Accomodating genomic Deoxyribonucleic acid in a comparatively little cell size necessitates its compression. Eukaryotes have a well defined packing machinery. In instance of procaryotes, nucleoid associated proteins ( NAPs ) aid in compression and planetary cistron ordinance. NAPs are dynamic in eubacterium and alter the form of nucleoid in conformity with growing phase. Structurally, NAPs compact DNA by bridging/bending mechanisms. NAPs of several eubacteriums other than Mycobacterium TB are good defined. A recent study for M.tuberculosis NAPs suggests their importance in important cellular and infective events. Few M.tuberculosis NAPs are really much different from other bacterial opposite numbers. It would be interesting to look at their constructions and DNA binding mechanisms which could besides reflect their possible regulative functions. These alone NAPs of M.tuberculosis and their possible functions in DNA compression and ordinance are discussed in this reappraisal.
Nucleoid organisation & A ; why it ‘s required:
Deoxyribonucleic acid size varies from being to being. Genome size in assorted beings may be every bit small as 0.5 Mb in Mycoplasma ( Su and Baseman, 1990 ) to every bit much as 3 X 103 Mb in worlds ( Venter et al. , 2001 ) . Deoxyribonucleic acid compression is therefore indispensable for adjustment in the limited infinite in the cell. In eucaryotes, DNA is arranged into good defined chromatin construction with the aid of histones and high mobility group proteins ( HMGs ) . Deoxyribonucleic acid agreement in bacteriums is non every bit specific as eucaryotes and is compacted into a slightly less organized equivalent nucleoid construction as shown in figure 1. The bacterial nucleoid is a extremely compact DNA which comprises of DNA associated with some histone-like proteins rich in basic residues and known as nucleoid associated proteins ( NAPs ) . Many different NAPs together interact with DNA and other DNA binding proteins and aid in compression of DNA.
Deoxyribonucleic acid Compaction in bacteriums:
Deoxyribonucleic acid compression in bacteriums may be achieved by three chief factors, viz. , DNA superhelicity, macromolecular crowding or by nucleoid associated proteins ( Luijsterburg et al. , 2006 ) . Deoxyribonucleic acid compression by DNA superhelicity is achieved by formation of plectonemic supercoils or by solenoid coiling ( Boles et al. , 1990 ) . In plectonemic coiling, DNA signifiers intertwined constructions while solenoid coiling is a sort of torroidal agreement of DNA on assorted protein surfaces. In procaryotes, plectonemic coiling is common because of round nature of DNA.
Macromolecular crowding is phenomena due to extra of supermolecules in the cytoplasmatic mileu. More than o.3 g RNA and protein/ml of E.coli surrounds the Deoxyribonucleic acid in a bacteria. Macromolecular crowding may assist in compression of DNA straight by prefering more compact DNA construction or indirectly by prefering the binding of proteins to DNA which consequence in its compression ( Zimmerman, 2006 ) .
Nucleoid associated proteins are proteins which are bound to DNA in specific or nonspecific mode. These proteins change the topology of DNA and convey approximately bending/ curving or other architectural alterations to pack DNA. These proteins are described in greater item below.
Figure 1: Cartoon representation of DNA compression in bacterial cell.
Nucleoid associated proteins ( NAPs ) in compression and ordinance:
Several nucleoid associated proteins e.g. fis, H-NS, IHF have been identified in assorted eubacteriums ( Azam and Ishihama, 1999 ; Basu et al. , 2009 ) . One of the of import functions of NAPs is compression of Deoxyribonucleic acid by organizing nucleoids. Apart from the architectural function associated with these DNA binding proteins, these proteins are besides known to play an of import portion in cistron ordinance, which may be brought approximately in a sequence-dependent ( e.g. IHF proteins in eubacterium ) or sequence-independent mode ( e.g. HU proteins of eubacterium ) . Many of the NAPs are reported to hold a function in planetary cistron ordinance e.g. fis ( factor for inversion stimulation ) acts as a written text activator during several cellular events ( Bradley et al. , 2007 ) . Another nucleoid associated protein, H-NS ( Histone-like nucleoid structuring ) , acts as transcriptional represser of appY ( Atlung et al. , 1996 ) bgl, proU, leuO operons ( Dorman, 2007 ) . At least, twelve NAPs have been reported in E. coli ( Azam and Ishihama, 1999 ) . These NAPs act together in modulating the construction of nucleoid at assorted phases of cell growing, which in bend regulates planetary cistron ordinance every bit good.
