Fluorescence Tools Adapted for Real-Time Monitoring of theBehaviors of Streptococcus Species

R. C. Shields,a J. R. Kaspar,a K. Lee,a S. A. M. Underhill,b R. A. Burnea

aDepartment of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USAbDepartment of Physics, University of Florida, Gainesville, Florida, USA

ABSTRACT Tagging of bacteria with fluorescent proteins has become an essentialcomponent of modern microbiology. Fluorescent proteins can be used to monitorgene expression and biofilm growth and to visualize host-pathogen interactions.Here, we developed a collection of fluorescent protein reporter plasmids for Strepto-coccus mutans UA159 and other oral streptococci. Using superfolder green fluores-cent protein (sfGFP) as a reporter for transcriptional activity, we were able to charac-terize four strong constitutive promoters in S. mutans. These promoter-sfgfp fusionsworked both for single-copy chromosomal integration and on a multicopy plasmid,with the latter being segregationally stable in the absence of selective pressure un-der the conditions tested. We successfully labeled S. mutans UA159, Streptococcusgordonii DL1, and Streptococcus sp. strain A12 with sfGFP, DsRed-Express2 (red), andcitrine (yellow). To test these plasmids under more challenging conditions, we per-formed mixed-species biofilm experiments and separated fluorescent populations us-ing fluorescence-activated cell sorting (FACS). This allowed us to visualize two strep-tococci at a time and quantify the amounts of each species simultaneously. Thesefluorescent reporter plasmids add to the genetic toolbox available for the study oforal streptococci.

IMPORTANCE Oral streptococci are the most abundant bacteria in the mouth andhave a major influence on oral health and disease. In this study, we designed andoptimized the expression of fluorescent proteins in Streptococcus mutans and otheroral streptococci. We monitored the levels of expression and noise (the variability influorescence across the population). We then created several fluorescent protein de-livery systems (green, yellow, and red) for use in oral streptococci. The data showthat we can monitor bacterial growth and interactions in situ, differentiating be-tween different bacteria growing in biofilms, the natural state of the organisms inthe human mouth. These new tools will allow researchers to study these bacteria innovel ways to create more effective diagnostic and therapeutic tools for ubiquitousinfectious diseases.

KEYWORDS Streptococcus mutans, biofilm, fluorescence microscopy, greenfluorescent protein, oral streptococci

Green fluorescent protein (GFP) is widely used to explore bacterial behaviors (1).GFP has been used in studies of protein localization and gene expression, for

examining spatial arrangements in biofilms, and for visualizing bacteria in host cells.The utility of GFP was first described in 1994 when Chalfie et al. (2) identified the gfpgene in the jellyfish (Aequorea victoria). Over the last 20 years, a number of fluorescentprotein (FP) variants have been described, including optimized versions of GFP (e.g.,superfolder GFP [sfGFP]) and homologs of GFP that emit light at different wavelengths(e.g., DsRed) (3). Besides altering the protein, there are a number of factors that caninfluence the fluorescence intensity. These include transcription rates, mRNA stability,

Citation Shields RC, Kaspar JR, Lee K, UnderhillSAM, Burne RA. 2019. Fluorescence toolsadapted for real-time monitoring of thebehaviors of Streptococcus species. ApplEnviron Microbiol 85:e00620-19. https://doi.org/10.1128/AEM.00620-19.

Editor Donald W. Schaffner, Rutgers, The StateUniversity of New Jersey

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to R. C. Shields,rshields@dental.ufl.edu.

R.C.S. and J.R.K. contributed equally to thisarticle.

Received 14 March 2019Accepted 11 May 2019

Accepted manuscript posted online 17 May2019Published

METHODS

crossm

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 1Applied and Environmental Microbiology

18 July 2019

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

and codon bias (4–6). To achieve stable and high levels of GFP fluorescence in anorganism, optimized reporter systems have to be designed (7). Once reporter systemshave been designed, they can be used to visualize the organism in diverse experimen-tal models (8).

Approximately 35% of the human population has untreated caries in permanentteeth, leading to significant reductions in quality of life through pain, disfigurement,and difficulty eating (9). Dental caries, which involves the loss of tooth mineral, iscaused by a compositional change in the oral microbiome (10). Streptococcus mutans isthe organism that is most consistently associated with carious lesions and has multiplevirulence traits that contribute to its ability to initiate and worsen carious lesions (11).Its interactions with other oral bacteria, including health-associated streptococci, areimportant for determining the health status of a person’s teeth (12). Despite themedical importance of these microorganisms, there is a lack of molecular tools availablefor their study, in contrast to the number of molecular tools available for the study ofthe more intensively studied model organisms. This includes fluorescence-based toolsthat could be used for the study of microbial interactions, host-microbe interactions,single-cell behaviors, and biofilm dynamics. Recent examples of fluorescent proteintagging in oral streptococci include the work of Vickerman et al. (13), who tagged eightoral streptococci with mTFP1 and mCherry, and Shabayek and Spellerberg (14), whoused the GFP derivatives enhanced GFP (EGFP) and Sirius to fluorescently tag oralstreptococci. Further refinements of the already available tools are required to realizethe full potential of fluorescence tagging in oral bacteria. Refinements include increas-ing expression of the fluorescent proteins, testing different delivery systems and theirimpact on fluorescence noise, and measuring the utility of fluorescent proteins in morecomplex experimental systems.

Here, we developed plasmid vectors that carry fluorescent protein markers to allowfor the visualization of S. mutans and other oral streptococci. We tested four constitu-tively active promoters fused to a superfolder gfp (sfgfp) gene as a way to comparepromoter strength. The intensity and noise of these promoters delivered in differentvectors were determined at the single-cell level. Next, we designed, tested, andconfirmed the effectiveness of sfgfp, DsRed-Express2, and citrine gene expression fromplasmid vectors in S. mutans, Streptococcus gordonii DL1, and Streptococcus sp. strainA12. Finally, we show that these plasmids can be used to visually discriminate oralstreptococci in coculture biofilms.

RESULTSRanking the strength of four constitutive promoters in S. mutans. The first goal

of this project was to construct a plasmid that would allow for the chromosomalintegration of a constitutively expressed fluorescent protein. To accomplish this, syn-thetic constructs were cloned into the integration vector pPMZ (P, phnA; M, mtlA; Z,lacZ) (15, 16). This plasmid contains DNA sequences flanking the cloning sites that allowfor the integration of the desired DNAs into the phnA-mtlA locus, which is required onlyfor mannitol metabolism and which has no effect on the fitness of S. mutans in theabsence of mannitol. To maximize the expression of gfp, we designed four constructswith four different constitutive promoters, P3, P23, Pldh, and Pveg. P3 has prokaryoticconsensus �35 (TTGACA) and �10 (TATAAT) boxes and is a strong constitutivepromoter in Streptococcus pneumoniae (17). P23 is a lactococcal promoter (Lactococcuslactis subsp. cremoris) with strong constitutive activity in S. mutans (18, 19). Pveg is aconstitutive promoter originally identified in Bacillus subtilis that has strong expressionin a variety of bacterial hosts (20). Pldh is the promoter of the gene encoding the lactatedehydrogenase (ldh, SMu.1115) of S. mutans and has been used as a constitutivepromoter in S. mutans (21). The sequences of the promoters are shown in Fig. 1A, withthe �35, �10, �1 (the transcription initiation site), and Shine-Dalgarno (SD) sequencesbeing highlighted in bold. For the Pveg and P23 promoters, we used an optimizedribosomal binding site (RBS) that contains a consensus SD (AGGAGG) sequence (22);this RBS displayed maximal translational efficiency in B. subtilis (22). For P3, the original

Shields et al. Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 2

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

RBS sequence described by Sorg et al. was retained (17), as was the native RBS of theldh gene. Next, we selected a superfolder GFP (sfGFP) that was codon optimized forBacillus subtilis but that was also highly expressed in S. pneumoniae (7). sfGFP maturesrapidly (6 min in Escherichia coli) and is highly stable, making it a useful fluorescentreporter (23). The sequences of each promoter-sfgfp fusion are available in the supple-mental material. For ease of cloning, we used a gene synthesis service provided byIntegrated DNA Technologies (IDT). Sequences were synthesized and provided in aplasmid cloning vector. SacI and SphI were used to excise the constructs from the IDTvector, with subsequent cloning into the pPMZ vector. These vectors were thentransformed into competent S. mutans cells.

