scholarly journals Characterization of the Alternative Sigma Factor σ54 and the Transcriptional Regulator FleQ of Legionella pneumophila, Which Are Both Involved in the Regulation Cascade of Flagellar Gene Expression

2004 ◽  
Vol 186 (9) ◽  
pp. 2540-2547 ◽  
Author(s):  
Sebastian Jacobi ◽  
Rüdiger Schade ◽  
Klaus Heuner
Keyword(s):  
Gene Regulation ◽  
Mutant Strain ◽  
Legionella Pneumophila ◽  
Consensus Sequence ◽  
Transcriptional Regulator ◽  
Alternative Sigma Factor ◽  
Flagellar Gene ◽  
Wild Type ◽  
Interaction Domain

ABSTRACT We cloned and analyzed Legionella pneumophila Corby homologs of rpoN (encoding σ54) and fleQ (encoding σ54 activator protein). Two other genes (fleR and pilR) whose products have a σ54 interaction domain were identified in the genome sequence of L. pneumophila. An rpoN mutant strain was nonflagellated and expressed very small amounts of the FlaA (flagellin) protein. Like the rpoN mutant, the fleQ mutant strain of L. pneumophila was also nonflagellated and expressed only small amounts of FlaA protein compared to the amounts expressed by the wild type. In this paper we show that the σ54 factor and the FleQ protein are involved in regulation of flagellar gene operons in L. pneumophila. RpoN and FleQ positively regulate the transcription of FliM and FleN, both of which have a σ54-dependent promoter consensus sequence. However, they seemed to be dispensable for transcription of flaA, fliA, or icmR. Our results confirmed a recently described model of the flagellar gene regulation cascade in L. pneumophila (K. Heuner and M. Steinert, Int. J. Med. Microbiol. 293:133-145, 2003). Flagellar gene regulation was found to be different from that of Enterobacteriaceae but seems to be comparable to that described for Pseudomonas or Vibrio spp.

scholarly journals Characterization of Acp, a Peptidoglycan Hydrolase of Clostridium perfringens with N-Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis

2010 ◽  
Vol 192 (9) ◽  
pp. 2373-2384 ◽  
Author(s):  
Emilie Camiade ◽  
Johann Peltier ◽  
Ingrid Bourgeois ◽  
Evelyne Couture-Tosi ◽  
Pascal Courtin ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Clostridium Perfringens ◽  
Vegetative Growth ◽  
Cell Separation ◽  
Catalytic Domain ◽  
Peptidoglycan Hydrolase ◽  
Wild Type ◽  
Induced Autolysis

ABSTRACT This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.

scholarly journals Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

2002 ◽  
Vol 70 (6) ◽  
pp. 3080-3084 ◽  
Author(s):  
Bhavna G. Gordhan ◽  
Debbie A. Smith ◽  
Heidi Alderton ◽  
Ruth A. McAdam ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Auxotrophic Mutant ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

scholarly journals The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii

1999 ◽  
Vol 181 (16) ◽  
pp. 4879-4889 ◽  
Author(s):  
Laura M. Hales ◽  
Howard A. Shuman
Keyword(s):  
Stationary Phase ◽  
Mutant Strain ◽  
Legionella Pneumophila ◽  
Regulatory Networks ◽  
Acanthamoeba Castellanii ◽  
Logarithmic Phase ◽  
Insertion Mutation ◽  
Wild Type ◽  
E Coli ◽  
Resistance To Stress

ABSTRACT To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of theEscherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoSmutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in therpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host,Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS inL. pneumophila is very different from what has previously been reported for E. coli rpoS.

Mutation of AN39-1 for production and characterization of constitutive, thermostable and pH-resistant dextransucrase / Konstitutif, sıcaklığa ve pH’ya dayanıklı dekstransukrazların üretimi ve karakterizasyonu için AN39-1’in mutasyonu

2016 ◽  
Vol 41 (2) ◽  
Author(s):  
Çiğdem Yamaner ◽  
Murat Kemal Avcı ◽  
Aziz Tanrıseven ◽  
İsmail Yavuz Sezen
Keyword(s):  
Mutant Strain ◽  
Parent Strain ◽  
Leuconostoc Mesenteroides ◽  
Industrial Applications ◽  
Wild Type ◽  
Methane Sulfonate ◽  
Ethyl Methane ◽  
Sucrose Medium ◽  
Higher Temperature

