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Research article

Comparison of transcriptomes of enlarged spheroplasts of Erythrobacter litoralis and Lelliottia amnigena

  • Received: 11 April 2016 Accepted: 20 May 2016 Published: 22 May 2016
  • Bacterial spheroplasts do not divide but they grow and enlarge with DNA replication in a broth containing an inhibitor of peptidoglycan synthesis and high salt concentration. The enlarged spheroplasts of Lelliottia amnigena, belonging to the family Enterobacteriaceae, formed vacuole-like structures, while those of the aerobic photosynthetic marine bacterium Erythrobacter litoralis did not form such structures. In addition, the enlarged spheroplasts of L. amnigena, which differ in the size of inner and outer membranes, were larger than those of E. litoralis. To elucidate the reason for these differences, we analyzed the transcriptome (RNA-seq) of spheroplasts at the beginning of growth and upon enlargement. After sequencing the RNAs expressed in normal and enlarged spheroplasts, we compared the gene expression levels of the 1100 orthologs in E. litoralis and L. amnigena. Among these, 347 and 213 genes were more than 2-fold upregulated in enlarged spheroplasts of E. litoralis and L. amnigena, respectively; 193 and 269 genes were less than 0.5-fold downregulated in those of E. litoralis and L. amnigena, respectively. Thirty six genes were upregulated in L. amnigena enlarged spheroplasts but were downregulated in E. litoralis enlarged spheroplasts, and may be related to vacuole-like structure generation. Fourteen of the 36 genes encoded a membrane protein. Our findings indicate that spheroplast enlargement varies between different organisms with respect to gene expression.

    Citation: Sawako Takahashi, Ayana Takayanagi, Yurika Takahashi, Taku Oshima, Hiromi Nishida. Comparison of transcriptomes of enlarged spheroplasts of Erythrobacter litoralis and Lelliottia amnigena[J]. AIMS Microbiology, 2016, 2(2): 152-189. doi: 10.3934/microbiol.2016.2.152

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  • Bacterial spheroplasts do not divide but they grow and enlarge with DNA replication in a broth containing an inhibitor of peptidoglycan synthesis and high salt concentration. The enlarged spheroplasts of Lelliottia amnigena, belonging to the family Enterobacteriaceae, formed vacuole-like structures, while those of the aerobic photosynthetic marine bacterium Erythrobacter litoralis did not form such structures. In addition, the enlarged spheroplasts of L. amnigena, which differ in the size of inner and outer membranes, were larger than those of E. litoralis. To elucidate the reason for these differences, we analyzed the transcriptome (RNA-seq) of spheroplasts at the beginning of growth and upon enlargement. After sequencing the RNAs expressed in normal and enlarged spheroplasts, we compared the gene expression levels of the 1100 orthologs in E. litoralis and L. amnigena. Among these, 347 and 213 genes were more than 2-fold upregulated in enlarged spheroplasts of E. litoralis and L. amnigena, respectively; 193 and 269 genes were less than 0.5-fold downregulated in those of E. litoralis and L. amnigena, respectively. Thirty six genes were upregulated in L. amnigena enlarged spheroplasts but were downregulated in E. litoralis enlarged spheroplasts, and may be related to vacuole-like structure generation. Fourteen of the 36 genes encoded a membrane protein. Our findings indicate that spheroplast enlargement varies between different organisms with respect to gene expression.


    1. Introduction

    Enlarged spheroplasts are generated by spheroplast cultivation in a broth containing an inhibitor of peptidoglycan synthesis (for example, penicillin) and high salt concentration after lysozyme treatment of bacterial cells [1,2,3,4,5]. In presence of penicillin, spheroplasts do not divide but grow and enlarge with DNA replication in the marine broth, whereas they divide in the absence of penicillin [6,7]. The rate of DNA increase in the absence of penicillin is higher than that observed in the presence of penicillin [6,7].

    Lelliottia amnigena(Enterobacter amnigenus) belongs to the family Enterobacteriaceae of Gram-negative bacteria [8]. Most enlarged spheroplasts of L. amnigena have quite different sizes of the inner and outer membrane vesicles [9]; the inner membrane shows a maximum size of approximately 15 μm in diameter, while the outer membrane has a maximum size of > 30 μm in diameter. Vacuole-like structures were formed in enlarged spheroplasts of L. amnigena. Based on the membrane specific protein distribution in Escherichia coli, the membrane of these vacuole-like structures is very similar but not identical to the inner membrane, which is completely different from the outer membrane [4].

    Erythrobacter litoralisis an aerobic, anoxygenic, and photosynthetic marine bacterium, belonging to Alphaproteobacteria, which generates bacteriochlorophyll a and carotenoids [10]. The size of E. litoralis spheroplasts is limited to a diameter of 6-7 μm [7]. Continuous exposure to light was shown to inhibit their enlargement [7]. In addition, they lacked vacuole-like structures. We hypothesized that the difference of enlarged spheroplast size and the lack of vacuole-like structure may be related to the difference of general metabolism in bacterial cells. In contrast, the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium E. coli are known to form vacuole-like structures and their cell size is approximately 15 μm in diameter [4,5]. Divergence between inner and outer membranes enlargements is not observed in E. litoralis enlarged spheroplasts.

    Thus, the giant spheroplasts of E. litoralis and L. amnigena are structurally different. In order to elucidate this difference, we sequenced the RNAs expressed in the spheroplasts at the beginning of growth and in the enlarged spheroplasts. We then compared the changes in expression of the orthologous genes between the enlarged spheroplasts of E. litoralis andL. amnigena.


    2. Methods


    2.1. Cultivation of E. litoralis and L. amnigena spheroplasts

    Cells of E. litoralis NBRC 102620 and L. amnigena (Enterobacter amnigenus) NBRC 105700 were grown on marine broth agar (Difco, Sparks, MD). The harvested cells (approximately 0.003 g) were suspended in a buffer (1 mL) consisting of 0.1 M Tris-HCl (pH 7.6) and 0.3 M sucrose. Lysozyme (200 μg/mL, Wako, Osaka) was added to the cell suspension and allowed to incubate at 25 °C (for E. litoralis) and 37 °C (for L. amnigena) for 15 min. After harvesting (centrifugation for 5 min at 3000 rpm) the suspension, the cells were suspended in marine broth (1 mL) containing 600 μg/mL penicillin G (Serva, München). The suspension was then diluted by adding 4 μL of suspension to marine broth (1 mL) containing penicillin G. The diluted suspension was then incubated at 25 °C in the dark. We used spheroplasts at two time points of growth: 0 h and 96 h for E. litoralis, and 0 h and 43 h for L. amnigena.


    2.2. DAPI staining

    Fluorescence images of enlarged L. amnigena cells stained with DAPI were acquired. DAPI (final concentration, 0.25 μg/mL) was added to the cell suspension and incubated at room temperature for 1 h. Fluorescent micrographs were taken using Keyence BZ-X710.


    2.3. RNA isolation

    Total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel, Düren). After removal of rRNA with the RiboMinus Transcriptome Isolation Kit for bacteria (Thermo Fisher Science, Waltham, MA), the RNAs were purified and concentrated with the NucleoSpin RNA Clean-up XS kit (Macherey-Nagel, Düren).


    2.4. RNA-seq

    Sequencing libraries were prepared from RiboMinus-treated RNAs by NEXTflex qRNA-Seq Kit (Bioo Scientific, Austin, TX). The resultant libraries were analyzed by TapeStation 2200 (Agilent, Waldbronn) equipped with a High Sensitivity D1000 tape (Agilent, Waldbronn). The concentration of each library was quantified with KAPA Library Quantification Kits. The four libraries were sequenced on a Miseq system (Illumina, San Diego, CA) with 2 × 75 base paired end sequencing.

