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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Microbiol.</journal-id>
<journal-title>Frontiers in Microbiology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Microbiol.</abbrev-journal-title>
<issn pub-type="epub">1664-302X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fmicb.2023.1189877</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Microbiology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Mutations identified in engineered <italic>Escherichia coli</italic> with a reduced genome</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Kotaka</surname>
<given-names>Yuto</given-names>
</name>
<xref rid="aff1" ref-type="aff"><sup>1</sup></xref>
<xref rid="aff2" ref-type="aff"><sup>2</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/2241064/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Hashimoto</surname>
<given-names>Masayuki</given-names>
</name>
<xref rid="aff3" ref-type="aff"><sup>3</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/858475/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Lee</surname>
<given-names>Ken-ichi</given-names>
</name>
<xref rid="aff2" ref-type="aff"><sup>2</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/191525/overview"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Kato</surname>
<given-names>Jun-ichi</given-names>
</name>
<xref rid="aff1" ref-type="aff"><sup>1</sup></xref>
<xref rid="c001" ref-type="corresp"><sup>&#x002A;</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/2251265/overview"/>
</contrib>
</contrib-group>
<aff id="aff1"><sup>1</sup><institution>Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University</institution>, <addr-line>Tokyo</addr-line>, <country>Japan</country></aff>
<aff id="aff2"><sup>2</sup><institution>Department of Bacteriology I, National Institute of Infectious Diseases</institution>, <addr-line>Tokyo</addr-line>, <country>Japan</country></aff>
<aff id="aff3"><sup>3</sup><institution>Institute of Molecular Medicine, College of Medicine, National Cheng Kung University</institution>, <addr-line>Tainan</addr-line>, <country>Taiwan</country></aff>
<author-notes>
<fn id="fn0001" fn-type="edited-by"><p>Edited by: Mamoru Yamada, Yamaguchi University, Japan</p></fn>
<fn id="fn0002" fn-type="edited-by"><p>Reviewed by: Michihisa Maeda, Meiji University, Japan; Toshihiko Kishimoto, Toho University, Japan</p></fn>
<corresp id="c001">&#x002A;Correspondence: Jun-ichi Kato, <email>jkato@tmu.ac.jp</email></corresp>
</author-notes>
<pub-date pub-type="epub">
<day>25</day>
<month>05</month>
<year>2023</year>
</pub-date>
<pub-date pub-type="collection">
<year>2023</year>
</pub-date>
<volume>14</volume>
<elocation-id>1189877</elocation-id>
<history>
<date date-type="received">
<day>20</day>
<month>03</month>
<year>2023</year>
</date>
<date date-type="accepted">
<day>08</day>
<month>05</month>
<year>2023</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#x00A9; 2023 Kotaka, Hashimoto, Lee and Kato.</copyright-statement>
<copyright-year>2023</copyright-year>
<copyright-holder>Kotaka, Hashimoto, Lee and Kato</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/"><p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>Characterizing genes that regulate cell growth and survival in model organisms is important for understanding higher organisms. Construction of strains harboring large deletions in the genome can provide insights into the genetic basis of cell growth compared with only studying wild-type strains. We have constructed a series of genome-reduced strains with deletions spanning approximately 38.9% of the <italic>E. coli</italic> chromosome. Strains were constructed by combining large deletions in chromosomal regions encoding nonessential gene groups. We also isolated strains &#x0394;33b and &#x0394;37c, whose growth was partially restored by adaptive laboratory evolution (ALE). Genome sequencing of nine strains, including those selected following ALE, identified the presence of several Single Nucleotide Variants (SNVs), insertions, deletions, and inversions. In addition to multiple SNVs, two insertions were identified in ALE strain &#x0394;33b. The first was an insertion at the promoter region of <italic>pntA</italic>, which increased cognate gene expression. The second was an insertion sequence (IS) present in <italic>sibE</italic>, encoding the antitoxin in a toxin-antitoxin system, which decreased expression of <italic>sibE</italic>. 5 strains of &#x0394;37c independently isolated following ALE harboring multiple SNVs and genetic rearrangements. Interestingly, a SNV was identified in the promoter region of <italic>hcaT</italic> in all five strains, which increased <italic>hcaT</italic> expression and, we predict, rescued the attenuated &#x0394;37b growth. Experiments using defined deletion mutants suggested that <italic>hcaT</italic> encodes a 3-phenylpropionate transporter protein and is involved in survival during stationary phase under oxidative stress. This study is the first to document accumulation of mutations during construction of genome-reduced strains. Furthermore, isolation and analysis of strains derived from ALE in which the growth defect mediated by large chromosomal deletions was rescued identified novel genes involved in cell survival.</p>
</abstract>
<kwd-group>
<kwd><italic>Escherichia coli</italic></kwd>
<kwd>genome-reduced strain</kwd>
<kwd>adaptive laboratory evolution</kwd>
<kwd>genome alteration</kwd>
<kwd>3-phenylpropionate</kwd>
<kwd>oxidative stress</kwd>
</kwd-group>
<counts>
<fig-count count="6"/>
<table-count count="2"/>
<equation-count count="0"/>
<ref-count count="38"/>
<page-count count="9"/>
<word-count count="5981"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-at-acceptance</meta-name>
<meta-value>Evolutionary and Genomic Microbiology</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="sec1" sec-type="intro">
<label>1.</label>
<title>Introduction</title>
