3/29/2023 - Open Access

Sequence analyses of a lipoprotein conserved with bacterial actins
responsible for swimming motility of wall-less helical Spiroplasma
Daichi Takahashi1, Makoto Miyata1,2§
1Graduate School of Science, Osaka Metropolitan University, Osaka, Japan.
2The OMU Advanced Research Center for Natural Science and Technology, Osaka Metropolitan University, Osaka, Japan.
§To whom correspondence should be addressed: miyata@omu.ac.jp

Abstract
Spiroplasma is a genus of pathogenic or commensal cell-wall-deficient helical bacterium. Spiroplasma-specific protein fibril
and five classes of bacterial actins, MreB1–5, are involved in a helical ribbon structure responsible for helical-cell morphology
and swimming motility. A gene for a hypothetical protein—SPE_1229, 7th protein—has been found in the locus coding
mreBs. In this study, we characterized the 7th protein using in silico methods and found that it could be a lipoprotein whosegene is encoded downstream of mreB3 and conserved in a clade of Spiroplasma.

Figure 1. Characterization of the 7th protein.:(A) (Left) Maximum likelihood phylogeny of 16S rRNA of Spiroplasma. Bacillus subtilis 16S rRNA sequence is used as theoutgroup. Species belonging to Apis and Citri clades are highlighted using cyan and magenta boxes, respectively. Bootstrap
support values were estimated from 500 alignment samples and indicated on each node. The scale bar is in units of thenucleotide substitution numbers per site. (Right) List summarizing the presence of genes for the 7th protein, Lgt, Lsp, and Lnt.

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The check and cross symbols indicate whether the corresponding gene is present or absent, respectively, on the genome.
Column G.O. indicates the patterns of the gene orders from mreB2 to mreB5 shown in panel B. Gene organization pattern of
Spiroplasma sp. Moj could not be determined owing to the insufficient length of the reported contig sequence including the
7th gene, denoted using “?” in the G.O. column. Three classifications in Citri clade (Mirum, Chrysopicola, and Citri) areindicated on the right of the list. (B) Four patterns of gene orders from mreB2 to mreB5 found in Citri clade species. The
nucleotide numbers of the mreB2 initiation and mreB5 termination points on the genomes are indicated below thecorresponding sites. (C) Amino acid sequence of the 7th protein of S. melliferum. Each amino acid is colored based on the four
conservation levels (purple for identical, magenta for strongly similar, cyan for weakly similar, and green for diverse)
estimated by the multiple alignment of 14 amino acid sequences of the 7th protein. The signal sequence region is indicatedusing a solid line. The lipobox region is indicated using a red box with its WebLogo on the sequence. The regions for the αhelix and β-strand predicted by AlphaFold2 are indicated using a magenta cylinder and yellow arrow, respectively. (D) Ribbon
representation of S. melliferum 7th protein structure predicted by AlphaFold2. The first 24 amino acids at the N-terminus
including the signal sequence and lipobox are excluded from the model. Each amino acid is colored by the predicted preresidue confidence score (LDDT). (E) Structural comparison among the 7th proteins of S. melliferum (beige), S. eriocheiris(cyan), and S. chrysopicola (magenta).

