Role of FlbT in flagellin production in Brucella melitensis

Microbiology ◽  
2011 ◽  
Vol 157 (5) ◽  
pp. 1253-1262 ◽  
Author(s):  
Jonathan Ferooz ◽  
Julien Lemaire ◽  
Jean-Jacques Letesson
Keyword(s):  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Marked Decrease ◽  
Brucella Melitensis ◽  
Body Structure ◽  
Wild Type ◽  
Reporter Construct ◽  
Flagellar Assembly

It was recently demonstrated that the pathogen Brucella melitensis produces a polar sheathed flagellum under the control of the master regulator FtcR. However, the regulatory mechanism controlling the flagellar assembly remains unknown. In this work, we investigate the flagellar hierarchy of B. melitensis as well as the flagellin FliC regulation. We show that a mutation in fliF or flgE (coding for the basal body structure and the hook, respectively) does not affect FliC synthesis, suggesting that production of FliC does not depend on the flagellar assembly. We demonstrate that FlbT is a FliC activator since inactivation of flbT causes a decrease in fliC expression by using a fliC–lacZ translational reporter construct. Moreover, the quantitative real-time PCR and Western blot analysis show a marked decrease in fliC mRNA and FliC protein level, respectively. Conversely, the B. melitensis wild-type strain overexpressing flaF fails to produce FliC, suggesting an opposite function. Interestingly, the expression of the flbT gene in an ftcR or an flbT mutant restores FliC production, demonstrating that FlbT plays a regulatory checkpoint role in FliC synthesis. This mechanism could be conserved in the Rhizobiales since complementation of an flbT or an ftcR mutant with flbT from Sinorhizobium meliloti restores FliC synthesis.

scholarly journals Role of Trehalose Transport and Utilization in Sinorhizobium meliloti-Alfalfa Interactions

2005 ◽  
Vol 18 (7) ◽  
pp. 694-702 ◽  
Author(s):  
John Beck Jensen ◽  
Osei Yaw Ampomah ◽  
Richard Darrah ◽  
N. Kent Peters ◽  
T. V. Bhuvaneswari
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Sinorhizobium Meliloti ◽  
Brucella Melitensis ◽  
Sole Source ◽  
Root Surface ◽  
Wild Type ◽  
Major Pathway ◽  
Mesorhizobium Loti ◽  
Infection Threads

Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for trehalose catabolism and are induced by trehalose but not by related structurally similar disaccharides like sucrose or maltose. S. meliloti strains mutated in either of these two genes were severely impaired in their ability to grow on trehalose as the sole source of carbon. ThuA and ThuB show no homology to any known enzymes in trehalose utilization. ThuA has similarity to proteins of unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis, and ThuB possesses homology to dehydrogenases containing the consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the root surface and in the infection threads but not in the symbiotic zone of the nodules. Even though thuA and thuB mutants were impaired in competitive colonization of Medicago sativa roots, these strains were more competitive than the wild-type Rm1021 in infecting alfalfa roots and forming nitrogen-fixing nodules. Possible reasons for their increased competitiveness are discussed.

scholarly journals Quorum Sensing Controls Exopolysaccharide Production in Sinorhizobium meliloti

2003 ◽  
Vol 185 (1) ◽  
pp. 325-331 ◽  
Author(s):  
Melanie M. Marketon ◽  
Sarah A. Glenn ◽  
Anatol Eberhard ◽  
Juan E. González
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Homoserine Lactone ◽  
Symbiotic Relationship ◽  
Wild Type ◽  
Sensing System ◽  
Quorum Sensing System

ABSTRACT Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by S. meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C16:1-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression. Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.

scholarly journals Formation of Methyl Mercaptan froml-Methionine by Porphyromonas gingivalis

2000 ◽  
Vol 68 (12) ◽  
pp. 6912-6916 ◽  
Author(s):  
Mamiko Yoshimura ◽  
Yoshio Nakano ◽  
Yoshihisa Yamashita ◽  
Takahiko Oho ◽  
Toshiyuki Saito ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Wild Type Strain ◽  
Marked Decrease ◽  
Oral Bacteria ◽  
Methyl Mercaptan ◽  
Wild Type ◽  
Oral Malodor ◽  
Gene Encoding ◽  
Degradation Activity