    FASTQ files were imported into the CLC Genomic Workbench (CLC bio, Germantown, MD) as paired-end reads, and read data were mapped on each genome by RNA-seq with the following parameters: Count paired reads as two = Yes, Auto-detect paired distances = No, Similarity fraction = 0.8, Length fraction = 0.5, Mismatch cost = 2, Insertion cost = 3, Deletion cost = 3.


    2.5. Gene expression comparison

    We performed reciprocal best hits using BLASTp (E-value < 0.001) to select orthologous genes between E. litoralis and L. amnigena. For orthologous genes, we calculated the ratio of reads per kilobase of gene per million mapped sequence reads (RPMK) in the enlarged spheroplasts per the RPKM in spheroplasts at 0 h of growth and selected the ratio of > 2 for the upregulated genes and the ratio of < 0.5 for the downregulated genes. The difference in expression was estimated by Fisher's exact test [11]. Considering that the mapped number of E. litoralis spheroplasts was 3 times greater than that of spheroplasts at 0 h of growth, when the RPKM = 0 (the mapped number = 0) in E. litoralis spheroplasts at 0 h of growth, we did not consider the genes with the mapped number 1, 2, and 3 in E. litoralis enlarged spheroplasts but considered the genes with the mapped number > 3 in enlarged spheroplasts as upregulated genes.


    3. Results and Discussion


    3.1. DNA location in the L. amnigena enlarged spheroplast

    In a previous study, it was observed that spheroplasts of L. amnigena elongated and divided in marine broth containing 3 μg/mL penicillin [9]. In contrast, in marine broth containing 300 μg/mL penicillin, they did not divide and became enlarged with differing sizes of the inner and outer membrane vesicles [9]. The fluorescence image of L. amnigena enlarged spheroplasts showed that the vacuole-like structures and the large periplasmic space were not stained with the DNA staining reagent DAPI (Figure 1). This indicates that the chromosomal DNA does not pass through the inner membrane of the enlarged spheroplasts or the membrane of the vacuole-like structures.

    Figure 1.A. Differential interference contrast microscopic images of enlarged spheroplasts of Lelliottia amnigena at 48 h of growth. The images are obtained using Olympus IX73. B. Microscopic image of an enlarged spheroplast of L. amnigena at 99 h of growth (left). Fluorescence image of the enlarged spheroplast (center). Merged image of the enlarged spheroplast (right). The images are obtained using Keyence BZ-X710.

    3.2. Mapped number of RNA sequences to orthologous genes in E. litoralis and L. amnigena

    We obtained 78934 and 62870 reads from the spheroplasts of E. litoralis at the beginning (0 h) of growth and after enlargement, respectively. Excluding the RNA sequences that map to the ribosomal RNA genes (61767 reads at the beginning; 31412 reads after enlargement) and did not map to the genomic DNA (10565 reads at the beginning; 8902 reads after enlargement), 6602 and 22556 RNA sequences were mapped from the spheroplasts of E. litoralis at the beginning (0 h) of growth and after enlargement, respectively. On the other hand, we obtained 6921404 and 957576 reads from the spheroplasts of L. amnigena at the beginning (0 h) of growth and after enlargement, respectively. Excluding the RNA sequences that map to the ribosomal RNA genes (6532071 reads at the beginning; 701391 reads after enlargement) and did not map to the genomic DNA (330459 reads at the beginning; 174806 reads after enlargement), 58874 and 81379 RNA sequences were mapped at the beginning (0 h) of growth and after enlargement from the spheroplasts of L. amnigena. The RPKMs were then calculated and compared at the gene expression level.

    The transcriptome data have been deposited in DDBJ under the accession number DRA004675.

    We identified 1100 orthologous gene pairs between E. litoralis and L. amnigena. Among these 1100 orthologs, 347 and 213 genes were more than 2-fold upregulated in the enlarged spheroplasts of E. litoralis and L. amnigena, respectively; whereas 193 and 269 genes were less than 0.5-fold downregulated in those of E. litoralis and L. amnigena, respectively (Figure 2, Supplementary Table 1).

    Figure 2.Venn diagram of gene expression changes in the enlarged spheroplasts of Erythrobacter litoralis and Lelliottia amnigena.

    3.3. Upregulated orthologous genes in E. litoralis and L. amnigena enlarged spheroplasts

    The 76 upregulated orthologous genes in both E. litoralis and L. amnigena enlarged spheroplasts are shown in Table 1. Of the 76 genes, 25 (32.9%) were homologous to membrane protein coding genes.

