Difference between revisions of "CH391L/S14/SmallRNAs"

From SynBioCyc
Jump to: navigation, search
Line 12: Line 12:
 
sRNAs can be classified in cis-encoded and trans-encoded. The former refers to those that are transcribed from the complementary strand of the genes that they target. This class represents the minority of the sRNAs that have been identified up to now. Additionally, cis-encoded sRNAs usually exert a tight control over single target messenger RNA (mRNA).  In contrast, trans-encoded sRNAs are transcribed from loci in the genome that are distant from where their mRNA targets are encoded. This class accounts for the great majority of sRNAs discovered to date. An astonishing feature is that these molecules can bind their mRNA partners by a minimal base-pairing requirement (8-9 nucleotides)<cite>Gottesman2004</cite>. Lastly but more importantly, this class of sRNAs can interact with multiple mRNAs<cite>DeLay2013</cite>. This property in turn enables the potential application of combinatorial gene knockdown in metabolic engineering.  
 
sRNAs can be classified in cis-encoded and trans-encoded. The former refers to those that are transcribed from the complementary strand of the genes that they target. This class represents the minority of the sRNAs that have been identified up to now. Additionally, cis-encoded sRNAs usually exert a tight control over single target messenger RNA (mRNA).  In contrast, trans-encoded sRNAs are transcribed from loci in the genome that are distant from where their mRNA targets are encoded. This class accounts for the great majority of sRNAs discovered to date. An astonishing feature is that these molecules can bind their mRNA partners by a minimal base-pairing requirement (8-9 nucleotides)<cite>Gottesman2004</cite>. Lastly but more importantly, this class of sRNAs can interact with multiple mRNAs<cite>DeLay2013</cite>. This property in turn enables the potential application of combinatorial gene knockdown in metabolic engineering.  
  
More specifically, these five chemoreceptors in charge of sensing attractants and repellants are sensory histidine kinases in turn coupled to phosphorylable response regulators (CheA, CheB, CheR, CheW, CheY, and CheZ) in charge of controlling the directional rotation of the flagellar motor (Figure 2). In the absence of the chemical gradient the flagellar motor rotates clockwise allowing individual cells to execute random walk. On the other hand, when rotating counterclockwise, the cell performs long runs up a chemoattractand or down a chemorepellent. Once the concentration of the chemical is constant, the cell resumes its random walk movement. An important feature of chemotaxis in E. coli is that individual cell do not only respond to absolute concentrations of a stimulus, they integrate such concentrations over time and respond accordingly based on the concentration differences between two time points.
+
Trans-encoded sRNAs can target proteins in addition to mRNAs, an example of that are sRNAs such as CsrB/C and 6S RNA. When controlling mRNA expression this class of sRNAs uses a diversity of mechanisms. They can (1) base-pair to their target mRNAs to enhance or attenuate transcription (Figure 1A), (2) directly block (Figure 1B i), or indirectly enhance or inhibit translation (Figure 1B ii), (3) sequester proteins (not shown), or (4) directly lead to mRNA and protein degradation (Figure 1B iii). In this article I will exclusively focus on those sRNAs that are trans-encoded and only target mRNAs. Hereafter, I will refer to them simply as sRNAs. This class of sRNAs, as aforementioned, accounts for the majority of discovered sRNAs and can target multiple genes. Consequently, these sRNAs have attracted much interest among the Synthetic Biology community as I will show in the remainder of this article.  
 +
 
 +
A particular feature that this class of sRNAs exhibit is the interaction with a major chaperone protein called Hfq. These interactions have been mainly observed in gram-negative bacteria. Hfq action leads to the stability sRNAs, assists their binding to target mRNAs and stabilizes interactions sRNA-mRNA (ref 43). Recent reports propose that Hfq can also exert negative regulation by delivering the sRNA-mRNA complex to the degradosome <cite>Storz2011</cite>. By engineering Hfq interaction gene expression control could potentially be greatly improved by enhancing its dynamic range. In addition, the introduction of Hfq domains into an already constructed sRNA-like device could bring about a very valuable multiple-target capability.  
  
