scholarly journals Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy-d-Xylulose-5-Phosphate Pathway inBacillus subtilis

2011 ◽  
Vol 77 (7) ◽  
pp. 2399-2405 ◽  
Author(s):  
Junfeng Xue ◽  
Birgitte K. Ahring
Keyword(s):  
Sigma Factor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Stress Factors ◽  
Alternative Sigma Factor ◽  
Wild Type ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Overexpression Strain ◽  
As Stress

ABSTRACTTo enhance the production of isoprene, a volatile 5-carbon hydrocarbon, in the Gram-positive spore-forming rod-shaped bacteriumBacillus subtilis, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr) were overexpressed inB. subtilisDSM 10. For the strain that overexpresses Dxs, the yield of isoprene was increased 40% over that by the wild-type strain. In the Dxr overexpression strain, the level of isoprene production was unchanged. Overexpression of Dxr together with Dxs showed an isoprene production level similar to that of the Dxs overproduction strain. The effects of external factors, such as stress factors including heat (48°C), salt (0.3 M NaCl), ethanol (1%), and oxidative (0.005% H2O2) stress, on isoprene production were further examined. Heat, salt, and H2O2induced isoprene production; ethanol inhibited isoprene production. In addition, induction and repression effects are independent of SigB, which is the general stress-responsive alternative sigma factor of Gram-positive bacteria.

scholarly journals Construction and Characterization of aMycobacterium tuberculosis Mutant Lacking the Alternate Sigma Factor Gene, sigF

2000 ◽  
Vol 68 (10) ◽  
pp. 5575-5580 ◽  
Author(s):  
Ping Chen ◽  
Rafael E. Ruiz ◽  
Qing Li ◽  
Richard F. Silver ◽  
William R. Bishai
Keyword(s):  
Stationary Phase ◽  
Sigma Factor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Axenic Culture ◽  
Wild Type ◽  
Intracellular Growth ◽  
Factor Gene ◽  
Time To Death

ABSTRACT The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain ofMycobacterium tuberculosis. The growth rate of the ΔsigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the ΔsigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the ΔsigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the ΔsigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

Effects of Acuc on the Growth Development and Spinosad Biosynthesis of Saccharopolyspora Spinosa

2021 ◽  
Author(s):  
Zhudong Liu ◽  
Jie Xiao ◽  
Jianli Tang ◽  
Yang Liu ◽  
Ling Shuai ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Type Strain ◽  
Wild Type Strain ◽  
Shuttle Vector ◽  
Type I ◽  
Wild Type ◽  
Saccharopolyspora Spinosa ◽  
Fermentation Products ◽  
Overexpression Strain ◽  
Mutant Strains

Abstract Background: The interaction between acuC and spinosad biosynthesis is complex. In this study, acetoin utilization protein (acuC) was characterized. It is a type I histone deacetylase that is highly conserved in bacteria. This study first explored the effect of acuC on the growth and development of secondary metabolites of S. spinosa. Results: The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The overexpression of the acuC gene affects the growth and phenotype of S. spinosa. Moreover, the spore production ability of the S. spinosa-acuC strain on solid medium was weaker than that of the wild-type strain. HPLC analysis of the fermentation products for the wild-type and mutant strains demonstrated that the yield of the overexpression strain was 87% higher than that of the wild-type strain. Conclusions: We concluded that the overexpression of acuC positively regulated the biosynthesis of spinosad and affected the acetylation pathway and the growth of S. spinosa. A comparative proteomic analysis between the wild-type and overexpression strains revealed related genes in different metabolic pathways that were affected. We envision that these results can be extended to other actinomycetes for secondary metabolite improvement.

scholarly journals The dlt Operon of Bacillus cereus Is Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects

2009 ◽  
Vol 191 (22) ◽  
pp. 7063-7073 ◽  
Author(s):  
Z. Abi Khattar ◽  
A. Rejasse ◽  
D. Destoumieux-Garzón ◽  
J. M. Escoubas ◽  
V. Sanchis ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Bacillus Cereus ◽  
Cell Walls ◽  
Type Strain ◽  
Pathogenic Bacteria ◽  
Wild Type Strain ◽  
Wild Type ◽  
Gram Positive ◽  
Cationic Antimicrobial Peptides ◽  
Humoral Immune

ABSTRACT The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The ΔdltBc mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. ΔdltBc was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in ΔdltBc ). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.

scholarly journals Transcription of the Bacillus subtilis gerK Operon, Which Encodes a Spore Germinant Receptor, and Comparison with That of Operons Encoding Other Germinant Receptors

2006 ◽  
Vol 188 (11) ◽  
pp. 4131-4136 ◽  
Author(s):  
Takao Igarashi ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Sigma Factor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Dipicolinic Acid ◽  
Mrna Levels ◽  
Wild Type ◽  
Putative Promoter ◽  
Rapid Fall ◽  
Sequence Similarities

ABSTRACT The gerA, gerB, and gerK operons, which encode germinant receptors in spores of Bacillus subtilis, were transcribed only in sporulation, and their mRNA levels peaked initially ∼3 h before the initiation of accumulation of the spore's dipicolinic acid. After a rapid fall, levels of these mRNAs peaked again ∼5 h later. In one wild-type strain (PS832), gerA mRNA was the most abundant, with levels of gerB and gerK mRNAs ∼50% of that of gerA mRNA, whereas gerB mRNA was the most abundant in another wild-type strain (PY79). The synthesis of gerK mRNA in sporulation was abolished by loss of the forespore-specific RNA polymerase sigma factor, σG, and induction of σG synthesis in vegetative cells led to synthesis of gerK mRNA. SpoVT, a regulator of σG-dependent gene expression, repressed gerK expression. The gerK promoter showed sequence similarities to σG-dependent promoters, and deletion of elements of this putative promoter abolished gerK expression in sporulation.

