Phase variation

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In biology, phase variation is a method for dealing with rapidly varying environments without requiring random mutation. It involves the variation of protein expression, frequently in an on-off fashion, within different parts of a bacterial population. As such the phenotype can switch at frequencies that are much higher (sometimes >1%) than classical mutation rates. Phase variation contributes to virulence by generating heterogeneity. Although it has been most commonly studied in the context of immune evasion, it is observed in many other areas as well and is employed by various types of bacteria, including Salmonella species.

Contents

Salmonella use this technique to switch between different types of the protein flagellin. As a result, flagella with different structures are assembled. Once an adaptive response has been mounted against one type of flagellin, or if a previous encounter has left the adaptive immune system ready to deal with one type of flagellin, switching types renders previously high affinity antibodies, TCRs, and BCRs ineffective against the flagella.

Site-specific recombination

Site-specific recombinations are usually short and occur at a single target site within the recombining sequence. For this to occur there are typically one or more cofactors (to name a few: DNA-binding proteins and the presence or absence of DNA binding sites) and a site-specific recombinase. [1] There is a change in orientation of the DNA that will affect gene expression or the structure of the gene product. [2] This is done by changing the spatial arrangement of the promoter or the regulatory elements. [1]

Inversion

Phase variation site specific recombination - inversion Phase variation site specific recombination - inversion.jpg
Phase variation site specific recombination - inversion

Through the utilization of specific recombinases, a particular DNA sequence is inverted, resulting in an ON to OFF switch and vice versa of the gene located within or next to this switch. Many bacterial species can utilize inversion to change the expression of certain genes for the benefit of the bacterium during infection. [1] The inversion event can be simple by involving the toggle in expression of one gene, like E. coli pilin expression, or more complicated by involving multiple genes in the expression of multiple types of flagellin by S. typhimurium. [3] Fimbrial adhesion by the type I fimbriae in E. coli undergoes site specific inversion to regulate the expression of fimA, the major subunit of the pili, depending on the stage of infection. The invertible element has a promoter within it that depending on the orientation will turn on or off the transcription of fimA. The inversion is mediated by two recombinases, FimB and FimE, and regulatory proteins H-NS, Integration Host Factor (IHF) and Leucine responsive protein (LRP). The FimE recombinase has the capability to only invert the element and turn expression from on to off while FimB can mediate the inversion in both directions. [4]

Insertion-excision

If excision is precise and the original sequence of DNA is restored, reversible phase variation can be mediated by transposition. Phase variation mediated by transposition targets specific DNA sequences. [5] P. atlantica contains an eps locus that encodes extracellular polysaccharide and the ON or OFF expression of this locus is controlled by the presence or absence of IS492. Two recombinases encoded by MooV and Piv mediate the precise excision and insertion, respectively, of the insertion element IS492 in the eps locus. When IS492 is excised it becomes a circular extrachromosomal element that results in the restored expression of eps. [5] [6]

Another, more complex example of site specific DNA rearrangement is used in the flagella of Salmonella typhimurium. In the usual phase, a promoter sequence promotes the expression of the H2 flagella gene along with a repressor of H1 flagella gene. Once this promoter sequence is inverted by the hin gene the repressor is turned off as is H2 allowing H1 to be expressed.

Gene conversion

Gene conversion is another example of a type of phase variation. Type IV pili of Neisseria gonorrhoeae are controlled in this way. There are several copies of the gene coding for these pili (the Pil gene) but only one is expressed at any given time. This is referred to as the PilE gene. The silent versions of this gene, PilS, can use homologous recombination to combine with parts of the PilE gene and thus create a different phenotype. This allows for up to 10,000,000 different phenotypes of the pili[ citation needed ].

