scholarly journals Genetic Characterization of Pseudomonas fluorescens SBW25 rsp Gene Expression in the Phytosphere and In Vitro

2005 ◽  
Vol 187 (24) ◽  
pp. 8477-8488 ◽  
Author(s):  
Robert W. Jackson ◽  
Gail M. Preston ◽  
Paul B. Rainey
Keyword(s):  
Pseudomonas Fluorescens ◽  
Ectopic Expression ◽  
Gene Clusters ◽  
Regulatory Genes ◽  
Regulatory Control ◽  
Lag Phase ◽  
Secretion Systems ◽  
General Role ◽  
Animal Pathogens

ABSTRACT The plant-colonizing Pseudomonas fluorescens strain SBW25 harbors a gene cluster (rsp) whose products show similarity to type III protein secretion systems found in plant and animal pathogens. Here we report a detailed analysis of the expression and regulation of the P. fluorescens rsp pathway, both in the phytosphere and in vitro. A combination of chromosomally integrated transcriptional reporter fusions, overexpressed regulatory genes, and specific mutants reveal that promoters controlling expression of rsp are actively transcribed in the plant rhizosphere but not (with the exception of the rspC promoter) in the phyllosphere. In synthetic medium, regulatory (rspL and rspR) and structural (rspU, plus the putative effector ropE) genes are poorly expressed; the rspC promoter is subject to an additional level of regulatory control. Ectopic expression of regulatory genes in wild-type and mutant backgrounds showed that RspR controls transcription of the alternate sigma factor, rspL, and that RspL controls expression of gene clusters encoding structural genes. Mutation of rspV did not affect RspR-mediated expression of rspU. A search for additional regulators revealed two candidates—one with a role in the conversion of alanine to pyruvate—suggesting that expression of rsp is partly dependent upon the metabolic status of the cell. Mutations in rsp regulators resulted in a significant reduction in competitive colonization of the root tips of sugar beet seedlings but also caused a marked increase in the lag phase of laboratory-grown cultures, indicating that rsp regulatory genes play a more significant general role in the function of P. fluorescens SBW25 than previously appreciated.

scholarly journals Disruption of c-di-GMP signaling networks unlocks cryptic expression of secondary metabolites during biofilm growth in Burkholderia pseudomallei

2021 ◽  
Author(s):  
Grace I Borlee ◽  
Mihnea R. Mangalea ◽  
Kevin H. Martin ◽  
Brooke A. Plumley ◽  
Samuel J. Golon ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Gene Clusters ◽  
Etiologic Agent ◽  
Regulatory Control ◽  
Colony Morphology ◽  
Secretion Systems ◽  
Biofilm Growth ◽  
Conditional Expression

The regulation and production of secondary metabolites during biofilm growth of Burkholderia spp. is not well understood. To learn more about the crucial role and regulatory control of cryptic molecules produced during biofilm growth, we disrupted c-di-GMP signaling in Burkholderia pseudomallei, a soil-borne bacterial saprophyte and the etiologic agent of melioidosis. Our approach to these studies combined transcriptional profiling with genetic deletions that targeted key c-di-GMP regulatory components to characterize responses to changes in temperature. Mutational analyses and conditional expression studies of c-di-GMP genes demonstrates their contribution to phenotypes such as biofilm formation, colony morphology, motility, and expression of secondary metabolite biosynthesis when grown as a biofilm at different temperatures. RNA-seq analysis was performed at varying temperatures in a ΔII2523 mutant background that is responsive to temperature alterations resulting in hypo- and hyper- biofilm forming phenotypes. Differential regulation of genes was observed for polysaccharide biosynthesis, secretion systems, and nonribosomal peptide and polyketide synthase (NRPS/PKS) clusters in response to temperature changes. Deletion mutations of biosynthetic gene clusters (BGCs) clusters 2, 11, 14 (syrbactin), and 15 (malleipeptin) in wild-type and ΔII2523 backgrounds also reveals the contribution of these BGCs to biofilm formation and colony morphology in addition to inhibition of Bacillus subtilis and Rhizoctonia solani. Our findings suggest that II2523 impacts the regulation of genes that contribute to biofilm formation and competition. Characterization of cryptic BGCs under differing environmental conditions will allow for a better understanding of the role of secondary metabolites in the context of biofilm formation and microbe-microbe interactions.

