Tuberculosis Nucleoid Associated Proteins Biology Essay

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Accomodating genomic DNA in a relatively small cell size necessitates its compaction. Eukaryotes have a well defined compacting machinery. In case of prokaryotes, nucleoid associated proteins (NAPs) help in compaction and global gene regulation. NAPs are dynamic in eubacteria and change the shape of nucleoid in accordance with growth stage. Structurally, NAPs compact DNA by bridging/bending mechanisms. NAPs of several eubacteria other than Mycobacterium tuberculosis are well defined. A recent report for M.tuberculosis NAPs suggests their importance in crucial cellular and pathogenic events. Few M.tuberculosis NAPs are very much different from other bacterial counterparts. It would be interesting to look at their structures and DNA binding mechanisms which could also reflect their potential regulatory roles. These unique NAPs of M.tuberculosis and their potential roles in DNA compaction and regulation are discussed in this review.

Nucleoid organization & why it's required:

DNA size varies from organism to organism. Genome size in various organisms may be as little as 0.5 Mb in Mycoplasma (Su and Baseman, 1990)to as much as 3 X 103 Mb in humans (Venter et al., 2001). DNA compaction is hence essential for accommodation in the limited space in the cell. In eukaryotes, DNA is arranged into well defined chromatin structure with the help of histones and high mobility group proteins (HMGs). DNA arrangement in bacteria is not as specific as eukaryotes and is compacted into a somewhat less organized equivalent nucleoid structure as shown in figure 1. The bacterial nucleoid is a highly 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 help in compaction of DNA.

DNA Compaction in bacteria:

DNA compaction in bacteria may be achieved by three main factors, namely, DNA superhelicity, macromolecular crowding or by nucleoid associated proteins (Luijsterburg et al., 2006). DNA compaction by DNA superhelicity is achieved by formation of plectonemic supercoils or by solenoid coiling (Boles et al., 1990). In plectonemic coiling, DNA forms intertwined structures while solenoid coiling is a kind of torroidal arrangement of DNA on various protein surfaces. In prokaryotes, plectonemic coiling is common because of circular nature of DNA.

Macromolecular crowding is phenomena due to excess of macromolecules in the cytoplasmic mileu. More than o.3 g RNA and protein/ml of E.coli surrounds the DNA in a bacterium. Macromolecular crowding may help in compaction of DNA directly by favoring more compact DNA structure or indirectly by favoring the binding of proteins to DNA which result in its compaction (Zimmerman, 2006).

Nucleoid associated proteins are proteins which are bound to DNA in specific or nonspecific manner. These proteins change the topology of DNA and bring about bending/ curving or other architectural changes to compact DNA. These proteins are described in greater detail below.

Figure 1: Cartoon representation of DNA compaction in bacterial cell.

Nucleoid associated proteins (NAPs) in compaction and regulation:

Several nucleoid associated proteins e.g. fis, H-NS, IHF have been identified in various eubacteria (Azam and Ishihama, 1999; Basu et al., 2009). One of the important roles of NAPs is compaction of DNA by forming nucleoids. Apart from the architectural role associated with these DNA binding proteins, these proteins are also known to play an important part in gene regulation, which may be brought about in a sequence-dependent (e.g. IHF proteins in eubacteria) or sequence-independent manner (e.g. HU proteins of eubacteria). Many of the NAPs are reported to have a role in global gene regulation e.g. fis (factor for inversion stimulation) acts as a transcription activator during several cellular events (Bradley et al., 2007). Another nucleoid associated protein, H-NS (Histone-like nucleoid structuring), acts as transcriptional repressor 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 regulating the structure of nucleoid at various stages of cell growth, which in turn regulates global gene regulation as well.

Dynamic nucleoid structure is associated with dynamic expression pattern of NAPs :

Bacterial life cycle is divided mainly in 3 phases; lag phase, log phase, stationary phase. During different phases of growth, differential gene expression of proteins required in each phase is required. This differential gene expression may be achieved by using different sigma factors which are stage specific. In addition, different regions in the bacterial nucleoid will be required to be accesible by RNA polymerases. This necessitates a change in nucleoid structure constantly during life cycle of bacteria to enable access and binding of various transcription factors.

