Structural, Functional and Molecular Basis of Cyanophage-Cyanobacterial Interactions and Its Significance

Review

Structural, functional and molecular basis of cyanophage-cyanobacterial interactions and its significance

P. Singh1, S. S. Singh2#, A.Srivastava1, A. Singh1 and A. K. Mishra1*

1Laboratory of Microbial Genetics, Department of Botany, Banaras Hindu University, Varanasi-221005, India. 2GGV,Blaspur,Chattisgarh,India

Accepted 5 May, 2011

The cyanophages are double-stranded deoxyribonucleic acid (ds-DNA) viruses, infecting cyanobacteria which are the first oxygenic photosynthesizers and significant nitrogen fixers of the biosphere. The evolutionary findings of the cyanophages do not truly reflect their actual time of origin. They show extreme diversification in morphology, habitat, host range and molecular attributes. They infect and establish an association with the cyanobacterial cells through one of the two modes of multiplication, that is, lytic or lysogenic type which might be dependent upon the environmental signals, such as nutritional status, the presence of any pathogenic condition and the relative concentration of either the control of repressor's operator/clear 1 (Cro/CI) protein that embody the bistable genetic switched regulators that are responsible to a large extent for the functioning of any of the above cycle. Various environmental factors that affect the stability and sustenance of the phages were meticulously reviewed. Genetic exchange and gene shuffling might be responsible for the enormous structural, functional, ecological and molecular diversity of the cyanophages. The cyanophages maintain the ecological equilibrium by keeping the nutrient cycling and microbial diversity at an appropriate level. Therefore, this review covers all the known, lesser acknowledged facets related to phage biology and clearly emphasizes the need for advanced molecular studies on the cyanophage-cyanobacterial association.

Key words: Cyanophage, cyanobacteria, cyanophages-cyanobacterial interaction.

INTRODUCTION

Viruses are the most common life forms in the aquatic the aquatic ecosystem. They are even known to mediate environment. They are known to be an abundant and gene transfer between microorganisms in aquatic dynamic component in the aquatic microbial communities ecosystems (Bergh et al., 1989; Suttle et al., 1990; that can regulate the biomass production and species Thingstad et al., 1993; Fuhrman, 1999). First time composition of varied microorganisms and other life isolation of a virus that infected filamentous cyano- forms like bacteria and phytoplankton. Due to their bacteria was reported by Safferman and Morris (1963). enormous abundance, ubiquity and significant impact on These viral agents have been previously named as both biogeochemical and ecological cycles through the phycoviruses (Schneider et al., 1964), algophages and infection and lysis of bacterial and phytoplankton blue-green algal viruses (Safferman and Morris, 1964; communities (Fuhrman, 1999; Wilhelm and Suttle, 1999; Luftig and Haselkorn, 1967). The term „Cyanophage‟ Hewson and Fuhrman, 2008; Sandaa, 2008; Thurber, seems to be the most appropriate because of the close 2009) they have been considered as a key component of resemblance with the term „bacteriophage‟ and there are many reports showing analogy of the cyanobacteria to the bacteria (Stanier and Van-Niel, 1962). The blue-green algae appeared in middle precambrian rock or even *Corresponding author. E-mail: [email protected] or earlier and are considered as one of the most ancient [email protected]. Tel: 91-542-3296111 or 91-542- organisms on the earth (Schopf, 1970). Before the advent 2307147. Fax: 91-542-2368174. of the cyanophages, the photoautotrophic metabolism 2592 Afr. J. Biotechnol.

was studied in the tissues of higher plants under viral (Luftig and Haselkorn, 1967; Cowie and Prager, 1969; infection but the results were far from satisfaction and Chen and Lu, 2002). There have been reports of the inconsistent because the plant system which is consisted homology among the highly conserved genes, that is, of different tissue systems, were difficult to isolate in deoxyribonucleic acid (DNA) polymerases of the host and tissue cultures and did not allow for homogeneous cyanophage (Braithwaite and Ito, 1993). The infection. cyanobacteria (first oxygenic photosynthesizers in the Due to the resemblance between cyanophages and planet) might have evolved before other bacteria and the the bacteriophages as well as the presence of common eukaryotes that‟s why cyanophages were considered as type of photosynthetic activity in blue-green algae and the possible original predator of cyanobacteria (Schopf higher plants provide an ideal and excellent model and Packer, 1987; Woese, 1987; Suttle, 2000). system to study the photosynthetic activity under viral Horizontal gene transfer especially the photosynthetic infection. Lots of works have been done related to the genes, was reported through infection of cyanophages on isolation and characterization of cyanophages system Prochlorococcus (host-strain specific) or closely related and also their potentiality to control cyanobacterial bloom marine Synechococcus (Sullivan et al., 2006). But still the in aquatic system (Padan and Shilo, 1973; Stewart and enormous diversity of the genetic material of the viruses Daft, 1977; Sherman and Brown, 1978; Martin and showed a polyphyletic origin whereas the three principal Benson, 1988; Suttle, 2000; Roth et al., 2008) but due to families of the tailed cyanophages; Podoviruses, the insignificant results the importance of the study was Myoviruses and the Siphoviruses that infect bacteria, diluted for two decades. Then, the work was mainly archaea and even non filamentous and filamentous focused on the significance of the cyanophages in the cyanobacteria are monophyletic in origin (Ackermann and photosynthesis of the cyanobacteria (Sullivan et al., DuBow, 1987b). Although the cyanophages seem to 2006) as well as in the cycling of the nutrients in the have arisen before the cyanobacteria but still the present aquatic ecosystem (Abedon et al., 2009). Their inter- day representatives of cyanophages; the Podoviridae, the action with the cyanobacteria has gained tremendous Myoviridae and the Siphoviridae seem to be of later importance due to cyanobacterial oxygenic photo- origin. But all these evolutionary findings are mere synthesis and nitrogen fixation. This cyanobacterial- speculations that need to be justified by extensive cyanophage association represents an ideal model morphological, biochemical and molecular charac- system for studying the mechanism of oxygenic terization of the cyanophages. photosynthesis under viral infection and also the corre- From the time of discovery of the cyanophages, the lation between the viral abundance and the surrounding nomenclature of the cyanophage was not sufficient environment. The cyanophages infected cyanobacteria enough to give an idea about the host, relatedness were also isolated from sea water (Proctor and Fuhrman, amongst the phages and the taxonomic position of the 1990; Suttle et al., 1990, 1992). phage. For example, N-1 and AN-10 were cyano- Apart from being the first oxygenic photosynthesizers, myoviruses while N-2 and AN-9 were cyanopodoviruses cyanobacteria are one of the most important and (Suttle, 2000). The taxonomy of cyanophages was significant nitrogen fixers and primary producers. Their proposed by Safferman et al. (1983) and also was relationship with the cyanophages must hence be extensively reviewed by a number of workers (Desjardins reviewed at each and every possible level so that our and Olson, 1983; Ackermann and DuBow, 1987a). The understanding of this cyanophage-cyanobacterial system members of tailed cyanophages having ds-DNA are is up to the mark. This review traverses through evolution classified on the basis of the contraction and size of the and diversity of the cyanophages, the interaction between tail; Myoviridae, Siphoviridae and Styloviridae and are cyanophages and cyanobacteria, the regulation of lytic known to infect bacteria, archaea, unicellular and and lysogenic cycle, cyanophage development and its filamentous cyanobacteria (Ackermann and DuBow, consequence on cyanobacterial metabolism, environ- 1987b). The cyanophages show extreme diversity in their mental factors affecting cyanophage development, structure, habitat and host range. They were diffe- molecular genetics of the cyanophage cyanobacterial rentiated into three major groups on the structural basis relationship and finally, the significance of the (Figure 1): cyanophages. 1 Cyanomyovirus were the first isolated, physically robust, remarkably versatile and easily adaptable viruses EVOLUTION AND DIVERSITY OF CYANOPHAGES belonging to the Myoviridae, are abundantly found in the marine waters and less frequent in the fresh waters Till date, the actual time of the origin of the cyanophages ((Safferman and Morris, 1963; Desjardins and Olson, is a matter of debate and the evolutionary studies are 1983; Suttle and Chan, 1993; Waterbury and Valois, generally based on the cynophageal morphology, its host 1993; Wilson et al., 1993; Tetart et al., 2001, Mann et al., specificity, genome size and its relatedness towards the 2005). Some cyanomyoviruses infect fresh watered highly diversified phage instead of the environment unicellular and filamentous cyanobacteria (Safferman et Singh et al. 2593