The levels of GFP expression for each individual construct were measured usingimmunodetection and a fluorescent plate reader. For Western blotting experiments, weused an anti-sfGFP polyclonal antibody (Sigma-Aldrich) against whole-cell lysates pre-pared from cells grown to the mid-exponential phase (optical density at 600 nm[OD600] � 0.5). We were able to detect sfGFP at this time point across all four constructs,with no evidence of degradation of sfGFP in the form of lower-molecular-weightimmunoreactive products (Fig. 1B). Using a plate reader, we were able to monitor thestrength of the fluorescence signals during the growth of S. mutans. For each construct,the absolute levels of GFP increased as the optical density of the population increased,consistent with promoter activity being constitutive. The fluorescence signals peakedduring the transition from late exponential to early stationary phase. In this assay,fluorescence levels were the highest for Pveg-sfgfp (33,000 relative fluorescence units[RFU]) and the lowest for Pldh-sfgfp (15,000 RFU) (Fig. 1C). None of the strains exhibitedany significant growth defects compared to wild-type S. mutans. Lastly, using fluores-

FIG 1 Fluorescence intensities of the four promoter-gfp fusion constructs. (A) Promoter sequences are shown, with the �35, �10, �1 (transcription initiationsite [TIS]), Shine-Dalgarno (ribosome binding site [RBS]), and sfgfp ATG sequences being indicated in bold. The promoter sequences were fused to thesuperfolder gfp gene, and these constructs were cloned into plasmids. To the best of our knowledge, for S. mutans Pldh the TIS has not been experimentallyvalidated. (B) Western blot detection of sfGFP using an anti-sfGFP antibody against whole-cell extracts collected at an OD600 of 0.5. (C) Fluorescence intensity(circles) and growth (lines) measured simultaneously using a microplate assay. (D) Single-cell fluorescence microscopy of S. mutans/pPM_Pveg-sfgfp. sfGFPfluorescence was detected by excitation at 488 nm, and emission was collected using a 525-nm (�25-nm)-band-pass filter. A 63� oil objective lens was usedto acquire the image.

Fluorescence Tools for Oral Streptococci Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 3

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

cence microscopy, S. mutans/Pveg-sfgfp cells could readily be visualized as greenovococci, the typical morphology of S. mutans (Fig. 1D).

Next, we cloned each promoter-sfgfp construct into the E. coli-streptococcal shuttlevector pDL278 (24). An advantage of this vector is that it can replicate in multiplestreptococcal species. The vector was constructed with a pVA380-1 backbone. PlasmidpVA380-1 was originally isolated from Streptococcus ferus and replicates via rolling-circle replication (RCR) (24–26). Plasmids that replicate via RCR can suffer from segre-gational instability, which has been attributed to the reliance of replication on single-stranded DNA intermediates, and the formation of linear high-molecular-weight (HMW)plasmid multimers (27). Plasmid-based fluorescence systems may not be useful ifselective pressure is required to avoid segregational instability. Addition of antibiotics(to increase plasmid stability) would limit the usefulness of GFP plasmids, as antibioticsare known to modify the physiology of bacteria in ways that alter important behaviors,including biofilm formation (28). To test for plasmid stability in the absence of specti-nomycin (pDL278 carries aad9), we grew pDL278 carrying Pveg-sfgfp in rich medium(brain heart infusion [BHI] medium) for approximately 50 generations. The strains weresubcultured (1:1,000) into fresh rich medium for a total of 5 serial passages, each ofwhich was incubated for 12 h. Prior to each passage, cells from the cultures were platedonto BHI agar with or without spectinomycin. After 48 h of incubation, the numbers ofCFU were counted and the total numbers of cells on selective and nonselective mediawere compared. Based on this plasmid stability assay, it was observed that pDL278carrying Pveg-sfgfp was retained over the entire course of the experiment, as there wereno significant differences (P � 0.6749) between the total number of cells that grew onBHI agar and the total number of cells that grew on BHI-spectinomycin agar (Fig. 2).

Confident that the GFP plasmids were segregationally stable under the conditionstested, we next tested the strength of GFP expression of each individual construct usingimmunodetection and plate reader measurements. As with the integrated reporters,Western blotting experiments confirmed the expression of sfGFP, with no visibledegradation of the protein, in the mid-exponential phase of growth (Fig. 3A). Next, sfgfpreporter strains were cultured in a microplate reader with fluorescence, and the opticaldensity was recorded for 16 h. The comparative strengths of the fluorescence signalsmatched those of the integrated constructs, with Pveg giving the strongest signal(330,000 RFU) and Pldh giving the weakest signal (150,000 RFU) (Fig. 3B).

Measuring the signal heterogeneity of the two GFP systems. While these GFPconstructs could be used for visualizing S. mutans, we were also interested in syntheticbiology applications from two perspectives: (i) using the sfgfp variants as readouts forpromoter activity and (ii) using the constitutive promoters for overexpression ofproteins or small RNAs. In both cases, it was important to know how heterogeneous thesignals were, as experimental outcomes can be altered if promoters and/or the type of

FIG 2 Stability of pDL278_Pveg-sfgfp in the presence and absence of spectinomycin. The numbers of spectinomycin-resistantS. mutans pDL278_Pveg-sfgfp cells cultured in the presence (A) or absence (B) of spectinomycin were compared. S. mutanscells harboring pDL278_Pveg-sfgfp were diluted 1:1,000 into BHI and cultured for 12 h. After 12 h the total numbers of S.mutans cells (blue) and spectinomycin-resistant S. mutans cells (red) were determined via serial dilution and agar plating. Thisassay was repeated five times for approximately 50 generations.

Shields et al. Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 4

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

construct (integrated versus plasmid borne) may influence the signal-to-noise ratios inthe system(s). Therefore, constructs were grown to an OD600 of 0.5, aliquots of thecultures were deposited on a microscope slide and covered with a coverslip, and thesfGFP intensity in single cells was measured for �1,000 cells per sample (see Materialsand Methods). The data for these experiments were plotted as histograms (Fig. 4), withthe number of cells per bin being provided on the x axis and the sfGFP intensity beingprovided on the y axis. When plotted in this manner, it was possible to observe therange of sfGFP fluorescence across the population. It was clear from these data that therange for sfGFP fluorescence was much larger for plasmid-borne sfgfp constructs thanfor constructs integrated into the chromosome (Fig. 4). Next, we calculated the Fanofactor (�p

2/�p�) for each construct, which provides a measure of the variance (squareof the standard deviation [�p

2]) over the mean value of expression (�p�) and can beused to measure gene expression noise (29). Phenotypic noise (Fano factor) wasincreased when the constructs were plasmid-borne (Table 1). Noise was also increasedwith increased promoter activity. For example, the Fano factor for the integratedversion of Pveg-sfgfp was almost 8 times that for the Pldh-sfgfp integrated construct.

