Enhanced Functionalisation of Major Facilitator Superfamily Transporters Via Fusion of C-Terminal Protein Domains Is Both Extensive and Varied in Bacteria

SHORT COMMUNICATION Willson et al., Microbiology 2019;165:419–424 DOI 10.1099/mic.0.000771

Enhanced functionalisation of major facilitator superfamily transporters via fusion of C-terminal protein domains is both extensive and varied in bacteria

Benjamin J. Willson, Lindsey Dalzell, Liam N. M. Chapman and Gavin H. Thomas*

Abstract The evolution of gene fusions that result in covalently linked protein domains is widespread in bacteria, where spatially coupling domain functionalities can have functional advantages in vivo. Fusions to integral membrane proteins are less widely studied but could provide routes to enhance membrane function in synthetic biology. We studied the major facilitator superfamily (MFS), as the largest family of transporter proteins in bacteria, to examine the extent and nature of fusions to these proteins. A remarkably diverse variety of fusions are identified and the 8 most abundant examples are described, including additional enzymatic domains and a range of sensory and regulatory domains, many not previously described. Significantly, these fusions are found almost exclusively as C-terminal fusions, revealing that the usually cytoplasmic C-terminal end of MFS protein would the permissive end for engineering synthetic fusions to other cytoplasmic proteins.

In nature, proteins commonly consist of multiple functional by Barabote et al. [6] in 2006 identified several novel fusions domains. This allows the combination of multiple functions from a range of transporter families and made several novel into a single protein, with fusions of catalytic domains predictions of function. Since then, thousands of bacterial potentially facilitating catalysis via substrate channeling [1], and archaeal genomes have been sequenced, providing a or fusions of regulatory domains allowing post-translational rich resource to mine for additional examples of fusions and control of activity [2]. Identification of these fusions is of assess systematically how widespread these are in biology. interest as they may allow the identification of functional For this study, we wished to identify the range and type of relationships between proteins through the Rosetta Stone fusions seen in the major facilitator superfamily (MFS), the method [3] or provide insights that enable optimisation of largest family of secondary transporters in bacteria. This is the development of synthetic fusions [4]. an ideal starting point as the vast majority of MFS trans- Membrane transporters are integral membrane proteins porters are single proteins [8], although they can sometimes involved in the active transport of chemicals across the lipid be found in complexes such as the EmrAB-TolC efflux bilayer. Many transporter families have been identified, and pump [9], are widespread, being found in all living organ- the strategies used for transport are diverse; transport can isms [10], and are identifiable in numerous architectures in be linked to ATP hydrolysis or to utilisation of proton or the InterPro database [11, 12]. + Na -ion gradients across the membrane [5]. A number of The InterPro database was searched for MFS domains with transport proteins are fused to other domains; these can be the identifier ‘IPR020846’, identifying 3527 architectures. To catalytic or regulatory, and can be located at the N-termi- survey the most commonly found, we selected architectures nus, C-terminus, or internally [6]. The value of studying with more than 100 examples, which also removed any these fusions to elucidate the function of transporters is chance of them being false positives from sequencing errors. well-established, and has enabled discoveries such as the Furthermore, as the study focussed on bacterial MFS trans- LplT lysophospholipid transporter [7]. This transporter was porters, we only included architectures with bacterial repre- identified through its fusion to domains involved in lyso- sentatives. Architectures containing particular domain phospholipid repair, and follow-up experiments confirmed fusions were assigned to groups; we added additional archi- a role in lysophospholipid transport. A subsequent analysis tectures containing these fused domains as part of multi-

Received 31 August 2018; Accepted 21 December 2018; Published 18 January 2019 Author affiliation: Department of Biology, University of York, Wentworth Way, York YO10 5DD, UK. *Correspondence: Gavin H. Thomas, [email protected] Keywords: major facilitator superfamily; fusion proteins; InterPro; protein evolution. Abbreviations: CBS, cystathionine b-synthase; CNBD, cyclic nucleotide binding domain; MFS, major facilitator superfamily; NTT, nucleotide transport protein; PABS, polyamine biosynthesis; PAS, Per-Arnt-Sim; PLP, patatin-like phospholipase; TM, transmembrane helix. Three supplementary tables and one supplementary figure are available with the online version of this article.

