- Email: [email protected]
Effector Molecules and Regulatory Proteins: Applications
TIBTEC 1383 No. of Pages 4
Forum
Effector Molecules and Regulatory Proteins: Applications J.M. Landete1,* Bacteria respond to their external environment by modulating gene expression in the presence of certain effector molecules. The adaptive responses are mediated by transcriptional regulators that, after binding to the DNA, recognize these effector molecules and modify transcription. Some applications of regulatory proteins are reviewed here. Effector Molecules and Regulatory Proteins To exist in a wide range of niches and cope with environmental stresses, bacteria must sense and respond to external signals. In response to environmental stresses, bacteria can evolve rapidly, allowing them to adapt to unfavorable environmental conditions [1]. Bacteria are highly sensitive to effector molecules such as sugars, organic acids, antibiotics, heavy metals, and bacteriocins (proteinaceous toxins produced by bacteria to inhibit the growth of related bacteria). Transcriptional regulators modify transcription after recognizing these effector molecules. Thus, effector molecules may be defined as compounds that trigger the activator or repressor function of a regulator (transcriptional regulators) thereby modifying gene expression. Effector molecules can also regulate the activity of some mRNA molecules (riboswitches) and/or modify enzyme activity. In some cases, proteins can be effector molecules, especially in cellular signal transduction cascades.
Organization of Prokaryotic Operons
recognize the effector molecules and subsequently bind DNA to modify In prokaryotic operons, we can distinguish transcription. structural genes that carry information for polypeptides and promoters. In some Components of Pathways cases we also find: (i) an operator, which Mediated by Regulatory Proteins is another control element, or a region of Two-Component Systems DNA with a sequence that is recognized Two-component regulatory systems are by a regulatory protein; (ii) a regulatory biological signaling systems. In the basic gene, the DNA sequence encoding the scheme of these systems, a histidine regulatory protein; (iii) the regulatory pro- kinase receives a signal that causes tein encoded by the regulatory gene, autophosphorylation. [4_TD$IF]Then the phoswhich binds to the operator region; and phate group is [5_TD$IF]ransferred to the respon(iv) effector molecules. sive regulatory protein to modify its activity. Thus, in two-component systems the sensing of signals that trigger Adaptive Response Mediated by a transcriptional process involves two Regulatory Proteins: Evidence proteins (Figure 1). from the Laboratory Evidence showing that regulatory proteins are necessary for the modification Two-Domain Proteins of transcription has been observed in the Most microbial regulators involved in tranlaboratory. These studies involved look- scriptional control are two-domain proing for molecules that were capable of teins, called transcriptional regulators, modulating (i.e., inducing or repressing) with a signal-receiving domain and a gene expression in lactic acid bacteria DNA-binding domain that transduces and bifidobacteria. In most cases, the the signal (Figure 2). Examples include inducing molecule in one species of lactic some mechanisms of resistance to antiacid bacterium did not work when the biotics or heavy metals. promoter was cloned into another species of lactic acid bacterium, and differ- Gene and Regulatory Protein ences were even found between strains of Applications the same species. For example, Lacto- Biosensors coccus lactis strains NZ9000 and Biosensors reflect changes in environMG1363 were transformed with a vector mental conditions in a dose-dependent containing a fluorescent protein gene manner. A greater knowledge of genes under the control of the nisin-inducible and regulatory proteins may allow us to promoter nisA in plasmid pNZ8048. Only use them as biosensors to detect effector including bacteriocins the NZ9000 strain showed fluorescence molecules, (Figure 1A), organic acids, sugars, and in the presence of nisin. The nisRK genes even biogenic amines [2]. of the NZ900 strain allow it to sense nisin and relay the signal to initiate transcription from the nisA promoter. NisK recognizes Bioreporters contain two essential genetic nisin and subsequently activates NisR elements, a promoter gene and a reporter through phosphorylation; the activated gene (e.g., GFP, Lux[6_TD$IF], etc). The promoter NisR then induces the nisin operon gene is turned on (transcribed) when the (Figure 1A). Similar results were observed effector molecule is present in the cell's with Lactobacillus strains for other com- environment. However, the effector molepounds such as bile, phenolic com- cule must also bind to a regulatory protein pounds, and polyols, showing that for that is capable of binding to the DNA, effector molecules to modulate gene modifying transcription. The regulatory expression it is necessary that the cell protein is either produced by the bactesynthesizes regulatory proteins that rium or supplied from a vector.