Dynamic nucleoid construction is associated with dynamic look form of NAPs:
Bacterial life rhythm is divided chiefly in 3 stages ; slowdown stage, log stage, stationary stage. During different stages of growing, differential cistron look of proteins required in each stage is required. This differential cistron look may be achieved by utilizing different sigma factors which are phase specific. In add-on, different parts in the bacterial nucleoid will be required to be accesible by RNA polymerases. This necessitates a alteration in nucleoid construction invariably during life rhythm of bacteriums to enable entree and binding of assorted written text factors.
Different NAPs have been found to be present at different phases of growing, although some NAPs may be present at all the phases at varying degrees. While certain NAPs, viz. histone-like HU protein and H-NS are expressed at all phases of growing in E. coli, some other NAPs e.g. fis, IHF are growing phase specific ( Azam and Ishihama, 1999 ) . The look degrees of all NAPs, nevertheless, alterations at different growing phases and are likely to play a function in cistron ordinance at that phase. In add-on, these NAPs bind to different parts on Deoxyribonucleic acid to consequence cistron look ; NAPs besides interact with other DNA associated proteins like RNA polymerases, DNA topoisomerases and modulate cistron look ( Berger et al. , 2009 ) . Many NAPs, for case the histone-like HU protein are specifically associated with the metabolically active DNA part in E. coli ( Durrenberger et al. , 1988 ) while few NAPs like H-NS are specifically associated with silenced part of DNA.
Structural facets of the reported Sleep:
Bacterial NAPs are divided into two major classs depending on their mechanism of DNA compression ; DNA bridging proteins and DNA bending proteins. Three-dimensional constructions of NAPs from both classs are available, lending to our apprehension of DNA binding and compression by these proteins.
Deoxyribonucleic acid bridging proteins:
Deoxyribonucleic acid bridging proteins consist of multiple DNA binding spheres. They form big multimeric assemblies and can bridge DNA by at the same time adhering two DNA semidetached houses. Bacterial NAPs, viz. , Lrp and H-NS belong to this class of proteins. Structures of Lrp from Pyrococcus furiosus, Mycobacterium TB and H-NS from Escherichia coli are available.
Crystal construction of Lrp consists of an oligomeric assembly dwelling of 8 fractional monetary units ( Fig. 2 ( B ) ) . The protein is besides known to organize larger hexadecamer assemblies during certain phases ( Luijsterburg et al. , 2006 ) .
Figure 2: Structures of bacterial NAPs. Structure of ( a ) H-NS spheres ( PDB ID 1LR1, 1HNR ) , ( B ) Lrp from Pyrococcus furiosus ( PDB:2GQQ ) , ( degree Celsius ) Lrp from M. TB ( PDB ID 2QZ8 ) , ( vitamin D ) IHF of E. coli ( PDB ID: 1IHF ) and ( vitamin E ) HU of Anabena ( PDB ID: 1P51 ) are shown. Structures of IHF and HU are available in complex with DNA, bespeaking the mechanism of DNA bending by these proteins. All figures were made in Chimera with co-ordinates downloaded from PDB.