 
== Engineering the chemotactic sensitivity in E. coli ==
 
== Engineering the chemotactic sensitivity in E. coli ==

Revision as of 17:31, 7 April 2014

Contents

Bacterial small RNAs: a powerful tool for metabolic engineering

Introduction

Bacterial small RNAs (sRNAs) are gene regulatory entities that range from 21 to 400 nucleotides in size. These RNAs are in charge of controlling expression of stress-response genes thus are essential for organism survival under different extreme environmental conditions (e.g. nutrient availability, osmolarity, pH and temperature)[1]. The presence of these regulatory molecules appears to be ubiquitous as they have been discovered in a wide range of bacterial species [2][3]. Their high modularity and orthogonally have risen interest among synthetic biologists for the construction of sRNA-like devices. In addition, sRNA capacity to simultaneously multiple genes has enabled the vision of sRNAs as a powerful tool for metabolic engineering applications. Hereby I will focus on a specific type of sRNA and its presence in synthetic biology.

Bacterial small RNAs

Figure 1: Gene Expression control mechanisms by bacterial sRNAs. (A) Transcription attenuation/enhancement. (A) sRNA binds to its target mRNA and causes a structural reconfiguration upon base-pairing, ultimately enhancing or attenuating transcription by the polymerase. (B) Translational control. Translational control is imparted by sRNAs in various ways: (1) A sRNA base-pairs to its target mRNA sequestering the Ribosome-Binding Site (RBS) and directly prevents translation initiation by the ribosomes. (2) A sRNA binds to the target mRNA at a distance from the RBS and the target mRNA suffers a structural change that indirectly affects ribosome binding. sRNA binding to its target can also enhance or inhibit mRNA decay by changing interactions with exonucleases and/or endonucleases.[4]

sRNAs can be classified in cis-encoded and trans-encoded. The former refers to those that are transcribed from the complementary strand of the genes that they target. This class represents the minority of the sRNAs that have been identified up to now. Additionally, cis-encoded sRNAs usually exert a tight control over single target messenger RNA (mRNA). In contrast, trans-encoded sRNAs are transcribed from loci in the genome that are distant from where their mRNA targets are encoded. This class accounts for the great majority of sRNAs discovered to date. An astonishing feature is that these molecules can bind their mRNA partners by a minimal base-pairing requirement (8-9 nucleotides)[1]. Lastly but more importantly, this class of sRNAs can interact with multiple mRNAs[5]. This property in turn enables the potential application of combinatorial gene knockdown in metabolic engineering.

Trans-encoded sRNAs can target proteins in addition to mRNAs, an example of that are sRNAs such as CsrB/C and 6S RNA. When controlling mRNA expression this class of sRNAs uses a diversity of mechanisms. They can (1) base-pair to their target mRNAs to enhance or attenuate transcription (Figure 1A), (2) directly block (Figure 1B i), or indirectly enhance or inhibit translation (Figure 1B ii), (3) sequester proteins (not shown), or (4) directly lead to mRNA and protein degradation (Figure 1B iii). In this article I will exclusively focus on those sRNAs that are trans-encoded and only target mRNAs. Hereafter, I will refer to them simply as sRNAs. This class of sRNAs, as aforementioned, accounts for the majority of discovered sRNAs and can target multiple genes. Consequently, these sRNAs have attracted much interest among the Synthetic Biology community as I will show in the remainder of this article.

A particular feature that this class of sRNAs exhibit is the interaction with a major chaperone protein called Hfq. These interactions have been mainly observed in gram-negative bacteria. Hfq action leads to the stability sRNAs, assists their binding to target mRNAs and stabilizes interactions sRNA-mRNA (ref 43). Recent reports propose that Hfq can also exert negative regulation by delivering the sRNA-mRNA complex to the degradosome [3]. By engineering Hfq interaction gene expression control could potentially be greatly improved by enhancing its dynamic range. In addition, the introduction of Hfq domains into an already constructed sRNA-like device could bring about a very valuable multiple-target capability.

