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1.
Kimihiro Abe Yuta Kawano Keito Iwamoto Kenji Arai Yuki Maruyama Patrick Eichenberger Tsutomu Sato 《PLoS genetics》2014,10(10)
Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection. 相似文献
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Six different restriction endonucleases were used to generate restriction fragment maps of the genome of the temperate Bacillus subtilis phage SPβ. AvaI and SalI each had six target sites in the phage DNA, AvaII had three, BamHI had seven, PstI had twenty, and SacI had sixteen. Restriction analysis and heteroduplex analysis were used to locate a 10-kb region of DNA that is deleted in the clear-plaque mutant, spβci. Thedeletion lay approx. 50 kb from the left end of the 126-kb phage genome. 相似文献
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Datta SA Temeselew LG Crist RM Soheilian F Kamata A Mirro J Harvin D Nagashima K Cachau RE Rein A 《Journal of virology》2011,85(9):4111-4121
Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch. 相似文献
7.
Physical and chemical characteristics of sediment were analyzed at eight sampling areas in the estuarine region of Cananéia (SP), Brazil. The samples were collected in 1995 during four periods; summer, autumn, winter and spring. Total nitrogen and phosphorus concentrations, redox potential, water and organic matter percentage, sand and silt percentage and grain size were determined in six different layers of 20 cm deep columns of sediment. In general, the nutrient concentration and the percentage of organic matter decreased with depth and greater variations were observed in the first centimeter of the samples. Differences in nutrient concentration and organic matter were also observed during the study period. The highest concentrations were recorded during the rainy season (February) as a consequence of an accumulation of organic matter brought into the system by rivers and adjacent flooded areas. In general, the sediments had oxidized characteristics until the 5 or 10 cm depth in areas near the rich coastal vegetation. However, in areas open to the ocean, the entire sediment columns were almost completely oxidized. 相似文献
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Nik A. B. N. Mahmood Esther Biemans-Oldehinkel Bert Poolman 《The Journal of biological chemistry》2009,284(21):14368-14376
We have previously shown that the C-terminal cystathionine β-synthase
(CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA,
in conjunction with an anionic membrane surface function, act as sensor of
internal ionic strength (Iin). Here, we show that a
surface-exposed cationic region in the CBS module domain is critical for ion
sensing. The consecutive substitution of up to five cationic residues led to a
gradual decrease of the ionic strength dependence of transport. In fact, a
5-fold mutant was essentially independent of salt in the range from 0 to 250
mm KCl (or NaCl), supplemented to medium of 30 mm
potassium phosphate. Importantly, the threshold temperature for transport was
lowered by 5–7 °C and the temperature coefficient
Q10 was lowered from 8 to ∼1.5 in the 5-fold mutant,
indicating that large conformational changes are accompanying the CBS-mediated
regulation of transport. Furthermore, by replacing the anionic C-terminal tail
residues that extend the CBS module with histidines, the transport of OpuA
became pH-dependent, presumably by additional charge interactions of the
histidine residues with the membrane. The pH dependence was not observed at
high ionic strength. Altogether the analyses of the CBS mutants support the
notion that the osmotic regulation of OpuA involves a simple biophysical
switching mechanism, in which nonspecific electrostatic interactions of a
protein module with the membrane are sufficient to lock the transporter in the
inactive state.In their natural habitats microorganisms are often exposed to changes in
the concentration of solutes in the environment
(1). A sudden increase in the
medium osmolality results in loss of water from the cell, loss of turgor, a
decrease in cell volume, and an increase in intracellular osmolyte
concentration. Osmoregulatory transporters such as OpuA in Lactococcus
lactis, ProP in Escherichia coli, and BetP in
Corynebacterium glutamicum diminish the consequences of the osmotic
stress by mediating the uptake of compatible solutes upon an increase in
extracellular osmolality
(2–4).
For the ATP-binding cassette
(ABC)5 transporter
OpuA, it has been shown that the system, reconstituted in proteoliposomes, is
activated by increased concentrations of lumenal ions (increased internal
ionic strength) (2,
5,
6). This activation is
instantaneous both in vivo and in vitro and only requires
threshold levels of ionic osmolytes. Moreover, the ionic threshold for
activation is highly dependent of the ionic lipid content (charge density) of
the membrane and requires the presence of so-called cystathionine
β-synthase (CBS) domains, suggesting that the ionic signal is transduced
to the transporter via critical interactions of the protein with membrane
lipids.The ABC transporter OpuA consists of two identical nucleotide-binding
domains (NBD) fused to CBS domains and two identical substrate-binding domains
fused to transmembrane domains. The NBD-CBS and substrate-binding
domain-transmembrane domain subunits are named OpuAA and OpuABC, respectively.
