Tat-Dependent Heterologous Secretion of Recombinant Tyrosinase by Pseudomonas fluorescens Is Aided by a Translationally Fused Caddie Protein

ABSTRACT Tyrosinase is a monooxygenase that catalyzes both the hydroxylation of p-hydroxyphenyl moieties to o-catechols and the oxidation of o-catechols to o-quinones. Apart from its critical functionality in melanogenesis and the synthesis of various neurotransmitters, this enzyme is also used in a variety of biotechnological applications, most notably mediating covalent cross-linking between polymers containing p-hydroxyphenyl groups, forming a hydrogel. Tyrosinases from the genus Streptomyces are usually secreted as a complex with their caddie protein. In this study, we report an increased secretion efficiency observed when the Streptomyces antibioticus tyrosinase gene melC2 was introduced into Pseudomonas fluorescens along with its caddie protein gene melC1, which has the DNA sequence for the Tat (twin-arginine translocation) signal. IMPORTANCE We observed that the S. antibioticus extracellular tyrosinase secretion level was even higher in its nonnatural translationally conjugated fusion protein form than in the natural complex of two separated polypeptides. The results of this study demonstrate that tyrosinase-expressing P. fluorescens can be a stable source of bacterial tyrosinase through exploiting the secretory machinery of P. fluorescens.

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Unpacking the self

Forum+ ◽

10.5117/forum2020.3.005.shar ◽

2020 ◽

Vol 27 (3) ◽

pp. 29-37

Author(s):

Manju Sharma

Keyword(s):

Visual Arts ◽

Personal Narrative ◽

The Self ◽

Cross Linking ◽

Artistic Practice ◽

Content Type ◽

Personal Stories ◽

The World ◽

Visual Artist ◽

Sensitive Issues

Abstract In this essay, visual artist and writer Manju Sharma reflects on the use of autobiography as a methodology for storytelling in the visual arts. She focuses on the methods that she uses to explore the self and its relatedness to the world that she wishes to grasp. She also sheds light on how autobiography fits into her artistic practice as a means of finding hidden narratives and to keep the personal narrative related to the world. The essay touches upon the use of personal stories, cross-linking and note-taking to unpack everyday sensitive issues that can allow people to find their voice and to speak out.

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Transcriptional Regulator PhlH Modulates 2,4-Diacetylphloroglucinol Biosynthesis in Response to the Biosynthetic Intermediate and End Product

Applied and Environmental Microbiology ◽

10.1128/aem.01419-17 ◽

2017 ◽

Vol 83 (21) ◽

Cited By ~ 9

Author(s):

Xu Yan ◽

Rui Yang ◽

Rui-Xue Zhao ◽

Jian-Ting Han ◽

Wen-Juan Jia ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Gene Cluster ◽

Plant Pathogens ◽

Feedback Regulation ◽

Biosynthetic Gene Cluster ◽

Sequence Motif ◽

Target Sequence ◽

Gene Encoding ◽

Negative Feedback Regulation ◽

Content Type

ABSTRACT Certain strains of biocontrol bacterium Pseudomonas fluorescens produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) to antagonize soilborne phytopathogens in the rhizosphere. The gene cluster responsible for the biosynthesis of 2,4-DAPG is named phlACBDEFGH and it is still unclear how the pathway-specific regulator phlH within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in Pseudomonas fluorescens strain 2P24 represses the expression of the phlG gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the −35 site recognized by σ70 factors. Through biochemical screening of PhlH ligands we identified the end product 2,4-DAPG and its biosynthetic intermediate monoacetylphloroglucinol (MAPG), which can act as signaling molecules to modulate the binding of PhlH to the target sequence and activate the expression of phlG. Comparison of 2,4-DAPG production between the ΔphlH, ΔphlG, and ΔphlHG mutants confirmed that phlH and phlG impose negative feedback regulation over 2,4-DAPG biosynthesis. It was further demonstrated that the 2,4-DAPG degradation catalyzed by PhlG plays an insignificant role in 2,4-DAPG tolerance but contributes to bacterial growth advantages under carbon/nitrogen starvation conditions. Taken together, our data suggest that by monitoring and down-tuning in situ levels of 2,4-DAPG, the phlHG genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation. IMPORTANCE 2,4-DAPG, which is synthesized by biocontrol pseudomonad bacteria, is a broad-spectrum antibiotic against bacteria, fungi, oomycetes, and nematodes and plays an important role in suppressing soilborne plant pathogens. Although most of the genes in the 2,4-DAPG biosynthetic gene cluster (phl) have been characterized, it is still not clear how the pathway-specific regulator phlH is involved in 2,4-DAPG metabolism. This work revealed the role of PhlH in modulating 2,4-DAPG levels by controlling the expression of 2,4-DAPG hydrolase PhlG in response to 2,4-DAPG and MAPG. Since 2,4-DAPG biosynthesis imposes a metabolic burden on biocontrol pseudomonads, it is expected that the fine regulation of phlG by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.

