Inactivation of the Crp/Fnr Family of Regulatory Genes in Listeria monocytogenes Strain F2365 Does Not Alter Its Heat Resistance at 60°C†

2006 ◽  
Vol 69 (11) ◽  
pp. 2758-2760 ◽  
Author(s):  
DARRELL O. BAYLES ◽  
GAYLEN A. UHLICH
Keyword(s):  
Listeria Monocytogenes ◽  
Heat Resistance ◽  
Thermal Tolerance ◽  
Type Strain ◽  
Transcriptional Control ◽  
Wild Type Strain ◽  
Gene Cassette ◽  
Wild Type ◽  
Mutant Strains ◽  
Virulence Regulator

A surprising facet of the Listeria monocytogenes genome is the presence of 15 genes that code for regulators in the Crp/Fnr family and include the virulence regulator PrfA. The genes under the transcriptional control of these regulators are currently undetermined, with the exception of some genes controlled by the major virulence regulator PrfA. Using 12 strains of L. monocytogenes, each with an inserted gene cassette that interrupts and renders nonfunctional a different L. monocytogenes strain F2365 Crp/Fnr regulator, we heat challenged each strain at 60°C with an immersed-coil heating apparatus, modeled the survivor data to calculate the underlying mean and mode of the heat resistance distribution for each strain, and compared the thermal tolerance of each mutant to the wild-type strain to determine if any of the Crp/Fnr mutants demonstrated altered heat tolerance. All 12 of the Crp/Fnr mutant strains tested had heat resistance characteristics similar to the wild-type strain (P > 0.05), indicating that mutations in these Crp/Fnr genes neither increased nor decreased the sensitivity of L. monocytogenes strain F2365 to mild heat.

scholarly journals Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

2005 ◽  
Vol 49 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
Ayush Kumar ◽  
Elizabeth A. Worobec
Keyword(s):  
Serratia Marcescens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Efflux Pump ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Diverse Range ◽  
Mutant Strains ◽  
Encoding Genes

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

scholarly journals Role of CpALS4790 and CpALS0660 in Candida parapsilosis Virulence: Evidence from a Murine Model of Vaginal Candidiasis

2020 ◽  
Vol 6 (2) ◽  
pp. 86
Author(s):  
Marina Zoppo ◽  
Fabrizio Fiorentini ◽  
Cosmeri Rizzato ◽  
Mariagrazia Di Luca ◽  
Antonella Lupetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Murine Model ◽  
Type Strain ◽  
Wild Type Strain ◽  
Candida Parapsilosis ◽  
Vaginal Candidiasis ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Mutant Strains

The Candida parapsilosis genome encodes for five agglutinin-like sequence (Als) cell-wall glycoproteins involved in adhesion to biotic and abiotic surfaces. The work presented here is aimed at analyzing the role of the two still uncharacterized ALS genes in C. parapsilosis, CpALS4790 and CpALS0660, by the generation and characterization of CpALS4790 and CpALS066 single mutant strains. Phenotypic characterization showed that both mutant strains behaved as the parental wild type strain regarding growth rate in liquid/solid media supplemented with cell-wall perturbing agents, and in the ability to produce pseudohyphae. Interestingly, the ability of the CpALS0660 null mutant to adhere to human buccal epithelial cells (HBECs) was not altered when compared with the wild-type strain, whereas deletion of CpALS4790 led to a significant loss of the adhesion capability. RT-qPCR analysis performed on the mutant strains in co-incubation with HBECs did not highlight significant changes in the expression levels of others ALS genes. In vivo experiments in a murine model of vaginal candidiasis indicated a significant reduction in CFUs recovered from BALB/C mice infected with each mutant strain in comparison to those infected with the wild type strain, confirming the involvement of CpAls4790 and CpAls5600 proteins in C. parapsilosis vaginal candidiasis in mice.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

scholarly journals Sigma B Contributes to Listeria monocytogenes Gastrointestinal Infection but Not to Systemic Spread in the Guinea Pig Infection Model