    Table 1.Orthologous genes upregulated (ratio > 2) in E. litoralis and L. amnigena enlarged spheroplasts.
    Protein ID of E. litoralisRPKM in E. litoralis spheroplasts at the beginning of growthRPKM in E. litoralis enlarged spheroplastsRatio of RPKMs of E. litoralisAnnotated functionProtein ID of L. amnigenaRPKM in L. amnigena spheroplasts at the beginning of growthRPKM in L. amnigena enlarged spheroplastsRatio of RPKMs of L. amnigenaAnnotated function
    Homologs of chaperone genes
    KEO89606.1*0.00 623.74 molecular chaperone GroESKDM56993.1*347.00 1300.00 3.75 chaperonin
    KEO92782.1*0.00 4575.93 phage shock proteinKDM46724.1*4670.00 27100.00 5.80 phage shock protein A
    KEO93197.1*0.00 2039.77 ATP-dependent proteaseKDM53259.1*178.00 608.00 3.42 ATP-dependent protease ATPase subunit HslU
    KEO93350.1*588.91 6885.08 11.69 ATPase AAAKDM51144.1*449.00 1660.00 3.70 chaperone ClpB
    KEO98976.1*265.47 587.82 2.21 molecular chaperone DnaJKDM57339.1*267.00 751.00 2.81 chaperone dnaJ
    Homologs of DNA replication and repair-related genes
    KEO92279.1*304.84 659.99 2.17 ATPase AAAKDM53654.1*0.00 8.08 Mg chelatase-like protein
    KEO92303.1*0.00 255.35 chromosomal replication initiator protein DnaAKDM52760.1*280.00 617.00 2.20 chromosomal replication initiator protein dnaA
    KEO92620.1*1142.64 8813.23 7.71 recombinase RecAKDM50414.1*401.00 893.00 2.23 protein recA
    Homologs of lipopolysaccharide synthesis and transport-related genes
    KEO89583.1*0.00 224.27 ABC transporter ATP-binding proteinKDM54743.1*46.80 339.00 7.24 lipopolysaccharide export system ATP-binding protein LptB
    KEO90699.1*309.18 1369.19 4.43 D-arabinose 5-phosphateKDM54739.1*34.40 74.70 2.17 arabinose 5-phosphate isomerase
    KEO92387.1*0.00 693.72 permeaseKDM54753.1*201.00 458.00 2.28 lipopolysaccharide export system permease lptF
    KEO96424.1*321.43 901.51 2.80 ABC transporterKDM55137.1*29.10 91.30 3.14 lipid A export ATP-binding/permease MsbA
    KEO98632.1*0.00 130.74 MFS transporterKDM50532.1*0.00 10.30 lysophospholipid transporter lplT
    Homologs of peptidoglycan synthesis-related genes
    KEO92316.1*0.00 1429.28 MltAKDM50514.1*46.40 123.00 2.65 membrane-bound lytic murein transglycosylase A
    KEO93302.1*0.00 801.96 peptidoglycan transglycosylaseKDM54749.1*0.00 16.90 monofunctional biosynthetic peptidoglycan transglycosylase
    KEO93307.1*0.00 252.66 N-acetylmuramoyl-L-alanine amidaseKDM55076.1*20.40 118.00 5.78 N-acetylmuramoyl-L-alanine amidase AmiD
    KEO93413.1*0.00 408.49 peptidoglycan glycosyltransferaseKDM57390.1*115.00 452.00 3.93 peptidoglycan synthase ftsI
    KEO93417.1*0.00 193.99 UDP-N-acetylmuramoylalanine--D-glutamate ligaseKDM57394.1*116.00 802.00 6.91 UDP-N-acetylmuramoylalanine-D-glutamate ligase
    Homologs of other membrane protein genes
    KEO89306.1*1090.43 2253.52 2.07 membrane protease subunit HflCKDM56960.1*135.00 599.00 4.44 protein HflC
    KEO89307.1*535.14 3554.84 6.64 peptidaseKDM56961.1*431.00 1380.00 3.20 protein HflK
    KEO89575.1*0.00 225.11 flagellar biosynthesis protein FliRKDM48637.1*0.00 31.30 flagellar biosynthetic protein fliR
    KEO89580.1*0.00 221.23 mechanosensitive ion channel protein MscSKDM55515.1*68.20 188.00 2.76 miniconductance mechanosensitive channel
    KEO92762.1*0.00 194.41 membrane proteinKDM51940.1*0.00 16.20 hypothetical protein
    KEO93138.1*0.00 505.31 ABC transporterKDM55480.1*321.00 823.00 2.56 hypothetical protein
    KEO93143.1*0.00 301.81 proline:sodium symporter PutPKDM51759.1*495.00 1180.00 2.38 sodium/proline symporter
    KEO96616.1*0.00 211.59 membrane proteinKDM48736.1*116.00 568.00 4.90 hypothetical protein
    KEO96782.1*0.00 281.12 MarC family transcriptional regulatorKDM46654.1*0.00 37.90 UPF0056 membrane protein yhcE
    KEO96802.1*0.00 526.58 hypothetical proteinKDM51307.1*54.80 674.00 12.30 manganese transporter mntH
    KEO98444.1*159.95 613.91 3.84 protoheme IX farnesyltransferaseKDM55407.1*612.00 1340.00 2.19 protoheme IX farnesyltransferase
    KEO98495.1*0.00 1075.18 hypothetical proteinKDM55130.1*24.50 70.90 2.89 UPF0702 transmembrane protein ycaP
    KEO98569.1*201.21 3148.42 15.65 histidine kinaseKDM51863.1*0.00 16.80 sensor protein phoQ
    KEO98609.1*288.10 612.40 2.13 membrane proteinKDM57482.1*316.00 742.00 2.35 outer membrane protein assembly factor yaeT
    KEO99117.1*0.00 227.97 membrane proteinKDM54035.1*14.10 51.10 3.62 hypothetical protein
    Others
    KEO89324.1*0.00 339.26 mannose-1-phosphate guanylyltransferaseKDM46222.1*118.00 1610.00 13.64 mannose-1-phosphate guanylyltransferase
    KEO89964.1*0.00 1086.92 taurine dioxygenaseKDM57053.1*19.90 115.00 5.78 alpha-ketoglutarate-dependent taurine dioxygenase
    KEO89994.1*0.00 480.32 aminomethyltransferaseKDM49721.1*264.00 696.00 2.64 aminomethyltransferase
    KEO90662.1*0.00 165.87 phosphoribosylaminoimidazole carboxylaseKDM55484.1*0.00 138.00 N5-carboxyaminoimidazole ribonucleotide synthase
    KEO90672.1*0.00 261.97 succinyl-CoA synthetase subunit betaKDM55689.1*480.00 1360.00 2.83 succinyl-CoA ligase [ADP-forming] subunit beta
    KEO91012.1*0.00 329.01 glutamyl-tRNA synthetaseKDM57451.1*0.00 13.80 glutamyl-Q tRNA(Asp) synthetase
    KEO92307.1*0.00 1173.54 2-octaprenylphenol hydroxylaseKDM52399.1*41.40 112.00 2.71 2-polyprenylphenol 6-hydroxylase
    KEO92322.1*1243.70 5084.10 4.09 alkyl hydroperoxide reductaseKDM55608.1*108.00 706.00 6.54 alkyl hydroperoxide reductase subunit F
    KEO92357.1*0.00 486.82 hypothetical proteinKDM55447.1*0.00 44.50 maltose O-acetyltransferase
    KEO92389.1*0.00 1032.40 Clp protease ClpSKDM55108.1*212.00 689.00 3.25 ATP-dependent Clp protease adapter protein ClpS
    KEO92432.1*0.00 225.11 acetyl-CoA acetyltransferaseKDM49670.1*0.00 10.40 hypothetical protein
    KEO92626.1*0.00 371.92 transcription elongation factor GreBKDM53922.1*0.00 25.90 transcription elongation factor greB
    KEO92766.1*0.00 499.00 pseudouridine synthaseKDM54709.1*53.60 142.00 2.65 tRNA pseudouridine synthase B
    KEO92773.1*238.61 1937.27 8.12 translation initiation factor IF-2KDM54711.1*645.00 1330.00 2.06 translation initiation factor IF-2
    KEO92898.1*0.00 183.68 oxidoreductaseKDM50444.1*0.00 20.20 formate hydrogenlyase subunit 2
    KEO92901.1*0.00 211.59 metallophosphoesteraseKDM54593.1*123.00 267.00 2.17 protein icc
    KEO93029.1*0.00 1061.69 Fur family transcriptional regulatorKDM55662.1*494.00 1760.00 3.56 ferric uptake regulation protein
    KEO93118.1*0.00 351.20 4-hydroxythreonine-4-phosphate dehydrogenaseKDM57359.1*86.00 299.00 3.48 4-hydroxythreonine-4-phosphate dehydrogenase
    KEO93158.1*0.00 513.67 ubiquinone biosynthesis protein UbiHKDM49723.1*43.20 93.80 2.17 2-octaprenyl-6-methoxyphenol hydroxylase
    KEO93247.1*0.00 269.73 TetR family transcriptional regulatorKDM55557.1*86.70 564.00 6.51 HTH-type transcriptional regulator BetI
    KEO93375.1*0.00 357.49 4-hydroxy-3-methylbut-2-enyl diphosphate reductaseKDM57347.1*53.60 142.00 2.65 4-hydroxy-3-methylbut-2-enyl diphosphate reductase
    KEO93411.1*0.00 566.68 16S rRNA methyltransferaseKDM57388.1*162.00 1000.00 6.17 ribosomal RNA small subunit methyltransferase H
    KEO96480.1*303.62 627.48 2.07 methionine sulfoxide reductase BKDM51893.1*41.00 208.00 5.07 peptide methionine sulfoxide reductase msrB
    KEO96529.1*0.00 210.35 phosphoribosylamine--glycine ligaseKDM52454.1*92.00 380.00 4.13 phosphoribosylamine-glycine ligase
    KEO96539.1*0.00 696.27 hypothetical proteinKDM51989.1*200.00 435.00 2.18 FeS cluster assembly protein sufD
    KEO96580.1*0.00 486.03 PTS IIA-like nitrogen-regulatory protein PtsNKDM54746.1*0.00 150.00 nitrogen regulatory protein
    KEO96608.1*0.00 1394.71 translation factor Sua5KDM53819.1*0.00 42.90 tRNA threonylcarbamoyladenosine biosynthesis protein RimN
    KEO96752.1*97.51 359.85 3.69 integraseKDM51758.1*94.30 425.00 4.51 putA protein
    KEO98470.1*0.00 124.49 hypothetical proteinKDM51955.1*11.80 59.60 5.05 UPF0061 protein ydiU
    KEO98596.1*0.00 814.69 LysR family transcriptional regulatorKDM57341.1*37.70 81.90 2.17 transcriptional activator protein nhaR
    KEO98607.1*0.00 192.91 1-deoxy-D-xylulose 5-phosphate reductoisomeraseKDM57478.1*42.50 1230.00 28.94 1-deoxy-D-xylulose 5-phosphate reductoisomerase
    KEO98608.1*0.00 1552.72 peptidaseKDM57481.1*264.00 854.00 3.23 RIP metalloprotease RseP
    KEO98641.1*0.00 1679.78 LysR family transcriptional regulatorKDM53240.1*130.00 321.00 2.47 hydrogen peroxide-inducible genes activator
    KEO98759.1*0.00 157.99 3-dehydroquinate synthaseKDM53905.1*46.80 192.00 4.10 3-dehydroquinate synthase
    KEO98794.1*0.00 345.72 branched-chain alpha-keto acid dehydrogenase subunit E2KDM57421.1*744.00 6140.00 8.25 dihydrolipoyllysine-residue acetyltransferase
    KEO98866.1*0.00 196.33 farnesyl-diphosphate synthaseKDM55391.1*75.50 519.00 6.87 farnesyl diphosphate synthase
    KEO98882.1*0.00 473.35 rRNA methyltransferaseKDM51417.1*107.00 496.00 4.64 tRNA (cytidine/uridine-2'-O-)-methyltransferase TrmJ
    KEO98935.1*0.00 3161.33 transcriptional regulatorKDM55471.1*0.00 120.00 HTH-type transcriptional regulator CueR
    KEO98999.1*0.00 246.42 nucleoside triphosphate hydrolaseKDM50489.1*85.80 233.00 2.72 nucleoside triphosphate pyrophosphohydrolase
    KEO99007.1*0.00 538.76 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthaseKDM50467.1*70.80 154.00 2.18 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase
    KEO99009.1*0.00 388.83 cyclaseKDM51170.1*35.60 155.00 4.35 hypothetical protein
    KEO99027.1*0.00 95.59 transketolaseKDM57420.1*1130.00 10900.00 9.65 pyruvate dehydrogenase E1 component
    KEO99055.1*0.00 1388.60 hypothetical proteinKDM55136.10.00 5.43 hypothetical protein
    *P-value < 0.05
     | Show Table
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    Peptidoglycan synthesis-related homologous genes (for example, mltA homolog) were significantly (p < 0.05) upregulated in both E. litoralis and L. amnigena enlarged spheroplasts (Table 1). Penicillin causes cell wall stress which might lead to upregulation of peptidoglycan synthesis.