H-NS ( Histone-like nucleoid structuring ) protein has a binding penchant for curved DNA ( Dame et al. , 2001 ) every bit good as Deoxyribonucleic acid with AT rich sequences. H-NS has been shown to modulate the laterally acquired cistrons including the major virulency cistrons of the Gram-negative bacteriums Salmonella, Shigella and Yersinia ( Dorman and Kane, 2009 ) . H-NS consists of two spheres which are separated by a flexible linker. Structures of both independent spheres have been solved. The C-terminal sphere binds to DNA while the N-terminal sphere helps in oligomerization ( Bloch et al. , 2003 ) . Dimerization of H-NS monomers requires first 65 aminic acids of N-terminal sphere while residues 65 to 91 are involved in oligomerization ( Fig 2a ) . N terminal sphere is made up of coiled spiral construction which has one big I±-helix H3 comprising residues 22 to 49 and two smaller spirals, spiral H1 comprising residues 2 to 7 and helix H2 10 to 16 residues ( Bloch et al. , 2003 ; Esposito et al. , 2002 ) . H-NS maps in dimeric signifier ( Falconi et al. , 1988 ; Smyth et al. , 2000 ) . The H-NS dimer ab initio binds to “ high affinity sites ” on DNA. This acts as a nucleation site for oligomerization of extra H-NS dimers at this site, later bridging two Deoxyribonucleic acid semidetached houses ( Lang et al. , 2007 ) . Deoxyribonucleic acid adhering motives in H-NS consists of one alpha spiral and two anti parallel beta sheets linked by short parts. The parts near to N-terminal of one beta sheet ( aminic acids A80 to K96 ) and between 2nd beta sheets and the 2nd alpha spiral are located near together and organize a positively charged surface which so interacts with the negatively charged Deoxyribonucleic acid in the major channel ( Shindo et al. , 1995 ; Shindo et al. , 1999 ) . H-NS binding constrains negative supercoils in DNA. H-NS overexpression in E. coli leads to strong compression of DNA and finally leads to cell decease ( Spurio et al. , 1992 ) .
Deoxyribonucleic acid flexing proteins:
Deoxyribonucleic acid flexing proteins may adhere to specific or non specific site on DNA and falsify the Deoxyribonucleic acid construction. Deoxyribonucleic acid flexing proteins tend to flex Deoxyribonucleic acid by bring oning a crick in Deoxyribonucleic acid construction upon adhering. IHF and HU belong to this class of NAPs.
IHF was ab initio identified as integrating host factor for lambda bacteriophage in E. coli. IHF binds in both sequence particular and nonspecific mode. IHF protects 25 bp but merely 9 bp on the right side of adhering site is conserved ( Goodrich et al. , 1990 ) , IHF identifies its binding site through contacts in the minor channel of DNA ( Rice et al. , 1996 ) and bends Deoxyribonucleic acid by more than 160A° ( Rice et al. , 1996 ) . IHF has two fractional monetary units ; an alpha fractional monetary unit and a beta fractional monetary unit ( Rice et al, 1996 ) ( Fig. 2d ) . Intertwined alpha spirals from the two fractional monetary units form the compact organic structure of IHF while a beta thread arm from each fractional monetary unit inserts into the minor channel of each DNA strand for binding ( Swinger and Rice, 2004 ) . These beta threads have a extremely conserved proline which intercalates between next base braces and induces DNA bending.
HU is a gay dimer that binds to DNA in a non-sequence specific mode through embolism of conserved proline residues to convey about DNA bending ( Swinger and Rice, 2004 ) HU non merely bends Deoxyribonucleic acid but at higher concentrations can besides organize a stiff superhelical fibril in which HU and DNA spiral around each other ( van Noort et al. , 2004 ) .
Nucleoid associated proteins in M. TB
Histone-like nucleoid-associated proteins of eubacteriums play of import functions non merely in compression of Deoxyribonucleic acid but besides in ordinance of assorted cellular procedures of DNA reproduction, recombination, written text etc. These proteins are involved in ordinance of cistrons that respond to cellular emphasis or environmental alterations in several eubacteriums ( Dorman and Deighan, 2003 ) . Nucleoid-associated proteins in mycobacterium, nevertheless, have been discovered merely late owing to hapless sequence similarities with other histone-like proteins. Of the 12 eubacterial NAPs, homologues of merely HU and LRP are present in M. TB, while a few others identified so far look to be specific to some mycobacteriums or to M. TB merely ( Table 1 ) . Little is understood about the alterations in cistron look brought about by these proteins during mycobacterial infection to do highly slow growing rates of mycobacteriums in response to environmental emphasis. Dissecting the interactions of these alone histone-like DNA-binding proteins of mycobacterium would assist understand their functions in cistron ordinance in greater inside informations.
Table I: Comparison of NAPs of Escherichia coli and Mycobacterium TB
In Mycobacterium TB, merely five NAPs are reported till day of the month, viz. , HU, lrp, lsr-2, H-NS and IHF. Recently, another protein, GroEL has been postulated to possess alone nucleoid-associated belongingss in M. TB ( Basu et al. , 2009 ) . HU and LRP of M. TB portion sequence similarity with other eubacterial opposite numbers. However, H-NS, Lsr-2 and IHF do non portion sequence similarity with any other eubacterial proteins ( except a few other mycobacterium ) and look to be alone NAPs. The NAPs identified in M. TB and their alone features are discussed in greater item below.
