**2. Fluorescent reporters and their applications for** *in vivo* **imaging in microbiomes**

The symbionts may also be involved in the detoxification of poisonous metabolites in food [1, 2]. The membranes of the intestinum carefully separate the bacterial symbionts from the host organism and prevent infection by invasive and deleterious members of the microbiome. Accordingly, the gut membrane is a complex structure that allows the exchange of nutrients with both high- and low-molecular weight (signaling) compounds and, on the other, blocks the entry of microbes and many of their macromolecular components [3]. The flux of nutrients and even more complex metabolites across the membrane is controlled by transport proteins expressed in the gut membrane [4, 5]. The microbiome also defends against parasites or pathogens [6–8]. The diverse functions provided by the microbial partners are vital for the

Although almost all organisms rely on core microbiomes [9], in many cases the gut community changes according to the insect's developmental stage. In early instars of *Spodoptera littoralis,* several *Enterococcus* spp. dominate, whereas in late instars, Clostridia contribute significantly (ca. 50%) to the microbial population [10]. A core community, consisting of Enterococci, Lactobacilli and Clostridia was revealed in the insect larvae. These bacteria were always present in the digestive tract at a relatively high frequency; although developmental stage and diet have a great impact on shaping bacterial communities, clearly the insect gut selects for particular bacterial phylotypes. Enterococci are also prominent in the gut of insects such as *Drosophila*, ground beetles and desert locusts [11, 12]. The strong dependence of the gut community on the developmental stage of the insect host suggests that unknown low- and high-molecular weight factors control symbiotic interactions among the partners. For example, in *Drosophila melanogaster*, the immune system not only plays a central role in preventing pathogen infection, but also controls the resident bacterial population. The intestinal homeobox gene *Caudal* regulates the resident gut microbial community by repressing the antimicrobial peptide genes that are dependent on the nuclear factor kappa B. Silencing the *Caudal* gene by RNAi resulted in the overexpression of antimicrobial peptides, which in turn

To monitor such developmentally controlled changes in the microbiome of *S. littoralis,* a fluorescent member of the gut symbionts—in particular, the dominant *Enterococcus mundtii* appeared to be an ideal reporting organism. A GFP-labeled *E. mundtii* [14] would easily integrate into the gut community and survive adverse conditions embedded in the community of enterococci, bacteria which are largely resistant to environmental stresses, such as antibiotic exposure, disinfection, desiccation and starvation [15]. As the transgenic *E. mundtii* are fluorescent, their presence can be monitored in all gut areas of the larvae and at developmental stages such as the pupa and the adult. Moreover, the reporter organisms can be easily recovered from the gut and used for transcriptomic analyses. By comparing transcriptomes from adjacent gut tissue and from the microbes, a "dialog" between the insect host and the symbiotic bacteria could be unraveled. This concept is generally applicable and can be used to holistically analyze host microbial interactions. The protocol of the approach based on the use of a fluorescent reporter organism—for example, using GFP-tagged *E. mundtii*—is described

insect's survival, especially in adverse ecological niches.

78 Metagenomics for Gut Microbes

reduced the microbial population in the gut [13].

in this chapter.

The green fluorescent protein (GFP) isolated from the jellyfish, *Aequorea victoria*, is widely used as a reporter for studying gene expression [16], and the localization and structure of living cells [17]. The GFP has a major excitation peak at about 395 nm and an emission peak at about 508 nm. The GFP contains 238 amino acids with a molecular weight of 26.9 kDa. It emits green fluorescence when exposed to light in the blue to ultraviolet range [18]. The GFP requires only oxygen as a cofactor for chromophore formation, which gives it an advantage over other reporter proteins [19]. It is sensitive and non-toxic, and does not affect cell growth [20, 21]. In addition, the GFP is stable at temperatures below 65°C and pH 6–11 [22]. Since the GFP was discovered, many mutants have been developed with modification in spectral and folding properties, or enhanced fluorescence intensity [23–26]. The choice of a GFP variant depends on several factors, such as pH, environmental temperature, toxicity, multimerization and photostability [26]. The first *gfp* gene was cloned in 1992 [27], and 2 years later, the gene was successfully expressed in both eukaryotes and prokaryotes [20]. Apart from GFP, many variants of red fluorescent proteins, such as mCherry and tdTomato have been developed based on DsRed originally isolated from *Discosoma* sp. [28]. Since then, over 40 coral fluorescent proteins with different colors, from cyan to chromo-red, have been described [29].

