**5. Transcriptomics**

**4.1. Amplification of RNA**

84 Metagenomics for Gut Microbes

*4.1.1. Polyadenylation of RNA*

*4.1.2. Synthesis of first-strand cDNA*

*4.1.3. Synthesis of second-strand cDNA*

with a T7 promoter sequence.

*4.1.4. Purification of cDNA*

hinder *in vitro* transcription.

which one wants to amplify the RNA.

At this point, the RNA has been amplified several fold: 1–2 ug.

*4.1.6. Purification of amplified RNA*

uct [100–104].

ably introduced.

*4.1.5. In Vitro transcription*

are as follows:

Amplification of RNA is required if the aim is to create an effective transcriptomic profile from a very low starting quantity of RNA. MessageAmp II aRNA amplification kit (Ambion) was used for amplification [96–99]. The principle is based on *in vitro* transcription. The steps

Bacterial RNA is devoid of a poly (A) tail. The *E. coli* poly (A) polymerase enables a poly (A)

Primers against the poly (A) stretch are used to synthesize the first strand of cDNA by reverse transcription. The primers are anchored with a bacteriophage promoter sequence: T7

RNaseH is used to degrade the RNA from the RNA-cDNA pair; DNA polymerase is required to synthesize the second strand of cDNA. The result is a double-stranded cDNA fragment

cDNA is cleaned by removing the fragmented RNA, enzymes and salts, all of which could

Multiple copies of antisense RNA are generated using DNA-dependent RNA polymerase. Linear amplification is employed for this. Depending on the bacteriophage promoter sequence attached to the cDNA, a polymerase is selected. Promoter-specific dNTPs are added to the reaction mix. 37°C is optimum for this reaction. The reaction time depends on the extent to

The residual enzymes, salts and unincorporated dNTPs must be removed from the final prod-

RNA amplification procedures have a drawback. When the concentration of RNA is brought to a point at which successful transcriptomic profiling is possible, certain biases are unavoid-

tail to be added at the ends of RNA. This stretch is required for cDNA synthesis.

oligo(dT) sequence, T3 or SP6. dNTPs are added to the reaction mix.

At this point, we have enough RNA to get a transcriptomic profiling of the bacterial cells done. The transcriptome is the entire set of genes expressed in a type of cell at a particular time point and/or condition. This is in contrast to a genome, which refers to the full complement of genes in a cell-type. Not all genes are constitutively induced. Information about transcripts, or genes expressed, may shed light on the developmental or physiological state of the cell. It also talks about other species of RNA, small RNAs and non-coding RNAs, novel transcripts, the transcriptional start sites, splicing regions, post-transcriptional modifications, and 3′ and 5′ ends. Another purpose of transcriptomic profiling is to quantify the expressed genes. One can judge the extent of regulation of a particular gene in the given conditions. As compared to one situation, when cells behave differently in another, one can now say which genes are differentially regulated to bring about the same.

In this chapter, our aim has been to investigate the survival and adaptation strategies of *E. mundtii* living inside the gut of *S. littoralis* as compared to in the laboratory. This unraveling has been done by cataloging the genes of *E. mundtii* which are differently regulated and which make it as one of the dominant bacterial species in the gut.

#### **5.1. RNASeq**

Transcriptome sequencing has improved dramatically over the past few years, starting with EST-based Sanger sequencing. The early method was mainly useful with the most abundant transcripts, whereas subsequent next-generation sequencing has been successfully carried out on all transcripts with sensitivity and accuracy even allowing the identification of low expressed genes. The situation has ameliorated with the advent of deep sequencing, which can increase the average number of times a nucleotide is sequenced. The deeper the sequencing is, the better the probability of detecting the less abundant transcripts. Next-generation sequencing has several hierarchies of its own. These days, RNA-seq is more widely used than the microarrays. The former gives us a base-pair level of resolution. Whereas microarrays can be used only when the reference genome sequence is available, RNA-seq can build the transcriptome *de novo.* Also, background noise is taken better care of in the case of RNA-seq. These days, sequencing is not confined to a larger number of cells. It is possible to obtain resolution up to a single cell. Naturally, the amount of RNA obtained from one single cell is in picograms and must be processed as discussed above. Among all the increased sensitivity of nextgeneration technologies, so far, Illumina allows us to start with the smallest amount of RNA.

**5.2. Adaptation and survival strategies of** *E. mundtii* **in the gut of the insect**

bial activities against them have been shown in the presence of *E. mundtii* [108].

analysis of differential gene expression were performed later.

