Ferric Uptake Regulator (FurA) is Required for Acidovorax citrulli Virulence on Watermelon

Acidovorax citrulli is the causal agent of bacterial fruit blotch, a serious threat to commercial watermelon and melon crop production worldwide. Ferric uptake regulator (Fur) is a global transcription factor that affects a number of virulence-related functions in phytopathogenic bacteria; however, the role of furA has not been determined for A. citrulli. Hence, we constructed an furA deletion mutant and a corresponding complement in the background of A. citrulli strain xlj12 to investigate the role of the gene in siderophore production, concentration of intracellular Fe2+, bacterial sensitivity to hydrogen peroxide, biofilm formation, swimming motility, hypersensitive response induction, and virulence on melon seedlings. The A. citrulli furA deletion mutant displayed increased siderophore production, intracellular Fe2+ concentration, and increased sensitivity to hydrogen peroxide. In contrast, biofilm formation, swimming motility, and virulence on melon seedlings were significantly reduced in the furA mutant. As expected, complementation of the furA deletion mutant restored all phenotypes to wild-type levels. In accordance with the phenotypic results, the expression levels of bfrA and bfrB that encode bacterioferritin, sodB that encodes iron/manganese superoxide dismutase, fliS that encodes a flagellar protein, hrcN that encodes the type III secretion system (T3SS) ATPase, and hrcC that encodes the T3SS outer membrane ring protein were significantly downregulated in the A. citrulli furA deletion mutant. In addition, the expression of feo-related genes and feoA and feoB was significantly upregulated in the furA mutant. Overall, these results indicated that, in A. citrulli, FurA contributes to the regulation of the iron balance system, and affects a variety of virulence-related traits.

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Ferric Uptake Regulator Fur Coordinates Siderophore Production and Defense against Iron Toxicity and Oxidative Stress and Contributes to Virulence in Chromobacterium violaceum

Applied and Environmental Microbiology ◽

10.1128/aem.01620-20 ◽

2020 ◽

Vol 86 (21) ◽

Author(s):

Renato Elias Rodrigues de Souza Santos ◽

Bianca Bontempi Batista ◽

José Freire da Silva Neto

Keyword(s):

Oxidative Stress ◽

Biofilm Formation ◽

Chromobacterium Violaceum ◽

Siderophore Production ◽

Phenotypic Characterization ◽

Ferric Uptake Regulator ◽

Content Type ◽

Swimming Motility ◽

Oxidative Stresses ◽

Major Player

ABSTRACT Iron is a highly reactive metal that participates in several processes in prokaryotic and eukaryotic cells. Hosts and pathogens compete for iron in the context of infection. Chromobacterium violaceum, an environmental Gram-negative bacterial pathogen, relies on siderophores to overcome iron limitation in the host. In this work, we studied the role of the ferric uptake regulator Fur in the physiology and virulence of C. violaceum. A Δfur mutant strain showed decreased growth and fitness under regular in vitro growth conditions and presented high sensitivity to iron and oxidative stresses. Furthermore, the absence of fur caused derepression of siderophore production and reduction in swimming motility and biofilm formation. Consistent with these results, the C. violaceum Δfur mutant was highly attenuated for virulence and liver colonization in mice. In contrast, a manganese-selected spontaneous fur mutant showed only siderophore overproduction and sensitivity to oxidative stress, indicating that Fur remained partially functional in this strain. We found that mutations in genes related to siderophore biosynthesis and a putative CRISPR-Cas locus rescued the Δfur mutant growth defects, indicating that multiple Fur-regulated processes contribute to maintaining bacterial cell fitness. Overall, our data indicated that Fur is conditionally essential in C. violaceum mainly by protecting cells from iron overload and oxidative damage. The requirement of Fur for virulence highlights the importance of iron in the pathogenesis of C. violaceum. IMPORTANCE Maintenance of iron homeostasis, i.e., avoiding both deficiency and toxicity of this metal, is vital to bacteria and their hosts. Iron sequestration by host proteins is a crucial strategy to combat bacterial infections. In bacteria, the ferric uptake regulator Fur coordinates the expression of several iron-related genes. Sometimes, Fur can also regulate several other processes. In this work, we performed an in-depth phenotypic characterization of fur mutants in the human opportunistic pathogen Chromobacterium violaceum. We determined that fur is a conditionally essential gene necessary for proper growth under regular conditions and is fully required for survival under iron and oxidative stresses. Fur also controlled several virulence-associated traits, such as swimming motility, biofilm formation, and siderophore production. Consistent with these results, a C. violaceum fur null mutant showed attenuation of virulence. Therefore, our data established Fur as a major player required for C. violaceum to manage iron, including during infection in the host.

