Research Plan
Introduction
Toxin-antitoxin (TA) systems incorporate pervasively in prokaryotic genomes that have also been suggested to perform a role in numerous critical cellular operations (Van, & Bast, 2009). These systems characteristically comprise of a two-gene operon encoding a toxic protein molecule which aims at an important cellular purpose and an antitoxin that connects to and hinders the toxin. Control of toxin action is attained via differential constancy of the stable toxin as well as the unstable antitoxin (Kamada & Hanaoka, 2005). In several occasions, the antitoxin also operates as a transcriptional autorepressor of the operon, whereby the degradation of the antitoxin leads to transcriptional introduction of the TA genes. Majority of what we understand about the structure and functioning of TA systems have originated from the founding experiments in E. coli, yet their role in bacterial physiology still remains debated. A portion of the genome-encoded systems are made active in as a result of environmental stress, prompting cell stasis whereby these cells can recuperate through more favorable growth environments (Kamada & Hanaoka, 2005). Conversely, there have been reports postulating that the MazEF TA system partakes in programmed cell death (apoptosis) (Nariya & Inouye, 2008). Significantly, the HipBA TA system has been associated with the generation of persistent cells, a subpopulation of the bacteria that manifest antibiotic forbearance in an otherwise vulnerable population (Nariya & Inouye, 2008) and might hence lead to the long treatment periods needed to cure particular pathogenic conditions. Since TA systems were revealed as plasmid strength components, it has also been suggested that the genomic TA loci might equally make stable or aide in the conservation of the genes encoded proximate to the genome (Van, & Bast, 2009). Eventually, it has also been hypothesized that these systems are basically self-centered genetic constituents that task only to ensure their own survival in a particular genome (Nariya & Inouye, 2008). Even though these examinations have availed crucial information concerning the purpose of TA systems in E. coli, their purpose in the physiology of different microbes are still unexploited to a greater extent (Nariya & Inouye, 2008).
The most prevalent means of TA system toxicity is arbitrated via mRNA cleavage, leading to inhibition of translation (Nariya & Inouye, 2008). The two well remarkable TA system groups of E. coli, MazEF as well as RelBE, have been exhibited to operate through this mechanism and also the cleave that is definite three-nucleotide sequences (Kamada & Hanaoka, 2005). The toxins of the biggest group of TA systems in pathogens, VapBC, contain PIN portions, a motif believed to be involved in ribonuclease role (Nariya & Inouye, 2008) and have been exposed to inhibit translation thru mRNA cleavage (Nariya & Inouye, 2008). The transient instigation of these mRNases might permit the bacteria to acclimatize to stress by inhibiting the replication process and similarly through degradation of present transcripts, tolerating an abrupt, rapid alteration in the metabolic conformation of the bacteria.
Provided that a TA system is necessary for the production of persisters in E. coli, it has been anticipated that the TA systems of Pathogenesis, Stress Responses may control cell division options during contagion (Kieser et al., 2000). In several individuals infected with the pathogens, the bacteria originally grow and then form a covert, asymptomatic condition which can persist for many years with the ability to reboot later in lifetime (Nariya & Inouye, 2008). These resilient bacteria are perceived to assume a reluctantly or non-replicating form in reaction to environmental stresses experienced in the host (Kamada & Hanaoka, 2005), though the mechanisms through which there is no replicating form is attained are not known. Since the majority of contemporary antimicrobials necessitate bacterial growth to exercise their killing act, these non-replicating insistent bacteria are believed to comprise an important subpopulation of bacteria that is refractory to antibiotic therapy (Van, & Bast, 2009). A comparable antibiotic-tolerant condition is prompted by TA system activation in different bacteria (Kieser et al., 2000), signifying that TA systems may contribute to the longtime of antibiotic administration needed to cure various pathogenic conditions (Nariya & Inouye, 2008).
Type ll Toxin - Antitoxin system are encoded in an operon made up of two small genes whose products, a toxin and its corresponding antitoxin produce a complex whereby the toxin is not active under ordinary growth situations (Van, & Bast, 2009). The Type II antitoxins possess a DNA connecting domain which binds directly the promoter part in the operon and performs a repressor function mostly together with the toxin. In particular circumstances, the antitoxin is degraded by the stress-proteases leading to the activation of toxin, inhibition of growth as well as the final death of the cell (Hallez et al., 2010). Free toxins from the type II TA system have been manifested to aim given important processes in the cell for instance, cell wall synthesis, cell division as well as translation (Gust et al., 2003).