Grouping of Cyanophages

Structural Basis Host range Specificity Ecological Basis

Cyanomyoviruses LPP Group Fresh Water

A, AN and NP Group Cyanopodoviruses Marine Water

Cyanostyloviruses AS and SM Group

Figure 1. Grouping of cyanophages based on different parameters.

al., 1972; Gromov, 1983; BakerFigure et al., 1. 2006).Grouping Variations of Cyanophages 1 (Luftigbased on and different Haselkorn, parameters 1967) to the mid 60s in SM-1 have also been found at the genetic and molecular level. (Mackenzie and Haselkorn, 1972a). The G+C content (mol %) of cyanomyoviruses was varied 3 Cyanostyloviruses also known as Styloviridae, from 37 to 55% while the genome molecular weight denote the Synechococcus infecting S-1 as the type varied from 24 x 106 to 57 x 106 Da (Adolph and species. It has an isometric head (about 50 nm in Haselkorn, 1971; Safferman et al., 1972; Sherman and diameter) and a rigid tail (140 nm) which may reach up to Connelly, 1976). 200 to 300 nm (Gromov, 1983; Suttle and Chan, 1993; 2 Cyanopodoviruses, also known as Podoviridae, Sode et al., 1994). There have been reports of wide have been isolated from both fresh water and marine variations in the number of structural proteins which may waste stabilization ponds (Safferman et al., 1969a; Singh vary from 13 to 23 (Adolph and Haselkorn, 1973; Wilson and Singh, 1967; Mendzhul et al., 1974; Gromov, 1983; et al., 1993). In comparison to the cyanomyoviruses, the Hu et al., 1981). LPP-1 is a cyanopodovirus that infects mol % G+C contents reported for these was typically three different genera of non-heterocystous filamentous higher (ca. 60 to 74%) (Adolph and Haselkorn, 1973; cyanobacteria, Lyngbya, Phormidium, Plectonema Khudyakov, 1977; Benson and Martin, 1984). (Schneider et al., 1964) and shows similarities with the order Oscillatoriales on the basis of the base ratio (46% On the basis of host range, the cyanophages have G+C) and density (1.705g/cm3) (Edelman et al.,1967; been categorized into three families (Figure 1): Padan and Shilo, 1973). They have an isometric head (around 59 nm in diameter) with a tail (15-20 nm long), A. LPP Group cyanophages which attack three different about 10 structural proteins and the genome (27 x 106 genera of non-heterocystous filamentous cyanobacteria; Da) with the mol % G+C varying from the low 50s in LPP- Lyngbya, Phormidium, Plectonema (Safferman and 2594 Afr. J. Biotechnol.

Morris, 1963; Safferman et al., 1969a; Gromov, 1983) been reported to contain 11 T7 like genes and hence and also showed similarities with the order Oscillatoriales appears most closely allied to the T7-like podophages. on the basis of the G+C ratio (46%) and density Hence the molecular parameter itself has not been (1.705g/cm3) (Edelman et al.,1967; Padan and Shilo, characterized to catalog the cyanophages suitably on its 1973). basis. B. The second group comprising of the A, AN, N and NP groups are known to infect a varied range of cyanobacteria. The A group of cyanophages infect mostly CYANOPHAGE-CYANOBACTERIAL INTERACTION filamentous cyanobacteria like Anabaena sp. and they Cyanophage development belong to either Podoviridae or Myoviridae. The AN group of cyanophages infect both Nostoc spp and Anabaena Nucleic acid of a phage contains only a small number of spp. and may be podovirus (AN series except AN-10 and the genes needed for the synthesis of new viruses, its AN-15) or myovirus (AN-10 and AN-15). The N groups of structural components such as the capsid proteins and cyanophages infect Nostoc spp and may be podovirus the enzymes used in the phage life cycle. The (N-2) or myovirus (N-1). The existence of the NP group, synthesized enzymes are entirely concerned with infecting Nostoc and Plectonema (Muradov et al., 1990) replicating or processing nucleic acids and are functional is still imprecise and has not been isolated properly. only when the phage is within the host cell (Sandaa, C. The third group comprising of AS and SM groups 2009). For synthesizing phage proteins and enzymes, the are known to infect Anacystis, Synechococcus and enzymes, ribosomes, transfer ribonucleic acid (t-RNA) Microcystis. Basically myoviruses belong to the AS group and energy production are supplied by the host cell of the cyanophages that infect unicellular cyanobacteria (Hyman and Abedon, 2009). Presence of characteristic Anacystis nidulans (UTEX 625 = PCC 6301) and structures inside the host cell after viral infection and their Synechococcus cedrorum (UTEX 1191 = PCC 6908) electron tomographic analysis to molecular resolutions (Safferman et al., 1972). But, the reports of the AS-1 has proved helpful in understanding the viral infection group infecting PP 6311, PCC 7942, PCC 7943 (Rippka mechanisms and thus studying the development of the and Herdman, 1992) created a lot of problems. Clarity cyanophages or other phages inside their host cells seems to be the rational that all of the independent (Iwasaki and Omura, 2010). isolates are belonging to a single species (Wilmotte and The two principal modes of multiplication of the phages Stam, 1984; Waterbury and Rippka, 1989). The same are the lytic cycle and the lysogenic cycle (Lenski, 1988; taxonomic confusion was found with the SM-1 group of Hanlon, 2007; Peduzzi and Luef, 2009; Hyman and cyanophages which are podoviruses and were first Abedon, 2009). We would discuss the lytic cycle in reported to infect Synechococcus elongatus (UTEX 563 = conjunction with the lysogenic cycle in this portion of the PCC 6907) and Microcystis aeruginosa (NRC-1) text focusing on the control mechanism of this intricate (Safferman et al., 1969b). Now, it has been known that pathway of gene regulation and expression. The cyano- AS-1 also infects NRC-1 and UTEX-563 (Safferman et phages show stark resemblance to the T phages in terms al., 1972). Later on, it was found that SM-1 infected a of morphological attributes and also the life cycle pattern number of closely associated groups of PCC 6907 like (Paracer and Ahmadjian, 2000). This resemblance in the PCC 6307, PCC 6713, PCC 6904, etc. life cycle has shaped the blueprint for much of the reports and facts that would be presented in this text. Altogether, the morphological and the physiological The lytic cycle ends with the lysis and the death of the characters are not too much reliable for the classification host cell. The lytic cycle depends a lot upon the activity and are subjected to a variety of changes under and the effectiveness of the enzyme lysozyme that is environmental stress conditions. Hence there is a need produced by the cyanophage itself. In case of non for the study of the genetics and molecular biology in production of lysozyme, it has been seen that the host‟s conjunction with morphological, physiological and respiration and metabolism are affected and may also be biochemical characters of the cyanophages so that a full ceased, but it is quite sure that the process of lyses does proof and sound classification of the phages can be done not occur in any case (Herskowitz and Hagen, 1980) (Allen and Wilson, 2008). Considerable differences have unless and until induced. There have been a lot of been reported on the morphological and genetical scale studies regarding the triggering of lyses prematurely in amongst the known families of the cyanophages different conditions like anaerobiosis or energy (Myoviridae, Siphoviridae and Podoviridae) (Lu et al., uncouplers or the protein synthesis inhibitors (Bisen et 2001; Yoshida et al., 2006). Till date only five al., 1985; Wilson et al., 1993; Sode et al., 1997; cyanophages have been characterized on the molecular Williamson et al., 2002; Boaz et al., 2006). It has been scale: P60, P-SSP7, P-SSM2, PSSM4, and S-PM2 reported that UV, mitomycin C and heavy metals such as (Chen and Lu, 2002; Mann et al., 2005; Sullivan et al., copper, cadmium can induce the release of cyanophage 2005). The first sequencing reports were obtained from in marine water The five distinct stages of the lytic cycle the Synechococcus WH7803 infecting phage P60. It has are as follows: Singh et al. 2595

