Abstract
Bacterial outer membrane proteins (Omps) are essential for environmental sensing, stress responses, and substance transport. Our previous study discovered that OmpA contributes to planktonic growth, biocide resistance, biofilm formation, and swimming motility in Citrobacter werkmanii, whereas the molecular functions of OmpF in this strain are largely unknown. Thus, in this study, the ompF gene was firstly knocked out from the genome of C. werkmanii using a homologous recombination method, and its phenotypical alternations of ∆ompF were then thoroughly characterized using biochemical and molecular approaches with the parental wild type (WT) and complementary (∆ompF-com) strains. The results demonstrated that the swimming ability of ∆ompF on semi-solid plates was reduced compared to WT due to the down-regulation of flgC, flgH, fliK, and fliF. Meanwhile, ompF deletion reduces biofilm formation on both glass and polystyrene surfaces due to decreased cell aggregation. Furthermore, ompF inactivation induced different osmotic stress (carbon sources and metal ions) responses in its biofilms when compared to WT and ∆ompF-com. Finally, a total of 6 maltose metabolic genes of lamB, malE, malK, malG, malM, and malF were all up-regulated in ∆ompF. The gene knockout and HPLC results revealed that the MalEFGK2 cluster was primarily responsible for maltose transport in C. werkmanii. Furthermore, we discovered for the first time that the upstream promoter of OmpF and its transcription can be combined with and negatively regulated by MalT. Overall, OmpF plays a role in a variety of biochemical processes and molecular functions in C. werkmanii, and it may even act as a targeted site to inhibit biofilm formation.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Availability of data and materials
All data generated or analyzed during this study are included in this article and its electronic supplementary materials. Additional files that support the findings of this study are available from the authors upon reasonable request.
References
Akiba T, Yoshimura H, Namba K (1991) Monolayer crystallization of flagellar LP rings by sequential addition and depletion of lipid. Science 252:1544–1546. https://doi.org/10.1126/science.2047860
Alphen W, Lugtenberg B (1977) Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli. J Bacteriol 131:623–630. https://doi.org/10.1128/jb.131.2.623-630.1977
Bari W, Lee K-M, Yoon SS (2012) Structural and functional importance of outer membrane proteins in Vibrio cholerae flagellum. J Microbiol 50:631–637. https://doi.org/10.1007/s12275-012-2116-3
Bible A, Russell MH, Alexandre G (2012) The Azospirillum brasilense Che1 chemotaxis pathway controls swimming velocity, which affects transient cell-to-cell clumping. J Bacteriol 194:3343–3355. https://doi.org/10.1128/JB.00310-12
Boos W, Shuman H (1998) Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol Mol Biol Rev 62:204–229. https://doi.org/10.1128/MMBR.62.1.204-229.1998
Bootman MD, Berridge MJ (1995) The elemental principles of calcium signaling. Cell 83:675–678. https://doi.org/10.1016/0092-8674(95)90179-5
Briones A, Lorca D, Cofre A, Cabezas C, Krüger G, Pardo-Esté C, Baquedano M, Salinas C, Espinoza M, Castro-Severyn J (2022) Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress. Biol Res 55:1–11. https://doi.org/10.1186/s40659-022-00377-3
Brown EM, Vassilev PM, Hebert SC (1995) Calcium ions as extracellular messengers. Cell 83:679–682. https://doi.org/10.1016/0092-8674(95)90180-9