AbstractObjective: Leuconostoc mesenteroides AN39-1 has recently been isolated from Crataegus orientalis var. Orientalis. It produces inducible extracellular dextransucrase (EC 2.4.1.5) forming dextran from sucrose. The aim of this study was (1) to obtain constitutive, pH-resistant and thermostable dextransucrase, (2) to characterization of these dextransucrase.Methods: Mutagenesis was carried out on the parent strain (AN39-1) using UV, ethyl methane sulfonate, and N- methyl- N´-nitro-N-nitrosoguanidine. Dextransucrases from wild type (AN39-1) and the mutant strain (A26-2/11) were purified by polyethylene glycol (PEG) precipitation and characterized.Results: Mutants (A26, A26-2, and A26-2/11) hyper producing and constitutive for dextransucrase were isolated. The mutants (A26, A26-2, A26-2/11) produced 7.2, 8.1, and 2.0 times more dextransucrase activity as compared to parent strain on sucrose medium, respectively. In addition, the mutants produced dextransucrase on glucose medium with higher activities (3.0-5.8 times) than what the parental strain produced on sucrose medium. The mutant enzyme (A26-2/11) was much more thermostable than the native enzyme and resistant to pH more than dextransucrase of AN39-1. The dextransucrase from mutant strain was stable up to 35°C and pH of 7.5 for 3 hr.Conclusion: The structures of dextrans produced by wild type and mutant enzymes were similar to commercially produced B-512 F dextran. Thus, the newly dextransucrases produced by mutant strain could find industrial applications at higher temperature and pH.

scholarly journals Isolation and characterization of a high iturin yielding Bacillus velezensis UV mutant with improved antifungal activity

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0234177
Author(s):  
Young Tae Kim ◽  
Sung Eun Kim ◽  
Won Jung Lee ◽  
Zhao Fumei ◽  
Min Sub Cho ◽  
...  
Keyword(s):  
Biological Control ◽  
Antifungal Activity ◽  
Mutant Strain ◽  
Fusarium Wilt ◽  
Potential Candidate ◽  
Wild Type ◽  
Bacillus Velezensis ◽  
Sclerotinia Rot ◽  
Isolation And Characterization

To isolate Bacillus velezensis mutants with improved antifungal activity for use in the biological control of phytopathogenic fungi, wild-type Bacillus velezensis KRF-001 producing iturin, surfactin, and fengycin was irradiated by ultraviolet (UV) rays. The in vitro and in vivo antifungal activities of UV mutants and characterization of the cyclic lipopeptides produced by a selected mutant were examined. A mutant strain yielding high levels of iturin showed over 2-fold higher antifungal activity than the wild-type against Fusarium oxysporum. A potent suppressive effect of the mutant was also observed on spore germination of Botrytis cinerea, the causative agent of cucumber gray mold, at different butanol extract concentrations. Further analysis of the mutant by real-time PCR and high-performance liquid chromatography revealed increased expression of iturin and surfactin biosynthesis genes as well as enhanced production of iturin and surfactin metabolites. However, the amounts of fengycin obtained from the mutant strain BSM54 were significantly lesser than those of iturin and surfactin. Particularly, iturin A production by the mutant was 3.5-fold higher than that of the wild-type, suggesting that the higher antifungal activity of the mutant against F. oxysporum resulted from the increased expression of biosynthesis genes associated with iturin production. The commercial greenhouse experiment using soil naturally infested with Sclerotinia sclerotiorum (sclerotinia rot) and F. oxysporum (fusarium wilt) showed that the mutant strain reduced sclerotinia rot and fusarium wilt diseases (P = 0.05) more effectively than the wild-type and commercially available product Cillus® in Korea. These results suggest that the mutant with high iturin yield is a potential candidate for the development of a biological control agent in agriculture.

scholarly journals Role of Ger Proteins in Nutrient and Nonnutrient Triggering of Spore Germination in Bacillus subtilis

2000 ◽  
Vol 182 (9) ◽  
pp. 2513-2519 ◽  
Author(s):  
Madan Paidhungat ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Mutant Strain ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Family Members ◽  
Wild Type ◽  
Receptor Proteins ◽  
Rich Media