Epigenetic modification – methylation

Unlike other mechanisms of phase variation, epigenetic modifications do not alter DNA sequence and therefore it is the phenotype that is altered not the genotype. The integrity of the genome is intact and the change incurred by methylation alters the binding of transcription factors. The outcome is the regulation of transcription resulting in switches in gene expression. [2] [5] An outer membrane protein Antigen 43 (Ag43) in E. coli is controlled by phase variation mediated by two proteins, DNA-methylating enzyme deoxyadenosine methyltransferase (Dam) and the oxidative stress regulator OxyR. Ag43, located on the cell surface, is encoded by the Agn43 gene (previously designated as flu) and is important for biofilms and infection. The expression of Agn43 is dependent on the binding of the regulator protein OxyR. When OxyR is bound to the regulatory region of Agn43, which overlaps with the promoter, it inhibits transcription. The ON phase of transcription is dependent upon Dam methylating the GATC sequences in the beginning of the Agn43 gene (which happens to overlap with the OxyR binding site). When the Dam methylates the GATC sites it inhibits the OxyR from binding, allowing transcription of Ag43. [7]

Nested DNA inversion

In this form of phase variation. The promoter region of the genome can move from one copy of a gene to another through homologous recombination. This occurs with Campylobacter fetus surface proteins. The several different surface antigen proteins are all silent apart from one and all share a conserved region at the 5' end. The promoter sequence can then move between these conserved regions and allow expression of a different gene[ citation needed ].

Slipped strand mispairing

Slipped strand mispairing (SSM) is a process that produces mispairing of short repeat sequences between the mother and daughter strand during DNA synthesis. [1] This RecA-independent mechanism can transpire during either DNA replication or DNA repair and can be on the leading or lagging strand. SSM can result in an increase or decrease in the number of short repeat sequences. The short repeat sequences are 1 to 7 nucleotides and can be homogeneous or heterogeneous repetitive DNA sequences. [3]

Phase variation slipped strand mispairing PV SSM.JPG
Phase variation slipped strand mispairing

Altered gene expression is a result of SSM and depending where the increase or decrease of the short repeat sequences occurs in relation to the promoter will either regulate at the level of transcription or translation. [8] The outcome is an ON or OFF phase of a gene or genes.

Transcriptional regulation (bottom portion of figure) occurs in several ways. One possible way is if the repeats are located in the promoter region at the RNA polymerase binding site, -10 and -35 upstream of the gene(s). The opportunistic pathogen H. influenzae has two divergently oriented promoters and fimbriae genes hifA and hifB. The overlapping promoter regions have repeats of the dinucleotide TA in the -10 and -35 sequences. Through SSM the TA repeat region can undergo addition or subtraction of TA dinucleotides which results in the reversible ON phase or OFF phase of transcription of the hifA and hifB. [3] [9] The second way that SSM induces transcriptional regulation is by changing the short repeat sequences located outside the promoter. If there is a change in the short repeat sequence it can affect the binding of a regulatory protein, such as an activator or repressor. It can also lead to differences in post-transcriptional stability of mRNA. [5]

Translation of a protein can be regulated by SSM if the short repeat sequences are in the coding region of the gene (top portion of the figure). Changing the number of repeats in the open reading frame can affect the codon sequence by adding a premature stop codon or by changing the sequence of the protein. This often results in a truncated (in the case of a premature stop codon) and/or nonfunctional protein.

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<span class="mw-page-title-main">Lambda phage</span> Bacteriophage that infects Escherichia coli

Enterobacteria phage λ is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.

<span class="mw-page-title-main">Promoter (genetics)</span> Region of DNA encouraging transcription

In genetics, a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The RNA transcript may encode a protein (mRNA), or can have a function in and of itself, such as tRNA or rRNA. Promoters are located near the transcription start sites of genes, upstream on the DNA . Promoters can be about 100–1000 base pairs long, the sequence of which is highly dependent on the gene and product of transcription, type or class of RNA polymerase recruited to the site, and species of organism.

A regulatory sequence is a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of specific genes within an organism. Regulation of gene expression is an essential feature of all living organisms and viruses.