Bystritskaya E, Stenkova A, Chistuylin D, Chernysheva N, Khomenko V, Anastyuk S, Novikova O, Rakin A, Isaeva M (2016) Adaptive responses of outer membrane porin balance of Yersinia ruckeri under different incubation temperature, osmolarity, and oxygen availability. Microbiologyopen 5:597–603. https://doi.org/10.1002/mbo3.354
Calderón IL, Morales E, Caro NJ, Chahúan CA, Collao B, Gil F, Villarreal JM, Ipinza F, Mora GC, Saavedra CP (2011) Response regulator ArcA of Salmonella enterica serovar Typhimurium downregulates expression of OmpD, a porin facilitating uptake of hydrogen peroxide. Res Microbiol 162:214–222. https://doi.org/10.1016/j.resmic.2010.11.001
Cruz LF, Cobine PA, De La Fuente L (2012) Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching motility. Appl Environ Microbiol 78:1321–1331. https://doi.org/10.1128/AEM.06501-11
Dardonville B, Raibaud O (1990) Characterization of malT mutants that constitutively activate the maltose regulon of Escherichia coli. J Bacteriol 172:1846–1852. https://doi.org/10.1128/jb.172.4.1846-1852.1990
Domka J, Lee J, Wood TK (2006) YliH (BssR) and YceP (BssS) regulate Escherichia coli K-12 biofilm formation by influencing cell signaling. Appl Environ Microbiol 72:2449–2459. https://doi.org/10.1128/AEM.72.4.2449-2459.2006
Dupont M, Dé E, Chollet R, Chevalier J, Pagès J-M (2004) Enterobacter aerogenes OmpX, a cation-selective channel mar-and osmo-regulated. FEBS Lett 569:27–30. https://doi.org/10.1016/j.febslet.2004.05.047
Enjalbert B, Smith DA, Cornell MJ, Alam I, Nicholls S, Brown AJ, Quinn J (2006) Role of the Hog1 stress-activated protein kinase in the global transcriptional response to stress in the fungal pathogen Candida albicans. Mol Biol Cell 17:1018–1032. https://doi.org/10.1091/mbc.e05-06-0501
Finlay JA, Allan VJM, Conner A, Callow ME, Basnakova G, Macaskie LE (1999) Phosphate release and heavy metal accumulation by biofilm-immobilized and chemically-coupled cells of a citrobacter sp. pre-grown in continuous culture. Biotechnol Bioeng 63:87–97. https://doi.org/10.1002/(SICI)1097-0290(19990405)63:1%3c87::AID-BIT9%3e3.0.CO;2-0
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30:1229–1235. https://doi.org/10.1093/molbev/mst012
Hirano T, Yamaguchi S, Oosawa K, Aizawa S-l (1994) Roles of FliK and FlhB in determination of flagellar hook length in Salmonella typhimurium. J Bacteriol 176:5439–5449. https://doi.org/10.1128/jb.176.17.5439-5449.1994
Hodges GR, Degener CE, Barnes WG (1978) Clinical significance of Citrobacter isolates. Am J Clin Pathol 70:37–40. https://doi.org/10.1093/ajcp/70.1.37
Jagmann N, Henke SF, Philipp B (2015) Cells of Escherichia coli are protected against severe chemical stress by co-habiting cell aggregates formed by Pseudomonas aeruginosa. Appl Microbiol Biotechnol 99:8285–8294. https://doi.org/10.1007/s00253-015-6726-7
Jeong BC, Hawes C, Bonthrone KM, Macaskie LE (1997) Localization of enzymically enhanced heavy metal accumulation by Citrobacter sp. and metal accumulation in vitro by liposomes containing entrapped enzyme. Microbiology 143:2497–2507. https://doi.org/10.1099/00221287-143-7-2497
Kao D-Y, Cheng Y-C, Kuo T-Y, Lin S-B, Lin C-C, Chow L-P, Chen W-J (2009) Salt-responsive outer membrane proteins of Vibrio anguillarum serotype O1 as revealed by comparative proteome analysis. J Appl Microbiol 106:2079–2085. https://doi.org/10.1111/j.1365-2672.2009.04178.x
Katzenellenbogen E, Kocharova NA, Korzeniowska-Kowal A, Bogulska M, Rybka J, Gamian A, Kachala VV, Shashkov AS, Knirel YA (2008) Structure of the glycerol phosphate-containing O-specific polysaccharide and serological studies on the lipopolysaccharides of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14). FEMS Immunol Med Mic 54:255–262. https://doi.org/10.1111/j.1574-695X.2008.00477.x