Trends in Biotechnology, Month Year, Vol. xx, No. yy
1
TIBTEC 1383 No. of Pages 4
MG 1363
NZ900
(A)
Nisin
Nis K Hs
PH
ATP
ADP
Anbiocs
(B)
Nisin H P
Hs
H P
ATP
P
ADP
H P
P
Nis R P
Possible targets Fluorescence signal
GFP gene
GFP gene
Pnis
Pnis
P
Anbioc resistance gene
Figure 1. Two-Component Systems. Only the strain containing the two-component system NisK/NisR detects the bacteriocin nisin and emits a [3_TD$IF]fluorescence signal (A). Two-component systems implicated in resistance to antibiotics. Knowledge of these regulatory proteins will allow us to use them as targets to inhibit resistance to antibiotics, inhibiting DNA binding by the response regulatory protein (B).
New Targets for the Development of Novel Antimicrobial Agents Many two-component systems are implicated in pathogenicity or resistance to antibiotics. Knowledge of these regulatory proteins will allow us to use them as targets to inhibit pathogenicity or resistance to antibiotics [3]. Examples include inhibiting DNA binding by the response regulator (Figure 1B). Additionally, quorum sensing is important in bacterial pathogenicity, and controlling or modifying quorum-sensing-regulated proteins would be particularly relevant in preventing pathogenicity [4]. Understanding the mechanisms behind bacterial cell responses to stress and knowledge of the transcribed genes will help us identify new targets for the development of novel antimicrobial agents.
sugar-, or biogenic amine-inducible promoters have been developed, but these require the gene encoding the regulatory protein, supplied from a vector [5].
their cell surface and can even be used as vectors for the production and delivery of oral vaccines against cholera, malaria, tetanus, or brucellosis [8].
We could control the expression of certain genes during fermentation by administering inductors such as malic acid or rhamnose and supplying the regulatory protein from a vector. Alternatively, biotechnologically interesting bacteria could be grown in a medium that does not contain sugars, using malic acid as the only carbon source using the two-component system for malic acid of Lactobacillus casei BL23 [6].
Food-Grade Vectors and Bioremediation Resistance mechanisms in bacteria may be of interest in the creation of food-grade vectors (i.e., vectors without antibiotic resistance) [9]. These resistance mechanisms are often mediated by two-component systems or two-domain proteins, including some mechanisms of resistance to heavy metals [10].
The LacI protein regulates the lactose catabolism operon in Escherichia coli by binding near the transcriptional start site, repressing transcription initiation [7]. When the effector molecule isopropyl-D-1-thiogalactopyranoside (IPTG) is present in the Protein Overexpression and Control of bacterium, it binds to the LacI protein and Biotechnological Processes for the LacI–IPTG complex disassociates from Metabolic Engineering Knowledge of effector molecules and reg- the DNA, allowing transcription (Figure 2A). ulatory proteins has resulted in their wide- Thus, IPTG is used extensively for protein spread use in applications ranging from overexpression. protein overexpression (Figure 2A) to control of biotechnological processes for met- These inducible systems controlled by abolic engineering (Figure 2B). Some regulatory proteins can be used to proexpression vectors based on bacteriocin-, duce proteins from pathogenic species on
2
Trends in Biotechnology, Month Year, Vol. xx, No. yy
Resistance mechanisms may be necessary to allow bacteria to grow in hostile environments or to eliminate toxic compounds. Therefore, the use of genetic engineering to create organisms specifically designed for bioremediation has great potential. The bacterium Deinococcus radiodurans has been modified to consume cobalt and ionic mercury from highly radioactive nuclear waste [11]. Many operon-clustered metal-resistance genes have been reported in bacterial systems, for cadmium, chromium, copper, lead, mercury and nickel resistance and detoxification [12]. These resistance
TIBTEC 1383 No. of Pages 4
IPTG
(A)
LacI-IPTG
LacI
Protein gene
Protein gene
P (B)
P Effector molecules
- Control of biotechnological processes - Producon and delivery of oral vaccines
Regulatory protein
Protein gene P
No toxic
(C)
Toxic molecule effector (heavy metals)
Detoxifying gene
Figure 2. Two-Domain Proteins. Isopropyl-D-1-thiogalactopyranoside (IPTG) binds to the LacI protein and the LacI–IPTG complex disassociates from the DNA, allowing transcription (A). These inducible systems regulated by regulatory proteins can be used to control biotechnological processes for metabolic engineering or as vectors for the production and delivery of oral vaccines (B). Mechanisms of resistance mediated by regulatory proteins that respond to toxic effector molecules initializing the resistance to these molecules, which could be used in bioremediation and in the creation of food-grade vectors (C).
