biology

Article Metabolites Produced by Kaistia sp. 32K Promote Biofilm Formation in Coculture with Methylobacterium sp. ME121

Yoshiaki Usui 1, Tetsu Shimizu 2, Akira Nakamura 2 and Masahiro Ito 1,3,* 1 Graduate School of Life Sciences, Toyo University, Oura-gun, Gunma 374-0193, Japan; [email protected] 2 Faculty of Life and Environmental Sciences, and Microbiology Research Center for Sustainability (MiCS), University of Tsukuba, Tsukuba, Ibaraki305-8572, Japan; [email protected] (T.S.); [email protected] (A.N.) 3 Bio-Nano Electronics Research Centre, Toyo University, Kawagoe, Saitama 350-8585, Japan * Correspondence: [email protected]; Tel.: +81-273-82-9202



Received: 31 July 2020; Accepted: 11 September 2020; Published: 13 September 2020


Abstract: Previously, we reported that the coculture of motile Methylobacterium sp. ME121 and non-motile Kaistia sp. 32K, isolated from the same soil sample, displayed accelerated motility of strain ME121 due to an extracellular polysaccharide (EPS) produced by strain 32K. Since EPS is a major component of biofilms, we aimed to investigate the biofilm formation in cocultures of the two strains. The extent of biofilm formation was measured by a microtiter dish assay with the dye crystal violet. A significant increase in the amount of biofilm was observed in the coculture of the two strains, as compared to that of the monocultures, which could be due to a metabolite produced by strain 32K. However, in the coculture with strain 32K, using Escherichia coli or Pseudomonas aeruginosa, there was no difference in the amount of biofilm formation as compared with the monoculture. Elevated biofilm formation was also observed in the coculture of strain ME121 with Kaistia adipata, which was isolated from a different soil sample. Methylobacterium radiotolerans, isolated from another soil sample, showed a significant increase in biofilm formation when cocultured with K. adipata, but not with strain 32K. We also found that the culture supernatants of strains 32K and K. adipata accelerated the motility of strains ME121 and M. radiotolerans, wherein culture supernatant of K. adipata significantly increased the motility of M. radiotolerans, as compared to that by the culture supernatant of strain 32K. These results indicated that there was a positive relationship between accelerated motility and increased biofilm formation in Methylobacterium spp. This is the first study to report that the metabolites from Kaistia spp. could specifically modulate the biofilm-forming ability of Methylobacterium spp. Methylobacterium spp. biofilms are capable of inhibiting the biofilm formation of mycobacteria, which are opportunistic pathogens that cause problems in infectious diseases. Thus, the metabolites from the culture supernatant of Kaistia spp. have the potential to contribute to the environment in which increased biofilm production of Methylobacterium is desired.

Keywords: coculture; biofilm; Methylobacterium; Kaistia; motility; Methylobacterium radiotolerans; Kaistia adipata

1. Introduction Methylobacterium sp. ME121 and Kaistia sp. 32K were isolated from the same soil sample, by chance, during screening for L-glucose-assimilating bacteria, and their growth was enhanced by coculture [1,2]. Methylobacterium species are characterized as Gram-negative non-spore-forming bacilli, facultative methylotrophs which form pink-pigmented colonies on agar plates and have been widely observed to live on plant leaves, in soil, and in chlorinated tap water [3,4]. Methylobacterium species are also found

Biology 2020, 9, 287; doi:10.3390/biology9090287 www.mdpi.com/journal/biology Biology 2020, 9, 287 2 of 14 in clinical samples as opportunist pathogens [4,5]. Kaistia species are characterized as Gram-negative non-spore-forming rod to coccus, strictly aerobic, chemoorganotrophic bacteria that form mucoid colonies on agar plates and have been observed to inhabit environments such as soil, wetland, and river sediments [6–9]. Strain ME121 is motile and has unipolar flagella, whereas strain 32K is non-motile and has no flagella [10]. In a previous study, we discovered that the motility of strain ME121 was accelerated in the coculture of strains ME121 and 32K because of an extracellular polysaccharide (EPS) produced by strain 32K [10]. The EPS derived from strain 32K was designated as K factor; it is composed of glucose and galactose in a ratio of ca. 1:1 and accounts for about 55% of the total weight of purified K factor. EPS is a major component of the biofilm complex [11,12]. A biofilm is a collection of microbial cells attached to a surface and surrounded by a matrix of exopolymer substances [13]. Biofilms are harmful to humans, as they cause decay of foods and are one of the causes of nosocomial infections [14]. In a medical field, such as a hospital, it is difficult to remove a biofilm formed inside an injection needle or a catheter, and this leads to nosocomial infections. In an actual medical field facing the problems, the development of a method for biofilm removal without using chemical agents, such as bactericides and antibiotics, is required, keeping in mind the health of people and resistance mechanisms of microorganisms [15]. Biofilm formation is closely related to bacterial motility, and its role in adhesion to solid surfaces [16,17]; during their life cycle, bacteria exist either as planktonic cells, wherein they explore the aqueous environments, or as biofilm complexes. The switch from the planktonic phase to the sessile phase involves a slowdown of metabolic activities and production of a complex mixture of the extracellular matrix, extracellular polysaccharides (EPS), proteins, and nucleic acids [18,19]. Cyclic di-GMP (c-di-GMP) is an intracellular signal transduction molecule that regulates the transition from the motile phase to the inactive phase in the bacterial cells [20]. High intracellular c-di-GMP concentrations promote biofilm formation, while low intracellular c-di-GMP levels promote motility [21]. The determinants of c-di-GMP-modulated biofilms include flagellar rotation to type IV pili contraction, EPS production, surface adhesion expression, antimicrobial resistance, and other stresses [20]. It also decreases biofilm-forming ability when motile microorganisms lose their motility [18]. Hence, we hypothesized that the biofilm-forming ability of motile microorganisms might increase with accelerated motility. Therefore, in this study, we investigated whether there was a difference in biofilm formation in monocultures and coculture of strains ME121 and 32K. In addition, we investigated whether the same effect was observed in cocultures with other Methylobacterium and Kaistia strains.

2. Materials and Methods

2.1. Bacterial Strains and Growth Media Bacterial strains Methylobacterium sp. ME121 [2], Methylobacterium radiotolerans [19], Kaistia sp. 32K [2], Kaistia adipata [6], E. coli W3110 [20], and P. aeruginosa PAO1 [21] were used in this study. Generally, Methylobacterium spp. has the chemotactic ability and also has chemotaxis-related genes [22]. E. coli W3110 was gifted from R. Aono. M. radiotolerans, K. adipata, and P. aeruginosa PAO1 were purchased from Japan Collection Microorganisms (JCM). Strains ME121 and M. radiotolerans were precultured in Met medium (10.0 g of peptone, 2.0 g of yeast extract, 1.0 g of MgSO4, and 5 mL of methanol per liter), and strains 32K and K. adipata were precultured in LM medium (10.0 g of tryptone, 5.0 g of yeast extract, and 1.0 g of d-mannitol per liter). Methanol was filter-sterilized with Millex-LG filters (Millipore, pore size: 0.2 µm). E. coli W3110 and P. aeruginosa PAO1 were precultured in Miller’s LB broth (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter). E. coli W3110 and P. aeruginosa PAO1 were used as the reference strains for the biofilm formation assay of strains ME121 and 32K. d-glucose synthetic medium (Glc medium) (1.07 g of NH4Cl, 0.81 g of MgCl2, 0.75 g of KCl, 1.74 g of KH2PO4, 1.36 g of K2HPO4, 2 mL of Hutner’s trace elements, and 0.90 g of d-glucose per liter) was used for the monocultures and cocultures. Hutner’s trace elements were prepared by dissolving Biology 2020, 9, 287 3 of 14

22.0 g of ZnSO 7H O, 11.4 g of H BO , 5.06 g of MnCl 7H O, 1.16 g of CoCl 5H O, 1.57 g of 4· 2 3 3 2· 2 2· 2 (NH ) Mo O 4H O, and 1.57 g of FeSO 7H O in 500 mL of sterile water. EDTA 2Na (50 g) was 4 6 7 24· 2 4· 2 · dissolved while heating 300 mL of Milli-Q water. The pH was adjusted within the 6.5–6.8 range with KOH after the addition of each component, and the final volume was adjusted to 1 L. The solution was stored at 4 ◦C, for approximately two weeks, until its color changed from light green to purple-red. It was then filter-sterilized and used.

