Abstract
The maintenance of the lipid asymmetry (Mla) system plays a critical role in facilitating the transport of phospholipids between the inner and outer membranes of the Gram-negative bacteria. In E. coli, the system consists of six proteins: MlaA-OmpF/C complex (outer membrane), MlaC (periplasm), and MlaFEDB complex (inner membrane). Despite extensive research on the core proteins (MlaFED) of the Mla system, the occurrence of Mla components like MlaA, MlaB, and MlaC in diderm remains uncertain. Therefore, this gap presents a significant opportunity for further investigation, particularly regarding MlaC, which serves as the sole mobile component of the Mla system. This has led to the identification of multiple copies of MlaC in 63 distinct genera of Proteobacteria and related phyla. Interestingly, amongst these genera, the genetic arrangements of the mla operon were observed to be varying and, thus, were further categorized into four distinct groups. The variations among the genetic organization of the mla operons suggest their evolution through various processes, such as duplications, losses, rearrangements, and fusions. Further, the results of this study highlight the MlaC’s substrate promiscuity, illuminating new avenues for the Mla system.
Highlights
• Multiple copies of MlaC and other Mla components were identified.
• A unique motif, “Ω-ζ-Φ/π-Φ-ζ-Φ” is located in the subdomain D1R2 of MlaC.
• The genetic arrangement of the mla operon is not conserved.
• MlaC duplicates demonstrate substrate promiscuity.
Similar content being viewed by others
Data availability
No datasets were generated or analysed during the current study.
Abbreviations
- CTD:
-
C-terminal domain
- LPS:
-
Lipopolysaccharide
- Mla:
-
Maintenance of lipid asymmetry
- MSA:
-
Multiple sequence alignment
- NBD:
-
Nucleotide-binding domain
- NTD:
-
N-terminal domain
- OM:
-
Outer membrane
- Omp:
-
Osmoporin
- PL:
-
Phospholipid
- SBP:
-
Substrate-binding protein
- TMD:
-
Transmembrane domain
References
Aasland R, Abrams C, Ampe C, Ball LJ, Bedford MT, Cesareni G, Gimona M, Hurley JH, Jarchau T, Lehto V, Lemmon MA, Linding R, Mayer BJ, Nagai M, Sudol M, Walter U, Winder SJ (2002) Normalization of nomenclature for peptide motifs as ligands of modular protein domains. FEBS Lett 513(1):141–144. https://doi.org/10.1016/S0014-5793(01)03295-1
Abellón-Ruiz J, Kaptan SS, Baslé A, Claudi B, Bumann D, Kleinekathöfer U, and, n den Berg B (2017) Structural basis for maintenance of bacterial outer membrane lipid asymmetry. Nat Microbiol 2(12):1616–1623. https://doi.org/10.1038/s41564-017-0046-x
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose I, Pupko T, Ben-Tal N (2016) ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res 44(W1):W344–W350. https://doi.org/10.1093/nar/gkw408
Ausmees N, Mayer R, Weinhouse H, Volman G, Amikam D, Benziman M, Lindberg M (2001) Genetic data indicate that proteins containing the GGDEF domain possess diguanylate cyclase activity. FEMS Microbiol Lett 204(1):163–167. https://doi.org/10.1111/j.1574-6968.2001.tb10880.x
Caporale LH (2003) Natural selection and the emergence of a mutation phenotype: an update of the evolutionary synthesis considering mechanisms that affect genome variation. Annual Reviews Microbiol 57(1):467–485. https://doi.org/10.1146/annurev.micro.57.030502.090855
Carpenter CD, Cooley BJ, Needham BD, Fisher CR, Trent MS, Gordon V, Payne SM (2014) The Vps/VacJ ABC transporter is required for intercellular spread of Shigella flexneri. Infect Immun 82(2):660–669. https://doi.org/10.1128/iai.01057-13
Chandravanshi M, Sharma A, Dasgupta P, Mandal SK, Kanaujia SP (2019) Identification and characterization of ABC transporters for carbohydrate uptake in Thermus thermophilus HB8. Gene 696:135–148. https://doi.org/10.1016/j.gene.2019.02.035