Type II systems of the chromosome are widely distributed in the prokaryotic genomes and mostly concentrated in the genomic islands. The distribution conforms to the fact that the TA modules are always located in the mobile genetic components such as plasmid, phages, or transposable elements; hence they mainly belong to prokaryotic mobilome (Van, & Bast, 2009).
Genetic conformations of common type II TA systems are quite preserved. Characteristically, these systems consist of two small genes arranged in an operon, the upstream gene encoding the antitoxin. General features of the components and the composition is also well conserved (Florek et a.l, 2008). The antitoxins comprise of two domains, an amino-terminal domain and the carboxy-terminal domain in charge of toxin interaction (Asada, 1994). Type ll Toxin - Antitoxin system is significant in the basic operation of organisms (Gust et al., 2003). Bacteria production takes place mainly via cell division as the main process and the Type ll Toxin - Antitoxin system is a fundamental process responsible for the encoding of proteins. Organisms depend on proliferation of proteins through encoding of poison as well as its corresponding antidote for a complete system (Kieser et al., 2000).
Organization of the type II TA systems rely on the amino acid sequence correspondence of the toxins, to each toxin family being associated with a particular antitoxin family. Therefore, type II systems are presently classified into several families (Van, & Bast, 2009). Nonetheless, the association of a given toxin from one family with the antitoxin from another family can be classified (Kieser et al., 2000). Hence extrapolations are that the type II TA systems are more abundant and differentiated as compared to the current description. According to the extrapolations, eighteen antitoxins and twenty three toxin sequences emanating from various bacterial species will be proved through experiments in E. coli, remarkably growing in the number of families of type II antitoxins and toxins. Hence, it is right to propose pore precise studies (Hallez et al., 2010).
Even though very numerous independently isolated streptomycetes are understood and have been designated, mainly with regards to their generation of antibiotics, only a few have established continuous attention in the scientific literature due to different reasons apart from the antibiotic production (Kamada & Hanaoka, 2005). However, data about other facets of an organism for instance, its ecology, physiology in addition to genetics may finally offer a rich background used as a platform to investigate antibiotic production. A remarkable instance is created by Streptomyces albus (Kamada & Hanaoka, 2005), the subject of massive genetic evaluation for over two decades before it will be realized to produce four variant antibiotics, three of the four have since constituted the focal point of molecular genetic investigations (Kamada & Hanaoka, 2005).
In the case of S. albus, there is a need for a comprehensive research since the isolation of restrictionless mutants for example 51074 (Hallez et al., 2010), which expedite its use as an acceptor of the DNA from other different origins, is essential. Protoplasts of mutants can be proficiently revived (Kieser et al., 2000), and can be changed by numerous plasmid and phage (Van, & Bast, 2009) vectors. The initial analyses of S. albus, nonetheless, will be biochemical (Kamada & Hanaoka, 2005): they regarded its bacteriolytic actions, depicted by its naming again as S. griseus subsp. Solvifaciens (Kamada & Hanaoka, 2005), though devotion to the initial name has remained in the literature. Section of the lytic norm, a DD-carboxypeptidase, has recently been intensively studied (Hallez et al., 2010). The initial studies (Kieser et al., 2000) will have revealed that the enzymatic lytic actions will be associated with ‘material of a lipoid nature that is bactericidal for the Gram-positive bacteria’. During the process of genetic studies, there will be a realization that a given proportion of mutants of S. albus will be sensitive to a diffusible component of the wild-type cultures (Nariya & Inouye, 2008). In the same study, the product will be filtered and recognized, and its reaction against other Gram-positive bacteria will be realized (Yamaguchi, Park & Inouye, 20E11).
Objectives
The main objective of m research will be to determine the Type II Toxin- Antitoxin system in Streptomyces albus. The information on Streptomyces albus is intended to widen the current scope of the study and enhance the likelihood of further understanding of the system in organisms (Hallez et al., 2010).