FIG 3 Characterization of plasmid-borne promoter-sfgfp constructs in S. mutans. (A) Western blot detection of sfGFP using an anti-sfGFP antibody againstwhole-cell extracts collected at an OD600 of 0.5. (B) Fluorescence intensity (circles) and growth (lines) measured simultaneously using a microplate assay.

FIG 4 Single-cell fluorescence intensities of integrated and plasmid-borne constructs. For each promoter-sfgfp fusion, the distribution of fluorescence strength (x axis) is plotted as a histogram, with the resultsfor both integrated and plasmid-borne constructs being shown on the same graph. sfGFP intensities arehigher and more widely distributed for the plasmid-borne constructs than for the integrated constructs.

Fluorescence Tools for Oral Streptococci Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 5

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

Fluorescent protein constructs for use in oral streptococci. We next wanted tobuild several plasmid-based constructs to aid the visualization of other oral strepto-cocci, choosing the E. coli-streptococcal shuttle vector pDL278 in anticipation that itwould replicate and be stable in a variety of streptococci. We also selected the P23promoter with the optimized RBS sequence, along with three different fluorescentprotein sequences to incorporate sfGFP, DsRed-Express2 (30), and a yellow fluorescentprotein variant, citrine, that is more resistant to acid quenching (31). Both the DsRed-Express2 and citrine gene sequences were downloaded from the SnapGene softwarewebsite, codon optimized for use in S. mutans using the IDT codon optimization tool,synthesized in combination with the P23 promoter, and cloned into pDL278, similarlyto the sfGFP constructs. The vectors pDL278_P23-sfgfp, pDL278_P23-DsRed-Express2,and pDL278_P23-citrine were then transformed into three different streptococci: S.mutans UA159; Streptococcus gordonii DL1; and Streptococcus A12, which is phyloge-netically situated between Streptococcus australis and Streptococcus parasanguinis (32).

We detected fluorescence signals from all three constructs in a microplate readerduring an 18-h incubation, as well as by fluorescence microscopy (Fig. 5). From theseobservations, we noted a strain-specific diversity in the fluorescence intensity, depend-ing on the reporter construct tested. For example, S. gordonii displayed higher levels ofthe sfGFP signal than S. mutans (1.5-fold greater) and strain A12 (4.4-fold greater) (Fig.5A and B), whereas S. mutans produced a higher citrine signal than S. gordonii (2.3-foldincrease) (Fig. 5A and B). In fact, A12 failed to express the P23-citrine construct underthe conditions tested. The signal production patterns of DsRed-Express2 for the threestrains were similar to those of sfGFP (Fig. 5A, B, and C). The growth of both S. mutansand S. gordonii was not substantially reduced by the reporters in either defined medium(Fig. 5D and E) or rich medium (see Fig. S1A and B in the supplemental material). Thereporters, most notably, pDL278_P23-citrine, did slow the growth of Streptococcus A12(Fig. 5F). Citrine expression in Streptococcus A12 appeared to be toxic to the organism,and this might explain the lack of yellow fluorescence detected during microscopy inthis organism (Fig. 5I). The stability of the pDL278_P23-citrine plasmid in StreptococcusA12 is also discussed below.

S. mutans UA159 and S. gordonii DL1 are model organisms for oral streptococcalresearch and are lab adapted, whereas Streptococcus A12 is a novel streptococcalisolate that has not been highly passaged. We wanted to determine if aberrant plasmidstability of pDL278 in Streptococcus A12 might be responsible for the lower fluores-cence signal from our constructed vectors. For these experiments, we also includedStreptococcus sanguinis SK150, which returned spectinomycin-resistant colonies aftertransformation with our vectors, but a fluorescent signal could not be detected for S.sanguinis SK150 when assayed, nor could fluorescence be observed by microscopy. Oursampling over a total of approximately 50 generations for S. mutans UA159, S. gordoniiDL1, S. sanguinis SK150, and Streptococcus A12 verified that the pDL278_P23-DsRed-Express2 plasmid was stable in all four organisms under the conditions tested, as thenumbers of CFU were similar on BHI and BHI-spectinomycin agar at all time points (Fig.S2A). When the presence of pDL278 after 50 generations was checked by PCR usingpDL278-specific primers, we found that only a faint band was present in S. sanguinisSK150, even at the beginning of our plasmid stability experiment (time zero) (Fig. S2B).

TABLE 1 Fano factor measurements of single-cell sfGFP heterogeneity

Promoter

Integrated Plasmid

Median intensity (A.U.)a Noiseb Median intensity (A.U.) Noise

Pldh 641 3.05 � 104 3,197 1.73 � 106

P3 1,235 1.38 � 105 6,085 8.03 � 106

P23 914 5.71 � 104 9,331 1.80 � 107

Pveg 1,677 2.39 � 105 10,782 1.71 � 107

aA.U., arbitrary units.bCalculated as the Fano factor (�p

2/�p�).

Shields et al. Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 6

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

For Streptococcus A12, we could not confirm the presence of pDL278_P23-citrine, eventhough the strain had become spectinomycin resistant (Fig. S3). This is the most likelyreason why no citrine could be detected in Streptococcus A12 in either our microplatereader or our microscopy experiments (Fig. 5G and I). These observations indicate thatthese particular constructs may not be suitable for all Streptococcus species. Therefore,constructs should be rigorously tested, and changes to the vector, promoter, orfluorescent reporter may be necessary prior to use in an isolate of interest.

Application of the fluorescent tools for biofilm studies. As we were able todetect fluorescence in all three species tested for both the sfGFP and DsRed-Express2constructs, we next moved to test whether we would be able to detect sufficientlystrong signals in a mixed-species biofilm model for future biofilm studies. We chose toexamine three different mixed-species biofilms grown over the course of 72 h, withimaging being performed every 24 h: S. mutans/pDL278_P23-sfgfp and S. mutans/pDL278_P23-DsRed-Express2 (S. mutans-only control biofilms), S. mutans/pDL278_P23-sfgfp and S. gordonii/pDL278_P23-DsRed-Express2, and S. mutans/pDL278_P23-sfgfpand Streptococcus A12/pDL278_P23-DsRed-Express2. For this experiment, biofilms weregrown in ibidi �-Slide 8-well chamber slides using chemically defined medium (CDM)supplemented with 20 mM glucose and 5 mM sucrose. After 24 h of incubation, thegrowth medium of the biofilm was replaced every 12 h (Fig. S4). Biofilms were analyzedby confocal microscopy in situ with no washing or medium replacement so as not todisrupt the biofilm structure.

Over the course of 72 h, we were able to visualize both bacterial species within thegrowing biofilms (Fig. 6). All three species tested were able to produce a strong, stable

FIG 5 Detection of fluorescence among different oral Streptococcus spp. in monoculture. The robustness of fluorescent reporter plasmids was evaluated in S.mutans UA159 (top row), S. gordonii DL1 (middle row), and Streptococcus A12 (bottom row). For each species, we measured the maximum fluorescence intensityof reporter plasmids using a Synergy 2 multimode plate reader after 18 h of growth (A, S. mutans; B, S. gordonii; C, Streptococcus A12) (A.U., arbitrary units).Growth measurements in defined medium were taken from the reporter strains using a Bioscreen C automated growth curve analysis system (D, S. mutans;E, S. gordonii; F, Streptococcus A12). Colored circles indicate strain backgrounds, as follows: wild-type strains in gray, pDL278_P23-sfgfp strains in green,pDL278_P23-citrine strains in yellow, and pDL278_P23-DsRed-Express2 strains in red. Fluorescence was also visualized using a confocal microscope, once thecell cultures had reached stationary phase (OD600, 0.7 to 0.8) in all three species (G, S. mutans; H, S. gordonii; I, Streptococcus A12). Images are overlays of thebright-field and fluorescence channels. We did not detect yellow fluorescence for Streptococcus A12/pDL278_P23-citrine.