**Gene/protein Pathway Function**

management

management

General stress protein Adaptation Various stress management

**Table 1.** Upregulation of genes and pathways in *E. mundtii* living in the gut of *S. littoralis*.

Universal stress protein Adaptation Adaptation to diverse stress sources

Phosphotransferase systems Sugar transport Regulates carbohydrate metabolism in diverse

Accessory gene regulator (*Agr*) Two-component system Virulence factor

fluorescent microscopic images (**Figure 4**).

as compared to the broth culture.

LPxTG-motif cell wall anchor domain

WxL domain surface cell wall-binding

Ferric (Fe+3) ABC superfamily ATP binding cassette transporter (*fetC*)

protein

protein

Superoxide dismutase (*SOD*) Oxidative stress

Catalase Oxidative stress

The GFP-tagged *E. mundtii* was fed to the *S. littoralis* larvae at early instars. The bacterial reporter was able to colonize the gut at various stages of the insect's life cycle, as seen in the

The Microbiome of *Spodoptera littoralis*: Development, Control and Adaptation to the Insect Host

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87

The production of antimicrobial substances from insects or their resident symbionts is a survival strategy to keep pathogens at bay. The dominant gut bacterium *E. mundtii* has been shown to produce an antimicrobial peptide called mundticin KS, which is a stable class IIa bacteriocin. It establishes a chemical barrier, which prevents colonization by competitors [108]. If allowed to persist, the early colonizers of the *S. littoralis* gut, *Enterococcus faecalis* and *Enterococcus casseliflavus*, could be potential pathogens for the insects. Successful antimicro-

The larvae were allowed to grow until the fifth instar, at which stag the guts were homogenized to retrieve the fluorescent *E. mundtii* by flow cytometry. The RNA of these sorted bacteria was used to probe their differential behavior inside the gut. RNA sequencing and

Numerous genes are differentially regulated in the *E. mundtii* obtained from the gut, when compared to the *E. mundtii* grown in bacterial culture under lab conditions (**Table 1**, **Figure 3**). Reactive oxygen species, such as superoxide radicals, hydrogen peroxide or hydroxyl radicals, from metabolic activities may cause oxidative stress and damage macromolecules. To survive the stress, resident bacteria have to come up with means to fight it. Superoxide dismutase and catalase are effective enzymes, over-produced by *E. mundtii* when inside the gut,

Quenching reactive oxidation species by partial

reduction of O2

Cell surface adhesion Signal peptide cleaved by sortase for cell surface

sources and adaptation

oxygen

adhesion

Cell surface adhesion Cell surface adhesion and adaptation

Iron transport Iron transporter permease

−

Quenching reactive oxidation species, converting hydrogen peroxide to water and

The fragmented and adapter-ligated cDNA is allowed to flow through a flow cell of the sequencer, which has oligonucleotides that complement the adapter sequences embedded in them. After hybridization, the oligonucleotides prime the polymerization process with the provided dNTPs and DNA polymerase. Each of the dNTPs is tagged with a fluorophore. As the nucleotide is incorporated, the resulting fluorescence is detected. With the addition of each nucleotide, the fluorophore is released, regenerating the 3′ hydroxyl group for the next nucleotide to join. This way, the fluorescent intensity is recorded and converted into nucleotide identity using an algorithm.

The amplified RNA from the fluorescent *E. mundtii* cells sorted by flow cytometry went through deep sequencing (Hiseq) to detect as many genes as possible to tell us the story of their adaptation to the gut environment of *S. littoralis* (**Figure 2**).

The complications arising from several different forms of RNA, alternate splicing, removal of introns, that is, the ones that are profound in eukaryotes are not required to be considered in the case bacteria. Although, there are several regulatory and non-coding RNAs in bacteria, but this particular case dictates one to follow a rather straightforward approach of unraveling the upregulated and downregulated transcripts only.

**Figure 2.** Overview of the workflow for bacterial RNA-seq. (A) Flow cytometry to sort fluorescent bacteria from gut homogenates. (B) Extraction of total bacterial RNA. (C) Amplification of the total RNA by *in vitro* amplification (unpublished).

#### **5.2. Adaptation and survival strategies of** *E. mundtii* **in the gut of the insect**

days, sequencing is not confined to a larger number of cells. It is possible to obtain resolution up to a single cell. Naturally, the amount of RNA obtained from one single cell is in picograms and must be processed as discussed above. Among all the increased sensitivity of nextgeneration technologies, so far, Illumina allows us to start with the smallest amount of RNA. The fragmented and adapter-ligated cDNA is allowed to flow through a flow cell of the sequencer, which has oligonucleotides that complement the adapter sequences embedded in them. After hybridization, the oligonucleotides prime the polymerization process with the provided dNTPs and DNA polymerase. Each of the dNTPs is tagged with a fluorophore. As the nucleotide is incorporated, the resulting fluorescence is detected. With the addition of each nucleotide, the fluorophore is released, regenerating the 3′ hydroxyl group for the next nucleotide to join. This way, the fluorescent intensity is recorded and converted into nucleotide identity using an algorithm. The amplified RNA from the fluorescent *E. mundtii* cells sorted by flow cytometry went through deep sequencing (Hiseq) to detect as many genes as possible to tell us the story of