In molecular biology and genetics, transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity. A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus mount a response. Some examples of this include producing the mRNA that encode enzymes to adapt to a change in a food source, producing the gene products involved in cell cycle specific activities, and producing the gene products responsible for cellular differentiation in multicellular eukaryotes, as studied in evolutionary developmental biology.

<span class="mw-page-title-main">DnaA</span>

DnaA is a protein that activates initiation of DNA replication in bacteria. Based on the Replicon Model, a positively active initiator molecule contacts with a particular spot on a circular chromosome called the replicator to start DNA replication. It is a replication initiation factor which promotes the unwinding of DNA at oriC. The DnaA proteins found in all bacteria engage with the DnaA boxes to start chromosomal replication. In addition to the DnaA protein, its concentration, binding to DnaA-boxes, and binding of ATP or ADP, we will cover the regulation of the DnaA gene, the unique characteristics of the DnaA gene expression, promoter strength, and translation efficiency. The onset of the initiation phase of DNA replication is determined by the concentration of DnaA. DnaA accumulates during growth and then triggers the initiation of replication. Replication begins with active DnaA binding to 9-mer (9-bp) repeats upstream of oriC. Binding of DnaA leads to strand separation at the 13-mer repeats. This binding causes the DNA to loop in preparation for melting open by the helicase DnaB.

<i>lac</i> operon Set genes encoding proteins and enzymes for lactose metabolism

The lactose operon is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.

<span class="mw-page-title-main">Nucleoid</span> Region within a prokaryotic cell containing genetic material

The nucleoid is an irregularly shaped region within the prokaryotic cell that contains all or most of the genetic material. The chromosome of a prokaryote is circular, and its length is very large compared to the cell dimensions, so it needs to be compacted in order to fit. In contrast to the nucleus of a eukaryotic cell, it is not surrounded by a nuclear membrane. Instead, the nucleoid forms by condensation and functional arrangement with the help of chromosomal architectural proteins and RNA molecules as well as DNA supercoiling. The length of a genome widely varies and a cell may contain multiple copies of it.

<span class="mw-page-title-main">Repressor</span> Sort of RNA-binding protein in molecular genetics

In molecular genetics, a repressor is a DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA. An RNA-binding repressor binds to the mRNA and prevents translation of the mRNA into protein. This blocking or reducing of expression is called repression.

<span class="mw-page-title-main">Two-hybrid screening</span> Molecular biology technique

Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.

E2F is a group of genes that encodes a family of transcription factors (TF) in higher eukaryotes. Three of them are activators: E2F1, 2 and E2F3a. Six others act as suppressors: E2F3b, E2F4-8. All of them are involved in the cell cycle regulation and synthesis of DNA in mammalian cells. E2Fs as TFs bind to the TTTCCCGC consensus binding site in the target promoter sequence.

Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. NIH Blueprint for Neuroscience Research has created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community.

<span class="mw-page-title-main">FLP-FRT recombination</span>

In genetics, Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase (Flp) derived from the 2 µ plasmid of baker's yeast Saccharomyces cerevisiae.

The gene rpoS encodes the sigma factor sigma-38, a 37.8 kD protein in Escherichia coli. Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. rpoS is transcribed in late exponential phase, and RpoS is the primary regulator of stationary phase genes. RpoS is a central regulator of the general stress response and operates in both a retroactive and a proactive manner: it not only allows the cell to survive environmental challenges, but it also prepares the cell for subsequent stresses (cross-protection). The transcriptional regulator CsgD is central to biofilm formation, controlling the expression of the curli structural and export proteins, and the diguanylate cyclase, adrA, which indirectly activates cellulose production. The rpoS gene most likely originated in the gammaproteobacteria.