Attachment required for DNA ejection from the head is provided mainly by the DNA pressure built inside the capsid, in Attachment site of the phage attaches to a com- which it is spooled in a quasicrystalline packing (Cerritelli plementary receptor site on the cyanobacterial cell et al., 1997). However, this often considered a simple through a chemical interaction in which weak bonds are mechanism is far more complex, as there is not a unique formed between the sites. Normally the presence of way for DNA injection and it has been found to differ integrase enzymes denote the functioning of temperate amongst the different hosts and the phages also. While life style of the cyanophages. Reports have shown that the above process is considered to be active and energy immunoglobulin-like (Ig-like) domains occur quite dependent in case of Bacillus subtilis phage  29, it has frequently on the surface of tailed dsDNA bacterio- also been proposed that the host metabolic machinery phages. Many of these Ig-like domains are added to does not have any significant role or infact plays no role cyanophage structural proteins via well coordinated in the process of injection of the phage DNA into the host ribosomal frameshifts (Fraser et al., 2007). cell in case of E. coli (Maltouf and Labedan, 1985). Their evolutionary patterns suggest that they can be Experiments conducted by workers throughout the world exchanged by horizontal gene transfer, independently of have indicated that even the use of metabolic inhibitors the protein to which they are attached in the host and uncouplers have not been able to disrupt or delay the cyanobacteria. These Ig-like domains on the cyano- first step transfer of the DNA segment. Hence it has also phages interact with carbohydrates on the cell surface been proposed that the transfer of the DNA across the and thus facilitate phage adsorption. Also, these Ig-like cytoplasmic membrane or the surrounding membranes of domains appear to be one of a number of conserved the host cell happens by the process of diffusion across domains displayed on phage surfaces that serve to the protein channels of the host cell (Maltouf and increase infectivity by binding to or degrading Labedan, 1983). Dusenbery and Uretz (1972) reported polysaccharides. Without any doubt, it is a well about the inhibition of bacteriophage DNA injection by understood fact that the tail of cyanophages is an dyes bound to the DNA, like proflavine, acridine orange, important structure that helps in the attachment process. or ethidium, but not polyamines. It seems that the Studies on the bacteriophage T4 (Rossmann et al., 2004) presence of the dye is required inside the permeability have shown the presence of a contractile sheath that barrier of the phage at the time of infection. The effect of surrounds a rigid tube and then terminates in a proflavine is reduced in the presence of polyamines, multiprotein baseplate, to which a mesh of long and short suggesting that the active site is certainly on DNA. With tail fibers of the phage are attached. These fibers assume the different modes of penetration, the rate of injection of importance as they are crucial in binding to the cell this process also varies a lot. One of the fastest studied receptors. The binding of these fibers to their cell modes of injection has been observed in the phage T4 in receptors, leads to a conformational change in the base which 169 Kb genome is transferred in about 30 s plate which, then initiates the contraction of the sheath through the help of the protonmotive force (Boulanger and finally culminates in transfer of the phage DNA from and Letellier, 1988; Letellier and Boulanger, 1989; Huici the capsid into the host cell through the tail tube. The et al., 2004) across the ion channels in the cytoplasmic structure of the base plate is a dome shaped six fold membrane. symmetric structures that are stabilized by a garland of six short tail fibers, running around the periphery of the dome. The centre of the dome has a membrane- Biosynthesis puncturing device, containing three lysozyme domains, Initially the phage uses the host cell‟s nucleotides and which disrupts the intermembrane peptidoglycan layer several of its enzymes to synthesize many copies of during infection. phage DNA. Later, the biosynthesis of phage proteins

begins. Any RNA transcribed in the cell is mRNA

transcribed from phage DNA for the biosynthesis of Penetration phage enzymes and DNA for the biosynthesis of phage

Phage particle functions like a hypodermic syringe to enzymes and capsid proteins. The host cell‟s ribosomes, inject its DNA into the bacterial cell. Although much enzymes and amino acids are used for translation. For proved reports about the cyanophages is still not present several minutes after infection, complete phages cannot but still tailed bacteriophages are quite adroit and efficient be found in the host cell. Only separate components-DNA in their ability to infect their hosts, and due to this reason and protein-can be detected. The period during phage they are also the most studied ones. It is basically a kind multiplication when complete and infective phages are of signal transduction mechanism in which the signal is not yet present is called the Eclipse Period (Amla, 1981). transmitted from the receptor binding site of the tail to the connector or the baseplate, causing the opening to allow Maturation the DNA ejection (Huici et al., 2004). Since it is an energy dependent process, hence it is assumed that the energy Assembly of the different components happens in the 2596 Afr. J. Biotechnol.