Chi X, Fan Q, Zhang Y, Liang K, Wan L, Zhou Q, Li Y (2020) Structural mechanism of phospholipids translocation by MlaFEDB complex. Cell Res 30(12):1127–1135. https://doi.org/10.1038/s41422-020-00404-6
Chong ZS, Woo WF, Chng SS (2015) Osmoporin OmpC forms a complex with MlaA to maintain outer membrane lipid asymmetry in Escherichia coli. Mol Microbiol 98(6):1133–1146. https://doi.org/10.1111/mmi.13202
Clifton LA, Skoda MW, Daulton EL, Hughes AV, Le Brun AP, Lakey JH, Holt SA (2013) Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic. J Royal Soc Interface 10(89):20130810. https://doi.org/10.1098/rsif.2013.0810
Coudray N, Isom GL, MacRae MR, Saiduddin MN, Bhabha G, Ekiert DC (2020) Structure of bacterial phospholipid transporter MlaFEDB with substrate bound. Elife 9:e62518. https://doi.org/10.7554/eLife.62518
Dasgupta P, Vinil K, Kanaujia SP (2024) Evolutionary trends indicate a coherent organization of Sap operons. Res Microbiol 104228. https://doi.org/10.1016/j.resmic.2024.104228
Delcour AH (2009) Outer membrane permeability and antibiotic resistance. Biochimica et biophysica acta (BBA)-Proteins and proteomics. 1794(5):808–816. https://doi.org/10.1016/j.bbapap.2008.11.005
Dutta A, Kanaujia SP (2022) MlaC belongs to a unique class of non-canonical substrate-binding proteins and follows a novel phospholipid-binding mechanism. J Struct Biol 214(4):107896. https://doi.org/10.1016/j.jsb.2022.107896
Ekiert DC, Bhabha G, Isom GL, Greenan G, Ovchinnikov S, Henderson IR, Cox JS, Vale RD (2017) Architectures of lipid transport systems for the bacterial outer membrane. Cell 169(2):273–285. https://doi.org/10.1016/j.cell.2017.03.019
El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, Qureshi M, Richardson LJ, Salazar GA, Smart A, Sonnhammer EL (2019) The Pfam protein families database in 2019. Nucleic Acids Res 47(D1):D427–D432. https://doi.org/10.1093/nar/gky995
Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr Sect D: Biol Crystallogr 66(4):486–501. https://doi.org/10.1107/S0907444910007493
Gasteiger J, Marsili M (1980) Iterative partial equalization of orbital electronegativity—a rapid access to atomic charges. Tetrahedron 36(22):3219–3228. https://doi.org/10.1016/0040-4020(80)80168-2
Gevers D, Vandepoele K, Simillion C, Van de Peer Y (2004) Gene duplication and biased functional retention of paralogs in bacterial genomes. Trends Microbiol 12(4):148–154. https://doi.org/10.1016/j.tim.2004.02.007
Gouet P, Robert X, Courcelle E (2003) ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 31(13):3320–3323. https://doi.org/10.1093/nar/gkg556
Grasekamp KP, Beaud Benyahia B, Taib N, Audrain B, Bardiaux B, Rossez Y, Lejeune M, Trivelli X, Chouit Z, Guerardel Y, Ghigo J, Gribaldo S, Beloin C (2023) The mla system of Diderm firmicute Veillonella parvula reveals an ancestral transenvelope Bridge for phospholipid trafficking. Nat Commun 14(1):1–15. https://doi.org/10.1038/s41467-023-43411-y
Guest RL, Lee MJ, Wang W, Silhavy TJ (2023) A periplasmic phospholipase that maintains outer membrane lipid asymmetry in Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences, 120(30), e2302546120. https://doi.org/10.1073/pnas.2302546120
Guinote IB, Moreira RN, Freire P, Arraiano CM (2012) Characterization of the BolA homolog IbaG: a new gene involved in acid resistance. J Microbiol Biotechnol 22(4):484–493. https://doi.org/10.4014/jmb.1107.07037
Henderson JC, Zimmerman SM, Crofts AA, Boll JM, Kuhns LG, Herrera CM, Trent MS (2016) The power of asymmetry: architecture and assembly of the Gram-negative outer membrane lipid bilayer. Annu Rev Microbiol 70:255–278. https://doi.org/10.1146/annurev-micro-102215-095308
Hezbri K, Ghodhbane-Gtari F, Montero-Calasanz MD, Sghaier H, Rohde M, Schumann P, Klenk HP, Gtari M (2015) Geodermatophilus Sabuli Sp. nov., a γ-radiation-resistant actinobacterium isolated from desert limestone. Int J Syst Evol MicroBiol 65(Pt10):3365–3372. https://doi.org/10.1099/ijsem.0.000422