Fluorescence Tools for Oral Streptococci Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 7

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

signal for imaging over the course of the experiment. Using this technique, weobserved differences in biofilm architecture between our three different dual-speciesgroups, especially the distribution of bacteria within the growing biofilms. Microcolonyformation was observed within our S. mutans-only control biofilm (Fig. 6A). S. mutanscells at 24 h were visualized within microcolonies and did not completely coat thesurface of the glass slides, leaving uncolonized sections of the substratum. Over thecourse of 72 h, these microcolonies grew in size to 20 to 30 �m in diameter. In contrast,no large microcolony formation for S. mutans was observed when it was coculturedeither with S. gordonii (Fig. 6B) or with Streptococcus A12 (Fig. 6C). We found that thedistribution of either S. gordonii or Streptococcus A12 differed when it was coculturedwith S. mutans. S. gordonii was found to cover much of the surface at 24 h, with S.mutans being found in only small microcolonies. The S. mutans microcolonies eventu-ally expanded over the course of the experiment, with S. gordonii ceding surface area.This was not the case in dual-species biofilms with Streptococcus A12. At 24 h, S. mutans

FIG 6 Images of dual-species biofilms. (A to C) Selected 3D reconstructions of maximum-intensity z-sectionconfocal microcopy images of dual-species biofilms of either S. mutans/pDL278_P23-sfgfp and S. mutans/pDL278_P23-DsRed-Express2 (control biofilm) (A), S. mutans/pDL278_P23-sfgfp and S. gordonii/pDL278_P23-DsRed-Express2 (B), and S. mutans/pDL278_P23-sfgfp and Streptococcus A12/pDL278_P23-DsRed-Express2 (C) atthe 24, 48-, and 72-h time points. Images are of 128-�m sections of the fluorescence range within the biofilmcollected at 1-�m intervals using a 63� oil objective lens (numerical aperture, 1.40). Biofilms were grown inchemically defined medium (CDM) supplemented with 20 mM glucose and 5 mM sucrose. At 24 h and every 12 hthereafter the spent medium was replaced with fresh medium for the length of the experiment. The biofilm imagesfrom time course experiment were reconstructed with Imaris software (6.4.0) using shadow projections. (D and E)The biofilm images from the time course experiment were analyzed by the Comstat2 (v2.1) program for biomass(D) and maximum thickness (E). Green line, S. mutans/pDL278_P23-sfgfp and S. mutans/pDL278_P23-DsRed-Express2 dual-species biofilms (control biofilm); orange line, S. mutans/pDL278_P23-sfgfp and S. gordonii/pDL278_P23-DsRed-Express2 dual-species biofilms; blue line, S. mutans/pDL278_P23-sfgfp and Streptococcus A12/pDL278_P23-DsRed-Express2 dual-species biofilms.

Shields et al. Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 8

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

predominately covered the substratum. We noted that Streptococcus A12 was visual-ized alongside of, but not within, the S. mutans microcolonies at early time points.Streptococcus A12 became associated with the sides of the S. mutans microcolonies asthey expanded over time but was not found within the microcolonies. Analysis of theseimages by the use of the Comstat2 program confirmed these observations, as we foundthe biomass to decrease over time for both of our S. gordonii DL1 and Streptococcus A12mixed-species biofilms but to increase in our S. mutans-only control biofilm (Fig. 6D).Similarly, the maximal thickness increased within our S. mutans single-species biofilmover time, whereas the thickness only moderately changed in our S. gordonii DL1 orStreptococcus A12 biofilm group (Fig. 6E). We were also able to obtain mixed-speciesbiofilm images from citrine- and DsRed-tagged bacteria (Fig. S5). The excitation andemission spectra of sfGFP and citrine overlap, which can make these proteins hard todetect with optimal brightness in the same experiment. However, a multispeciesbiofilm of bacteria colored by GFP, yellow fluorescent protein (YFP), and red fluorescentprotein (RFP) might be possible with a confocal microscope that is capable of spectralimaging and linear unmixing.

Another attractive option for research studies is the direct quantification of bacteriawithin the biofilm using flow cytometry for detection of these fluorescent reportersrather than using more traditional and labor-intensive methods, such as enumerationof the CFU on selective media. The use of flow cytometry can save time, as datacollection is extremely rapid and can be more cost-effective because of the reductionin the amount of supplies, e.g., agar and plastics, used. We analyzed our 24-h dual-species biofilms by flow cytometry to determine if our constructed reporters would beviable for such an approach (Fig. 7). Indeed, a clear separation between our sfGFP- andDsRed-producing strains could be observed, such that we were able to determine theproportion of each species within our biofilm samples. Additionally, the total numberof cells within the population that were analyzed as containing either sfGFP or DsRedwas �99%, suggesting that the constructs and fluorescent proteins were stable enoughduring biofilm growth to allow for accurate counting. Our S. mutans cocultured controlbiofilm contained a roughly equal proportion of sfGFP and DsRed, as expected (Fig. 7A).

FIG 7 Quantification of dual-species biofilms by flow cytometry. Histogram of cell counts from flowcytometry analysis of the pDL278_P23-DsRed-Express2 reporter on the left and a colored dot plot of boththe pDL278_P23-DsRed-Express2 and pDL278_P23-sfgfp fluorescence intensity on the right of thefollowing biofilms grown for 24 h: S. mutans/pDL278_P23-sfgfp and S. mutans/pDL278_P23-DsRed-Express2 (control biofilm) (A), S. mutans/pDL278_P23-sfgfp and S. gordonii/pDL278_P23-DsRed-Express2(B), and S. mutans/pDL278_P23-sfgfp and Streptococcus A12/pDL278_P23-DsRed-Express2 (C). The greenand red colored markers in the histogram denote the DsRed-negative and DsRed-positive cell popula-tions that were used to determine the percentage of each within the sample. A total of 50,000 cells werecounted in three independent replicates for each experiment.

Fluorescence Tools for Oral Streptococci Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 9

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

However, the proportions of S. gordonii DL1 (Fig. 7B) and Streptococcus A12 (Fig. 7C)were different when they were grown with S. mutans: S. gordonii DL1 cells represented83% of the biofilm cells at 24 h when S. gordonii DL1 was grown with S. mutans,whereas Streptococcus A12 cells comprised only 2% of the total cells measured at thesame time point. These data correlate with what was observed by confocal microscopyat 24 h. In all, these observations highlight the feasibility of the use of the constructedfluorescent reporter strains for biofilm research not only to reveal differences in biofilmarchitecture or spatial arrangement when coculturing different streptococcal speciestogether but also to allow direct quantification by flow cytometry.

DISCUSSION

With this study we aimed to provide new fluorescence visualization tools for S.mutans and other oral streptococci. We also used this study as an opportunity to testthe relative strength of four constitutive promoters and determine how consistentlythese vectors produced fluorescence across a population of cells (noise). Lastly, we usedthese fluorescence tools to label oral streptococci so that they could be differentiatedby using microscopy in a coculture biofilm system. Collectively, we achieved theconstruction of fluorescent reporter plasmids that are conformationally and segrega-tionally stable with high FP expression for use in a broad range of oral streptococci andthat should be valuable for gathering information on bacterial biofilm behaviors andinterspecies interactions.