Engineering the chemotactic sensitivity in E. coli

Figure 2: Engineered chemotaxis pathway. (A) The pathway incorporates an enzyme that converts the target molecule (gold spheres) into a product (gold triangles) that is a ligand for the chemotaxis receptor. (B) Two ligand–target molecule pairs used in this study. Asparagine is converted to aspartate, the ligand for the wild-type Tar chemoreceptor, by asparaginase. Phenylacetyl glycine is converted to phenylacetic acid, the ligand for the engineered chemoreceptor TarPA, by penicillin acylase.[6]

Goulian and co-workers [7] back in 2006 developed a simple method to obtain variants of chemoreceptor proteins that enable cells to swim towards target attractants. The method is based solely on a diffusive concentration gradient of target attractant in semi-solid agar media (by spreading a thin line of attractant in the center of an agar plate). The researchers focused on the E. coli aspartate receptor tar. They confirmed the relative plasticity of the protein as some of the tar alleles, specifically the variant that responded to cysteic acid still showed strong sensitivity to aspartate. Tar mutants with increased sensitivity to phenylalanine, N-methyl aspartate and glutamate showed a considerable decrease in their response to aspartate.

Grounded on this previous work, Goulian and co-workers continued to develop methods for increasing the chemotactic sensitivity E. coli for novel molecules. Goldberg et al. [6] engineered E. coli chemotactic response by synthetically introduce enzymatic activity in the periplasm that converts the target molecule to the native chemoreceptor ligand (Figure 2). This approach was used for the design of two systems. In the first system asparagine responsive strain was engineered by using the E. coli asparaginase II, a hydrolase that degrades asparagine to aspartate. The strain was genetically engineered for the enzyme to be produce at normal conditions and since the strain lacks the chromosomal genes encoding all five chemoreceptors, the receptor Tar was expressed from a plasmid. In the second system, the researchers introduce phenylacetyl glycine (PAG) sensitivity by co-expressing from a plasmid the penicillin acylase (Pac) gene from E. coli strain W (with its native promoter) and the previously selected phenylacetic acid tar receptor (TarPA).

Engineering chemotactic intracellular pathways in E. coli via RNA molecules

Figure 3:(Top) Proteins involved in wild-type E. coli chemotaxis. The direction of rotation of the flagellar motor is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motor rotates counterclockwise (CCW). When CheY is phosphorylated (CheY-P), it can bind to the flagellar motor protein FliM, causing the cell to tumble. Wild-type E. coli can migrate on semisolid agar (top right; cells grown for 10 h at 37 °C). (Bottom) Cells lacking the protein CheZ (strain RP1616) cannot dephosphorylate CheY-P and these cells tumble incessantly (bottom right; cells grown for 10 h at 37 °C).[8]
Figure 4:(a) Diagram of plates containing semisolid media patterned with solutions of various ligands. Cells were plated at the location shown and grown for 10 h at 37 °C. (b) Motility of wild-type cells expressing GFP.(c) Motility of RP1616 (¢cheZ) cells expressing a red fluorescent protein.(d) Motility of reprogrammed cells expressing GFP.[6]

Topp and Gallivan [8] demonstrated that by engineering a riboswitch to control E. coli chemotaxis rather than selecting for a chemoreceptor with modified sensitivity could be an efficient method to accomplish novel chemotactic responses. Riboswitches are non-coding RNAs that regulate gene expression via structural reconfiguration upon binding of a ligand. Located at the 5’ untranslated (and les often at the 3’ UTR) of the messenger RNA that they control, these structures are sensitive to ligands, usually small molecules such a metabolite (an amino acid) of the same pathway that they control. The researchers introduced the one of the chemotaxis genes that regulates the directional rotation of the flagellar motor, CheZ , under the control of theophylline-sensitive riboswitch. They sought to bypass the chemoreceptor approach entirely by reprogramming how the intracellular chemotactic pathway works. In E. coli, the 5 different chemoreceptors are linked to the chemotaxis proteins CheB, R and W, which collectively control CheA phosphorylation. CheA phosphorylates CheY that in turn controls the rotational direction of the flagellar motor by binding to the flagellar switch protein FliM. When CheY is phospholyrated the flagellar motor rotates clockwise, which causes the cell to tumble. In contrast, when CheY is dephosphorylated by CheZ, the flagellar motor changes direction to counterclockwise rotation allowing E. coli to execute smooth swims. E, coli lacking the CheZ gene stumbles permanently, thus becoming non-motile. It has been demonstrated previously that inducing CheZ expression restores chemotaxis in a CheZ knockout (Figure 3).

