scholarly journals Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum

2009 ◽  
Vol 418 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Claudia S. López ◽  
R. Sean Peacock ◽  
Jorge H. Crosa ◽  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Solution Structure ◽  
Bacterial Species ◽  
Vibrio Anguillarum ◽  
Fish Pathogen ◽  
E Coli ◽  
Terminal Domain ◽  
C Terminus

In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.

scholarly journals The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function

2003 ◽  
Vol 185 (13) ◽  
pp. 3821-3827 ◽  
Author(s):  
Elisabeth Enggist ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heme Binding ◽  
Membrane Anchor ◽  
Terminal Domain ◽  
Conserved Domain ◽  
C Terminus ◽  
Helical Turn ◽  
Low Activity

ABSTRACT CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence 131DENYTPP137 was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.

scholarly journals Inhibition of Vibrio anguillarum byPseudomonas fluorescens AH2, a Possible Probiotic Treatment of Fish

1999 ◽  
Vol 65 (3) ◽  
pp. 969-973 ◽  
Author(s):  
Lone Gram ◽  
Jette Melchiorsen ◽  
Bettina Spanggaard ◽  
Ingrid Huber ◽  
Torben F. Nielsen
Keyword(s):  
Iron Concentration ◽  
Vibrio Anguillarum ◽  
Fish Farming ◽  
Fish Pathogen ◽  
Probiotic Treatment ◽  
Treated Fish ◽  
Culture Supernatants ◽  
Probiotic Effect

ABSTRACT To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strainPseudomonas fluorescens strain AH2 against the fish-pathogenic bacterium Vibrio anguillarum. As iron is important in virulence and bacterial interactions, the effect ofP. fluorescens AH2 was studied under iron-rich and iron-limited conditions. Sterile-filtered culture supernatants from iron-limited P. fluorescens AH2 inhibited the growth ofV. anguillarum, whereas sterile-filtered supernatants from iron-replete cultures of P. fluorescens AH2 did not.P. fluorescens AH2 inhibited the growth of V. anguillarum during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1,000 times greater that of the fish pathogen. These in vitro results were successfully repeated in vivo. A probiotic effect in vivo was tested by exposing rainbow trout (Oncorynchus mykissWalbaum) to P. fluorescens AH2 at a density of 105 CFU/ml for 5 days before a challenge with V. anguillarum at 104 to 105 CFU/ml for 1 h. Some fish were also exposed to P. fluorescens AH2 at 107 CFU/ml during the 1-h infection. The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12°C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont.

scholarly journals Adaptation in a Mouse Colony Monoassociated with Escherichia coli K-12 for More than 1,000 Days

2010 ◽  
Vol 76 (14) ◽  
pp. 4655-4663 ◽  
Author(s):  
Sean M. Lee ◽  
Aaron Wyse ◽  
Aaron Lesher ◽  
Mary Lou Everett ◽  
Linda Lou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Bacterial Species ◽  
Intestinal Flora ◽  
Average Density ◽  
Host Bacterium ◽  
E Coli ◽  
K 12

ABSTRACT Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

scholarly journals Functions of the C-Terminal Domain of Varicella-Zoster Virus Glycoprotein E in Viral Replication In Vitro and Skin and T-Cell Tropism In Vivo

2004 ◽  
Vol 78 (22) ◽  
pp. 12406-12415 ◽  
Author(s):  
Jennifer Moffat ◽  
Chengjun Mo ◽  
Jason J. Cheng ◽  
Marvin Sommer ◽  
Leigh Zerboni ◽  
...  
Keyword(s):  
T Cell ◽  
Viral Replication ◽  
Varicella Zoster Virus ◽  
Point Mutations ◽  
Glycoprotein E ◽  
Varicella Zoster ◽  
Terminal Domain ◽  
C Terminus