All four of the promoters tested in this study were constitutive and gave measurablelevels of gene expression in S. mutans. Of the four, the veg promoter (Pveg) of B. subtilis,which included an optimized SD sequence, was the strongest. In a previous study byBiswas et al. (19), Pveg was also shown to have high activity in S. mutans. The other threepromoters, P23, P3, and Pldh, were also active but showed moderately lower levels oftranscriptional activity (approximately half the fluorescence intensity) compared withthat yielded by the Pveg constructs. For synthetic biology applications, it would beuseful to characterize weakly, moderately, and highly expressed promoters in S. mutans.The promoters that we tested here exhibit moderate to high levels of gene expression,but the platforms that we have developed could be used to screen for weaker orstronger promoters in a more comprehensive approach. It is important to note thatoptimal expression for one protein (e.g., GFP) might not correlate with that for anotherbecause of differences in 5=-end mRNA folding affecting mRNA stability and translationinitiation (33). This is an especially important consideration here, as we did not use thesame RBS for each promoter-sfgfp fusion. Therefore, it is difficult to determine whichpart of the promoter/5= untranslated region is responsible for the changes in sfGFPlevels across the four constructs. Despite these limitations, the data should be valuablein selecting which promoter-FP construct would be most helpful for particular appli-cations in S. mutans.

The location of the promoter-sfgfp constructs, whether integrated in the chromo-some or on a multicopy plasmid, did not alter the promoter strength rankings (Pveg

displayed the highest activity). There was a measurable increase in the signal intensityand noise for the plasmid constructs compared with those for the integrated versions.Plasmids are typically present in multiple copies per cell, and this is the most likelyexplanation for the increased sfGFP fluorescence compared with that achieved withsingle-copy genome integration. pDL278 is based on the streptococcal plasmidpVA380-1, which has a plasmid copy number of 25 (34). The wider distribution ofsfGFP fluorescence between cells in a population for pDL278 constructs is not aconcern for strain marking. However, it could add stochasticity to experiments whereit is not needed (e.g., promoter-reporter or overexpression studies). Increased popula-tion heterogeneity is most likely associated with variability of the actual plasmid copynumber per cell, which could be influenced by segregational instability (e.g., randomtransmission to daughter cells or the formation of HMW multimers) or structural issues(e.g., replicative infidelity or illegitimate recombination, leading to plasmid rearrange-

Shields et al. Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 10

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

ments) (27, 35). Our single-cell analysis should aid researchers in selecting the optimalvectors and/or promoters for synthetic biology applications in S. mutans.

Having determined the relative expression from the promoter-RBS combinationsand compared the noise levels between cells in a population from chromosomal andextrachromosomal sfGFP constructs in S. mutans, we were next able to use thisknowledge to create various GFP, RFP, and YFP reporters in S. mutans and other oralstreptococci. We selected the E. coli-streptococcal shuttle vector pDL278 as the back-bone for reporter constructs because it replicates in oral streptococci and cloning canbe performed in E. coli (24). While these constructs worked well in most of the strainsunder the conditions tested, we did observe species- and gene-specific stability issues.Segregational and/or conformational plasmid stability differences between bacterialspecies or gene inserts have been observed by others (36). However, we anticipate thatthe constructs will function well across the majority of transformable oral streptococciand will allow timely fluorescent protein tagging of strains of interest. The reportermethods developed here offer several advantages compared to other techniques,including the imaging of bacteria without addition of exogenous dyes (e.g., LIVE/DEADstaining or fluorescent in situ hybridization), live cell imaging, visualization of biofilmcells without disruption of the biofilm structure, and straightforward dual labeling ofstrains in coculture experiments. Fluorescent reporter systems also have some weak-nesses. Fluorescent proteins are sensitive to certain environmental conditions (e.g.,acidic conditions or low oxygen concentrations), and they can photobleach duringextended excitation periods (the rate is protein specific) (37). It would be of interest totest these reporters in more diverse environments to determine their activities undermore conditions that could conceivably be encountered in a human host.

Using our newly designed reporters, we were able to visualize two Streptococcusspp. simultaneously within biofilms. There is a renewed interest in the study ofmicrobial interactions within different microbiome communities that colonize thehuman body. One recent example is the exploration of the community structure withinsupragingival dental plaque (38). Of interest are the interactions between the earlycolonizers of the oral biofilm, consisting largely of Streptococcus and Actinomycesspecies that efficiently attach to the salivary pellicle. An inverse association existsbetween the abundance of these commensal species and that of cariogenic bacteria,such as mutans group streptococci. An increase in the proportions of cariogenicbacteria under acidic conditions promotes the lower diversity of organisms within thebiofilms and demineralization of the tooth enamel (12). Exploring the interactionsbetween these health-associated commensals and acidogenic bacteria is critical for ourunderstanding of the pathogenic potential of S. mutans and for the development ofnovel therapeutic interventions, e.g., probiotic strains that antagonize the growth of S.mutans through multiple pathways (32, 39). The construction and use of the variousstreptococcal fluorescent strains described here should allow for better insight into thebehaviors of multispecies biofilms.

In summary, this study provides new tools that should enable researchers to shedlight on critical traits of S. mutans and microbe-microbe interactions. Our detailedinvestigation of several constitutive promoters should serve as a basis for the correctselection of tools for synthetic biology applications in S. mutans. In addition, theplasmid-borne fluorescence reporters will allow the timely tagging of streptococci andbe beneficial in various experimental systems.

MATERIALS AND METHODSStrains and growth conditions. The strains of Streptococcus mutans and other Streptococcus spp.

(Table 2) were cultured in brain heart infusion (BHI) broth (Difco) or chemically defined medium (FMC)(40) supplemented with either 25 mM glucose (for planktonic growth) or 20 mM glucose and 5 mMsucrose (for growth as biofilms). Streptococci were grown in a 5% CO2 aerobic environment at 37°C,unless stated otherwise. Strains of Escherichia coli (Table 2) were routinely grown in lysogeny broth (LB)with slight modifications (Lennox LB; 10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaCl) at 37°C withaeration. The following antibiotics were added to the growth media at the indicated concentrations:kanamycin, 1.0 mg/ml for S. mutans and 50 �g/ml for E. coli; spectinomycin, 1.0 mg/ml for S. mutans and50 �g/ml for E. coli; and ampicillin, 100 �g/ml for E. coli. The strains and plasmids are listed in Table 2.

Fluorescence Tools for Oral Streptococci Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 11

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

Construction of plasmids. Each construct was created using gene synthesis (Integrated DNATechnologies) and restriction enzyme cloning. Previously published promoter sequences were combinedwith an sfgfp sequence (which is codon optimized for Bacillus subtilis but which has a good intensity inStreptococcus pneumoniae). At the 5= end of the sequence we included a SacI recognition site, and at the3= end we included an SphI recognition site. These restriction sites facilitated the cloning into themulticopy E. coli-streptococcal shuttle plasmid pDL278 (24) and the integration plasmid pPMZ (16). ForpPMZ, restriction digestion removed the PaguR promoter and the lacZ gene. Cloning into these plasmidswas carried out in NEB 10-beta competent E. coli (New England BioLabs). After cloning, the plasmids werepurified using a QIAprep spin miniprep kit (Qiagen) and transformed into competent S. mutans UA159with selection on the appropriate antibiotic (for pDL278, spectinomycin; for pPMZ, kanamycin). For theDsRed-Express2 and citrine constructs, a similar protocol was followed, except that the sfGFP sequencewas replaced with the sequence for DsRed2-Express2 or citrine.