2.2. Monoculture and Coculture Conditions

Strains ME121 and 32K were cultured in 10 mL of Met and LM medium, respectively (28 ◦C, 300 rpm, and 48 h), harvested by centrifugation (4 C, 9100 g, 5 min), and then each pellet was washed ◦ × with saline and suspended in 2 mL of Glc medium. For monoculture, the cells were inoculated in Φ24 mm test tubes containing 10 mL of Glc medium to ensure an initial optical density (OD600) of 0.08, and the tubes were incubated at 28 ◦C and 300 rpm for 24 h. For the coculture, 5 mL of each bacterial suspension (OD600 = 0.08) was mixed in the same test tube and incubated at 28 ◦C and 300 rpm for 24 h.

2.3. Preparation of the Culture Supernatant of Strain ME121

Strain ME121 was cultured in 50 mL Met medium at 28 ◦C and 200 rpm for 48 h. The culture was then centrifuged (4 C, 9100 g, 5 min), and the cell pellet was resuspended in 50 mL of saline and ◦ × centrifuged again (4 C, 9100 g, 5 min); the pellet was inoculated in a 500 mL conical flask containing ◦ × 200 mL of Glc medium to achieve an initial OD600 of 0.08 and incubated at 28 ◦C and 200 rpm for 48 h. The culture supernatant was harvested by centrifugation (4 C, 9100 g, 30 min), and it (200 mL) was ◦ × sterilized by using a Nalgene Rapid-Flow Polyethersulfone (PES) membrane filter unit (pore diameter: 0.2 µm; Thermo Fisher Scientific).

2.4. Preparation of Culture Supernatant of Strains 32K and K. adipata

Strains 32K and K. adipata were cultured in 100 mL of LM medium at 28 ◦C and 200 rpm for 48 h; the cells were then centrifuged (4 C, 9100 g, 5 min), and each cell culture was resuspended in 50 mL ◦ × of saline and centrifuged again (4 C, 9100 g, 5 min). Each cell pellet was inoculated in a 500 mL ◦ × conical flask containing 200 mL of Glc medium to achieve an initial OD600 of 0.08 and incubated at 28 C and 200 rpm for 48 h. The culture supernatant was harvested by centrifugation (4 C, 9100 g, ◦ ◦ × 30 min), and it was sterilized by using a Nalgene Rapid-Flow Polyethersulfone (PES) membrane filter unit (pore diameter: 0.2 µm; Thermo Fisher Scientific).

2.5. Biofilm Formation Assay A single colony of strains ME121 and M. radiotolerans inoculated in Met medium, and a single colony of strains 32K and K. adipata inoculated in LM medium were subjected to reciprocal shaking at 28 ◦C and 300 rpm for 24 h. E. coli W3110 and P. aeruginosa PAO1 were subjected to reciprocal shaking at 37 ◦C and 200 rpm, for 16 h, in 2 mL of LB medium. Glc medium, culture supernatant of strain 32K, culture supernatant of strain ME121, and culture supernatant of K. adipata were each added, separately, in 5 mL batches, to a Falcon 24-well flat-bottom microplate (polystyrene, CORNING), and the culture medium was adjusted to an initial OD600 of 0.01. The compartment containing only the medium served as the negative control. Glc medium was used for the dilution of the supernatant. After culturing, 0.5 mL of the culture broth was removed, and 0.5 mL of 0.1% crystal violet solution was added dropwise. Growth was evaluated by measuring the OD600 of the culture solution. Staining of the biofilm was carried out for 30 min, under static conditions, at 25 ◦C. The removal of the supernatant and the dropwise addition of the solution were performed gently, to prevent any damage to the biofilm. The crystal violet solution was removed, and the biofilm was washed by adding and removing 0.75 mL of sterilized water. This process was performed three times. The microplate was placed in a 55 ◦C dry heater, for 10 min, to remove excess water. Then, 1 mL of 99.5% ethanol solution was Biology 2020, 9, 287 4 of 14 added dropwise, and pipetting was performed. After leaving the plate still for 30 min, crystal violet decolorized from the biofilm. Then, Abs570 was measured to determine the amount of biofilm formed. The same independent experiment was performed at least more than three times.

2.6. Swimming Speed of Strain ME121 and M. radiotolerans in the Culture Supernatant of Strain 32K and K. Adipata Single colonies of strains ME121 and M. radiotolerans were inoculated in 10 mL of Met medium (separately) and incubated at 28 ◦C and 300 rpm for 48 h; the preculture broth (50 µL) was inoculated into a test tube containing 10 mL of Met medium and incubated at 28 ◦C and 300 rpm for 24 h. The culture broth (1 mL) was centrifuged (room temperature, 9100 g, 3 min), and the cells of strains × ME121 and M. radiotolerans were resuspended in 1 mL of the swimming assay medium. Three types of swimming assay media were tested: (i) Glc medium, (ii) culture supernatant of strain 32K, and (iii) culture supernatant of K. adipata. Immediately after suspension, the microbial cells were kept at 28 ◦C, on a glass heater; the motility of the bacteria was observed with a dark-field microscope. We recorded a video of the observed movements for about 5 s and the moving distance per second (40 frames), using a digital color camera (Leica DF310 FX). The speed of each swimming cell was calculated by using 2D movement measurement capture 2D-PTV software (Digimo, Tokyo, Japan) and analysis of the captured movie [23].

2.7. Statistical Analysis All the results were analyzed by appropriate statistical methods (one-way analysis of variance (ANOVA), two-way ANOVA, and post hoc analysis (Tukey’s multiplex test) by the BellCurve for Excel, version 3.21, Social Survey Research Information Co., Ltd., Tokyo, Japan). Tukey test data for post hoc analysis of the results of Figures1–6 are listed in Supplementary Materials Tables S1–S6.