The reporter proteins provide important tools with which to monitor gene expression from within the cells in real time and in the *in vivo* environment, such as the gastrointestinal tract. For a gene to be selected as a reporter, it must be able to easily detect signals secreted by the expressed reporter gene in the cells [30]. The *lux* gene derived from bacteria, and luciferase from the firefly and click beetle (*luc*), are two other common reporter genes used in bioluminescence imaging. Fluorescence imaging is commonly associated with the use of green and red fluorescence proteins [31, 32]. Rats and mice are popular model organisms which study the proliferation and colonization of lactic acid bacteria (LAB) [33, 34]. LAB has been tagged with green fluorescent protein (GFP) and mCherry to study their colonization of the intestinal tract of chickens, mice and zebrafish [35–39].

#### **2.1. Construction of a GFP fluorescent system for** *E. mundtii*

LAB is widely used as probiotics due to the benefits they bring to human and animal health by balancing the gut microbiome and by eliminating pathogenic microorganisms through the production of antimicrobial peptides [40, 41]. Due to the importance of LAB in many applications, it is essential to study how they survive and colonize by monitoring their metabolic activities *in vivo* through the development of fluorescent reporter microorganisms. It is important that the reporter gene in the fluorescent bacteria is stably expressed [42].

Plasmids are present in most of the members of LAB, including *Enterococcus*, *Lactobacillus*, *Lactococcus*, *Bifidobacterium*, *Streptococcus*, *Pediococcus, Leuconostoc*, etc. Plasmids found in LAB vary in size (0.87 kb to more than 250 kb), copy number (1 or more per cell) and gene content [43–46].

Therefore, the use of constitutive or native-based promoters would be favorable, as these promoters could ensure the constant production of the target protein, especially in the gut environment. Several studies using homologous promoters have been reported to achieve efficient gene expression [71, 72], as the transcriptional signal induced by native promoters is recognized by the host bacteria. Bacteria with the *gfp* gene cloned downstream of a native

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Several methods have been used to introduce exogenous DNA into microbial cells; these include chemical treatment, electroporation, the use of a biolistic gun, ultrasound, polyethylene glycol, microwave and hydrogel [73]. Of all the methods, electroporation most efficiently transforms a broad array of microorganisms [74] by introducing foreign DNA-like plasmid into bacteria. Electroporation is one of the transformation techniques for rapid introduction of foreign DNA-like plasmid into bacteria. The method uses an electric pulse that forms pores on the bacterial cell walls so that DNA can pass into the cell. In recent years, numerous lactic acid bacteria have been transformed using electroporation [75]. The success rate of electrotransformation depends on the cell wall becoming sufficiently permeable to allow DNA to enter. In some cases, to improve a cell's electro-transformation efficiency, the cell wall is pretreated with chemicals such as lysozyme [76, 77], threonine [78, 79], penicillin G [80], ethanol [81] and glycine [82, 83]. These weaken the cell walls only for certain bacteria species. It has been shown that the efficiency of electro-transformation of *Lactococcus lactis* was affected by several parameters, such as the cell's growth phase and density, the medium, the plasmid

The choice of method in the preparation of the competent cells is important for a successful transformation. Although competent *E. coli* cells have reportedly been prepared with ice-cold calcium chloride [85], the transformation achieved with this method is less successful than that achieved using the electroporation method [86]. The electrocompetent cells, the equipment and the washing buffers all have to be prepared at cold temperatures

In this chapter, we report the use of a conventional method to transform *E. mundtii* based on the modified protocol of *Escherichia coli* [89]. The electrocompetent cells and electroporation protocol for *E. mundtii* have been published [14]. Briefly, the bacterial cells were grown to the exponential phase and then washed with ice-cold water for two rounds to remove salts from the growth medium. Glycerol at a final concentration of 10% was added to the bacterial suspension so that the cells could be preserved and stored frozen. A concentration of plasmids between 0.15 and 0.2 μg worked fine for us. The competent cells were mixed with the plasmid DNA and then transferred to a 0.2 cm plastic cuvette for electroporation at a pulse of 1.8 kV, 600 Ω parallel resistance and 10 μF capacitance. The pulsed cells were recovered with fresh broth medium, and the cell suspension was incubated at 37°C for 2 hours before plating on plates containing antibiotic erythromycin. After 2 days, the bacterial transformants were

constitutive promoter express GFP efficiently in broth culture (**Figure 1B**).