Microtiter plate assays. For monitoring of cell density and GFP fluorescence over time, S. mutans orstrains of commensal streptococci were processed as follows. Strains were cultured to an OD600 of 0.5and then diluted 1:100 into FMC with glucose as the sole carbohydrate source. In quadruplicate, 200 �lof the cultures was placed in dark-sided, 96-well microtiter plates. For each fluorescent strain, a controlthat harbored the plasmid or integrated cassette without the gene for the fluorescent protein was usedto allow subtraction of the background fluorescence of the cells and/or medium components. Afterloading of the cultures, the 96-well plate was placed in a Synergy HT microtiter plate reader (BioTek). Thefluorescence and optical density (absorbance at 600 nm) were measured at 30-min intervals using Gen5software (BioTek). The fluorescence settings were as follows: for GFP, excitation at 485 nm, emission at525 nm, and a sensitivity of 65; for DsRed-Express2, excitation at 560 nm, emission at 590 nm, and asensitivity of 65; and for citrine, excitation at 510 nm, emission at 530 nm, and a sensitivity of 65. Datareadings were collected, and the background fluorescence or OD600 was subtracted prior to datavisualization using GraphPad Prism (v7) software (GraphPad Software).

Western blotting. The bacterial strains were grown in FMC at 37°C to an OD600 of 0.5. Cells wereharvested by centrifugation at 3,500 � g for 10 min, spent medium was discarded, and cell pellets werestored overnight at �20°C. On the following day, the pellets were resuspended in 100 �l lysis buffer(60 mM Tris, pH 6.8, 2% sodium dodecyl sulfate [SDS]) and transferred to a screw-cap microcentrifugetube that contained 100 �l 0.1-mm ice-cold glass beads. Samples were homogenized in a bead beaterfor 30 s three times with 5-min intervals on ice. The samples were then centrifuged at 8,000 � g for10 min at 4°C. The supernatants, which contained the S. mutans proteins, were carefully removed andplaced into a fresh 1.5-ml microcentrifuge tube. The protein concentration was determined using abicinchoninic acid (BCA) assay following the supplier’s protocol (Pierce), with purified bovine serumalbumin used as the standard. Ten micrograms of each lysate was diluted in 4� SDS loading buffer, andthe mixture was boiled for 10 min. Protein samples were separated by SDS-polyacrylamide gel electro-phoresis (PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane using aTrans-Blot Turbo transfer system (Bio-Rad). Green fluorescent protein was detected using a poly-clonal anti-GFP antibody (1:5,000 dilution; Millipore Sigma) and a goat-anti-rabbit immunoglobulinG (IgG) antibody (1:5,000 dilution; SeraCare Life Sciences, USA). Western blot signals were detected

TABLE 2 Strains and plasmids used in this study

Strain or plasmid Genotype or descriptiona Reference or source

StrainsS. mutans UA159 Wild type ATCC 700610S. gordonii DL1 Wild type ATCC 35105S. australis-like strain A12 Wild type 32E. coli NEB 10-beta Cloning host, derivative of DH10B New England Biolabs

PlasmidspPMZ S. mutans integration vector 16pDL278 E. coli-streptococcal shuttle vector; Spr 24pRCS5 Pveg-sfgfp in IDT ampicillin vector This studypRCS6 P23-sfgfp in IDT ampicillin vector This studypRCS7 Pldh-sfgfp in IDT ampicillin vector This studypRCS16 P3-sfgfp in IDT ampicillin vector This studypPM_Pveg-sfgfp phnA= Pveg-sfgfp km =mtlA Kmr Emr This studypPM_P23-sfgfp phnA= P23-sfgfp km =mtlA Kmr Emr This studypPM_Pldh-sfgfp phnA= Pldh-sfgfp km =mtlA Kmr Emr This studypPM_P3-sfgfp phnA= P3-sfgfp km =mtlA Kmr Emr This studypDL278_Pveg-sfgfp E. coli-streptococcal shuttle vector; Spr This studypDL278_P23-sfgfp E. coli-streptococcal shuttle vector; Spr This studypDL278_Pldh-sfgfp E. coli-streptococcal shuttle vector; Spr This studypDL278_P3-sfgfp E. coli-streptococcal shuttle vector; Spr This studypDL278_P23-DsRed-Express2 E. coli-streptococcal shuttle vector; Spr This studypDL278_P23-citrine E. coli-streptococcal shuttle vector; Spr This study

akm, polar kanamycin resistance; Kmr, kanamycin resistance; Spr, spectinomycin resistance; Emr,erythromycin resistance.

Shields et al. Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 12

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

using a SuperSignalWest Pico chemiluminescent substrate kit (Thermo Fisher Scientific) and visual-ized with a FluorChem 8900 imaging system (Alpha Innotech, USA).

Plasmid stability assay. Streptococcal strains carrying derivatives of pDL278 were cultured in BHIwith or without spectinomycin for 12 h. Every 12 h (for a total of 5 passages, or approximately 50generations), the strains were subcultured 1:1,000 into fresh medium. At each passage time point, analiquot from the 12-h culture was serially diluted onto BHI agar with and without selection (spectino-mycin). After 48 h, the colonies on these plates were enumerated and the numbers of CFU in thepresence or absence of selective pressure were calculated. Experiments were repeated at least threetimes.

Single-cell GFP measurements. S. mutans cells were washed twice in phosphate-buffered saline(PBS), pH 7.2, from overnight cultures and diluted 20-fold into FMC growth medium. At an OD600 of 0.5,the culture was gently sonicated using a Fisher Scientific FB120 sonic dismembrator probe in order todechain the cells, and then 4 �l of each sample was pipetted onto a glass coverslip and imaged on aNikon TE2000-U inverted phase-contrast microscope using a Nikon C-FL GFP HC HISN zero-shift filtercube with a Nikon Intensilight mercury arc lamp source for GFP fluorescence excitation and detection.Images were collected on a Photometrics Prime CMOS camera and analyzed by a previously describedmethod (41).

Biofilm time course imaging and analysis. To begin the biofilm experiments, overnight cultures ofselected strains grown in BHI medium with appropriate antibiotics were washed and diluted 1:20 intofresh CDM (42) with glucose as the sole carbohydrate source and were then grown to mid-exponentialphase (OD600 � 0.5). An aliquot (10 �l) of each strain was added to 1 ml of CDM supplemented with20 mM glucose and 5 mM sucrose, and 350 �l of these mixtures was used to inoculate one chamber ofan ibidi �-Slide 8-well chamber slide (catalog number 80826; ibidi GmbH). CDM is used for theseexperiments because it is more strongly buffered than FMC. The use of CDM rather than FMC reducesthe impact of the acidification of the medium on fluorescent protein stability and diminishes theantagonistic capabilities of S. mutans against commensal organisms. The samples were incubated at 37°Cin a 5% CO2 aerobic atmosphere, and the time of inoculation was represented as time zero. Every 24 h(times of 24, 48, and 72 h), the biofilms were removed from the incubator and imaged by confocalmicroscopy (see Fig. S2 in the supplemental material). At 24 h and every 12 h thereafter, the spentmedium was gently removed from the biofilms and replenished with fresh medium. Biofilm images wereacquired using a spinning-disk confocal system connected to a Leica DM IRB inverted fluorescencemicroscope equipped with a Photometrics cascade-cooled electron-multiplying charge-coupled-device(EMCCD) camera. GFP fluorescence was detected by excitation at 488 nm, and emission was collectedusing a 525-nm (�25-nm)-band-pass filter. Detection of DsRed-Express2 fluorescence (RFP) was per-formed using a 545-nm excitation laser and a 590-nm (�25-nm)-band-pass filter. All z-sections werecollected at 1-�m intervals using a 63� oil objective lens (numerical aperture, 1.40). Image acquisitionand processing were performed using VoxCell software (VisiTech International). The three-dimensional(3D) reconstruction of selected biofilm images was completed using Imaris software (v6.4.0; Bitplane),and analysis was completed using the Comstat2 program (v2.1; www.comstat.dk) (43). For each biofilmand time point, five images were acquired from different parts of the biofilm and used for image analysis.