scholarly journals A Novel Automethylation Reaction in the Aspergillus nidulans LaeA Protein Generates S-Methylmethionine

2013 ◽  
Vol 288 (20) ◽  
pp. 14032-14045 ◽  
Author(s):  
Alexander N. Patananan ◽  
Jonathan M. Palmer ◽  
Graeme S. Garvey ◽  
Nancy P. Keller ◽  
Steven G. Clarke
Keyword(s):  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Point Mutations ◽  
Pathogenic Fungi ◽  
Gene Clusters ◽  
Methionine Residue ◽  
Cell Extracts ◽  
Animal Pathogens

The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that can adapt to their environment by producing various secondary metabolites, including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are globally regulated by the LaeA protein. Null mutants of LaeA in all pathogenic fungi examined to date show decreased virulence coupled with reduced secondary metabolism. Although the amino acid sequence of LaeA contains the motifs characteristic of seven-β-strand methyltransferases, a methyl-accepting substrate of LaeA has not been identified. In this work we did not find a methyl-accepting substrate in Aspergillus nidulans with various assays, including in vivo S-adenosyl-[methyl-3H]methionine labeling, targeted in vitro methylation experiments using putative protein substrates, or in vitro methylation assays using whole cell extracts grown under different conditions. However, in each experiment LaeA was shown to self-methylate. Amino acid hydrolysis of radioactively labeled LaeA followed by cation exchange and reverse phase chromatography identified methionine as the modified residue. Point mutations show that the major site of modification of LaeA is on methionine 207. However, in vivo complementation showed that methionine 207 is not required for the biological function of LaeA. LaeA is the first protein to exhibit automethylation at a methionine residue. These findings not only indicate LaeA may perform novel chemistry with S-adenosylmethionine but also provide new insights into the physiological function of LaeA.

scholarly journals Infection-Blocking Genes of a Symbiotic Rhizobium leguminosarum Strain That Are Involved in Temperature-Dependent Protein Secretion

2003 ◽  
Vol 16 (1) ◽  
pp. 53-64 ◽  
Author(s):  
M. R. Bladergroen ◽  
K. Badelt ◽  
H. P. Spaink
Keyword(s):  
Nitrogen Fixation ◽  
Protein Secretion ◽  
Rhizobium Leguminosarum ◽  
Signal Sequence ◽  
Gene Clusters ◽  
Secreted Proteins ◽  
Type I ◽  
Secretion Systems ◽  
Temperature Dependent ◽  
Animal Pathogens

Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation. The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea. A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea. We have sequenced a 33-kb region around the phage-transductable Tn5 insertion. The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus. Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens. Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain. Other proteins show similarity to proteins involved in type III protein secretion. Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5. The production of these proteins in the wild-type strain is temperature dependent. Sequencing of two of these proteins revealed that their first 20 amino acids are identical. This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V. cholerae. Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems. Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen. Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype. These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants.

scholarly journals Enterococcal PrgU mitigates PrgB overexpression toxicity by binding to intergenic RNA downstream of the PQ promoter

2020 ◽  
Author(s):  
Lena Lassinantti ◽  
Martha I Camacho ◽  
Rebecca J B Erickson ◽  
Julia L E Willett ◽  
Nicholas R. De Lay ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Rna Structure ◽  
Rna Binding ◽  
Fine Tuning ◽  
Conjugative Plasmid ◽  
Regulatory Control ◽  
Sequence Specificity ◽  
Secretion Systems

AbstractEfficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the sex pheromone cCF10, which induces the expression of the Type 4 Secretion System (T4SS) genes controlled by the PQ promoter. The pheromone responsive PQ promoter is strictly regulated to prevent overproduction of the prgQ operon, which contains the T4SS, and to limit the cell toxicity caused by overproduction of PrgB, a T4SS adhesin involved in cellular aggregation. PrgU plays an important role in regulating this toxicity by decreasing PrgB production. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. With a combination of lacZ reporter fusion, northern blot, and RNAseq analyses, we provide evidence that PrgU binds a specific RNA sequence within the intergenic region (IGR), ca 400 bp downstream of the PQ promoter. PrgU-IGR binding reduces levels of downstream transcripts, with the strongest decrease seen for prgB messages. Consistent with these findings, we determined that pCF10-carrying cells expressing prgU decreased transcript levels more rapidly than isogenic cells deleted of prgU. Finally, purified PrgU bound RNA in vitro, but without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors to bind its RNA target in vivo. Together, our results support a working model where PrgU binding to the IGR serves to recruit RNase(s) for targeted degradation of downstream transcripts.ImportanceBacteria utilize Type 4 Secretion Systems (T4SS) to efficiently transfer DNA from donor to recipient cells, thereby spreading genes encoding for antibiotic resistance as well as various virulence factors. The conjugative plasmid pCF10 from Enterococcus faecalis, originally isolated from clinical isolates, serves as a model system for these processes in Gram-positive bacteria. It is very important to strictly regulate the expression of the T4SS proteins for the bacteria, as some of these proteins are highly toxic to the cell. Here, we identify the mechanism by which PrgU performs its delicate fine tuning of the expression levels. As prgU genes are present in various conjugative plasmids and transposons, this provides an important new insight into the bacterial repertoire of regulation mechanisms of these clinically important systems.

scholarly journals Draft Genome Sequence of Pseudomonas fluorescens ML11A, an Endogenous Strain from Brook Charr with Antagonistic Properties against Aeromonas salmonicida subsp. salmonicida

2017 ◽  
Vol 5 (9) ◽  
Author(s):  
Jeff Gauthier ◽  
Steve J. Charette ◽  
Nicolas Derome
Keyword(s):  
Pseudomonas Fluorescens ◽  
Draft Genome ◽  
Gene Clusters ◽  
Inhibitory Effect ◽  
Aeromonas Salmonicida ◽  
Control Strain ◽  
Fish Pathogen ◽  
Brook Charr ◽  
Content Type

ABSTRACT Pseudomonas fluorescens ML11A, isolated from brook charr, showed a strong in vitro inhibitory effect against Aeromonas salmonicida subsp. salmonicida, a bacterial fish pathogen. Its genome harbors gene clusters for siderophore and bacteriocin biosynthesis and shares 99% whole-genome identity with P. fluorescens A506, a biological control strain used in agriculture.

Hindbrain patterning: FGFs regulate Krox20 and mafB/kr expression in the otic/preotic region

Development ◽  
2000 ◽  
Vol 127 (22) ◽  
pp. 4925-4935 ◽  
Author(s):  
F. Marin ◽  
P. Charnay
Keyword(s):  
Growth Factor ◽  
Neural Crest ◽  
Fibroblast Growth Factor ◽  
Neural Tube ◽  
Ectopic Expression ◽  
Regulatory Genes ◽  
Chick Embryos ◽  
Fibroblast Growth ◽  
General Role ◽  
Fgf Signalling

Krox20 and mafB/kr are regulatory genes involved in hindbrain segmentation and anteroposterior (AP) patterning. They are expressed in rhombomeres (r) r3/r5 and r5/r6 respectively, as well as in the r5/r6 neural crest. Since several members of the fibroblast growth factor (FGF) family are expressed in the otic/preotic region (r2-r6), we investigated their possible involvement in the regulation of Krox20 and mafB/kr. Application of exogenous FGFs to the neural tube of 4- to 7-somite chick embryos led to ectopic expression in the neural crest of the somitic hindbrain (r7 and r8) and to the extension of the Krox20- or mafB/kr-positive areas in the neuroepithelium. Application of an inhibitor of FGF signalling led to severe and specific downregulation of Krox20 and mafB/kr in the hindbrain neuroepithelium and neural crest. These data indicate that FGFs are involved in the control of regional induction and/or maintenance of Krox20 and mafB/kr expression, thus identifying a novel function for these factors in hindbrain development, besides their proposed more general role in early neural caudalisation.