3. Results

3.1. Biofilm Formation during the Coculture of Bacterial Strains ME121 and 32K Biofilm formation assay was performed, using Glc medium (Figure1A). No biofilm was observed in the monoculture of strain ME121. On the other hand, biofilm was formed in the monoculture of strain 32K. A significant increase in the amount of biofilm formed was observed in the coculture of strains ME121 and 32K, as compared to that in the monoculture of strain 32K. In a similar experiment, wherein strain ME121 was replaced with E. coli W3110 or P. aeruginosa PAO1 in cocultures with strain 32K, an increase in biofilm formation was not observed (Figure1B,C). Tukey’s multiplex test was used to test the difference between the amount of biofilm formed (mean values) in each coculture and the monocultures of their constituent strains (p < 0.05). A significant difference was observed between the amount of biofilm formed in the coculture of strains ME121 and 32K and the monocultures of strains ME121 and 32K, but no significant difference was observed between the control (Glc medium) and the amount of biofilm formed in the monoculture of strain ME121 (Figure1A). Figure1B shows that a significant difference was observed between the amount of biofilm formed in the coculture of E. coli and strain 32K and E. coli monoculture, but no significant difference was observed between the amount of biofilm formed in monoculture of strain 32K and coculture of E. coli and strain 32K (Figure1B). We observed a significant di fference in the amount of biofilm formed between the coculture of P. aeruginosa and strain 32K and monoculture of strain 32K, but not in the amount of biofilm formed between the monoculture of P. aeruginosa and coculture of P. aeruginosa and strain 32K (Figure1C). Biology 2020, 9, 287 5 of 14 Biology 2020, 9, x FOR PEER REVIEW 5 of 14

Figure 1. Biofilm formation during cocultivation of strain 32K with test strains. (A) Coculture of strains Figure 1. Biofilm formation during cocultivation of strain 32K with test strains. (A) Coculture of ME121 and 32K, (B) coculture of E. coli W3110 and strain 32K, and (C) coculture of P. aeruginosa PAO1 strains ME121 and 32K, (B) coculture of E. coli W3110 and strain 32K, and (C) coculture of P. aeruginosa and strain 32K. The vertical axis is Abs570. Error bars are standard deviations. One-way ANOVA PAO1 and strain 32K. The vertical axis is Abs570. Error bars are standard deviations. One-way ANOVA test was performed for equality of all means; ME121 + 32K: F(3, 59) = 98.90, p < 0.01. E. coli + 32K: test was performed for equality of all means; ME121 + 32K: F(3, 59) = 98.90, P < 0.01. E. coli + 32K: F(3, 59) F(3, 59) = 63.96, p < 0.01, P. aeruginosa + 32K: F(3, 31) = 31.97, p < 0.01. Tukey’s test was performed for = 63.96, P < 0.01, P. aeruginosa + 32K: F(3, 31) = 31.97, P < 0.01. Tukey’s test was performed for post hoc post hoc analysis, p < 0.05. Different superscript letters (a, b, c) denote significant difference from each otheranalysis, in all combinations. P < 0.05. Different superscript letters (a, b, c) denote significant difference from each other in all combinations. 3.2. Biofilm Formation Using Culture Supernatants of Strains ME121 and 32K 3.2. Biofilm Formation Using Culture Supernatants of Strains ME121 and 32K Based on the results from Section 3.1, we hypothesized that strain ME121 produced a biofilm-promotingBased on the factor results which from promoted Sectionthe 3.1, biofilm we hypothesized formation of that strain strain 32K ME121 or vice produced versa. Therefore, a biofilm- a biofilmpromoting formation factor assay which was promoted carried out the by biofilm obtaining formatio culturen supernatantof strain 32K of or strain vice ME121versa. grownTherefore, in a Glcbiofilm medium formation and culture assay supernatant was carried of strainout by 32K obtaining grown culture in Glc medium supernatant (Figure of strain2). ME121 grown in GlcTukey’s medium multiple and culture comparison supernatant test was of stra performedin 32K grown to assess in Glc the medium differences (Figure between 2). the effects of only GlcTukey’s medium multiple and additioncomparison of the test two was culture performed supernatants to assess onthe the differences amount ofbetween biofilm the formed, effects of afteronly two-way Glc medium ANOVA and analysis. addition The of results the two showed culture no supernatants significant di ffonerence the amou betweennt of the biofilm amount formed, of biofilmafter formed two-way in monocultureANOVA analysis. of strain The ME121 results in Glcshowed medium no significant with no inoculum difference and between in the presence the amount of cultureof biofilm supernatant formed of strainin monoculture ME121 (Figure of strain2A,B). ME121 In the casein Glc of themedium culture with supernatant no inoculum of strain and 32K, in the therepresence was no significantof culture supernatant difference between of strain the ME121 amount (Figure of biofilm 2A,B). formed In the in case the monoculturesof the culture ofsupernatant strains ME121of strain and 32K32K, (Figure there 2wasC). No no significantsignificant di differencefference was between observed the inamount all three of conditions biofilm formed (only Glcin the medium,monocultures culture supernatant of strains ME121 of strain and 32K, 32K and (Figure culture 2C). supernatant No significant of strain difference ME121) when was noobserved inoculum in all wasthree added. conditions In addition, (only there Glc wasmedium, a significant culturedi supernatantfference between of strain the 32K, amount and of culture biofilm supernatant formed in of monoculturesstrain ME121) of strains when ME121no inoculum and 32K was (except added. in In only addition, Glc medium there was and a culture significant supernatant difference of strainbetween ME121),the amount but there of wasbiofilm a significant formed in di monoculturesfference in the of amount strains of ME121 biofilm and formed 32K in(except all other in only combinations. Glc medium andNo culture improvement supernatant in the of amountstrain ME121), of biofilm but formedthere was by a strain significant 32K wasdifference observed in the when amount Glc of mediumbiofilm or formed culture in supernatant all other combinations. of strain ME121 was added (Figure2A,B). On the other hand, when theNo culture improvement supernatant in ofthe strain amount 32K of was biofilm used, theformed amount by ofstrain biofilm 32K formed was observed by strain ME121when Glc increasedmedium (Figure or culture2A,C). supernatant This result suggestedof strain ME121 that strain was 32Kadded produced (Figure the2A,B). biofilm-promoting On the other hand, factor, when whichthe promotedculture supernatant the biofilm formationof strain 32K of strain was ME121.used, the amount of biofilm formed by strain ME121 increased (Figure 2A,C). This result suggested that strain 32K produced the biofilm-promoting factor, which promoted the biofilm formation of strain ME121.