Flow cytometry. Dual-species bacterial biofilms were grown for 24 h before being harvested,washed, and resuspended in 1� PBS. Cells were sonicated in a water bath sonicator for 4 intervals of 30 seach while resting on ice in 5-ml polystyrene round-bottom tubes. Samples were analyzed using an LSRII (BD Biosciences) flow cytometer. Forward and side scatter signals were set stringently to allow sortingof single cells. In total, 5 � 104 cells were counted from each event at a maximum rate of 2 � 103 cellsper second, and each experiment was performed in triplicate. Data were acquired for unstained cells andsingle-color-positive controls so that the data collection parameters and compensation could be properlyset. The data were collected using FACSDiva software (BD Biosciences) and analyzed with FCS Express(v4) software (De Novo Software). Gating for quadrant analysis was selected by using a dot density plotwith forward and side scatter, with the gates being set to capture the densest section of the plot. x- andy-axis data represent logarithmic scales of fluorescence intensity (arbitrary units).

Addgene availability. The following plasmids are stored on Addgene, the not-for-profit plasmidrepository: pPM_Pveg-sfgfp (Addgene number 121503; Pveg-sfgfp integration vector for S. mutans),pDL278_P23-sfgfp (Addgene number 121504; P23-sfgfp E. coli-streptococcal shuttle vector), pDL278_P23-DsRed-Express2 (Addgene number 121505; P23-DsRed-Express2 E. coli-streptococcal shuttle vector),and pDL278_P23-citrine (Addgene number 121506; P23-citrine E. coli-streptococcal shuttle vector).

Accession number(s). The DNA sequences of each synthetic construct are deposited at NCBI underaccession numbers MK301203 for Pveg-gfp, MK301204 for P23-gfp, MK301205 for Pldh-sfgfp, MK301206 forP3-sfgfp, MK301207 for P23-DsRed-Express2, and MK301208 for P23-citrine. These sequences are alsoavailable in the supplemental material.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/AEM

.00620-19.SUPPLEMENTAL FILE 1, PDF file, 0.4 MB.

ACKNOWLEDGMENTSThe research reported in this publication was supported by the National Institute of

Dental and Craniofacial Research of the National Institutes of Health under award

Fluorescence Tools for Oral Streptococci Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 13

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

numbers R01 DE013239, R01 DE025832, R01 DE023339, T90 DE21990, F32 DE028469,and F30 DE028184.

We declare that there are no potential conflicts of interest.

REFERENCES1. Southward CM, Surette MG. 2002. The dynamic microbe: green fluores-

cent protein brings bacteria to light. Mol Microbiol 45:1191–1196.https://doi.org/10.1046/j.1365-2958.2002.03089.x.

2. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. 1994. Greenfluorescent protein as a marker for gene expression. Science 263:802– 805. https://doi.org/10.1126/science.8303295.

3. Rodriguez EA, Campbell RE, Lin JY, Lin MZ, Miyawaki A, Palmer AE, ShuX, Zhang J, Tsien RY. 2017. The growing and glowing toolbox of fluo-rescent and photoactive proteins. Trends Biochem Sci 42:111–129.https://doi.org/10.1016/j.tibs.2016.09.010.

4. Kudla G, Murray AW, Tollervey D, Plotkin JB. 2009. Coding-sequencedeterminants of gene expression in Escherichia coli. Science 324:255–258. https://doi.org/10.1126/science.1170160.

5. Agashe D, Martinez-Gomez NC, Drummond DA, Marx CJ. 2013. Goodcodons, bad transcript: large reductions in gene expression and fitnessarising from synonymous mutations in a key enzyme. Mol Biol Evol30:549 –560. https://doi.org/10.1093/molbev/mss273.

6. Boël G, Letso R, Neely H, Price WN, Wong K-H, Su M, Luff J, Valecha M,Everett JK, Acton TB, Xiao R, Montelione GT, Aalberts DP, Hunt JF. 2016.Codon influence on protein expression in E. coli correlates with mRNAlevels. Nature 529:358 –363. https://doi.org/10.1038/nature16509.

7. Overkamp W, Beilharz K, Detert Oude Weme R, Solopova A, Karsens H,Kovacs AT, Kok J, Kuipers OP, Veening J-W. 2013. Benchmarking variousgreen fluorescent protein variants in Bacillus subtilis, Streptococcus pneu-moniae, and Lactococcus lactis for live cell imaging. Appl Environ Micro-biol 79:6481– 6490. https://doi.org/10.1128/AEM.02033-13.

8. Sullivan MJ, Ulett GC. 2018. Stable expression of modified green fluo-rescent protein in group B streptococci to enable visualization in exper-imental systems. Appl Environ Microbiol 84:e01262-18. https://doi.org/10.1128/AEM.01262-18.

9. Pitts NB, Zero DT, Marsh PD, Ekstrand K, Weintraub JA, Ramos-Gomez F,Tagami J, Twetman S, Tsakos G, Ismail A. 2017. Dental caries. Nat Rev DisPrimers 3:17030. https://doi.org/10.1038/nrdp.2017.30.

10. Espinoza JL, Harkins DM, Torralba M, Gomez A, Highlander SK, Jones MB,Leong P, Saffery R, Bockmann M, Kuelbs C, Inman JM, Hughes T, CraigJM, Nelson KE, Dupont CL. 2018. Supragingival plaque microbiomeecology and functional potential in the context of health and disease.mBio 9:e01631-18. https://doi.org/10.1128/mBio.01631-18.

11. Lemos JA, Quivey RG, Koo H, Abranches J. 2013. Streptococcus mutans: anew Gram-positive paradigm? Microbiology 159:436 – 445. https://doi.org/10.1099/mic.0.066134-0.

12. Bowen WH, Burne RA, Wu H, Koo H. 2018. Oral biofilms: pathogens,matrix, and polymicrobial interactions in microenvironments. TrendsMicrobiol 26:229 –242. https://doi.org/10.1016/j.tim.2017.09.008.

13. Vickerman MM, Mansfield JM, Zhu M, Walters KS, Banas JA. 2015. Codon-optimized fluorescent mTFP and mCherry for microscopic visualizationand genetic counterselection of streptococci and enterococci. J Micro-biol Methods 116:15–22. https://doi.org/10.1016/j.mimet.2015.06.010.

14. Shabayek S, Spellerberg B. 2017. Making fluorescent streptococci andenterococci for live imaging, p 141–159. In Nordenfelt P, Collin M (ed),Bacterial pathogenesis: methods and protocols. Springer, New York, NY.

15. Zeng L, Wen ZT, Burne RA. 2006. A novel signal transduction system andfeedback loop regulate fructan hydrolase gene expression in Strepto-coccus mutans. Mol Microbiol 62:187–200. https://doi.org/10.1111/j.1365-2958.2006.05359.x.