    Chaperone homologous genes (for example, groES homolog) were also significantly upregulated in enlarged spheroplasts of both bacteria (Table 1), suggesting that a number of proteins may exist and be maintained in the enlarged spheroplasts. The expression of the phage shock protein homologous gene was very high in the enlarged spheroplasts (Table 1), suggesting that these cells were under stress.

    Homologs of lipopolysaccharide synthesis and transport-related genes (for example, lptB and lptF homologs) were upregulated in both E. litoralis and L. amnigena enlarged spheroplasts (Table 1). It is strongly suggested that these genes may be required to maintain the enlarged inner and outer membranes of these bacteria [12].

    Homologs of DNA replication initiator dnaA and recombinase recA genes were upregulated in both E. litoralis and L. amnigena enlarged spheroplasts (Table 1). DNA replication was not inhibited in the enlarged spheroplasts [6,7]. Thus, multiple chromosomal DNAs exist in a single cell. The DnaA and RecA homologs might function to maintain the multiple DNAs in the enlarged cells of both bacteria. The fact that enlarged spheroplasts have multiple copies of chromosome per cell supports the notion that chromosomal copy number is determined by the balance between DNA replication and cell division time [13].


    3.4. Downregulated orthologous genes in E. litoralis and L. amnigena enlarged spheroplasts

    The 51 downregulated genes in both the E. litoralis and L. amnigena enlarged spheroplasts are shown in Table 2. Of these 51 genes, 7 (13.7%) were homologous to inner and outer membrane protein coding genes, including 3 flagellar protein-encoding genes.