maturation phase. All the important proteins that are sophisticated and compact continuous meshwork of the needed for the phage progenies to survive outside the peptidoglycan layer which nearly renders the outer host cell are manufactured and assembled into their surroundings out of bounds for the newly formed phage respective sites inside the phage genome (Moisa et al., particles. Even the huge amount of freshly formed 1981b). On a whole, the late messenger ribonucleic acid phages is unable to build up sufficient pressure for even (mRNA) synthesized in the biosynthesis stage directs the the osmotic break up of the peptidoglycan layer (Young synthesis of three kinds of proteins: phage structural et al., 2000). Hence, in order to escape out of the cell wall proteins, proteins that help with phage assembly without the phage needs specialized mechanisms for lysing the becoming part of the virion structure, and proteins cyanobacterial cell wall. Amongst the strategies known involved in cell lysis and phage release (Barrell and Air, and observed till now, two are particularly important 1976; Pollock et al., 1978; Atkins et al., 1979; Beremand (Young, 1992; Young et al., 2000). The phage may and Blumenthal, 1979; Bernhardt et al., 2002). At the proceed with release process either through the time of packaging and assembly of the phage endolysin mediated mechanism or through the holing components, the DNA is translocated into already mediated lysis (Wang et al., 2000; Mann, 2003). The use preformed protein shells. During the process of of the above two mechanisms has been observed in most packaging, irrespective of the type of cyanophage or of the phage particles with the use varying to different bacteriophage, there is a change in the conformation and extents (Mann, 2003). The need for the holin mediated the properties of DNA (Rao and Feiss, 2008). As a result pathway is due to the fact that endolysins lack a of massive compaction and compression, the highly secretory signal sequence. Hence the endolysins need negatively charged DNA is brought at the same density the assistance of holins to gain access to the murein of as that of crystalline DNA (Earnshaw and Casjens, 1980; the cyanobacterial cell wall (Mann, 2003). The holins Rao and Feiss, 2008). This process of packaging is not a have tremendous capacity to somehow pemeabilize the spontaneous process, but is powered by adenosine membrane and thus assist the endolysin in the phage triphosphate (ATP) hydrolysis accomplished by the help release process (Young et al., 2000). Hence they are of a translocating motor. This translocating motor is important in the regulation of the timing of the process of actually responsible for the compaction of the DNA lysis and release. The endolysin accumulates in the molecule into a compact shell. This preformed shell is cytosol but even this is not sufficient enough to attack the usually an icosahedron that is formed by many copies of murein layer (Young et al., 2000). The holin, at a the major capsid protein. The process of translocation is genetically programmed moment, forms a lethal achieved by the enzyme terminase (Szpirer and Brachet, membrane lesion that terminates respiration and thus 1970; Rao and Feiss, 2008). It is basically an allows the endolysin to get access to the murein layer. amalgamation of hetero oligomers of a small protein The term “lesion”, here denotes simply a kind of hole that involved in DNA recognition and the larger protein allows the endolysin to cross the cytoplasmic membrane involved in translocation ATPase, an endonuclease (Young et al., 2000) enzyme and a motif for docking at the portal vertex (Black, 1989; Rao and Feiss, 2008). The endonucleases are used in the cutting of the large concatemers of DNA THE CHOICE FOR LYTIC CYCLE OR LYSOGENIC into the smaller lengths. The ATPase associated with the CYCLE terminase assists in the powering of the translocation machine. The smaller subunit associated with the The lytic lysogeny pronouncement has long been regar- terminase becomes very useful during the DNA ded as a standard for the works in developmental genetic recognition and the differentiation of the phage DNA from networks. This intricate understanding is exemplified by the host DNA (Rao and Feiss, 2008). three key features and their operation in different conditions. Firstly, it depends on the environment signals and the extent of phage infection per cell which cycle has Release to be operated. Secondly, the stability of the lysogenic prophage is without any doubt, very high. Thirdly, the The mature phage particles are released after the lyses highly stable lysogenic prophage enters the lytic cycle of the cyanobacterial cell. The process of release is swiftly when it encounters conditions that are detrimental important as the cyanophages need to escape from the to them or to their genetic material. This means that the host cyanobacteria and let their new progenies be decision that whether lytic cycle or lysogenic cycle has to dispersed so that more of the host can be parasitized be operated depends upon the nutritional status or the (Knobler and Gelbart, 2009). This process though sounds presence of any pathogenic condition to which the simple, is associated with a lot of complexities for the bacterium is susceptible The CI and the Cro proteins new virions as the cell wall of the bacteria is nearly embody the bistable genetic switched regulators that are impregnable for the phage particles (Tzlil et al., 2003). responsible to a large extent for the functioning of any of The primary obstacle encountered is the highly the above cycle. While the CI protein is associated with Singh et al. 2597

Tetramerization Dimerization

Activating Region

DNA Binding

Figure 2. Structural components of the Lambda. N indicates the amino and C the carboxy domains.

Figure 2. Structural components of the lambda repressor. N indicates the amino domain and C the carboxy the maintenance ofdomain the stable lysogenic phase, the Cro is the crucial connector between the two monomers protein sets the lytic cycle into action by lowering the whose lysis is responsible for the induction of the lytic level of clear II (CII) protein that in turn fails to activate cycle (Carlson and Koudelka, 1994). The connection the expression of the CI protein. This regulation is between the residues 111 and 113 is broken down and governed by the combined and synchronized action of a this is the phase when the lytic cycle is induced. The large number of proteins and promoters that are factors responsible for induction may be a variety of responsible for the lytic or lysogenic decision. The adverse conditions like UV irradiation or any pathogenic decision is hence not dependent upon a single individual condition which ultimately leads to the proteolytic factor or protein but instead relies on a large number of inactivation of the repressor. On induction of the lytic factors and their coordination with each other. The cycle, the connection between the dimers is cleaved and various proteins and the promoter‟s alongwith their only the N-terminal domain remains in the monomeric structural and functional aspects were disccussed in the form. This type of formation upsets the equilibrium coming sections in depth and detail. The CI Protein obtained in the dimeric state. Due to this, the repressor encodes the Lambda repressor (Figure 2), a protein of dissociates from DNA, thus starting the lytic cycle. Also, two domains joined by a flexible linker region of about 40 the disturbed dimeric state upsets the cooperativity residues (Pabo et al., 1979). Here the N-terminal domain, between the adjacent dimmers and thus this also residues 1-92 are responsible for DNA binding whereas contributes to the induction of the lytic cycle. The binding the C-terminal domain, residues 132-236 participate in to the DNA done by the repressor dimer through the dimerization (Bell et al., 2000; Friedman and Court, central base pair is recognized. Half sites are also 2001). The process of dimerization is important as the present on each side of the central base pair and thus binding to the operator needs the dimeric form so that the each N-terminal region contacts a half site. The N- two DNA binding domains can contact the operator terminal domain of the lambda repressor consists of simultaneously (Bell et al., 2000). This is important as several stretches of α-helix. Out of the total five stretches although the information for DNA binding lies only in the present, two of them are responsible for binding DNA. N-terminal domain but the efficiency of the process is The type of structural arrangement is called a helix-turn- greatly enhanced by the attachment and dimerization of helix model. In each monomeric form, the α-helix-3 the C-terminal domain. The dimeric structure of the consists of nine amino acids which lie at fixed angle to repressor is very crucial for maintenance of lysogeny as it seven amino acids of the α-helix-2 (Branden and Tooze, 2598 Afr. J. Biotechnol.