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FigureFigure 2. 2.Biofilm Biofilm formation formation of of strains strains ME121, ME121, 32K, 32K, and and ME121 ME121+ +32K 32K in in the the presence presence of of culture culture supernatantssupernatants of of strains strains ME121 ME121 or 32K.or 32K. (A) (A) Glc Glc medium, medium, (B) culture(B) culture supernatant supernatant of strain of strain ME121, ME121, and ( Cand) culture(C) culture supernatant supernatant of strain of strain 32K. 32K. The verticalThe vertical axis axis is Abs is Abs570, and570, and the the error error bar bar is standardis standard deviation. deviation. Two-way ANOVA test was performed for equality of all means; Glc medium: F = 64.44, p < 0.01. Two-way ANOVA test was performed for equality of all means; Glc medium:(3, F 19)(3, 19) = 64.44, P < 0.01. ME121 culture supernatant: F = 98.24, p < 0.01, 32K culture supernatant: F = 44.65, p < 0.01. ME121 culture supernatant:(3, F(3, 19) 19) = 98.24, P < 0.01, 32K culture supernatant:(3, F(3, 19) 19) = 44.65, P < 0.01. Tukey’sTukey’s test test was was performed performed for for post post hoc hoc analysis, analysis,p < P 0.05.< 0.05. Di Differentfferent superscript superscript letters letters (a, (a, b, b, c) c) denote denote significantsignificant di ffdifferenceserences from from each each other other in in all all combinations. combinations. 3.3. Relationship between Growth and Biofilm Formation during Coculture of Strains ME121 and 32K 3.3. Relationship between Growth and Biofilm Formation during Coculture of Strains ME121 and 32K Since the biofilm production of strain ME121 increased due to the metabolites present in the cultureSince supernatant the biofilm of strain production 32K, the of relationshipstrain ME121 between increased the due growth to the and metabolites biofilm formation present in was the investigatedculture supernatant by diluting of thestrain culture 32K, supernatant the relationship of strain between 32K with the growth Glc medium and biofilm (Figure 3formation). was investigatedTukey’s multiple by diluting comparison the culture test supernatant was performed of strain to assess 32K the with di ffGlcerence medium in proliferation (Figure 3). between each cultureTukey’s (monoculture multiple comparison of strain ME121, test monoculturewas perform ofed strain to assess 32K, andthe coculturedifference of in strains proliferation ME121 andbetween 32K) aftereach additionculture (monoculture of different dilutions of strain of ME1 culture21, monoculture supernatant of of strain strain 32K, 32K. and No significantcoculture of distrainsfference ME121 in the and growth 32K) was after observed addition between of different the monoculturesdilutions of culture of strains supernatant ME121 andof strain 32K in32K. only No Glcsignificant medium, difference 100-fold,10-fold, in the growth 5-fold, andwas 2-foldobserved diluted between culture the supernatant monocultures of strain of strains 32K. Moreover,ME121 and there32K wasin only no significantGlc medium, diff 100-fold,erence between 10-fold, the5-fold, amount and 2-fold of biofilm diluted formed culture in thesupernatant monoculture of strain of strain32K. 32KMoreover, and the there coculture was ofno strains significant ME121 difference and 32K atbetween 100-fold the dilution amount of theof biofilm culture formed supernatant. in the Allmonoculture other combinations of strain were 32K significantlyand the coculture different of (Figurestrains 3ME121A,C,E). and 32K at 100-fold dilution of the cultureThe esupernatant.ffects of the dilutionsAll other ofcombinations culture supernatant were significantly of strain 32K different on the (Figure amount 3A,C,E). of biofilm formed were alsoThe investigated effects of the for dilutions monoculture of culture of strains supernatant ME121 of and strain 32K 32K and on their the coculture. amount of We biofilm observed formed a were also investigated for monoculture of strains ME121 and 32K and their coculture. We observed significant difference in the ratio of the amount of biofilm formed in all dilutions of culture supernatant a significant difference in the ratio of the amount of biofilm formed in all dilutions of culture of strain 32K (Figure3B,D,F). Two-way ANOVA analysis was used to evaluate whether there is a supernatant of strain 32K (Figure 3B,D,F). Two-way ANOVA analysis was used to evaluate whether relationship between bacterial growth and biofilm formation. The analysis showed that the difference there is a relationship between bacterial growth and biofilm formation. The analysis showed that the between the amount of biofilm formed in the monoculture of strain ME121 and the coculture was difference between the amount of biofilm formed in the monoculture of strain ME121 and the significant under all dilutions of culture supernatant of strain 32K. There was a significant difference in coculture was significant under all dilutions of culture supernatant of strain 32K. There was a the amount of biofilm formed between monoculture of strain 32K and the coculture (except in case significant difference in the amount of biofilm formed between monoculture of strain 32K and the of only Glc medium and 10-fold dilution), and monocultures of strains ME121 and 32K in only Glc coculture (except in case of only Glc medium and 10-fold dilution), and monocultures of strains medium. No significant difference was observed in the other combinations (Figure3B,D,F). ME121 and 32K in only Glc medium. No significant difference was observed in the other The growth and amount of biofilm formed in monoculture of strain ME121 improved when culture combinations (Figure 3B,D,F). supernatant of strain 32K was added (Figure3A,B). On the other hand, in the case of monoculture of The growth and amount of biofilm formed in monoculture of strain ME121 improved when strain 32K, its culture supernatant showed no significant difference in improving its growth, but it culture supernatant of strain 32K was added (Figure 3A,B). On the other hand, in the case of decreased the amount of biofilm formed (Figure3C,D). Growth of the strain also improved when strains monoculture of strain 32K, its culture supernatant showed no significant difference in improving its ME121 and 32K were cocultured, but the amount of the biofilm formed was not significantly different growth, but it decreased the amount of biofilm formed (Figure 3C,D). Growth of the strain also (Figure3E,F). It has been reported that Pseudomonas aeruginosa produces an EPS-degrading enzyme improved when strains ME121 and 32K were cocultured, but the amount of the biofilm formed was not significantly different (Figure 3E,F). It has been reported that Pseudomonas aeruginosa produces an

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EPS-degrading enzyme in a nutrient-starved state and uses the degraded polysaccharide as a nutrient in asource nutrient-starved (32). After statethe process and uses of the biofilm degraded formation polysaccharide is completed, as a nutrientthe biofilm source is decomposed, (32). After the and processbacteria of biofilm are eliminated formation according is completed, to the thesurroundi biofilmng is nutritional decomposed, conditions and bacteria and stress are eliminated environment according(23, 24, to26). the Therefore, surrounding in our nutritional study, after conditions 48 h of and cocultivation, stress environment many cells (23, continued 24, 26). Therefore, to form the in ourbiofilm, study, and after the 48 number h of cocultivation, of plankton manyic bacteria cells was continued not large. to formHowever, the biofilm, in the culture and the supernatant number of of planktonicstrain 32K, bacteria the cells was that not originally large. However, formed inbiofilm the culture were detached supernatant from of the strain biofilm 32K, after the 48 cells h, and that the originallyremaining formed cells biofilm existed were as planktonic detached from bacteria. the biofilm As a result, after 48even h, and when the only remaining strain cells32K was existed cultured as planktonicor strains bacteria. ME121 Asand a 32K result, were even cocultured, when only the strain cells 32Kin the was biofilm cultured obtained or strains nutrients ME121 from and the 32K aged werebiofilm cocultured, and were the cells released in the from biofilm the obtained biofilm nutrientsin search from of a the new aged desirable biofilm andenvironment. were released It was fromspeculated the biofilm that in searchthe amount of a new of bi desirableofilm formed environment. in the supernatant It was speculated was reduced that the or amountunchanged. of biofilm Another formedpossibility in the is supernatant that the metabolites was reduced contained or unchanged. in the culture Another supernatant possibility of is strain that the32K metabolites regulated the containedamount in of the biofilm culture formed supernatant under ofeach strain culture 32K regulatedcondition. the amount of biofilm formed under each culture condition.

FigureFigure 3. Growth 3. Growth and and biofilm biofilm formation formation in monocultures in monocultures of strains of strains ME121 ME121 (A, B(A,), 32KB), 32K (C,D (C,), cocultureD), coculture of strainsof strains ME121 ME121 and and 32K 32K (E,F (E,), usingF), using culture culture supernatant supernatant of strain of strain 32K 32K diluted diluted with with Glc Glc medium. medium. ErrorError bars bars are standardare standard deviations. deviations. Two-way Two-way ANOVA ANOV testA test was was performed performed for equalityfor equality of all of means;all means;