    Table 2.Orthologous genes downregulated (ratio < 0.5) in E. litoralis and L. amnigena enlarged spheroplasts.
    Protein ID of E. litoralisRPKM in E. litoralis spheroplasts at the beginning of growthRPKM in E. litoralis enlarged spheroplastsRatio of RPKMs of E. litoralisAnnotated functionProtein ID of L. amnigenaRPKM in L. amnigena spheroplasts at the beginning of growthRPKM in L. amnigena enlarged spheroplastsRatio of RPKMs of L. amnigenaAnnotated function
    Homologs of amino acid synthesis and metabolism-related genes
    KEO88954.1*616.47 136.50 0.22 L-asparaginaseKDM51897.1*33.40 0.00 0.00 L-asparaginase 1
    KEO89592.1*365.66 143.94 0.39 S-adenosylmethionine synthetaseKDM49649.1*1070.00 255.00 0.24 S-adenosylmethionine synthase
    KEO89916.1*363.48 0.00 0.00 2,3,4,5-tetrahydropyridine-2,6-carboxylate N-succinyltransferaseKDM57471.1*1050.00 432.00 0.41 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase
    KEO90596.1*298.26 88.06 0.30 threonine aldolaseKDM55097.1*509.00 172.00 0.34 low specificity L-threonine aldolase
    KEO92299.1*201.21 0.00 0.00 4-hydroxy-tetrahydrodipicolinate reductaseKDM57348.1*269.00 14.90 0.06 dihydrodipicolinate reductase
    KEO92640.1*250.39 0.00 0.00 argininosuccinate synthaseKDM54714.1*2220.00 219.00 0.10 argininosuccinate synthase
    KEO92798.1*507.05 149.70 0.30 3-isopropylmalate dehydrataseKDM57378.1*196.00 60.80 0.31 3-isopropylmalate dehydratase small subunit
    KEO93106.1*831.23 245.41 0.30 serine acetyltransferaseKDM52905.1*331.00 149.00 0.45 serine acetyltransferase
    KEO93391.1*1078.83 318.51 0.30 glutamine synthetaseKDM53324.1*385.00 174.00 0.45 glutamine synthetase
    KEO93480.1*1248.41 595.39 0.48 3-phosphoglycerate dehydrogenaseKDM49615.1*964.00 369.00 0.38 D-3-phosphoglycerate dehydrogenase
    KEO98853.1*596.53 0.00 0.00 ketol-acid reductoisomeraseKDM53647.1*3330.00 450.00 0.14 ketol-acid reductoisomerase
    KEO99046.1*292.53 0.00 0.00 anthranilate synthase component IKDM46686.1*1870.00 7.86 0.00 anthranilate synthase component 1
    KEO99050.1*459.56 45.23 0.10 anthranilate phosphoribosyltransferaseKDM46685.1*2870.00 53.90 0.02 anthranilate synthase component II
    Homologs of flagellar protein genes
    KEO89561.1*22111.90 337.67 0.02 flagellinKDM48656.1*124.00 44.90 0.36 hypothetical protein
    KEO90993.1*740.22 0.00 0.00 flagellar basal body rod protein FlgCKDM51808.1*41.90 0.00 0.00 flagellar basal-body rod protein flgC
    KEO92484.1*806.97 79.42 0.10 flagellar motor protein MotBKDM51720.1*24600.00 1390.00 0.06 outer membrane protein A
    Homologs of nucleotide synthesis and pentose phosphate pathway-related genes
    KEO91063.1*325.03 95.96 0.30 phosphoribosylpyrophosphate synthetaseKDM47000.1*985.00 298.00 0.30 ribose-phosphate pyrophosphokinase
    KEO93239.1*414.76 61.23 0.15 glucose-6-phosphate dehydrogenaseKDM48740.1*345.00 150.00 0.43 glucose-6-phosphate 1-dehydrogenase
    KEO93278.1*293.09 0.00 0.00 aspartate carbamoyltransferase catalytic subunitKDM56880.1*1090.00 65.90 0.06 aspartate carbamoyltransferase
    KEO96500.1*688.30 0.00 0.00 ribulose-phosphate 3-epimeraseKDM53902.1*200.00 54.40 0.27 ribulose-phosphate 3-epimerase
    KEO96518.1*349.69 103.24 0.30 nicotinate-nucleotide pyrophosphorylaseKDM57413.1*57.00 27.50 0.48 nicotinate-nucleotide pyrophosphorylase [carboxylating]
    KEO98799.1*610.90 45.09 0.07 transketolaseKDM49630.1*844.00 278.00 0.33 transketolase 1
    Homologs of other membrane protein genes
    KEO92879.1*12676.20 0.00 0.00 membrane proteinKDM46678.1*80.50 0.00 0.00 outer membrane protein W
    KEO93429.1*12522.20 4363.68 0.35 membrane proteinKDM54978.1*216.00 86.80 0.40 inner membrane protein YbhL
    KEO98493.1*726.95 268.28 0.37 ABC transporter ATP-binding proteinKDM57319.1*479.00 192.00 0.40 hypothetical protein
    KEO99262.1*1240.06 162.72 0.13 hypothetical proteinKDM52093.1*131.00 11.80 0.09 AI-2 transporter tqsA
    Others
    KEO89629.1*824.47 121.71 0.15 3-demethylubiquinone-9 3-methyltransferaseKDM46348.1*116.00 16.90 0.15 3-demethylubiquinone-9 3-methyltransferase
    KEO90016.1*338.03 49.90 0.15 hypothetical proteinKDM52077.1*18.60 0.00 0.00 HTH-type transcriptional regulator AbgR
    KEO90690.1*737.52 0.00 0.00 enoyl-ACP reductaseKDM46715.1*495.00 156.00 0.32 enoyl-[acyl-carrier-protein] reductase [NADH]
    KEO92246.1*756.79 111.72 0.15 glucose-1-phosphate thymidylyltransferaseKDM46209.1*58.80 28.30 0.48 glucose-1-phosphate thymidylyltransferase 2
    KEO92477.1*572.94 0.00 0.00 molybdopterin biosynthesis protein BKDM54974.1*232.00 47.90 0.21 molybdenum cofactor biosynthesis protein B
    KEO92492.1*852.18 125.80 0.15 oxidoreductaseKDM46695.1*312.00 80.60 0.26 hypothetical protein
    KEO92638.1*479.48 0.00 0.00 cyclopropane-fatty-acyl-phospholipid synthaseKDM52012.1*148.00 0.00 0.00 cyclopropane-fatty-acyl-phospholipid synthase
    KEO92662.1*1438.43 212.34 0.15 50S ribosomal protein L17KDM53831.1*354.00 128.00 0.36 50S ribosomal protein L17
    KEO92699.1*771.18 113.84 0.15 LysR family transcriptional regulatorKDM50509.1*37.00 0.00 0.00 glycine cleavage system transcriptional activator
    KEO93102.1*223.37 0.00 0.00 bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferaseKDM52744.1*372.00 108.00 0.29 glmU protein
    KEO93190.1*831.23 409.01 0.49 single-stranded DNA-binding proteinKDM56126.1*679.00 140.00 0.21 single-stranded DNA-binding protein
    KEO93395.1*438.05 64.66 0.15 cytosol aminopeptidaseKDM51408.1*158.00 47.70 0.30 peptidase B
    KEO96463.1*111.07 32.79 0.30 glutamate-ammonia-ligase adenylyltransferaseKDM54608.1*214.00 64.50 0.30 glutamate-ammonia-ligase adenylyltransferase
    KEO96716.1*396.13 116.95 0.30 GntR family transcriptional regulatorKDM52778.1*68.20 0.00 0.00 hypothetical protein
    KEO96769.1*581.14 171.57 0.30 ribonucleotide-diphosphate reductase subunit betaKDM46350.1*481.00 130.00 0.27 ribonucleoside-diphosphate reductase 1 subunit beta
    KEO98584.1*1114.39 0.00 0.00 transcriptional regulator HU subunit alphaKDM52452.1*2050.00 630.00 0.31 DNA-binding protein HU-alpha
    KEO98612.1*1352.13 0.00 0.00 50S ribosomal protein L31KDM53254.1*159.00 0.00 0.00 50S ribosomal protein L31
    KEO98687.1*228.14 84.19 0.37 phosphoenolpyruvate carboxylaseKDM53245.1*621.00 236.00 0.38 phosphoenolpyruvate carboxylase
    KEO98733.1*470.03 173.46 0.37 DNA topoisomerase IKDM46698.1*569.00 255.00 0.45 DNA topoisomerase 1
    KEO98800.1*603.63 0.00 0.00 glyceraldehyde-3-phosphate dehydrogenaseKDM51892.1*2900.00 1360.00 0.47 glyceraldehyde-3-phosphate dehydrogenase
    KEO98996.1*96.21 0.00 0.00 methionine--tRNA ligaseKDM46274.1*576.00 115.00 0.20 methionyl-tRNA synthetase
    KEO99011.1*473.88 139.91 0.30 carbonate dehydrataseKDM52008.1*26.70 0.00 0.00 hypothetical protein
    KEO99037.1*378.39 0.00 0.00 inorganic polyphosphate kinaseKDM51166.1*96.60 0.00 0.00 hypothetical protein
    KEO99041.1*4328.46 2008.15 0.46 heat-shock proteinKDM52775.1*41.30 0.00 0.00 small heat shock protein ibpA
    KEO99260.1*277.08 81.80 0.30 aminotransferaseKDM51415.1*1540.00 637.00 0.41 cysteine desulfurase
    *P-value < 0.05
     | Show Table
    DownLoad: CSV

    Homologs of amino acid and nucleotide synthesis-related genes and pentose phosphate pathway genes were significantly downregulated in both E. litoralis and L. amnigena enlarged spheroplasts (Table 2). This indicated that amino acid and nucleotide synthesis were repressed in enlarged spheroplasts. Repression of nucleotide synthesis is consistent with the reduced speed of DNA replication [6,7]. In addition, homologs of flagellar protein genes were significantly downregulated in both E. litoralis and L. amnigena enlarged spheroplasts (Table 2), which is consistent with absence of motility in the spheroplasts of both bacteria.