**2.2. Transformation of** *E. mundtii* **KD251 using electroporation**

concentration and the strength of the electrical field [84].

screened for the plasmid-containing *gfp* gene.

[87, 88].

Enterococci harbor plasmids that are resistant to a wide range of antibiotics, including erythromycin, tetracycline, gentamicin and vancomycin [47–50]. Some of these plasmids encode bacteriocins [51–53], virulence factors [54, 55], toxins [56] and sex pheromones [57]. Plasmids replicate via rolling circle replication (RCR) and theta replication [58]. Theta-replicating plasmids can carry large DNA fragments and are more stable than RCR plasmids [59]. The enterococci plasmid pAMβ1 replicates via theta mode. In the early 1990s, shuttle vectors in the pTRK family of high and low copy number carrying the origin of replication of pAMβ1 for LAB and p15A for *E. coli* were developed [60]. The plasmids carrying the replicon pAMβ1 isolated from *Enterococcus faecalis* [61] have been reported to replicate in Gram-positive bacteria [62].

The choice of a reliable expression vector depends on several factors, such as the mode of replication, copy number and stability [63]. The expression vector used in this study is derived from pTRKH3 plasmid with a broad host range. pTRKH3 is a shuttle vector for *E. coli*, *Enterococcus*, *Lactococcus*, *Streptococcus* and *Lactobacillus* [60]. The vector has a copy number (30–40) in *E. coli*, and a somewhat higher copy number (45–85) in *Lactococcus* and *Streptococcus* species [64]. It carries a gene for erythromycin resistance, which is expressed in *E. coli* and LAB. In this chapter, we report the expression of mutated *gfp* (mgfp5) on a pTRKH3 plasmid controlled by a strong constitutive promoter, erythromycin ribosomal methylase (*ermB*) [61], in *E. mundtii* (**Figure 1A**). The lactate dehydrogenase (*ldh*) promoter from *Lactobacillus acidophilus* [65] has also been used to control the expression of GFP. In contrast, the use of a surface-layer (*slp*) promoter from *L. acidophilus* [66] was not able to induce the expression of GFP [14]. Accordingly, the selection of an appropriate promoter to achieve a high level of GFP expression is crucial. Nisin-inducible promoters have been used for heterologous gene expression in lactobacilli [67, 68]. Nisin that can be degraded within the intestinal environment is a drawback of this inducible expression system [69, 70].

**Figure 1.** Construction of GFP-tagged *E. mundtii* by electroporation. (A) Plasmid map of pTRKH3 harboring the *mgfp5* gene regulated by an erythromycin ribosomal methylase (*ermB*) promoter. The plasmid is an *E. coli*-LAB shuttle vector with p15A and pAMβ1 as the origins of replication. (B) The *ermB* promoter was used to increase the expression of GFP from *E. mundtii* grown in Todd-Hewitt Bouillon (THB) broth culture. Scale bar: 10 μm [14].

Therefore, the use of constitutive or native-based promoters would be favorable, as these promoters could ensure the constant production of the target protein, especially in the gut environment. Several studies using homologous promoters have been reported to achieve efficient gene expression [71, 72], as the transcriptional signal induced by native promoters is recognized by the host bacteria. Bacteria with the *gfp* gene cloned downstream of a native constitutive promoter express GFP efficiently in broth culture (**Figure 1B**).

#### **2.2. Transformation of** *E. mundtii* **KD251 using electroporation**

vary in size (0.87 kb to more than 250 kb), copy number (1 or more per cell) and gene content

Enterococci harbor plasmids that are resistant to a wide range of antibiotics, including erythromycin, tetracycline, gentamicin and vancomycin [47–50]. Some of these plasmids encode bacteriocins [51–53], virulence factors [54, 55], toxins [56] and sex pheromones [57]. Plasmids replicate via rolling circle replication (RCR) and theta replication [58]. Theta-replicating plasmids can carry large DNA fragments and are more stable than RCR plasmids [59]. The enterococci plasmid pAMβ1 replicates via theta mode. In the early 1990s, shuttle vectors in the pTRK family of high and low copy number carrying the origin of replication of pAMβ1 for LAB and p15A for *E. coli* were developed [60]. The plasmids carrying the replicon pAMβ1 isolated from *Enterococcus faecalis* [61] have been reported to replicate in Gram-positive

