Role of the N-terminal region and of β-sheet residue Thr29 on the activity of the ω2 global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035

2005 ◽  
Vol 386 (9) ◽  
Author(s):  
Karin Welfle ◽  
Florencia Pratto ◽  
Rolf Misselwitz ◽  
Joachim Behlke ◽  
Juan C. Alonso ◽  
...  
Keyword(s):  
Host Range ◽  
Streptococcus Pyogenes ◽  
Regulatory Protein ◽  
Terminal Region ◽  
Broad Host Range ◽  
Wild Type ◽  
Global Regulator ◽  
Β Sheet

AbstractThe dimeric regulatory protein wild-type ω (wt ω

scholarly journals Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3433
Author(s):  
Noramirah Bukhari ◽  
Adam Thean Chor Leow ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Fairolniza Mohd Shariff
Keyword(s):  
Rational Design ◽  
Thermal Denaturation ◽  
Temperature Stability ◽  
Industrial Applications ◽  
Terminal Region ◽  
Wild Type ◽  
Functional Region ◽  
Target Region ◽  
Residue Substitution

Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.

scholarly journals The N-Terminus ofDictyosteliumScar Interacts with Abi and HSPC300 and Is Essential for Proper Regulation and Function

2007 ◽  
Vol 18 (5) ◽  
pp. 1609-1620 ◽  
Author(s):  
Diana Caracino ◽  
Cheryl Jones ◽  
Mark Compton ◽  
Charles L. Saxe
Keyword(s):  
Actin Polymerization ◽  
Terminal Region ◽  
Wild Type ◽  
Large Molecular Weight ◽  
Weight Protein ◽  
And Function ◽  
Necessary And Sufficient

Scar/WAVE proteins, members of the conserved Wiskott-Aldrich syndrome (WAS) family, promote actin polymerization by activating the Arp2/3 complex. A number of proteins, including a complex containing Nap1, PIR121, Abi1/2, and HSPC300, interact with Scar/WAVE, though the role of this complex in regulating Scar function remains unclear. Here we identify a short N-terminal region of Dictyostelium Scar that is necessary and sufficient for interaction with HSPC300 and Abi in vitro. Cells expressing Scar lacking this N-terminal region show abnormalities in F-actin distribution, cell morphology, movement, and cytokinesis. This is true even in the presence of wild-type Scar. The data suggest that the first 96 amino acids of Scar are necessary for participation in a large-molecular-weight protein complex, and that this Scar-containing complex is responsible for the proper localization and regulation of Scar. The presence of mis-regulated or unregulated Scar has significant deleterious effects on cells and may explain the need to keep Scar activity tightly controlled in vivo either by assembly in a complex or by rapid degradation.

scholarly journals The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of Streptococcus pyogenes

2002 ◽  
Vol 70 (9) ◽  
pp. 4968-4976 ◽  
Author(s):  
Susanna Ricci ◽  
Robert Janulczyk ◽  
Lars Björck
Keyword(s):  
Oxidative Stress ◽  
Streptococcus Pyogenes ◽  
Stress Responses ◽  
Metal Binding ◽  
Iron Homeostasis ◽  
Bacterial Species ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Allelic Replacement

ABSTRACT Ferric uptake regulator (Fur) and Fur-like proteins form an important family of transcriptional regulators in many bacterial species. In this work we have characterized a Fur-like protein, the peroxide regulator PerR, in an M1 serotype of Streptococcus pyogenes. To determine the role of PerR in S. pyogenes, we inactivated the gene by allelic replacement. PerR-deficient bacteria showed 48% reduction of 55Fe incorporation from the culture medium. Transcriptional analysis revealed that mtsA, encoding a metal-binding protein of an ABC transporter in S. pyogenes, was transcribed at lower levels than were wild-type cells. Although total iron accumulation was reduced, the growth of the mutant strain was not significantly hampered. The mutant showed hyperresistance to hydrogen peroxide, and this response was induced in wild-type cells by growth in aerobiosis, suggesting that PerR acts as an oxidative stress-responsive repressor. PerR may also participate in the response to superoxide stress, as the perR mutant was more sensitive to the superoxide anion and had a reduced transcription of sodA, which encodes the sole superoxide dismutase of S. pyogenes. Complementation of the mutation with a functional perR gene restored 55Fe incorporation, response to peroxide stress, and transcription of both mtsA and sodA to levels comparable to those of wild-type bacteria. Finally, the perR mutant was attenuated in virulence in a murine air sac model of infection (P < 0.05). These results demonstrate that PerR is involved in the regulation of iron homeostasis and oxidative stress responses and that it contributes to the virulence of S. pyogenes.

