2/5/2020 - Open Access

OP50, a bacterial strain conventionally used as food for laboratory
maintenance of C. elegans, is a biofilm formation defective mutant
Yukinobu Arata1§, Taku Oshima2, Yusaku Ikeda1,3, Hiroshi Kimura3 and Yasushi Sako1
1Cellular Informatics Laboratory, RIKEN, 2-1 Wako, Saitama, 351-0198, Japan2

Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan

3Department of Mechanical Engineering, School of Engineering, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292,

Japan§To whom correspondence should be addressed: arata@riken.jp

Figure 1: Biofilm formation, the establishment of OP50-derived fliC and fimH knockout strains, and bacterial motility
assay. Biofilm formation on glass (A) and polystyrene (B). Biofilm formed by indicated E. coli strains on glass and2/5/2020 - Open Accesspolystyrene quantified by CV staining. Optical absorbance at 600 nm was measured in three independent experiments andcompared with paired student t-tests; *p < 0.05, ** p < 0.005. Biofilm formation ability of OP50 fliC::Km, OP50 fimH::Km,and BL21 should be studied further with rigorous experiments and statistical testing. (C and D) Isolation of OP50 fimH::Kmand OP50 fliC::Km. (C) Schematic structure of fliC and fimH knockout alleles with position and direction of primers for PCR.
Km cassette was integrated in gene locus to form fimH::Km and fliC::Km. (D) Confirmation of the presence of fimH::Km andfliC::Km in OP50-derived E. coli colonies via P1 transduction. Bacteria obtained from Km-resistant colonies were subjected to
PCR with the indicated primers. DNA fragments of expected sizes from the genomic structure of the fliC::Km and fimH::Km
knockout alleles were amplified with a primer set with K1 but not K2 primer. (E) Motility of E. coli strains. Motility ofindicated E. coli strains was compared by colony size after cultivation on soft agar LB plates.