Hypothesis
Chromosomal type II TA systems are stress-response elements: The recent research prompted this assumption that will be then set open for resultant debate and possibly the consequential experimental proofs. The type II Toxin-Antitoxin systems will be realized in low copy numbers and perceived to be addiction modules whose prime role will be to make the systems stable (Hallez et al., 2010). Toxin–antitoxin (TA) systems incorporate minute genetic modules present in bacterial mobile genetic components and bacterial chromosomes (Gust et al., 2003). They give the impression of being specific of the eubacterial as well as archaebacterial worlds as no homologous chromosomal sequences are discovered in eukaryotic genomes apart from the PIN toxin domain that is found in eukaryotes (Nariya & Inouye, 2008). Toxins are at all times proteins though based on the nature of the antitoxin, as well as its mode of operation. TA systems are presently categorized into three main classes: There are Antitoxins of type I and III systems. These are small RNAs that hinder either toxin manifestation (type I) or activity (type III) (Kieser et al., 2000). Antitoxins of type II systems that are also our main concern in this case are proteins, which inactivate toxins through the formation of protein–protein (Hallez et al, 2010).
As per the hypothesis outlined, in situations of hostile circumstances, antitoxins could be degenerated by stress induced proteases (Gust et al., 2003). The stress factors result into relief of transcriptional repression of TA operons and release of toxins from TA complexes (Kieser et al, 2000). As a result, the free toxins would prompt reversible growth arrest or cell death by hampering an indispensable cellular activity, such as protein synthesis or else DNA replication (Kamada & Hanaoka, 2005). This postulation has been boosted by most of the current experiment findings. For instance, in E. coli, stimulation of TA systems are prompted by various stresses and ectopic manifestation of nearly all characterized chromosomal toxins could improve the capability to confer resistance to stress (Nariya & Inouye, 2008). Therefore, there is need for a careful selection of the strains and the sporulation medium and its maintenance at desirable conditions of temperature (Kieser et al., 2000). The extraction of medium, seed culture, fermentation, chromatography and mass spectroscopy are among the methods desirable for consideration in order to obtain the best findings together with the consequential inferences (Leplae et al., 2011)
Methods
1-Bacterial strains and growth conditions
For cloning, isolation of plasmids, express, and purify proteins, we will use E. coli BL21 (DE3). All strains will grow in Luria-Bertani (LB) liquid broth or on LB aga S. albus will grow on solid R2YE medium, on MSA medium , and in liquid YES medium (1% yeast extract 10.3% sucrose [pH 7.2] supplemented with 0.5% glucose, 5 Mm MgCl2 and 0.5% glycine). Liquid cultures will be carried out in baffled flasks at 28uC and 200 rpm.
2-Identifying putative TA systems
In order to widely search the S.albus genome to detect putative TA, PSI BLAST examines of the S.albus genome will be conducted. Toxin and antitoxin protein sequences derived from each of the following eight major families of TA system will be applied in this case: HigBA, CcdBA HipBA, ParDE, MazEF Doc/PhD, RelBE, and VapBC. When possible, sequences from both distantly-related organisms (Gram-negative) and more closely-related organisms (Gram-positive and high-GC) are used in searching for homologs. The method is similar to the approach used for identifying novel TA systems in E. coli. Consequently, it includes constraints on orientation, size, and spacing of putative TA pairs.
Following of the identification of toxin genes will help find out the presence of the gene smaller than the putative toxin gene and adjacent upstream. On the other hand, following of the identification of antitoxin genes will help in determining if an adjacent downstream gene larger than the putative toxin was present. At most 150 bp between the putative toxin and antitoxin was required in both cases. Homologs that meet certain conditions will be excluded: those for which adjacent cognate toxin or antitoxin could not be found and those in which the adjacent genes failed to meet either size or distance between genes criteria. In cases where the putative toxin and antitoxin of an adjacent pair were homologous to dissimilar TA system families, will allocate the pair centered on the homologous conformation of the toxin gene. Finally, a fully sequenced as well as marked up genomes from the NCBI database were downloaded.