Mutational analysis of the global regulator KorA of broad-host-range plasmid RK2

1998 ◽  
Vol 281 (3) ◽  
pp. 453-463 ◽  
Author(s):  
Kalliopi Kostelidou ◽  
Grazyna Jagura-Burdzy ◽  
Christopher M Thomas
Keyword(s):  
Host Range ◽  
Mutational Analysis ◽  
Broad Host Range ◽  
Global Regulator ◽  
Broad Host Range Plasmid ◽  
Plasmid Rk2

scholarly journals Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

1998 ◽  
Vol 180 (22) ◽  
pp. 6023-6030 ◽  
Author(s):  
Carla L. Easter ◽  
Helmut Schwab ◽  
Donald R. Helinski
Keyword(s):  
Host Range ◽  
Plasmid Stability ◽  
Broad Host Range ◽  
E Coli ◽  
Broad Host Range Plasmid ◽  
Resolution System ◽  
Stable Maintenance ◽  
Plasmid Stabilization ◽  
Plasmid Rk2

ABSTRACT The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons,parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, andparCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or bothparB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss ofparCBA activity. For P. aeruginosaPAO1161Rifr, the parCBA operon provided little if any plasmid stability, but for P. aeruginosaPAC452Rifr, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parAand res alone were sufficient for stabilization. Thecer resolvase system of plasmid ColE1 and theloxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all threeE. coli strains. These observations indicate that theparA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of theparC and parB genes but that in at least oneE. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or thecer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.

scholarly journals Dissecting the Role of the N-Terminal Region of the Escherichia coli Global Transcription Factor FNR

2008 ◽  
Vol 190 (24) ◽  
pp. 8230-8233 ◽  
Author(s):  
Aixin Yan ◽  
Patricia J. Kiley
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Terminal Region ◽  
Wild Type ◽  
Fnr Protein

ABSTRACT The role of the N-terminal region of the transcription factor FNR, which immediately precedes the first ligand (Cys20) of the [4Fe-4S] cluster, was investigated. We found that truncation mutants that removed residues 2 to 16 and 2 to 17 had wild-type levels of FNR protein but surprisingly altered O2 regulation.

scholarly journals Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range PromoterPm

2011 ◽  
Vol 77 (8) ◽  
pp. 2648-2655 ◽  
Author(s):  
Rahmi Lale ◽  
Laila Berg ◽  
Friederike Stüttgen ◽  
Roman Netzer ◽  
Marit Stafsnes ◽  
...  
Keyword(s):  
Host Range ◽  
Untranslated Region ◽  
Micrococcus Luteus ◽  
Background Level ◽  
Broad Host Range ◽  
Continuous Control ◽  
Wild Type ◽  
Content Type ◽  
Broad Host Range Plasmid ◽  
Genes Encoding

ABSTRACTThe induciblePmpromoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in thePmDNA region corresponding to the 5′ untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding β-lactamase, luciferase, and phosphoglucomutase) inEscherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter,P1(Pantitet), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C50carotenoid sarcinaxanthin in an engineered strain ofE. colithat produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing threeMicrococcus luteusderived genes from the promoterPm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.