    The nucleoid protein HU homologous gene was significantly downregulated in both E. litoralis and L. amnigena enlarged spheroplasts (Table 2). Cross-talk between HU and topoisomerase I has been reported in E. coli [14]. Interestingly, topoisomerase I homologous gene was also significantly downregulated in both enlarged spheroplasts (Table 2). The downregulation of both HU and topoisomerase I homologous genes may be one of the characteristics of enlarged spheroplasts. Thus, in enlarged spheroplasts, recombination may be repressed in multiple copies of chromosomal DNAs.


    3.5. Orthologous genes with differential expression between E. litoralis and L. amnigena spheroplasts

    The genes with differential expression between E. litoralis and L. amnigena enlarged spheroplasts are shown in Tables 3 and 4.

    Table 3.Orthologous genes upregulated in E. litoralis enlarged spheroplasts and downregulated in L. amnigena enlarged spheroplasts.
    Protein ID of E. litoralisRPKM in E. litoralis spheroplasts at the beginning of growthRPKM in E. litoralis enlarged spheroplastsRatio of RPKMs of E. litoralisAnnotated functionProtein ID of L. amnigenaRPKM in L. amnigena spheroplasts at the beginning of growthRPKM in L. amnigena enlarged spheroplastsRatio of RPKMs of L. amnigenaAnnotated function
    Homologs of membrane protein genes
    KEO89564.1*0.00 106.93 hypothetical proteinKDM48649.1*40.40 14.60 0.36 flagellar M-ring protein
    KEO89607.1*0.00 129.05 multidrug transporterKDM52010.1*150.00 45.10 0.30 multidrug resistance protein mdtK
    KEO89895.1*0.00 634.32 biopolymer transporter ExbBKDM55699.1*564.00 177.00 0.31 protein tolQ
    KEO89973.1*0.00 184.81 magnesium transporter CorAKDM52382.1*661.00 168.00 0.25 magnesium transporter CorA
    KEO92632.1*0.00 146.76 hypothetical proteinKDM46187.1*175.00 72.30 0.41 inner membrane transporter YeeF
    KEO92658.1*0.00 1301.72 hypothetical proteinKDM57464.1*136.00 39.40 0.29 UPF0126 inner membrane protein yadS
    KEO92812.1*0.00 172.56 type VI secretion proteinKDM54463.1*51.90 12.50 0.24 hypothetical protein
    KEO93024.1*262.72 2171.79 8.27 iron transporterKDM53929.1*413.00 171.00 0.41 Fe/S biogenesis protein nfuA
    KEO93116.1*0.00 227.97 organic solvent tolerance proteinKDM57361.1*483.00 224.00 0.46 LPS-assembly protein lptD
    KEO93384.1*352.12 1143.53 3.25 MttB family proteinKDM52402.1*220.00 15.90 0.07 sec-independent protein translocase tatC
    KEO98525.1*0.00 128.22 hypothetical proteinKDM52772.1*118.00 38.00 0.32 D-galactonate transporter
    KEO98837.1*0.00 215.39 phosphate ABC transporter permeaseKDM52748.1*724.00 152.00 0.21 phosphate transport system permease pstA
    KEO98838.1*0.00 194.41 phosphate ABC transporter permeaseKDM52747.1*1310.00 230.00 0.18 phosphate transport system permease pstC
    KEO98907.1*243.77 539.78 2.21 3,4-dihydroxy-2-butanone 4-phosphate synthaseKDM54605.1*1220.00 432.00 0.35 3,4-dihydroxy-2-butanone 4-phosphate synthase
    Others
    KEO88945.1*1061.88 3056.67 2.88 peptide deformylaseKDM53823.1*699.00 313.00 0.45 peptide deformylase
    KEO88946.1*0.00 300.90 recombinase RecRKDM55460.1*336.00 101.00 0.30 recombination protein recR
    KEO88958.1*0.00 546.11 argininosuccinate lyaseKDM53241.1*1610.00 116.00 0.07 argininosuccinate lyase
    KEO88960.1*484.06 1822.10 3.76 diaminopimelate decarboxylaseKDM50540.1*780.00 19.50 0.03 diaminopimelate decarboxylase
    KEO89297.1*0.00 160.53 histidinol-phosphate aminotransferaseKDM46192.1*240.00 92.60 0.39 histidinol-phosphate aminotransferase
    KEO89579.1*0.00 507.45 tRNA (guanine-N7)-methyltransferaseKDM54472.1*802.00 154.00 0.19 tRNA (guanine-N(7)-)-methyltransferase
    KEO89635.1*0.00 207.20 formyltetrahydrofolate deformylaseKDM46646.1*80.60 14.60 0.18 formyltetrahydrofolate deformylase
    KEO89998.1*0.00 437.71 ArsR family transcriptional regulatorKDM55050.1*52.90 0.00 0.00 hypothetical protein
    KEO90041.1*0.00 118.57 hypothetical proteinKDM48627.1*48.00 0.00 0.00 DNA-cytosine methyltransferase
    KEO90698.1*0.00 438.68 3-deoxy-manno-octulosonate cytidylyltransferaseKDM55141.1*91.00 0.00 0.00 3-deoxy-manno-octulosonate cytidylyltransferase
    KEO91020.1*0.00 391.37 molecular chaperone DnaKKDM57452.1*5740.00 2210.00 0.39 DnaK suppressor protein
    KEO91036.1*0.00 883.47 molecular chaperone Hsp33KDM53917.1*77.30 28.00 0.36 chaperonin
    KEO91037.1*0.00 191.92 ornithine carbamoyltransferaseKDM56877.1*16.90 0.00 0.00 ornithine carbamoyltransferase, catabolic
    KEO92302.1*0.00 465.99 molybdopterin biosynthesis protein MoeBKDM52444.1*112.00 16.30 0.15 sulfur carrier protein ThiS adenylyltransferase
    KEO92334.1*113.82 285.62 2.51 aconitate hydrataseKDM46701.1*305.00 152.00 0.50 aconitate hydratase 1
    KEO92372.1*0.00 272.18 phosphomethylpyrimidine kinaseKDM46258.1*42.40 15.30 0.36 hydroxymethylpyrimidine/phosphomethylpyrimidine kinase
    KEO92402.1*0.00 190.30 glutamyl-tRNA synthetaseKDM51312.1*216.00 86.80 0.40 glutamyl-tRNA synthetase
    KEO92434.1*0.00 266.53 2-hydroxyacid dehydrogenaseKDM52411.1*69.70 25.20 0.36 glyoxylate/hydroxypyruvate reductase B
    KEO92503.1*0.00 462.99 inosine-5-monophosphate dehydrogenaseKDM51383.1*417.00 117.00 0.28 inosine-5'-monophosphate dehydrogenase
    KEO92549.1*0.00 490.81 30S ribosomal protein S6KDM56939.1*944.00 217.00 0.23 30S ribosomal protein S6
    KEO92597.1*0.00 368.72 tryptophan synthase subunit betaKDM46683.1*3260.00 134.00 0.04 tryptophan synthase beta chain
    KEO92634.1*0.00 460.61 oxidoreductaseKDM52065.1*23.50 0.00 0.00 dihydrofolate reductase folM
    KEO92667.1*0.00 278.51 adenylate kinaseKDM55462.1*1950.00 914.00 0.47 adenylate kinase
    KEO92711.1*0.00 424.08 3-isopropylmalate dehydrogenaseKDM57380.1*280.00 113.00 0.40 3-isopropylmalate dehydrogenase
    KEO92758.1*0.00 1060.85 DNA-binding proteinKDM51729.1*7260.00 966.00 0.13 heat shock protein hspQ
    KEO92776.1*0.00 1339.41 ribosome maturation protein RimPKDM54713.1*187.00 81.40 0.44 ribosome maturation factor rimP
    KEO92780.1*0.00 511.79 ATPase AAAKDM46723.1*52.10 25.10 0.48 psp operon transcriptional activator
    KEO92795.1*0.00 399.20 thioesteraseKDM55422.1*298.00 61.60 0.21 long-chain acyl-CoA thioesterase tesC
    KEO92811.1*0.00 81.47 polyphosphate kinaseKDM51368.1*437.00 107.00 0.24 polyphosphate kinase
    KEO93025.1*499.56 1179.89 2.36 malonic semialdehyde reductaseKDM51751.1*57.50 20.80 0.36 hypothetical protein
    KEO93125.1*0.00 144.29 succinylarginine dihydrolaseKDM51916.1*89.70 27.80 0.31 N-succinylarginine dihydrolase
    KEO93177.1*0.00 687.39 aromatic amino acid aminotransferaseKDM55151.1*385.00 124.00 0.32 aspartate aminotransferase
    KEO93181.1*0.00 286.50 riboflavin synthase subunit alphaKDM52011.1*317.00 76.60 0.24 riboflavin synthase alpha chain
    KEO93285.1*1214.49 3764.87 3.10 bacterioferritinKDM53859.1*463.00 77.30 0.17 bacterioferritin
    KEO93379.1*0.00 350.17 beta-hexosaminidaseKDM51842.1*248.00 71.90 0.29 beta-hexosaminidase
    KEO93387.1*0.00 283.79 threonine dehydrataseKDM54671.1*51.50 0.00 0.00 threonine dehydratase catabolic
    KEO93441.1*0.00 293.53 cystathionine beta-lyaseKDM54516.1*285.00 92.90 0.33 cystathionine beta-lyase
    KEO93476.1*0.00 3077.91 hypothetical proteinKDM54633.1*68.60 24.80 0.36 hypothetical protein
