Marianna Patrauchan - Academia.edu (original) (raw)

Papers by Marianna Patrauchan

Cell Calcium, Mar 1, 2015

Ca 2+ efflux •P-ATPases •Electrochemical potential driven transporters Ca 2+ binding proteins Two... more Ca 2+ efflux •P-ATPases •Electrochemical potential driven transporters Ca 2+ binding proteins Two-component systems •PA2656/57 •PhoP/Q •CarS/R •AtoSC Ca 2+ influx •Channels External Ca 2+ Internally stored Ca 2+ Resting [Ca 2+ ] in 0.09-0.3µM Specific sensors or transporters Other external stimuli •Quorum sensing (HSL) •Heat and cold shock •Carbohydrate metabolites •Oxidative Stress (H 2 O 2) •Red light Intracellular messengers Increased [Ca 2+ ] in 1-5.4 µM External Ca 2+ Internally stored Ca 2+ Resting [Ca 2+ ] in 0.09-0.3µM Increased [Ca 2+ ] in 1-5.4 µM Calcium networks in bacteria (Adapted from Berridge et al. (2000). Domínguez, D.C, M. Guragain, and M. A. Patrauchan. Cell Calcium Special issue "Evolution of Calcium Signaling"

Cell Calcium, 2017

Highlights: 1. Global proteomic approach identified several RND transporters, whose abundance was... more Highlights: 1. Global proteomic approach identified several RND transporters, whose abundance was affected during Pseudomonas aeruginosa PAO1 growth at elevated Ca2+. 2. Six out of twelve RND transporters encoded in the PAO1 genome are involved in Ca2+-induced tobramycin. 3. Elevated Ca2+ induces the transcription of several RND transporters, and this effect is mediated by changes in the intracellular Ca2+ homeostasis. 4. The roles of RND transporters in maintaining intracellular Ca2+ homeostasis and Ca2+induced plant infectivity in P. aeruginosa were elucidated. Overall, this is the first report of the regulatory relationship between intracellular Ca2+ homeostasis and Ca2+-induced antibiotic resistance.

Cell Calcium, Nov 1, 2013

Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infect... more Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca 2+) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca 2+ , we characterized Ca 2+ homeostasis in P. aeruginosa PAO1 cells. By using Ca 2+-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca 2+ ([Ca 2+ ] in) is 0.14±0.05 μM. In response to external Ca 2+ , the [Ca 2+ ] in quickly increased at least 13 fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca 2+ modulated this response. Treatment with inhibitors known to affect Ca 2+ channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca 2+ ] in response, suggesting the importance of the corresponding mechanisms in Ca 2+ homeostasis. To identify Ca 2+ transporters maintaining this homeostasis, bioinformatic and LC-MS/MS-based membrane proteomic analyses were used. [Ca 2+ ] in homeostasis was monitored for seven Ca 2+-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca 2+ homeostasis. The lack of PA3920 and vanadate treatment abolished Ca 2+-induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca 2+ .

Journal of Bacteriology, Mar 15, 2016

Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening in... more Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca 2؉) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca 2؉ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca 2؉ level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca 2؉-induced virulence factor production and Ca 2؉ homeostasis are not known. Here, we characterized the genomewide transcriptional response of P. aeruginosa to elevated [Ca 2؉ ] in both planktonic cultures and biofilms. Among the genes induced by CaCl 2 in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators phoPQ and pmrAB were repressed by CaCl 2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR and performed transcriptome analysis of the mutant strain at low and high [Ca 2؉ ]. Among the genes regulated by CarSR in response to CaCl 2 are the predicted periplasmic OB-fold protein, PA0320 (here called carO), and the inner membrane-anchored five-bladed ␤-propeller protein, PA0327 (here called carP). Mutations in both carO and carP affected Ca 2؉ homeostasis, reducing the ability of P. aeruginosa to export excess Ca 2؉. In addition, a mutation in carP had a pleotropic effect in a Ca 2؉-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca 2؉ and responding through its regulatory targets that modulate Ca 2؉ homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin. IMPORTANCE During infectious disease, Pseudomonas aeruginosa encounters environments with high calcium (Ca 2؉) concentrations, yet the cells maintain intracellular Ca 2؉ at levels that are orders of magnitude less than that of the external environment. In addition, Ca 2؉ signals P. aeruginosa to induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca 2؉ homeostasis or how Ca 2؉ acts as a signal. In this study, we identified a two-component regulatory system in P. aeruginosa PAO1, termed CarRS, that is induced at elevated Ca 2؉ levels. CarRS modulates Ca 2؉ signaling and Ca 2؉ homeostasis through its regulatory targets, CarO and CarP. The results demonstrate that P. aeruginosa uses a two-component regulatory system to sense external Ca 2؉ and relays that information for Ca 2؉-dependent cellular processes.