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Organization of the Flagellar Switch Complex ofBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00626-18 ◽

2018 ◽

Vol 201 (8) ◽

Cited By ~ 13

Author(s):

Elizabeth Ward ◽

Eun A Kim ◽

Joseph Panushka ◽

Tayson Botelho ◽

Trevor Meyer ◽

...

Keyword(s):

Bacillus Subtilis ◽

Protein Interaction ◽

Protein Complex ◽

Cross Linking ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Middle Domain ◽

Flagellar Rotation

ABSTRACTWhile the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied inEscherichia coliandSalmonella, less is known about the switch complex inBacillus subtilisor other Gram-positive species. Two component proteins (FliG and FliM) are shared betweenE. coliandB. subtilis, but in place of the protein FliN found inE. coli, theB. subtiliscomplex contains the larger protein FliY. Notably, inB. subtilisthe signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action inE. coli. Here, we have examined the architecture and function of the switch complex inB. subtilisusing targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, theB. subtilisswitch complex appears to be organized similarly to that inE. coli. The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that ofE. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences fromE. coliinvolve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCEFlagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex inBacillus subtilisor other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas inE. coliorSalmonellaCheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of theB. subtilisswitch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control inB. subtilisand other Gram-positive species.

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Hyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at Alkaline pH

Applied and Environmental Microbiology ◽

10.1128/aem.02250-17 ◽

2017 ◽

Vol 84 (3) ◽

Cited By ~ 3

Author(s):

James E. Hennessy ◽

Melissa J. Latter ◽

Somayeh Fazelinejad ◽

Amy Philbrook ◽

Daniel M. Bartkus ◽

...

Keyword(s):

Carbon Dioxide ◽

Hyperthermophilic Archaea ◽

High Yield ◽

Water Runoff ◽

High Catalytic Activity ◽

Alkaline Ph ◽

Biotechnological Applications ◽

Carbamoyl Phosphate ◽

Content Type ◽

Carbamate Kinase

ABSTRACT Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus , the previously unreported enzymes from the hyperthermophiles Thermococcus sibiricus and Thermococcus barophilus , the thermophiles Fervidobacterium nodosum and Thermosipho melanesiensis , and the mesophile Clostridium tetani were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4). Thermococcus barophilus and Thermococcus sibiricus carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the Thermococcus barophilus kinase is efficiently converted in situ to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate. IMPORTANCE Much of the nitrogen in animal wastes and used in fertilizers is commonly lost as ammonia in water runoff, from which it must be removed to prevent downstream pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl phosphate may be converted into other valuable compounds, the kinases provide a route for useful sequestration of ammonia, as well as of carbon dioxide, another greenhouse gas. At the same time, recycling the ammonia in chemical synthesis reduces the need for its energy-intensive production. However, robust catalysts are required for such biotransformations. Here we show that carbamate kinases from hyperthermophilic archaea display remarkable stability and high catalytic activity across broad ranges of pH and temperature, making them promising candidates for biotechnological applications. We also show that carbamoyl phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate.

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Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

Journal of Bacteriology ◽

10.1128/jb.00016-21 ◽

2021 ◽

Vol 203 (9) ◽

Author(s):

Hiroyuki Terashima ◽

Seiji Kojima ◽

Michio Homma

Keyword(s):

Escherichia Coli ◽

Cross Linking ◽

Physical Interaction ◽

Flagellar Motor ◽

Intact Cells ◽

Content Type ◽

Bacterial Flagellum ◽

Cross Links ◽

Bacterial Flagellar Motor ◽

Rotary Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

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RNase III Is Required for Actinomycin Production in Streptomyces antibioticus