Description
Spiroplasma belongs to the class Mollicutes. Spiroplasma are pathogenic or commensal to plants and arthropods and possess awall-less helical cell morphology (Cole et al., 1973; Daniels et al., 1973; Paredes et al., 2015; Tsai et al., 2018). They show
characteristic swimming motility, in which cells are driven by the continuous switching of cell helicities (Shaevitz et al., 2005;
Wada and Netz 2009). Compared to the conventional types of bacterial motility such as flagellar and pili motilities, theswimming force of Spiroplasma is generated by conformational changes in an intracellular ribbon structure composed of fibril,a cytoskeletal protein specific to Spiroplasma, and bacterial actin MreB (Kürner et al., 2005; Trachtenberg et al., 2008; Liu etal., 2017; Miyata et al., 2020; Sasajima et al., 2022). Spiroplasma possesses five classes of MreBs (MreB1–5), which are
divided into three functional groups based on sequence similarity: MreB1&4, MreB2&5, and MreB3. Previous experiments on
Spiroplasma MreB expression in Escherichia coli cells and in vitro polymerization suggested the following roles of these
MreBs: MreB1 and/or MreB4 form a static backbone interacting with fibril filaments; MreB2 and/or MreB5 change the ribbon
forms by polymerization dynamics, and MreB3 forms static filaments to anchor MreB1 and/or MreB4 onto the cell membrane(Masson et al., 2021; Takahashi et al., 2022). Previous studies showed that these genes are involved in the swimming motility(Harne et al., 2020a; Harne et al., 2020b; Kiyama et al., 2022; Lartigue et al., 2022). The five classes of MreBs and fibril areconserved in the Spiroplasma genus (Ku et al., 2014; Takahashi et al., 2020), suggesting that the swimming mechanism isconserved in Spiroplasma, although previous studies on swimming in this genus have focused mostly on four species (S.
melliferum, S. citri, S. eriocheiris, and S. poulsonii) (Shaevitz et al., 2005; Wada and Netz 2009; Liu et al., 2017; Boudet et al.,2018; Harne et al., 2020a; Nakane et al., 2020; Masson et al., 2021; Kiyama et al., 2022; Lartigue et al., 2022; Sasajima et al.,2022). The hypothetical gene SPE_1229 was found to be encoded by a locus encoding mreBs. Although poorly characterized,
SPE_1229 and its homologs are found in many Spiroplasma species with a corresponding position (Harne et al., 2020a;
Kiyama et al., 2022). In this study, we analyzed SPE_1229 using in silico methods and in the following sections, we havetentatively named SPE_1229 and its gene as “7th protein” and “7th gene,” respectively, because the gene position is adjacent
to one of the “six” cytoskeletal proteins responsible for Spiroplasma swimming.
We first performed a BLAST search to collect amino acid sequences of the 7th protein (Extended Data 1) and searched for its
distribution (Figure 1A). Homologs from species other than Spiroplasma were not found, suggesting that the 7th protein is
specific to Spiroplasma. The Spiroplasma genus is divided into two clades based on phylogenetic relationships: Apis and CitriChrysopicola-Mirum (Citri) (Ku et al., 2014; Paredes et al., 2015; Tsai et al., 2018; Takahashi et al., 2020). The 7th protein isconserved in the Citri clade but not in the Apis clade. Later, we examined the order of genes coding for the 7th protein and thecytoskeletal proteins related to Spiroplasma swimming, that is, fibril and the five classes of MreBs. The 7th gene is always
found in a locus that includes mreB2, mreB3, mreB4, and mreB5. The gene order from mreB2 to mreB5 was classified into four
patterns (Figure 1B). In the genomes of 8 out of 10 species in the same clade as S. citri (species labeled as Citri in Figure 1A),
mreB2, mreB3, 7th gene, mreB4, and mreB5 were tandemly encoded in the order shown (Figure 1B-3). The S. melliferum
genome encoded another hypothetical gene immediately before mreB3 (Figure 1B-4). In the genomes of three species (S.
eriocheiris, S. mirum, and S. atrichopogonis), alkA (at the minus strand), adaA, and adaB all of which are responsible fordemethylations of DNA (Sedgwick and Lindahl 2002) are encoded between mreB2 and mreB3 (Figure 1B-1). S. chrysopicola
and S. syrphidicola encode a gene for NADH-flavin deaminase and adaA between the 7th gene and mreB4 (Figure 1B-2).
Although there is diversity in gene orders at the loci, the 7th gene is always encoded downstream of mreB3 (Figure 1B).

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To characterize the 7th protein, its amino acid sequence was analyzed using Phobius, a prediction tool for membrane proteins(Käll et al., 2004). All the 7th protein sequences were projected to possess signal sequences for extracellular secretion on the
N-terminal 18–23 amino acid residues (Figure 1C, Extended Data 1). At the 21st to 25th position, around the end region of thesignal sequence, the [V/L]-[V/T]-S-C-[S/L] motif was conserved in 13 out of 14 sequences of the 7th protein (Figure 1C redbox). This motif matches the consensus sequence of the lipobox ([L/V/I]-[A/S/T/V/I]-[G/A/S]-C-X) conserved at the end of asignal sequence in bacterial prolipoproteins (Kovacs-Simon et al., 2011). The structure of the 7th protein predicted by
AlphaFold2 (Mirdita et al., 2022) excluding the signal sequence, showed a globular fold enriched with α-helices (Figure 1D).
The globular fold of the 7th protein is well conserved among those of phylogenetically distant species (S. melliferum, S.
eriocheiris, and S. chrysopicola) (Figure 1A and E). Altogether, we predict that the 7th protein is a lipoprotein that takes theglobular fold on the extracellular side of the membrane. To predict the lipid modifications of the 7th protein, we examined the
distribution of genes encoding three key enzymes responsible for the maturation of bacterial lipoproteins: Lgt, which adds a
diacylglycerol group on the cysteine residue in the lipobox of a prolipoprotein; Lsp, which cleaves off the signal sequence ofthe prolipoprotein; and Lnt, acylating the amino group of the N-terminus of the prolipoprotein to mature up to the lipoprotein(Kovacs-Simon et al., 2011; Buddelmeijer 2015). Lgt and Lsp were conserved in Spiroplasma, whereas Lnt was absent as wellas other Mollicutes species (Figure 1A) (Jan et al., 1995; Serebryakova et al., 2011; Buddelmeijer 2015), suggesting that the
7th protein undergoes the diacylglycerol modification and the signal sequence cleavage. Regarding N-acylation, further studies
are needed for conclusion because many lipoproteins are reported for N-acylation in Mollicutes species lacking the Lnt gene
(Serebryakova et al., 2011), including S. melliferum (Le Hénaff and Fontenelle 2000).
In this study, we predicted that the 7th protein is a lipoprotein whose gene is encoded downstream of mreB3 in the Citri clade.
Bacterial genes encoded closely in the same locus are often functionally related (Cohen et al., 2019; Toyonaga et al., 2021;
Megrian et al., 2022; Yamamoto et al., 2023). Our analyses suggest that the 7th protein localizes to the extracellular side,
whereas MreBs localize to the cells (Kürner et al., 2005; Trachtenberg et al., 2008; Liu et al., 2017; Sasajima et al., 2022).
These suggest that the 7th protein and MreBs do not interact with each other for their functions. However, the position of the
7th gene downstream of mreB3 was conserved in every Citri clade species. Possibly this protein gives positive effects on Citriclade's survival through swimming, because Mollicutes genomes code for limited number of genes (Andersson and Kurland
1998). We also found that the 7th protein was conserved in the Citri clade species but not in the Apis clade (Figure 1A),
suggesting differences in survival strategies between these clades.