000771 ã 2019 The Authors Downloaded from www.microbiologyresearch.org by This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. IP: 54.70.40.11 419 On: Thu, 23 May 2019 23:34:27 Willson et al., Microbiology 2019;165:419–424 domain fusions when more than 30 representatives were [16]. NarK1 encodes a nitrate/proton symporter, and identified. NarK2 a nitrate/nitrite antiporter; it has been proposed that, during denitrification, NarK1 allows the import of nitrate, From this conservative analysis, we identified a broad range which is reduced to nitrite and then exchanged with nitrate of fusions (Fig. 1 and Table 1), several of which are as yet via NarK2 [15]. The fusion is not essential for function, but uncharacterised. To obtain further data, gene/protein iden- may allow cross-talk between the two domains, and main- tifiers were used to search UniProt [13], providing the pre- tains the stoichiometry of the two transporters at 1 : 1 [16]. dicted function, gene name, host species, taxonomic However, a significant limitation of our strategy is that this distribution, and length (Table S1, is available in the online category of fusions includes all proteins that are annotated version of this article). Finally, the NCBI BLAST server [14] as having two separate MFS domains by InterPro, regardless was used to identify homology between proteins and spe- of the size of the domains. This means that this group is cific domains. By combining these analyses with available likely to have a high rate of false positives; for example, 12- literature, it was possible to predict functions for some of transmembrane helix (TM) proteins with extended loops the unidentified fusions. between helix 6 and 7 (e.g. UniProt: A0A031GPQ9) may be The three most prevalent fusions we identified are all well- falsely considered as two separate MFS domains. studied. The largest apparent group (Group 1.) consists of The second group contains fusions of MFS to a phospho- fusions of two full-length MFS transporters, which includes lipid/glycerol acyltransferase domain (PlsC, IPR002123), the fused nitrate/nitrite transporter NarK, as characterised with three architectures: fusion to PlsC alone (Group 2a.), in Paracoccus pantrophus [15] and Paracoccus denitrificans with an additional AMP-dependent synthetase/ligase

Fig. 1. Schematic diagram of the eight most prevalent groups of MFS fusion proteins in bacteria. Dashed lines indicate domain bound- aries where multiple architectures are observed. Numbers in parentheses indicate the numbers of results in InterPro for each domain architecture. For group 5, numbers of HEAT repeat containing and non-HEAT repeat containing architectures are combined (annotated as † and ‡) as these are not separated in InterPro. Blue highlights represent regulatory domains, and red highlights represent catalytic domains. The results presented here are accurate as of 07/03/18.

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domain (ACS, IPR000873) (Group 2b.) or with an ACS domain and an AMP-binding enzyme, C-terminal domain (IPR025110) (Group 2c.). These represent different degrees of fusion of the flippase LplT to the lysophospholipid repair system; LplT transfers the lysophospholipid 2-acylglycero- phosphoethanolamine to the internal leaflet of the inner membrane, where it is acylated by PlsC/ACS to form phos- phatidylethanolamine [7]. The two-domain fusion was only identified in g-proteobacteria, whereas the three-domain fusion was identified in a-, d-, and "-proteobacteria and the g-proteobacterium Microbulbifer degradans. Our analysis reveals a significantly wider distribution, with the two- domain fusion also being widespread within the b-proteo- Known flippase coupled to lysophospholipid repair HEAT repeat-containing members of Groups 5a and 5b are known ATP importers, possibly linked toPredicted cAMP nitrate level transporter, possibly redox-sensitive Known osmosensitive transporter of compatible solutes Possible spermidine synthase linked to putrescine transporter Known nitrate/nitrite transporter Known bile salt efflux pumps, role of CBS unknown Possible involvement in stress response Function bacterial Burkholderiales order, whereas the three-domain fusion has examples in all groups of proteobacteria, the PVC superphylum, and even some Gram-positives. The third group consists of a fusion to an ‘osmosensory transporter coiled coil’ (Osmo_CC, IPR015041) domain (Group 3.), a short sequence found specifically at the C-terminus of MFS transporters. This fusion is found almost exclusively in Gram-negative bacteria, primarily Enterobac- terales and Pseudomonales (g-proteobacteria), but with