Applied and Environmental Microbiology ◽

10.1128/aem.02272-13 ◽

2013 ◽

Vol 79 (20) ◽

pp. 6447-6451 ◽

Cited By ~ 6

Author(s):

Jung-Hoon Lee ◽

Marcha L. Gatewood ◽

George H. Jones

Keyword(s):

Parental Strain ◽

Insertional Mutagenesis ◽

Southern Blotting ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Production Medium ◽

Wild Type ◽

Rnase Iii ◽

Content Type ◽

Streptomyces Antibioticus

ABSTRACTUsing insertional mutagenesis, we have disrupted the RNase III gene,rnc, of the actinomycin-producing streptomycete,Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of therncdisruption with the wild-typerncgene fromS. antibioticusrestored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case inStreptomyces coelicolor, RNase III is required for antibiotic production inS. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of theS. antibioticus rncmutant and its parental strain.

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Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

Journal of Bacteriology ◽

10.1128/jb.00760-17 ◽

2018 ◽

Vol 200 (14) ◽

Cited By ~ 7

Author(s):

Satya Deo Pandey ◽

Shilpa Pal ◽

Ganesh Kumar N ◽

Ankita Bansal ◽

Sathi Mallick ◽

...

Keyword(s):

Cell Surface ◽

Mycobacterium Smegmatis ◽

Biofilm Formation ◽

Cross Linking ◽

Surface Lipid ◽

Cross Link ◽

Content Type ◽

Murine Macrophages ◽

Cross Links ◽

Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

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Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes

Journal of Bacteriology ◽

10.1128/jb.00543-19 ◽

2019 ◽

Vol 201 (23) ◽

Cited By ~ 4

Author(s):

Germán E. Piñas ◽

John S. Parkinson

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Kinase Activity ◽

Critical Role ◽

Active State ◽

Dynamics Simulation ◽

Cross Linking ◽

Atp Binding ◽

Content Type

ABSTRACT Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, “dipped” and “undipped,” for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units. IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called “dipped” CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

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PFM-Like Enzymes Are a Novel Family of Subclass B2 Metallo-β-Lactamases from Pseudomonas synxantha Belonging to the Pseudomonas fluorescens Complex

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01700-19 ◽

2019 ◽

Vol 64 (2) ◽

Cited By ~ 1

Author(s):

Laurent Poirel ◽

Mattia Palmieri ◽

Michael Brilhante ◽

Amandine Masseron ◽

Vincent Perreten ◽

...

Keyword(s):

Amino Acid ◽

Pseudomonas Fluorescens ◽

Bacterial Species ◽

Amino Acid Identity ◽

Chicken Meat ◽

The Novel ◽

Content Type ◽

Acid Identity ◽

Carbapenem Resistant ◽

Encoding Genes

ABSTRACT A carbapenem-resistant Pseudomonas synxantha isolate recovered from chicken meat produced the novel carbapenemase PFM-1. That subclass B2 metallo-β-lactamase shared 71% amino acid identity with β-lactamase Sfh-1 from Serratia fonticola. The blaPFM-1 gene was chromosomally located and likely acquired. Variants of PFM-1 sharing 90% to 92% amino acid identity were identified in bacterial species belonging to the Pseudomonas fluorescens complex, including Pseudomonas libanensis (PFM-2) and Pseudomonas fluorescens (PFM-3), highlighting that these species constitute reservoirs of PFM-like encoding genes.

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Draft Genome Sequence of the Industrially Significant Bacterium Pseudomonas fluorescens ATCC 13525

Microbiology Resource Announcements ◽

10.1128/mra.01368-18 ◽

2018 ◽

Vol 7 (17) ◽

Cited By ~ 3

Author(s):

M. J. Meier ◽

R. M. Subasinghe ◽

L. A. Beaudette

Keyword(s):

Antibiotic Resistance ◽

Pseudomonas Fluorescens ◽

Virulence Factors ◽

Draft Genome ◽

Antibiotic Resistance Genes ◽

Strain Atcc ◽

Negative Bacterium ◽

Gram Negative ◽

Content Type ◽

Metabolic Functions

Pseudomonas fluorescens is a Gram-negative bacterium with versatile metabolic functions and potential industrial uses. We sequenced P. fluorescens strain ATCC 13525 with the goal of determining virulence factors and antibiotic resistance genes to predict the potential impacts on human and environmental health in the event of exposure.

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