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV replication. To further analyze the functions of gE in VZV replication, a full deletion and point mutations were made in the 62-amino-acid (aa) C-terminal domain. Targeted mutations were introduced in YAGL (aa 582 to 585), which mediates gE endocytosis, AYRV (aa 568 to 571), which targets gE to the trans-Golgi network (TGN), and SSTT, an “acid cluster” comprising a phosphorylation motif (aa 588 to 601). Substitutions Y582G in YAGL, Y569A in AYRV, and S593A, S595A, T596A, and T598A in SSTT were introduced into the viral genome by using VZV cosmids. These experiments demonstrated a hierarchy in the contributions of these C-terminal motifs to VZV replication and virulence. Deletion of the gE C terminus and mutation of YAGL were lethal for VZV replication in vitro. Mutations of AYRV and SSTT were compatible with recovery of VZV, but the AYRV mutation resulted in rapid virus spread in vitro and the SSTT mutation resulted in higher virus titers than were observed for the parental rOka strain. When the rOka-gE-AYRV and rOka-gE-SSTT mutants were evaluated in skin and T-cell xenografts in SCIDhu mice, interference with TGN targeting was associated with substantial attenuation, especially in skin, whereas the SSTT mutation did not alter VZV infectivity in vivo. These results provide the first information about how targeted mutations of this essential VZV glycoprotein affect viral replication in vitro and VZV virulence in dermal and epidermal cells and T cells within intact tissue microenvironments in vivo.

scholarly journals Fusion of the N-terminal 119 amino acids with the RelA-CTD renders its growth inhibitory effects (p)ppGpp-dependent

2021 ◽  
Author(s):  
Jayashree Pohnerkar ◽  
Krishma Tailor ◽  
Prarthi Sagar ◽  
Keyur Dave
Keyword(s):  
Amino Acids ◽  
Feedback Regulation ◽  
Inhibitory Effects ◽  
E Coli ◽  
Growth Inhibitory ◽  
In The Wild ◽  
Regulatory Aspect ◽  
And Function

The guanosine nucleotide derivatives ppGpp and pppGpp are central to the remarkable capacity of bacteria to adapt to fluctuating environment and metabolic perturbations. These alarmones are synthesized by two proteins, RelA and SpoT in E. coli and the activities of each of the two enzymes are highly regulated for homeostatic control of (p)ppGpp levels in the cell. Although the domain structure and function of RelA are well defined, the findings of this study unfold the regulatory aspect of RelA that is possibly relevant in vivo. We uncover here the importance of the N-terminal 1-119 amino acids of the enzymatically compromised (p)ppGpp hydrolytic domain (HD) of monofunctional RelA for the (p)ppGpp mediated regulation of RelA-CTD function. We find that even moderate level expression of RelA appreciably reduces growth when the basal levels of (p)ppGpp in the cells are higher than in the wild type, an effect independent of its ability to synthesize (p)ppGpp. This is evidenced by the growth inhibitory effects of oversynthesis of the RelA-CTD in the relA+ strain but not in relA null mutant, suggesting the requirement of the functional RelA protein for basal level synthesis of (p)ppGpp, accordingly corroborated by the restoration of the growth inhibitory effects of the RelA-CTD expression in the relA1 spoT202 mutant. The N-terminal 119 amino acids of RelA fused in-frame with the RelA-CTD, both from 406-744 amino acids (including TGS) and from 454-744 amino acids (sans TGS) caused growth inhibition only in spoT1 and spoT202 relA1 mutants, uncovering the hitherto unrealized (p)ppGpp-dependent regulation of RelA-CTD function. An incremental rise in the (p)ppGpp levels is proposed to progressively modulate the interaction of RelA-CTD with the ribosomes, with possible implications in the feedback regulation of the N-terminal (p)ppGpp synthesis function, a proposal that best explains the nonlinear relationship between (p)ppGpp synthesis and increased ratio of RelA:ribosomes, both in vitro as well as in vivo.

scholarly journals A Rationally Designed Aminoacyl-tRNA Synthetase for Genetically Encoded Fluorescent Amino Acids

