Role of Simple Sequence Repeats in Bacterial Phase Variation - Coursework Example

Describe the Role of Simple Sequence Repeats in Bacterial Phase Variation and Discuss the Proposed Mechanism Underlying Repeat Variability INTRODUCTION DNA repeats are repetitive sequences found extensively and repeatedly in a genome. Most organisms are found to have such repetitive sequences in their genomes. Although not found as abundantly as a Eukaryotic cell, these repetitive sequences in the bacterial genome has been well studied. Their mechanism, dynamics, functions and structural constraints are also well established. The taxonomy or the genome size of the bacteria does not correlate with the density of the sequence repeats (Chang and Chang, 2003). In a wide range of bacteria these sequence repeats range to nearly less than about 400bps. They are found in multiple copies some are known to exist to about 1,600 copies and are found in the intergenic regions of the chromosome. Some repeats which are known to produce a mobile elements in abundance are known to be parasitic or a potential hazard to the organism. These evolutionary species of these organisms are now known to have fewer mobile elements in their genomes. These sequences are known to be found in the site of plasticity in the genome of the bacteria (Delihas, 2011). Today it is well understood that the evolution is the main reason for the abundance in the genomic repeats in a species. These repeat sequences may either prove as beneficial or posses a disadvantage to the species by its location in the genome. But the real question is, is there any real role of these simple sequence repeats in the bacterial genome? Are these repeats of any importance to the bacterial species? What are these short sequence repeats? How many types are present and what is their actual role in evolution? These are the questions that have provoked the researchers and scientists today to better understand the bacterial genome and further to analyse in depth the importance and role of these sequence repeats within the genome. SIMPLE SEQUENCE REPEATS Simple sequence repeats (SSR’s) also known as microsatellites are known to occur in both the genome as well as the plasmid of a bacterium. These repeats contribute to the plasticity of bacterial genome such as amplification, translocations, deletions, inversions and also transposition. These repeats are also responsible for the high-frequency genetic variation in both interspecies as well as inter-strain levels (Romero et al, 1999). Simple sequence repeats are short DNA repeats of about 2 – 6 nucleotides in length and are found to be distributed throughout the genome repetitively. DNA repeats of nearly 400 bps are found within the genome of the bacteria and are known to have as many as 1,600 copies. They are co-dominant and highly variable and are widely used for biotechnological techniques today such as finger-printing, marker assisted back-crossing and marker assisted selections (Queller, Strassmann and Hughes, 1993). Understanding and analyzing the implications of sequence repeats in the organism has given insights on evolutionary adaptations and evolutionary control of mutation processes. In higher organisms and human beings they are found to be the cause of many diseases. Especially in human beings they are known as a cause of degenerative neurodegenerative diseases (Kashi, King, Soller, 1997). The simple sequence repeats has been extensively studied and is now known as an informative marker for bacterial genomes. These repeats are also known to have a role to play in bacterial adaptation. They also contribute to phenotypic variations since repeats in the regulatory region acts as an on/off trigger for gene expression at the transcriptional level. Similarly, repeats found in the coding region may act as a translational blocker during mutation. However, much is yet to be defined and understood about these simple sequence repeats and thus the work continues (Chang and Chang, 2003). BACTERIAL PHASE VARIATION MECHANISM Simple Sequence Repeat’s (SSR) or mini-satellites generally range about 1 – 6 bps. Their most salient feature is their high degree of polymorphism in their repeat numbers. They are sequences, highly susceptible to mutations such as deletion, insertion and substitution. Today, researches have established that the micro-satellites are more than just repetitive sequences since they are known to play an active role in gene expression. They are also quite common in genes that contribute to the virulence of the bacteria and are important factors for phase variation. They are also responsible for adaptation to certain environmental conditions. A region with a high concentration of mini-satellites is termed as contingency loci. Although as stated above these simple sequence