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Genetic Analysis of the AdnA Regulon in Pseudomonas fluorescens: Nonessential Role of Flagella in Adhesion to Sand and Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.185.2.453-460.2003 ◽

2003 ◽

Vol 185 (2) ◽

pp. 453-460 ◽

Cited By ~ 39

Author(s):

Eduardo A. Robleto ◽

Inmaculada López-Hernández ◽

Mark W. Silby ◽

Stuart B. Levy

Keyword(s):

Pseudomonas Aeruginosa ◽

Transcription Factor ◽

Pseudomonas Fluorescens ◽

Biofilm Formation ◽

Deletion Mutant ◽

Open Reading Frames ◽

Cell Processes ◽

Reading Frames ◽

Insertion Mutants

ABSTRACT AdnA is a transcription factor in Pseudomonas fluorescens that affects flagellar synthesis, biofilm formation, and sand adhesion. To identify the AdnA regulon, we used a promoterless Tn5-lacZ element to study the phenotypes of insertion mutants in the presence and absence of AdnA. Of 12,000 insertions, we identified seven different putative open reading frames (ORFs) activated by AdnA (named aba for activated by AdnA). aba120 and aba177 showed homology to flgC and flgI, components of the basal body of the flagella in Pseudomonas aeruginosa. Two other insertions, aba18 and aba51, disrupted genes affecting chemotaxis. The mutant loci aba160 (possibly affecting lipopolysaccharide synthesis) and aba175 (unknown function) led to loss of flagella. The mutant bearing aba203 became motile when complemented with adnA, but the mutated gene showed no similarity to known genes. Curiously, aba18, aba51, aba160, and aba203 mutants formed biofilms even in the absence of AdnA, suppressing the phenotype of the adnA deletion mutant. The combined findings suggest that flagella are nonessential for sand attachment or biofilm formation. Sequence and promoter analyses indicate that AdnA affects at least 23 ORFs either directly or by polar effects. These results support the concept that AdnA regulates cell processes other than those directly related to flagellar synthesis and define a broader cadre of genes in P. fluorescens than that described so far for its homolog, FleQ, in P. aeruginosa.

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Elastase LasB of Pseudomonas aeruginosa promotes biofilm formation partly through rhamnolipid-mediated regulation

Canadian Journal of Microbiology ◽

10.1139/cjm-2013-0667 ◽

2014 ◽

Vol 60 (4) ◽

pp. 227-235 ◽

Cited By ~ 29

Author(s):

Hua Yu ◽

Xiaomei He ◽

Wei Xie ◽

Junzhi Xiong ◽

Halei Sheng ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Deletion Mutant ◽

Gene Expression Analysis ◽

Bacterial Attachment ◽

Transcriptional Level ◽

Complementary Strain ◽

Host Infection ◽

The Impact

Elastase LasB, an important extracellular virulence factor, is shown to play an important role in the pathogenicity of Pseudomonas aeruginosa during host infection. However, the role of LasB in the life cycle of P. aeruginosa is not completely understood. This report focuses on the impact of LasB on biofilm formation of P. aeruginosa PAO1. Here, we reported that the lasB deletion mutant (ΔlasB) displayed significantly decreased bacterial attachment, microcolony formation, and extracellular matrix linkage in biofilm associated with decreased biosynthesis of rhamnolipids compared with PAO1 and lasB complementary strain (ΔlasB+). Nevertheless, the ΔlasB developed restored biofilm formation with supplementation of exogenous rhamnolipids. Further gene expression analysis revealed that the mutant of lasB could result in the downregulation of rhamnolipid synthesis at the transcriptional level. Taken together, these results indicated that LasB could promote biofilm formation partly through the rhamnolipid-mediated regulation.

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Deletion Mutant Library for Investigation of Functional Outputs of Cyclic Diguanylate Metabolism in Pseudomonas aeruginosa PA14

Applied and Environmental Microbiology ◽

10.1128/aem.00299-14 ◽

2014 ◽

Vol 80 (11) ◽

pp. 3384-3393 ◽

Cited By ~ 48

Author(s):

Dae-Gon Ha ◽

Megan E. Richman ◽

George A. O'Toole

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Deletion Mutant ◽

Deletion Mutants ◽

Mutant Library ◽

Potential Utility ◽

Cyclic Diguanylate ◽

Swarming Motility ◽

Content Type ◽

Swimming Motility

ABSTRACTWe constructed a library of in-frame deletion mutants targeting each gene inPseudomonas aeruginosaPA14 predicted to participate in cyclic di-GMP (c-di-GMP) metabolism (biosynthesis or degradation) to provide a toolkit to assist investigators studying c-di-GMP-mediated regulation by this microbe. We present phenotypic assessments of each mutant, including biofilm formation, exopolysaccharide (EPS) production, swimming motility, swarming motility, and twitch motility, as a means to initially characterize these mutants and to demonstrate the potential utility of this library.