<p>Genome sequencing has facilitated the identification of essential genes and minimal gene sets for model micro-organisms such as yeast, <italic>Bacillus subtilis</italic>, and <italic>Escherichia coli</italic>. <italic>Mycoplasma</italic> encode a small genome, and subsequently all essential genes have been identified from the isolation and analysis of many transposon insertion mutants (<xref ref-type="bibr" rid="ref12">Glass et al., 2006</xref>). A genome-reduced strain of <italic>Mycoplasma</italic> (JCVI-syn3.0) with a 531&#x2009;kb genome approximately 49.2% the size of the wild-type genome was constructed, and the minimum gene set was experimentally characterized (<xref ref-type="bibr" rid="ref16">Hutchison et al., 2016</xref>). This 473 gene set includes essential, quasi-essential, and nonessential genes, but the functions of 149 of these genes remain unknown (<xref ref-type="bibr" rid="ref16">Hutchison et al., 2016</xref>). <italic>E. coli</italic> and <italic>B. subtilis</italic> have larger genomes than <italic>Mycoplasma</italic>, and all essential genes in these species were first identified by generating many gene-disrupted strains (<xref ref-type="bibr" rid="ref2">Baba et al., 2006</xref>; <xref ref-type="bibr" rid="ref21">Kato and Hashimoto, 2007</xref>). Functions of approximately 65.4% of all genes in <italic>E. coli</italic> have been experimentally characterized and those of only about 2.4% have not yet been estimated (<xref ref-type="bibr" rid="ref11">Ghatak et al., 2019</xref>). Furthermore, the functions of almost all essential genes have been experimentally characterized (<xref ref-type="bibr" rid="ref24">Kurata et al., 2015</xref>). Instead, it has not been possible to identify the minimum gene set by combining synthetic genes, as in studies of <italic>Mycoplasma</italic>. Genome-reduced strains have, however, been constructed by combining large-scale chromosomal deletions.</p>
<p>Genome-reduced strains of <italic>E. coli</italic> have been constructed using different strategies by several groups (<xref ref-type="bibr" rid="ref25">Kurokawa and Ying, 2020</xref>). Blattner et al. compared the genomes of several <italic>E. coli</italic> strains with the goal of deleting genes that were introduced during evolution. Strain MDS43 was first constructed and harbors a deletion of approximately 15.27% of the wild-type genome (<xref ref-type="bibr" rid="ref31">P&#x00F3;sfai et al., 2006</xref>). Strain MDS69 was later constructed by deleting foreign gene clusters identified through comparison of additional genomes and harbors deletion of 20.3% of the wild-type genome (<xref ref-type="bibr" rid="ref20">Karcagi et al., 2016</xref>). Ogasawara et al. compared the genomes of <italic>E. coli</italic> with those of Buchnera spp., an insect symbiotic bacterium with a small genome and deleted chromosomal regions found only in <italic>E. coli</italic> to construct strain DGF-298, which lacks 36% of the wild-type genome (<xref ref-type="bibr" rid="ref15">Hirokawa et al., 2013</xref>). An additional study described construction of strain MS56, which lacks 23% of the wild-type genome, by deletion of all ISs, K-islands, flagellar genes, ciliated genes, and lipopolysaccharide genes identified from the <italic>E. coli</italic> data bank (<xref ref-type="bibr" rid="ref30">Park et al., 2014</xref>). <italic>B. subtilis</italic> strain PG38 strain, which harbors deletion of approximately 40% of the wild-type genome, has been described (<xref ref-type="bibr" rid="ref26">Michalik et al., 2021</xref>).</p>
<p>We have previously constructed strains with global deletion mutations spanning the entire <italic>E. coli</italic> chromosome with the aim of identifying trans- and cis-acting genetic information essential for growth (<xref ref-type="bibr" rid="ref21">Kato and Hashimoto, 2007</xref>). We subsequently identified that <italic>oriC</italic> is the only unique cis-acting genetic information (<xref ref-type="bibr" rid="ref21">Kato and Hashimoto, 2007</xref>). We have also analyzed individual essential genes with unknown functions leading to identification of DNA topoisomerase IV, which is essential for chromosome segregation; Hda, which is essential for chromosomal replication initiation; and YqgF, which is essential for rRNA processing and so on (<xref ref-type="bibr" rid="ref23">Kato et al., 1990</xref>; <xref ref-type="bibr" rid="ref22">Kato and Katayama, 2001</xref>; <xref ref-type="bibr" rid="ref14">Hashimoto et al., 2005</xref>; <xref ref-type="bibr" rid="ref24">Kurata et al., 2015</xref>). By combining large-scale deletion mutations, we have constructed a series of genome-reduced strains (&#x0394;1&#x2013;&#x0394;16) in which approximately 29.7% of the wild-strain chromosome was deleted and examined cell size, shape, and nucleoid organization (<xref ref-type="bibr" rid="ref14">Hashimoto et al., 2005</xref>). We have also constructed a series of genome-reduced strains (&#x0394;17&#x2013;&#x0394;33a) with approximately 38.9% deletion of the chromosome, examined their resistance to oxidative stress (<xref ref-type="bibr" rid="ref14">Hashimoto et al., 2005</xref>; <xref ref-type="bibr" rid="ref18">Iwadate et al., 2011</xref>), and identified genes involved in growth and survival. We later introduced large-scale chromosomal deletions into a series of genome-reduced strains, and by identifying genes displaying synthetic lethality, we identified a novel gene involved in DNA repair (<xref ref-type="bibr" rid="ref36">Watanabe et al., 2016</xref>). Lastly, through analysis of a series of genome-reduced strains with differential resistances to the redox-cycling drugs menadione, we identified genes involved in oxidative stress resistance (<xref ref-type="bibr" rid="ref17">Iwadate et al., 2017</xref>; <xref ref-type="bibr" rid="ref19">Iwadate and Kato, 2017</xref>).</p>
<p>In this work, we constructed a series of genome-reduced strains (&#x0394;33b-&#x0394;41c) by introducing further deletions into the genome of strain &#x0394;33a. During this process, we isolated strains whose growth rate was partially rescued by ALE. By sequencing nine strains, we clarified mutations commonly occurring during ALE and identified a novel gene involved in cell survival.</p>
</sec>
<sec id="sec2" sec-type="materials|methods">
<label>2.</label>
<title>Materials and methods</title>
<sec id="sec3">
<label>2.1.</label>
<title>Bacterial strains and culture media</title>