The authors accomplished a process called pseudotaxis by controlling CheZ with a theophylline-sensitive riboswitch. Pseudotaxis differs from chemotaxis in that cell motility is dictated by the absolute theophylline concentration as opposed to changes in concentration over time. Figure 4 shows not only that the authors accomplished pseudotaxis triggered by theophylline absolute concentration but also that the cell migration can be guided across a specific path on a semisolid media plate.

Control of E. coli cell localization within a consortium via the artificial manipulation of chemotaxis

Figure 5:An engineered cell consortium consisting of two interacting strains that produce a mutually interdependent chemotactic response. The red cell is sensitive to molecules that are produced by the blue cell, and the blue cell is sensitive to compounds produced by the red cell..[9]

In a representative example, Goldberg et al. [6] in the same study described in section above exploited the “hitchhiker” effect observed in which cells lacking the enzymatic activity can be induced to motility by cells that produce the proper ligand. They engineered two E. coli strains to form a consortium in which each of them can produce the ligand that induces chemotaxis in the other strain. Specifically, one strain was engineered to produce the native aspartate chemoreceptor and penicillin acylase, the other strain was designed to express a PAA-responsive mutant chemoreceptor and asparaginase II. The observed behavior of the consortium was that of an “AND” Boolean gate since when each strain was isolated and put in contact with the appropriate ligands, no chemotaxis was observed. In contrast, when the two strains were plated in close proximity, the two chemical signals were present and the strains were motile (Figure 5). This research work advances the possibility of utilizing these engineered strains in a real environment since bacterial populations are usually better fit to thrive when living in consortia.

Engineering the intracellular chemotactic pathway vs the manipulation of chemoreceptor sensitivity

The advantages and disadvantages of both approaches presented above are summarized in the following table (information taken from [9] ):

Intracellular engineering by RNA molecules Chemoreceptor manipulation
Advantages 1. Ability to introduce RNA molecules into organisms with minimal modification

2. Capability to evolve RNA molecules (riboswitches) to recognize a completely novel ligand

1. Intracellular machinery remains in place facilitating adaptation responses

2. Response times are dictated by the native schemes and are expected to be lower than the RNA counterpart.

Disadvantages Longer response times since ligand has to diffuse inside the cell which translates into a reduced capacity of adaptation Reduced plasticity since chemoreceptors are limited by a structural scaffold and marginal range of modification is expected.

Potential applications: the paradoxic “therapeutic bacterium” and more

In a proof of concept of a medical application, Anderson et al. [10] engineered E. coli to invade cancer cells depending on the local environment. The researchers control E. coli behavior by introducing the gene that allows cell invasion, inv gene, under the control of native promoters that act under specific conditions of hypoxia or cell density. By this approach they were able to control when and where cell invasion take place. This example and the others described above enable the vision of almost surreal applications such as a “therapeutic E. coli” in which its chemoctactic activity is engineered to detect disease sites and then release drugs for whose production the organism had been rewired a priori. This means that the field of potential applications is unlimited in areas such as target cell therapy and regenerative medicine, drug delivery, and even other non-medical applications such as biosafety and anti-biofouling in pipes, boats, implantable devices and surgical instruments [4].