Lsr-2: Lsr-2 ( Rv3597c ) is a little 12 KDa protein, found in all mycobacterial genomes. It is an indispensable cistron for the endurance of M. TB as described by Sassetti et Al, 2003. Lsr-2 exhibits no homology with any other known NAPs in other eubacterium. Lsr-2 exhibits no conserved sphere or sequence repetitions as checked in pfam. AFM surveies have shown that Lsr-2 is a nucleoid associated protein and achieves its map of compression and planetary cistron ordinance by a Deoxyribonucleic acid bridging mechanism. ( Chen et al. , 2008 ) . Recent studies suggest its physical interaction in oligomeric signifier with DNA every bit good as protection of Deoxyribonucleic acid from ROS harm ( Colangeli et al. , 2009 ) . It is besides involved in the transcriptional ordinance of antibiotic opposition operon ( Colangeli et al. , 2007 ) and may be involved in regulative map during multiple cellular events. Lsr-2 complements E. coli H-NS mutation ( Gordon et al. , 2008 ) , corroborating its function as a NAP with a similar map in M. TB. Further penetrations into the DNA adhering mechanisms of this protein would assist understand the regulative effects of this alone NAP of M. TB in greater inside informations.
H-NS: H-NS in M. TB ( H-NS-Mtb ) ( Rv3852 ) is a 134 amino acid protein with an approx MW of 13.8 KDa. Although it shows no homology to E. coli H-NS, it has some similarity with N-terminal of eukaroyotic histones as it contains 7 to 8 PAKK, KAKK repeats. These basic residues and a net positive charge on H-NS-Mtb are likely to help its interaction with DNA. While H-NS of other eubacterium has a penchant for AT rich sequences every bit good as curved Deoxyribonucleic acid, the penchant of H-NS-Mtb for curved DNA has been demonstrated ( Werlang et al. , 2009 ) but a sequence penchant, if any remains to be determined. Mtb-H-NS regulates proU Operon ( Werlang et al. , 2009 ) . An E. coli H-NS mutation, nevertheless, could non be complemented with Mtb-H-NS, so it is speculated that the ORF annotated as putative “ H-NS ” of Mtb does non match to this household of proteins and may hold alone belongingss. Further probe in this facet is necessary to understand the molecular events involved in DNA binding, compression and ordinance by this putative NAP of M. TB.
m-IHF: . A fresh integrating host factor of M. smegmatis and M. TB ( Rv1388 ) that stimulates the integrating of a mycobacteriophage has been reported earlier ( Pedulla et al. , 1996 ) . The mycobacterial IHF ( m-IHF ) has several alone belongingss. It bears no sequence similarity to other eubacterial integrating host factors ( IHF ) and shows no penchant for integrating at the attP site. m-IHF of both M. TB and M. smegmatis are indispensable for endurance of the several mycobacterium ( Pedulla and Hatfull, 1998 ; Sassetti and Rubin, 2003 ) . The function of m-IHF in regulative functions during assorted physiological procedures remains a mostly undiscovered field. IHF of other eubacteriums have of import functions in cistron ordinance and one would theorize similar functions for m-IHF. Interestingly, the IHF proteins of Salmonella typhimurium ( Mangan et al. , 2006 ) and Vibrio cholerae ( Stonehouse et al. , 2008 ) modulate the protein look of stationary stage and virulency cistrons. A similar function for m-IHF has non yet been demonstrated. In add-on, in the deficiency of a sequence penchant for adhering in M. TB it is fascinating how a specific mycobacteriophage integrates into its genome. Clearly, sequence penchants and molecular inside informations involved in this event demand to be investigated in much inside informations to implicate this protein in its nucleoid associated and possible cistron regulative maps.
HU: HU or the histone-like protein ( Hlp ) of M. TB ( Rv2986c ) , is present in all mycobacterial species investigated boulder clay day of the month. Transposon-based cistron knock-out experiments have shown Hlp to be indispensable for the endurance of M. TB ( Sassetti and Rubin, 2003 ) . Although hlp smasher of M. smegmatis grows good in anaerobiotic civilizations at 37A°C, these cells could non turn at 10A°C ( Shires and Steyn, 2001 ) , proposing that mycobacterial Hlp proteins play an of import function in stress response. It is interesting to observe that the extremely reduced M. leprae genome besides encodes the hlp cistron ( Cole et al. , 2001 ) underscoring its importance. Using an antisense attack, ( Lewin et al. , 2008 ) have late shown that M. bovis cells harbouring antisense Deoxyribonucleic acid to hlp turn ill corroborating the importance of this protein in keeping proper growing of mycobacterial cells.