gi13883156 [30] (UniProt: Q8VJ44) some examples in a- and b-proteobacteria. Observed sub- NarK [16] (UniProt: Q93PW1) BL0920 [39] (UniProt: Q8G5T3) NarK [17] (UniProt: A1B9V7) OusA [19] (UniProt: Q47421) BBr_0838 [38] (UniProt: C4NXS7) strates of these transporters are compatible solutes such as proline, glycine betaine, taurine, ectoine, and pipecolate ProP [18, 20] (UniProt: P0C0L7) [17, 18]. The Osmo_CC domain is a regulatory domain that allosterically activates the transporter under osmotic stress; E. coli ProP loses function when the Osmo_CC domain is deleted [19], but can still respond to high levels of osmotic Multiple examples of HEAT repeat- containing proteins [29 ] (e.g.A0A0E9CVD3, UniProt: D0NEC5) Mycobacterium tuberculosis Multiple examples of 2- andfusions 3-domain [7] (e.g. UniProt: A0A085VKZ6, A0A1A5IBS3) Escherichia coli Erwinia chrysanthemi Bifidobacterium breve Bifidobacterium longum Paratrophus pantrophus Paracoccus denitrificans stress if the domain is truncated [20]. However, the activity of OusA from Erwinia chrysanthemi, a ProP homologue 99 121 953 75 32 168 45 101 2289 962 56 [18], is not affected by osmolarity, suggesting that the domain is not necessarily indicative of osmoregulation. R R C C C R R R/C C C C Several variants of polyamine biosynthesis (PABS) domain (IPR030374)-MFS fusions were observed. The most prevalent has an MFS domain immediately followed by the PABS domain (Group 4a.). This group contains MFS domains with 13 TM helices and ‘short’ variants with seven TM helices. Another variant (Group 4b.) consists of the 13TM MFS and PABS domain followed by a tetratricopeptide repeat-containing domain (TPR, IPR013026). There are 32 examples of a PABS domain with a C-terminal fused 7TM MFS transporter (Group 4c.); these proteins also typically contain seven TM helices at the N-terminus (as identified by TMHMM MFS_IPR000014 MFS_IPR000014_IPR000700 MFS_IPR030374 MFS_IPR030374_IPR013026 IPR030374_MFS MFS_IPR000595 MFS_IPR000595_IPR000595 MFS_IPR000595_IPR002641 MFS_IPR002123 MFS_IPR002123_IPR000873 MFS_IPR002123_IPR000873_ IPR025110 [21]) which are unrecognised as a domain. In the Transporter Classification Database [22], both 7TM and 13TM variants are considered to be MFS of the Uncharacterised Major Facil- itator-30 (UMF30) family. Whether or not the 7TM MFS forms a functional transporter is uncertain. The Neisseria meningitidis protein NMB0240 appears to be a 7TM member of this group, and is immediately preceded on the genome by a 6TM protein recognised as an MFS [23]. Given that homo- MFS_PAS MFS_PAS_PAC MFS_PABS MFS_PABS_TPR (7TM)_PABS_MFS MFS_CNBD MFS_CNBD_CNBD MFS_CNBD_PLP MFS_PlsC MFS_PlsC_ACS MFS_PlsC_ACS_ACS-Cterm logues of these two proteins are fused in some organisms (e.g. Most abundant fused MFS architectures identified in this study. Architectures have been organised into groups according to fused domain identities. Individual architectures have been C9PRR1 from Pasteurella dagmatis), it is possible that these two proteins could function as a split MFS transporter, as 3. MFS_Osmo_CC MFS_IPR015041 R 1235 6a. 6b. 7. MFS_CBS_CBS MFS_IPR000644_IPR000644 R 208 4a. 4b 4c. 5a. 5b. 5c. 2a. 2b. 2c. 8. MFS_UspA MFS_IPR006016 R 122 Group Architectures1. MFS_MFS IPR identifiers MFS_MFS Category Hits Notable examples R? 5286 characterised as Regulatoryunclassified fusions 7-transmembrane (R) helix region. or Catalytic fusions (C). Notable examples are those that have been characterised or identified previously, and are discussed in the main text. 7TM: Table 1. MFS transporters can be ‘split’ and expressed as two discrete

Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11421 On: Thu, 23 May 2019 23:34:27 Willson et al., Microbiology 2019;165:419–424 proteins while retaining function [24, 25]. This genomic conditions for use as an electron acceptor [30]; under anaer- arrangement seems to be unusual in the wider group, obic conditions, NarK is significantly upregulated [31]. although some 7TM examples have small 2TM or 4TM pro- Denitrification has also been observed in other Bacillales teins immediately upstream. NMB0240 has been suggested [32]. PAS domains are involved in the sensing of a range of to be involved in arginine uptake [23]; furthermore, knock- signals [33], including redox states, oxygen, and light. PAS out of this protein leads to a significant growth impairment domains have been observed to play a role in regulation of on minimal medium, which can be rescued by addition of nitrogen metabolism. In some nitrogen-fixing bacteria, the leucine or isoleucine. It is possible that the proteins of Group nif nitrogen fixation operon is repressed under aerobic con- 4c constitute functional 14TM transporters with the PABS ditions by the NifL/NifA system; under anaerobic condi- domain located in the linker region that joins the two helical tions, the flavin mononucleotide bound by the PAS domain bundles. While several MFS transporters have an additional of NifL is reduced, leading to derepression [34]. Given the two TM helices at this location [26], to our knowledge, there role of PAS domains in oxygen sensing, and the oxygen- are no examples with a functional soluble domain inserted in sensitive regulation of B. subtilis NarK, it is tempting to this region. speculate that the PAS domain of MFS-PAS proteins may provide post-translational regulation of transporter activity In bacteria, PABS domain-containing proteins are typically based on oxygen and/or redox levels. involved in the production of spermidine from putrescine or thermospermine from spermidine [27]. However, as Fusions of MFS to two C-terminal cystathionine b-synthase these transporters have ‘extra’ helices, the PABS domain is (CBS, IPR000644) domains (Group 7.) are found primarily unusually located in the periplasm. An interesting alterna- in Actinobacteria, but also a handful of Firmicutes, a-pro- tive explanation could be that the PABS domain in this teobacteria, and even archaea. The presence of two CBS group functions as a sensory domain; the transporter could domains is consistent with the usual arrangement found in then be modulated by PABS substrate binding, or simply CBS-containing proteins [35]. These domains are able to serve to transmit the signal into the cytoplasm. bind nucleotides, typically ATP, allowing regulation of enzymatic activity and/or oligomerisation state in relation MFS can also be fused to one (Group 5a.) or two (Group to the internal concentration of ATP (and potentially also 5b.) cyclic nucleotide binding domains (CNBD, inhibitory nucleotides, such as GTP in eukaryotic IMPDH IPR000595). Some examples contain an un-annotated enzymes) [36]. Two homologous CBS-containing MFS region with an Armadillo-type fold signature between the transporters, Bifidobacterium breve BBr_0838 and Bifido- MFS domain and