The complications arising from several different forms of RNA, alternate splicing, removal of introns, that is, the ones that are profound in eukaryotes are not required to be considered in the case bacteria. Although, there are several regulatory and non-coding RNAs in bacteria, but this particular case dictates one to follow a rather straightforward approach of unraveling

**Figure 2.** Overview of the workflow for bacterial RNA-seq. (A) Flow cytometry to sort fluorescent bacteria from gut homogenates. (B) Extraction of total bacterial RNA. (C) Amplification of the total RNA by *in vitro* amplification

their adaptation to the gut environment of *S. littoralis* (**Figure 2**).

the upregulated and downregulated transcripts only.

86 Metagenomics for Gut Microbes

(unpublished).

The GFP-tagged *E. mundtii* was fed to the *S. littoralis* larvae at early instars. The bacterial reporter was able to colonize the gut at various stages of the insect's life cycle, as seen in the fluorescent microscopic images (**Figure 4**).

The production of antimicrobial substances from insects or their resident symbionts is a survival strategy to keep pathogens at bay. The dominant gut bacterium *E. mundtii* has been shown to produce an antimicrobial peptide called mundticin KS, which is a stable class IIa bacteriocin. It establishes a chemical barrier, which prevents colonization by competitors [108]. If allowed to persist, the early colonizers of the *S. littoralis* gut, *Enterococcus faecalis* and *Enterococcus casseliflavus*, could be potential pathogens for the insects. Successful antimicrobial activities against them have been shown in the presence of *E. mundtii* [108].

The larvae were allowed to grow until the fifth instar, at which stag the guts were homogenized to retrieve the fluorescent *E. mundtii* by flow cytometry. The RNA of these sorted bacteria was used to probe their differential behavior inside the gut. RNA sequencing and analysis of differential gene expression were performed later.

Numerous genes are differentially regulated in the *E. mundtii* obtained from the gut, when compared to the *E. mundtii* grown in bacterial culture under lab conditions (**Table 1**, **Figure 3**). Reactive oxygen species, such as superoxide radicals, hydrogen peroxide or hydroxyl radicals, from metabolic activities may cause oxidative stress and damage macromolecules. To survive the stress, resident bacteria have to come up with means to fight it. Superoxide dismutase and catalase are effective enzymes, over-produced by *E. mundtii* when inside the gut, as compared to the broth culture.


**Table 1.** Upregulation of genes and pathways in *E. mundtii* living in the gut of *S. littoralis*.

The ability to adapt to variable living conditions is very much attributed to "two-component systems." These systems form a class of signal-transduction mechanisms that are induced when the insect senses stress in the environment. The main players in the system are auto-inducing proteins (AIPs), histidine protein kinases (HPKs) and response regulators. AIPs, which interact with the HPKs, are produced in response to stress. The signal is relayed to the response regulators. This cascade ultimately produces certain factors or proteins that aid *E. mundtii* to survive in the stressful environment [111]. Accordingly, the agr family of genes was found upregulated

The Microbiome of *Spodoptera littoralis*: Development, Control and Adaptation to the Insect Host

http://dx.doi.org/10.5772/intechopen.72180

89

Quorum sensing is a phenomenon where the bacterial cells interact and communicate with one another for survival. AIPs are also key players for quorum sensing. In addition, also several quorum-sensing strategies are two-component systems. AIPs accumulate in response to increases in bacterial cell density; these increases are followed by a signaling cascade and lead

Stress proteins are adaptive factors that are induced when living conditions become stressful. There exist general and universal stress proteins. General stress proteins help bacteria deal with oxidative stress, heat stress, salt stress or oxygen limitation [113]. Universal stress proteins are induced in response to temperature fluctuations, heat or oxidative stress and hypoxia. Both of these protein classes were upregulated in *E. mundtii* in response to the insect

The type of sugar transport system expressed by bacteria depends on the types of carbon sources available. Phosphotransferase systems form a class of sugar transporters that sense the sugar source available in the environment and allow the respective transporters for fructose, glucose, mannose or cellobiose to act on it. Using energy from phosphoenolpyruvate, the transport system utilizes a cascade of cytoplasmic protein components with an accompanying phosphorylation of each component [115]. These transporters are generally sugar specific and because they help bacteria to survive in presence of complex carbohydrate conditions, they are said to help in their adaptation. Several of these PTS systems are upregulated by *E. mundtii*

