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1.
Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC‐homologue HgdD in protein secretion
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Alexander Hahn Mara Stevanovic Eva Brouwer Daniela Bublak Joanna Tripp Tobias Schorge Michael Karas Enrico Schleiff 《Environmental microbiology》2015,17(3):767-780
Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC‐homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst‐forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss‐of‐function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild‐type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin‐tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD‐mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD‐dependent pathway. 相似文献
2.
Alexander Hahn Mara Stevanovic Oliver Mirus Iryna Lytvynenko Klaas Martinus Pos Enrico Schleiff 《The Journal of biological chemistry》2013,288(43):31192-31205
The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component “AB-D” systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox− phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120. 相似文献
3.
Fenglong Liu Hongbin Zhang Dingji Shi Zhidi Shang Chen Lin Ning Shao Guohong Peng Xueyan Zhang Haixia Zhang Jinyin Wu Jie Wang Xudong Xu Yuehua Jiang Zepu Zhong Shujin Zhao Min Wu Chengkui Zeng 《中国科学C辑(英文版)》1999,42(1):25-33
The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacteriumAnabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDGTNF. The expression of the rhTNF gene inEscherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced intoAnabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with α-32P labeled hTNF cDNA probes, while the expression of the hTNF gene inAnabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic c~anobacteriumAnabaena sp. PCC 7120. 相似文献
4.
TonB dependent transporters (TBDT) are an essential protein family in bacteria involved in the uptake of a broad variety of molecules such as siderophore-chelated iron, which was the first described substrate. Meanwhile it is known that TBDTs are involved in the uptake of many metals, sugars and polypeptides. The action of TBDTs is regulated and energized by the plasma membrane anchored TonB, which is charged by a proton pump. The number of the genes coding for TBDTs varies in different species, which might reflect environmental adaptations or evolutionary variations of the system. For example, in the cyanobacterium Anabaena sp. PCC 7120 the large number of 22 genes coding for TBDTs has been identified and the expression of these genes has been explored in the absence of iron or copper as well as under nitrogen starvation. We describe the analysis of the expression of the TBDT genes and the according cytoplasmic-membrane localized components; the latter appear to have a lower degree of complexity in Anabaena sp. PCC 7120. This analysis unravels that the response is not sole dependent on the metal supply, but also on cell culture densities. In addition, we present a large group of FhuA-like genes which is expressed highest under standard conditions suggesting a function distinct from iron or copper transport. The genes are clustered according to the expression profile and the consequences for our understanding of the transport systems in Anabaena sp. PCC 7120 are discussed. 相似文献
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6.
《Bioscience, biotechnology, and biochemistry》2013,77(7):1113-1117
Genomes of many eubacterial strains have been shown to encode for multiple rpoD-related genes. In this report, we describe the identification of the multiple rpoD-related genes of cyanobacterial strains. DNAs of three cyanobacterial strains, Anabaena sp. PCC7120, Synechococcus sp. PCC7942, and Synechocystis sp. PCC6803, were examined by Southern hybridization, using a synthetic probe designed for detecting rpoD or rpoD-related genes. Four or five hybridization signals were found in each DNA. Four DNA regions of Synechococcus sp. PCC7942 corresponding to the hybridization signals were cloned and partially sequenced. The sequence data indicate the presence of genes, named rpoDl, rpoD2, rpoD3, and rpoD4, whose products are highly similar to the basic structure of the principal σ factors of eubacterial strains. The rpoDl gene showed the greatest similarity to the sigA gene of Anabaena sp. PCC7120. 相似文献
7.
Butachlor, a commonly used herbicide adversely affects the nitrogen fixing capability of Anabaena, an acclaimed nitrogen fixer in
the Indian paddy fields. The nitrogen fixation in Anabaena is triggered by the excision of nifD element by xisA gene leading to
rearrangement of nifD forming nifHDK operon in the heterocyst of Anabaena sp. PCC7120. Functional elucidation adjudged
through in-silico analysis revealed that xisA belongs to integrase family of tyrosine recombinase. The predicted functional partners
with XisA protein that have shown cooccurence with this protein in a network are mainly hypothetical proteins with unknown
functions except psaK1 whose exact function in photosystem I is not yet known. The focus of this study was to find out the relation
between XisA and butachlor using in-silico approaches. The XisA protein was modeled and its active sites were identified. Docking
studies revealed that butachlor binds at the active site of XisA protein hampering its excision ability vis-à-vis nif genes in Anabaena
sp. PCC7120. This study reveals that butachlor is not directly involved in hampering the nitrogen fixing ability of Anabaena sp.
PCC7120 but by arresting the excision ability of XisA protein necessary for the functioning of nif gene and nitrogen fixation. 相似文献
8.
Prashanth S Raghavan Gagan D Gupta Hema Rajaram Vinay Kumar 《Journal of biosciences》2016,41(4):589-600
The hypothetical protein ‘Alr3200’ of Anabaena sp. strain PCC7120 is highly conserved among cyanobacterial species. It is a member of the DUF820 (Domain of Unknown Function) protein family, and is predicted to have a DNase domain. Biochemical analysis revealed a Mg(II)-dependent DNase activity for Alr3200 with a specific activity of 8.62×104 Kunitz Units (KU) mg?1 protein. Circular dichroism analysis predicted Alr3200 to have ~40% β-strands and ~9% α-helical structures. Anabaena PCC7120 inherently expressed Alr3200 at very low levels, and its overexpression had no significant effect on growth of Anabaena under control conditions. However, Analr3200 +, the recombinant Anabaena strain overexpressing Alr3200, exhibited zero survival upon exposure to 6 kGy of γ-radiation, which is the LD50 for wild type Anabaena PCC7120 as well as the vector control recombinant strain, AnpAM. Comparative analysis of the two recombinant Anabaena strains suggested that it is not the accumulated Alr3200 per se, but its possible interactions with the radiation-induced unidentified DNA repair proteins of Anabaena, which hampers DNA repair resulting in radiosensitivity. 相似文献
9.
