© 2016, Springer Science+Business Media New York.Human opportunistic pathogen Serratia marcescens secrete siderophores to enable growth under iron-limiting conditions. Iron acquisition genes are among a few known virulence factors of S. marcescens. Siderophore export systems in Gram-negative bacteria are not fully understood. There is some evidence for involvement of efflux pumps in the export of synthesized enterobactin-like molecules. The goal of this study was to characterize siderophore production by two different strains of S. marcescens, SM6 and SR41-8000, and to evaluate the role of efflux pump MacAB in siderophore secretion by these strains. We showed that both strains produced siderophores in CAS agar assay. We further showed that both strains were able to secrete catecholate siderophores in response to iron starvation. MacAB efflux pump played a role in siderophore secretion of S. marcescens SR41-8000 strain but was dispensable for accumulation of these molecules in the culture supernatant of S. marcescens strain SM6

Bogomolnaya Lydia Mikhailovna

Khilyas Irina V

Matrosova Liliya Evgenevna

Sharipova Margarita Rashidovna

Shirshikova Tatiana Vladimirovna

Sorokina Alena Vitalevna

BioNanoScience

Khilyas I.

Shirshikova T.

Matrosova L.

Sorokina A.

Sharipova M.

Bogomolnaya L.

Kazan Federal University Digital Repository

Production of Siderophores by Serratia marcescens and the Role of MacAB Efflux Pump in Siderophores Secretion