Methods
Sequences of the 7th protein were obtained by a BLAST search as the reference for S. eriocheiris (WP_047791952.1) with an
E-value threshold of 0.05, on August 22, 2022. Sequences that were duplicated and misannotated were excluded from thesequence set for analysis (WP_004028916.1, CAK98229.1, and WP_252157057.1). Thus, the sequence set contained 14
amino acid sequences of the 7th protein (Extended Data 1). A phylogenetic tree of 16S rRNA sequences was constructed using
MEGA-X, as previously described (Kumar et al., 2018; Takahashi et al., 2020). The sequence of the 7th protein from
Spiroplasma endosymbiont of Phyllotreta cruciferae, whose 16S rRNA sequence was not reported, was excluded from thephylogenetic and gene organization analyses. Signal peptide regions were predicted using Phobius (Käll et al., 2004). The
conservation level of each amino acid residue was evaluated by multiple alignment of the 7th protein sequence using Clustal
Omega (Sievers and Higgins 2018). The amino acid conservation of the lipobox is further evaluated by WebLogo (Crooks etal., 2004). The structures of the 7th proteins were predicted from their full-length sequences using the CoLab version of
AlphaFold2 (Mirdita et al., 2022), and structural visualizations and removal of the signal sequence were performed using
UCSF Chimera ver. 1.13.1 (Meng et al., 2006).
Acknowledgements: We thank Hana Kiyama at the Graduate School of Science, Osaka Metropolitan University, Japan, forthe helpful discussions.

Extended Data
Description: The accession IDs used in this study and the summary of the analyses are shown in an Excel file containing two
sets of extended data. Extended Data 1: Accession IDs and the source species of 7th proteins obtained by the BLAST searchand their predicted lengths of signal sequences. Extended Data 2: Accession IDs and the source species of the genome orcontig DNA sequences refer to gene orders from mreB2 to mreB5. Resource Type: Dataset. File: ExtendedData_202212
07.xlsx. DOI: 10.22002/ba1pp-vk861
Description: PDB file predicted by AlphaFold2. Resource Type: Dataset. File: melliferum_009b2_unrelaxed_rank_1_model_4.pdb. DOI: 10.22002/zx8y9-4b517

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Funding: This study was supported by JST CREST (grant number JPMJCR19S5 to MM). DT was a recipient of the Research
Fellowship of the Japan Society for the Promotion of Science (22J10345).
Author Contributions: Daichi Takahashi: conceptualization, data curation, formal analysis, investigation, funding
acquisition, validation, visualization, writing - original draft, writing - review editing. Makoto Miyata: funding acquisition,
project administration, supervision, validation, writing - original draft, writing - review editing.
Reviewed By: Sofya Lushchekina, Laure Beven
History: Received December 13, 2022 Revision Received February 7, 2023 Accepted March 9, 2023 Published Online
March 15, 2023 Indexed April 5, 2023
Copyright: © 2023 by the authors. This is an open-access article distributed under the terms of the Creative Commons
Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.

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Citation: Takahashi, D; Miyata, M (2023). Sequence analyses of a lipoprotein conserved with bacterial actins responsible forswimming motility of wall-less helical Spiroplasma. microPublication Biology. 10.17912/micropub.biology.000713