<p>All <italic>E. coli</italic> strains described in this study are derivatives of MG1655. Cells were grown in LB medium (1% Bacto tryptone, 0.5% Bacto yeast extract, and 1% NaCl) or Antibiotic Medium 3 (AM3, Becton Dickinson), unless otherwise stated. The approximate composition of AM3 is 1.5&#x2009;gL<sup>&#x2212;1</sup> of beef extract, 1.5&#x2009;gL<sup>&#x2212;1</sup> of yeast extract, 5&#x2009;gL<sup>&#x2212;1</sup> of peptone, 1&#x2009;gL<sup>&#x2212;1</sup> of dextrose, 3.5&#x2009;gL<sup>&#x2212;1</sup> of sodium chloride, 3.68&#x2009;gL<sup>&#x2212;1</sup> of dipotassium phosphate, and 1.32&#x2009;gL<sup>&#x2212;1</sup> of monopotassium phosphate.</p>
</sec>
<sec id="sec4">
<label>2.2.</label>
<title>Construction of genome-reduced strains</title>
<p>Deletion mutants were constructed using the FRT4 system with some modifications (<xref ref-type="bibr" rid="ref18">Iwadate et al., 2011</xref>, <xref ref-type="supplementary-material" rid="SM4">Supplementary Figure S1</xref>). Deletion mutants were constructed by introducing an Ap resistance cassette into the MG1655 <italic>red</italic> strain using the lambda phage red homologous recombination system. DNA fragments flanked by short regions of homology to chromosomal regions (25&#x2013;40&#x2009;bp) were prepared by PCR and introduced into cells by electroporation. Deletions were confirmed by PCR before preparation of P1 phage from the resulting cells. The Ap resistance gene was replaced with a Cm resistance gene by introducing Cm-FRT PCR fragment flanked with 25&#x2013;40&#x2009;bp sequences with homology to the Ap resistance gene. The positive selection marker was removed using a suicide vector encoding flanking sequences of the deleted region, an <italic>rpsL</italic><sup>+</sup> gene for negative selection, and the Ap resistance gene.</p>
</sec>
<sec id="sec5">
<label>2.3.</label>
<title>Isolation of ALE strains</title>
<p>The genome-reduced strain was aerobically cultured in 2&#x2009;mL of AM3 medium until stationary phase. Cultures were subsequently diluted 1/20 with fresh medium, and the process was manually repeated until strains with increased growth were isolated.</p>
</sec>
<sec id="sec6">
<label>2.4.</label>
<title>Cell growth measurement</title>
<p>Genome-reduced strains were inoculated from an overnight culture and grown in AM3 at 37&#x00B0;C with vigorous shaking. Optical densities were measured and generation time calculated.</p>
</sec>
<sec id="sec7">
<label>2.5.</label>
<title>SNVs detection</title>
<p>Genomic DNA of <italic>E. coli</italic> was purified using a DNA extraction Kit (NucleoSpin Microbial DNA). Genomic DNA was enzymatically fragmented, and a DNA library was constructed using the Celero PCR Workflow and an Enzymatic Fragmentation DNA Seq Kit (TECAN) with a bead-based enrichment step to isolate library fragments greater than 400&#x2009;bp. Paired-end sequencing was subsequently performed on the Illumina MiSeq system. Sequencing reads were processed using the CLC Genomics Workbench v10 (CLC Bio). Sequencing reads were trimmed of low-quality reads (limit&#x2009;=&#x2009;0.1), ambiguous nucleotides, and adapter sequences (TruSeq universal and indexed adapter; GCTCTTCCGATCT). Trimmed reads were mapped to reference sequences with the following parameters: Minimum length fraction&#x2009;=&#x2009;0.5, minimum similarity fraction&#x2009;=&#x2009;0.8, match score&#x2009;=&#x2009;1, mismatch cost&#x2009;=&#x2009;2, insertion cost&#x2009;=&#x2009;3, and deletion cost&#x2009;=&#x2009;3. Sequences were considered variants if mutations were present in more than 50% of the mapped reads. The effects of variants were detected using SnpEff (<xref ref-type="bibr" rid="ref6">Cingolani et al., 2012</xref>), and <italic>rrn</italic> variants were removed manually.</p>
</sec>
<sec id="sec8">
<label>2.6.</label>
<title>Construction and annotation of complete genomes</title>
<p>Genomic DNA for long-read sequencing was extracted using a KingFisher Duo Prime (Thermo Fisher Scientific) with the MagMAX DNA Multi-Sample Ultra 2.0 Kit. Sequencing libraries were prepared using a Rapid Barcoding Sequencing Kit (SQK-RBK004, Oxford Nanopore Technologies, Oxford, United Kingdom). A MinION R9.4.1 flow cell (Oxford Nanopore) was used for 48&#x2009;h sequencing. Base calling was performed using the &#x2018;super accurate&#x2019; algorithm. Long- and short-read sequences were subject to hybrid assembly by using Unicycler v.0.4.8 (<xref ref-type="bibr" rid="ref37">Wick et al., 2017</xref>). Annotation of the complete genomes was performed using DFAST (<xref ref-type="bibr" rid="ref34">Tanizawa et al., 2018</xref>) with manual curation.</p>
</sec>
<sec id="sec9">
<label>2.7.</label>
<title>Visualization of genomic changes</title>
<p>Complete genomes from short- and long-read hybrid assemblies were compared by BLAST (<xref ref-type="bibr" rid="ref1">Altschul et al., 1990</xref>). Results were visualized using Kablammo (<xref ref-type="bibr" rid="ref38">Wintersinger and Wasmuth, 2015</xref>).</p>
</sec>
<sec id="sec10">
<label>2.8.</label>
<title>Promoter activity assay</title>
<p>A strain was constructed in which the upstream portion of the gene to be examined was inserted upstream of the <italic>lacZ</italic> gene on the chromosome so that the start codon of the gene exactly matched the start codon of the <italic>lacZ</italic> gene (<xref ref-type="supplementary-material" rid="SM4">Supplementary Figure S2</xref>). Overnight cultures in LB medium were diluted 1/100 in fresh LB with supplements. Cultures were incubated in test tubes for 3&#x2009;h at 37&#x00B0;C with shaking (130&#x2009;r.p.m.). &#x03B2;-galactosidase activities were measured as previously described (<xref ref-type="bibr" rid="ref27">Miller, 1972</xref>).</p>
</sec>
<sec id="sec11">
<label>2.9.</label>
<title>Phenylpropionate transporter colorimetric assay</title>
<p>Overnight cultures in LB medium were diluted 1/100 in fresh LB containing 1&#x2009;mM 3-phenylpropionate. Cultures were incubated in test tubes for 24&#x2009;h at 37&#x00B0;C with shaking (130&#x2009;r.p.m.). Cells were subsequently pelleted by centrifugation, and the absorbance of the supernatant was measured at 500&#x2009;nm.</p>
</sec>
<sec id="sec12">
<label>2.10.</label>
<title>Competition assay</title>
<p>Mutant and control strains were incubated overnight at 37&#x00B0;C until stationary phase and were then combined in a 1:1 ratio. The presence of equal numbers of viable bacteria of each strain was confirmed using a spot test. Mixed cultures were subsequently diluted 1/100,000 and incubated at 37&#x00B0;C with or without menadione. After 1, 3, and 5&#x2009;days, the number of viable bacteria was enumerated by diluting samples 1/50, 1/2,500, and 1/125,000; spotting them on AM3 plates; and culturing them at 37&#x00B0;C.</p>