Two tandem CBS domains are linked to the C-terminal end of the NBD; each
domain (CBS1 and CBS2) has a β-α-β-β-α secondary
structure (5)
(Fig. 1A). The CBS
domains are widely distributed in most if not all species of life but their
function is largely unknown. Most of the CBS domains are found as tandem
repeats but data base searches have also revealed tetra-repeat units
(5). The crystal structures of
several tandem CBS domains have been elucidated
(7–9,
32), and in a number of cases
it has been shown that two tandem CBS domains form dimeric structures with a
total of four CBS domains per structural module (hereafter referred to as CBS
module). The crystal structures of the full-length MgtE Mg2+
transporter confirm the dimeric configuration and show that the CBS domains
undergo large conformational changes upon Mg2+ binding or release
(10,
11). In general, ABC
transporters are functional as dimers, which implies that two tandem CBS
domains are present in the OpuA complex. Preliminary experiments with
disulfides engineered at the interface of two tandem CBS domains in OpuA
suggest that large structural rearrangements (association-dissociation of the
interfaces) play a determining role in the ionic strength-regulated transport.
Finally, a subset of CBS-containing proteins has a C-terminal extension, which
in OpuA is highly anionic (sequence: ADIPDEDEVEEIEKEEENK) and modulates the
ion sensing activity (6).Open in a separate windowFIGURE 1.Domain structure of CBS module of OpuA. A, sequence of
tandem CBS domains. The predicted secondary structure is indicated
above the sequence. The residues modified in this study are
underlined. The amino acid sequence end-points of OpuAΔ61 and
OpuAΔ119 are indicated by vertical arrows. B, homology
model of tandem CBS domain of OpuA. The CBS domains were individually modeled
on the crystal structure of the tandem CBS protein Ta0289 from T.
acidophilum (PDB entry 1PVM), using Phyre. Ta0289 was used for the
initial modeling, because its primary sequence was more similar to the CBS
domains of OpuA than those of the other crystallized CBS proteins. The
individual domain models were then assembled with reference to the atomic
coordinates of the tandem CBS domains of IMPDH from Streptococcus
pyogenes (PDB entry 1ZFJ) to form the tandem CBS pair, using PyMOL
(DeLano). The positions of the (substituted) cationic residues are
indicated.In this study, we have engineered the surface-exposed cationic residues of
the CBS module and the C-terminal anionic tail of OpuA
(Fig. 1B). The ionic
strength and lipid dependence of the OpuA mutants were determined in
vivo and in vitro. We show that substitution of five cationic
residues for neutral amino acids is sufficient to inactivate the ionic
strength sensor and convert OpuA into a constitutively active transporter.
Moreover, by substituting six anionic plus four neutral residues of the
C-terminal anionic tail for histidines, the transport reaction becomes
strongly pH-dependent. 相似文献
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Intracellular β-Carbonic Anhydrase of the Unicellular Green
Alga Coccomyxa
: Cloning of the cDNA and Characterization of the Functional
Enzyme Overexpressed in Escherichia coli
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Thomas Hiltonen Harry Bj?rkbacka Cecilia Forsman Adrian K. Clarke G?ran Samuelsson 《Plant physiology》1998,117(4):1341-1349
Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic. 相似文献
13.
SP—A的分子生物学 总被引:2,自引:0,他引:2
陈政良 《国外医学:分子生物学分册》1997,19(6):241-245
肺表面活性物质蛋白A(SP-A)是一个约650kD的寡聚体巨分子糖蛋白,人SP-A基因座位由2个功能基因和1个假基因所组成并表现极大的变异性,其表达调控机制极其复杂,它不权对正常肺功能的维持起关键作用,而且在肺部局部防御及免疫调节中扮演重要角色。 相似文献
14.