Trends in Biotechnology, Month Year, Vol. xx, No. yy
3
TIBTEC 1383 No. of Pages 4
mechanisms are mediated by regulatory proteins that respond to the effector molecules initializing the resistance to these molecules. This strategy could be used in bioremediation through genetic engineering (Figure 2C).
Concluding Remarks and Perspectives Bacteria, in response to environmental stress, have evolved by adapting response mechanisms, developing highly sensitive systems able to detect effector molecules. Generally, transcription factors regulate gene expression, allowing bacteria to quickly adapt to unfavorable environmental conditions. The presence of the regulatory protein is necessary for the function of the effector molecule. Understanding the mechanisms behind bacterial cell response to stress and knowing the genes of transcriptional regulators would
4
Trends in Biotechnology, Month Year, Vol. xx, No. yy
be of great biotechnological interest as mentioned[2_TD$IF]. Regulatory protein genes could be introduced by genetic engineering. Knowledge of regulatory protein genes would allow these genes to be cloned in bacteria with biotechnological interest. 1
Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de la Coruña Km 7.5, 28040 Madrid, Spain *Correspondence: [email protected] (J.M. Landete). http://dx.doi.org/10.1016/j.tibtech.2016.04.011 References 1. Sasson, V. et al. (2012) Mode of regulation and the insulation of bacterial gene expression. Mol. Cell 46, 399–407
2. Linares, D.M. et al. (2015) Implementation of the agmatinecontrolled expression system for inducible gene expression in Lactococcus lactis. Microb. Cell Fact. 14, 208 3. Worthington, R.J. and Melander, C. (2013) Combination approaches to combat multidrug-resistant bacteria. Trends Biotechnol. 31, 177–184
4. Bassler, B. (2015) Manipulating quorum sensing to control bacterial pathogenicity. FASEB J. 29, S88.1 5. Linares, D.M. (2014) An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis. Microb. Cell Fact. 13, 169 6. Landete, J.M. et al. (2010) Requirement of the Lactobacillus casei MaeKR two-component system for L-malic acid utilization via a malic enzyme pathway. Appl. Environ. Microbiol. 76, 84–85 7. Goodson, K.A. et al. (2013) LacI–DNA–IPTG loops: equilibria among conformations by single-molecule FRET. J. Phys. Chem. B 117, 4713–4722 8. Zamri, H.F. et al. (2012) Oral vaccination with Lactococcus lactis expressing the Vibrio cholerae Wzm protein to enhance mucosal and systemic immunity. Vaccine 30, 3231–3238 9. Landete, J.M. (2016) A review of food-grade cloning vectors in lactic acid bacteria: from the laboratory to their application. Crit. Rev. Biotechnol. 26, 1–13 10. Bondarczuk, K. and Piotrowska-Seget, Z. (2013) Molecular basis of active copper resistance mechanisms in Gramnegative bacteria. Cell Biol. Toxicol. 29, 397–405 11. Gogada, R. et al. (2015) Engineered Deinococcus radiodurans R1 with NiCoT genes for bioremoval of trace cobalt from spent decontamination solutions of nuclear power reactors. Appl. Microbiol. Biotechnol. 99, 9203–9213 12. Das, S. et al. (2016) Genetic basis and importance of metal resistant genes in bacteria for bioremediation of contaminated environments with toxic metal pollutants. Appl. Microbiol. Biotechnol. 100, 2967–2984
Forum
Effector Molecules and Regulatory Proteins: Applications J.M. Landete1,* Bacteria respond to their external environment by modulating gene expression in the presence of certain effector molecules. The adaptive responses are mediated by transcriptional regulators that, after binding to the DNA, recognize these effector molecules and modify transcription. Some applications of regulatory proteins are reviewed here. Effector Molecules and Regulatory Proteins To exist in a wide range of niches and cope with environmental stresses, bacteria must sense and respond to external signals. In response to environmental stresses, bacteria can evolve rapidly, allowing them to adapt to unfavorable environmental conditions [1]. Bacteria are highly sensitive to effector molecules such as sugars, organic acids, antibiotics, heavy metals, and bacteriocins (proteinaceous toxins produced by bacteria to inhibit the growth of related bacteria). Transcriptional regulators modify transcription after recognizing these effector molecules. Thus, effector molecules may be defined as compounds that trigger the activator or repressor function of a regulator (transcriptional regulators) thereby modifying gene expression. Effector molecules can also regulate the activity of some mRNA molecules (riboswitches) and/or modify enzyme activity. In some cases, proteins can be effector molecules, especially in cellular signal transduction cascades.