1999; Kenny et al., 2006). This strategic placing of the α- promoters, one fact that becomes highly significant and helices places the two helix-3 regions at a distance of 34 imperative is that the decision for lytic cycle or lysogeny Å and thus makes them suitable to fit into the major depends on the relative concentration of either the Cro grooves of DNA. In the dimeric form, the helix-2 regions protein or the CI protein (Dodd et al., 2005; Kenny et al., lie at a point that places them across the grooves 2006). Hence the ratio of the above two proteins seems (Branden and Tooze, 1999; Kenny et al., 2006). This to play one of the most important roles in the regulation of symmetrical positioning of the dimer to the sites lytic and the lysogenic cycle. Alongwith the Cro protein ultimately causes the binding of each N-terminal domain and the CI protein, their affinities with the OR protein is of the dimer to a similar set of bases in its half site. Now important. The OR has three sites viz., OR1, OR2, and the question that may come to the mind of most of the OR3 (Court et al., 2007). The CI proteins order of affinity readers would be to define the role of both the helices towards the OR sites is OR1 > OR2> OR3. Similarly, the and their importance in this context. The helix-3 is Cro proteins order of affinity towards the OR sites is OR3> responsible for making contact between the exposed OR2 > OR1. If CI binds to OR1 then OR2 and then RNA hydrogen bonds of the amino acid side chains and the polymerase cannot bind to PR and genes on PR cannot exposed part of the base pairs. This contact makes the be synthesized. Hence lytic cycle does not happen recognition of the appropriate part of the DNA double (Figure 3). helix possible (Branden and Tooze, 1999; Kenny et al., 2006).Hence this helix is also known as the recognition Helix. The helix-2 contacts the phosphate backbone THE CONTROL OF LYTIC AND THE LYSOGENIC through the hydrogen bonds. This serves the purpose of CYCLE binding although it seems to have no role in specifying the recognition of the target sequences (Branden and The basic strategy of the phage is always to incorporate Tooze, 1999; Kenny et al., 2006). It represses PR and PL the least number of genes and proteins that are needed which are termed as the major rightward promoter and inside the cyanobacteria and thereafter to insert these at major leftward promoter. It also interacts with PRM and strategic places of need. The phage genome is itself of a this interaction depends on the concentration of the CI very small size and hence proper selection of the genes protein itself (Dodd et al., 2005; Kenny et al., 2006). If the and proteins is very much necessary. As a means of concentration of the CI protein is high then it represses survival, the phage does need three imperative phases: PRM. At low concentrations, PRM is activated by CI protein replication, transcription and translation. As far as the (Dodd et al., 2005; Kenny et al., 2006). The Cro Protein is replication of the DNA is concerned, the phage relies on an imperative protein which is known to prevent the its own genes that are needed for replication inside the synthesis of the repressor and secondly it also turns off host. These genes are accountable for the initiation of the expression of the immediate early genes. Thus, replication and may even include a new DNA through its dual functions, it finally leads to the repression polymerase. Coming to the transcriptional level, the of lysogeny and establishment of lytic cycle. The CII preferential transcription of the phage mRNAs is the protein is the activator of PRE and Pint. The N protein basic driving force and it is accomplished by either the functions as an antiterminator factor. The N protein, just changes at the host cell or substitution of the RNA like Cro, is a decisive lytic regulator but acts by a polymerase or even modification at the initiation or the completely different mechanism. It antiterminates termination level. During the process of protein synthesis, transcription at termination signals, allowing expression the phage relies a lot on the host protein synthesis of distal genes in the PL and PR operons (Oppenheim et apparatus except that the cyanobacterial mRNA is al., 2005). Its action at the nut sites allows transcription to replaced by the phage mRNA. The development and the proceed into the delayed early genes (Court et al., 2007). expression of the phage genes always happen in a The Q protein also functions as an antiterminator factor sequential manner in which there are basically two that is responsible for mediating the transcription of late phases of expression of the genes: the early phase and genes through the host RNA polymerase (Court et al., the late phase. The genes of the early phase are 2007). The CIII protein stabilizes/protects the CII protein. concerned with the production of enzymes involved in the The above proteins help in deciding the fate of the cell reproduction of DNA (Eisen et al., 1968; Young 1992). that whether it would go for lytic cycle or lysogenic cycle. These are needed for DNA synthesis, recombination and Alongwith these proteins, a number of promoters are also sometimes even modification. All these fast paced needed to aid and assist the proteins in the decision activities of these genes lead to an assembly and making process viz. PL: major leftward promoter; PR: accretion of genes and gene products. Now during the major rightward promoter; PRE: promoter for repressor late phase, the assemblage of the protein components of establishment; PRM: promoter for repressor maintenance the phage starts. In this phase, both the structural and Pint: promoter for integration; PR: secondary rightward the assembly proteins are needed by the phage particle. promoter. The assembly proteins are needed for the construction of After this detailed discussion about the proteins and the the phage particles although they themselves are not . Singh et al. 2599

N CI Cro CII

PL PRM PR

CI Cro

OL OR Lytic Cycle Lysogenic Cycle

PRM PR Transcription Transcription

CI Cro

OR3 OR2 OR1

Figure 3. The regulation of the lytic and the lysogenic pathway.

Figure 3. The regulation of the lytic and the lysogenic pathway incorporated in the genome of the phage. Now it must be chances of the lysogenic pathway after single infection kept in mind that the starting point for both lytic or the (Kourilsky and Knapp, 1974). Cells raised in nutrient rich lysogenic cycle is the same. The pathway for both the media have higher concentration of the host global cycles is sort of interlocked with each other. The lysis- regulator RNase III, which leads to elevated rates of lysogeny pronouncement is subjected by the number of expression of the protein N favoring lytic growth (Wilson phage particles infecting the cell as well as by the cell et al., 2002). In carbon-starved cells, on the other hand, physiology (Kourilsky, 1973; Kourilsky and Knapp, 1974; RNase III and consequently N concentrations are low. Oppenheim et al., 2005). High multiplicity of infection is Under these conditions N translation is repressed. This known to favor lysogeny. Also the nutritional status of the reduction of N concentration would reduce Q expression cells that has been discussed earlier is important. In to a level that provides more opportunity for lysogenic nutrient rich media, the lytic course predominates, response. When the phage DNA enters the host cell, whereas growing cells in poor media increases the then there are options for the operation of both lytic and 2600 Afr. J. Biotechnol.

Temperature Depth Solar Radiation

Environmental Factors affecting Cyanophage Cyanobacterial Interactions

Seasonal pH variations

Cation Concentrations Salinity

Figure 4. Environmental factors affecting cyanobacterial interactions.