(A)F(A)(5, 21)F(5,= 21)10.75, = 10.75,p < P0.001. < 0.001. (B ):(B): F(5, F 21)(5, 21)= =23.05, 23.05,p

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3.4. Comparison of the Amount of Biofilm Formed due to Difference in Initial Inoculum Size 3.4. Comparison of the Amount of Biofilm Formed due to Difference in Initial Inoculum Size There is a specific relationship between dominant and rare microbial species in their natural habitats.There Therefore, is a specific we investigated relationship the between effect of dominant different andinitial rare inoculum microbial size species of strains in theirME121 natural and habitats.32K during Therefore, biofilm formation. we investigated Tukey’s the multiplex effect of te distff erentwas performed initial inoculum to assess size the of difference strains ME121 between and 32Kthe duringamount biofilm of biofilm formation. formed Tukey’s in each multiplex coculture test (mean was performedvalues) when to assessthe initial the di inoculumfference between of the thebacterial amount strains of biofilm was varied formed (P in < each0.05). coculture The results (mean are values)shown in when Figure the 4. initial inoculum of the bacterial strainsWhen was variedthe initial (p < inoculum0.05). The of results strain are ME121 shown was in Figurereduced4. to 1/10 or increased to 10 times the originalWhen inoculum, the initial it did inoculum not affect of strainbiofilm ME121 formation was during reduced coculture to 1/10 with or increased strain 32K to (Figure 10 times 4A). the originalOn the other inoculum, hand, it when did not the a ffinitialect biofilm inoculum formation of strain during 32K was coculture reduced with 1/10-fold, strain 32Kthe (Figureamount4 A).of Onbiofilm the otherformed hand, in the when monoculture the initial of inoculum strain 32K of decreased, strain 32K but was the reduced amount 1 /of10-fold, biofilm the formed amount was of biofilmthe same formed in coculture in themonoculture with strain ME121 of strain when 32K the decreased, initial inoculum but theamount of strain of 32K biofilm was formedvaried (Figure was the same4B). We in coculture found that with the strain viable ME121 cell count when of thestrains initial ME121 inoculum and of32K strain at OD 32K600 wasof 0.01 varied was (Figure6.15 × 104B).6 6 WeCFU/mL found and that 1.31 the viable× 107 CFU/mL, cell count respectively. of strains ME121 It wasand speculated 32K at ODthat,600 whenof 0.01 the was bacterial 6.15 cell10 numberCFU/mL × andof strain1.31 32K107 exceedsCFU/mL a, certain respectively. threshold It was value, speculated the production that, when of EPS the bacterialand other cell compounds number of starts, strain × 32Kand exceedsa large amount a certain of threshold biofilm can value, be formed the production in association of EPS with and strain other ME121. compounds starts, and a large amount of biofilm can be formed in association with strain ME121.

FigureFigure 4.4. BiofilmBiofilm formation in monocultures monocultures of of strains strains ME121 ME121 and and 32K, 32K, and and coculture coculture of of strains strains ME121 ME121 andand 32K32K whenwhen ((A)A) thethe initial inoculum of strain ME121 ME121 was was changed changed and and (B) (B )wh whenen the the initial initial inoculum inoculum

ofof strainstrain 32K 32K was was changed. changed. The The vertical vertical axis axis is is Abs Abs570570, and, and the the error error bar bar is standardis standard deviation. deviation. One-way One-

ANOVAway ANOVA test was test performed was performed for equality for equality of all means; of all means; (A): F(7, (A): 39) =F(7,140.00, 39) = 140.00,p < 0.001, P < 0.001, (B): F(7, (B): 39) F=(7,69.90, 39) = p69.90,< 0.001. P < 0.001. Tukey’s Tukey’s test was test performed was performed for post for hocpost analysis, hoc analysis,p < 0.05. P < 0.05. Diff erentDifferent superscript superscript letters letters (a, b, c,(a, d) b, denote c, d) denote significant significant differences differenc fromes eachfrom othereach other in all in combinations. all combinations.

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3.5. Comparison of Biofilm Formation during Coculture with Another Methylobacterium and Kaistia Strain 3.5. Comparison of Biofilm Formation during Coculture with Another Methylobacterium and Kaistia Strain A significant increase in the amount of biofilm formed was observed when strains ME121 and 32K wereA significant cocultured. increase We examined in the amount whether of this biofilm phenomenon formed was occurred observed even when when strains the strains ME121 were and cocultured32K were cocultured.with another We Methylobacterium examined whether or Kaistia this phenomenon strain. M. radiotolerans occurred even (hereafter when the referred strains to were as Mr)cocultured and K. adipata with another (hereafterMethylobacterium referred to asor Ka)Kaistia werestrain. the twoM. radiotoleransother strains(hereafter used for this referred experiment. to as Mr) [6,19].and K. In adipata this study,(hereafter we used referred Mr and to as Ka Ka) as wererepresentative the two other Methylobacterium strains used forand this Kaistia experiment. bacteria [from6,19]. differentIn this study, isolation we used sites. Mr Tukey’s and Ka asmultiplex representative test wasMethylobacterium performed to andtest Kaistiathe differencebacteria frombetween different the biofilmisolation formed sites. Tukey’s(mean values) multiplex in each test wascoculture performed (P < 0.05). to test The the results difference are shown between in the Figure biofilm 5. formed (meanAn values) increase in in each the cocultureamount of (p biofilm< 0.05). formed The results was arealso shown observed in Figure during5. coculture of strains Mr and KaAn compared increase into the monoculture amount of of biofilm Ka (Figure formed 5B). was In also addition, observed an increase during coculturewas also ofseen strains during Mr cocultureand Ka compared of strains to ME121 monoculture and Ka of compared Ka (Figure to5B). monoculture In addition, of an Ka increase (Figure was 5C). also However, seen during no significantcoculture ofincrease strains in ME121 the amount and Ka of compared biofilm form to monocultureed was observed of Ka (Figurein coculture5C). However, of strains no Mr significant and 32K comparedincrease in to the monoculture amount of biofilm of 32K formed (Figure was 5D). observed Thus, inwe coculture concluded of strainsthat the Mr amount and 32K of compared biofilm increasedto monoculture even in of the 32K coculture (Figure5D). of Thus,strain weME121 concluded and Kaistia that the spp. amount bacteria of biofilmthat were increased isolated even from in differentthe coculture soil samples. of strain ME121 and Kaistia spp. bacteria that were isolated from different soil samples.

FigureFigure 5. BiofilmBiofilm formationformation during during coculture coculture of di offferent different combinations combinations of bacterial of bacterial strains. (Astrains.) Coculture (A) Cocultureof strains ME121of strains and ME121 32K, (Band) coculture 32K, (B) ofcocultureM. radiotolerans of M. radiotolerans(Mr) and K. (Mr) adipata and(Ka), K. adipata (C) coculture (Ka), (C) of coculturestrain ME121 of strain and K.ME121 adipata and(Ka), K. andadipata (D) coculture(Ka), and of(D)M. coculture radiotolerans of M.(Mr) radiotolerans and strain 32K.(Mr) Theand verticalstrain axis is Abs , and the error bar is standard deviation. One-way ANOVA test was performed for 32K. The vertical570 axis is Abs570, and the error bar is standard deviation. One-way ANOVA test was equality of all means; (A): F(3, 19) = 98.90, p < 0.001. (B): F(3, 19) = 49.47, p < 0.001. (C): F(3, 19) = 389.05, performed for equality of all means; (A): F(3, 19) = 98.90, P < 0.001.(B): F(3, 19) = 49.47, P < 0.001. (C): F(3, 19) p < 0.001.(D): F(3, 19) = 10.90, p < 0.001. Tukey’s test was performed for post hoc analysis, p < 0.05. = 389.05, P < 0.001.(D): F(3, 19) = 10.90, P < 0.001. Tukey’s test was performed for post hoc analysis, P < Different superscript letters (a, b, c) denote significant difference from each other in all combinations. 0.05. Different superscript letters (a, b, c) denote significant difference from each other in all combinations.