</sec>
<sec id="sec13">
<label>2.11.</label>
<title>Statistical analysis</title>
<p>Normality and variances in each dataset were determined <italic>a priori</italic> using the Shapiro&#x2013;Wilk or Bartlett tests. Welch&#x2019;s <italic>t</italic>-test was used to analyze differences between two groups. An analysis of variance (ANOVA) test was used to analyze differences between three groups, followed by a Tukey&#x2019;s multiple comparison correction. A Monte Carlo simulation with 100,000 replicates was used for the chi-square test for goodness of fit. <italic>p</italic>-values smaller than 0.05 were considered significant.</p>
</sec>
</sec>
<sec id="sec14" sec-type="results">
<label>3.</label>
<title>Results and discussion</title>
<sec id="sec15">
<label>3.1.</label>
<title>Construction of genome-reduced strain &#x0394;33b&#x2013;&#x0394;41c</title>
<p>Previously constructed genome-reduced strains did not lack essential genes but had attenuated growth compared with the WT (<xref ref-type="bibr" rid="ref14">Hashimoto et al., 2005</xref>). The doubling time of the genome-reduced &#x0394;33a strain, which lacks approximately 38.9% of the WT chromosome, was 170.0&#x2009;min (<xref ref-type="bibr" rid="ref14">Hashimoto et al., 2005</xref>, <xref rid="tab1" ref-type="table">Table 1</xref>). To construct genome-reduced strains with further chromosomal deletions, we first attempted to isolate strains with enhanced growth by ALE. After sequentially culturing the &#x0394;33a parent strain for approximately 1,800 generations, we isolated a faster growing variant, termed strain &#x0394;33b, with a doubling time of 113.3&#x2009;min (<xref rid="tab1" ref-type="table">Table 1</xref>).</p>
<table-wrap position="float" id="tab1">
<label>Table 1</label>
<caption>
<p>Doubling time of genome-reduced strains.</p>
</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th align="left" valign="top">Strain</th>
<th align="center" valign="top">Doubling time (min)</th>
<th align="center" valign="top">SE</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left" valign="middle">&#x0394;33a</td>
<td align="char" valign="middle" char=".">170.0</td>
<td align="char" valign="middle" char=".">9.4</td>
</tr>
<tr>
<td align="left" valign="middle">&#x0394;33b</td>
<td align="char" valign="middle" char=".">113.3</td>
<td align="char" valign="middle" char=".">7.2</td>
</tr>
<tr>
<td align="left" valign="middle">&#x0394;37b</td>
<td align="char" valign="middle" char=".">133.3</td>
<td align="char" valign="middle" char=".">7.2</td>
</tr>
<tr>
<td align="left" valign="middle">&#x0394;37c13</td>
<td align="char" valign="middle" char=".">53.3</td>
<td align="char" valign="middle" char=".">2.7</td>
</tr>
<tr>
<td align="left" valign="middle">&#x0394;37c16</td>
<td align="char" valign="middle" char=".">40.0</td>
<td align="char" valign="middle" char=".">0.0</td>
</tr>
<tr>
<td align="left" valign="middle">&#x0394;37c143</td>
<td align="char" valign="middle" char=".">86.7</td>
<td align="char" valign="middle" char=".">5.4</td>
</tr>
<tr>
<td align="left" valign="middle">&#x0394;37c145</td>
<td align="char" valign="middle" char=".">43.3</td>
<td align="char" valign="middle" char=".">2.7</td>
</tr>
<tr>
<td align="left" valign="middle">&#x0394;37c146</td>
<td align="char" valign="middle" char=".">50.0</td>
<td align="char" valign="middle" char=".">0.0</td>
</tr>
<tr>
<td align="left" valign="middle">&#x0394;41c</td>
<td align="char" valign="middle" char=".">136.7</td>
<td align="char" valign="middle" char=".">29.9</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>By introducing additional nonessential chromosomal deletions into strain &#x0394;33b, we constructed additional genome-reduced strains (&#x0394;33b to &#x0394;37b; <xref rid="fig1" ref-type="fig">Figures 1A</xref>&#x2013;<xref rid="fig1" ref-type="fig">C</xref>). To sequentially introduce chromosomal deletions, we optimized the FRT4 system to remove positive selection markers (<xref ref-type="bibr" rid="ref18">Iwadate et al., 2011</xref>, <xref ref-type="supplementary-material" rid="SM4">Supplementary Figure S1</xref>). First, several large-scale deletions were introduced to intergenic regions between essential genes of the &#x0394;33b strain in the MG1655 <italic>red</italic> strain using an Ap resistance gene. Next, these new deletion mutations were introduced into genome-reduced strains using P1 transduction, and mutations that did not significantly attenuate growth were selected. The positive selection marker was subsequently exchanged with a Cm resistance gene with a FLP-FRT recombination site in the MG1655 <italic>red</italic> strain. Chromosomal regions on both sides of the deletion, in addition to the negative selection marker gene <italic>rpsL</italic>+, and the Ap resistance gene were cloned into a suicide vector containing FRT, and the plasmid was inserted into the chromosome by homologous recombination. Lastly, deletion mutations were introduced into genome-reduced strains using P1 transduction and FLP-FRT site-specific recombination after introduction of a plasmid expressing the FLP recombinase. Sm-resistant, Cm-, and Ap-sensitive strains were then isolated, and the introduction of the deletion was confirmed by PCR.</p>
<fig position="float" id="fig1">
<label>Figure 1</label>
<caption>
<p>Construction of genome-reduced strains. <bold>(A)</bold> Genetic units deleted in the construction of genome-reduced strains. The genomes of &#x0394;41c, &#x0394;33a, and MG1655 are represented from the center of the circle, respectively. The closed boxes indicate deleted regions. <bold>(B)</bold> Schematic depicting the relationship between constructed genome-reduced strains. <bold>(C)</bold> Table describing the number and length of deletion units.</p>
</caption>
<graphic xlink:href="fmicb-14-1189877-g001.tif"/>
</fig>