Description
OP50 is an Escherichia coli strain conventionally used as a bacterial food in the laboratory maintenance of Caenorhabditis
elegans on agar plates. It has also been used to feed C. elegans in longitudinal cultures within microfluidic devices (MFDs)(Hulme et al., 2010; Li et al., 2015), where it has been subject to killing by ultraviolet irradiation or pasteurization performed
to suppress clogging due to biofilm formation and aggregation (Li et al., 2015; Zhuo et al., 2017). However, the killedbacterial food can change C. elegans aging dynamics, likely due to influences on C. elegans physiology (Saul et al., 2009;
Gruber et al.;, 2007; Garigan et al., 2002). Further development of longitudinal culturing systems for C. elegans in MFDs
requires elucidation of the mechanisms that underlie food bacteria clogging and delineation of culture conditions in whichliving bacterial food can be incorporated without clogging. Bacteria switch from planktonic growth to aggregated growthunder conditions of environmental stress, in the presence of toxins (e.g. antibiotics), and when there is a lack of nutrients(Trunk et al., 2018). Biofilms, such as dental plaque, are bacterial communities that are organized in a film-like form in which
they are embedded in a self-produced polymeric matrix on biotic or abiotic surfaces; pellicles are floating biofilms that form at
liquid-air interfaces. Meanwhile, autoaggregations are aggregated communities of bacteria suspended in solution, such asbacterial flocs formed in activated sludge. Biofilms and autoaggregations are formed by both shared and independent genetic
and physico-chemical mechanisms (Trunk et al., 2018; Berne et al., 2018; Berne et al., 2015). In this study, we examined
OP50 biofilm formation.
Biofilm formation is mediated by flagellin proteins (e.g. FliC), which form flagella, and the adhesion protein FimH, which islocated at the tips of type I pili (Berne et al., 2018, Jones et al., 1995; Pratt and Kolter, 1998; Friedlander et al., 2013). We
compared the biofilm formation ability of OP50 with that of the biofilm-forming (Wood et al., 2006) wild-type BW251113 E.
coli strain as well as that of two BW251113-derived knockouts produced with a kanamycin (Km) cassette characterized as
biofilm formation defective mutants: JW4283: BW25113 fimH::Km (a fimH knockout) and JW1908: BW25113 fliC::Km (afliC knockout) (Baba et al., 2006). Compared to the original BW251113 strain, BW251113 fliC::Km had a significantly
reduced ability to form biofilm on glass and polystyrene (Fig. 1A and 1B, p < 0.05) and BW25113 fimH::Km had asignificantly reduced ability to form biofilm on glass (Fig. 1A, p < 0.05; biofilm formation on polystyrene showed a nearsignificant reduction trend Fig. 1B, p = 0.0574). Compared with the original BW251113 strain, we found that OP50 had asignificantly reduced biofilm formation ability on polystyrene (Fig. 1B, p < 0.05; biofilm formation on glass showed a nearsignificant reduction trend, Fig. 1A, p = 0.0507). The biofilm formation ability of OP50 was as low as that seen with theBW251113 biofilm formation defective mutants, and similar to that of OP50 fliC::Km and OP50 fimH::Km mutants (Fig. 1A
and 1B), which were constructed by transferring fliC::Km and fimH::Km alleles to OP50 by P1 transduction (Fig. 1C and 1D).
Therefore, we conclude that the original OP50 strain is itself a biofilm formation defective mutant.
Bacterial motility is required for biofilm formation. Despite of the presence of flagella, mutants of a motor protein that enables
flagellar rotation have been shown to have a reduced biofilm formation ability (Pratt and Kolter, 1998; Friedlander et al.,2013; Wood et al., 2006). To study the mechanism of impaired biofilm formation ability of OP50, we performed a motility
assay that showed that OP50 exhibited much lower motility than BW251113 (Fig. 1E). Motility was similar among theoriginal OP50, OP50 fliC::Km, and OP50 fimH::Km strains (Fig. 1E). Preliminary mapping of a draft genome sequence ofOP50 to that of a reference E. coli strain (REL606) identified non-synonymous nucleic acid substitutions in flagellar hookprotein E, flagellar P-ring protein precursor I, and flagellar hook-associated protein K (personal communication with Robin C.
May
atUniversityofBirmingham)
(see
Excel
file
in
WormBase,
ftp://ftp.wormbase.org/pub/wormbase/species/e_coli/op50/annotation/op50_annotated_snps.xls). These results suggest that
OP50’s impaired biofilm formation is likely caused by a lack of functional flagella due to these point mutations on flagella
genes.
In our experiments, the motility and biofilm formation ability on glass and polystyrene of BL21 strain E. coli was similar tothat seen with BW25113 fliC::Km (Fig. 1A, 1B, and 1E). Our preliminary experiments suggest that BL21 is also a biofilm
formation defective mutant with impaired motility. Impaired biofilm formation in BL21 and OP50 due to impaired motility is2/5/2020 - Open Accessnot necessarily caused by maintenance in nutrient-rich laboratory conditions, where bacteria can survive without active
chemotaxis by flagella. It has been reported that flagellar regulon is frequently deleted secondarily to experimental genetic
manipulation (Hobman et al., 2007). Additionally, E. coli mutants with no functional flagella are selected in mouse intestine
under certain conditions (Leatham et al., 2005). These phenomena are thought to be caused by high selective pressure fromextracellular nutrient conditions on the flagellum regulon, because flagella are high energy consumers. Given that nonflagellar mutants are frequently selected in response to energy balance conditions, OP50 and BL21 may be ordinary examplesof biofilm defective mutants among laboratory E. coli strains.
We found that motility was greatly enhanced in BW25113 fimH::Km, compared to original BW25113 (Fig. 1E). It has beenshown that down-regulation of the expression of genes responsible for the formation of pili, including fimH, result in an
upregulation of fliC, leading to enhanced motility (Leatham et al., 2005). The high motility of BW25113 fimH::Km is likely
caused by anticorrelated control between fimH and fliC. Even in high-motility BW25113 fimH::Km, FimH is required forbiofilm formation (Fig. 1A). The lack of motility enhancement in OP50 fimH::Km (Fig. 1E) is likely due to OP50’s nonfunctional flagella.
Our finding that OP50 is a biofilm formation-deficient mutant suggests that a genetic mechanism of biofilm formation isunlikely to be a direct cause of OP50 clogging in MFDs. Experimentally, autoaggregation has been studied by examining
bacterial sedimentation in a culture solution (Trunk et al., 2018). Autoaggregation has been shown to be promoted by Van der
Waals forces between adhesion proteins, such as Flu/Ag43 (Lane et al., 2007) and TibA (Hasman et al., 1999), and bydepletion forces between bacterial cells that have been defined in colloid physics (Sherlock et al., 2005; Schwarz-Linek et al.,2010; Roux et al., 2005). Thus, it would be prudent to study how MFD clogging is affected by genetic and physicalmechanisms of auto-aggregation in efforts to optimize conditions for using living bacteria for food in culture systems in
MFDs.