Applied and Environmental Microbiology, Sep 15, 2012

Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-t... more Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ϳ250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO 2 utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ⌬sigG deletion mutant showed only minor changes in cell survival. Stationaryphase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.

Journal of Bacteriology, 2008

Journal of Bacteriology, Mar 1, 2008

The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpe... more The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinategrown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 ؎ 0.01 M) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.

Journal of Bacteriology, Jun 15, 2005

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rh... more Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the ␤-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the ␤-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent ␤-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.

Journal of Biological Chemistry, Aug 1, 2010

Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no ... more Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. The ANHase structural genes anhA and anhB are separated by anhE, predicted to encode an 11.1-kDa polypeptide. An anhE deletion mutant did not grow on acetonitrile but grew on acetamide, the ANHase reaction product. Growth on acetonitrile was restored by providing anhE in trans. AnhA could be used to assemble ANHase in vitro, provided the growth medium was supplemented with 50 M CoCl 2. Ten-to 100-fold less CoCl 2 sufficed when anhE was co-expressed with anhA. Moreover, AnhA contained more cobalt when produced in cells containing AnhE. Chromatographic analyses revealed that AnhE existed as a monomer-dimer equilibrium (100 mM phosphate, pH 7.0, 25°C). Divalent metal ions including Co 2؉ , Cu 2؉ , Zn 2؉ , and Ni 2؉ stabilized the dimer. Isothermal titration calorimetry studies demonstrated that AnhE binds two half-equivalents of Co 2؉ with K d of 0.12 ؎ 0.06 nM and 110 ؎ 35 nM, respectively. By contrast, AnhE bound only one half-equivalent of Zn 2؉ (K d ‫؍‬ 11 ؎ 2 nM) and Ni 2؉ (K d ‫؍‬ 49 ؎ 17 nM) and did not detectably bind Cu 2؉. Substitution of the sole histidine residue did not affect Co 2؉ binding. Holo-AnhE had a weak absorption band at 490 nm (⑀ ‫؍‬ 9.7 ؎ 0.1 M ؊1 cm ؊1), consistent with hexacoordinate cobalt. The data support a model in which AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. This study provides insight into the maturation of NHases and metallochaperone function.

PLOS ONE, Jun 11, 2014

Pseudomonas aeruginosa is a facultative human pathogen, and a major cause of nosocomial infection... more Pseudomonas aeruginosa is a facultative human pathogen, and a major cause of nosocomial infections and severe chronic infections in endocarditis and in cystic fibrosis (CF) patients. Calcium (Ca 2+) accumulates in pulmonary fluids of CF patients, and plays a role in the hyperinflamatory response to bacterial infection. Earlier we showed that P. aeruginosa responds to increased Ca 2+ levels, primarily through the increased production of secreted virulence factors. Here we describe the role of putative Ca 2+-binding protein, with an EF-hand domain, PA4107 (EfhP), in this response. Deletion mutations of efhP were generated in P. aeruginosa strain PAO1 and CF pulmonary isolate, strain FRD1. The lack of EfhP abolished the ability of P. aeruginosa PAO1 to maintain intracellular Ca 2+ homeostasis. Quantitative high-resolution 2D-PAGE showed that the efhP deletion also affected the proteomes of both strains during growth with added Ca 2+. The greatest proteome effects occurred when the pulmonary isolate was cultured in biofilms. Among the proteins that were significantly less abundant or absent in the mutant strains were proteins involved in iron acquisition, biosynthesis of pyocyanin, proteases, and stress response proteins. In support, the phenotypic responses of FRD1 DefhP showed that the mutant strain lost its ability to produce pyocyanin, developed less biofilm, and had decreased resistance to oxidative stress (H 2 O 2) when cultured at high [Ca 2+ ]. Furthermore, the mutant strain was unable to produce alginate when grown at high [Ca 2+ ] and no iron. The effect of the DefhP mutations on virulence was determined in a lettuce model of infection. Growth of wild-type P. aeruginosa strains at high [Ca 2+ ] causes an increased area of disease. In contrast, the lack of efhP prevented this Ca 2+-induced increase in the diseased zone. The results indicate that EfhP is important for Ca 2+ homeostasis and virulence of P. aeruginosa when it encounters host environments with high [Ca 2+ ].