In comparing to other eubacterium, mycobacterial Hlps are alone proteins as these consist of an extra C-terminal sphere rich in lysine and proline residues with weak homology to the eucaryotic histone H1 proteins downstream of the N-terminal HU-like sphere. The N-terminal sphere portions homology with other bacterial HU proteins ( Figure 3a ) and besides contains a similar overall 3-dimensional crease ( Fig 3b ) ( Ramagopal et al. , 2009. , unpublished informations )
( a )
( B )
Fig. 3: HU of M.tuberculosis. ( A ) Domain organisation of M. TB Hlp-protein ( obtained from Pfam ) . The C-terminal sphere is characteristic of Mycobacterial proteins merely. ( B ) Crystal construction of N-terminal Domain of M.tuberculosis ( PDB ID: ) Structure is really similar to Anabena HU. ( besides see figure 2e )
Like other HU-like sequences, M. TB Hlp has DNA adhering belongingss ( Prabhakar et al. , 1998 ) . This Deoxyribonucleic acid adhering belongings, nevertheless, has been shown to be present in both N- and C-terminal parts in at least the M. smegmatis ortholog. Both spheres appear to adhere Deoxyribonucleic acid with different affinities ( Mukherjee et al. , 2008 ) . Although the C-terminus spheres of mycobacterial sequences are variable, it is non clear whether these differences are responsible for differential emphasis response in different mycobacteriums.
M. TB Hlp has late been shown to intercede transportation of mycolic acids during cell wall biogenesis ( Katsube et al. , 2007 ) . Cell wall of mycobacterium is alone and its correct assembly is indispensable for its continuity in the utmost host cell environment. Understanding the function of mycobacterial HU, and its alone C-terminal sphere hence assumes an even of import function as a possible drug mark.
Lrp: Lrp ( Rv3291c ) of M. TB ( Lrp-Mtb ) is a 150 amino acid protein and portions homology with E. coli Lrp. The construction of Lrp-Mtb is available ( Reddy et al. , 2008 ; Shrivastava et al. , 2009 ) at high declaration and is really similar to that of E. coli ( Fig. 2c ) Lrp-Mtb binds to DNA with high specificity and is expected to be a general regulator written text as its E. coli opposite number.
Decisions and Positions
Deoxyribonucleic acid organisation and compression serves for the adjustment of long DNA into cell compartment in add-on to playing an of import function in planetary ordinance in eubacterium. Several eubacterial NAPs have been studied in item and their structural information has revealed the nature of DNA compression and their differing mechanisms of DNA adhering. Apart from the architectural function, eubacterial NAPs play an of import function in planetary cistron ordinance. Temporal look form of bacterial NAPs reveals the differential look degrees of assorted NAPs at different phases of growing. In M. TB, merely five Sleeps have been reported compared to twelve of E. coli and other eubacterial opposite numbers. Of these, merely two, HU and LRP portion sequence similarity with other eubacteriums. It is therefore expected that there may be structural and mechanistic differences in DNA binding, compression and ordinance by M. TB NAPs. Further probe for dissecting the structural mechanism of DNA binding and interaction is necessary to understand these events at a molecular degree. It is fascinating to observe that Lsr 2, m-IHF, H-NS of M. TB have been implicated in cistron ordinance of several of import operons, including the multidrug opposition operon, cell wall biogenesis, etc. Furthermore, the G: C content of M. TB is higher compared to many other eubacteriums. While some eubacterial NAPs exhibit sequence penchants for adhering to DNA, sequence penchants of mycobacterial NAPs remains to be investigated. It is plausible that some of the alone NAPs in M. TB may hold evolved to adhere to certain parts of the higher Gram: C rich genome of M. TB. Word picture of M. TB NAPs and their structural and mechanistic differences with their eubacterial opposite numbers therefore form an of import measure to look into some of these challenging inquiries.