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1.
The electrically conductive pili (e-pili) of Geobacter species enable extracellular electron transfer to insoluble metallic minerals, electrodes and other microbial species, which confers biogeochemical significance and global prevalence on Geobacter in diverse anaerobic environments. E-pili are constructed by truncated PilA which is considered to have evolved from full-length pilin by gene fission under positive evolutionary selection. However, this hypothesis is based on phylogenetic analysis and has not yet been experimentally confirmed. Here, we reconstructed an ancestral strain of G. sulfurreducens (designated COMB) carrying full-length PilA by combining genes GSU1496 and GSU1497. The results demonstrated that strain COMB expressed and assembled the full-length fused PilA and exhibited an outer membrane c-type cytochrome profile similar to the wild-type strain. Surprisingly, the generated COMB-pili were also conductive, indicating the evolution of truncated PilA did not occur for conductivity. Moreover, strain COMB minimally reduced Fe(III) oxides but maintained its ability to respire electrodes, demonstrating the truncation of pilin enables iron respiration. This study provides the first experimental evidence that the truncation of pilin in Geobacter species confers adaption to Fe(III)-mineral-mediated selective pressures, and suggests an evolutionary event during which the separation of the GSU1497 gene helped Geobacter survive and thrive in natural environments.  相似文献   

2.
The general secretion pathway (GSP), found in a wide range of bacteria, is responsible for extracellular targeting of a subset of proteins from the periplasm. In Pseudomonas aeruginosa, the GSP requires the participation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homologues of PilA, the major subunit of type IV pili. The interaction between the pilin-like Xcp proteins was investigated using bifunctional cross-linking reagents. Cross-linking analysis of whole cells of wild-type P. aeruginosa, followed by immunoblot analysis, revealed a 34-kDa XcpT-containing complex. This complex was shown to consist of XcpT/PilA heterodimers. The role of PilA in the GSP was examined, using P. aeruginosa mutants in the pilA gene, or in rpoN, a gene regulating pilA expression. Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells. The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other components of the secretion machinery, nor did it require the pilus biogenesis factors PilB and PilC. The dimeric XcpT/PilA complex was also formed in a pilD mutant, which lacks the leader peptidase enzyme, demonstrating that the leader peptide at the N-terminus of PilA or XcpT did not have to be removed for the dimerization to occur. XcpW and XcpU can also be cross-linked to form dimeric complexes with PilA. When expression of XcpT is increased, its homodimers, as well as XcpT/XcpW heterodimers, can be detected. Finally, an oligohistidine-tagged XcpT was shown to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co-purified by nickel-affinity chromatography. The results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the secretion apparatus. Alternatively, these may represent dynamic intermediates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretion argues for a critical role played by PilA in two related processes during P. aeruginosa infections: formation of an adhesive pilus organelle and secretion of exoenzymes.  相似文献   

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The natural transformation system of Thermus thermophilus has become a model system for studies of the structure and function of DNA transporter in thermophilic bacteria. The DNA transporter in T. thermophilus is functionally linked to type IV pili (T4P) and the major pilin PilA4 plays an essential role in both systems. However, T4P are dispensable for natural transformation. In addition to pilA4, T. thermophilus has a gene cluster encoding the three additional pilins PilA1–PilA3; deletion of the cluster abolished natural transformation but retained T4P biogenesis. In this study, we investigated the roles of single pilins PilA1, PilA2 and PilA3 in natural transformation by mutant studies. These studies revealed that each of these pilins is essential for natural transformation. Two of the pilins, PilA1 and PilA2, were found to bind dsDNA. PilA1 and PilA3 were detected in the inner membrane (IM) but not in the outer membrane (OM) whereas PilA2 was present in both membranes. All three pilins where absent in pilus fractions. This suggests that the pilins form a short DNA binding pseudopilus anchored in the IM. PilA1 was found to bind to the IM assembly platform of the DNA transporter via PilM and PilO. These data are in line with the hypothesis that a DNA binding pseudopilus is connected via an IM platform to the cytosolic motor ATPase PilF.  相似文献   

