scholarly journals Effect of Mutations in GvpJ and GvpM on Gas Vesicle Formation of Halobacterium salinarum

2021 ◽  
Vol 12 ◽  
Author(s):  
Alisa Jost ◽  
Regine Knitsch ◽  
Kerstin Völkner ◽  
Felicitas Pfeifer
Keyword(s):  
Halobacterium Salinarum ◽  
Haloferax Volcanii ◽  
Vesicle Formation ◽  
Gas Vesicles ◽  
Structural Protein ◽  
Homologous Proteins ◽  
Partner Protein ◽  
Terminal Fragment ◽  
Comparison Of The Results

The two haloarchaeal proteins, GvpM and GvpJ, are homologous to GvpA, the major gas vesicle structural protein. All three are hydrophobic and essential for gas vesicle formation. The effect of mutations in GvpJ and GvpM was studied in Haloferax volcanii transformants by complementing the respective mutated gene with the remaining gvp genes and inspecting the cells for the presence of gas vesicles (Vac+). In case of GvpJ, 56 of 66 substitutions analyzed yielded Vac– ΔJ + Jmut transformants, indicating that GvpJ is very sensitive to alterations, whereas ten of the 38 GvpM variants resulted in Vac– ΔM + Mmut transformants. The variants were also tested by split-GFP for their ability to interact with their partner protein GvpL. Some of the alterations leading to a Vac– phenotype affected the J/L or M/L interaction. Also, the interactions J/A and J/M were studied using fragments to exclude an unspecific aggregation of these hydrophobic proteins. Both fragments of GvpJ interacted with the M1–25 and M60–84 fragments of GvpM, and fragment J1–56 of GvpJ interacted with the N-terminal fragment A1–22 of GvpA. A comparison of the results on the three homologous proteins indicates that despite their relatedness, GvpA, GvpJ, and GvpM have unique features and cannot substitute each other.

scholarly journals Eight of Fourteen gvp Genes Are Sufficient for Formation of Gas Vesicles in Halophilic Archaea

2000 ◽  
Vol 182 (15) ◽  
pp. 4328-4336 ◽  
Author(s):  
Sonja Offner ◽  
Annette Hofacker ◽  
Gerhard Wanner ◽  
Felicitas Pfeifer
Keyword(s):  
Phase Contrast ◽  
Gene Deletion ◽  
Streptomyces Coelicolor ◽  
Halophilic Archaea ◽  
Halobacterium Salinarum ◽  
Vesicle Formation ◽  
Gas Vesicles ◽  
Minimal Set ◽  
Number Of Genes ◽  
Contrast Microscopy

ABSTRACT The minimal number of genes required for the formation of gas vesicles in halophilic archaea has been determined. Single genes of the 14 gvp genes present in the p-vac region on plasmid pHH1 ofHalobacterium salinarum (p-gvpACNO and p-gvpDEFGHIJKLM) were deleted, and the remaining genes were tested for the formation of gas vesicles in Haloferax volcanii transformants. The deletion of six gvp genes (p-gvpCN, p-gvpDE, and p-gvpHI) still enabled the production of gas vesicles in H. volcanii. The gas vesicles formed in some of thesegvp gene deletion transformants were altered in shape (ΔI, ΔC) or strength (ΔH) but still functioned as flotation devices. A minimal p-vac region (minvac) containing the eight remaining genes (gvpFGJKLM-gvpAO) was constructed and tested for gas vesicle formation in H. volcanii. The minvac transformants did not form gas vesicles; however, minvac/gvpJKLM double transformants contained gas vesicles seen as light refractile bodies by phase-contrast microscopy. Transcript analyses demonstrated that minvac transformants synthesized regular amounts of gvpA mRNA, but the transcripts derived from gvpFGJKLM were mainly short and encompassed onlygvpFG(J), suggesting that thegvpJKLM genes were not sufficiently expressed. SincegvpAO and gvpFGJKLM are the onlygvp genes present in minvac/JKLM transformants containing gas vesicles, these gvp genes represent the minimal set required for gas vesicle formation in halophilic archaea. Homologs of six of these gvp genes are found in Anabaena flos-aquae, and homologs of all eight minimal halobacterialgvp genes are present in Bacillus megateriumand in the genome of Streptomyces coelicolor.

Effect of Anoxic Conditions and Temperature on Gas Vesicle Formation in Halobacterium salinarum

2011 ◽  
pp. 237-248
Author(s):  
Felicitas Pfeifer ◽  
Regina Frommherz ◽  
Karin Faist ◽  
Torsten Hechler ◽  
Katharina Teufel ◽  
...  
Keyword(s):  
Halobacterium Salinarum ◽  
Vesicle Formation ◽  
Anoxic Conditions

Glucose inhibits the formation of gas vesicles in Haloferax volcanii transformants

2007 ◽  
Vol 0 (0) ◽  
pp. 070816220133001-??? ◽  
Author(s):  
Torsten Hechler ◽  
Miriam Frech ◽  
Felicitas Pfeifer
Keyword(s):  
Haloferax Volcanii ◽  
Gas Vesicles

scholarly journals Acoustic Behavior of Halobacterium salinarum Gas Vesicles in the High-Frequency Range: Experiments and Modeling