ABSTRACT Dormant Bacillus subtilis spores germinate in the presence of particular nutrients called germinants. The spores are thought to recognize germinants through receptor proteins encoded by the gerA family of operons, which includesgerA, gerB, and gerK. We sought to substantiate this putative function of the GerA family proteins by characterizing spore germination in a mutant strain that contained deletions at all known gerA-like loci. As expected, the mutant spores germinated very poorly in a variety of rich media. In contrast, they germinated like wild-type spores in a chemical germinant, a 1-1 chelate of Ca2+ and dipicolinic acid (DPA). These observations showed that proteins encoded bygerA family members are required for nutrient-induced germination but not for chemical-triggered germination, supporting the hypothesis that the GerA family encodes receptors for nutrient germinants. Further characterization of Ca2+–DPA-induced germination showed that the effect of Ca2+–DPA on spore germination was saturated at 60 mM and had a Km of 30 mM. We also found that decoating spores abolished their ability to germinate in Ca2+–DPA but not in nutrient germinants, indicating that Ca2+–DPA and nutrient germinants probably act through parallel arms of the germination pathway.

scholarly journals Secreted Enzymatic Activities of Wild-Type andpilD-Deficient Legionella pneumophila

2000 ◽  
Vol 68 (4) ◽  
pp. 1855-1863 ◽  
Author(s):  
Virginia Aragon ◽  
Sherry Kurtz ◽  
Antje Flieger ◽  
Birgid Neumeister ◽  
Nicholas P. Cianciotto
Keyword(s):  
Fatty Acids ◽  
Free Fatty Acids ◽  
Legionella Pneumophila ◽  
Wild Type ◽  
Intracellular Infection ◽  
Type Iv ◽  
Nitrophenyl Phosphate ◽  
Pilus Biogenesis ◽  
Legionnaires Disease

ABSTRACT Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by apilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release ofp-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.

scholarly journals Expression of Multiple Pili by Legionella pneumophila: Identification and Characterization of a Type IV Pilin Gene and Its Role in Adherence to Mammalian and Protozoan Cells

1998 ◽  
Vol 66 (4) ◽  
pp. 1768-1775 ◽  
Author(s):  
Barbara J. Stone ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Insertion Mutation ◽  
Wild Type ◽  
Type Iv ◽  
Transmission Electron ◽  
Wild Type Phenotype ◽  
Type Iv Pilin ◽  
Legionnaires Disease ◽  
Pilin Gene

ABSTRACT Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 μm) or short (0.1 to 0.6 μm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL ). An insertion mutation was constructed in pilEL and introduced into theL. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that thepilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires’ disease and invasion of amoebas in the environment.

scholarly journals Influence of the Alternative σ28 Factor on Virulence and Flagellum Expression of Legionella pneumophila

2002 ◽  
Vol 70 (3) ◽  
pp. 1604-1608 ◽  
Author(s):  
Klaus Heuner ◽  
Claudia Dietrich ◽  
Carina Skriwan ◽  
Michael Steinert ◽  
Jörg Hacker
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Mutant Strain ◽  
Legionella Pneumophila ◽  
Blot Analysis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Kanamycin Resistance Cassette

ABSTRACT The fliA gene of Legionella pneumophila encoding the alternative σ28 factor was inactivated by introducing a kanamycin resistance cassette. Electron microscopy and Western blot analysis revealed that the fliA mutant strain is aflagellate and expresses no flagellin. Reporter gene assays indicated that the flaA promoter is not active in the fliA mutant strain. The fliA mutant strain multiplied less effectively in coculture with amoebae than the wild-type strain and was not able to replicate in coculture with Dictyostelium discoideum.

scholarly journals Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

2015 ◽  
Vol 83 (6) ◽  
pp. 2382-2395 ◽  
Author(s):  
Misu Sanson ◽  
Nishanth Makthal ◽  
Maire Gavagan ◽  
Concepcion Cantu ◽  
Randall J. Olsen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Mutant Strain ◽  
Group A Streptococcus ◽  
Global Gene Expression ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Content Type ◽  
Group A

Whole-genome sequencing analysis of ∼800 strains of group AStreptococcus(GAS) found that the gene encoding themultiple virulencegene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenicmgamutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidinesin vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations inmga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis.

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