The DSM-IV major depression "bereavement exclusion" (BE), which recognizes that depressive symptoms are sometimes normal in recently bereaved individuals, is proposed for elimination in DSM-5. Evidence cited for the BE's invalidity comes from two 2007 reviews purporting to show that bereavement-related depression is similar to other depression across various validators, and a 2010 review of subsequent research. We examined whether the 2007 and 2010 reviews and subsequent relevant literature support the BE's invalidity. Findings were: a) studies included in the 2007 reviews sampled bereavement-related depression groups most of whom were not BE-excluded, making them irrelevant for evaluating BE validity; b) three subsequent studies cited by the 2010 review as supporting BE elimination did examine BE-excluded cases but were in fact inconclusive; and c) two more recent articles comparing recurrence of BE-excluded and other major depressive disorder cases both support the BE's validity. We conclude that the claimed evidence for the BE's invalidity does not exist. The evidence in fact supports the BE's validity and its retention in DSM-5 to prevent false positive diagnoses. We suggest some improvements to increase validity and mitigate risk of false negatives. 相似文献
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The action of the environmental toxic Pb2+ on photosynthetic electron transport was studied in thylakoid membranes isolated from spinach leaves. Fluorescence and thermoluminescence techniques were performed in order to determine the mode of Pb2+ action in photosystem II (PSII). The invariance of fluorescence characteristics of chlorophyll a (Chl a) and magnesium tetraphenylporphyrin (MgTPP), a molecule structurally analogous to Chl a, in the presence of Pb2+ confirms that Pb cation does not interact directly with chlorophyll molecules in PSII. The results show that Pb interacts with the water oxidation complex thus perturbing charge recombination between the quinone acceptors of PSII and the S2 state of the Mn4Ca cluster. Electron transfer between the quinone acceptors QA and QB is also greatly retarded in the presence of Pb2+. This is proposed to be owing to a transmembrane modification of the acceptor side of the photosystem. 相似文献
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Nidhi Ahuja Dmitry Korkin Rachna Chaba Brent O. Cezairliyan Robert T. Sauer Kyeong Kyu Kim Carol A. Gross 《The Journal of biological chemistry》2009,284(8):5403-5413
The Escherichia coli envelope stress response is controlled by the
alternative sigma factor, σE, and is induced when unfolded
outer membrane proteins accumulate in the periplasm. The response is initiated
by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB
is an important negative regulator of envelope stress response that exerts its
negative effects onσE activity through its binding to RseA.
In this study, we analyze the interaction between RseA and RseB. We found that
tight binding of RseB to RseA required intact RseB. Using programs that
performed global and local sequence alignment of RseB and RseA, we found
regions of high similarity and performed alanine substitution mutagenesis to
test the hypothesis that these regions were functionally important. This
protocol is based on the hypothesis that functionally dependent regions of two
proteins co-evolve and therefore are likely to be sequentially conserved. This
procedure allowed us to identify both an N-terminal and C-terminal region in
RseB important for binding to RseA. We extensively analyzed the C-terminal
region, which aligns with a region of RseA coincident with the major RseB
binding determinant in RseA. Both allele-specific suppression analysis and
cysteine-mediated disulfide bond formation indicated that this C-terminal
region of similarity of RseA and RseB identifies a contact site between the
two proteins. We suggest a similar protocol can be successfully applied to
pairs of non-homologous but functionally linked proteins to find specific
regions of the protein sequences that are important for establishing
functional linkage.The Escherichia coli σE-mediated envelope stress
response is the major pathway to ensure homeostasis in the envelope
compartment of the cell
(1-3).
σE regulon members encode periplasmic chaperones and
proteases, the machinery for inserting β-barrel proteins into the outer
membrane and components controlling the synthesis and assembly of LPS
(4-6).
This pathway is highly conserved among γ-proteobacteria
(6).The σE response is initiated when periplasmic protein
folding and assembly is compromised
(7-9).
During steady state growth, σE is inhibited by its antisigma
factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to
σE with picomolar affinity
(10-13).
Accumulation of unassembled porin monomers serves as a signal to activate the
DegS protease to cleave RseA in its periplasmic domain
(14,
15). This initiates a
proteolytic cascade in which RseP cleaves periplasmically truncated RseA near
or within the cytoplasmic membrane to release the
RseAcytoplasmic-σE complex, and cytoplasmic
ATP-dependent proteases complete the degradation of RseA thereby releasing
active σE
(16-19).RseB, a second negative regulator of the envelope stress response
(11,
20,
21), binds to the periplasmic
domain of RseA with nanomolar affinity. RseB is an important regulator of the
response (2,
22,
23). It prevents RseP from
degrading intact RseA, thereby ensuring that proteolysis is initiated only
when the DegS protease is activated by a stress signal
(21). Additionally, RseB
prevents activated DegS from cleaving RseA, suggesting that interaction of
RseB with RseA must be altered before the signal transduction cascade is
activated (23).The goal of the present studies was to explore how RseB binds to RseA. The
interaction partner of RseB is the unstructured periplasmic domain of RseA
(RseA-peri). Within RseA-peri, amino acids ∼169-186 constitute a major
binding determinant to RseB
(23,
24). This peptide alone binds
RseB with 6 μm affinity, and deleting this region abrogates
binding to RseB (23).