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Deletion of the Ferric Uptake Regulator Fur Impairs the In Vitro Growth and Virulence of Actinobacillus pleuropneumoniae

Infection and Immunity ◽

10.1128/iai.73.6.3740-3744.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3740-3744 ◽

Cited By ~ 30

Author(s):

Ilse Jacobsen ◽

Jörg Gerstenberger ◽

Achim D. Gruber ◽

Janine T. Bossé ◽

Paul R. Langford ◽

...

Keyword(s):

Deletion Mutant ◽

Constitutive Expression ◽

Actinobacillus Pleuropneumoniae ◽

Infection Model ◽

Ferric Uptake Regulator ◽

In Vitro Growth ◽

Transferrin Binding Proteins ◽

Aerosol Infection

ABSTRACT In order to investigate the role of the ferric uptake regulator Fur in the porcine lung pathogen Actinobacillus pleuropneumoniae, we constructed an isogenic in-frame deletion mutant, A. pleuropneumoniae Δfur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.

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Identification of the sigB Operon inStaphylococcus epidermidis: Construction and Characterization of a sigB Deletion Mutant

Infection and Immunity ◽

10.1128/iai.69.12.7933-7936.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7933-7936 ◽

Cited By ~ 22

Author(s):

Stefanie Kies ◽

Michael Otto ◽

Cuong Vuong ◽

Friedrich Götz

Keyword(s):

Staphylococcus Epidermidis ◽

Sigma Factor ◽

Biofilm Formation ◽

Deletion Mutant ◽

Alternative Sigma Factor ◽

Global Regulator ◽

Profound Influence ◽

Cytoplasmic Proteins

ABSTRACT The role of the alternative sigma factor ςB inStaphylococcus epidermidis was investigated by the construction, complementation, and characterization of asigB deletion mutant. Electrophoretic analyses confirmed a profound influence of ςB on the expression of exoproteins and cytoplasmic proteins. Detailed investigation revealed reduced lipase and enhanced protease activity in the ςB mutant. Furthermore, no significant influence of ςB on heterologous biofilm formation or on the activity of the global regulator agr was detected.

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Altered proteome of a Burkholderia pseudomallei mutant defective in short-chain dehydrogenase affects cell adhesion, biofilm formation and heat stress tolerance

PeerJ ◽

10.7717/peerj.8659 ◽

2020 ◽

Vol 8 ◽

pp. e8659 ◽

Cited By ~ 1

Author(s):

Onrapak Reamtong ◽

Nitaya Indrawattana ◽

Amporn Rungruengkitkun ◽

Tipparat Thiangtrongjit ◽

Taksaon Duangurai ◽

...

Keyword(s):

Heat Stress ◽

Biofilm Formation ◽

Burkholderia Pseudomallei ◽

Deletion Mutant ◽

Stress Conditions ◽

Host Cells ◽

Short Chain ◽

Wild Type ◽

Short Chain Dehydrogenase

Burkholderia pseudomallei is a Gram-negative bacillus that causes melioidosis and is recognized as an important public health problem in southeast Asia and northeast Australia. The treatment of B. pseudomallei infection is hampered by resistance to a wide range of antimicrobial agents and no vaccine is currently available. At present, the underlying mechanisms of B. pseudomallei pathogenesis are poorly understood. In our previous study, we reported that a B. pseudomallei short-chain dehydrogenase (SDO; BPSS2242) mutant constructed by deletion mutagenesis showed reduced B. pseudomallei invasion and initial intracellular survival. This indicated that SDO is associated with the pathogenesis of melioidosis. In the present study, the role of B. pseudomallei SDO was further investigated using the SDO deletion mutant by a proteomic approach. The protein profiles of the SDO mutant and wild-type K96243 were investigated through gel-based proteomic analysis. Quantitative intensity analysis of three individual cultures of the B. pseudomallei SDO mutant revealed significant down-regulation of five protein spots compared with the wild-type. Q-TOF MS/MS identified the protein spots as a glutamate/aspartate ABC transporter, prolyl-tRNA synthetase, Hsp70 family protein, quinone oxidoreductase and a putative carboxypeptidase. Functional assays were performed to investigate the role of these differentially expressed proteins in adhesion to host cells, biofilm induction and survival under heat stress conditions. The SDO deletion mutant showed a decreased ability to adhere to host cells. Moreover, biofilm formation and the survival rate of bacteria under heat stress conditions were also reduced in the mutant strain. Our findings provide insight into the role of SDO in the survival and pathogenesis of B. pseudomallei at the molecular level, which may be applied to the prevention and control of B. pseudomallei infection.