2016 ◽  
Author(s):  
Ximena Steinberg ◽  
Jason Galpin ◽  
Gibran Nasir ◽  
Jose Sepulveda-Ugarte ◽  
Romina V. Sepúlveda ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Structure ◽  
Structure Function ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
E Coli ◽  
Promising Strategy ◽  
Fluorescent Amino Acids

AbstractThe incorporation of non-canonical amino acids into proteins has emerged as a promising strategy to manipulate and study protein structure-function relationships with superior precision in vitro and in vivo. To date, fluorescent non-canonical amino acids (f-ncAA) have been successfully incorporated in proteins expressed in bacterial systems, Xenopus oocytes, and HEK-293T cells. Here, we describe the rational generation of an orthogonal aminoacyltRNA synthetase based on the E. coli tyrosine synthetase that is capable of encoding the f-ncAA tyr-coumarin in HEK-293T cells.

scholarly journals FtsW exhibits distinct processive motions driven by either septal cell wall synthesis or FtsZ treadmilling in E. coli

2019 ◽  
Author(s):  
Xinxing Yang ◽  
Ryan McQuillen ◽  
Zhixin Lyu ◽  
Polly Phillips-Mason ◽  
Ana De La Cruz ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Single Molecule ◽  
Bacterial Cell ◽  
Spatial Information ◽  
Bacterial Species ◽  
E Coli ◽  
Slow Moving

AbstractDuring bacterial cell division, synthesis of new septal peptidoglycan (sPG) is crucial for successful cytokinesis and cell pole morphogenesis. FtsW, a SEDS (Shape, Elongation, Division and Sporulation) family protein and an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a new monofunctional peptidoglycan glycosyltransferase (PGTase). FtsW and its cognate monofunctional transpeptidase (TPase) class B penicillin binding protein (PBP3 or FtsI in E. coli) may constitute the essential, bifunctional sPG synthase specific for new sPG synthesis. Despite its importance, the septal PGTase activity of FtsW has not been documented in vivo. How its activity is spatiotemporally regulated in vivo has also remained unknown. Here we investigated the septal PGTase activity and dynamics of FtsW in E. coli cells using a combination of single-molecule imaging and genetic manipulations. We show that FtsW exhibits robust activity to incorporate an N-acetylmuramic acid analog at septa in the absence of other known PGTases, confirming FtsW as the essential septum-specific PGTase in vivo. Notably, we identified two populations of processive moving FtsW molecules at septa. A fast-moving population is driven by the treadmilling dynamics of FtsZ and independent of sPG synthesis. A slow-moving population is driven by active sPG synthesis and independent of FtsZ’s treadmilling dynamics. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving, sPG synthesis-dependent population. Our results support a two-track model, in which inactive sPG synthase molecules follow the fast treadmilling “Z-track” to be distributed along the septum; FtsN promotes their release from the “Z-track” to become active in sPG synthesis on the slow “sPG-track”. This model explains how the spatial information is integrated into the regulation of sPG synthesis activity and suggests a new mechanistic framework for the spatiotemporal coordination of bacterial cell wall constriction.

scholarly journals Peptide Purification, Complementary Deoxyribonucleic Acid (DNA) and Genomic DNA Cloning, and Functional Characterization of Ghrelin in Rainbow Trout

Endocrinology ◽  
2003 ◽  
Vol 144 (12) ◽  
pp. 5215-5226 ◽  
Author(s):  
Hiroyuki Kaiya ◽  
Masayasu Kojima ◽  
Hiroshi Hosoda ◽  
Shunsuke Moriyama ◽  
Akiyoshi Takahashi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rainbow Trout ◽  
Functional Characterization ◽  
Somatic Growth ◽  
Decanoic Acid ◽  
C Terminus ◽  
Ghrelin Gene ◽  
High Level