Methods
Request a detailed protocol
Biofilm assay
Biofilm was quantified by a crystal violet (CV) (Tokyo Chemical Industry Co., LTD. Japan) quantitation method (Pratt and
Kolter, 1998, Roux et al., 2005). A single E. coli strain colony was inoculated in 1 ml LB medium in a polystyrene 24-wellplate (Multidish 24 wells Nunclon Delta SI, 142475, Nunc, Denmark, and 24-well Cell Culture Plate, 3524, Corning, USA) or
glass tube (IWAKI 10X 100 mm 9832-1310 Asahi Glass Co. LTD., Japan), and cultured at room temperature for 2 d. After
cultivation, the plate or glass tube was washed twice with 2 ml phosphate buffered saline and then incubated with 1% CV
solution for 1 h. After CV staining, the plate or glass tube was washed with 2 ml phosphate buffered saline, and CV wasresolved by 200 µl ethanol:acetone (8:2) solution. CV in ethanol:acetone solution was quantified by optical density at 600 nm(Microplate reader, Epoch2, BioTek, USA).
Isolation of OP50 fliC::Km and OP50 fimH::Km
Km cassettes, fliC::Km and fimH::Km in JW1908: BW25113 fliC::Km or JW4283: BW25113 fimH::Km, were transferred toOP50 by P1 transduction. The presence of fliC::Km and fimH::Km in OP50-derived colonies was checked by polymerase
chain reaction (PCR) using a primer set between K1 or K2 primers (Datsenko and Wanner, 2000) and fimH confirming F
primer (CACAATCAGCGCACTTCCCGTTACAG) or fliC confirming F primer (AGAAAAGAGTATTTCGGCGACTAAC)
with bacteria picked up from a colony of candidates OP50 fimH::Km and OP50 fliC::Km. Bacteria obtained from all colonies
of candidate OP50 fimH::Km and OP50 fliC::Km were subjected to PCR with the indicated primers; PCRs amplified DNA
fragments of expected size (~700 base pairs) when a primer set containing K1 (but K2) primer was used. We used clone 1 forOP50 fimH::Km and OP50 fliC::Km.
Motility assay
Motility of bacteria was compared by quantitation of colony size after culturing on LB plates with 0.3% agar (Eaves-Piles etal., 2008; Rashid and Kornberg, 2000). One-microliter aliquots of overnight culture solution of LB media were spotted onto
soft agar LB plates and incubate for 2 d at room temperature.
Statistical analysis
Statistical testing was performed with paired Student’s t-tests and p-values in multiple comparison were determined after
Bonferroni correction. We considered p < 0.05 to be statistically significant.

Reagents
BW25113 fliC::Km and JW4283: BW25113 fimH::Km were obtained from the National Institute of Genetics, Japan.

2/5/2020 - Open AccessAcknowledgments: We thank the Caenorhabditis Genetics Center (CGC) for the bacterial strain OP50. The CGC is funded
by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank the National BioResource Project-E.coli at
NIG for E. coli strains.

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Funding: Y. Arata is supported by a research grant, Challenging Research (Pioneering), Grants-in-Aid for Scientific Research(18H05300).
Author Contributions: Yukinobu Arata: Conceptualization, Investigation, Writing - original draft. Taku Oshima:
Investigation, Writing - review and editing. Yusaku Ikeda: Investigation. Hiroshi Kimura: Project administration, Writing review and editing. Yasushi Sako: Project administration.
Reviewed By: Anonymous
History: Received January 8, 2020 Accepted January 25, 2020 Published February 5, 2020
Copyright: © 2020 by the authors. This is an open-access article distributed under the terms of the Creative Commons
Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.
Citation: Arata, Y; Oshima, T; Ikeda, Y; Kimura, H; Sako, Y (2020). OP50, a bacterial strain conventionally used as food forlaboratory maintenance of C. elegans, is a biofilm formation defective mutant. microPublication Biology.
https://doi.org/10.17912/micropub.biology.000216