    KEO96380.1*0.00 130.17 protein-PII uridylyltransferaseKDM57472.1*108.00 32.10 0.30 [protein-PII] uridylyltransferase
    KEO96408.1*0.00 407.90 GTP cyclohydrolaseKDM46702.1*287.00 104.00 0.36 GTP cyclohydrolase-2
    KEO96434.1*131.11 377.39 2.88 glutamate synthaseKDM54762.1*2310.00 320.00 0.14 glutamate synthase [NADPH] large chain
    KEO96435.1*0.00 156.26 glutamate synthaseKDM54763.1*1780.00 320.00 0.18 glutamate synthase [NADPH] small chain
    KEO96528.1*0.00 211.70 exodeoxyribonuclease VII large subunitKDM51384.1*74.20 0.00 0.00 exodeoxyribonuclease 7 large subunit
    KEO96545.1*0.00 814.69 AsnC family transcriptional regulatorKDM51416.1*2800.00 150.00 0.05 HTH-type transcriptional regulator iscR
    KEO98460.1*0.00 1164.32 50S ribosomal protein L27KDM54727.1*1840.00 667.00 0.36 50S ribosomal protein L27
    KEO98556.1*0.00 200.94 citrate lyaseKDM54480.1*19.40 0.00 0.00 citrate lyase subunit beta
    KEO98575.1*300.92 1066.10 3.54 farnesyltranstransferaseKDM54729.1*175.00 25.30 0.14 octaprenyl-diphosphate synthase
    KEO98712.1*0.00 412.01 thioredoxin reductaseKDM55115.1*351.00 88.80 0.25 thioredoxin reductase
    KEO98796.1*0.00 1374.78 5-formyltetrahydrofolate cyclo-ligaseKDM49727.1*85.40 41.20 0.48 5-formyltetrahydrofolate cyclo-ligase
    KEO98834.1*0.00 260.35 chemotaxis protein CheYKDM55825.1*2140.00 142.00 0.07 phosphate regulon transcriptional regulatory protein phoB
    KEO98839.1*0.00 180.36 histidine kinaseKDM55824.1*380.00 47.40 0.12 phosphate regulon sensor protein phoR
    KEO98906.1*0.00 424.68 6,7-dimethyl-8-ribityllumazine synthaseKDM55386.1*325.00 157.00 0.48 6,7-dimethyl-8-ribityllumazine synthase
    KEO98922.1*0.00 653.71 50S ribosomal protein L25KDM46330.1*477.00 129.00 0.27 50S ribosomal protein L25
    KEO98968.1*0.00 712.85 nucleoside-triphosphate diphosphataseKDM54467.1*57.20 0.00 0.00 nucleoside-triphosphatase rdgB
    KEO98978.1*0.00 229.80 DNA repair protein RadAKDM57315.1*282.00 106.00 0.38 DNA repair protein radA
    KEO99042.1*0.00 438.68 CTP synthaseKDM50488.1*871.00 353.00 0.41 CTP synthase
    *P-value < 0.05
     | Show Table
    DownLoad: CSV
    Table 4.Orthologous genes downregulated in E. litoralis enlarged spheroplasts and upregulated in L. amnigena enlarged spheroplasts.
    Protein ID of E. litoralisRPKM in E. litoralis spheroplasts at the beginning of growthRPKM in E. litoralis enlarged spheroplastsRatio of RPKMs of E. litoralisAnnotated functionProtein ID of L. amnigenaRPKM in L. amnigena spheroplasts at the beginning of growthRPKM in L. amnigena enlarged spheroplastsRatio of RPKMs of L. amnigenaAnnotated function
    Homologs of membrane protein genes
    KEO89304.1*629.44 247.78 0.39 penicillin-binding proteinKDM57456.1*296.00 1010.00 3.41 penicillin-binding protein 1B
    KEO89573.1*405.64 59.88 0.15 flagellar biosynthesis protein flipKDM48639.1*0.00 16.70 flagellar biosynthetic protein fliP
    KEO90985.1*453.73 133.96 0.30 hypothetical proteinKDM51816.1*0.00 22.50 flagellar hook-associated protein 1
    KEO90987.1*2625.04 163.16 0.06 flagellar P-ring protein FlgIKDM51814.1*15.50 56.00 3.61 flagellar P-ring protein
    KEO90994.1*859.40 0.00 0.00 flagellar basal body rod protein FlgBKDM51807.1*0.00 147.00 flagellar basal-body rod protein flgB
    KEO92489.1*458.87 135.47 0.30 ABC transporterKDM51852.1*72.60 175.00 2.41 lipoprotein-releasing system ATP-binding protein LolD
    KEO92547.1*320.92 0.00 0.00 ABC transporterKDM48630.1*0.00 13.50 inner membrane protein yedI
    KEO92700.1*2204.56 0.00 0.00 magnesium transporter ApaGKDM57357.1*89.90 488.00 5.43 protein ApaG
    KEO93367.1*2069.58 111.09 0.05 secretion system proteinKDM53908.1*0.00 9.92 hypothetical protein
    KEO93416.1*284.06 0.00 0.00 phospho-N-acetylmuramoyl-pentapeptide-transferaseKDM57393.1*204.00 817.00 4.00 phospho-N-acetylmuramoyl-pentapeptide-transferase
    KEO93419.1*495.89 109.80 0.22 UDP-diphospho-muramoylpentapeptide beta-N-acetylglucosaminyltransferaseKDM57396.1*79.70 415.00 5.21 undecaprenyldiphospho-muramoylpentapeptide beta-N-acetylglucosaminyltransferase
    KEO93424.1*682.13 134.26 0.20 cell division protein FtsAKDM57400.1*865.00 2250.00 2.60 cell division protein ftsA
    KEO98787.1*1321.30 373.84 0.28 preprotein translocase subunit SecAKDM57404.1*69.00 559.00 8.10 protein translocase subunit secA
    KEO98980.1*184.72 54.53 0.30 major facilitator transporterKDM46178.1*64.50 159.00 2.47 shikimate transporter
    Others
    KEO89313.1*2157.65 0.00 0.00 peptide methionine sulfoxide reductaseKDM56920.1*0.00 19.10 peptide methionine sulfoxide reductase msrA
    KEO89605.1*7190.86 2640.13 0.37 molecular chaperone GroELKDM56992.1*1220.00 4660.00 3.82 chaperonin
    KEO89995.1*402.88 33.98 0.08 5-methyltetrahydrofolate--homocysteine methyltransferaseKDM55766.1*111.00 223.00 2.01 methionine synthase
    KEO90687.1*897.43 132.48 0.15 pyridoxamine 5'-phosphate oxidaseKDM52037.1*0.00 37.40 pyridoxine/pyridoxamine 5'-phosphate oxidase
    KEO91004.1*607.24 0.00 0.00 hypothetical proteinKDM49677.1*375.00 1380.00 3.68 protein yciF
    KEO91044.1*382.68 0.00 0.00 glucose-methanol-choline oxidoreductaseKDM55555.1*51.00 185.00 3.63 choline dehydrogenase
    KEO91076.1*644.55 190.30 0.30 phosphomannomutaseKDM46221.1*99.10 1120.00 11.30 phosphomannomutase
    KEO92378.1*1931.61 855.42 0.44 RNA polymerase sigma70KDM51449.1*2570.00 7420.00 2.89 RNA polymerase sigma-E factor
    KEO92529.1*224.36 66.24 0.30 hypothetical proteinKDM57043.1*0.00 6.34 hypothetical protein
    KEO92652.1*1365.79 554.44 0.41 succinate--CoA ligaseKDM55690.1*566.00 1450.00 2.56 succinyl-CoA ligase [ADP-forming] subunit alpha
    KEO92654.1*2162.76 141.89 0.07 dihydrolipoamide succinyltransferaseKDM55688.1*415.00 1100.00 2.65 hypothetical protein
    KEO92656.1*2572.76 822.87 0.32 dihydrolipoamide dehydrogenaseKDM57422.1*775.00 3320.00 4.28 dihydrolipoyl dehydrogenase
    KEO92681.1*1049.06 206.48 0.20 50S ribosomal protein L16KDM53849.1*537.00 2360.00 4.39 50S ribosomal protein L16
    KEO93425.1*1877.96 607.24 0.32 cell division protein FtsZKDM57401.1*1180.00 5490.00 4.65 cell division protein ftsZ
    KEO93475.1*113.94 0.00 0.00 ATPase AAAKDM55119.1*88.50 311.00 3.51 replication-associated recombination protein A
    KEO96455.1*712.48 35.06 0.05 UDP-N-acetylglucosamine 1-carboxyvinyltransferaseKDM54731.1*162.00 351.00 2.17 UDP-N-acetylglucosamine 1-carboxyvinyltransferase
    KEO96456.1*1034.79 101.84 0.10 UTP--glucose-1-phosphate uridylyltransferaseKDM46650.1*206.00 678.00 3.29 UTP-glucose-1-phosphate uridylyltransferase
    KEO96517.1*762.48 0.00 0.00 chemotaxis protein CheYKDM46276.1*0.00 68.30 hypothetical protein
    KEO96548.1*291.41 86.03 0.30 dihydroorotate dehydrogenaseKDM51709.1*16.80 72.90 4.34 dihydroorotate dehydrogenase
    KEO98611.1*618.35 0.00 0.00 3-hydroxyacyl-ACP dehydrataseKDM57485.1*74.50 350.00 4.70 (3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase
    KEO99053.1*249.16 73.56 0.30 molybdopterin biosynthesis proteinKDM55029.1*68.90 149.00 2.16 molybdopterin molybdenumtransferase
    KEO99263.1*1114.39 0.00 0.00 glutamine amidotransferaseKDM54696.1*327.00 852.00 2.61 protein yhbO
    *P-value < 0.05
     | Show Table
    DownLoad: CSV