7.
Myxococcus xanthus is a Gram‐negative bacterium capable of complex developmental processes involving vegetative swarming and fruiting body formation. Social (S‐) gliding motility, one of the two motility systems used by M. xanthus, requires at least two cell surface structures: type IV pili (TFP) and extracellular polysaccharides (EPS). Extended TFP that are composed of thousands of copies of PilA retract upon binding to EPS and thereby pull the cell forward. TFP also act as external sensor to regulate EPS production. In this study, we generated a random PilA mutant library and identified one derivative, SW1066, which completely failed to undergo developmental processes. Detailed characterization revealed that SW1066 produced very little EPS but wild‐type amounts of PilA. These mutated PilA subunits, however, are unable to assemble into functional TFP despite their ability to localize to the membrane. By preventing the mutated PilA of SW1066 to translocate from the cytoplasm to the membrane, fruiting body formation and EPS production were restored to the levels observed in mutant strains lacking PilA. This apparent connection between PilA membrane accumulation and reduction in surface EPS implies that specific cellular PilA localization are required to maintain the EPS level necessary to sustain normal S‐motility in M. xanthus.  相似文献   

8.
The conductive pili of Geobacter species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of Geobacter sulfurreducens have a metal-like conductivity, independent of the presence of c-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of G. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of Pseudomonas aeruginosa PAO1. Strain PA expressed and properly assembled P. aeruginosa PilA subunits into pili and exhibited a profile of outer surface c-type cytochromes similar to that of a control strain expressing the G. sulfurreducens PilA. Surprisingly, the strain PA pili were decorated with the c-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the G. sulfurreducens PilA that are necessary for conductivity.  相似文献   

9.
Cyanobacteria require large quantities of iron to maintain their photosynthetic machinery; however, in most environments iron is present in the form of insoluble iron oxides. Whether cyanobacteria can utilize these sources of iron, and the potential molecular mechanisms involved remains to be defined. There is increasing evidence that pili can facilitate electron donation to extracellular electron acceptors, like iron oxides in non-photosynthetic bacteria. In these organisms, the donation of electrons to iron oxides is thought to be crucial for maintaining respiration in the absence of oxygen. Our study investigates if PilA1 (major pilin protein) may also provide a mechanism to convert insoluble ferric iron into soluble ferrous iron. Growth experiments supported by spectroscopic data of a strain deficient in pilA1 indicate that the presence of the pilA1 gene enhances the ability to grow on iron oxides. These observations suggest a novel function of PilA1 in cyanobacterial iron acquisition.  相似文献   

10.
Many cyanobacteria exhibit surface motility powered by type 4 pili (T4P). In the model filamentous cyanobacterium Nostoc punctiforme, the T4P systems are arrayed in static, bipolar rings in each cell. The chemotaxis‐like Hmp system is essential for motility and the coordinated polar accumulation of PilA on cells in motile filaments, while the Ptx system controls positive phototaxis. Using transposon mutagenesis, a gene, designated hmpF, was identified as involved in motility. Synteny among filamentous cyanobacteria and the similar expression patterns for hmpF and hmpD imply that HmpF is part of the Hmp system. Deletion of hmpF produced a phenotype distinct from other hmp genes, but indistinguishable from pilB or pilQ. Both an HmpF‐GFPuv fusion protein, and PilA, as assessed by in situ immunofluorescence, displayed coordinated, unipolar localization at the leading pole of each cell. Reversals were modulated by changes in light intensity and preceded by the migration of HmpF‐GFPuv to the lagging cell poles. These results are consistent with a model where direct interaction between HmpF and the T4P system activates pilus extension, the Hmp system facilitates coordinated polarity of HmpF to establish motility, and the Ptx system modulates HmpF localization to initiate reversals in response to changes in light intensity.  相似文献   