<p>The doubling time of the genome-reduced strain &#x0394;37b was 133.3&#x2009;min, longer than that of strain &#x0394;33b (<xref rid="tab1" ref-type="table">Table 1</xref>). To construct genome-reduced strains with further deletion, we first isolated strains with enhanced growth by ALE by performing five independent subcultures, allowing for approximately 1,960&#x2013;2,300 generations. Following this process, we isolated strains &#x0394;37c-13, 16, 143, 145, and 146 with doubling times of 53.3, 40.0, 86.7, 43.3, and 50.0&#x2009;min, respectively (<xref rid="tab1" ref-type="table">Table 1</xref>). We next introduced large-scale chromosome deletions into strain &#x0394;37c-16 to generate &#x0394;41c, which harbors deletion of approximately 44% (~2&#x2009;Mb) of the wild-type chromosome (<xref rid="fig1" ref-type="fig">Figures 1A</xref>&#x2013;<xref rid="fig1" ref-type="fig">C</xref>) and has a doubling time of 136.7&#x2009;min (<xref rid="tab1" ref-type="table">Table 1</xref>).</p>
<p>We next subjected nine genome-reduced strains (&#x0394;33a, the ALE strain &#x0394;33b, &#x0394;37b, the five ALE strains &#x0394;37c, and &#x0394;41c) to whole genome sequencing. We first performed sequencing with the Illumina MiSeq system, followed by long-read sequencing using the MinION system, and identified several SNVs, insertions, and deletions (<xref rid="tab2" ref-type="table">Table 2</xref>; <xref ref-type="supplementary-material" rid="SM1">Supplementary Table S1</xref>). SNVs, insertions, and deletions were already present in strain &#x0394;33a, indicating that mutations had occurred during construction of the genome-reduced strains. When we examined the types of mutation present in the nine strains, we found that missense and synonymous mutations were most common (<xref ref-type="supplementary-material" rid="SM4">Supplementary Figure S3</xref>). Insertions, deletions, and genomic rearrangements occurred in all strains, but we found no evidence of common mutational hotspots (<xref ref-type="supplementary-material" rid="SM4">Supplementary Figure S4</xref>). Genome-reduced strains have previously been identified by the length of the deleted region and the presence or absence of genes, but it is also necessary to classify them based on genomic alterations.</p>
<table-wrap position="float" id="tab2">
<label>Table 2</label>
<caption>
<p>Mutations identified by genomic resequencing.</p>
</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th/>
<th/>
<th align="center" valign="top">&#x0394;33a</th>
<th align="center" valign="top">&#x0394;33b</th>
<th align="center" valign="top">&#x0394;37b</th>
<th align="center" valign="top">&#x0394;37c13</th>
<th align="center" valign="top">&#x0394;37c16</th>
<th align="center" valign="top">&#x0394;37c143</th>
<th align="center" valign="top">&#x0394;37c145</th>
<th align="center" valign="top">&#x0394;37c146</th>
<th align="center" valign="top">&#x0394;41c</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left" valign="middle">SNV</td>
<td align="left" valign="middle">Missense</td>
<td align="center" valign="middle">39</td>
<td align="center" valign="middle">51</td>
<td align="center" valign="middle">59</td>
<td align="center" valign="middle">108</td>
<td align="center" valign="middle">143</td>
<td align="center" valign="middle">114</td>
<td align="center" valign="middle">122</td>
<td align="center" valign="middle">87</td>
<td align="center" valign="middle">159</td>
<td align="left" valign="middle">Synonymous</td>
<td align="center" valign="middle">14</td>
<td align="center" valign="middle">17</td>
<td align="center" valign="middle">17</td>
<td align="center" valign="middle">54</td>
<td align="center" valign="middle">69</td>
<td align="center" valign="middle">53</td>
<td align="center" valign="middle">63</td>
<td align="center" valign="middle">42</td>
<td align="center" valign="middle">83</td>
<td align="left" valign="middle">Frameshift</td>
<td align="center" valign="middle">2</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">11</td>
<td align="center" valign="middle">11</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">7</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">22</td>
<td align="left" valign="middle">Stop gained</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">5</td>
<td align="center" valign="middle">6</td>
<td align="center" valign="middle">8</td>
<td align="center" valign="middle">19</td>
<td align="center" valign="middle">8</td>
<td align="center" valign="middle">11</td>
<td align="center" valign="middle">9</td>
<td align="center" valign="middle">21</td>
<td align="left" valign="middle">Stop lost</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">2</td>
<td align="left" valign="middle">Upstream</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">8</td>
<td align="center" valign="middle">20</td>
<td align="center" valign="middle">16</td>
<td align="center" valign="middle">16</td>
<td align="center" valign="middle">18</td>
<td align="center" valign="middle">17</td>
<td align="center" valign="middle">17</td>
<td align="left" valign="middle">RNA</td>
<td align="center" valign="middle">2</td>
<td align="center" valign="middle">2</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">2</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">2</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">1</td>
<td align="left" valign="middle">Intergene</td>
<td align="center" valign="middle">14</td>
<td align="center" valign="middle">14</td>
<td align="center" valign="middle">14</td>
<td align="center" valign="middle">23</td>
<td align="center" valign="middle">26</td>
<td align="center" valign="middle">23</td>
<td align="center" valign="middle">19</td>
<td align="center" valign="middle">20</td>
<td align="center" valign="middle">35</td>
</tr>
<tr>
<td/>
<td align="left" valign="middle">SNV Total</td>
<td align="center" valign="middle">78</td>
<td align="center" valign="middle">97</td>
<td align="center" valign="middle">107</td>
<td align="center" valign="middle">227</td>
<td align="center" valign="middle">286</td>
<td align="center" valign="middle">221</td>
<td align="center" valign="middle">244</td>
<td align="center" valign="middle">183</td>
<td align="center" valign="middle">340</td>
</tr>
<tr>
<td align="left" valign="middle" colspan="2">Insertion</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">5</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">5</td>
</tr>
<tr>
<td align="left" valign="middle" colspan="2">Deletion</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">1</td>
<td align="center" valign="middle">2</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">5</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">6</td>