    As mentioned above, the recA homolog was upregulated in both enlarged spheroplasts (Table 1). However, the DNA recombinase radA homolog was upregulated in E. litoralis enlarged spheroplasts, but was downregulated in L. amnigena enlarged spheroplasts (Table 3). In the enlarged spheroplasts, the expression level of the E. litoralis recA homolog was much higher than that of L. amnigena, suggesting that higher requirement for DNA recombinase in E. litoralis. Although we should estimate the functional difference between RecA and RadA homologs [15], it is suggested that the RadA homolog might be needed in addition to the RecA homolog in E. litoralis enlarged spheroplasts.

    The secA homolog was upregulated in L. amnigena enlarged spheroplasts but was downregulated in E. litoralis enlarged spheroplasts (Table 4). In a previous study [4], SecY, a major component of the Sec complex, was strongly detected in the vacuole-like structure membrane of E. coli. In our findings, the expression level of the secY homolog was upregulated (1.57-fold) in L. amnigena spheroplasts but was downregulated (0.84-fold) in E. litoralis enlarged spheroplasts (Supplementary Table 1). Thus, it is strongly suggested that the SecA homolog (and the SecY homolog) is associated with generation of the vacuole-like structure in L. amnigena.

    The homologs of cell division-related genes ftsA and ftsZ were upregulated in L. amnigena enlarged spheroplasts but were downregulated in E. litoralis enlarged spheroplasts (Table 4), suggesting that these two genes may be also associated with the generation of vacuole-like structures.

    Penicillin binding protein 1B homologous gene was upregulated in L. amnigena enlarged spheroplasts and was downregulated in E. litoralis (Table 4). E. coli spheroplasts resynthesize their cell wall and shape without penicillin [16], and the penicillin binding protein 1B was required for the shape recovery [16]. Upon transferring the enlarged spheroplasts to a penicillin-free broth, the shape recovery rate of L. amnigena may be higher than that of E. litoralis.

    The Mg2+ transport system was contrasting between the E. litoralis and L. amnigena enlarged spheroplasts. The Mg2+ influx-related gene corA [17] was upregulated in E. litoralis enlarged spheroplasts but was downregulated in L. amnigena enlarged spheroplasts (Table 3). On the other hand, the Mg2+ efflux-related gene apaG (corD) [17] was downregulated in E. litoralis but was upregulated in L. amnigena (Table 4). These results strongly suggest that E. litoralis enlarged spheroplasts require Mg2+ but the L. amnigena enlarged spheroplasts discard it.


    4. Conclusion

    We showed that spheroplast enlargement varies between E. litoralis and L. amnigena. The changes in expression of the orthologous genes also vary between enlarged spheroplasts of the two bacteria. Based on the different gene expression pattern, the candidate genes related to the cell structural differences were extracted in this study.


    Acknowledgements

    We thank Dr. Shinji Kondo for his valuable comments and suggestion. This work was supported by grant from The Cannon Foundation.


    Conflict of Interest

    The authors declare that there is no conflict of interest regarding the publication of this paper.


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