The choice of a reliable expression vector depends on several factors, such as the mode of replication, copy number and stability [63]. The expression vector used in this study is derived from pTRKH3 plasmid with a broad host range. pTRKH3 is a shuttle vector for *E. coli*, *Enterococcus*, *Lactococcus*, *Streptococcus* and *Lactobacillus* [60]. The vector has a copy number (30–40) in *E. coli*, and a somewhat higher copy number (45–85) in *Lactococcus* and *Streptococcus* species [64]. It carries a gene for erythromycin resistance, which is expressed in *E. coli* and LAB. In this chapter, we report the expression of mutated *gfp* (mgfp5) on a pTRKH3 plasmid controlled by a strong constitutive promoter, erythromycin ribosomal methylase (*ermB*) [61], in *E. mundtii* (**Figure 1A**). The lactate dehydrogenase (*ldh*) promoter from *Lactobacillus acidophilus* [65] has also been used to control the expression of GFP. In contrast, the use of a surface-layer (*slp*) promoter from *L. acidophilus* [66] was not able to induce the expression of GFP [14]. Accordingly, the selection of an appropriate promoter to achieve a high level of GFP expression is crucial. Nisin-inducible promoters have been used for heterologous gene expression in lactobacilli [67, 68]. Nisin that can be degraded within the intestinal environment is a drawback of this inducible expression system [69, 70].

**Figure 1.** Construction of GFP-tagged *E. mundtii* by electroporation. (A) Plasmid map of pTRKH3 harboring the *mgfp5* gene regulated by an erythromycin ribosomal methylase (*ermB*) promoter. The plasmid is an *E. coli*-LAB shuttle vector with p15A and pAMβ1 as the origins of replication. (B) The *ermB* promoter was used to increase the expression of GFP

from *E. mundtii* grown in Todd-Hewitt Bouillon (THB) broth culture. Scale bar: 10 μm [14].

[43–46].

80 Metagenomics for Gut Microbes

bacteria [62].

Several methods have been used to introduce exogenous DNA into microbial cells; these include chemical treatment, electroporation, the use of a biolistic gun, ultrasound, polyethylene glycol, microwave and hydrogel [73]. Of all the methods, electroporation most efficiently transforms a broad array of microorganisms [74] by introducing foreign DNA-like plasmid into bacteria. Electroporation is one of the transformation techniques for rapid introduction of foreign DNA-like plasmid into bacteria. The method uses an electric pulse that forms pores on the bacterial cell walls so that DNA can pass into the cell. In recent years, numerous lactic acid bacteria have been transformed using electroporation [75]. The success rate of electrotransformation depends on the cell wall becoming sufficiently permeable to allow DNA to enter. In some cases, to improve a cell's electro-transformation efficiency, the cell wall is pretreated with chemicals such as lysozyme [76, 77], threonine [78, 79], penicillin G [80], ethanol [81] and glycine [82, 83]. These weaken the cell walls only for certain bacteria species. It has been shown that the efficiency of electro-transformation of *Lactococcus lactis* was affected by several parameters, such as the cell's growth phase and density, the medium, the plasmid concentration and the strength of the electrical field [84].

The choice of method in the preparation of the competent cells is important for a successful transformation. Although competent *E. coli* cells have reportedly been prepared with ice-cold calcium chloride [85], the transformation achieved with this method is less successful than that achieved using the electroporation method [86]. The electrocompetent cells, the equipment and the washing buffers all have to be prepared at cold temperatures [87, 88].

In this chapter, we report the use of a conventional method to transform *E. mundtii* based on the modified protocol of *Escherichia coli* [89]. The electrocompetent cells and electroporation protocol for *E. mundtii* have been published [14]. Briefly, the bacterial cells were grown to the exponential phase and then washed with ice-cold water for two rounds to remove salts from the growth medium. Glycerol at a final concentration of 10% was added to the bacterial suspension so that the cells could be preserved and stored frozen. A concentration of plasmids between 0.15 and 0.2 μg worked fine for us. The competent cells were mixed with the plasmid DNA and then transferred to a 0.2 cm plastic cuvette for electroporation at a pulse of 1.8 kV, 600 Ω parallel resistance and 10 μF capacitance. The pulsed cells were recovered with fresh broth medium, and the cell suspension was incubated at 37°C for 2 hours before plating on plates containing antibiotic erythromycin. After 2 days, the bacterial transformants were screened for the plasmid-containing *gfp* gene.