Klebensberger J, Lautenschlager K, Bressler D, Wingender J, Philipp B (2007) Detergent-induced cell aggregation in subpopulations of Pseudomonas aeruginosa as a preadaptive survival strategy. Environ Microbiol 9:2247–2259. https://doi.org/10.1111/j.1462-2920.2007.01339.x
Li L-J, Zhou G, Shi Q-S, Chen Y-C, Chen Y-B, ouyang Y-S, Hu W-F, (2014) Identification and biofilm formation characterization of Citrobacter werkmanii isolated from industrial spoilage. Microbiology China 41:2–7
Li Y, Hao G, Galvani CD, Meng Y, De La Fuente L, Hoch H, Burr TJ (2007) Type I and type IV pili of Xylella fastidiosa affect twitching motility, biofilm formation and cell-cell aggregation. Microbiology 153:719–726. https://doi.org/10.1099/mic.0.2006/002311-0
Liu X, Ferenci T (2001) An analysis of multifactorial influences on the transcriptional control of ompF and ompC porin expression under nutrient limitation. Microbiology 147:2981–2989. https://doi.org/10.1099/00221287-147-11-2981
Long Y, Fu W, Wang S, Deng X, Jin Y, Bai F, Cheng Z, Wu W (2020) Fis contributes to resistance of Pseudomonas aeruginosa to ciprofloxacin by regulating pyocin synthesis. J Bacteriol 202:e00064-e120. https://doi.org/10.1128/JB.00064-20
Macaskie LE, Empson RM, Lin F, Tolley MR (1995) Enzymatically-mediated uranium accumulation and uranium recovery using a Citrobacter sp. Immobilised as a biofilm within a plug-flow reactor. J Chem Technol Biotechnol 63:1–16. https://doi.org/10.1002/jctb.280630102
Macnab RM (2003) How bacteria assemble flagella. Annu Rev Microbiol 57:77. https://doi.org/10.1146/annurev.micro.57.030502.090832
Maervoet VE, Beauprez J, De Maeseneire SL, Soetaert WK, De Mey M (2012) Citrobacter werkmanii, a new candidate for the production of 1, 3-propanediol: strain selection and carbon source optimization. Green Chem 14:2168–2178
Maervoet VE, De Maeseneire SL, Avci FG, Beauprez J, Soetaert WK, De Mey M (2014) 1, 3-propanediol production with Citrobacter werkmanii DSM17579: effect of a dhaD knock-out. Microb Cell Fact 13:1–11. https://doi.org/10.1186/1475-2859-13-70
Maervoet VE, De Maeseneire SL, Avci FG, Beauprez J, Soetaert WK, De Mey M (2016) High yield 1, 3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii. Microb Cell Fact 15:1–11. https://doi.org/10.1186/s12934-016-0421-y
Martinez RM, Dharmasena MN, Kirn TJ, Taylor RK (2009) Characterization of two outer membrane proteins, FlgO and FlgP, that influence Vibrio cholerae motility. J Bacteriol 191:5669–5679. https://doi.org/10.1128/JB.00632-09
Minamino T, Saijo-Hamano Y, Furukawa Y, González-Pedrajo B, Macnab RM, Namba K (2004) Domain organization and function of Salmonella FliK, a flagellar hook-length control protein. J Mol Biol 341:491–502. https://doi.org/10.1016/j.jmb.2004.06.012
Mizuno T, Mizushima S (1987) Isolation and characterization of deletion mutants of ompR and envZ, regulatory genes for expression of the outer membrane proteins OmpC and OmpF in Escherichia coli. J Biochem 101:387–396. https://doi.org/10.1093/oxfordjournals.jbchem.a121923
Nakamura S, Minamino T (2019) Flagella-driven motility of bacteria. Biomolecules 9:279. https://doi.org/10.3390/biom9070279
Otto K, Hermansson M (2004) Inactivation of ompX causes increased interactions of type 1 fimbriated Escherichia coli with abiotic surfaces. J Bacteriol 186:226–234. https://doi.org/10.1128/JB.186.1.226-234.2004
Patrauchan M, Sarkisova S, Sauer K, Franklin M (2005) Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp. Microbiology 151:2885–2897. https://doi.org/10.1099/mic.0.28041-0