repeats are known to have many roles to play in a bacterial genome their role in the phase variation altering the virulence of the bacteria is the most important and prominent one (Chun Li, Korol, Fahima, Beiles and Nevo, 2002). In pathogenic bacteria, the presence of these mini-satellites in the coding region may lead to drastic changes in the gene products as a result of premature termination due to shifting of the coding frame. In the case of the virulence genes, the frame shift mechanism leads to the production of greater defensive capacity the pathogens which help to escape the hostile host environment. The actual phase variation mechanism is associated with the expression or non-expression that is the on/off switch mechanism of the cell surface pili-adhesion complexes. These phase variations in the pili is brought about by site-specific and homologous recombination mechanisms (Sreenu, Kumar, Nagaraju and Nagarajaram, 2007). PAP PHASE VARIATION MODEL The pili-phase variation is brought about by a large group of pili-operons which includes the pyelonephritis-associated pili (pap) which is controlled by DNA methylation pattern formation and is regulated by an epigenetic switch. The expression of pap include a number of proteins and they are, 1) papI and papB, the local regulators, 2) Leucine responsive regulatory proteins (Lrp) and the catabolic gene activator protein (CAP) which are the global regulators, 3) for pap transcription DNA adenine methylase (Dam). Any knock out mutation in any of these genes may lead to perpetual phase ON switch in the pap gene. In addition, to all the above proteins the H-NS which is the histone-like nucleoid structural protein is important for the switching phase in pap since its mutants affects the OFF switch state (Hernday, Krabbe, Braaten and Low, 2002). A simple E.Coli pap Operon Model (Kawamura, Vartanian, Zhou and Dahlquist, 2010) The regulatory region of the pap operons consist of the papI and papB genes along with the 416-bps intergenic region. The pap regulatory site consists of 6 Lrp binding sites. The GATC sites on the DNA are found to be the target sites for Dam, which methylates the adenosine of the GATC. Mutations in the Lrp binding sites are also known to affect the ON/OFF switch. Binding of Lrp near the papBA promoter inhibits transcription and when Lrp binds to the distal end it activates transcription. DNA footprint analysis indicated that Lrp had higher affinity to Lrp binding sites 1 – 3 and any mutational modification in site 1 or 2 lead to a permanent ON phase for transcription whereas sites 4 – 6 showed lesser Lrp affinity. Moreover, DNA methylation pattern examination proved that in the OFF phase cells GATCprox are non-methylated whereas the GATCdist was methylated whereas in the ON phased cells the opposite was true. Lrp is known to influence the methylation patterns since its binding in the pap regulatory sequence influences the ON/OFF phase. Thus, Lrp plays a dual role as a regulator for pap transcription and repressor when bound to the proximal papBA pili promoter (Hernday, Krabbe, Braaten and Low, 2002). The self-perpetuating phase off state During the OFF phase methylation occurs only at the distal GATC site of the pap regulatory region and is found to be important to block transcription. Although Dam is important for the methylation process, Lrp was also found to have minor affinity towards it. But it has been found out that over methylation or over production of Dam may lead to perpetual OFF state and may prevent the transition from OFF phase to ON phase to occur. The Lrp affinity for its sites 1 – 3 is found to be equally important to maintain the OFF phase and may act as a signal amplification mechanism. Binding of Lrp to sites 1 – 3 inhibits transcription and directly blocks pap transcription (Weyand and Low, 2000). In vitro analysis using RNA polymerase on a super-coiled pap DNA template with the papBA promoter resulted in both Lrp and cAMP-CAP independent transcription. Thus binding of Lrp to the GATCprox region leads to inhibit pap transcription and therefore, Lrp is important to maintain the OFF phase (Braaten et al, 1992). The phase OFF to phase ON transition Steps in the transition of OFF to ON phase in pap transcription in E.coli (Hernday et al, 2004) The pap OFF to ON phase is found to occur at a 100 times lower rate when compared to the ON to the OFF phase. This is mostly common to most of the pili operons and regardless of the switch mechanism (Hernday et al, 2004). This phase generally occurs during DNA replication since the Lrp is removed from the binding site 1 – 3. Due to transient hemi-methylation of GATCdist, the Lrp binds to the binding site 4 – 6 along with papI which assists in the methylation of GATCdist which in turn leads to the transition of the OFF phase