Hughes GW, Hall SC, Laxton CS, Sridhar P, Mahadi AH, Hatton C, Piggot TJ, Wotherspoon PJ, Leney AC, Ward DG, Jamshad M (2019) Evidence for phospholipid export from the bacterial inner membrane by the mla ABC transport system. Nat Microbiol 4(10):1692–1705. https://doi.org/10.1038/s41564-019-0481-y
Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, Li Q, Shoemaker BA, Thiessen PA, Yu B, Zaslavsky L (2021) PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Res 49(D1):D1388–D1395. https://doi.org/10.1093/nar/gkaa971
Kolich LR, Chang YT, Coudray N, Giacometti SI, MacRae MR, Isom GL, Teran EM, Bhabha G, Ekiert DC (2020) Structure of MlaFB uncovers novel mechanisms of ABC transporter regulation. Elife 9:e60030. https://doi.org/10.7554/eLife.60030
Kumar S, Stecher G, Tamura K (2016) Mol Biol Evol 33(7):1870–1874. https://doi.org/10.1093/molbev/msw054. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets
Kumar N, Su C, Chou H, Radhakrishnan A, Delmar JA, Rajashankar KR, Yu EW (2017) Crystal structures of the burkholderia multivorans hopanoid transporter HpnN. Proc Natl Acad Sci USA 114(25):6557–6562. https://doi.org/10.1073/pnas.1619660114
Laskowski RA, Jabłońska J, Pravda L, Vařeková RS, Thornton JM (2017) PDBsum: structural summaries of PDB entries. Protein Science: Publication Protein Soc 27(1):129–134. https://doi.org/10.1002/pro.3289
Low WY, Thong S, Chng S (2021) ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry. Proc Natl Acad Sci 118(50):e2110055118. https://doi.org/10.1073/pnas.2110055118
Malinverni JC, Silhavy TJ (2009) An ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane. Proc Natl Acad Sci 106(19):8009–8014. https://doi.org/10.1073/pnas.0903229106
Mangiarotti A, Genovese DM, Naumann CA, Monti MR, Wilke N (2019) Hopanoids, like sterols, modulate dynamics, compaction, phase segregation and permeability of membranes. Biochim Et Biophys Acta (BBA) - Biomembr 1861(12):183060. https://doi.org/10.1016/j.bbamem.2019.183060
Manson MD, Boos W, Bassford PJ Jr, Rasmussen BA (1985) Dependence of maltose transport and chemotaxis on the amount of maltose-binding protein. J Biol Chem 260(17):9727–9733. https://doi.org/10.1016/S0021-9258(17)39299-2
Mitchell AL, Attwood TK, Babbitt PC, Blum M, Bork P, Bridge A, Brown SD, Chang HY, El-Gebali S, Fraser MI, Gough J (2019) InterPro in 2019: improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res 47(D1):D351–D360. https://doi.org/10.1093/nar/gky1100
Moreno-Hagelsieb G (2015) The power of Operon rearrangements for predicting functional associations. Comput Struct Biotechnol J 13:402–406. https://doi.org/10.1016/j.csbj.2015.06.002
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated Docking with selective receptor flexibility. J Comput Chem 30(16):2785–2791. https://doi.org/10.1002/jcc.21256
Munguia J, LaRock DL, Tsunemoto H, Olson J, Cornax I, Pogliano J, Nizet V (2017) The mla pathway is critical for Pseudomonas aeruginosa resistance to outer membrane permeabilization and host innate immune clearance. J Mol Med 95:1127–1136. https://doi.org/10.1007/s00109-017-1579-4
Nikaido H, Vaara M (1985) Molecular basis of bacterial outer membrane permeability. Microbiol Rev 49(1):1–32. https://doi.org/10.1128/mr.49.1.1-32.1985
Powers MJ, Trent MS (2018) Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics. Proc Natl Acad Sci 115(36):E8518–E8527. https://doi.org/10.1073/pnas.1806714115
Powers MJ, Simpson BW, Trent MS (2020) The mla pathway in acinetobacter baumannii has no demonstrable role in anterograde lipid transport. Elife 9:e56571. https://doi.org/10.7554/eLife.56571
Riehle MM, Bennett AF, Long AD (2001) Genetic architecture of thermal adaptation in Escherichia coli. Proceedings of the National Academy of Sciences, 98(2), 525–530. https://doi.org/10.1073/pnas.98.2.525