Additional regions of RseA-peri also contribute to RseB binding, as intact
RseA-peri binds with 20 nm affinity to RseB
(23). Much less is known about
the regions of RseB required for interaction with RseA. RseB is homodimeric
two-domain protein, whose large N-terminal domain shares structural homology
with LolA, a protein that transports lipoproteins to outer membrane
(24,
25). The smaller C-terminal
domain is connected to the N-terminal domain by a linker, and the two domains
share a large interface, which may facilitate interdomain signaling.
Glutaraldehyde cross-linking studies indicate that the C-terminal domain
interacts with RseA, but the regions of interaction were not identified
(25).In the present report, we study the interaction of RseB and RseA. We
establish that both domains of RseB interact with RseA-peri. Using a global
sequence alignment, we discovered several regions in RseA and RseB that had
high sequence similarity, despite the low overall sequence similarity between
these two proteins, a finding that was independently confirmed by a local
sequence similarity algorithm. This suggested that these regions were
functionally dependent, and we performed a set of mutagenesis experiments
designed to test this idea. Our studies of the binding properties of these
mutants revealed that regions in both the N terminus and C terminus of RseB
modulate interaction with RseA. Moreover, genetic suppression analysis and
cysteine-mediated disulfide bond formation suggest that the region of RseA/B
with highest similarity (RseA residues 165-191 (major binding determinant in
RseA) and RseB residues 233-258) are interacting partners. 相似文献
19.
Mar��a-Natalia Lisa Lars Hemmingsen Alejandro J. Vila 《The Journal of biological chemistry》2010,285(7):4570-4577
Metallo-β-lactamases (MβLs) stand as one of the main
mechanisms of bacterial resistance toward carbapenems. The rational design of an
inhibitor for MβLs has been limited by an incomplete knowledge of
their catalytic mechanism and by the structural diversity of their active sites.
Here we show that the MβL GOB from Elizabethkingia
meningoseptica is active as a monometallic enzyme by using
different divalent transition metal ions as surrogates of the native Zn(II) ion.
Of the metal derivatives in which Zn(II) is replaced, Co(II) and Cd(II) give
rise to the most active enzymes and are shown to occupy the same binding site as
the native ion. However, Zn(II) is the only metal ion capable of stabilizing an
anionic intermediate that accumulates during nitrocefin hydrolysis, in which the
C–N bond has already been cleaved. This finding demonstrates that the
catalytic role of the metal ion in GOB is to stabilize the formation of this
intermediate prior to nitrogen protonation. This role may be general to all
MβLs, whereas nucleophile activation by a Zn(II) ion is not a
conserved mechanistic feature. 相似文献
20.
Bdellovibrio bacteriovorus is a Gram-negative bacterium that is a pathogen of other Gram-negative bacteria, including many bacteria which are pathogens of humans, animals and plants. As such Bdellovibrio has potential as a biocontrol agent, or living antibiotic. B. bacteriovorus HD100 has a large genome and it is not yet known which of it encodes the molecular machinery and genetic control of predatory processes. We have tried to fill this knowledge-gap using mixtures of predator and prey mRNAs to monitor changes in Bdellovibrio gene expression at a timepoint of early-stage prey infection and prey killing in comparison to control cultures of predator and prey alone and also in comparison to Bdellovibrio growing axenically (in a prey-or host independent “HI” manner) on artificial media containing peptone and tryptone. From this we have highlighted genes of the early predatosome with predicted roles in prey killing and digestion and have gained insights into possible regulatory mechanisms as Bdellovibrio enter and establish within the prey bdelloplast. Approximately seven percent of all Bdellovibrio genes were significantly up-regulated at 30 minutes of infection- but not in HI growth- implicating the role of these genes in prey digestion. Five percent were down-regulated significantly, implicating their role in free-swimming, attack-phase physiology. This study gives the first post- genomic insight into the predatory process and reveals some of the important genes that Bdellovibrio expresses inside the prey bacterium during the initial attack. 相似文献