Organization of Prokaryotic Operons
recognize the effector molecules and subsequently bind DNA to modify In prokaryotic operons, we can distinguish transcription. structural genes that carry information for polypeptides and promoters. In some Components of Pathways cases we also find: (i) an operator, which Mediated by Regulatory Proteins is another control element, or a region of Two-Component Systems DNA with a sequence that is recognized Two-component regulatory systems are by a regulatory protein; (ii) a regulatory biological signaling systems. In the basic gene, the DNA sequence encoding the scheme of these systems, a histidine regulatory protein; (iii) the regulatory pro- kinase receives a signal that causes tein encoded by the regulatory gene, autophosphorylation. [4_TD$IF]Then the phoswhich binds to the operator region; and phate group is [5_TD$IF]ransferred to the respon(iv) effector molecules. sive regulatory protein to modify its activity. Thus, in two-component systems the sensing of signals that trigger Adaptive Response Mediated by a transcriptional process involves two Regulatory Proteins: Evidence proteins (Figure 1). from the Laboratory Evidence showing that regulatory proteins are necessary for the modification Two-Domain Proteins of transcription has been observed in the Most microbial regulators involved in tranlaboratory. These studies involved look- scriptional control are two-domain proing for molecules that were capable of teins, called transcriptional regulators, modulating (i.e., inducing or repressing) with a signal-receiving domain and a gene expression in lactic acid bacteria DNA-binding domain that transduces and bifidobacteria. In most cases, the the signal (Figure 2). Examples include inducing molecule in one species of lactic some mechanisms of resistance to antiacid bacterium did not work when the biotics or heavy metals. promoter was cloned into another species of lactic acid bacterium, and differ- Gene and Regulatory Protein ences were even found between strains of Applications the same species. For example, Lacto- Biosensors coccus lactis strains NZ9000 and Biosensors reflect changes in environMG1363 were transformed with a vector mental conditions in a dose-dependent containing a fluorescent protein gene manner. A greater knowledge of genes under the control of the nisin-inducible and regulatory proteins may allow us to promoter nisA in plasmid pNZ8048. Only use them as biosensors to detect effector including bacteriocins the NZ9000 strain showed fluorescence molecules, (Figure 1A), organic acids, sugars, and in the presence of nisin. The nisRK genes even biogenic amines [2]. of the NZ900 strain allow it to sense nisin and relay the signal to initiate transcription from the nisA promoter. NisK recognizes Bioreporters contain two essential genetic nisin and subsequently activates NisR elements, a promoter gene and a reporter through phosphorylation; the activated gene (e.g., GFP, Lux[6_TD$IF], etc). The promoter NisR then induces the nisin operon gene is turned on (transcribed) when the (Figure 1A). Similar results were observed effector molecule is present in the cell's with Lactobacillus strains for other com- environment. However, the effector molepounds such as bile, phenolic com- cule must also bind to a regulatory protein pounds, and polyols, showing that for that is capable of binding to the DNA, effector molecules to modulate gene modifying transcription. The regulatory expression it is necessary that the cell protein is either produced by the bactesynthesizes regulatory proteins that rium or supplied from a vector.