</tr>
<tr>
<td align="left" valign="middle" colspan="2">Total</td>
<td align="center" valign="middle">80</td>
<td align="center" valign="middle">101</td>
<td align="center" valign="middle">113</td>
<td align="center" valign="middle">234</td>
<td align="center" valign="middle">295</td>
<td align="center" valign="middle">229</td>
<td align="center" valign="middle">252</td>
<td align="center" valign="middle">191</td>
<td align="center" valign="middle">351</td>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec id="sec16">
<label>3.2.</label>
<title>Genome alteration from &#x0394;33a to &#x0394;33b</title>
<p>In strain &#x0394;33a, 78 SNVs, 1 insertion, and 1 deletion were identified, whereas in strain &#x0394;33b isolated by ALE, we found 97 SNVs, 3 insertions, and 1 deletion (<xref rid="tab2" ref-type="table">Table 2</xref>; <xref ref-type="supplementary-material" rid="SM1">Supplementary Table S1</xref>). This indicates that 19 SNVs and 2 insertions occurred during the process of ALE (<xref rid="tab2" ref-type="table">Table 2</xref>; <xref ref-type="supplementary-material" rid="SM1">Supplementary Table S1</xref>). Some SNVs occurred in essential genes. Most SNVs in essential genes were missense mutations, with the exception of a nonsense mutation in <italic>yceQ</italic> that introduced a stop codon at amino acid position 8. A previous study using transposon mutagenesis reported that <italic>yceQ</italic> itself was not an essential gene but rather contained a promoter for the essential gene <italic>rne</italic> (<xref ref-type="bibr" rid="ref13">Goodall et al., 2018</xref>). Therefore, all SNVs that occurred in essential genes were missense mutations. If all genes were mutated randomly, we would expect the ratio of mutations in essential genes to nonessential genes to decrease as mutations in essential genes would include lethal mutations. However, in strains &#x0394;33a and &#x0394;33b, we found a significantly higher proportion of mutations in essential genes (<xref ref-type="supplementary-material" rid="SM2">Supplementary Table S2</xref>). These results suggest that during the process of constructing strain &#x0394;33a and that of isolating strain &#x0394;33b by ALE, mutations in essential genes may have enhanced growth.</p>
<p>We found two IS insertion mutations within the <italic>pntA</italic> promoter region and the <italic>sibE</italic> gene (<xref rid="fig2" ref-type="fig">Figures 2A</xref>,<xref rid="fig2" ref-type="fig">B</xref>). Using a <italic>lacZ</italic> reporter gene, we found that the expression of <italic>pntA</italic> was increased by the promoter insertion (<xref rid="fig2" ref-type="fig">Figure 2C</xref>). We also found that the <italic>sibE</italic> insertion decreased expression of the gene (<xref rid="fig2" ref-type="fig">Figure 2C</xref>). PntA is a proton transporter that synthesizes NADPH from NADH by proton influx (<xref ref-type="bibr" rid="ref7">Clarke and Bragg, 1985</xref>). SibE, which produces a small RNA, is the antitoxin component of a toxin-antitoxin system and suppresses expression of the toxin gene <italic>ibsE</italic>, which exists in the form of reverse overlap within the <italic>sibE</italic> gene (<xref ref-type="bibr" rid="ref10">Fozo et al., 2008</xref>). There are five chromosomal <italic>ibs</italic> homologues (ibsA, B, C, D, E) that encode putative membrane proteins. Although their exact functions are unknown, their overexpression is known to cause depolarization (<xref ref-type="bibr" rid="ref10">Fozo et al., 2008</xref>). Both of the two insertions identified in &#x0394;33b are distinct from simply destructive insertions. This suggests that there is a functional benefit conferred by the insertion that may pertain to growth rate. One potential mechanism for the insertion-mediated rescue of growth is that antitoxin is diminished by the <italic>sibE</italic> insertion, causing overexpression of the IbsE toxin, which subsequently increases membrane potential. Furthermore, insertion-mediated overexpression of PntA may increase levels of NADPH, promoting growth or resistance to oxidative stress.</p>
<fig position="float" id="fig2">
<label>Figure 2</label>
<caption>
<p>Insertions identified in strain &#x0394;33b. <bold>(A)</bold> Schematic depicting the location of the identified insertions. <bold>(B)</bold> Alignment showing the sequences adjacent to the identified insertions. <bold>(C)</bold> Bar chart depicting the effects of the identified insertions on gene expression. The bar chart shows the mean&#x2009;&#x00B1;&#x2009;standard error values (<italic>n</italic>&#x2009;=&#x2009;3). The insertion significantly increased the expression from the <italic>pntA</italic> promoter (<italic>p</italic>&#x2009;&#x003C;&#x2009;0.01), while the expression of <italic>sibE</italic> was significantly decreased (<italic>p</italic>&#x2009;=&#x2009;0.021).</p>
</caption>
<graphic xlink:href="fmicb-14-1189877-g002.tif"/>
</fig>
</sec>
<sec id="sec17">
<label>3.3.</label>
<title>Genome alterations in &#x0394;37c relative to &#x0394;37b</title>
<p>In strain &#x0394;37b, we identified 107 SNVs, 4 insertions, and 2 deletions. Furthermore, we identified increased SNVs, insertions, and deletions in five &#x0394;37c strains (<xref rid="tab2" ref-type="table">Table 2</xref>; <xref ref-type="supplementary-material" rid="SM1">Supplementary Tables S1</xref>, <xref ref-type="supplementary-material" rid="SM3">S3</xref>). Since the five &#x0394;37c strains were isolated independently, we predicted that mutations common to all strains underpin the rescued growth phenotype. We identified 12 genes with SNVs present in 2 or more strains (<xref rid="fig3" ref-type="fig">Figure 3A</xref>; <xref ref-type="supplementary-material" rid="SM3">Supplementary Table S3</xref>). Interestingly, the <italic>hcaT</italic> gene contained SNV in all five strains (<xref rid="fig3" ref-type="fig">Figure 3A</xref>; <xref ref-type="supplementary-material" rid="SM3">Supplementary Table S3</xref>). Furthermore, all SNVs were present upstream of <italic>hcaT</italic>, most commonly in the &#x2212;10 region of the promoter (<xref rid="fig3" ref-type="fig">Figures 3A</xref>,<xref rid="fig3" ref-type="fig">C</xref>). Using a LacZ reporter assay, we found that all SNVs increased gene expression (<xref rid="fig3" ref-type="fig">Figure 3B</xref>). We also found SNVs in the region upstream of the <italic>thiB</italic> gene, but these mutations did not increase expression except for 37c145 (<xref ref-type="supplementary-material" rid="SM4">Supplementary Figure S5</xref>). In addition to SNV, we identified an inversion between <italic>rrn</italic> loci in one out of five strains (<xref rid="fig4" ref-type="fig">Figure 4A</xref>), and deletions in f-Met tRNA (<xref rid="fig4" ref-type="fig">Figure 4B</xref>). Three tandem copies of f-Met tRNA are present on the wild-type chromosome, and we observed deletion of one or two copies through homologous recombination (<xref rid="fig4" ref-type="fig">Figure 4B</xref>). Although three out of five strains showed reduced copy number, it remains unclear whether this altered growth of the genome-reduced strains.</p>