CNBD. These represent HEAT repeat- bacterium longum BL0920, have been characterised. Both containing nucleotide transporters (NTTs) [28] involved in were shown to be bile salt efflux pumps involved in bile uptake of ATP from the environment; NTT proteins are resistance, although the role of the CBS domains was not part of the AAA family, which is a distant member of the investigated [37, 38]. CBS domains have been identified in MFS superfamily [26]. The CNBD is proposed to modulate other transporters, where they play a regulatory role. A transporter activity in response to levels of cAMP, which is group of osmoprotectant ABC transporters, found in B. sub- produced from ATP. Of the other MFS-CNBD fusions, the tilis and Lactococcus lactis as well as in pseudomonads, relies majority are from Actinobacteria such as Nocardioides, on CBS domains for osmoregulation [39]. Additionally, in Microbacterium and Agromyces; the two-CNBD variant is the Mg2+ channel MgtE, ATP binding to the CBS domain found only in Mycobacterium triplex, Mycobacterium lenti- seems to increase the sensitivity of the channel to internal flavum and Mycobacterium iranicum. Another architecture Mg2+, promoting channel closure at physiological Mg2+ lev- (Group 5c.) contains a CNBD and a patatin-like phospholi- els [40]. In the human CNNM2 transporter, CBS domains pase (PLP) domain (IPR002641) at the C-terminus. This bind ATP and Mg2+ and are required for Mg2+ efflux [41]. architecture is primarily found in mycobacteria, although some are found in Nocardiodes and Knoellia isolates. One Finally, there are 122 examples (Group 8.) of MFS trans- example has been identified in Mycobacterium. tuberculosis porters fused to UspA (IPR006016) domains. Almost all of [29], but no assessment has been made of its function. these are derived from Actinobacteria, primarily Mycobacte- rium and Streptomyces species, although a few are found in Two architectures contain PAS (Per/Arnt/Sim, IPR000014) proteobacteria and archaea. To our knowledge, no MFS- domains, one (Group 6a.) with an associated PAS-associ- UspA fusion has been identified; while some fusions to APC ated C-terminal domain (IPR000700), and one without transporters have been identified [6], the role of the UspA (Group 6b.). Almost all are found in the order Bacillales, domains in these proteins is unknown. Indeed, while Usp primarily in the families Bacillaceae and Paenibacillaceae. proteins are known to be involved in stress response and Many are annotated as nitrate/nitrite transporters. The MFS tolerance, the underlying mechanism is not fully understood domains of these proteins are homologous to the B. subtilis [42]. Thus, it is difficult to predict the role of these fusions. nitrite efflux protein NarK; the closest is Bacillus vireti NarK (UniProt: W1SEL7), the MFS domain of which shares 60 % Almost all of the fusions identified according to our param- identity with Bacillus subtilis NarK. In B. subtilis, NarK pre- eters are C-terminal, with the exception of the internal vents the toxic accumulation of nitrite, which is produced PABS domain-MFS fusion, and the MFS-MFS fusions of from nitrate during denitrification under anaerobic Group 1, which can be considered as both N- and