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Biology 2020, 9, x FOR PEER REVIEW 10 of 14 3.6. Comparison of Swimming Speed during Coculture with Another Methylobacterium and Kaistia Bacteria 3.6. Comparison of Swimming Speed during Coculture with Another Methylobacterium and Kaistia Bacteria Previously, we reported that the swimming speed of strain ME121 increases when culture Previously, we reported that the swimming speed of strain ME121 increases when culture supernatant of strain 32K is added. As a mechanism for accelerating the swimming of strain ME121, supernatant of strain 32K is added. As a mechanism for accelerating the swimming of strain ME121, it was found that the flagellar motor of strain ME121 had a simultaneous increase in motor driving it was found that the flagellar motor of strain ME121 had a simultaneous increase in motor driving force when exposed to the K factor contained in the 32K culture supernatant [10]. It is known that the force when exposed to the K factor contained in the 32K culture supernatant [10]. It is known that the flagellarflagellar motor motor stator stator constantly constantly exchanges exchanges its its number number of of units units in in a a motor motor [[24].24]. In this way, way, the the torque torque outputoutput of the of the flagellar flagellar motor motor is adjustedis adjusted by by the the number number of of stator stator units. units. WhenWhen strain ME121 is is also also exposedexposed to the to the K factor, K factor, the the number number of of stators stators taken taken into into the the motor motor increases, increases, andand we believe that that the the swimmingswimming speed speed increases increases due todue the to increase the increase in motor in motor torque. torque. Therefore, Therefore, swimming swimming speeds speeds of strains of ME121strains and ME121 Mr were and measured Mr were measured when Glc when medium, Glc m cultureedium, supernatant culture supernatant of strain of 32K, strain and 32K, culture and supernatantculture supernatant of strain Ka of were strain added Ka were (Supplementary added (Suppl Materialsementary VideosMaterials S1 Videos and S2). S1 Tukey’s and S2). multiplex Tukey’s test wasmultiplex performed test was to testperformed the diff erenceto test betweenthe differen thece swimming between the speeds swimming (mean speeds values) (mean of strains values) ME121 of andstrains Mr in theME121 presence and Mr of in culture the presence supernatant of culture of strain supernatant ME121 orof strain ME121 Ka (p < or0.05). strain The Ka results(P < 0.05). are shownThe in results Figure are6. Characteristically,shown in Figure 6. weCharacteristically, observed that we both observed strains ME121that both and strains Mr had ME121 accelerated and Mr swimminghad accelerated speeds, swimming regardless speeds, of the cultureregardless supernatant of the culture used. supernatant Culture supernatantused. Culture of supernatant strain 32K acceleratedof strain the 32K swimming accelerated speed the ofswimming strain ME121 speed more of strain than thatME121 by themore culture than supernatantthat by the ofculture strain Ka.supernatant On the other of hand,strain Ka. significant On the other acceleration hand, significant in the swimming acceleration speed in the of swimming strain Mr speed was observedof strain whenMr culture was observed supernatant when of culture strain supernatant Ka was added. of strain Ka was added. TheThe cocultivation cocultivation of strains of strains 32K and32K Mrand did Mr not did contribute not contribute to the to increase the increase in the amountin the amount of biofilm of formed,biofilm as compared formed, as to compared that of the to cocultivationthat of the cocultivation of strains Ka of andstrains Mr Ka (Figure and Mr5D). (Figure Similarly, 5D). in Similarly, terms of in terms of swimming speed, the acceleration of swimming speed with culture supernatant of strain swimming speed, the acceleration of swimming speed with culture supernatant of strain 32K was lower, 32K was lower, as compared to that with the culture supernatant of strain Ka. Figures 5 and 6 suggest as compared to that with the culture supernatant of strain Ka. Figures5 and6 suggest that the biofilm that the biofilm formation and acceleration of swimming speed are positively correlated between formation and acceleration of swimming speed are positively correlated between Methylobacterium spp. Methylobacterium spp. and the Kaistia spp. and the Kaistia spp. A schematic diagram of the effect of different cocultures on biofilm formation and motility is shownA schematic in Figure diagram 7. Except of for the the eff ectcoculture of diff erentof strains cocultures Mr and on32K, biofilm increased formation biofilm andformation motility and is shownaccelerated in Figure motility7. Except was for observed the coculture in all other of strains combinationsMr and of 32K, cocultures. increased biofilm formation and accelerated motility was observed in all other combinations of cocultures.

FigureFigure 6. Swimming6. Swimming speed speed of of strain strain ME121 ME121 and andM. M. radiotolerans radiotolerans(Mr) (Mr)when when D-glucoseD-glucose synthetic medium,medium, culture culture supernatant supernatant of of strain strain 32K, 32K, and and culture culture supernatant supernatant ofof K.K. adipataadipata (Ka) were were added. added. HalfHalf of the of totalthe total values values are are collected collected in in the the box, box, and and the the thick thick line line in in the the centercenter ofof thethe box shows the the averageaverage value. value. The The lines lines that th extendat extend vertically vertically are are the the remaining remaining values, values, andand thethe edgesedges indicate the the maximummaximum and and minimum minimum values, values, respectively. respectively. The The vertical vertical axis axis showsshows thethe swimmingswimming speed speed (μ (µm/s).m/s).

One-wayOne-way ANOVA ANOVA test test was was performed performed for for equality equality of of all all means;means; ME121: F F(2,269)(2,269) = 225.29,= 225.29, P < p0.001.< 0.001. M. M. radiotoleransradiotolerans:: FF(2,239)(2,239) == 100.20,100.20 ,Pp << 0.001.0.001. Tukey’s Tukey’s test test was was pe performedrformed for for post post hoc hoc analysis, analysis, Pp << 0.05.0.05. DifferentDifferent superscript superscript letters letters (a, b,(a, c)b, denotec) denote significant significant di differencesfferences from from each each other other inin all combinations.

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Figure 7. Schematic diagram of the effects on biofilm formation and motility of each strain. “↑” shows Figure 7. Schematic diagram of the effects on biofilm formation－ and motility of each strain. “ ” shows↗ Figureincreased 7. Schematic biofilm formation diagram andof the accelerated effects on motility.biofilm formation “ ” shows and no motility effect on of biofilmeach strain. formation. “↑”↑ shows “ ” increased biofilm formation and accelerated motility. “—” shows no effect on biofilm formation. “ ” increasedshows moderately biofilm formation accelerated and motility. accelerated motility. “－” shows no effect on biofilm formation. “↗”% shows moderately accelerated motility. shows moderately accelerated motility. 4. Discussion 4. Discussion 4. Discussion Relationship4.1. Relationship between between Motility Mot Accelerationility Acceleration of Strain of Strain ME121 ME121 and and Biofilm Biofilm Formation Formation 4.1. RelationshipAfter strain between ME121 Mot recognizesility Acceleration the K factor of Strain prod ME121uced by and strain Biofilm 32K, Formation the rotational force of the After strain ME121 recognizes the K factor produced by strain 32K, the rotational force of the flagellar motor of strain ME121 is accelerated [10]. The advantages of motility acceleration for strain flagellarAfter motor strain of strainME121 ME121 recognizes is accelerated the K factor [10 ].prod Theuced advantages by strain of 32K, motility the rotational acceleration force for of strainthe ME121 are speculated to be faster access to the biofilm of strain 32K and enhanced invasion into the ME121flagellar are motor speculated of strain to beME121 faster is accessaccelerated to the [10]. biofilm The advantages of strain 32K of andmotility enhanced acceleration invasion for intostrain the strain 32K biofilm complex. We elucidated that the growth of strain ME121 also improved due to the strainME121 32K are biofilm speculated complex. to be Wefaster elucidated access to that the thebiofilm growth of strain of strain 32K ME121and enhanced also improved invasion dueinto tothe the growth enhancer produced by strain 32K (Figure 3). Strain ME121 may also be capable of producing growthstrain enhancer32K biofilm produced complex. by We strain elucidated 32K (Figure that the3). growth Strain ME121of strain may ME121 also also be capableimproved of due producing to the a strain 32K-growth-improving factor and -biofilm-promoting factor in a coculture with strain 32K. a straingrowth 32K-growth-improving enhancer produced by strain factor 32K and (Figure -biofilm-promoting 3). Strain ME121 factor may inalso a coculturebe capable with of producing strain 32K. aStrain strain 32K 32K-growth-improving forms biofilms in a monoculture, factor and -biofilm-pro but in a coculturemoting with factor strain in a ME121, coculture it enhances with strain biofilm 32K. Strain 32K forms biofilms in a monoculture, but in a coculture with strain ME121, it enhances biofilm Strainproduction. 32K forms Therefore, biofilms it is in considered a monoculture, that both but stinrains a coculture ME121 withand 32Kstrain affect ME121, each it other, enhances and abiofilm larger production. Therefore, it is considered that both strains ME121 and 32K affect each other, and a larger production.biofilm can be Therefore, formed in it theiris considered cocultures that (Figure both st8).rains It is ME121conceivable and 32K that affect strains each ME121 other, and and 32K a larger grow biofilm can be formed in their cocultures (Figure8). It is conceivable that strains ME121 and 32K biofilmin a symbiotic can be formed relationship, in their such cocultures as coevolution (Figure 8). in It nature.is conceivable Future that prospe strainscts ME121of the studyand 32K include grow grow in a symbiotic relationship, such as coevolution in nature. Future prospects of the study include inobservation a symbiotic of therelationship, distribution such and as abundance coevolution ratios in nature. of the Futuretwo strains prospe andcts the of thebiofilm study formation include observation of the distribution and abundance ratios of the two strains and the biofilm formation observationprocess over of time the in distribution the coculture and of abundancestrains ME121 ratios and of 32K. the two strains and the biofilm formation processprocess over over time time in in the the coculture coculture of of strains strains ME121ME121 andand 32K.

