution of the
PKD1
gene family in primates. BMC Evolutionary Biology 2008, 8 :263 (doi:10.1186/1471-2148-8-263) 相似文献
946.
Panos N Papapanou Jan H Behle Moritz Kebschull Romanita Celenti Dana L Wolf Martin Handfield Paul Pavlidis Ryan T Demmer 《BMC microbiology》2009,9(1):221
Background
Periodontitis is a chronic inflammatory disease caused by the microbiota of the periodontal pocket. We investigated the association between subgingival bacterial profiles and gene expression patterns in gingival tissues of patients with periodontitis. A total of 120 patients undergoing periodontal surgery contributed with a minimum of two interproximal gingival papillae (range 2-4) from a maxillary posterior region. Prior to tissue harvesting, subgingival plaque samples were collected from the mesial and distal aspects of each tissue sample. Gingival tissue RNA was extracted, reverse-transcribed, labeled, and hybridized with whole-genome microarrays (310 in total). Plaque samples were analyzed using checkerboard DNA-DNA hybridizations with respect to 11 bacterial species. Random effects linear regression models considered bacterial levels as exposure and expression profiles as outcome variables. Gene Ontology analyses summarized the expression patterns into biologically relevant categories. 相似文献947.
Martin Frenkel Carsten Külheim Hanna Johansson Jänkänpää Oskar Skogström Luca Dall'Osto Jon Ågren Roberto Bassi Thomas Moritz Jon Moen Stefan Jansson 《BMC plant biology》2009,9(1):12-16
Background
Plant performance is affected by the level of expression of PsbS, a key photoprotective protein involved in the process of feedback de-excitation (FDE), or the qE component of non-photochemical quenching, NPQ. 相似文献948.
Background
Differentiation of long and short shoots is an important developmental trait in several species of the Rosaceae family. However, the physiological mechanisms controlling this differentiation are largely unknown. We have studied the role of gibberellin (GA) in regulation of shoot differentiation in strawberry (Fragaria × ananassa Duch.) cv. Korona. In strawberry, differentiation of axillary buds to runners (long shoot) or to crown branches (short shoot) is promoted by long-day and short-day conditions, respectively. Formation of crown branches is a prerequisite for satisfactory flowering because inflorescences are formed from the apical meristems of the crown. 相似文献949.
Johannes Hirrlinger Anja Scheller Petra G. Hirrlinger Beate Kellert Wannan Tang Michael C. Wehr Sandra Goebbels Andreas Reichenbach Rolf Sprengel Moritz J. Rossner Frank Kirchhoff 《PloS one》2009,4(1)
Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting. 相似文献
950.
Stefano Di Santo Zijiang Yang Moritz Wyler von Ballmoos Jan Voelzmann Nicolas Diehm Iris Baumgartner Christoph Kalka 《PloS one》2009,4(5)