scholarly journals The Actinobacillus actinomycetemcomitans Autotransporter Adhesin Aae Exhibits Specificity for Buccal Epithelial Cells from Humans and Old World Primates

2005 ◽  
Vol 73 (4) ◽  
pp. 1947-1953 ◽  
Author(s):  
Daniel H. Fine ◽  
Kabilan Velliyagounder ◽  
David Furgang ◽  
Jeffrey B. Kaplan
Keyword(s):  
Epithelial Cells ◽  
Host Range ◽  
Mutant Strain ◽  
Actinobacillus Actinomycetemcomitans ◽  
Broad Host Range ◽  
Wild Type ◽  
New World Primates ◽  
Old World ◽  
Broad Host Range Plasmid ◽  
Buccal Epithelial Cells

ABSTRACT Cells of the gram-negative periodontopathogen Actinobacillus actinomycetemcomitans express a surface-exposed, outer membrane autotransporter protein, designated Aae, which has been implicated in epithelial cell binding. We constructed a mutant strain of A. actinomycetemcomitans that contained a transposon insertion in the Aae structural gene (aae) and tested the mutant to determine its ability to bind to buccal epithelial cells (BECs) isolated from healthy volunteers. Significantly fewer mutant cells than wild-type cells bound to BECs. A broad-host-range plasmid that contained an intact aae gene driven by a heterologous tac promoter restored the ability of the mutant strain to bind to BECs at wild-type levels. This plasmid also conferred upon Escherichia coli the ability to express the Aae protein on its surface and to bind to human BECs. Aae-expressing E. coli also bound to BECs isolated from six Old World primates but not to BECs isolated from four New World primates or nine other nonprimate mammals, as well as to human gingival epithelial cells but not to human pharyngeal, palatal, tongue, bronchial, or cervical epithelial cells. Our findings indicate that Aae mediates binding of A. actinomycetemcomitans to BECs from humans and Old World primates and that this process may contribute to the host range specificity and tissue tropism exhibited by this bacterium.

scholarly journals Rgg Regulates Growth Phase-Dependent Expression of Proteins Associated with Secondary Metabolism and Stress in Streptococcus pyogenes

2004 ◽  
Vol 186 (21) ◽  
pp. 7091-7099 ◽  
Author(s):  
Michelle A. Chaussee ◽  
Eduardo A. Callegari ◽  
Michael S. Chaussee
Keyword(s):  
Secondary Metabolism ◽  
Mutant Strain ◽  
Streptococcus Pyogenes ◽  
Growth Phase ◽  
Stress Responses ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Protein ◽  
Wild Type ◽  
Proteomic Approach

ABSTRACT The transcriptional regulatory protein Rgg coordinates amino acid catabolism and virulence factor expression in Streptococcus pyogenes. We used a proteomic approach to compare cytoplasmic proteins isolated from S. pyogenes wild-type strain NZ131 (serotype M49) to proteins isolated from an rgg mutant strain during the exponential and stationary phases of growth. Proteins were separated by two-dimensional gel electrophoresis, and 125 protein spots of interest were identified by tandem mass spectrometry. Comparative analysis of proteins isolated from the isogenic strains revealed that growth phase-associated regulation of enzymes involved in the metabolism of arginine (ArcABC), histidine (HutI), and serine (SdhA) was abrogated in the rgg mutant strain, which synthesized the proteins in the exponential phase of growth. In contrast, the enzymes were detected only among wild-type proteins isolated from organisms in the stationary phase of growth. The differences in protein composition were correlated with previously described metabolic changes. In addition, proteins associated with thermal and oxidative stress responses, including ClpE and ClpL, were present in samples isolated from the rgg mutant strain but not in samples isolated from the wild-type strain. The rgg mutant strain was more tolerant to elevated temperature and puromycin than the wild-type strain; however, the mutant was less tolerant to paraquat. We concluded that Rgg is a global regulatory factor that contributes to growth phase-dependent synthesis of proteins associated with secondary metabolism and oxidative and thermal stress responses.

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