<fig position="float" id="fig3">
<label>Figure 3</label>
<caption>
<p>SNVs common to &#x0394;37c ALE strains. <bold>(A)</bold> Schematic depicting mutation sites. Green boxes indicate nonessential genes, and blue boxes indicate essential genes. Red arrows indicate the location of mutations. <bold>(B)</bold> Bar chart depicting the effects of <italic>hcaT</italic> mutations on gene expression. The bar chart shows the mean&#x2009;&#x00B1;&#x2009;standard error values (<italic>n</italic>&#x2009;=&#x2009;3). Both mutations significantly increased gene expression (<italic>p</italic>&#x2009;&#x003C;&#x2009;0.01). Numbers indicate base positions calculated relative to the transcription start site as +1. <bold>(C)</bold> Sequence alignment of identified <italic>hcaT</italic> mutations. &#x2212;10 and &#x2212;35 indicate promoter regions for sigma 24, and gray arrows represent transcription.</p>
</caption>
<graphic xlink:href="fmicb-14-1189877-g003.tif"/>
</fig>
<fig position="float" id="fig4">
<label>Figure 4</label>
<caption>
<p>Inversions and deletions identified in &#x0394;37c ALE strains. <bold>(A)</bold> Schematic showing the inversion between <italic>rrnD</italic> and <italic>rrnE</italic> in strain &#x0394;37c16. <bold>(B)</bold> Deletion in the initiating methionine tRNA gene cluster. Two initiating methionine tRNAs (&#x0394;37c13, 145) or one initiating methionine tRNA (&#x0394;37c146) were deleted from the <italic>metZWV</italic> region by homologous recombination.</p>
</caption>
<graphic xlink:href="fmicb-14-1189877-g004.tif"/>
</fig>
</sec>
<sec id="sec18">
<label>3.4.</label>
<title>Analysis of <italic>hca</italic> and <italic>mhp</italic> mutant strains</title>
<p>Although <italic>hcaT</italic> was implicated in the growth rescue of strain &#x0394;37b, the function of this gene remains poorly understood. We constructed and phenotypically characterized deletion strains lacking <italic>hcaT</italic>, <italic>hcaR</italic>, and <italic>hcaE</italic>-<italic>hcaD</italic> genes, as well as <italic>mhp</italic> related genes. The sequence of HcaT indicates this protein is a putative transporter, but its substrate is not known. The <italic>hcaE</italic>-<italic>hcaD</italic> and <italic>mhp</italic> gene clusters are encoded close to <italic>hcaT</italic> and are involved in acetyl-CoA synthesis by breaking down 3-phenylpropionate (<xref ref-type="bibr" rid="ref29">Pao et al., 1998</xref>; <xref ref-type="bibr" rid="ref9">D&#x0131;&#x0301;az et al., 2001</xref>). Since <italic>hcaR</italic>, which is encoded immediately upstream of <italic>hcaT</italic>, is an activator of the <italic>hca</italic> gene cluster, <italic>hcaT</italic> has also been speculated as a 3-phenylpropionate transporter (<xref ref-type="bibr" rid="ref8">D&#x00ED;az et al., 1998</xref>). Auto-oxidation of 3-(2,3-dihydroxyphenyl) propionate, which accumulates when the <italic>mhp</italic> gene cluster is deleted, oxidizes proline in the growth medium, causing a color change to red (<xref ref-type="bibr" rid="ref4">Burlingame et al., 1986</xref>) (<xref rid="fig5" ref-type="fig">Figure 5A</xref>). Double mutant strains were constructed lacking the <italic>mhp</italic> gene cluster and either <italic>hcaT</italic> or <italic>hcaR</italic>. The <italic>mhp hcaR</italic> double mutant did not cause pigmentation of the medium although the <italic>mhp hcaT</italic> mutant showed redder medium than the <italic>mhp hcaR</italic> double mutant but not as much as the <italic>mhp</italic> single mutant (<xref rid="fig5" ref-type="fig">Figures 5B</xref>,<xref rid="fig5" ref-type="fig">C</xref>). Since pigmentation of the medium does not occur unless 3-phenylpropionate is present, these results suggest that HcaT is a 3-phenylpropionate transporter (<xref rid="fig5" ref-type="fig">Figures 5B</xref>,<xref rid="fig5" ref-type="fig">C</xref>; <xref ref-type="supplementary-material" rid="SM4">Supplementary Figure S6</xref>). In addition, we examined the growth of constructed strains using 3-phenylpropionate as a carbon source. On minimal medium supplemented with 3-phenylpropionate, wild-type strains were able to grow, but <italic>hcaR</italic>, <italic>hcaE</italic>-<italic>hcaD</italic>, <italic>mhp</italic>T-<italic>mhp</italic>R, and <italic>mhp</italic>T-<italic>mhp</italic>R <italic>hcaT</italic> deletion strains were not (<xref ref-type="supplementary-material" rid="SM4">Supplementary Figure S7</xref>). Since <italic>hcaT</italic> deletion mutants were able to grow, these results also suggest the presence of additional 3-phenylpropionate transporters other than HcaT.</p>
<fig position="float" id="fig5">
<label>Figure 5</label>
<caption>
<p><italic>hca</italic> genes are involved in phenylpropionate degradation. <bold>(A)</bold> The phenylpropionic acid (I) degradation pathway. Phenylpropionate is degraded to succinate (V) and acetyl CoA (VI) <italic>via</italic> several metabolic intermediates (II-IV). <bold>(B)</bold> Color of culture supernatants after growth of <italic>&#x0394;mhpR-T</italic>, <italic>&#x0394;mhpR</italic>-<italic>T &#x0394;hcaT</italic>, and <italic>&#x0394;mhpR-T &#x0394;hcaR</italic> mutants in the presence of phenylpropionate. <bold>(C)</bold> Absorbance of culture supernatants at OD500&#x2009;nm. The bar chart shows the mean&#x2009;&#x00B1;&#x2009;standard error of absorbance measurements (<italic>n</italic>&#x2009;=&#x2009;3, &#x0394;<italic>mhpRT</italic> is <italic>n</italic>&#x2009;=&#x2009;4). The deletion of <italic>hcaT</italic> significantly suppressed pigmentation of the medium, but the color change was still observed even in the presence of <italic>hcaT</italic> deletion (<italic>p</italic>&#x2009;&#x003C;&#x2009;0.01).</p>