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C-terminal. Apart from these, the most prevalent non-C- examples. An MFS_3-thymidylate kinase fusion, with 47 terminal fusion to a 12TM MFS in bacteria is a protein examples in Pfam, is not included as InterPro does not rec- kinase (IPR000719) fusion found in Streptomyces species. ognise thymidylate kinase as a ‘domain’. Searching for thy- This architecture has 89 examples, although many are fun- midylate kinase (IPR018094) ‘family’ architectures revealed gal. The rarity of N-terminal fusions suggests that the C-ter- 553 MFS transporters annotated as single MFS domains. minus is the most permissive site for addition of new We also examined the Protein Data Bank [47] for crystal domains to MFS proteins in bacteria. Conversely, there are structures of MFS fusions. However, we were unable to find N-terminal fusions with wider distributions in eukaryotes. structures for any of our identified fusions. This makes it Four hundred and forty examples are available of SPX difficult to analyse other properties of the fused domains domain-MFS fusions; these are found in plants and mediate such as surface charge. The only structure available of a phosphate transport between the vacuole and cytoplasm fused MFS protein is for the E. coli transporter YajR [48]; [43, 44]. Another architecture with 158 examples, contain- however, the YAM domain of this protein is not considered ‘ ’ ing N-terminal glycosyl hydrolase and starch synthase as a domain in InterPro, and hence it has not been domains, appears to represent AGS-1, a glucan synthase included in our analysis. Future analyses would benefit by involved in aerial hyphae and conidia formation in fungi integrating analyses from multiple databases, including [45]. The fusion of domains seems to be highly permissive structural databases, in order to further develop and refine with regards to size, with fusions ranging from around 30– the dataset. 40 amino acids for the Osmo_CC domain fusions, up to The primary aim of this research was specifically to assess around 600 amino acids for the PlsC_ACS fusions, and the suitability of using InterPro for identification of trans- even to around 1000 amino acids for the HEAT-containing porter fusions, rather than the identification of novel MFS_CNBD_CNBD fusions. fusions, and many of the fusions that we have identified sys- In order to obtain a greater understanding of linker struc- tematically have been previously characterised. Neverthe- ture, we analysed a small selection of linkers from Groups 2 less, we have also been able to identify previously and 7 (Tables S2 and S3). In Group 2, the analysed linkers undescribed fusions, such as the MFS-PABS and MFS-PAS were typically around 35 amino acids in length, although fusions, and identifying the roles of these fusions could be an interesting avenue for further study. some were as short as 29 amino acids, and one example was as long as 61 amino acids. In the g-proteobacterial examples, these linkers typically consisted of a conserved a-helix Funding information and area of extended secondary structure, followed by a The present work was funded by a BBSRC IB Catalyst Grant Project poorly conserved unstructured region (Table S2, Fig. S1). DETOX (BB/N01040X/1) to BJW and GHT. The linkers of Group 7 (Table S3) show greater variability, Acknowledgements with some being around 50 amino acids long, and others We would like to acknowledge Dr Vicki Springthorpe from the Univer- being around 15 amino acids. Secondary structure in these sity of York for her invaluable assistance with bioinformatics, and Dr linkers is less apparent, although the linkers from Bifidobac- Alex Bateman from EBI for helpful discussions on the architecture of terium proteins show an a-helical region towards the C-ter- InterPro. minal end of the linker. Conflicts of interest The Pfam database [46] also allows the identification of The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict domain architectures, and it is thus possible to carry out a of interest. similar analysis using the MFS clan (CL0015). Pfam divides the MFS clan into 24 members, which makes the dataset References 1. Wheeldon I, Minteer SD, Banta S, Barton SC, Atanassov P et al. more complex, but allows some fusions to be examined in Substrate channelling as an approach to cascade reactions. Nat more detail; for example, while InterPro identifies 5286 Chem 2016;8:299–309. MFS-MFS fusions, Pfam identifies 33368 MFS_1-MFS_1 2. Laskowski RA, Gerick F, Thornton JM. The structural basis of fusions, 4469 Sugar_tr_Sugar_tr fusions, 1480 PTR2_PTR2 allosteric regulation in proteins. FEBS Lett 2009;583:1692–1698. fusions, and several others. Other common examples in 3. Marcotte EM, Pellegrini M, Ng HL, Rice DW, Yeates TO et al. InterPro have fewer hits in Pfam. For example, MFS_PlsC, Detecting protein function and protein-protein interactions from genome sequences. Science 1999;285:751–753. with 2289 representatives in InterPro, has only 318 in Pfam; 4. Liu C, Chin JX, Lee DY. SynLinker: an integrated system for MFS-PlsC-ACS has only 147 representatives in Pfam but designing linkers and synthetic fusion proteins. Bioinformatics has 962 in InterPro. Thus, it may be harder to distinguish 2015;31:3700–3702. less common fusions from false positives using Pfam. We 5. Alberts B, Johnson A, Lewis J. Carrier proteins and active mem- also identified fusions that were overlooked in the InterPro brane transport. Molecular Biology of the Cell. Garland Science. pp. analysis; for example, the MFS domain of the MFS_2-phos- 1–8. phoenolpyruvate-dependent sugar phosphotransferase sys- 6. Barabote RD, Tamang DG, Abeywardena SN, Fallah NS, Fu JY, Jyc F et al. Extra domains in secondary transport carriers and tem, EIIA 1 fusion, with 79 examples in Pfam, is annotated channel proteins. Biochim Biophys Acta 2006;1758:1557–1579. as a sodium:galactoside symporter (IPR001927) by InterPro 7. Harvat EM, Zhang YM, Tran CV, Zhang Z, Frank MW et al. Lyso- and was not included in our analysis despite having 479 phospholipid flipping across the Escherichia coli inner membrane

Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11423 On: Thu, 23 May 2019 23:34:27 Willson et al., Microbiology 2019;165:419–424

catalyzed by a transporter (LplT) belonging to the major facilitator 29. Banerji S, Flieger A. Patatin-like proteins: a new family of lipolytic superfamily. J Biol Chem 2005;280:12028–12034. enzymes present in bacteria? Microbiology 2004;150:522–525. 8. Yan N. Structural advances for the major facilitator superfamily 30. Hoffmann T, Frankenberg N, Marino M, Jahn D. Ammonification (MFS) transporters. Trends Biochem Sci 2013;38:151–159. in Bacillus subtilis utilizing dissimilatory nitrite reductase is 9. Hinchliffe P, Greene NP, Paterson NG, Crow A, Hughes C et al. dependent on resDE. J Bacteriol 1998;180:186–189. Structure of the periplasmic adaptor protein from a major facilita- 31. Cruz Ramos H, Boursier L, Moszer I, Kunst F, Danchin A et al. tor superfamily (MFS) multidrug efflux pump. FEBS Lett 2014;588: Anaerobic transcription activation in Bacillus subtilis: identification 3147–3153. of distinct FNR-dependent and -independent regulatory mecha- 10. Pao SS, Paulsen IT, Saier MH. Major facilitator superfamily. nisms. EMBO J 1995;14:5984–5994. Microbiol Mol Biol Rev 1998;62:1–34. 32. Verbaendert I, de Vos P, Boon N, Heylen K. Denitrification in 11. Apweiler R, Attwood TK, Bairoch A, Bateman A, Birney E et al. Gram-positive bacteria: an underexplored trait. Biochem Soc Trans The InterPro database, an integrated documentation resource for 2011;39:254–258. protein families, domains and functional sites. Nucleic Acids Res 33. Taylor BL, Zhulin IB. PAS domains: internal sensors of oxygen, 2001;29:37–40. redox potential, and light. Microbiol Mol Biol Rev 1999;63:479–506. et al. 12. Finn RD, Attwood TK, Babbitt PC, Bateman A, Bork P Inter- 34. Martinez-Argudo I, Little R, Shearer N, Johnson P, Dixon R. The Pro in 2017-beyond protein family and domain annotations. NifL-NifA System: a multidomain transcriptional regulatory complex Nucleic Acids Res 2017;45:D190–D199. that integrates environmental signals. J Bacteriol 2004;186:601– 13. Bateman A, Martin MJ, O’Donovan C, Magrane M, Alpi E et al. Uni- 610. Prot: the universal protein knowledgebase. Nucleic Acids Res 35. Baykov AA, Tuominen HK, Lahti R. The CBS domain: a protein 2017;45:D158–D169. module with an emerging prominent role in regulation. ACS Chem 14. NCBI Resource Coordinators. Database resources of the National Biol 2011;6:1156–1163. Center for Biotechnology Information. Nucleic Acids Res 2016;44:D7– 36. Anashkin VA, Baykov AA, Lahti R. Enzymes regulated via cysta- D19. thionine b-synthase domains. Biochem 2017;82:1079–1087. 15. Wood NJ, Alizadeh T, Richardson DJ, Ferguson SJ, Moir JW. Two 37. Ruiz L, O’Connell-Motherway M, Zomer A, de Los Reyes-Gavilan CG, domains of a dual-function NarK protein are required for nitrate et al. uptake, the first step of denitrification in Paracoccus pantotro- Margolles A A bile-inducible membrane protein mediates bifi- phus. Mol Microbiol 2002;44:157–170. dobacterial bile resistance. Microb Biotechnol 2012;5:523–535. 38. Gueimonde M, Garrigues C, van Sinderen D, de Los Reyes- 16. Goddard AD, Moir JW, Richardson DJ, Ferguson SJ. Interdepen- dence of two NarK domains in a fused nitrate/nitrite transporter. Gavilan CG, Margolles A. Bile-inducible efflux transporter from Mol Microbiol 2008;70:667–681. Bifidobacterium longum NCC2705, conferring bile resistance. Appl Environ Microbiol 2009;75:3153–3160. 17. MacMillan SV, Alexander DA, Culham DE, Kunte HJ, Marshall EV et al. The ion coupling and organic substrate specificities of osmo- 39. Chen C, Beattie GA. Characterization of the osmoprotectant trans- regulatory transporter ProP in Escherichia coli. Biochim Biophys porter OpuC from Pseudomonas syringae and demonstration that Acta 1999;1420:30–44. cystathionine-beta-synthase domains are required for its osmo- regulatory function. J Bacteriol 2007;189:6901–6912. 