Piepenbreier H, Fritz G, Gebhard S (2017) Transporters as information processors in bacterial signalling pathways. Mol Microbiol 104:1–15. https://doi.org/10.1111/mmi.13633
Pratt LA, Hsing W, Gibson KE, Silhavy TJ (1996) From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli. Mol Microbiol 20:911–917. https://doi.org/10.1111/j.1365-2958.1996.tb02532.x|
Raczkowska A, Brzóstkowska M, Brzostek K (2008) Regulation of Yersinia enterocolitica mal genes by MalT and Mic proteins. Pol J Microbiol 57:19
Richet E, Raibaud O (1987) Purification and properties of the MalT protein, the transcription activator of the Escherichia coli maltose regulon. J Biol Chem 262:12647–12653. https://doi.org/10.1016/S0021-9258(18)45255-6
Saier M Jr, Crasnier M (1996) Inducer exclusion and the regulation of sugar transport. Res Microbiol 147:482–489. https://doi.org/10.1016/S0923-2508(96)90150-3
Salton M (1967) Structure and function of bacterial cell membranes. Annu Rev Microbiol 21:417–442. https://doi.org/10.1146/annurev.mi.21.100167.002221
Sarkisova S, Patrauchan M, Berglund D, Nivens D, Franklin M (2005) Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. J Bacteriol 187:4327–4337. https://doi.org/10.1128/JB.187.13.4327-4337.2005
Schaechter M (2015) A brief history of bacterial growth physiology. Front Microbiol 6:289. https://doi.org/10.3389/fmicb.2015.00289
Schoenhals GJ, Macnab RM (1996) Physiological and biochemical analyses of FlgH, a lipoprotein forming the outer membrane L ring of the flagellar basal body of Salmonella typhimurium. J Bacteriol 178:4200–4207. https://doi.org/10.1128/jb.178.14.4200-4207.1996
Shanks RM, Meehl MA, Brothers KM, Martinez RM, Donegan NP, Graber ML, Cheung AL, O’Toole GA (2008) Genetic evidence for an alternative citrate-dependent biofilm formation pathway in Staphylococcus aureus that is dependent on fibronectin binding proteins and the GraRS two-component regulatory system. Infect Immun 76:2469–2477. https://doi.org/10.1128/IAI.01370-07
Singh R, Olson MS (2008) Application of bacterial swimming and chemotaxis for enhanced bioremediation Emerging Environmental Technologies. Springer, pp 149–172
Smith R (1995) Calcium and bacteria. Adv Microb Physiol 37:83–133. https://doi.org/10.1016/S0065-2911(08)60144-7
Song B, Leff LG (2006) Influence of magnesium ions on biofilm formation by Pseudomonas fluorescens. Microbiol Res 161:355–361. https://doi.org/10.1016/j.micres.2006.01.004
Stepanović S, Vuković D, Dakić I, Savić B, Švabić-Vlahović M (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Meth 40:175–179. https://doi.org/10.1016/S0167-7012(00)00122-6
Stoorvogel J, Van Bussel M, Van De Klundert J (1991) Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX). J Bacteriol 173:161–167. https://doi.org/10.1128/jb.173.1.161-167.1991
Sun Z, Shi J, Liu C, Jin Y, Li K, Chen R, Jin S, Wu W, Payne SM (2014) PrtR homeostasis contributes to Pseudomonas aeruginosa pathogenesis and resistance against ciprofloxacin. Infect Immun 82:1638–1647. https://doi.org/10.1128/IAI.01388-13
Tang Q, Feng M (2007) DPS data processing system: experimental design, statistical analysis and data mining. Science Press, Beijing
Terashima H, Kojima S, Homma M (2008) Flagellar motility in bacteria: structure and function of flagellar motor. Int Rev Cell Mol Biol 270:39–85. https://doi.org/10.1016/S1937-6448(08)01402-0
Ueno T, Oosawa K, Aizawa S-I (1992) M ring, S ring and proximal rod of the flagellar basal body of Salmonella typhimurium are composed of subunits of a single protein, FliF. J Mol Biol 227:672–677. https://doi.org/10.1016/0022-2836(92)90216-7