Kerstin Nicolaisen Marianne Valdebenito Anastazia Samborski Iris Maldener Ana Valladares Klaus Hantke 《生物化学与生物物理学报:生物膜》2010,1798(11):2131-2140
Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (Ptrc) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying Ptrc-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120. 相似文献
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11.
Lopes Pinto F Erasmie S Blikstad C Lindblad P Oliveira P 《Journal of plant physiology》2011,168(16):1934-1942
In prokaryotes, cell division is normally achieved by binary fission, and the key player FtsZ is considered essential for the complete process. In cyanobacteria, much remains unknown about several aspects of cell division, including the identity and mechanism of the various components involved in the division process. Here, we report results obtained from a search of the players implicated in cell division, directly associating to FtsZ in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Histidine tag pull-downs were used to address this question. However, the main observation was that FtsZ is a target of proteolysis. Experiments using various cell-free extracts, an unrelated protein, and protein blot analyses further supported the idea that FtsZ is proteolytically cleaved in a specific manner. In addition, we show evidence that both FtsZ termini seem to be equally prone to proteolysis. Taken together, our data suggest the presence of an unknown player in cyanobacterial cell division, opening up the possibility to investigate novel mechanisms to control cell division in Anabaena PCC 7120. 相似文献
12.
LIU Feng-Long ZHANG Hong-BinSHI Ding-Ji SHANG Zhi-Di LIN Chen SHAO Ning PENG GuohongZHANG Xue-Yan ZHANG Hai-Xia WU Jin-Yin XU Xu-Dong WANG JieJIANG Yue-Hua ZHONG Ze-PuZHAO Shu-JinWU Min CENG Cheng-Kui 《中国科学:生命科学英文版》1999,42(1):25-33
The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in
a cyanobacteriumAnabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream
of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the
shuttle vector pDC-8 to get the shuttle, expression vector pDGTNF. The expression of the rhTNF gene inEscherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two
vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression
level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids,
pDC-TNF has been introduced intoAnabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the
introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis
with the extracted plasmid samples and Southern blotting with α-32P labeled hTNF cDNA probes, while the expression of the hTNF gene inAnabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal
antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude
extracts from the transgenic c~anobacteriumAnabaena sp. PCC 7120.
Project supported by the National Natural Science Foundation of China (Grant No. 39280016). 相似文献
13.
Nicolaisen K Moslavac S Samborski A Valdebenito M Hantke K Maldener I Muro-Pastor AM Flores E Schleiff E 《Journal of bacteriology》2008,190(22):7500-7507
Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell. 相似文献
14.
Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and
sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the
noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N2-fixing and non-N2-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of
Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible
hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme.
Received: 20 August 1997 / Accepted: 24 November 1997 相似文献
15.
María B. Pascual Alejandro Mata-Cabana Francisco J. Florencio Marika Lindahl Francisco J. Cejudo 《The Journal of biological chemistry》2010,285(45):34485-34492
In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system. 相似文献
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In Gram-negative bacteria, trans-envelope efflux pumps have periplasmic membrane fusion proteins (MFPs) as essential components. MFPs act as mediators between outer membrane factors (OMFs) and inner membrane factors (IMFs). In this study, structure–function relations of the ATP-driven glycolipid efflux pump DevBCA-TolC/HgdD from the cyanobacterium Anabaena sp. PCC 7120 were analyzed. The binding of the MFP DevB to the OMF TolC absolutely required the respective tip-regions. The interaction of DevB with the IMF DevAC mainly involved the β-barrel and the lipoyl domain. Efficient binding to DevAC and TolC, substrate recognition and export activity by DevAC were dependent on stable DevB hexamers. 相似文献
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19.
Katerina Kourpa Eftychia Manarolaki Alexandros Lyratzakis Vasso Strataki Fiona Rupprecht Julian D. Langer Georgios Tsiotis 《Proteomics》2019,19(19)
Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N2‐fixing conditions are investigated. Using a label‐free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120. 相似文献
20.
In natural habitats, organisms especially phytoplankton are not always continuously subjected to ultraviolet-B radiation (UVBR). By simulation of the natural situation, the N2-fixing cyanobacterium Anabaena sp. PCC 7120 was subjected to UV-B exposure and recovery cycles. A series of morphological and physiological changes were observed in Anabaena sp. PCC 7120 under repeated UVBR when compared with controls. Such as the breakage of filaments, intervals between heterocysts, heterocyst frequency, total carbohydrate, and carotenoids were increased, while the nitrogenase activity and photosynthetic activity were inhibited by repeated UVBR; however, these activities could recover when UV-B stress was removed. Unexpectedly, the over-compensatory growth was observed at the end of the second round of exposure and recovery cycle. Our results showed that discontinuous UVBR could increase the growth rate and the tolerance as well as repair capacity of Anabaena sp. PCC 7120. These results indicate that moderate UVBR may increase the growth of cyanobacteria in natural habitats. 相似文献