Production of Siderophores by Serratia marcescens and the Roleof MacAB Efflux Pump in Siderophores SecretionIrina V. Khilyas1 & Tatiana V. Shirshikova1 & Lilia E. Matrosova1 & Alyona V. Sorokina1 &Margarita R. Sharipova1 & Lydia M. Bogomolnaya1,2# Springer Science+Business Media New York 2016Abstract Human opportunistic pathogen Serratia marcescenssecrete siderophores to enable growth under iron-limiting con-ditions. Iron acquisition genes are among a few known viru-lence factors of S. marcescens. Siderophore export systems inGram-negative bacteria are not fully understood. There is someevidence for involvement of efflux pumps in the export ofsynthesized enterobactin-like molecules. The goal of this studywas to characterize siderophore production by two differentstrains of S. marcescens, SM6 and SR41-8000, and to evaluatethe role of efflux pump MacAB in siderophore secretion bythese strains. We showed that both strains producedsiderophores in CAS agar assay. We further showed that bothstrains were able to secrete catecholate siderophores inresponse to iron starvation. MacAB efflux pump played a rolein siderophore secretion of S. marcescens SR41-8000 strainbut was dispensable for accumulation of these molecules inthe culture supernatant of S. marcescens strain SM6.Keywords Serratia marcescens . Siderophores .2,3-Dihydroxybenzoic acid . Efflux pump .MacAB1 IntroductionSerratia marcescens is a member of the Enterobacteriaceaefamily. This bacterium can cause infections of numerousorgan systems and represents a growing public health prob-lem. Virulence factors of S. marcescens include secretion ofhydrolytic enzymes [1], lipopolysaccharide biosynthesis,hemolysin production, and iron uptake [2]. Iron is an essentialelement for many biological processes. To overcome irondeficiency, many bacteria synthesize siderophores, lowmolecular weight iron-chelating compounds [3]. Relatedbacterium E. coli secretes catecholate siderophoreenterobactin. The systems involved in synthesis anduptake of enterobactin are well characterized [4]. In con-trast, enterobactin export system is not fully understood.In addition to previously identified ABC-type transpoterEntS [5], several RND efflux pump, AcrB, AcrD, andMdtABC recently have been shown to be involved inexport of enterobactin [6]. Our understanding of produc-tion and secretion of siderophores by S. marcescens isvery limited. Thus, the aim of this study was to charac-terize siderophore production by two wild-type strains ofS. marcescens, SM6 and SR41-8000, and to investigatethe role of ABC-type efflux pump MacAB in secretionof siderophores by these strains.2 Material and MethodsS. marcescens strains SM6 and SR41-8000 were obtainedfrom Michael Benedik (Texas A&M University). IsogenicΔmacAB strains were generated using modified lambda redrecombination method [7] (will be described elsewhere).Bacterial strains were initially screened for siderophore pro-duction using the CAS assay method [8]. The Arnow assaywas used to detect catecholate-type siderophores [9]. 2,3-Dihydroxybenzoic acid (2.3-DHBA) (Acros) was used as astandard for calibration curve. The Atkin assay was used totest for the presence of hydroxamate-type siderophores [10].* Lydia M. BogomolnayaLMBogomolnaya@kpfu.ru1 Laboratory of Microbial Biotechnology, Institute of FundamentalMedicine and Biology, Kazan Federal University, 9 ParizhskoyCommuny str., Kazan 420021, Russia2 Texas A&M University Health Science Center, Bryan 77807, TX,USABioNanoSci.DOI 10.1007/s12668-016-0264-3Desferrioxamine B (Sigma) was used as a standard for cali-bration curve.Siderophore detection in solution was done using CASliquid assay [11]. Bacteria were cultivated in MM9 (with100 mM PIPES, 3 % casamino acids and 20 % glucose).Detection of siderophores in supernatants was performed byusing CAS assay solution (2 mM aqueous CAS, 1 mM FeCl3in 10 mM HCl, 10 mM HDTMA, 4.307 g anhydrous pi-perazine). Equal volumes of supernatant and CAS assaysolution were mixed and incubated for 5 min, followedby addition of 1/20th of volume 0.2 M 5-sulfosalicylicacid. Absorbance of the solution was measured at630 nm. Results were presented in siderophore units cal-culated as described in [11].S. marcescens strains were grown at 30 °C with shaking(200 rpm) for 12 h in 5 mL of LB medium containing (per L)10 g tryptone, 5 g yeast extract 5 g NaCl. Bacterial cells wereharvested by centrifugation, washed with 16 mM phosphatebuffer (pH 7.0), and resuspended in 15 ml M9 minimal medi-um. The M9 medium contained (per L) 6.77 g Na2HPO4, 3 gKH2PO4, 1 g NH4Cl, 0.5 g NaCl, and 20–100 mM glucose or2–10 % glycerine, pH 6.9–7.0. Stock solutions of MgSO4 ×7H2O and CaCl2 were added to final concentration 246 and100 μM, respectively. M9 medium was supplemented withBIP (2.2-bipiridyl) (Sigma) to final concentration 50 μM.Control experiments were performed in the presence of100 μM of FeCl3. Bacterial growth was monitored at 600 nm(iMark™ microplate absorbance reader, BioRad, USA).Each experiment was performed on at least three separateoccasions. Statistical significance was determined by usingStudent’s t test.3 Results and DiscussionThe results of the CAS assay showed that both S. marcescenswild-type strains, SM6 and SR41-8000, as well as their iso-genicΔmacAB were able to produce siderophores. After 12 hof growth of S. marcescens strains on CAS agar plate, themedium color has changed from blue to yellow. Productionof siderophores depended on the strain background.S. marcescens strain SR41-8000 produced more siderophoreson CAS plates compared to strain SM6 (Fig. 1a).CAS agar plate assay is a universal method for siderophoredetection, but it cannot provide any information about chemicalproperties of iron-chelating compounds. To characterize syn-thesized molecules in greater details, we determined concentra-tion of catecholate and hydroxamate siderophores in the culturesupernatants using methods of Arnow and Atkin, respectively.The analysis of siderophore accumulation in the culture super-natant over 30 h of growth under iron-limiting conditionsshowed that initial production of catecholate siderophores byCB*p=0.06AFig. 1 Production ofsiderophores by Serratiamarcescens strains SM6,SR41-8000 and their isogenicΔmacAB mutants. a AllS. marcescens strains producesiderophores on CAS agar.b, c Secretion of siderophoresby S. marcescens strains wasevaluated in liquid CAS assay.b SM6—closed circles; SM6ΔmacAB—open circles. cSR41-8000—closed squares;SR41-8000 ΔmacAB—opensquares. Asterisk indicatesp < 0.05 in two-tailed unpairedStudent’s t testBioNanoSci.S. marcescens SM6 coincided with transition to the early expo-nential growth phase. Siderophore concentration gradually in-creased over 30 h of growth and remained constant after that. Itwas found that low concentrations of glucose (20 mM) andglycerine (2 %) stimulated a siderophore secretion byS. marcescens SM6 strain at 30 °C (Table 1). Production ofcatecholate siderophores by S. marcescens SR41-8000 strainunder used conditions was low, regardless of the temperatureand carbon source (Table 1). Growth of bacteria in the presenceof 100 μM FeSO4 did not result in siderophore secretion.Production of hydroxamate-type of siderophores was notdetected in neither of the S. marcescens strains.To evaluate the impact of MacAB efflux pump on the se-cretion of siderophores, we used liquid CAS assay [11]. Inagreement with CAS plate assay S. marcescens SM6 and itsisogenic ΔmacAB mutant strain produced significantly lesssiderophores compared to strain SR41-8000 (Fig. 1b, c). Therole of MacAB efflux pump in secretion of siderophores byS. marcescens was strain-specific. Deletion of MacAB effluxpump did not play a role in accumulation of siderophores inthe culture supernatant of S. marcescens SM6 over 48 h ofgrowth (Fig. 1b). In contrast, secretion of siderophores byΔmacAB mutant strain was significantly reduced comparedto its parent, S. marcescens SR41-8000 strain (Fig. 1c). Thisfinding illustrates remarkable intraspecies diversity previouslyobserved for Serratia marcescens [12].4 ConclusionsThus, both Serratia marcescens strains, SM6 and SR41-8000,produced catecholate siderophores under iron-limiting growthconditions. Efflux pumpMacABwas involved in the secretionof these iron-chelating molecules in the strain SR41-8000 butnot in the strain SM6.Acknowledgments This work was supported by the RussianFoundation for Basic Research projects 15-04-02110 to LMB and 16-34-60200 to IVKh and performed in accordance with the Russian GovernmentProgram of Competitive Growth of Kazan Federal University.References1. Hines, D. A., Saurugger, P. N., Ihler, G. M., Benedik, M. J. (1988).Genetic analysis of extracellular proteins of Serratia marcescens.Journal of Bacteriology, 170(9), 4141–4146.2. Kurz, C. L., Chauvet, S., Andres, E., Aurouze, M., Vallet, I., Michel,G. P., et al. (2003). Virulence factors of the human opportunisticpathogen Serratia marcescens identified by in vivo screening. TheEMBO Journal, 22(7), 1451–1460. doi:10.1093/emboj/cdg159.3. Payne, S. M. (1993). Iron acquisition in microbial pathogenesis.Trends in Microbiology, 1(2), 66–69.4. Saha, R., Saha, N., Donofrio, R. S., Bestervelt, L. L. (2013).Microbial siderophores: a mini review. Journal of BasicMicrobiology, 53(4), 303–317. doi:10.1002/jobm.201100552.5. Furrer, J. L., Sanders, D. N., Hook-Barnard, I. G., McIntosh, M. A.(2002). Export of the siderophore enterobactin in Escherichia coli:involvement of a 43 kDa membrane exporter. MolecularMicrobiology, 44(5), 1225–1234.6. Horiyama, T., & Nishino, K. (2014). AcrB, AcrD, and MdtABCmultidrug efflux systems are involved in enterobactin export inEscherichia coli. PloS One, 9(9), e108642. doi:10.1371/journal.pone.0108642.7. Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products.Proceedings of the National Academy of Sciences of the UnitedStates of America, 97(12), 6640–6645. doi:10.1073/pnas.120163297.8. Schwyn, B., & Neilands, J. B. (1987). Universal chemical assay forthe detection and determination of siderophores. AnalyticalBiochemistry, 160(1), 47–56.9. Arnow, L. E. (1937). Colorimetric determination of the componentsof 3,4-dihydroxyphenylalanine-tyrosine mixtures. Journal ofBiological Chemistry, 118.10. Atkin, C. L., Neilands, J. B., Phaff, H. J. (1970). Rhodotorulic acidfrom species of Leucosporidium, Rhodosporidium, Rhodotorula,Sporidiobolus, and Sporobolomyces, and a new alanine-containing ferrichrome from Cryptococcus melibiosum. Journal ofBacteriology, 103(3), 722–733.11. Payne, S. M. (1994). Detection, isolation, and characterization ofsiderophores. Methods in Enzymology, 235, 329–344.12. Iguchi, A., Nagaya, Y., Pradel, E., Ooka, T., Ogura, Y., Katsura, K.,et al. (2014). Genome evolution and plasticity of Serratiamarcescens, an importantmultidrug-resistant nosocomial pathogen.Genome Biology and Evolution, 6(8), 2096–2110. doi:10.1093/gbe/evu160.Table 1 Production of siderophores by Serratia marcescens strainsSM6 and SR41-8000 after 30 h under iron-limiting conditionsGrowth temperature Carbon source Siderophores concentration, μMSM6 SR41-800030 °C 20 mM glucose 78.0 ± 5.69 10.0 ± 4.02 % glycerin 127.0 ± 7.1 11.0 ± 3.037 °C 20 mM glucose ND ND2 % glycerin ND NDND not detectedBioNanoSci.