ABSTRACT Methyl mercaptan production by oral bacteria is thought to be one of the main causes of oral malodor. We examined the ability of periodontopathic Porphyromonas gingivalis to produce methyl mercaptan from l-methionine and found that the invasive strains W83 and W50 produced large amounts of methyl mercaptan. We cloned and sequenced the mgl gene encodingl-methionine-α-deamino-γ-mercaptomethane-lyase (METase) from P. gingivalis W83. The structural mgl gene consisted of 1,200 bp and encoded a 43.3-kDa protein. To examine the role of methyl mercaptan in the pathogenesis ofP. gingivalis, a METase-deficient mutant of P. gingivalis W83 was constructed. The methionine degradation activity and virulence of the mutant (M1217) and the parent strain (W83) in mice were compared. M1217 showed a marked decrease in the formation of methyl mercaptan from l-methionine and decreased virulence compared with the wild-type strain W83. These results suggest that methyl mercaptan not only is one of the sources of oral malodor, but may also play a role in the pathogenicity of P. gingivalis.

scholarly journals Functional Characterization of the Sinorhizobium meliloti Acetate Metabolism Genes aceA, SMc00767, and glcB

2007 ◽  
Vol 189 (16) ◽  
pp. 5875-5884 ◽  
Author(s):  
J. A. Ramírez-Trujillo ◽  
S. Encarnación ◽  
E. Salazar ◽  
A. García de los Santos ◽  
M. F. Dunn ◽  
...  
Keyword(s):  
Type Strain ◽  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Isocitrate Lyase ◽  
Functional Characterization ◽  
Malate Synthase ◽  
Acetate Metabolism ◽  
Transcriptional Level ◽  
Wild Type

ABSTRACT The genes encoding malate synthase (glcB) and isocitrate lyase (aceA) and a 240-bp open reading frame (SMc00767) located downstream of aceA were isolated and functionally characterized in Sinorhizobium meliloti. Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed. aceA mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient in glcB grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that aceA and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants. aceA-glcB, aceA-SMc00767, and glcB-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type aceA-glcB or aceA-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of aceA and SMc00767 and show that glcB, in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains. aceA and glcB were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulates aceA expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, including aceA; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in S. meliloti. Genetic and functional analyses indicated that aceA and SMc00767 constitute a functional two-gene operon, which is conserved in other α-proteobacteria. Alfalfa plants infected with the aceA and glcB mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the Rhizobium-legume symbiosis.

scholarly journals Salmonella enterica Serovar Kentucky Flagella Are Required for Broiler Skin Adhesion and Caco-2 Cell Invasion

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Sanaz Salehi ◽  
Kevin Howe ◽  
Mark L. Lawrence ◽  
John P. Brooks ◽  
R. Hartford Bailey ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Flagellar Motor ◽  
Structural Genes ◽  
Wild Type ◽  
Content Type ◽  
Flagellar Assembly ◽  
Adhesion And Invasion

ABSTRACT Nontyphoidal Salmonella strains are the main source of pathogenic bacterial contamination in the poultry industry. Recently, Salmonella enterica serovar Kentucky has been recognized as the most prominent serovar on carcasses in poultry-processing plants. Previous studies showed that flagella are one of the main factors that contribute to bacterial attachment to broiler skin. However, the precise role of flagella and the mechanism of attachment are unknown. There are two different flagellar subunits (fliC and fljB) expressed alternatively in Salmonella enterica serovars using phase variation. Here, by making deletions in genes encoding flagellar structural subunits (flgK, fliC, and fljB), and flagellar motor (motA), we were able to differentiate the role of flagella and their rotary motion in the colonization of broiler skin and cellular attachment. Utilizing a broiler skin assay, we demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables Salmonella motility, but this subunit is unable to mediate tight attachment. Deletion of the flgK gene prevents proper flagellar assembly, making Salmonella significantly less adherent to broiler skin than the wild type. S. Kentucky with deletions in all three structural genes, fliC, fljB, and flgK, as well as a flagellar motor mutant (motA), exhibited less adhesion and invasion of Caco-2 cells, while an fljB mutant was as adherent and invasive as the wild-type strain. IMPORTANCE In this work, we answered clearly the role of flagella in S. Kentucky attachment to the chicken skin and Caco-2 cells. We demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables Salmonella motility, but this subunit is unable to mediate strong attachment. Deletion of the flgK gene prevents proper flagellar assembly, making Salmonella significantly less adherent to broiler skin than the wild type. S. Kentucky with deletions in all three structural genes, fliC, fljB, and flgK, as well as a flagellar motor mutant (motA), exhibited less adhesion and invasion of Caco-2 cells, while an fljB mutant was as adherent and invasive as the wild-type strain. We expect these results will contribute to the understanding of the mechanisms of Salmonella attachment to food products.