PERBANDINGAN DAYA HAMBAT BAKTERI Pseudomonas fluorescens ASAL TOMAT, KEDELAI, DAN JAGUNG TERHADAP Ralstonia solanacearum SECARA IN VITRO

Agrotek ◽  
2018 ◽  
Vol 5 (6) ◽  
Author(s):  
Aco Roni Kirihio ◽  
Ivonne Fitria Mariay ◽  
Cipta Meliala
Keyword(s):  
Pseudomonas Fluorescens ◽  
Filter Paper ◽  
Ralstonia Solanacearum ◽  
Randomized Design ◽  
Completely Randomized Design

<em>Inhibition of Pseudomonas fluorescens isolates the origin of tomato, soybean and corn against Ralstonia solanacearum tested using a completely randomized design (CRD).        P. fluorescens growth was measured at King's B medium by way of suspension antagonist put on filter paper of 0.5 cm in diameter. Inhibition of P. fluorescens is done by placing the antagonist suspension of 0.5 cm diameter filter paper on NA media that has been deployed R. solanacearum. The results showed that: (a) the growth of P. fluorescens origin of tomato, soybean and corn on King's B media were not significantly different, (b) the inhibition of P. fluorescens isolates against R. solanacearum not significantly different and, (c) the inhibition of isolates P. fluorescens origin of tomato, soybean and corn against R. solanacearum in vitro relatively strong</em>

scholarly journals Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Kkabi Son ◽  
Yangmee Kim
Keyword(s):  
Fatty Acid ◽  
Acinetobacter Baumannii ◽  
Gene Cluster ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Carrier Protein ◽  
Aryl Group ◽  
Visible Absorption

AbstractSome Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure–function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host–pathogen interaction mechanisms and novel antibiotics discovery.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

scholarly journals Antibiotic sensitivity and in vitro antimicrobial activity of plant extracts to pseudomonas fluorescens isolates collected from diseased fish

1970 ◽  
Vol 1 (4) ◽  
pp. 82-88 ◽  
Author(s):  
MJ Foysal ◽  
MM Rahman ◽  
M Alam
Keyword(s):  
Antibacterial Activity ◽  
Pseudomonas Fluorescens ◽  
Plant Extracts ◽  
Antibiotic Sensitivity ◽  
Diffusion Method ◽  
Biochemical Tests ◽  
Leaf Extracts ◽  
Sensitivity Pattern ◽  
Medicinal Plant Extracts

Studies were conducted to identify Pseudomonas fluorescens isolates from a collection of bacteria isolated from bacterial haemorrhagic septicaemia infected carp and catfish, evaluate their antibiotic sensitivity pattern and screen the antibacterial activity of some medicinal plant extracts against the isolates.. A total of 10 isolates were identified as P. fluorescens by morphological, physiological and biochemical tests. In vitro antibiotic sensitivity test of the P. fluorescens isolates were conducted by disc diffusion method for seven antibiotics where, all of the isolates were found to be sensitive only against streptomycin and gentamycin but, most of the isolates (80%) were found resistant to chloramphenicol (C). Moreover, eighty percent of the isolates showed resistance to multiple antibiotics. A total of 118 plant extracts were screened for their antibacterial activity against the P. fluorescens isolates where the isolates exhibited sensitivity to 30 samples. Leaf extracts of Tamarindus indicus, Terminalia chebula, Citrus aurantifolia, Eugenia caryophyllata and Spondias pinnata were found to inhibit the growth of all of the P. fluorescens isolates. DOI: http://dx.doi.org/10.3329/ijns.v1i4.9733 IJNS 2011 1(4): 82-88

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