#### **2.3. Colonization of GFP-tagged bacterium in the gut of** *S. littoralis*

The fluorescent reporter *E. mundtii* has been integrated into the gut microbiome across all developmental stages of *S. littoralis* [14], indicating its symbiotic relationship with the insect host. Microorganisms have the ability to face environmental stresses, particularly those within the gastrointestinal environment. Constructing the fluorescent reporter *E. mundtii*, we explored the mechanisms these bacteria use to adapt to stress; we recovered the reporter bacteria from the gut of *S. littoralis* using the state-of-the-art technology of flow cytometry. The dominance and persistence of *E. mundtii* in the gut motivates us to look deeper into their gene expression system. Therefore, it is important to unravel the mechanisms used by microorganisms living within the gastrointestinal environment. Construction of the fluorescent reporter *E. mundtii* is one of the strategies to find out those mechanisms, since it has been possible to recover the reporter bacteria from the gut of the very same insects using the *state-of-the-art* technology of flow cytometry. Fluorescence-activated cell sorting (FACS) enabled us to pick out the GFP-tagged reporter *E. mundtii* from a mixture of insect and other bacterial cells.

a set of photo multiplier tubes that have specific filters to select for certain wavelengths of

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Once the larvae are fed with the fluorescent *E. mundtii*, the number of larvae that survive can be determined and eventually recovered for further studies. The *E. mundtii* cells are sorted and their transcriptomes can be studied. This technology has made it possible to focus on a

The GFP-tagged *E. mundtii* are sorted by the flow cytometer and collected in a RNA-protective reagent (RNAlater®). The role of such reagents is twofold: first, they preserve the integrity of RNA, which has a very short half-life, for a few minutes. We need the RNA to be intact and of good quality in order to process it for sequencing. Second, addition of protective reagents minimizes subsequent changes from being introduced when the cells are handled. As soon as the cells are collected in a Falcon tube filled with the protective reagent (RNA Protect or RNA Later), the reagent percolates into the cells and prevents an alteration in the gene expression [91]. Additionally, the entire process is maintained at 4°C, as all metabolic activities slow down at low temperatures. The Falcon tube is centrifuged at a high speed to pellet down the

RNA is very sensitive to exogenous and endogenous RNases. The entire extraction procedure is done in an area free of RNase. Moreover, RNase inhibitors are used to clean all equipment, ranging from gloves to microcentrifuge tubes to get rid of RNase. *E. mundtii* is a Gram-positive bacterium with a cell wall containing a thick layer of peptidoglycan and lipoteichoic acid, followed by a single lipid membrane. The cell wall is anchored to the membrane by diacylglycerols. To release the nucleic acid from the cell, it has to be detached from its peptidoglycancontaining cell wall and membrane. Lysozyme is a glycoside hydrolase that hydrolyzes the 1,4-beta linkages between *N*-acetylmuramic acid and *N*-acetyl-D-glucosamine residues of the peptidoglycan. Additionally, guanidium thiocyanate, beta-mercaptoethanol and a detergent called dithiothreitol help in cell lysis and deproteinization. Proteinase K frees the RNA from

Following lysis, the RNA is separated by density gradient centrifugation using phenol, chloroform and isoamyl alcohol, and further precipitation with ethanol. The RNeasy® Mini Kit (Qiagen) based on silica-matrix RNA extraction was used in our work. Several studies have reported on extraction of high quality bacterial RNA using this kit [92–94]. Thus, RNA is

The extraction of total RNA from the low number of bacterial cells that remain after sorting by the flow cytometer is challenging. The concentration of RNA was as low as a few picograms to 50 ng. The minimum threshold quantity for a successful RNA library preparation is 100 ng. This is too low an amount to proceed with RNA sequencing. Hence, the total RNA must be

single cell or cells of interest, to study their function or their physiological state.

cells, and care is taken not to disturb it while draining the supernatant.

the bound proteins and endogenous RNase.

amplified before we can use it further.

obtained from the cells of the sorted *E. mundtii* [95].

the beam and are set at the excitation range to view GFP.

**4. RNA extraction**