2017 ◽  
Vol 43 (5) ◽  
pp. 1016-1030 ◽  
Author(s):  
Emmanuel Cherin ◽  
Johan M. Melis ◽  
Raymond W. Bourdeau ◽  
Melissa Yin ◽  
Dennis M. Kochmann ◽  
...  
Keyword(s):  
High Frequency ◽  
Halobacterium Salinarum ◽  
Acoustic Behavior ◽  
High Frequency Range ◽  
Gas Vesicles ◽  
Frequency Range

Structure of the gas vesicle protein GvpF from the cyanobacteriumMicrocystis aeruginosa

2014 ◽  
Vol 70 (11) ◽  
pp. 3013-3022 ◽  
Author(s):  
Bo-Ying Xu ◽  
Ya-Nan Dai ◽  
Kang Zhou ◽  
Yun-Tao Liu ◽  
Qianqian Sun ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Gene Cluster ◽  
Microcystis Aeruginosa ◽  
Immunogold Labelling ◽  
Gas Vesicles ◽  
Structural Protein ◽  
Apparent Variation ◽  
Vesicle Protein

Gas vesicles are gas-filled proteinaceous organelles that provide buoyancy for bacteria and archaea. A gene cluster that is highly conserved in various species encodes about 8–14 proteins (Gvp proteins) that are involved in the formation of gas vesicles. Here, the first crystal structure of the gas vesicle protein GvpF fromMicrocystis aeruginosaPCC 7806 is reported at 2.7 Å resolution. GvpF is composed of two structurally distinct domains (the N-domain and C-domain), both of which display an α+β class overall structure. The N-domain adopts a novel fold, whereas the C-domain has a modified ferredoxin fold with an apparent variation owing to an extension region consisting of three sequential helices. The two domains pack against each otherviainteractions with a C-terminal tail that is conserved among cyanobacteria. Taken together, it is concluded that the overall architecture of GvpF presents a novel fold. Moreover, it is shown that GvpF is most likely to be a structural protein that is localized at the gas-facing surface of the gas vesicle by immunoblotting and immunogold labelling-based tomography.

scholarly journals Opsin-Mediated Inhibition of Bacterioruberin Synthesis in Halophilic Archaea

2017 ◽  
Vol 199 (21) ◽  
Author(s):  
Ronald F. Peck ◽  
Alexandru M. Pleşa ◽  
Serena M. Graham ◽  
David R. Angelini ◽  
Emily L. Shaw
Keyword(s):  
Organic Molecules ◽  
Regulatory Mechanism ◽  
Colorimetric Assay ◽  
Halophilic Archaea ◽  
Halobacterium Salinarum ◽  
Haloferax Volcanii ◽  
Content Type ◽  
Colony Color ◽  
Ion Pumping ◽  
Limited Oxygen

ABSTRACT Halophilic archaea often inhabit environments with limited oxygen, and many produce ion-pumping rhodopsin complexes that allow them to maintain electrochemical gradients when aerobic respiration is inhibited. Rhodopsins require a protein, an opsin, and an organic cofactor, retinal. We previously demonstrated that in Halobacterium salinarum, bacterioopsin (BO), when not bound by retinal, inhibits the production of bacterioruberin, a biochemical pathway that shares intermediates with retinal biosynthesis. In this work, we used heterologous expression in a related halophilic archaeon, Haloferax volcanii, to demonstrate that BO is sufficient to inhibit bacterioruberin synthesis catalyzed by the H. salinarum lycopene elongase (Lye) enzyme. This inhibition was observed both in liquid culture and in a novel colorimetric assay to quantify bacterioruberin abundance based on the colony color. Addition of retinal to convert BO to the bacteriorhodopsin complex resulted in a partial rescue of bacterioruberin production. To explore if this regulatory mechanism occurs in other organisms, we expressed a Lye homolog and an opsin from Haloarcula vallismortis in H. volcanii. H. vallismortis cruxopsin-3 expression inhibited bacterioruberin synthesis catalyzed by H. vallismortis Lye but had no effect when bacterioruberin synthesis was catalyzed by H. salinarum or H. volcanii Lye. Conversely, H. salinarum BO did not inhibit H. vallismortis Lye activity. Together, our data suggest that opsin-mediated inhibition of Lye is potentially widespread and represents an elegant regulatory mechanism that allows organisms to efficiently utilize ion-pumping rhodopsins obtained through lateral gene transfer. IMPORTANCE Many enzymes are complexes of proteins and nonprotein organic molecules called cofactors. To ensure efficient formation of functional complexes, organisms must regulate the production of proteins and cofactors. To study this regulation, we used bacteriorhodopsin from the archaeon Halobacterium salinarum. Bacteriorhodopsin consists of the bacterioopsin protein and a retinal cofactor. In this article, we further characterize a novel regulatory mechanism in which bacterioopsin promotes retinal production by inhibiting a reaction that consumes lycopene, a retinal precursor. By expressing H. salinarum genes in a different organism, Haloferax volcanii, we demonstrated that bacterioopsin alone is sufficient for this inhibition. We also found that an opsin from Haloarcula vallismortis has inhibitory activity, suggesting that this regulatory mechanism might be found in other organisms.