Vidal-Ingigliardi D, Ricbet E, Raibaud O (1991) Two MalT binding sites in direct repeat: a structural motif involved in the activation of all the promoters of the maltose regulons in Escherichia coli and Klebsiella pneumoniae. J Mol Biol 218:323–334. https://doi.org/10.1016/0022-2836(91)90715-I
Wang T, Flint S, Palmer J (2019) Magnesium and calcium ions: roles in bacterial cell attachment and biofilm structure maturation. Biofouling 35:959–974. https://doi.org/10.1080/08927014.2019.1674811
Whitman WB (2015) Genome sequences as the type material for taxonomic descriptions of prokaryotes. Syst Appl Microbiol 38:217–222. https://doi.org/10.1016/j.syapm.2015.02.003
Zhao X, Chen Y, Zhang L, Li Z, Wu X, Chen J, Wang F (2021) Molecular cloning and biochemical characterization of a trehalose synthase from Myxococcus sp. strain V11. Protein Expr Purif 183: 105865. https://doi.org/10.1016/j.pep.2021.105865
Zhou G, Li L, Shi Q, Ouyang Y, Chen Y, Hu W (2013) Effects of nutritional and environmental conditions on planktonic growth and biofilm formation for Citrobacter werkmanii BF-6. J Microbiol Biotechnol 23:1673–1682. https://doi.org/10.4014/jmb1307.07041
Zhou G, Peng H, Wang Y-S, Li C-L, Shen P-F, Huang X-M, Xie X-B, Shi Q-S (2019) Biological functions of nirS in Pseudomonas aeruginosa ATCC 9027 under aerobic conditions. J Ind Microbiol Biotechnol 46:1–12. https://doi.org/10.1007/s10295-019-02232-z
Zhou G, Shi Q-S, Huang X-M, Xie X-B (2016a) Comparison of transcriptomes of wild-type and isothiazolone-resistant Pseudomonas aeruginosa by using RNA-seq. Mol Biol Rep 43:527–540. https://doi.org/10.1007/s11033-016-3978-y
Zhou G, Shi Q-S, Huang X-M, Xie X-B (2016b) Proteome responses of Citrobacter werkmanii BF-6 planktonic cells and biofilms to calcium chloride. J Proteom 133:134–143. https://doi.org/10.1016/j.jprot.2015.12.019
Zhou G, Wang Y-S, Peng H, Huang X-M, Xie X-B, Shi Q-S (2018) Role of ttca of Citrobacter werkmanii in bacterial growth, biocides resistance, biofilm formation and swimming motility. Int J Mol Sci 19:2644. https://doi.org/10.3390/ijms19092644
Acknowledgements
We would like to thank Prof. Hai-hong Wang of South China Agricultural University for the generous provision of E. coli S17-1 and pSRK-GM. We are also grateful to Prof. Yan-guang Cong of Hospital of Traditional Chinese Medicine Affiliated to Southwest Medical University for providing plasmid pYG4 to us along with some valuable experimental guidance.
Funding
This work was funded by the National Natural Science Foundation of China (No. 31770091), Natural Science Foundation of Guangdong Province (No. 2020A151501848), Research and Development Plan for Key Fields of Guangdong Province (No. 2022B1111040002), GDAS’ Project of Science and Technology Development (No. 2022GDASZH-2022010202), and Guangdong Science and Technology Program (No. 2017B030314045).
Author information
Authors and Affiliations
Contributions
GZ, QS, and XX conceived and designed the research. GZ, YW, HP, SL, TS, and PS performed experiments. GZ, XX, QS, YW, and HP analyzed the data. GZ wrote the manuscript. All authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethics approval and consent to participate
Not applicable.
Consent for publication
All the authors agreed to publish the paper.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Zhou, G., Wang, YS., Peng, H. et al. Outer membrane protein of OmpF contributes to swimming motility, biofilm formation, osmotic response as well as the transcription of maltose metabolic genes in Citrobacter werkmanii. World J Microbiol Biotechnol 39, 15 (2023). https://doi.org/10.1007/s11274-022-03458-3
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-022-03458-3