Trends in Biotechnology, Month Year, Vol. xx, No. yy
1
TIBTEC 1383 No. of Pages 4
MG 1363
NZ900
(A)
Nisin
Nis K Hs
PH
ATP
ADP
Anbiocs
(B)
Nisin H P
Hs
H P
ATP
P
ADP
H P
P
Nis R P
Possible targets Fluorescence signal
GFP gene
GFP gene
Pnis
Pnis
P
Anbioc resistance gene
Figure 1. Two-Component Systems. Only the strain containing the two-component system NisK/NisR detects the bacteriocin nisin and emits a [3_TD$IF]fluorescence signal (A). Two-component systems implicated in resistance to antibiotics. Knowledge of these regulatory proteins will allow us to use them as targets to inhibit resistance to antibiotics, inhibiting DNA binding by the response regulatory protein (B).
New Targets for the Development of Novel Antimicrobial Agents Many two-component systems are implicated in pathogenicity or resistance to antibiotics. Knowledge of these regulatory proteins will allow us to use them as targets to inhibit pathogenicity or resistance to antibiotics [3]. Examples include inhibiting DNA binding by the response regulator (Figure 1B). Additionally, quorum sensing is important in bacterial pathogenicity, and controlling or modifying quorum-sensing-regulated proteins would be particularly relevant in preventing pathogenicity [4]. Understanding the mechanisms behind bacterial cell responses to stress and knowledge of the transcribed genes will help us identify new targets for the development of novel antimicrobial agents.
sugar-, or biogenic amine-inducible promoters have been developed, but these require the gene encoding the regulatory protein, supplied from a vector [5].
their cell surface and can even be used as vectors for the production and delivery of oral vaccines against cholera, malaria, tetanus, or brucellosis [8].
We could control the expression of certain genes during fermentation by administering inductors such as malic acid or rhamnose and supplying the regulatory protein from a vector. Alternatively, biotechnologically interesting bacteria could be grown in a medium that does not contain sugars, using malic acid as the only carbon source using the two-component system for malic acid of Lactobacillus casei BL23 [6].
Food-Grade Vectors and Bioremediation Resistance mechanisms in bacteria may be of interest in the creation of food-grade vectors (i.e., vectors without antibiotic resistance) [9]. These resistance mechanisms are often mediated by two-component systems or two-domain proteins, including some mechanisms of resistance to heavy metals [10].
The LacI protein regulates the lactose catabolism operon in Escherichia coli by binding near the transcriptional start site, repressing transcription initiation [7]. When the effector molecule isopropyl-D-1-thiogalactopyranoside (IPTG) is present in the Protein Overexpression and Control of bacterium, it binds to the LacI protein and Biotechnological Processes for the LacI–IPTG complex disassociates from Metabolic Engineering Knowledge of effector molecules and reg- the DNA, allowing transcription (Figure 2A). ulatory proteins has resulted in their wide- Thus, IPTG is used extensively for protein spread use in applications ranging from overexpression. protein overexpression (Figure 2A) to control of biotechnological processes for met- These inducible systems controlled by abolic engineering (Figure 2B). Some regulatory proteins can be used to proexpression vectors based on bacteriocin-, duce proteins from pathogenic species on
2
Trends in Biotechnology, Month Year, Vol. xx, No. yy
Resistance mechanisms may be necessary to allow bacteria to grow in hostile environments or to eliminate toxic compounds. Therefore, the use of genetic engineering to create organisms specifically designed for bioremediation has great potential. The bacterium Deinococcus radiodurans has been modified to consume cobalt and ionic mercury from highly radioactive nuclear waste [11]. Many operon-clustered metal-resistance genes have been reported in bacterial systems, for cadmium, chromium, copper, lead, mercury and nickel resistance and detoxification [12]. These resistance
TIBTEC 1383 No. of Pages 4
IPTG
(A)
LacI-IPTG
LacI
Protein gene
Protein gene
P (B)
P Effector molecules
- Control of biotechnological processes - Producon and delivery of oral vaccines
Regulatory protein
Protein gene P
No toxic
(C)
Toxic molecule effector (heavy metals)
Detoxifying gene
Figure 2. Two-Domain Proteins. Isopropyl-D-1-thiogalactopyranoside (IPTG) binds to the LacI protein and the LacI–IPTG complex disassociates from the DNA, allowing transcription (A). These inducible systems regulated by regulatory proteins can be used to control biotechnological processes for metabolic engineering or as vectors for the production and delivery of oral vaccines (B). Mechanisms of resistance mediated by regulatory proteins that respond to toxic effector molecules initializing the resistance to these molecules, which could be used in bioremediation and in the creation of food-grade vectors (C).