scholarly journals The nsp1, nsp13, and M Proteins Contribute to the Hepatotropism of Murine Coronavirus JHM.WU

2015 ◽  
Vol 89 (7) ◽  
pp. 3598-3609 ◽  
Author(s):  
Rong Zhang ◽  
Yize Li ◽  
Timothy J. Cowley ◽  
Adam D. Steinbrenner ◽  
Judith M. Phillips ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Human Populations ◽  
Cdna Clones ◽  
Virulence Determinants ◽  
Structural Protein ◽  
Human Pathogens ◽  
Homologous Proteins ◽  
Content Type ◽  
Murine Coronavirus

ABSTRACTMouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these full-length cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replicationin vitroand in the liverin vivo. The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect onin vitroreplication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-β expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is aBetacoronavirusvirulence factor.IMPORTANCETheBetacoronavirusgenus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus, it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies.

scholarly journals PhANNs, a fast and accurate tool and web server to classify phage structural proteins

2020 ◽  
Vol 16 (11) ◽  
pp. e1007845
Author(s):  
Vito Adrian Cantu ◽  
Peter Salamon ◽  
Victor Seguritan ◽  
Jackson Redfield ◽  
David Salamon ◽  
...  
Keyword(s):  
Large Fraction ◽  
Web Server ◽  
Structural Proteins ◽  
Metagenomic Data ◽  
Test Accuracy ◽  
Neural Network Ensemble ◽  
Structural Protein ◽  
Homologous Proteins ◽  
New Approach ◽  
Artificial Neural

For any given bacteriophage genome or phage-derived sequences in metagenomic data sets, we are unable to assign a function to 50–90% of genes, or more. Structural protein-encoding genes constitute a large fraction of the average phage genome and are among the most divergent and difficult-to-identify genes using homology-based methods. To understand the functions encoded by phages, their contributions to their environments, and to help gauge their utility as potential phage therapy agents, we have developed a new approach to classify phage ORFs into ten major classes of structural proteins or into an “other” category. The resulting tool is named PhANNs (Phage Artificial Neural Networks). We built a database of 538,213 manually curated phage protein sequences that we split into eleven subsets (10 for cross-validation, one for testing) using a novel clustering method that ensures there are no homologous proteins between sets yet maintains the maximum sequence diversity for training. An Artificial Neural Network ensemble trained on features extracted from those sets reached a test F1-score of 0.875 and test accuracy of 86.2%. PhANNs can rapidly classify proteins into one of the ten structural classes or, if not predicted to fall in one of the ten classes, as “other,” providing a new approach for functional annotation of phage proteins. PhANNs is open source and can be run from our web server or installed locally.

scholarly journals Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO

2016 ◽  
Vol 198 (7) ◽  
pp. 1077-1086 ◽  
Author(s):  
Tatsuya Hattori ◽  
Hiromichi Shiba ◽  
Ken-ichi Ashiki ◽  
Takuma Araki ◽  
Yoh-kow Nagashima ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Promoter Sequence ◽  
Haloferax Volcanii ◽  
Anaerobic Growth ◽  
Halophilic Archaeon ◽  
Anaerobic Conditions ◽  
Homologous Proteins ◽  
Promoter Assay ◽  
Content Type ◽  
Extremely Halophilic Archaeon

ABSTRACTThe extremely halophilic archaeonHaloferax volcaniigrows anaerobically by denitrification. A putative DNA-binding protein, NarO, is encoded upstream of the respiratory nitrate reductase gene ofH. volcanii. Disruption of thenarOgene resulted in a loss of denitrifying growth ofH. volcanii, and the expression of the recombinant NarO recovered the denitrification capacity. A novel CXnCXCX7C motif showing no remarkable similarities with known sequences was conserved in the N terminus of the NarO homologous proteins found in the haloarchaea. Restoration of the denitrifying growth was not achieved by expression of any mutant NarO in which any one of the four conserved cysteines was individually replaced by serine. A promoter assay experiment indicated that thenarOgene was usually transcribed, regardless of whether it was cultivated under aerobic or anaerobic conditions. Transcription of the genes encoding the denitrifying enzymes nitrate reductase and nitrite reductase was activated under anaerobic conditions. A putativeciselement was identified in the promoter sequence of haloarchaeal denitrifying genes. These results demonstrated a significant effect of NarO, probably due to its oxygen-sensing function, on the transcriptional activation of haloarchaeal denitrifying genes.IMPORTANCEH. volcaniiis an extremely halophilic archaeon capable of anaerobic growth by denitrification. The regulatory mechanism of denitrification has been well understood in bacteria but remains unknown in archaea. In this work, we show that the helix-turn-helix (HTH)-type regulator NarO activates transcription of the denitrifying genes ofH. volcaniiunder anaerobic conditions. A novel cysteine-rich motif, which is critical for transcriptional regulation, is present in NarO. A putativeciselement was also identified in the promoter sequence of the haloarchaeal denitrifying genes.

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