scholarly journals Redefining the Clostridioides difficile σB regulon: σB activates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

2020 ◽  
Author(s):  
Ilse M. Boekhoud ◽  
Annika-Marisa Michel ◽  
Jeroen Corver ◽  
Dieter Jahn ◽  
Wiep Klaas Smits
Keyword(s):  
Hydrogen Peroxide ◽  
Stress Response ◽  
Sigma Factor ◽  
Gene Activation ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Alternative Sigma Factor ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Sigma B

AbstractIn many gram-positive bacteria the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile σB has been implicated in protection against stressors such as reactive oxygen species and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allowed for the refinement of the previously proposed σB regulon. At least 32% the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro run-off transcription of specific target genes (cd0350, cd3614, cd3605, cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation.ImportanceSigma B is the alternative sigma factor governing stress response in many gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together our data suggest that σB is a key player in dealing with toxic radicals.

scholarly journals Redefining the Clostridioides difficile σB Regulon: σB Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Ilse M. Boekhoud ◽  
Annika-Marisa Michel ◽  
Jeroen Corver ◽  
Dieter Jahn ◽  
Wiep Klaas Smits
Keyword(s):  
Hydrogen Peroxide ◽  
Stress Response ◽  
Sigma Factor ◽  
Gene Activation ◽  
Luciferase Reporter ◽  
Alternative Sigma Factor ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Sigma B

ABSTRACT In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation. IMPORTANCE Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.

scholarly journals The Extra Cytoplasmic Function Sigma Factor σE Is Essential for Mycobacterium tuberculosis Virulence in Mice

2004 ◽  
Vol 72 (5) ◽  
pp. 3038-3041 ◽  
Author(s):  
Riccardo Manganelli ◽  
Lanfranco Fattorini ◽  
Dejiang Tan ◽  
Elisabetta Iona ◽  
Graziella Orefici ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Animal Models ◽  
Sigma Factor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Immunocompetent Mice ◽  
Different Characteristics

ABSTRACT The virulence of a Mycobacterium tuberculosis H37Rv sigE mutant was studied in immunodeficient and immunocompetent mice. The mutant was strongly attenuated in both animal models and induced formation of granulomas with different characteristics than those induced by the wild-type strain.

scholarly journals Identification of the σB Regulon of Bacillus cereus and Conservation of σB-Regulated Genes in Low-GC-Content Gram-Positive Bacteria

2007 ◽  
Vol 189 (12) ◽  
pp. 4384-4390 ◽  
Author(s):  
Willem van Schaik ◽  
Menno van der Voort ◽  
Douwe Molenaar ◽  
Roy Moezelaar ◽  
Willem M. de Vos ◽  
...  
Keyword(s):  
Heat Shock ◽  
Bacillus Cereus ◽  
Sigma Factor ◽  
Phylogenetic Analyses ◽  
Gc Content ◽  
Alternative Sigma Factor ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Food Borne ◽  
Lower Expression

ABSTRACT The alternative sigma factor σB has an important role in the acquisition of stress resistance in many gram-positive bacteria, including the food-borne pathogen Bacillus cereus. Here, we describe the identification of the set of σB-regulated genes in B. cereus by DNA microarray analysis of the transcriptome upon a mild heat shock. Twenty-four genes could be identified as being σB dependent as witnessed by (i) significantly lower expression levels of these genes in mutants with a deletion of sigB and rsbY (which encode the alternative sigma factor σB and a crucial positive regulator of σB activity, respectively) than in the parental strain B. cereus ATCC 14579 and (ii) increased expression of these genes upon a heat shock. Newly identified σB-dependent genes in B. cereus include a histidine kinase and two genes that have predicted functions in spore germination. This study shows that the σB regulon of B. cereus is considerably smaller than that of other gram-positive bacteria. This appears to be in line with phylogenetic analyses where σB of the B. cereus group was placed close to the ancestral form of σB in gram-positive bacteria. The data described in this study and previous studies in which the complete σB regulon of the gram-positive bacteria Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus were determined enabled a comparison of the sets of σB-regulated genes in the different gram-positive bacteria. This showed that only three genes (rsbV, rsbW, and sigB) are conserved in their σB dependency in all four bacteria, suggesting that the σB regulon of the different gram-positive bacteria has evolved to perform niche-specific functions.

scholarly journals PorA Represents the Major Cell Wall Channel of the Gram-Positive Bacterium Corynebacterium glutamicum

2003 ◽  
Vol 185 (16) ◽  
pp. 4779-4786 ◽  
Author(s):  
Noelia Costa-Riu ◽  
Andreas Burkovski ◽  
Reinhard Krämer ◽  
Roland Benz
Keyword(s):  
Cell Wall ◽  
Corynebacterium Glutamicum ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Deletion Strain ◽  
Wild Type ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Reverse Transcription Pcr

ABSTRACT The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA. porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032ΔporA. Southern blot analysis demonstrated that porA was deleted. Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain. Searches within the known chromosome of C. glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain. The porA deletion strain exhibited slower growth and longer growth times than the C. glutamicum wild-type strain. Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C. glutamicum strain. The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C. glutamicum contains cell wall channels which are not related to PorA.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

Sign in / Sign up

Export Citation Format

Share Document