<span class="mw-page-title-main">Eukaryotic transcription</span> Transcription is heterocatalytic function of DNA

Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes comes in three variations, each translating a different type of gene. A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. The complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control.

fis

fis is an E. coli gene encoding the Fis protein. The regulation of this gene is more complex than most other genes in the E. coli genome, as Fis is an important protein which regulates expression of other genes. It is supposed that fis is regulated by H-NS, IHF and CRP. It also regulates its own expression (autoregulation). Fis is one of the most abundant DNA binding proteins in Escherichia coli under nutrient-rich growth conditions.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors have an origin of replication, a multicloning site, and a selectable marker.

RNA polymerase II holoenzyme is a form of eukaryotic RNA polymerase II that is recruited to the promoters of protein-coding genes in living cells. It consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins.

Post-transcriptional regulation is the control of gene expression at the RNA level. It occurs once the RNA polymerase has been attached to the gene's promoter and is synthesizing the nucleotide sequence. Therefore, as the name indicates, it occurs between the transcription phase and the translation phase of gene expression. These controls are critical for the regulation of many genes across human tissues. It also plays a big role in cell physiology, being implicated in pathologies such as cancer and neurodegenerative diseases.

Oxidation response is stimulated by a disturbance in the balance between the production of reactive oxygen species and antioxidant responses, known as oxidative stress. Active species of oxygen naturally occur in aerobic cells and have both intracellular and extracellular sources. These species, if not controlled, damage all components of the cell, including proteins, lipids and DNA. Hence cells need to maintain a strong defense against the damage. The following table gives an idea of the antioxidant defense system in bacterial system.

The fim switch in Escherichia coli is the mechanism by which the fim gene cluster, encoding Type I Pili, is transcriptionally controlled.

References

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  2. 1 2 Bayliss CD (2009). "Determinants of phase variation rate and the fitness implications of differing rates for bacterial pathogens and commensals". FEMS Microbiol Rev. 33 (3): 504–520. doi: 10.1111/j.1574-6976.2009.00162.x . PMID   19222587.
  3. 1 2 3 Wisniewski-Dyé F, Vial L (2008). "Phase and antigenic variation mediated by genome modifications". Antonie van Leeuwenhoek. 94 (4): 493–515. doi:10.1007/s10482-008-9267-6. PMID   18663597. S2CID   25378695.
  4. ↑ Gally DL, Bogan JA, Eisenstein BI, Blomfield IC (1993). "Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12: effects of temperature and media". J Bacteriol. 175 (19): 6186–93. doi:10.1128/jb.175.19.6186-6193.1993. PMC   206713 . PMID   8104927.
  5. 1 2 3 4 van der Woude MW, Bäumler AJ (2004). "Phase and antigenic variation in bacteria". Clin Microbiol Rev. 17 (3): 581–611. doi:10.1128/CMR.17.3.581-611.2004. PMC   452554 . PMID   15258095.
  6. ↑ Higgins BP, Carpenter CD, Karls AC (2007). "Chromosomal context directs high-frequency precise excision of IS492 in Pseudoalteromonas atlantica". Proc Natl Acad Sci U S A. 104 (6): 1901–1906. doi: 10.1073/pnas.0608633104 . PMC   1794265 . PMID   17264213.
  7. ↑ van der Woude MW, Henderson IR (2008). "Regulation and function of Ag43 (flu)". Annu Rev Microbiol. 62: 153–169. doi:10.1146/annurev.micro.62.081307.162938. PMID   18785838.
  8. ↑ Torres-Cruz J, van der Woude MW (2003). "Slipped strand mispairing can function as a phase variation mechanism in Escherichia coli". J Bacteriol. 185 (23): 6990–6994. doi:10.1128/JB.185.23.6990-6994.2003. PMC   262711 . PMID   14617664.
  9. ↑ van Ham SM, van Alphen L, Mooi FR, van Putten JP (1993). "Phase variation of H. influenzae fimbriae: transcriptional control of two divergent genes through a variable combined promoter region". Cell. 73 (6): 1187–96. doi:10.1016/0092-8674(93)90647-9. PMID   8513502. S2CID   40980720.
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