Lactic acid bacteria are important in the production of fermented foods, such as dairy products. LAB is potential probiotics that provide benefits to human health [116]. Modified LAB could also be used as live vaccines or vaccine delivery systems [117]. It has been shown that the genetically modified *L. lactis* can survive and colonize the digestive tract of humans [118] and gnotobiotic mice [119]. In this chapter, we report the use of GFP to tag *E. mundtii* to monitor the bacteria's survival and activities in the intestinal tract of cotton leafworm, *S. littoralis*. It has been shown that spatial and temporal distribution of fluorescent *E. mundtii* was observed across all developmental stages (**Figure 4**), as well as in the foregut, midgut and hindgut of *S. littoralis*. Data from the colony forming units (CFUs) show that the midgut houses the most

in *E. mundtii* living in the insect gut.

gut's living conditions [114].

living in the gut of *S. littoralis*.

**6. Discussion**

to cooperative gene expression by the bacteria [112].

**Figure 3.** The gut microbiome of *S. littoralis* was dominated by *E. mundtii* and *Clostridia* sp. (A) Overview of the gut structure of fifth-instar larva of *S. littoralis*. (B) Illustration from within the gut space, which harbors major symbionts *E. mundtii*, *Clostridia* sp. and other bacteria. Bacteria adhere to the mucus layer of insect gut epithelium. Unknown interactions occur between microbe-microbe and host-microbe. (C) Illustration of some major expressed pathways *E. mundtii* used for survival in the gut. (i) Secretion of mundticin, an antimicrobial peptide, keeps pathogens at bay and helps the *E. mundtii* dominate the colonization process. (ii) A two-component system involving the accessory gene regulator (*agr*) system, which directs a histidine kinase to phosphorylate the response regulator, leads to the activation of transcription factors required for adaption. (iii) The induction of superoxide dismutase and catalase to manage oxidative stress leads to the conversion of superoxide radicals to water and oxygen. (iv) General or universal stress proteins help to overcome different kinds of stresses, such as oxygen starvation, heat or oxidative stress (unpublished).

Adhesion to the host gut epithelial surface is another key to successful colonization. Endosymbionts employ certain proteins (motifs and domains) for this purpose. These are mostly surface proteins associated with the cell wall and employing certain motifs, which act as the signal peptide for attaching to the cell wall. For example, the motif called LPXTG is a sorting peptide. The endopeptidase sortase cleaves it at the site between threonine and glycine residues, and links the peptide covalently to the peptidoglycan of the cell wall [109]. There is up-regulation in the genes encoding this motif and also in the sortase enzymes, indicating attachment of *E. mundtii* to the insect gut wall and biofilm formation. The up-regulation of the WxL domain hints at the increased colonization of the bacteria by their adherence to the gut epithelium. The WxL domain proteins are also crucial for adapting to varying environmental conditions [110].

The ability to adapt to variable living conditions is very much attributed to "two-component systems." These systems form a class of signal-transduction mechanisms that are induced when the insect senses stress in the environment. The main players in the system are auto-inducing proteins (AIPs), histidine protein kinases (HPKs) and response regulators. AIPs, which interact with the HPKs, are produced in response to stress. The signal is relayed to the response regulators. This cascade ultimately produces certain factors or proteins that aid *E. mundtii* to survive in the stressful environment [111]. Accordingly, the agr family of genes was found upregulated in *E. mundtii* living in the insect gut.

Quorum sensing is a phenomenon where the bacterial cells interact and communicate with one another for survival. AIPs are also key players for quorum sensing. In addition, also several quorum-sensing strategies are two-component systems. AIPs accumulate in response to increases in bacterial cell density; these increases are followed by a signaling cascade and lead to cooperative gene expression by the bacteria [112].

Stress proteins are adaptive factors that are induced when living conditions become stressful. There exist general and universal stress proteins. General stress proteins help bacteria deal with oxidative stress, heat stress, salt stress or oxygen limitation [113]. Universal stress proteins are induced in response to temperature fluctuations, heat or oxidative stress and hypoxia. Both of these protein classes were upregulated in *E. mundtii* in response to the insect gut's living conditions [114].

The type of sugar transport system expressed by bacteria depends on the types of carbon sources available. Phosphotransferase systems form a class of sugar transporters that sense the sugar source available in the environment and allow the respective transporters for fructose, glucose, mannose or cellobiose to act on it. Using energy from phosphoenolpyruvate, the transport system utilizes a cascade of cytoplasmic protein components with an accompanying phosphorylation of each component [115]. These transporters are generally sugar specific and because they help bacteria to survive in presence of complex carbohydrate conditions, they are said to help in their adaptation. Several of these PTS systems are upregulated by *E. mundtii* living in the gut of *S. littoralis*.