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icaA Gene of Staphylococcus aureus Responds to NaCl, Leading to Increased Biofilm Formation

Journal of Food Protection ◽

10.4315/0362-028x.jfp-17-238 ◽

2018 ◽

Vol 81 (3) ◽

pp. 412-416 ◽

Cited By ~ 3

Author(s):

Soomin Lee ◽

Sejeong Kim ◽

Heeyoung Lee ◽

Jimyeong Ha ◽

Jeeyeon Lee ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Deletion Mutant ◽

Microtiter Plate ◽

Temperature Sensitive ◽

Wild Type ◽

Microtiter Plate Assay ◽

Nacl Concentration ◽

Icaa Gene

ABSTRACT The objective of this study was to elucidate the role of the icaA gene in biofilm formation of Staphylococcus aureus exposed to NaCl. The icaA-deletion mutant of S. aureus ATCC 13565 was constructed with the temperature-sensitive plasmid pIMAY. Microtiter plate assays were performed to confirm biofilm formation for both the wild type and the mutant at 0% (control), 2, 4, and 6% NaCl. The microtiter plate assay revealed that biofilm formation by the wild type increased (P < 0.05) as NaCl concentration increased, but biofilm formation of the mutant was not affected by NaCl concentration. Biofilm formation by the mutant was lower (P < 0.05) than that by the wild type. These results indicate that icaA plays an important role in biofilm formation by S. aureus when the pathogen is exposed to NaCl.

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A Candida parapsilosis Overexpression Collection Reveals Genes Required for Pathogenesis

Journal of Fungi ◽

10.3390/jof7020097 ◽

2021 ◽

Vol 7 (2) ◽

pp. 97

Author(s):

Sára E. Pál ◽

Renáta Tóth ◽

Joshua D. Nosanchuk ◽

Csaba Vágvölgyi ◽

Tibor Németh ◽

...

Keyword(s):

Biofilm Formation ◽

Deletion Mutant ◽

Candida Parapsilosis ◽

Galleria Mellonella ◽

Mutant Library ◽

Human Pathogen ◽

Opportunistic Human Pathogen ◽

Mutant Collection

Relative to the vast data regarding the virulence mechanisms of Candida albicans, there is limited knowledge on the emerging opportunistic human pathogen Candida parapsilosis. The aim of this study was to generate and characterize an overexpression mutant collection to identify and explore virulence factors in C. parapsilosis. With the obtained mutants, we investigated stress tolerance, morphology switch, biofilm formation, phagocytosis, and in vivo virulence in Galleria mellonella larvae and mouse models. In order to evaluate the results, we compared the data from the C. parapsilosis overexpression collection analysis to the results derived from previous deletion mutant library characterizations. Of the 37 overexpression C. parapsilosis mutants, we identified eight with altered phenotypes compared to the controls. This work is the first report to identify CPAR2_107240, CPAR2_108840, CPAR2_302400, CPAR2_406400, and CPAR2_602820 as contributors to C. parapsilosis virulence by regulating functions associated with host-pathogen interactions and biofilm formation. Our findings also confirmed the role of CPAR2_109520, CPAR2_200040, and CPAR2_500180 in pathogenesis. This study was the first attempt to use an overexpression strategy to systematically assess gene function in C. parapsilosis, and our results demonstrate that this approach is effective for such investigations.

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Role of Ttca of Citrobacter Werkmanii in Bacterial Growth, Biocides Resistance, Biofilm Formation and Swimming Motility

International Journal of Molecular Sciences ◽

10.3390/ijms19092644 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2644

Author(s):

Gang Zhou ◽

Ying-Si Wang ◽

Hong Peng ◽

Xiao-Mo Huang ◽

Xiao-Bao Xie ◽

...

Keyword(s):

Growth Rate ◽

Homologous Recombination ◽

Biofilm Formation ◽

The Other ◽

Swimming Motility ◽

Tn5 Transposon ◽

Phenotypic Changes ◽

Resistant Strains ◽

First Time

To screen, identify and study the genes involved in isothiazolone resistance and biofilm formation in Citrobacter werkmanii strain BF-6. A Tn5 transposon library of approximately 900 mutants of C. werkmanii strain BF-6 was generated and screened to isolate 1,2-benzisothiazolin-3-one (BIT) resistant strains. In addition, the tRNA 2-thiocytidine (32) synthetase gene (ttcA) was deleted through homologous recombination and the resulting phenotypic changes of the ΔttcA mutant were studied. A total of 3 genes were successfully identified, among which ΔttcA mutant exhibited a reduction in growth rate and swimming motility. On the other hand, an increase in biofilms formation in ΔttcA were observed but not with a significant resistance enhancement to BIT. This work, for the first time, highlights the role of ttcA gene of C. werkmanii strain BF-6 in BIT resistance and biofilm formation.

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