Trends in Biotechnology, Month Year, Vol. xx, No. yy
3
TIBTEC 1383 No. of Pages 4
mechanisms are mediated by regulatory proteins that respond to the effector molecules initializing the resistance to these molecules. This strategy could be used in bioremediation through genetic engineering (Figure 2C).
Concluding Remarks and Perspectives Bacteria, in response to environmental stress, have evolved by adapting response mechanisms, developing highly sensitive systems able to detect effector molecules. Generally, transcription factors regulate gene expression, allowing bacteria to quickly adapt to unfavorable environmental conditions. The presence of the regulatory protein is necessary for the function of the effector molecule. Understanding the mechanisms behind bacterial cell response to stress and knowing the genes of transcriptional regulators would
4
Trends in Biotechnology, Month Year, Vol. xx, No. yy
be of great biotechnological interest as mentioned[2_TD$IF]. Regulatory protein genes could be introduced by genetic engineering. Knowledge of regulatory protein genes would allow these genes to be cloned in bacteria with biotechnological interest. 1
Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de la Coruña Km 7.5, 28040 Madrid, Spain *Correspondence: [email protected] (J.M. Landete). http://dx.doi.org/10.1016/j.tibtech.2016.04.011 References 1. Sasson, V. et al. (2012) Mode of regulation and the insulation of bacterial gene expression. Mol. Cell 46, 399–407
2. Linares, D.M. et al. (2015) Implementation of the agmatinecontrolled expression system for inducible gene expression in Lactococcus lactis. Microb. Cell Fact. 14, 208 3. Worthington, R.J. and Melander, C. (2013) Combination approaches to combat multidrug-resistant bacteria. Trends Biotechnol. 31, 177–184
4. Bassler, B. (2015) Manipulating quorum sensing to control bacterial pathogenicity. FASEB J. 29, S88.1 5. Linares, D.M. (2014) An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis. Microb. Cell Fact. 13, 169 6. Landete, J.M. et al. (2010) Requirement of the Lactobacillus casei MaeKR two-component system for L-malic acid utilization via a malic enzyme pathway. Appl. Environ. Microbiol. 76, 84–85 7. Goodson, K.A. et al. (2013) LacI–DNA–IPTG loops: equilibria among conformations by single-molecule FRET. J. Phys. Chem. B 117, 4713–4722 8. Zamri, H.F. et al. (2012) Oral vaccination with Lactococcus lactis expressing the Vibrio cholerae Wzm protein to enhance mucosal and systemic immunity. Vaccine 30, 3231–3238 9. Landete, J.M. (2016) A review of food-grade cloning vectors in lactic acid bacteria: from the laboratory to their application. Crit. Rev. Biotechnol. 26, 1–13 10. Bondarczuk, K. and Piotrowska-Seget, Z. (2013) Molecular basis of active copper resistance mechanisms in Gramnegative bacteria. Cell Biol. Toxicol. 29, 397–405 11. Gogada, R. et al. (2015) Engineered Deinococcus radiodurans R1 with NiCoT genes for bioremoval of trace cobalt from spent decontamination solutions of nuclear power reactors. Appl. Microbiol. Biotechnol. 99, 9203–9213 12. Das, S. et al. (2016) Genetic basis and importance of metal resistant genes in bacteria for bioremediation of contaminated environments with toxic metal pollutants. Appl. Microbiol. Biotechnol. 100, 2967–2984