18. Gouesbet G, Trautwetter A, Bonnassie S, Wu LF, Blanco C. Char- acterization of the Erwinia chrysanthemi osmoprotectant trans- 40. Tomita A, Zhang M, Jin F, Zhuang W, Takeda H et al. ATP-depen- porter gene ousA. J Bacteriol 1996;178:447–455. dent modulation of MgtE in Mg2+ homeostasis. Nat Commun 2017; 19. Culham DE, Tripet B, Racher KI, Voegele RT, Hodges RS et al. The 8:148. role of the carboxyl terminal alpha-helical coiled-coil domain in 41. Hirata Y, Funato Y, Takano Y, Miki H. Mg2+-dependent interactions osmosensing by transporter ProP of Escherichia coli. J Mol of ATP with the cystathionine-b-synthase (CBS) domains of a mag- Recognit 2000;13:309–322. nesium transporter. J Biol Chem 2014;289:14731–14739. 20. Tsatskis Y, Khambati J, Dobson M, Bogdanov M, Dowhan W et al. 42. Kvint K, Nachin L, Diez A, Nyström T. The bacterial universal The osmotic activation of transporter ProP is tuned by both its C- stress protein: function and regulation. Curr Opin Microbiol 2003;6: terminal coiled-coil and osmotically induced changes in phospho- 140–145. lipid composition. J Biol Chem 2005;280:41387–41394. 43. Wang C, Huang W, Ying Y, Li S, Secco D et al. Functional charac- 21. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting terization of the rice SPX-MFS family reveals a key role of OsSPX- transmembrane protein topology with a hidden Markov model: MFS1 in controlling phosphate homeostasis in leaves. New Phytol application to complete genomes. J Mol Biol 2001;305:567–580. 2012;196:139–148. 22. Saier MH, Reddy VS, Tsu BV, Ahmed MS, Li C et al. The trans- 44. Liu TY, Huang TK, Yang SY, Hong YT, Huang SM et al. Identifica- porter classification database (TCDB): recent advances. Nucleic tion of plant vacuolar transporters mediating phosphate storage. Acids Res 2016;44:D372–D379. Nat Commun 2016;7:11095. 23. Chu AJ. Characterising Two Genomic Islands Involved in Metabolism 45. Fu C, Tanaka A, Free SJ. Neurospora crassa 1,3-a-glucan syn- in Neisseria meningitidis. University of York; 2017. thase, AGS-1, is required for cell wall biosynthesis during mac- 24. Zen KH, McKenna E, Bibi E, Hardy D, Kaback HR. Expression of roconidia development. Microbiology 2014;160:1618–1627. lactose permease in contiguous fragments as a probe for mem- 46. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J et al. The brane-spanning domains. Biochemistry 1994;33:8198–8206. Pfam protein families database: towards a more sustainable 25. Zomot E, Yardeni EH, Vargiu AV, Tam HK, Malloci G et al. A new future. Nucleic Acids Res 2016;44:D279–D285. critical conformational determinant of multidrug efflux by an MFS 47. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN et al. transporter. J Mol Biol 2018;430:1368–1385. The protein data bank. Nucleic Acids Res 2000;28:235–242. 26. Reddy VS, Shlykov MA, Castillo R, Sun EI, Saier MH. The major facili- 48. Jiang D, Zhao Y, Wang X, Fan J, Heng J et al. Structure of the YajR – tator superfamily (MFS) revisited. FEBS J 2012;279:2022 2035. transporter suggests a transport mechanism based on the con- 27. Minguet EG, vera-Sirera F, Marina A, Carbonell J, Blazquez MA. served motif A. Proc Natl Acad Sci USA 2013;110:14664–14669. Evolutionary diversification in polyamine biosynthesis. Mol Biol Evol 2008;25:2119–2128. 28. Major P, Embley TM, Williams TA. Phylogenetic diversity of NTT nucleotide transport proteins in free-living and parasitic bacteria and eukaryotes. Genome Biol Evol 2017;9:480–487. Edited by: S. Gebhard and C. Dahl

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