Figure 4. Environmental factors affecting cyanophage cyanobacterial interactions the lysogenic cycle. Both the processes require the Temperature synthesis and also the expression of the immediate early and the delayed early genes. But after this point, there It is one of the most important factors that affect the occurs the point of deviation of both the processes: lytic stability of phages. Most of the reports regarding the process occurs if the late genes are expressed and effect of temperature on the cyanophages have been lysogeny happens if the repressor gets expressed. from isolates and only few from naturally occurring cyanophage communities. Temperature is an imperative inducer for AS-1 cyanophage conversion from lysogenic ENVIRONMENTAL FACTORS AFFECTING to lytic cycle and could have applied significance in the CYANOPHAGE-CYANOBACTERIAL INTERACTION modulation of cyanobacterial populations in freshwater aquatic environments (Chu et al., 2010). Although free- Being a component of the ecosystem, it is natural that zing affects the cyanophages yet in genera, cyanophages cyanophages do get affected immensely by the changes can be stored for a month or even more than 4°C in environmental conditions(Shilo,1972;Jessup and (Desjardins and Olson, 1983; Safferman and Morris, Forde, 2008). This section would deal one by one with 1964b; Safferman et al., 1969b). It was shown that at the possible factors that may affect the sustenance of the higher temperatures it depended upon the thermo- cyanophages (Figure 4). tolerance capacity of the cyanophages; whether they Singh et al. 2601

could survive or not. The thermally tolerant strains lipid phosphate phosphatase-1 (LPP-1) and in their survived at greater than 40°C (Safferman and Morris, stability in comparison to normal deionized water 1964 b; Safferman et al., 1969b) while the sensitive ones (Safferman and Morris, 1964b; Schneider et al., 1964) were unable to survive and no plaque formation was and SM-1 (Martin and Benson, 1988). At or above 1mM seen even at 35°C (Padan et al., 1971). Above 50°C, Mg2+, the phage remained infective while at 0.1mM con- there was a loss of infectivity at the temperatures centrations, the phage lost its infectivity (Goldstein et al., (Safferman and Morris, 1964b; Padan et al., 1971; Sarma 1967). and Singh, 1995). Also AS-1 was reported to be more stable in the presence of cations (Amla, 1981; Desjardins and Olson, 1983). An important conclusion that could be drawn from Solar radiation the above three reports is that even though the three phages belong to three different families all of them need The intense solar radiation on the infectivity of the cations for sustenance. Hence a generalized concept cyanophages has been studied and is one of the major about the need of a particular cation for a particular causes that accounts for the loss of the phages in the taxonomic group cannot be made. In addition to these natural environment (Suttle and Chen, 1992; Wommack reports about the need of cations, there are reports about et al., 1996; Nobel and Fuhrman, 1997; Wilhelm et al., the phage SM-1 that has no cation requirements and they 1998). Solar powered circulation (SPC) has been recently remain infectious when resuspended in distilled water reported to be a cure for suppression of freshwater (Safferman et al., 1969b). Other cations that are reported harmful algal blooms (Hudnell et al., 2010). The rate of to play important roles for phage infectivity are Mn2+ and removal of the natural phage communities were Na+ (Amla, 1981). An interesting observation made by measured to be 0.53 and 0.75 day-1 when they were Bancroft and Smith (1992) was that the cyanophage AS- integrated over the surface mixed layer and subsequently 1 could not tolerate NaCl concentrations above 0.05M. averaged over 24 h. It is well known that the rate of decay varied inversely with the depth. It seems that the decay rates also depend on the season. pH According to a general consensus, phage communities are more resistant to damaging effects of sunlight in the There seem to be no clear cut and conclusive reports summers as compared to the springs and the winters. about the effect of pH on the sustenance of infectivity of When the natural phage communities and isolates were cyanophages. It is well known that cyanophages can compared, decay rates of the isolates were about 2-fold tolerate wide variations in the pH and hence this may not higher than the rate of natural phage communities in be a very important factor in the distribution of phages. summers. The loss of the infectivity of the phages in Freshwater cyanophages are known to have greater pH sunlight is the result of the pyrimidine dimer formation. It tolerance than other bacteriophages and the later loose is well known that cyanobacteria have a very efficient their infectivity at pH greater than 8 or 9 (Adams, 1959; DNA repair mechanism and the same is also helpful Ackerman and DuBow, 1987a). LPP-1, AS-1 and SM-1 repairing the damaged phage DNA (Wu et al., 1967; showed pH range from pH 5 to 11 (Safferman and Morris, Singh, 1975; Asato, 1976; Amla, 1979; Hwang-Lee et al., 1964b; Safferman et al., 1969b, 1972). Hence in terms of 1985; Levine and Thiel, 1987). It is very clear that the pH tolerance, cyanophages show better adaptivity than damage due to the infectivity is basically based on the the bacteriophages and its broad pH tolerance also DNA damage and not in the adsorption or injection of the reflects the pH of their hosts, that is, the cyanobacteria phage DNA into the host. The environmental factors that can tolerate. can influence the phage infectivity and the survival in sunlight are solar angle and insulation, water trans- parency and depth of the mixed layer (Suttle, 2000). Salinity

Little information is gained regarding the effects of salinity Cation concentrations on cyanophages distribution. Most of the studies only are focused only on marine ecosystems rather than fresh The concentration of cations seems to influence the water systems. Lu et al. (2001) studied the effects of cyanophage infectivity a lot. A specific or universal salinity on the distribution, isolation, host specificity and response is not associated towards the effect of cations diversity of cyanophages and reported that salinity play a on the cyanophages. There are many cases where there significant impact on both the cyanophage or host is no effect of the cations while there may be cases population structure. The growth of different strains of where the cation concentrations may play an important Synechococcus was studied at different salinities. It was role. Presence of Mg2+ is reported to play an important observed that PE strains did not grow well when the role in maintaining the integrity of the phage capsid of salinity of the medium was less than 28% while the PC 2602 Afr. J. Biotechnol.