3-Assessing toxin and antitoxin activity
In order to conditionally express the putative toxin genes, S. albus will be identified then all putative S. albus toxin genes and toxin-antitoxin gene pairs will be inserted downstream of the inducible acetamidase promoter in E. coli BL21(DE3 ) plasmid. Then, Toxin and antitoxin activity will be assessed by growing E. coli BL21 (DE3) carrying the appropriate vector at 37uC on solid media with Tween-80, kanamycin, and acetamide to induce gene expression. Then growth after incubation will be assessed. Results of putative TA system testing in previous studies were products of Genes encoding a toxic protein repressed growth of the cultures on the plates together with the inducer. Nevertheless, they never affected the growth of bacteria on the plates deprived of the inducer. Therefore, with regard to each gene that was toxic, toxin and antitoxin will then be co-expressed. Same inducible promoter will be used in this case in determining if it allowed cell growth in the presence of inducer. Then toxic proteins that inactivated by their putative antitoxins will considered functional TAsystems.
4-Overexpression, purification and identification of putative toxin –antitoxin
Overproduction of Purify antitoxin –toxin -His6sl complex will be observed in E. coli BL21 (DE3) that is transformed with the corresponding plasmids. Five hours after induction with IPTG, cells will be harvested at 50006g at 4uC for 10 min. The cell pellet will re-suspended in lysis buffer (Na2HPO4/NaH2PO4, pH 7.5, NaCl, Triton X100, imidazole), and then sonicate and centrifuged for 30 min. at 100.0006g. The supernatant will apply to a column containing NTA-Ni resin. Then wash the column three times with washing buffer 1 (Na2HPO4/NaH2PO4, pH 7.5, NaCl, Triton X100, imidazole) and twice with washing buffer 2 (Na2HPO4/NaH2PO4, pH 7.5, NaCl, Triton X100, imidazole).
Tagged proteins will elute 3 times with elution buffer 1 (Na2HPO4/NaH2PO4, pH 7.5, NaCl, Triton X100, imidazole) and twice with elution buffer 2 (Na2HPO4/NaH2PO4, pH 7.5, NaCl, Triton X100, imidazole). Fractions containing the highest concentrations of proteins will pool and dialyzed with D-Tube TM Dialyzer Maxi for 48 h against dialysis buffer ( Tris, pH 7.5, NaCl, glycerol).(SM)
The purified products will separate with SDS–PAGE, and then excise co-purified proteins from gel and determine by MALDI-TOF mass spectrometry (MS) analysis. The proteins embedded in the gel slice will digest with trypsase and analyte to determine the mass/charge (m/z) values according to the manufacturer’s instructions. The experimental peptide masses will be compared to a theoretical cleavage with trypsin using the MS-DIGEST program (AN).
5-the response of TA systems to the Macrophage infection
And for macrophage infection, will use isolated Bone marrow-derived macrophages from C57BL/6 mice and culture for 6 d in media containing L-cell supernatant in the presence of antibiotics. Macrophages will stimulate with recombinant mouse IFN-c for 24 hours prior to infection. Macrophages will be infected using DMEM containing 10% horse serum and incubate for 2 h then wash and fresh medium will add. At the indicate time points, S.albus RNA from inside macrophages will isolate and amplify using qPCR.
It is interesting that previous studies monitored two TA systems in M. tuberculosis are activated during hypoxia are never activated throughout the infection of macrophage, provided the efficiently hypoxic conditions created through nitric oxide signaling due to IFN-c encouragement of the macrophages. Definitely, these two conditions both result in induction of the dormancy
Regulon (MT).
Summary
The activity defined in this proposal will encompass the application of different methods in evaluating the Type II toxin-antitoxin system in Streptomyces albus. The tools to be applied in the research are in are critical for both academic and industrial scenarios and will be very relevant for my students as well as technicians (those who will use my research) in fostering their scientific careers. Through these experimental methodologies, there is hope for the achievement of a more absolute understanding of type II toxin-antitoxin system in Streptomyces albus. Learning proteins incorporated in cell bacteria and defining how the TA systems influence the conformations will aide in providing insight into the way the entire system operates. At least the functionality of the system in other microorganisms has been elucidated hence this would provide increased probability of considering their tendencies.
References
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