11.
The kinetochore is assembled during mitotic and meiotic divisions within the centromeric region of chromosomes. It is composed of more than eighty different proteins. Spc105 (also designated as Spc7, KNL‐1 or Blinkin in different eukaryotes) is a comparatively large kinetochore protein, which can bind to the Mis12/MIND and Ndc80 complexes and to the spindle assembly checkpoint components Bub1 and BubR1. Our genetic characterization of Drosophila Spc105 shows that a truncated version lacking the rapidly evolving, repetitive central third still provides all essential functions. Moreover, in comparison with Cenp‐C that has previously been observed to extend from the inner to the outer kinetochore region, full‐length Spc105 is positioned further out and is not similarly extended along the spindle axis. Thus, our results indicate that Spc105 forms neither an extended link connecting inner Cenp‐A chromatin with outer kinetochore regions nor a scaffold constraining kinetochore subcomplexes and spindle assembly checkpoint components together into a geometrically rigid supercomplex. Spc105 seems to provide a platform within the outer kinetochore allowing independent assembly of various kinetochore components.  相似文献   

12.
γ-Tubulin Small Complex (γ-TuSC) is the universally-conserved complex in eukaryotes that contains the microtubule (MT) nucleating protein: γ-tubulin. γ-TuSC is a heterotetramer with two copies of γ-tubulin and one copy each of Spc98p and Spc97p. Previously, the structure of γ-TuSC was determined by single particle electron microscopy (EM) at 25 Å resolution. γ-TuSC is Y-shaped with a single flexible arm that could be the key to regulating MT nucleation. EM gold labeling revealed the locations of γ-tubulin at the top of the Y. In vivo Fluorescence Resonance Energy Transfer (FRET) suggested the relative orientations of Spc98p and Spc97p but did not distinguish which large subunit formed the flexible arm. Here, using fluorescent proteins as covalently attached tags, we used class averages and 3-D random conical tilt reconstructions to confirm the in vivo FRET results, clearly demonstrating that the Spc98p/97p C-termini interact directly with γ-tubulin. Most significantly we have determined that the flexible arm belongs to Spc98p and our data also suggests that the N-termini of Spc98p and Spc97p are crossed. More generally, our results confirm that despite their small size, covalently-attached fluorescent proteins perform well as subunit labels in single particle EM.  相似文献   

13.
D A Stirling  K A Welch    M J Stark 《The EMBO journal》1994,13(18):4329-4342
NUF1/SPC110, encoding a nuclear filament-related protein which is a component of the yeast spindle pole body (SPB), has been identified in a screen designed to isolate genes encoding targets of yeast calmodulin. Spc110p interacts with calmodulin by two different criteria and the calmodulin interacting region has been localized within the C-terminus of the protein. Point mutations between residues 898 and 917 further define the calmodulin binding site within this region. Mutations in this domain which abolish calmodulin binding in vitro prevent Spc110p function in vivo, demonstrating that calmodulin binding by Spc110p has important functional consequences. In keeping with a role for calmodulin in Spc110p function, we show that calmodulin localizes to the yeast SPB when cells are prepared under appropriate conditions. Non-functional mutant Spc110 proteins which cannot bind calmodulin are present at lowered steady-state levels in the cell; when their level is increased by elevated gene dosage, partial recovery of Spc110p function is seen. Overexpression of calmodulin suppresses the defect(s) associated with the mutant Spc110 proteins, supporting the notion that Spc110p stability is a consequence of its ability to bind calmodulin and pointing to a direct role for calmodulin in Spc110p function.  相似文献   

14.
Mushroom‐forming basidiomycetes colonize large areas in nature. Their hyphae are compartmentalized by perforated septa, which are usually covered by a septal pore cap (SPC). Here, we describe, for the first time, the composition and function of SPCs using the model system Schizophyllum commune. The SPC of S. commune was shown to consist of a proteinaceous matrix covered by a lipid membrane. The matrix was demonstrated to define the ultrastructure of the SPC and to consist of two main proteins, Spc14 and Spc33. Gene spc14 encodes a protein of 86 amino acids, which lacks known domain, signal or localization sequences. Gene spc33 encodes a 239 and a 340 amino acid variant. Both forms contain a predicted signal anchor that targets them to the ER. Immuno‐localization showed the presence of Spc33 in the SPC but not in ER. From this and previous reports it is concluded that the SPC is derived from this organelle. Inactivation of spc33 resulted in loss of SPCs and the inability to close septa. The latter may well explain why vegetative growth and mushroom formation were severely reduced in strains in which spc33 was inactivated.  相似文献   