Production of siderophores by Serratia marcescens and the role of MacAB efflux pump in siderophores secretion

BioNanoScience 2016 vol.6 N4, pages 480-482Production of Siderophores by Serratia marcescens andthe Role of MacAB Efflux Pump in Siderophores SecretionKhilyas I., Shirshikova T., Matrosova L., Sorokina A., Sharipova M., Bogomolnaya L.Kazan Federal University, 420008, Kremlevskaya 18, Kazan, RussiaAbstract© 2016, Springer Science+Business Media New York.Human opportunistic pathogen Serratiamarcescens  secrete  siderophores  to  enable  growth  under  iron-limiting  conditions.  Ironacquisition genes are among a few known virulence factors of S. marcescens. Siderophoreexport systems in Gram-negative bacteria are not fully understood. There is some evidence forinvolvement of efflux pumps in the export of synthesized enterobactin-like molecules. The goalof  this  study  was  to  characterize  siderophore  production  by  two  different  strains  of  S.marcescens, SM6 and SR41-8000, and to evaluate the role of efflux pump MacAB in siderophoresecretion by these strains. We showed that both strains produced siderophores in CAS agarassay. We further showed that both strains were able to secrete catecholate siderophores inresponse to iron starvation. MacAB efflux pump played a role in siderophore secretion of S.marcescens SR41-8000 strain but was dispensable for accumulation of these molecules in theculture supernatant of S. marcescens strain SM6.http://dx.doi.org/10.1007/s12668-016-0264-3Keywords2,3-Dihydroxybenzoic acid, Efflux pump, MacAB, Serratia marcescens, Siderophores

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Production of siderophores by Serratia marcescens and the role of MacAB efflux pump in siderophores secretion

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