16. Liu Y, Zeng L, Burne RA. 2009. AguR is required for induction of theStreptococcus mutans agmatine deiminase system by low pH and ag-matine. Appl Environ Microbiol 75:2629 –2637. https://doi.org/10.1128/AEM.02145-08.

17. Sorg RA, Kuipers OP, Veening J-W. 2015. Gene expression platform forsynthetic biology in the human pathogen Streptococcus pneumoniae.ACS Synth Biol 4:228 –239. https://doi.org/10.1021/sb500229s.

18. van der Vossen JM, van der Lelie D, Venema G. 1987. Isolation andcharacterization of Streptococcus cremoris Wg2-specific promoters.Appl Environ Microbiol 53:2452–2457.

19. Biswas I, Jha JK, Fromm N. 2008. Shuttle expression plasmids for genetic

studies in Streptococcus mutans. Microbiology 154:2275–2282. https://doi.org/10.1099/mic.0.2008/019265-0.

20. Lam KH, Chow KC, Wong WK. 1998. Construction of an efficient Bacillussubtilis system for extracellular production of heterologous proteins. JBiotechnol 63:167–177. https://doi.org/10.1016/S0168-1656(98)00041-8.

21. Palmer SR, Burne RA. 2015. Post-transcriptional regulation by distalShine-Dalgarno sequences in the grpE-dnaK intergenic region of Strep-tococcus mutans. Mol Microbiol 98:302–317. https://doi.org/10.1111/mmi.13122.

22. Guiziou S, Sauveplane V, Chang H-J, Clerté C, Declerck N, Jules M, BonnetJ. 2016. A part toolbox to tune genetic expression in Bacillus subtilis.Nucleic Acids Res 44:7495–7508. https://doi.org/10.1093/nar/gkw624.

23. Pédelacq J-D, Cabantous S, Tran T, Terwilliger TC, Waldo GS. 2006.Engineering and characterization of a superfolder green fluorescentprotein. Nat Biotechnol 24:79 – 88. https://doi.org/10.1038/nbt1172.

24. LeBlanc DJ, Lee LN, Abu-Al-Jaibat A. 1992. Molecular, genetic, andfunctional analysis of the basic replicon of pVA380-1, a plasmid of oralstreptococcal origin. Plasmid 28:130 –145. https://doi.org/10.1016/0147-619X(92)90044-B.

25. Macrina FL, Wood PH, Jones KR. 1980. Genetic transformation of Strep-tococcus sanguis (Challis) with cryptic plasmids from Streptococcus ferus.Infect Immun 28:692– 699.

26. Macrina FL, Tobian JA, Jones KR, Evans RP, Clewell DB. 1982. A cloningvector able to replicate in Escherichia coli and Streptococcus sanguis.Gene 19:345–353. https://doi.org/10.1016/0378-1119(82)90025-7.

27. Kiewiet R, Kok J, Seegers J, Venema G, Bron S. 1993. The mode ofreplication is a major factor in segregational plasmid instability in Lac-tococcus lactis. Appl Environ Microbiol 59:358 –364.

28. Ranieri MR, Whitchurch CB, Burrows LL. 2018. Mechanisms of biofilmstimulation by subinhibitory concentrations of antimicrobials. Curr OpinMicrobiol 45:164 –169. https://doi.org/10.1016/j.mib.2018.07.006.

29. Ozbudak EM, Thattai M, Kurtser I, Grossman AD, van Oudenaarden A.2002. Regulation of noise in the expression of a single gene. Nat Genet31:69 –73. https://doi.org/10.1038/ng869.

30. Strack RL, Strongin DE, Bhattacharyya D, Tao W, Berman A, BroxmeyerHE, Keenan RJ, Glick BS. 2008. A noncytotoxic DsRed variant for whole-cell labeling. Nat Methods 5:955–957. https://doi.org/10.1038/nmeth.1264.

31. Heikal AA, Hess ST, Baird GS, Tsien RY, Webb WW. 2000. Molecularspectroscopy and dynamics of intrinsically fluorescent proteins: coral red(dsRed) and yellow (Citrine). Proc Natl Acad Sci U S A 97:11996 –12001.https://doi.org/10.1073/pnas.97.22.11996.

32. Huang X, Palmer SR, Ahn S-J, Richards VP, Williams ML, Nascimento MM,Burne RA. 2016. A highly arginolytic Streptococcus species that potentlyantagonizes Streptococcus mutans. Appl Environ Microbiol 82:2187–2201. https://doi.org/10.1128/AEM.03887-15.

33. Sauer C, Ver Loren van Themaat E, Boender L, Groothuis D, Cruz R,Hamoen LW, Harwood C, van Rij T. 2018. Exploring the non-conservedsequence space of synthetic expression modules in Bacillus subtilis. ACSSynth Biol 7:1773–1784. https://doi.org/10.1021/acssynbio.8b00110.

34. Macrina FL, Jones KR, Wood PH. 1980. Chimeric streptococcal plasmidsand their use as molecular cloning vehicles in Streptococcus sanguis(Challis). J Bacteriol 143:1425–1435.

35. Gruss A, Ehrlich SD. 1989. The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev 53:231–241.

36. Leer RJ, van Luijk N, Posno M, Pouwels PH. 1992. Structural and func-tional analysis of two cryptic plasmids from Lactobacillus pentosusMD353 and Lactobacillus plantarum ATCC 8014. Mol Gen Genet 234:265–274. https://doi.org/10.1007/BF00283847.

37. Shaner NC, Steinbach PA, Tsien RY. 2005. A guide to choosing fluorescentproteins. Nat Methods 2:905–909. https://doi.org/10.1038/nmeth819.

38. Mark Welch JL, Rossetti BJ, Rieken CW, Dewhirst FE, Borisy GG. 2016.Biogeography of a human oral microbiome at the micron scale. Proc NatlAcad Sci U S A 113:E791–E800. https://doi.org/10.1073/pnas.1522149113.

39. Huang X, Browngardt CM, Jiang M, Ahn S-J, Burne RA, Nascimento MM.2018. Diversity in antagonistic interactions between commensal oral

Shields et al. Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 14

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

streptococci and Streptococcus mutans. Caries Res 52:88 –101. https://doi.org/10.1159/000479091.

40. Terleckyj B, Willett NP, Shockman GD. 1975. Growth of several cariogenicstrains of oral streptococci in a chemically defined medium. InfectImmun 11:649 – 655.

41. Kwak IH, Son M, Hagen SJ. 2012. Analysis of gene expression levels inindividual bacterial cells without image segmentation. Biochem BiophysRes Commun 421:425– 430. https://doi.org/10.1016/j.bbrc.2012.03.117.

42. Chang JC, LaSarre B, Jimenez JC, Aggarwal C, Federle MJ. 2011. Twogroup A streptococcal peptide pheromones act through opposing Rggregulators to control biofilm development. PLoS Pathog 7:e1002190.https://doi.org/10.1371/journal.ppat.1002190.

43. Givskov M, Hentzer M, Ersbøll BK, Heydorn A, Sternberg C, Nielsen AT,Molin S. 2000. Quantification of biofilm structures by the novel com-puter program Comstat. Microbiology 146:2395–2407. https://doi.org/10.1099/00221287-146-10-2395.

Fluorescence Tools for Oral Streptococci Applied and Environmental Microbiology

August 2019 Volume 85 Issue 15 e00620-19 aem.asm.org 15

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from