Figure 8. Schematic representation of biofilm formation of strains ME121 and 32K. In the synthetic Figure 8. Schematic representation of biofilm formation of strains ME121 and 32K. In the synthetic medium used in this experiment, strain ME121 does not form a biofilm when cultured alone, medium used in this experiment, strain ME121 does not form a biofilm when cultured alone, whereas whereasFigure 8. monoculture Schematic representation of strain 32K of forms biofilm a biofilm.formation It of was strains speculated ME121 and that 32K. a larger In the biofilm synthetic was monoculture of strain 32K forms a biofilm. It was speculated that a larger biofilm was formed in the formedmedium in theused coculture in this experiment, of strains ME121strain ME121 and 32K. does not form a biofilm when cultured alone, whereas monoculturecoculture of strains of strain ME121 32K formsand 32K. a biofilm. It was speculated that a larger biofilm was formed in the Thecoculture amount of strains of biofilm ME121 formed and 32K. was significantly increased in the coculture of strains ME121 and 32K, and strains ME121 and Ka. However, there was no increase in the amount of biofilm formed

Biology 2020, 9, 287 12 of 14 in the coculture of strains Mr and 32K (Figures5 and7). In addition, the motility of strain Mr was moderately accelerated in the swimming assay when the culture supernatant of strain 32K was added (Figures6 and7). Strains ME121 and 32K were isolated from the same soil [ 2], whereas strain Mr was isolated from brown rice in Japan [19], and strain Ka was isolated from soil in Korea [6]. This suggests that increased biofilm formation and acceleration of motility in Methylobacterium spp. and Kaistia spp. depend on the combination of the bacterial strains and not on the source of the bacterium. Since a biofilm is more resistant to various stress conditions than the planktonic cells, it was postulated that strains ME121 and 32K both grow to form a biofilm to survive in nature. It was considered that the metabolites produced by strain 32K cause an increase in the growth and motility of strain ME121 and vice versa. In the future, it is expected that the specific factors involved in the process will be identified by conducting more cocultivation tests of Methylobacterium spp. and Kaistia spp., using accelerated motility and improvement in biofilm formation as indicators. In nature, microorganisms do not coexist with the same cell number for each species, but they exist as dominant and rare species in a habitat. Thus, we varied the initial inoculum size to investigate the effects of inoculum size on the biofilm formation of strains ME121 and 32K. As a result, even when the initial OD of strain 32K was reduced to OD600 of 0.001, the amount of biofilm increased significantly during the coculture (Figure4). The results showed that when the initial inoculum of strain 32K exceeded a certain threshold value, the production of EPS and other molecules began, and a large biofilm was formed together with strain ME121. We also observed that even a small number of strain 32K cells could interact with the cells of strain ME121 to form a large biofilm. We previously reported that the accelerated motility of strain ME121 cocultured with strain 32K was related to EPS produced by strain 32K [10]. EPS is the main component of a biofilm. During biofilm formation, information is exchanged between cells, using quorum sensing and autoinducers [25,26]. Acyl homoserine lactone compounds are used as common quorum sensing substances in Gram-negative bacteria [26,27]. It is inferred that, in the current study also, information was exchanged by these substances during the biofilm formation of the coculture of strains ME121 and 32K. As the genome sequence of strain ME121 has been elucidated [2], in the future, we will search for genes related to quorum sensing and quantify the expression levels of those genes, using monoculture of strain ME121 and its coculture with strain 32K, using real-time PCR, qPCR, etc. By analyzing not only the genes involved in quorum sensing but also the genes related to polysaccharide production and quantifying the expression of these genes, biofilm formation during coculture of strains ME121 and 32K can be analyzed at the expression level. This is expected to lead to the elucidation of the mechanism of biofilm formation. Through a series of studies, we would like to discuss new findings in the symbiotic relationship between Methylobacterium spp. and Kaistia spp. and the possibility of controlling bacterial biofilm formation. Methylobacterium spp. biofilms are known to inhibit mycobacterial biofilm formation, which causes hospital-related infection problems [28]. Based on the results of this study, it is expected that the metabolites contained in the culture supernatant of Kaistia spp. can be used to increase the production of Methylobacterium spp. biofilms as a biological material, and to modulate the amount of metabolites of Methylobacterium spp. biofilm as a microbial material. It is expected that the investigation of the relationship between biofilms formed by complex microbial systems and their motility can be applied to various fields, such as drug discovery [29]. If future studies can elucidate the mechanism of motility improvement and identify the motility improvement factor, then biofilm formation can be modulated artificially.

Supplementary Materials: The following data are available online at http://www.mdpi.com/2079-7737/9/9/287/s1. Video S1: Swimming motility of ME121 with Glc medium, 32K supernatant and K. adipata (Ka) supernatant. Video S2: Swimming motility of M. radiotolerans with Glc medium, 32K supernatant and K. adipata (Ka) supernatant. Table S1: Tukey test data for post hoc analysis of the results in Figure1. Table S2: Tukey test data for post hoc analysis of the results in Figure2. Table S3: Tukey test data for post hoc analysis of the results in Figure3. Table S4: Tukey test data for post hoc analysis of the results in Figure4. Table S5: Tukey test data for post hoc analysis of the results in Figure5. Table S6: Tukey test data for post hoc analysis of the results in Figure6. Biology 2020, 9, 287 13 of 14

Author Contributions: A.N. and M.I. designed the research; Y.U. and M.I. conducted the research; A.N., T.S., and M.I. analyzed the data; and Y.U. and M.I. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by a Grant-in-Aid for Scientific Research on Innovative Areas of the Ministry of Education, Culture, Sports, Science and Technology of Japan, grant number JP24117005 (MI). Acknowledgments: We would like to thank Arthur A. Guffanti for the critical discussions and reading the manuscript. Conflicts of Interest: The authors declare that they have no conflicts of interest. Ethical approval: This article does not describe any studies with human participants or animals performed by any of the authors.