2006 ◽  
Vol 74 (2) ◽  
pp. 876-886 ◽  
Author(s):  
M. R. Garner ◽  
B. L. Njaa ◽  
M. Wiedmann ◽  
K. J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Type Strain ◽  
Wild Type Strain ◽  
Critical Role ◽  
Host Cells ◽  
Gastrointestinal Infection ◽  
Wild Type ◽  
Systemic Spread

ABSTRACT Contributions of the alternative sigma factor σB to Listeria monocytogenes infection were investigated using strains bearing null mutations in sigB, prfA, or inlA or in selected inlA or prfA promoter regions. The ΔP4 inlA strain, which has a deletion in the σB-dependent P4 inlA promoter, and the ΔsigB strain had significantly reduced invasion efficiencies relative to that of the wild-type strain in the Caco-2 human colorectal epithelial cell line, while the invasion efficiency of a strain bearing a deletion in the partially σB dependent P2 prfA promoter region did not differ from that of the wild type. The virulence of the ΔsigB and ΔP4 inlA strains was attenuated in intragastrically inoculated guinea pigs, with the ΔsigB strain showing greater attenuation, while the virulence capacity of the ΔP2 prfA strain was similar to that of the wild-type strain, suggesting that attenuation of virulence due to the ΔsigB mutation does not result from loss of σB-dependent prfA transcription. Our results show that σB-dependent activation of inlA is important for cell invasion and gastrointestinal infection and suggest that σB-regulated genes in addition to inlA appear to contribute to gastrointestinal infection. Interestingly, the virulence of the ΔsigB strain was not attenuated in intravenously infected guinea pigs. We conclude that (i) L. monocytogenes σB plays a critical role in invasion of human host cells, (ii) σB-mediated contributions to invasion are, in part, due to direct effects on inlA transcription but not on prfA transcription, and (iii) σB plays a critical role during the gastrointestinal stage of listeriosis in the guinea pig but is not important for systemic spread of the organism.

scholarly journals An Antibiotic Complex from Lysobacter enzymogenes Strain C3: Antimicrobial Activity and Role in Plant Disease Control

2008 ◽  
Vol 98 (6) ◽  
pp. 695-701 ◽  
Author(s):  
S. Li ◽  
C. C. Jochum ◽  
F. Yu ◽  
K. Zaleta-Rivera ◽  
L. Du ◽  
...  
Keyword(s):  
Biological Control ◽  
Fusarium Head Blight ◽  
Tall Fescue ◽  
Leaf Spot ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Head Blight ◽  
Lysobacter Enzymogenes ◽  
Mutant Strains

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 μg/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 μg/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and ΔNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.

scholarly journals Contributions of the 8-Methoxy Group of Gatifloxacin to Resistance Selectivity, Target Preference, and Antibacterial Activity against Streptococcus pneumoniae

2001 ◽  
Vol 45 (6) ◽  
pp. 1649-1653 ◽  
Author(s):  
Hideyuki Fukuda ◽  
Ryuta Kishii ◽  
Masaya Takei ◽  
Masaki Hosaka
Keyword(s):  
Antibacterial Activity ◽  
Streptococcus Pneumoniae ◽  
Type Strain ◽  
Methoxy Group ◽  
Wild Type Strain ◽  
Dna Gyrase ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Mutant Strains

ABSTRACT Gatifloxacin (8-methoxy, 7-piperazinyl-3′-methyl) at the MIC selected mutant strains that possessed gyrA mutations at a low frequency (3.7 × 10−9) from wild-type strainStreptococcus pneumoniae IID553. AM-1147 (8-methoxy, 7-piperazinyl-3′-H) at the MIC or higher concentrations selected no mutant strains. On the other hand, the respective 8-H counterparts of these two compounds, AM-1121 (8-H, 7-piperazinyl-3′-methyl) and ciprofloxacin (8-H, 7-piperazinyl-3′-H), at one and two times the MIC selected mutant strains that possessed parC mutations at a high frequency (>2.4 × 10−6). The MIC of AM-1147 increased for the gyrA mutant strains but not for theparC mutant strains compared with that for the wild-type strain. These results suggest that fluoroquinolones that harbor 8-methoxy groups select mutant strains less frequently and prefer DNA gyrase, as distinct from their 8-H counterparts. The in vitro activities of gatifloxacin and AM-1147 are twofold higher against the wild-type strain, eight- and twofold higher against the first-stepparC and gyrA mutant strains, respectively, and two- to eightfold higher against the second-step gyrA andparC double mutant strains than those of their 8-H counterparts. These results indicate that the 8-methoxy group contributes to enhancement of antibacterial activity against target-altered mutant strains as well as the wild-type strain. It is hypothesized that the 8-methoxy group of gatifloxacin increases the level of target inhibition, especially against DNA gyrase, so that it is nearly the same as that for topoisomerase IV inhibition in the bacterial cell, leading to potent antibacterial activity and a low level of resistance selectivity.