strains grew well at salinities ranging from 18 to 30%. concentration of viruses was reported to fall from about 5 Thus, on the basis of the above results it could be x 106 per ml to 104 per ml during winter seasons in the concluded that the PC strains had much better capacity Norwegian coastal waters. Viral abundances shifted to adapt at high salinity than the PE strains. Another significantly during the winter and the summer seasons in interesting inference that could be drawn from the above the Tampa Bay and the Northern Adriatic Sea. From the work was that the cyanophages that resided at high low levels in the winters (106 per ml) to the summer highs salinity water (coastal water) were had better and much of around 107 per ml or more, the shifts were certainly more diverse morphotypes than those which inhabited significant (Jiang and Paul, 1994; Weinbauer et al., 1995; low salinity water (upstream water). At the end, Lu et al. Cochran and Paul, 1998). Around 12 X 106 to 61 X 106 (2001) concluded that the abundance of cyanophages on per ml of viral particles are reported from the freshwater Synechococcus spp. was directly proportional to the ecosystems in the Danube River (Mathias et al., 1995). salinity in three Georgia coastal rivers. Normally, the viral abundance is at its peak in the autumn and the spring season but the peaks in the autumn season show greater viral abundances than the spring Depth season. In the coastal waters of the Rhode Island, temporal variations of cyanophages were reported by The depth of the water column has significant impact on Marston and Sallee (2003). Extinction dilution enrichment the planktonic life especially that of the cyanophages. and restriction fragment length polymorphism (RFLP) Various cyanophages having different hosts show were used to detect the relative abundance of cyano- different preferences for water depth. It is obvious that phages in June, July and August, 2002 and observed water temperature and salinity gradients vary gradually result showed that the abundance of cyanophages varied with the depth of the water column. This variation would according to season and specific cyanophages were certainly lead to a kind of stratification, ultimately leading dominant in different seasons. to different kinds of microenvironments within the same Apart from the above listed factors there are more water body. Studies conducted at the Chesapeake Bay factors which may certainly affect the cyanophages showed that during the calm summer weather, the bottom abundance at different locations and at different times. water body which was more saline, becames anoxic and But, the above were some of the most important factors thus affected the various life forms present there (Officer that cause changes in the abundance, distribution and et al., 1984; Tuttle et al., 1987). Work done at different diversity of cyanophages. sites proved that the abundance of the virioplankton significantly decreases below the euphotic zone in the open ocean water (200 m). According to estimates, the MOLECULAR GENETICS OF THE CYANOPHAGE abundances can be as low as around 106 viruses per ml CYANOBACTERIAL INTERACTION (Paul et al., 1991; Bird and Maranger, 1993; Boehme et al., 1993; Hara et al., 1996; Steward et al., 1996). Recent works and observations suggested that cyano- Frederickson et al. (2003) studied the effects of the phages directly control the photosynthetic behaviour of physical environment on cyanophage communities in the cyanobacteria. Prochlorococcus and Synechococcus British Columbia inlets. Denaturing gradient gel are important in light of their share in the process of electrophoresis (DGGE) of polymerase chain reaction global carbon fixation. It is estimated that they account for (PCR) amplified g20 gene fragments were analyzed to nearly 30% of the fixed carbon around the planet. Like examine the variations that were encountered in three the other photosynthesizers they also use the photo- inlets. According to their observations, some of the systems I and II for carrying out their photosynthesis. It cyanophages were very abundant and were found in the has been reported that cyanophages not only possess entire sample while some were found only at specific genes for photosynthesis but are also involved in the depths. Above variation certainly reflected that some exchange of genetic material with the cyanobacteria phages show preferences for certain depths in a water (Sullivan et al., 2006). The two core photosystem II column. This may be due to the salinity or the reaction centre proteins D1 and D2 are encoded by the temperature gradients at the depths. At greater depths, psbA and psbD genes. The psbA is one of the most the phages show a decline in their abundance and a sort rapidly turned over core protein in all oxygenic evolving of preferential occurrence. photosynthesizers. The psbD is involved in a kind of protein complexation with psbA. During stress, D1 is rapidly turned over. This also happens during phage Seasonal variations infection (Falkowski and Raven, 1997). The phages switch on its lytic cycle in the presence of any kind of Seasonal variations influence the abundance of the stress, that is, it needs to do phage gene transcription cyanophage and the existence of viruses in seawater. It rather than the supporting host gene transcription. The certainly affects the distribution of the phages. The D1 genes continue to produce the protein which helps in Singh et al. 2603

maintaining the process and level of photosynthesis case in Prochlorococcus and in one case in (Mann et al., 2003; Lindell et al., 2004, Millard et al., Synechococcus. 2004). Since D1 is encoded by psbA, hence the essential Astonishing were the findings that the sequences in the role of psbA during infection is beyond any doubt and Prochlorococcus phages matched with the homologues cannot be questioned (Lindell et al., 2004). found in the hosts (Lindell et al., 2004). This indicates Moreover, evidences have emerged from the ubiquity directly that the genes are functional. Now if it is assumed of psbA amongst the cyanophages, their similarity to that the genes are functional then they can maintain the cyanobacterial hosts and at last the conserved amino activity of the PSII reaction centre in two ways. In case of acid sequences. It has been reported that although the electron deficit they can provide the back up electrons psbA and psbD genes are wide apart on the away from PSII (pet genes). They can also support the cyanobacterial genome they are very close to each other photosynthesis in plants by stabilizing the PSII reaction on the cyanophage genome. In context to the algal- centre (PsbD and HliP (Lindell et al., 2004). cyanophage photosynthetic machinery, it has been Thus the genetics of the cyanophages has mostly been reported that 88% of the cyanophages of Sargasso Sea studied by taking the photosynthetic genes as the models and the Red Sea possess psbA while 50% had psbD which revealed a lot about the evolution of the cyano- (Sullivan et al., 2006). psbA gene was also found in all phages. Recent reports have indicated the role of a myoviruses and Prochlorococcus podoviruses. Although certain antisense RNA (asRNA) that is co-regulated with psbD was not present in all and it was less prolific but still psbA. Analysis of scaffolds from global ocean survey it was present in some cases; 20 Prochlorococcus datasets confirms that this asRNA seems to be myoviruses and 17 Synechococcus myoviruses. Another commonly associated with the 3′ end of cyanophage interpretation of the findings is that in cases where psbA psbA genes, indicating that this proposed mechanism of was present, psbD may or may not be present but in regulation of marine „viral‟ photosynthesis is evolutionarily cases where psbD was present psbA was undoubtedly conserved and thus gains phylogenetic eminence (Millard present. The myoviruses cyanophages have the maxi- et al., 2010). mum abundance and the broadest host range. Later on, This process of microbial evolution has occurred a heterodimer protein complex was formed by psbA-psbD through ages and certainly represents one of the best which had the broadest host range. Thus it seems that examples of horizontal gene transfer. Thus, the whole this complex formation is involved in charge separation work reveals a lot about the diversity of cyanophages and which tends to help the cyanophages for better infectivity their potential for evolution but still better results are by diversifying their host range. There is the existence of needed for proper studying of the phage diversity. There lot of conserved sequences in the cyanophages and is a need for better approach using proper molecular these sequences help the phages to acclimatize and biology tools and bioinformatics. The metagenomic infect in a much better way. The source and the time of approach can be an ideal way for proper and complete transfer of the PSII genes into the cyanophages was also understanding of the enormous, uncharacterized diversity investigated (Sullivan et al., 2006). Nucleotide sequences of the phages. This can be done by the sequence of psbA and psbD from Prochlorococcus and analysis of the shotgun libraries of the total viral DNA. Synechococcus were analyzed by sequencing and the Chen and Lu (2002) sequenced the genome of use of statistics which clearly indicated horizontal gene cyanophage P60 infecting marine Synechococcus transfer where psbA was transferred four times from the WH7803 which has 47872 bp‟s with around 80 ORF‟s. cyanobacteria genome to the cyanophage genome. The The podoviruses have a more conserved DNA replication transfer was two times for psbD from the cyanobacteria system than the siphoviruses and the myoviruses. In to the cyanophage. The exchange was generally host phylogenetic analyses, the coding regions for nucleotide specific but cases where these generalizations were not metabolism were very helpful having a lot of similarities followed were also seen. These exceptions indicate with the cyanophage P60 and marine unicellular towards a type of cross-exchange between two different cyanobacteria. The DNA structure of LPP-3 was peculiar phages during co-infection of the same host. This in having protruding 3‟-ends with the ends showing no intragenic recombination seems to favour the cyano- complementarity (Syrchin and Mendzhul, 2002) phages in their efficiency of cross-infectivity. Also, this Restriction analysis of separate fragments has indicated gene-shuffling helps the phage to efficiently co-ordinate the possible use of genome of LPP-3 as a new cloning with the photosynthetic machinery of the host cyano- vector in cyanobacterium. Reports of the similarities in bacteria especially during the stress conditions when the sequences from g18 to g23 between the marine lytic cycle needs to be operated. The genes responsible cyanomyovirus S-PM2 and coliphages T4 was given by for photosynthetic electron transport thylakoid associated Hambly et al. (2001). The order of the genes has been genes were also transferred from the cyanobacteria to reported to be similar except for the presence of some the cyanophage. The PET proteins petE and petF were insertions and deletions of small ORF‟s of unknown found in Prochlorococcus phages (Lindell et al., 2004) function. The studies reported that just like the T4 phage and high light inducible proteins hliP were found in one where g18 codes for the tail sheath, g19 for the tail tube, 2604 Afr. J. Biotechnol.