Polymers, 2019

Biofilms are surface-associated microbial communities known for their increased resistance to ant... more Biofilms are surface-associated microbial communities known for their increased resistance to antimicrobials and host factors. This resistance introduces a critical clinical challenge, particularly in cases associated with implants increasing the predisposition for bacterial infections. Preventing such infections requires the development of novel antimicrobials or compounds that enhance bactericidal effect of currently available antibiotics. We have synthesized and characterized twelve novel silver(I) cyanoximates designated as Ag(ACO), Ag(BCO), Ag(CCO), Ag(ECO), Ag(PiCO), Ag(PICO) (yellow and red polymorphs), Ag(BIHCO), Ag(BIMCO), Ag(BOCO), Ag(BTCO), Ag(MCO) and Ag(PiPCO). The compounds exhibit a remarkable resistance to high intensity visible light, UV radiation and heat and have poor solubility in water. All these compounds can be well incorporated into the light-curable acrylate polymeric composites that are currently used as dental fillers or adhesives of indwelling medical dev...

In nature, bacteria reside in biofilms - multicellular differentiated communities held together b... more In nature, bacteria reside in biofilms - multicellular differentiated communities held together by extracellular matrix. In this work, we identified a novel subpopulation essential for biofilm formation – mineral-forming cells. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community.The molecular mechanisms promoting calcite scaffold formation were conserved between three distant phyla – the Gram-positive Bacillus subtilis, Gram-negative Pseudomonas aeruginosa and the actinobacterium Mycobacterium abscessus. Biofilm development of all three species was similarly impaired by inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization both significantly reduced the attachment of P. aeruginosa to...

iScience

Highlights 3D microCT X-ray reveals mineral scaffolds in Pseudomonas aeruginosa Calcium carbonate... more Highlights 3D microCT X-ray reveals mineral scaffolds in Pseudomonas aeruginosa Calcium carbonate production promotes the formation of Mycobacterium abscessus biofilms Calcium carbonate production is associated with lung colonization by P. aeruginosa Targeting biomineralization reduced the damage inflicted by biofilms to the lung

Microscopy and Microanalysis

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial human pathogen that is responsi... more Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial human pathogen that is responsible for severe acute and chronic infections worldwide. The ubiquitous nature of P. aeruginosa and ability to survive on abiotic surfaces, makes it one of the leading causes of nosocomial infections [1, 2]. Due to its capability to adapt, survive and possess resistance to multiple broad-spectrum antibiotics, P. aeruginosa infections are posing a serious public health threat with approximately 51,000 healthcare associated infections in US every year (Center of Disease Control, 2019 report). It is one of the primary organisms that infects airways of cystic fibrosis (CF) patient's lungs and a leading cause of morbidity and mortality [3]. CF is a genetic disorder that occurs because of the defect in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR is responsible for regulating the flow of chloride ions in the lung secretions, aiding in mucocilliary clearance. In CF, the mutations in CFTR disrupt its functioning leading to decreased chloride excretion and deposition of thick mucus in the lung airways [4]. Pulmonary fluids of CF patients contain an elevated concentration of ions such as Na + , Mg 2+ , and Ca 2+ [5]. Studies suggest that Ca 2+ , an important cellular signaling molecule, is known to enhance the virulence capability of P. aeruginosa [6], however little is known about how it regulates the host-pathogen interactions of P. aeruginosa with lung epithelial cells.

Odisha to study the effect of climate on weed management practices in elephant foot yam [Amorphop... more Odisha to study the effect of climate on weed management practices in elephant foot yam [Amorphophallus paeoniifolius (Dennst.) Nicolson]. Higher average monthly maximum temperature was noticed during early crop growth period (vegetative phase) in 2017 compared to 2016. The mean minimum temperature was higher during the cropping period of 2017 compared to 2016. During 2017, Celosia argentea was observed more number and grown more vigourously compared to 2016. The weed biomass was higher during the year 2017 compared to 2016 in all weed management treatments. Higher average monthly maximum and minimum temperatures during early crop growth period (vegetative phase) favoured for more number of weeds to grow vigourously during 2017. The effect of weather was noticed more in pre (1 DAP) and post emergence (45 DAP) herbicide application treatments and weedy check. During 2016 recorded higher corm yield than 2017 irrespective of weed management practices. The weedy check treatment resulted in lower corm yield in 2017 than 2016. It can be concluded that higher rainfall and average monthly maximum temperature during crop growing period led to lower herbicide efficacy or weed management efficiency under high rainfall sub-humid zone. K e y w o r d s Amorphophallus paeoniifolius argentea

Journal of Bacteriology, 2016

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen that causes severe, life-threate... more ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections,P. aeruginosaoften encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed thatP. aeruginosaresponds to externally added Ca2+through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+homeostasis are not known. Here, we characterized the genome-wide transcriptional response ofP. aeruginosato elevated [Ca2+] in both planktonic cultures and biofilms. Among the genes induced by CaCl2in strain PAO1 was an operon containing the two-component regulator PA...

Acta Crystallographica Section F Structural Biology and Crystallization Communications, Aug 1, 2013