to the ON phase. Since Lrp is dissociated from its binding sites 1 – 3, Dam methylates GATCprox leading to the commencement of pap transcription. Thus, these are the 3 steps involved in the transition of the OFF phase to the ON phase of pap transcription (Hernday, Krabbe, Braaten and Low, 2002). Role of papI A small co-regulatory protein with a molecular weight of 8Kda, papI is found in most of the bacterial species with pili operons. The two important functions of papI are specific binding to Lrp and DNA sequence specific binding to Lrp sites 2 – 5. PapI is known to have low affinity to free Lrp and pap DNA alone but has high affinity for Lrp bound to binding sites 2 – 5. The binding of the papI to DNA is stabilized by Lrp by binding to adjacent or overlapping sites. PapI increases the affinity of Lrp to bind with binding sites 2 – 5 forming the papI-Lrp-DNA complexes (Hernday, Krabbe, Braaten and Low, 2002). Role of Dam PapI is found to increase the affinity of Lrp to Lrp binding site 1 – 3 as well as 4 – 6 but the actual transition of the Lrp molecule is not very prominent. The answer for this question lies in DNA methylation. In the absence of Dam the pap is transcriptionally inactive. Thus, in the presence of Dam pap moves from the OFF state to the ON phase because GATCprox is methylated by the Dam molecules moving the Lrp to the 4 – 6 binding site. Hence, this proves that methylation of GATCprox by Dam leads to the transcription of pap DNA (Hernday, Krabbe, Braaten and Low, 2002). Role of CAP Activation of both papBA and papI promoters requires CAP, which binds in a single specific site between both papBA and papI promoters leading to their transcription. The transcription of papBA promoter by CAP is quite direct whereas in the case of papI promoter transcription occurs by the expression of papB regulatory protein. The activation of papI generally occurs without CAP when an independent promoter expresses papB. It is also known that the switching phase of pap is affected by CAP since it affects the movement of Lrp from sites 1 – 3 to 4 – 6 is affected by the transcription of papI. Thus, it shows that CAP directly activates the papBA promoter and indirectly influences the transcription of papI by papB regulatory proteins (Hernday, Krabbe, Braaten and Low, 2002). Role of papB PapB binds as an 8 – 10 subunit multimer and has a molecular weight of 12 kDa and binds specifically to a minor groove of DNA. PapB as such is not important for pap transcription. The main role of papB is to produce regulatory proteins which in turn bring about the transcription of papI. This activation of papI promoter is done by binging to a specific site in between CAP binding site and the papI promoter. This site has a high affinity for papB and binding to the site leads to the production of papB regulatory protein which activates the papI promoter leading to the transcription of papI which ultimately results in pap pili expression. Thus papB acts as a link between papBA and papI promoters switching the OFF phase of the pap pili expression to the ON phase. Role of H-NS H-NS is not directly involved in pap phase variation but seems to control the process by environmental signals such as low temperature. H-NS is a 15.5 kDa global regulatory protein and is known to methylate both the GATCdist as well as the GATCprox at a temperature of 26oC. Thus at a lower temperature the H-NS protein disrupts methylation and the transcription is said to be stopped even before reaching the OFF state, which indicates that in the ON phase at a lower temperature the nucleotide-protein repression complex is formed. At room temperature, H-NS both activates and represses transcription of pap DNA. H-NS is known to promote the ON phase of the phase variation by specifically binding with pap regulatory proteins. Thus, H-NS promotes OFF to ON switching by reducing the Lrp affinity to sites 1 – 3 and by negatively regulating the transcription of papBA using RNA polymerases interaction with its promoters (Hernday, Krabbe, Braaten and Low, 2002). The self-perpetuating phase ON state For the transition of the ON phase from the OFF phase the addition of Lrp and cAMP-CAP are important which activates the papBA transcription. This activation of papBA in turn leads to the binding of papB which activates papI transcription. PapI helps the movement of Lrp from site 1 – 3 to site 4 – 6 which changes the OFF phase to ON phase. Thus, according to this model the transcription of papI and papBA is important for the phase variation of pap pili expression. The auto-regulation of papB is responsible for prevention of over transcription of papI and papB. The auto-regulation of papB is brought about by a low-affinity papB binding site overlapping the RNA polymerase binding site in the papBA promoter. Under certain