References

1. Shimizu, T.; Takaya, N.; Nakamura, A. An L-glucose catabolic pathway in Paracoccus species 43P. J. Biol. Chem. 2012, 287, 40448–40456. [CrossRef][PubMed] 2. Fujinami, S.; Takeda-Yano, K.; Onodera, T.; Satoh, K.; Shimizu, T.; Wakabayashi, Y.; Narumi, I.; Nakamura, A.; Ito, M. Draft Genome Sequence of Methylobacterium sp. ME121, Isolated from Soil as a Mixed Single Colony with Kaistia sp. 32K. Genome Announc. 2015, 3, e01005-15. [CrossRef][PubMed] 3. Green, P.N.; Ardley, J.K. Review of the genus Methylobacterium and closely related organisms: A proposal that some Methylobacterium species be reclassified into a new genus, Methylorubrum Gen. Nov. Int. J. Syst. Evol. Microbiol. 2018, 68, 2727–2748. [CrossRef][PubMed] 4. Kovaleva, J.; Degener, J.E.; van der Mei, H.C. Methylobacterium and its role in health care-associated infection. J. Clin. Microbiol. 2014, 52, 1317–1321. [CrossRef][PubMed] 5. Sanders, J.W.; Martin, J.W.; Hooke, M.; Hooke, J. Methylobacterium mesophilicum infection: Case report and literature review of an unusual opportunistic pathogen. Clin. Infect. Dis. 2000, 30, 936–938. [CrossRef] [PubMed] 6. Im, W.T.; Yokota, A.; Kim, M.K.; Lee, S.T. Kaistia adipata gen. nov., sp. nov., a novel alpha-proteobacterium. J. Gen. Appl. Microbiol. 2004, 50, 249–254. [CrossRef] 7. Jin, L.; Kim, K.K.; Lee, H.G.; Ahn, C.Y.; Oh, H.M. Kaistia defluvii sp. nov., isolated from river sediment. Int. J. Syst. Evol. Microbiol. 2012, 62, 2878–2882. [CrossRef][PubMed] 8. Weon, H.Y.; Lee, C.M.; Hong, S.B.; Kim, B.Y.; Yoo, S.H.; Kwon, S.W.; Go, S.J. Kaistia soli sp. nov., isolated from a wetland in Korea. Int. J. Syst. Evol. Microbiol. 2008, 58, 1522–1524. [CrossRef] 9. Glaeser, S.P.; Galatis, H.; Martin, K.; Kampfer, P. Kaistia hirudinis sp. nov., isolated from the skin of Hirudo verbana. Int. J. Syst. Evol. Microbiol. 2013, 63, 3209–3213. [CrossRef] 10. Usui, Y.; Wakabayashi, Y.; Shimizu, T.; Tahara, Y.O.; Miyata, M.; Nakamura, A.; Ito, M. A factor produced by Kaistia sp. 32K accelerated the motility of Methylobacterium sp. ME121. Biomolecules 2020, 10, 618. [CrossRef] 11. Fulaz, S.; Vitale, S.; Quinn, L.; Casey, E. Nanoparticle-Biofilm Interactions: The Role of the EPS Matrix. Trends Microbiol. 2019, 27, 915–926. [CrossRef] 12. Flemming, H.C.; Wingender, J. The biofilm matrix. Nat. Rev. Microbiol. 2010, 8, 623–633. [CrossRef][PubMed] 13. Donlan, R.M.; Costerton, J.W. Biofilms: Survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 2002, 15, 167–193. [CrossRef][PubMed] 14. Colagiorgi, A.; Di Ciccio, P.; Zanardi, E.; Ghidini, S.; Ianieri, A. A look inside the Listeria monocytogenes biofilms extracellular matrix. Microorganisms 2016, 4, 22. [CrossRef][PubMed] 15. Petrova, O.E.; Sauer, K. Escaping the biofilm in more than one way: Desorption, detachment or dispersion. Curr. Opin. Microbiol. 2016, 30, 67–78. [CrossRef] 16. Kilmury, S.L.N.; Burrows, L.L. The Pseudomonas aeruginosa PilSR two-component system regulates both twitching and swimming motilities. mBio 2018, 9, e01310-18. [CrossRef] 17. Dufrene, Y.F.; Persat, A. Mechanomicrobiology: How bacteria sense and respond to forces. Nat. Rev. Microbiol. 2020, 18, 227–240. [CrossRef] 18. Ito, M.; Hicks, D.B.; Henkin, T.M.; Guffanti, A.A.; Powers, B.D.; Zvi, L.; Uematsu, K.; Krulwich, T.A. MotPS is the stator-force generator for motility of alkaliphilic Bacillus, and its homologue is a second functional Mot in Bacillus subtilis. Mol. Microbiol. 2004, 53, 1035–1049. [CrossRef][PubMed] 19. Ito, H.; Iizuka, H. Taxonomic Studies on a Radio-resistant Pseudomonas. Agric. Biol. Chem. 1971, 35, 1566–1571. [CrossRef] Biology 2020, 9, 287 14 of 14

20. Bachmann, B.J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 1972, 36, 525–557. [CrossRef] 21. Holloway, B.W. Genetic recombination in Pseudomonas aeruginosa. J. Gen. Microbiol. 1955, 13, 572–581. [CrossRef][PubMed] 22. Tola, Y.H.; Fujitani, Y.; Tani, A. Bacteria with natural chemotaxis towards methanol revealed by chemotaxis fishing technique. Biosci. Biotechnol. Biochem. 2019, 83, 2163–2171. [CrossRef][PubMed] 23. Takahashi, Y.; Koyama, K.; Ito, M. Suppressor mutants from MotB-D24E and MotS-D30E in the flagellar stator complex of Bacillus subtilis. J. Gen. Appl. Microbiol. 2014, 60, 131–139. [CrossRef][PubMed] 24. Leake, M.C.; Chandler, J.H.; Wadhams, G.H.; Bai, F.; Berry, R.M.; Armitage, J.P. Stoichiometry and turnover in single, functioning membrane protein complexes. Nature 2006, 443, 355–358. [CrossRef][PubMed] 25. Parsek, M.R.; Greenberg, E.P. Sociomicrobiology: The connections between quorum sensing and biofilms. Trends Microbiol. 2005, 13, 27–33. [CrossRef][PubMed] 26. Whiteley, M.; Diggle, S.P.; Greenberg, E.P. Progress in and promise of bacterial quorum sensing research. Nature 2017, 551, 313–320. [CrossRef][PubMed] 27. Laganenka, L.; Colin, R.; Sourjik, V. Chemotaxis towards autoinducer 2 mediates autoaggregation in Escherichia coli. Nat. Commun. 2016, 7, 12984. [CrossRef] 28. García-Coca, M.; Rodríguez-Sevilla, G.; Pérez-Domingo, A.; Aguilera-Correa, J.-J.; Esteban, J.; Muñoz-Egea, M.-C. Inhibition of Mycobacterium abscessus, M. chelonae, and M. fortuitum biofilms by Methylobacterium sp. J. Antibiot. 2020, 73, 40–47. [CrossRef] 29. Onaka, H.; Mori, Y.; Igarashi, Y.; Furumai, T. Mycolic acid-containing bacteria induce natural-product biosynthesis in Streptomyces species. Appl. Environ. Microbiol. 2011, 77, 400–406. [CrossRef]

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