</caption>
<graphic xlink:href="fmicb-14-1189877-g005.tif"/>
</fig>
<p>3-phenylpropionate is considered a quality control measure during protein synthesis. 3-phenylpropionate competitively inhibits the ligation of phenylalanine to tRNA<sup>Phe</sup> by phenylalanine aminoacyl-tRNA synthetase (PheRS) (<xref ref-type="bibr" rid="ref28">Mulivor and Rappaport, 1973</xref>). Tyrosine can be erroneously ligated to tRNA<sup>Phe</sup> by PheRS but is subsequently hydrolyzed and removed by the proofreading activity of PheRS (<xref ref-type="bibr" rid="ref32">Roy et al., 2004</xref>). Cytotoxic meta-tyrosine is generated from tyrosine in the presence of oxidative stress (<xref ref-type="bibr" rid="ref3">Bullwinkle et al., 2014</xref>). The fact that the proofreading activity of PheRS increases under oxidative stress suggests an important role for the removal of meta-tyrosine (<xref ref-type="bibr" rid="ref33">Steiner et al., 2019</xref>). In strain &#x0394;37c, in addition to deletion of the <italic>mhp</italic> gene cluster, we predict that levels of HcaT increase due to mutation of the <italic>hcaT</italic> promoter, which subsequently increases the levels of intracellular 3-phenylpropionate. 3-phenylpropionate may subsequently reduce levels of meta-tyrosine-tRNA<sup>Phe</sup>, improving the quality of protein synthesis and restoring growth.</p>
<p>HcaR is involved in expression of scavenger enzymes, which remove reactive oxygen species, and has been associated with the oxidative stress response (<xref ref-type="bibr" rid="ref35">Turlin et al., 2005</xref>). Since the <italic>mhp</italic> gene cluster is deleted in strain &#x0394;37b, we constructed a double mutant harboring both a <italic>mhp</italic> deletion and a SNV mutation (<italic>hcaT</italic> (37c)) that increases <italic>hcaT</italic> expression. We investigated the survival of long-term stationary phase cultures in the presence of oxidative stress and the redox-cycling drug menadione. When the <italic>mhp</italic> deletion mutant and the <italic>mhp hcaT</italic> (37c) double mutant strain were cultured alone, we observed no significant difference in growth (<xref ref-type="supplementary-material" rid="SM4">Supplementary Figure S8</xref>). However, when those strains were cultured together for 5&#x2009;days, the <italic>mhp</italic> mutant showed enhanced survival (<xref rid="fig6" ref-type="fig">Figure 6A</xref>). The same results were also obtained in the <italic>mhp</italic>&#x2009;+&#x2009;background, albeit to a lesser extent (<xref rid="fig6" ref-type="fig">Figure 6B</xref>). Survival of not only of the <italic>mhp hcaT</italic> (37c) double mutant but also the mixed <italic>mhp</italic> deletion strain suggested a change to the composition of the medium. Increased expression of <italic>hcaT</italic> in the <italic>mhp hcaT</italic> (37c) double mutant may change the composition of the medium by taking up and metabolizing 3-phenylpropionate, promoting survival of the strain in the presence of oxidative stress at least in the conditions where a large number of genes were deleted like the genome-reduced strain constructed in this work. Further characterization of the functions of 3-phenylpropionate and related compounds will elucidate the mechanism underpinning this phenotype.</p>
<fig position="float" id="fig6">
<label>Figure 6</label>
<caption>
<p>Competition assay with the strain harboring the <italic>hcaT</italic> mutation identified in &#x0394;37c-16. <bold>(A)</bold> Double mutant, <italic>hcaT</italic> (37c) &#x0394;<italic>mhpAE</italic>, and control, <italic>hcaT</italic> (WT) &#x0394;<italic>mhpAE</italic>. Spotting cultures are shown after day 1 and day 5 of growth. <bold>(B)</bold> Mutant, <italic>hcaT</italic> (37c) and control, <italic>hcaT</italic> (WT) are shown after day 1 and day 5 of growth.</p>
</caption>
<graphic xlink:href="fmicb-14-1189877-g006.tif"/>
</fig>
<p>A recent study demonstrated rewiring of imbalanced metabolism in genome-reduced strains through isolation and analysis of strains with growth restored by ALE (<xref ref-type="bibr" rid="ref5">Choe et al., 2019</xref>). This may enable characterization of the stress resistance mechanism, which promotes survival in stationary phase. Analysis of genome-reduced strains in which the various systems are imbalanced as a resource reveals aspects that are different from research with wild-type strains and individual gene deletion mutants. These studies will shed fundamental insights into cell proliferation and survival.</p>
</sec>
</sec>
<sec id="sec19" sec-type="data-availability">
<title>Data availability statement</title>
<p>The datasets presented in this study have been deposited in the DNA Data Bank of Japan, accession number PRJDB15441: <ext-link xlink:href="https://ddbj.nig.ac.jp/resource/bioproject/PRJDB15441" ext-link-type="uri">https://ddbj.nig.ac.jp/resource/bioproject/PRJDB15441</ext-link>.</p>
</sec>
<sec id="sec20">
<title>Author contributions</title>
<p>YK, MH, KL, and JK designed the study and performed the experiment. YK analyzed and visualized the data. JK supervised the study. YK and JK wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.</p>
</sec>
<sec id="sec21" sec-type="funding-information">
<title>Funding</title>
<p>This work was supported by Grants of Tokyo Metropolitan University to JK.</p>
</sec>
<sec id="conf1" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="sec100" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
</body>
<back>
<ack>
<p>The authors thank K. Tominaga and S. Iyoda for discussions and advice.</p>
</ack>
<sec id="sec23" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary material for this article can be found online at: <ext-link xlink:href="https://www.frontiersin.org/articles/10.3389/fmicb.2023.1189877/full#supplementary-material" ext-link-type="uri">https://www.frontiersin.org/articles/10.3389/fmicb.2023.1189877/full#supplementary-material</ext-link></p>
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<supplementary-material xlink:href="Data_Sheet_1.PDF" id="SM4" mimetype="application/pdf" xmlns:xlink="http://www.w3.org/1999/xlink"/>
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