circumstances, papI may also act as an auto-regulator under conditions of over expression. This condition mainly occurs after DNA replication since the GATCprox occurs in a semi-methylated state before it is re-methylated by Dam. Hence, in this semi-methylated state, the papI dependent Lrp may not be inhibited even at higher papI levels. It is also understood that higher papI levels has a higher OFF phase switch rate (Braaten et al, 1992). Switch inputs The environmental factors are known to play an important role in pap-pili expression. Such as, 1) Under decreased glucose levels the phase variation occurs from the OFF phase to the ON phase because cAMP levels are lower which in turn prevents the activation of papBA and papI transcription. 2) At a lower temperature and a richer media the pap pili transcription is found to be repressed. 3) H-NS may be responsible for the repression of pap transcription but the exact process is not well known. 4) cpxA and cpxR a double component membrane sensor and regulator system is known to increase the OFF stage to ON phase by binding to the Lrp binding site 1 – 3 and increasing the rate of transcription of pap DNA. It is also known that a number of other non-pap pili operons are found to have similar regulatory features like pap which leads to the conclusion that additional regulatory inputs may control the expression of pap DNA. Outputs PapB and papI which is known to be important for the ON phase of pap pili expression is found to control genes other than the pap operons in E.coli. There are multiple pili operons in E.coli which share the core mechanism of papI and papB as cross-contemplating homologues. For example, the pap-17 and pap-21 pili operons have different gene expressions but have single operons in operation. Hence, this proves that there is cross-talk between pili like operons but their mechanisms and interactions are not exactly well known and need to be better understood with further research. Another important example of regulatory cross-talk is the decreased expression of type 1 pili by papB. Two DNA recominbinases FimE and FimB are known to catalyse the phase variation in type 1 pili. FimB enhances both phase variations whereas FimE mediates only ON to OFF switching. PapB is known to enhance the OFF phase by rapidly converting the ON phase to OFF phase. Although the exact mechanism is not known it is understood that pap pili and type 1 pili are exclusive (Hernday, Krabbe, Braaten and Low, 2002). CONCLUSION The mechanism of phase variation in pili phase expression is a highly complicated process since it involves a number of global as well as a number of regulatory components. Not only that the environmental factors such as pH, temperature, media and glucose levels are also known to affect the phase variation switch rates. They are not only complex but also tightly regulated as many of the components involved in the switch mechanism are auto-regulators and can self restrict the OFF or ON phase according to the conditions required. Moreover, there are a number of operons for pili phase expression and most of them are known to share sequence homologues with papI and papB promoters. Some are even known to share their components making them two different operations but with similar components (Hernday, Krabbe, Braaten and Low, 2002). The environments in which the bacteria survive also play a major role in cell pili phase variation process. For example, if in a specific environment only a small group of E.coli is expressing phase variation then the entire population converts to the ON phase since papI is a highly adaptable and heritable component of the pili phase expression genes. And hence this helps to promote E.coli cell colonization. However, when the environmental condition does not require the bacteria to produce pili phase variation then the energy required for the process is used for other cellular activity. Cell differentiation is also known to be controlled by pap pili phase variation switch mechanism as it is highly susceptible to the environment surrounding the bacteria. When pap pili expression is in the ON phase it tends to influence to switch of type 1 pili expression but the exact mechanism of the process and its importance to the bacteria is not yet established or understood. But it is well understood that the pap pili and the type 1 pili are known to bind to different receptors since they are found in different environment of the urinary tract. Type 1 pili are known to colonize the lower urinary tract while the pap pili are known to colonize the upper urinary tract. This is mainly to avoid detection by the immune system hence the pili expression is highly regulated and controlled mechanism (Baga, Goransson, Normark, Uhlin, 1985). 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