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 Development of Mutants of Coniothyrium Minitans with Improved Efficiency for Control of Sclerotinia Sclerotiorum

2011 ◽  
Vol 51 (2) ◽  
pp. 179-183 ◽  
Author(s):  
Itamar Melo ◽  
Alex Moretini ◽  
Ana Cassiolato ◽  
Jane Faull
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Plant Tissue ◽  
Type Strain ◽  
Mycelial Growth ◽  
Wild Type Strain ◽  
Wild Type ◽  
Coniothyrium Minitans ◽  
High Concentration ◽  
Mutant Strains ◽  
Chitinase Production

Development of Mutants ofConiothyrium Minitanswith Improved Efficiency for Control ofSclerotinia SclerotiorumConiothyrium minitans(CM) is hyperparasitic toSclerotinia sclerotiorum(SS), a pathogen of many economically important crops. In this paper, we describe the isolation of improved mutants of CM, using a UV - irradiation regime, with altered chitinase production and tolerance to high concentration of iprodione, which are effective against SS. Three out of the 59 mutants obtained inhibited the mycelial growth of CM. Infectivity of sclerotia by the new mutants was assayed by the plant-tissue-based system using carrot segments. More than 80% of sclerotia were colonized by the mutants and the wild-type CM. The mutant strains retained ability to produce significant amounts of chitinase. The mutants differed from their wild-type strain in appearance, morphology and sporulation. In conclusion, the results presented here provide evidence that the new biotypes ofC. minitansare effective in controllingS. sclerotiorum.

scholarly journals Transcriptional Control by A-Factor of Two Trypsin Genes in Streptomyces griseus

2005 ◽  
Vol 187 (1) ◽  
pp. 286-295 ◽  
Author(s):  
Jun-ya Kato ◽  
Won-Jae Chi ◽  
Yasuo Ohnishi ◽  
Soon-Kwang Hong ◽  
Sueharu Horinouchi
Keyword(s):  
Type Strain ◽  
Transcriptional Control ◽  
Time Course ◽  
Wild Type Strain ◽  
Streptomyces Griseus ◽  
Trypsin Activity ◽  
Wild Type ◽  
Regulatory Cascade ◽  
Transcriptional Start Point ◽  
In The Wild

ABSTRACT AdpA is the key transcriptional activator for a number of genes of various functions in the A-factor regulatory cascade in Streptomyces griseus, forming an AdpA regulon. Trypsin-like activity was detected at a late stage of growth in the wild-type strain but not in an A-factor-deficient mutant. Consistent with these observations, two trypsin genes, sprT and sprU, in S. griseus were found to be members of the AdpA regulon; AdpA activated the transcription of both genes by binding to the operators located at about −50 nucleotide positions with respect to the transcriptional start point. The transcription of sprT and sprU, induced by AdpA, was most active at the onset of sporulation. Most trypsin activity exerted by S. griseus was attributed to SprT, because trypsin activity in an sprT-disrupted mutant was greatly reduced but that in an sprU-disrupted mutant was only slightly reduced. This was consistent with the observation that the amount of the sprT mRNA was much greater than that of the sprU transcript. Disruption of both sprT and sprU (mutant ΔsprTU) reduced trypsin activity to almost zero, indicating that no trypsin genes other than these two were present in S. griseus. Even the double mutant ΔsprTU grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain.

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