Engineering other types of motility

Synthetic Cilia-like structure engineered to help understand the functioning of biological cilia

Cilia are highly conserved eukaryotic structures essential for reproduction and survival of many biological organisms. In this work, Sanchez and collaborators [11], molecularly engineered cilia to study how the biological version functions. The authors describe a minimal model system composed by densely packed, actively bending bundles of microtubules and molecular motors, spontaneously synchronize their beating patterns. This simplified in vitro model of eukaryotic cilia could bring insights into the beating of isolated cilia and the synchronization of their beatings.

iGEM projects on chemotaxis

A couple of examples of iGEM projects associated with chemotaxis and motility engineering are as follows:

Back in 2012, team Göttingen developed a selection method to engineer a strain that they called “Homing E. coli”. Through genetic manipulation and directed mutagenesis, they were able to optimize E. Coli’s capacity to quickly travel through a chemo-attractive gradient of aspartate. They identified FlhDC as a cellular factor for flagella enhancement and elongation, and created a library of receptors that they will soon explore to modify specificity of the receptor and, ultimately, motility of the E. Coli. Specifically they applied directed evolution to chemoreceptors by targeting five amino acid residues in the ligand binding site. In order to select for the appropriate phenotype, a new special "swimming" plate was developed by using a low concentration of agar (0.3%). Several tests were run to determine incubation times and temperature, nutrients and chemoattractant concentrations, and even the right strain. They chose BL21 cells in 0.3% swimming agar at 33 C for 1-2 days. After determining the appropriate "swimming" assay, the team started investigating genes involved in the flagellar function of E. coli and their effect on bacterial motility when over-express from a plasmid. They found that gene yhjH had a relatively important positive effect in the "swimming" behavior of the cells since by over-expressing from plasmids, the ability of cells to brake was considerably diminished and biofilm formation appears to repressed. These results are in accordance to the functions that gene yhjH regulates in E. coli. fliC (flagellin) encodes for the structural protein that builds up the filament of the bacterial flagellum. By overexpressing it the authors observed a similar behavior to yhjH versions. A higher motility than the control was observed in general when overexpressing flhDC (master regulator of motility and chemotaxis), however inconsistent results prevent the authors from obtaining definitive conclusions. In general, by overexpressing genes such as yhjH (releases brake and spreads individual cells), fliC (makes stronger and elongated flagella) and flhDC (increases number of flagella), a relatively positive effect on E. coli motility was observed [12].

In 2010, team UPO-Sevilla explored their capacity to concentrate populations of bacteria, or enhance a phenomena termed “Bacterial Crowding”. This fundamentally requires a strain (containing the Prh system) to sense a non-diffusive chemical attractant and subsequently release signals to attract other bacteria to crowd there. This team used two chemo-attractants: aspartate and salicylate, in the hope of demonstrating that they can induce crowding of a strain that is not chemo-sensitive to salicylate.

References

Error fetching PMID 15487940:
Error fetching PMID 20980440:
Error fetching PMID 21925377:
Error fetching PMID 23362267:
Error fetching PMID 17480075:
Error fetching PMID 20576425:
Error fetching PMID 16330045:
Error fetching PMID 24356572:
Error fetching PMID 21778400:
Error fetching PMID 4598187:
  1. Error fetching PMID 15487940: [Gottesman2004]
    Comprehensive review on bacterial small RNAs
  2. Error fetching PMID 20980440: [Gottesman2011]
    A more recent review on bacterial small RNAs.
  3. Error fetching PMID 21925377: [Storz2011]
    Another recent review on bacterial small RNAs.
  4. Error fetching PMID 24356572: [Vazquez2013]
    A thorough review on synthetic regulatory RNAs.
  5. Error fetching PMID 23362267: [DeLay2013]
    A review on sRNA negative regulation.
  6. Error fetching PMID 17480075: [Topp2006]
    Engineering of E. coli chemotaxis via RNA molecules.
  7. Error fetching PMID 20576425: [Mishler2010]
    Review on synthetic biology efforts to engineer chemotaxis.
  8. Error fetching PMID 16330045: [Anderson2006]
    A very "cool" proof of concept of engineered chemotaxis towards medical applications.
  9. Error fetching PMID 21778400: [Sanchez2011]
    Engineered cilia for studying biological motility.
  10. [1] http://2012.igem.org/Team:Goettingen/Project [iGEMGottingen2012]
    A iGEM chemotaxis project for the engineering of "Homing Coli".
  11. Error fetching PMID 4598187: [Adler1974]
    Paper describing thoroughly the chemotaxis phenomenon.
All Medline abstracts: PubMed | HubMed