Rivero J, Cutillas C, Callejón R (2021) Trichuris Trichiura (Linnaeus, 1771) from human and Non-human primates: morphology, biometry, host specificity, molecular characterization, and phylogeny. Front Veterinary Sci 7:626120. https://doi.org/10.3389/fvets.2020.626120
Roier S, Zingl FG, Cakar F, Durakovic S, Kohl P, Eichmann TO, Schild S (2016) A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria. Nat Commun 7(1):1–13. https://doi.org/10.1038/ncomms10515
Saha S, Mandal SK, Kanaujia SP (2024) Distinct characteristics of putative archaeal 5-methylcytosine RNA methyltransferases unveil their substrate specificities and evolutionary ancestries. J Biomol Struct Dynamics 1–18. https://doi.org/10.1080/07391102.2024.2325670
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Research, 13(11), 2498–2504. https://doi.10.1101/gr.1239303
Sievers F, Higgins DG (2014) Clustal Omega, accurate alignment of very large numbers of sequences. Multiple Seq Alignment Methods 105–116. https://doi.org/10.1007/978-1-62703-646-7_6
Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M (2015) STRING v10: protein–protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43(D1):D447–D452. https://doi.org/10.1093/nar/gku1003
Tang X, Chang S, Qiao W, Luo Q, Chen Y, Jia Z, Dong H (2021) Structural insights into outer membrane asymmetry maintenance in Gram-negative bacteria by MlaFEDB. Nat Struct Mol Biology 28(1):81–91. https://doi.org/10.1038/s41594-020-00532-y
The UniProt Consortium (2023) UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res 51(D1). https://doi.org/10.1093/nar/gkac1052. D523-D531
Thomsen MCF, Nielsen M (2012) Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, Pseudo counts and two-sided representation of amino acid enrichment and depletion. Nucleic Acids Res 40(W1):W281–W287. https://doi.org/10.1093/nar/gks469
Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, Yuan D, Stroe O, Wood G, Laydon A, Žídek A (2022) AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res 50(D1):D439–D444. https://doi.org/10.1093/nar/gkab1061
Yamanaka K, Fang L, Inouye M (1998) The CspA family in Escherichia coli: multiple gene duplication for stress adaptation. Mol Microbiol 27(2):247–255. https://doi.org/10.1046/j.1365-2958.1998.00683.x
Yero D, Díaz-Lobo M, Costenaro L, Conchillo-Solé O, Mayo A, Ferrer-Navarro M, Vilaseca M, Gibert I, Daura X (2021) The Pseudomonas aeruginosa substrate-binding protein Ttg2D functions as a general glycerophospholipid transporter across the periplasm. Commun Biology 4(1):448. https://doi.org/10.1038/s42003-021-01968-8
Yu J, Zhou Y, Tanaka I, Yao M (2010) Roll: a new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics 26(1):46–52. https://doi.org/10.1093/bioinformatics/btp599
Acknowledgements
The work was supported by the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India (Grant number: EEQ/2021/000060). The authors acknowledge the facilities provided by the Indian Institute of Technology Guwahati, Assam, India. The authors would like to express their gratitude to the members of the Structural and Computational Biology Laboratory (SCBL) for all the timely support. RT acknowledges the Ministry of Education, Government of India, for providing the research fellowship.
Author information
Authors and Affiliations
Contributions
SPK conceived the project and guided the research. RT and DA collected the data. RT performed the formal data analysis. RT and SPK analyzed the data. RT and SPK wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Communicated by Yusuf Akhter.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
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
Tripathi, R., Ayekpam, D. & Kanaujia, S.P. Unveiling multiple copies of MlaC highlights its multifaceted nature. Arch Microbiol 207, 107 (2025). https://doi.org/10.1007/s00203-025-04308-0
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00203-025-04308-0