15.
Kinetochore (KTs) are macromolecular protein assemblies that attach sister chromatids to spindle microtubules (MTs) and mediate accurate chromosome segregation during mitosis. The outer KT consists of the KMN network, a protein super-complex comprising Knl1 (yeast Spc105), Mis12 (yeast Mtw1), and Ndc80 (yeast Ndc80), which harbours sites for MT binding. Within the KMN network, Spc105 acts as an interaction hub of components involved in spindle assembly checkpoint (SAC) signalling. It is known that Spc105 forms a complex with KT component Kre28. However, where Kre28 physically localizes in the budding yeast KT is not clear. The exact function of Kre28 at the KT is also unknown. Here, we investigate how Spc105 and Kre28 interact and how they are organized within bioriented yeast KTs using genetics and cell biological experiments. Our microscopy data show that Spc105 and Kre28 localize at the KT with a 1 : 1 stoichiometry. We also show that the Kre28–Spc105 interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the KTs. We created several truncation mutants of kre28 that affect Spc105 loading at the KTs. When over-expressed, these mutants sustain the cell viability, but SAC signalling and KT biorientation are impaired. Therefore, we conclude that Kre28 contributes to chromosome biorientation and high-fidelity segregation at least indirectly by regulating Spc105 localization at the KTs.  相似文献   

16.
Type IV pilins are bacterial proteins that are small in size but have a broad range of functions, including motility, transformation competence and secretion. Although pilins vary in sequence, they possess a characteristic signal peptide that has to be removed by the prepilin peptidase PilD during pilin maturation. We generated a pilD (slr1120) null mutant of the cyanobacterium Synechocystis 6803 that accumulates an unprocessed form of the major pilin PilA1 (pPilA1) and its non‐glycosylated derivative (NpPilA1). Notably, the pilD strain had aberrant membrane ultrastructure and did not grow photoautotrophically because the synthesis of Photosystem II subunits was abolished. However, other membrane components such as Photosystem I and ATP synthase were synthesized at levels comparable to the control strain. Proliferation of the pilD strain was rescued by elimination of the pilA1 gene, demonstrating that PilA1 prepilin inhibited the synthesis of Photosystem II. Furthermore, NpPilA1 co‐immunoprecipitated with the SecY translocase and the YidC insertase, and both of these essential translocon components were degraded in the mutant. We propose that unprocessed prepilins inactivate an identical pool of translocons that function in the synthesis of both pilins and the core subunits of Photosystem II.  相似文献   

17.
Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.  相似文献   

18.
Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.  相似文献   

19.

Background

Stress-activated protein kinases regulate multiple cellular responses to a wide variety of intracellular and extracellular conditions. The conserved, multifunctional, ATF/CREB protein Atf1 (Mts1, Gad7) of fission yeast binds to CRE-like (M26) DNA sites. Atf1 is phosphorylated by the conserved, p38-family kinase Spc1 (Sty1, Phh1) and is required for many Spc1-dependent stress responses, efficient sexual differentiation, and activation of Rec12 (Spo11)-dependent meiotic recombination hotspots like ade6-M26.

Methodology/Principal Findings

We sought to define mechanisms by which Spc1 regulates Atf1 function at the ade6-M26 hotspot. The Spc1 kinase was essential for hotspot activity, but dispensable for basal recombination. Unexpectedly, a protein lacking all eleven MAPK phospho-acceptor sites and detectable phosphorylation (Atf1-11M) was fully proficient for hotspot recombination. Furthermore, tethering of Atf1 to ade6 in the chromosome by a heterologous DNA binding domain bypassed the requirement for Spc1 in promoting recombination.

Conclusions/Significance

The Spc1 protein kinase regulates the pathway of Atf1-promoted recombination at or before the point where Atf1 binds to chromosomes, and this pathway regulation is independent of the phosphorylation status of Atf1. Since basal recombination is Spc1-independent, the principal function of the Spc1 kinase in meiotic recombination is to correctly position Atf1-promoted recombination at hotspots along chromosomes. We also propose new hypotheses on regulatory mechanisms for shared (e.g., DNA binding) and distinct (e.g., osmoregulatory vs. recombinogenic) activities of multifunctional, stress-activated protein Atf1.  相似文献   

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