Abstract We have identified ghrelin from the stomach of rainbow trout. Four isoforms of ghrelin peptide were isolated: the C-terminal amidated type of rainbow trout ghrelin (rt ghrelin) composed of 24 amino acids (GSSFLSPSQKPQVRQGKGKPPRV-amide) is a basic form; des-VRQ-rt ghrelin, which deleted three amino acids (V13R14Q15) from rt ghrelin; and further two types of rt ghrelin that retained the glycine residue at the C terminus, rt ghrelin-Gly, and des-VRQ-rt ghrelin-Gly. The third serine residue was modified by octanoic acid, decanoic acid, or the unsaturated form of those fatty acids. In agreement with the isolated peptides, two cDNAs of different lengths were isolated. The rt ghrelin gene has five exons and four introns, and two different mRNA molecules are predicted to be produced by alternative splicing of the gene. A high level of ghrelin mRNA expression was detected in the stomach, and moderate levels were detected in the brain, hypothalamus, and intestinal tracts. Des-VRQ-rt ghrelin stimulated the release of GH in the rat in vivo. Furthermore, des-VRQ-rt ghrelin stimulated the release of GH, but not the release of prolactin and somatolactin in rainbow trout in vivo and in vitro. These results indicate that ghrelin is a novel GH secretagogue in rainbow trout that may affect somatic growth or osmoregulation through GH. Because ghrelin is expressed in various tissues other than stomach, it may play important role(s) in cellular function as a local regulator.

scholarly journals Topogenesis in membranes of the NTB–VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain

2004 ◽  
Vol 85 (2) ◽  
pp. 535-545 ◽  
Author(s):  
Aiming Wang ◽  
Sumin Han ◽  
Hélène Sanfaçon
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Proteinase K ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Microsomal Membranes ◽  
Hydrophobic Amino Acids ◽  
Definition Of

The putative NTP-binding protein (NTB) of Tomato ringspot nepovirus (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB–VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB–VPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and adopts a topology similar to that observed in vivo in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB–VPg polyprotein acts as a membrane-anchor for the replication complex.

scholarly journals Inducible degradation of I kappa B alpha in vitro and in vivo requires the acidic C-terminal domain of the protein.

1995 ◽  
Vol 15 (5) ◽  
pp. 2413-2419 ◽  
Author(s):  
M S Rodriguez ◽  
I Michalopoulos ◽  
F Arenzana-Seisdedos ◽  
R T Hay
Keyword(s):  
Amino Acids ◽  
Proteolytic Activity ◽  
Cell Activation ◽  
Nf Kappa B ◽  
Free System ◽  
In Vitro System ◽  
I Kappa B Alpha ◽  
Terminal Domain

After exposure of cells to tumor necrosis factor (TNF), I kappa B alpha is rapidly degraded by a proteolytic activity that is required for nuclear localization and activation of transcription factor NF-kappa B. To investigate this problem, we have developed a cell-free system to study the degradation of I kappa B alpha initiated in vivo. In this in vitro system, characteristics of endogenous I kappa B alpha degradation were comparable to those observed in vivo. Recombinant I kappa B alpha, when added to lysates from cells exposed to TNF, was specifically degraded by a cellular proteolytic activity; however, it was stable in extracts from unstimulated cells. Inhibition characteristics of the proteolytic activity responsible for I kappa B alpha degradation suggest the involvement of a serine protease. Analysis of mutated forms of I kappa B alpha in the in vitro system demonstrated that an I kappa B alpha species which was unable to interact with NF-kappa B was still efficiently degraded. In contrast, deletion of the C-terminal 61 amino acids from I kappa B alpha rendered the protein resistant to proteolytic degradation. Expression of I kappa B alpha mutated forms in COS-7 cells confirmed the importance of the C-terminal domain for the degradation of the protein in vivo following cell activation. Thus, it is likely that the acidic, negatively charged region represented by the C-terminal 61 amino acids of the protein contains residues critical for TNF-inducible degradation of I kappa B alpha.

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