scholarly journals Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals Role Of Selected Fimbrial Adheins In Pathogenesis Of Acid-Induced Eneterohemorrhagic Escherichia Coli 0157:H7

2021 ◽  
Author(s):  
Shahnaz Haque
Keyword(s):  
Fatty Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Short Chain Fatty Acid ◽  
Acid Stress ◽  
Chain Fatty Acid ◽  
Short Chain ◽  
Wild Type ◽  
Food Borne

Enterohemorrhagic Escherichia coli (EHEC) 0157:H7 is a food-borne pathogen that causes hemolytic uremic syndrome and hemorrhagic colitis. The mechanisms underlying the adhesion of EHEC 0157:H7 to intestinal epithelial cells are not well understood. Like other food-borne pathogens, ECEC 0157:H7 must survive the acid stress of the gastric juice in the stomach and short chain fatty acid in the intestine in order to colonize the large intestine. We have found that acid stress and short chain fatty acid stress significantly enhance host-adhesion of EHEC 0157:H7 and also upregulates expression of EHEC fimbrial genes, lpfA1, lpfA2 and yagZ, as demonstrated by our DNA microarray. We now report that disruption of the yagZ (also known as the E. coli common pilus A) gene results in loss of the acid-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the sress-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the stress-induced adhesion pehnotype is restored, confirming the role of yagZ in the acid as well as short chain fatty acid induced adhesion to HEp-2 cells. On the other hand, neither disruption in the long polar fimbria genes lpfA1 or lpfA2 in the wild type showed any effect in adherence to HEp-2 cells; rather displaying a hyperadherant phenotype to HEp-2 cells after acid-induced or short chain fatty acid-induced stress. The results also indicate that acid or short chain fatty acid stress, which is a part of the host's natural defense mechanism against pathogens, may regulate virulence factors resulting in enhanced bacteria-host attachment during colonization in the human or bovine host.

Identification of Crp as a novel regulator of the Std fimbrial expression in Salmonella

Microbiology ◽  
2021 ◽  
Author(s):  
Karine Dufresne ◽  
France Daigle
Keyword(s):  
Wild Type Strain ◽  
Distal Region ◽  
Transcriptional Factor ◽  
Major Influence ◽  
Wild Type ◽  
Content Type ◽  
Link Type ◽  
Serovar Typhimurium ◽  
Metabolic Regulators

The Salmonella enterica serovar Typhi genome contains 14 putative fimbrial systems. The Std fimbriae belong to the chaperone-usher family and its regulation has not been investigated in S. Typhi. Several regulators of Std were previously identified in the closely related serovar Typhimurium. We hypothesize that regulators of S. Typhimurium may be shared with S. Typhi, but that several other regulators remain to be discovered. Here, we describe the role of more than 50 different candidate regulators on std expression. Three types of regulators were investigated: known regulators in S. Typhimurium, in silico predicted regulators and virulence/metabolic regulators. Expression of std was determined in the regulator mutants and compared with the wild-type strain. Overall, 21 regulator mutations affect std promoter expression. The role of Crp, a newly identified factor for std expression, was further investigated. Crp acted as an activator of std expression on a distal region of the std promoter region. Together, our results demonstrate the major influence of Crp as a novel transcriptional factor on std promoter expression and later production of Std fimbriae in Salmonella .

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.

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