g20 for the head portal protein, g21 for the prohead core keeping the nutrient cycling and microbial diversity at an protein, g22 for a scaffolding protein, and g23 for the appropriate level. The lytic cycle performed by different major capsid protein, the cyanophages also do not phages at different stages of their life cycle is also an deviate on a very big scale. Thus, the differences and the important mechanism through which the balance in the similarities indicate a kind of genetic shuffling that may population structure is maintained. Dammeyer et al. have occurred or is still occurring progressively by the (2008) in their findings state that the cyanophages sharing of these types of conserved modules. mediate the biosynthesis of pigments in the oceanic cyanobacteria. Such phage mediated biosynthesis of pigments certainly needs to be tested further so that SIGNIFICANCE OF THE CYANOPHAGE- positive results may have some biotechnological CYANOBACTERIAL INTERACTION applications. This would also enhance the growing belief about the cyanophages a Living laboratory of conti- The significance of the cyanophages was initially related nuously increasing discoveries. Bailey et al. (2004) in to the prospects of their use in the control of algal blooms their report state that the cyanophages may help the but later on it was reported that the cyanophages cyanobacteria against photo-inhibition in high sunlight exhibited specificities for the cyanobacteria they predated thereby protecting the host against the harmful aftermath on and it became apparent that every cyanophage of intense light intensity. This would in turn assure the cannot infect and control every cyanobacteria. Moreover cyanobacteria of a proper energy supply for processes some recent findings indicated the enhanced dispersal of such as replication and thus help the cell maintain its toxic filamentous cyanobacteria on using lake side integrity. Thus it is apparent that at the present time the phages which further advocates towards the inefficiency field of phage biology is going through a period of of cyanophages in the control of algal blooms (Pollard renaissance in which new and exciting innovative ideas and Young, 2010). are coming from all parts of the globe. There is a need for Also some fungal strains have been reported to be very a proper understanding of these reports so that they can competent in the degradation of phytoplankton species be implemented for the welfare of all the life forms on the and thus have opened new avenues for the biological planet (Figure 5). control of algal blooms (Jia et al., 2010 a,b). That is why there was a loss of interest in the studies related to cyanophages. After a long gap, they again became a hot FUTURE PROSPECTS topic of research mainly due to their enormous abundance of Synechococcus and Prochlorococcus in Cyanophages represent one of the most abundant and the marine ecosystem. Due to most significant carbon also the most diverse life-forms whose diversity is still and nitrogen fixers, Synechococcus and Prochlorococcus untapped and uncharacterized. The genetic diversity they is an important matter of research because the exhibit is simply awesome and it certainly throws a cyanophages are associated with them and it is challenge to the microbiologists so that they can tap its speculated that there must be a kind of inter-relationship true potential. The significance of the cyanophages gains with their hosts. Research related to this association later importance in view of the horizontal gene transfer that on has led to some very appealing facts that gain has been reported in the case of the photosynthetic prominence in view of the role of the cyanophages in the genes of cyanophages. Apart from this, reports are process of genetic exchange and a kind of gene shuffling. coming on a regular basis about the various systems Reports about the homology of the photosynthetic genes where there has been a recombination or an exchange of and the assembly genes amid cyanophages and their genes between the phages and the cyanobacteria. Thus hosts exemplify the phylogenetic importance of the the importance of the cyanophages cannot be questioned cyanophages. The enormous diversity of the cyano- in the field of phylogenetic and evolutionary science. phages certainly indicates the exchange process that Apart from this, the various roles played by the phages in might occur with their hosts and this is a topic that needs bloom control, pigment biosynthesis, photo-inhibition a sound application of molecular biology and techniques protection and maintenance of the community structure along with a sound application of bioinformatics tools. needs to be further assessed with enhanced focus on the The study of photosynthesis under viral infection has use of molecular biology. More and more use of specific been understood significantly by experiments on the primers along with suitable techniques like RFLP, DGGE, cyanophage cyanobacterial system. The seasonal fingerprinting etc. are the need of the hour so that a abundance of the cyanophages indicates their possible better understanding at the physiological, biochemical, role in maintaining the ecological poise of the ecosystem genetic and molecular level is reached. An important role and in the maintenance of the biogeochemical cycling can be played by the field of the new and emerging and nutritional status of the surroundings. Hence grazing stream of molecular evolution. Better tools of of the cyanobacteria by the cyanophages does help to bioinformatics and computational biology are needed for maintain the population structure of the ecosystem a sound interpretation of the field data. Singh et al. 2605

Nutrient Cycling Biosynthesis of pigments

Study of photosynthesis Protection from under Viral Infection photo inhibition

Maintenance of Microbial Diversity Roles in Physiological Marine Significance Ecosystem

Significance of Cyanophages

Evolutionary Control of Significance Algal Blooms

Genetic Exchange

Gene Shuffling

Figure 5. Significance of cyanophages.Figure 5. Significance of cyanophages

Hence as a concluding note, people from physiology, ABBREVIATIONS genetics, microbiology, evolution, molecular biology, biochemistry and bioinformatics need to work together to ds-DNA, Double-stranded deoxyribonucleic acid; Cro/CI, explore and understand the various unknown facets of control of repressor's operator/clear 1; t-RNA, transfer these small but immensely important group of organisms. ribonucleic acid; Ig-like, Immunoglobulin-like; mRNA, In short, the whole of science needs to go hand in hand messenger ribonucleic acid; ATP, adenosine to understand this living laboratory of innovation. triphosphate; SPC, solar powered circulation; LPP-1, lipid phosphate phosphatase-1; DGGE, denaturing gradient gel electrophoresis; PCR, polymerase chain reaction; ACKNOWLEDGEMENTS RFLP, restriction fragment length polymorphism; asRNA, antisense ribonucleic acid. We are thankful to the Council of Scientific and Industrial Research, New Delhi, India for the financial support. The Head of the Department of Botany, BHU, Varanasi, India REFERENCES is gratefully acknowledged for providing the laboratory Abedon ST, Duffy S, Turner PE (2009). Bacteriophage Ecology. facilities. Encyclopedia Microbiol. pp. 42-57. 2606 Afr. J. Biotechnol.

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