Different NAPs have been found to be present at different stages of growth, although some NAPs may be present at all the stages at varying levels. While certain NAPs, namely histone-like HU protein and H-NS are expressed at all stages of growth in E. coli, some other NAPs e.g. fis, IHF are growth stage specific (Azam and Ishihama, 1999). The expression levels of all NAPs, however, changes at different growth stages and are likely to play a role in gene regulation at that stage. In addition, these NAPs bind to different regions on DNA to effect gene expression; NAPs also interact with other DNA associated proteins like RNA polymerases, DNA topoisomerases and regulate gene expression (Berger et al., 2009). Many NAPs, for instance the histone-like HU protein are specifically associated with the metabolically active DNA region in E. coli (Durrenberger et al., 1988)while few NAPs like H-NS are specifically associated with silenced region of DNA.

Structural aspects of the reported NAPs:

Bacterial NAPs are divided into two major categories depending on their mechanism of DNA compaction; DNA bridging proteins and DNA bending proteins. Three-dimensional structures of NAPs from both categories are available, contributing to our understanding of DNA binding and compaction by these proteins.

DNA bridging proteins:

DNA bridging proteins consist of multiple DNA binding domains. They form large multimeric assemblies and can bridge DNA by simultaneously binding two DNA duplexes. Bacterial NAPs, namely, Lrp and H-NS belong to this category of proteins. Structures of Lrp from Pyrococcus furiosus, Mycobacterium tuberculosis and H-NS from Escherichia coli are available.

Crystal structure of Lrp consists of an oligomeric assembly consisting of 8 subunits (Fig. 2 (b)). The protein is also known to form larger hexadecamer assemblies during certain stages (Luijsterburg et al., 2006).

Figure 2: Structures of bacterial NAPs. Structure of (a) H-NS domains (PDB ID 1LR1, 1HNR), (b) Lrp from Pyrococcus furiosus (PDB:2GQQ), (c) Lrp from M. tuberculosis (PDB ID 2QZ8), (d) IHF of E. coli (PDB ID: 1IHF) and (e) HU of Anabena (PDB ID: 1P51) are shown. Structures of IHF and HU are available in complex with DNA, indicating the mechanism of DNA bending by these proteins. All figures were made in Chimera with coordinates downloaded from PDB.

H-NS (Histone-like nucleoid structuring) protein has a binding preference for curved DNA (Dame et al., 2001)as well as DNA with AT rich sequences. H-NS has been shown to regulate the laterally acquired genes including the major virulence genes of the Gram-negative bacteria Salmonella, Shigella and Yersinia (Dorman and Kane, 2009). H-NS consists of two domains which are separated by a flexible linker. Structures of both independent domains have been solved. The C-terminal domain binds to DNA while the N-terminal domain helps in oligomerization (Bloch et al., 2003). Dimerization of H-NS monomers requires first 65 amino acids of N-terminal domain while residues 65 to 91 are involved in oligomerization (Fig 2a). N terminal domain is made up of coiled coil structure which has one large α-helix H3 comprising residues 22 to 49 and two smaller helices, helix H1 comprising residues 2 to 7 and helix H2 10 to 16 residues (Bloch et al., 2003; Esposito et al., 2002). H-NS functions in dimeric form (Falconi et al., 1988; Smyth et al., 2000). The H-NS dimer initially binds to "high affinity sites" on DNA. This acts as a nucleation site for oligomerization of additional H-NS dimers at this site, subsequently bridging two DNA duplexes (Lang et al., 2007). DNA binding motif in H-NS consists of one alpha helix and two anti parallel beta sheets linked by short regions. The regions close to N-terminal of one beta sheet (amino acids A80 to K96) and between second beta sheets and the second alpha helix are located close together and form a positively charged surface which then interacts with the negatively charged DNA in the major groove(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 compaction of DNA and ultimately leads to cell death (Spurio et al., 1992).

DNA bending proteins:

DNA bending proteins may bind to specific or non specific site on DNA and distort the DNA structure. DNA bending proteins tend to bend DNA by inducing a kink in DNA structure upon binding. IHF and HU belong to this category of NAPs.

IHF was initially identified as integration host factor for lambda bacteriophage in E. coli. IHF binds in both sequence specific and nonspecific manner. IHF protects 25 bp but only 9 bp on the right side of binding site is conserved (Goodrich et al., 1990), IHF identifies its binding site through contacts in the minor groove of DNA (Rice et al., 1996)and bends DNA by more than 160° (Rice et al., 1996). IHF has two subunits; an alpha subunit and a beta subunit (Rice et al, 1996) (Fig. 2d). Intertwined alpha helices from the two subunits form the compact body of IHF while a beta ribbon arm from each subunit inserts into the minor groove of each DNA strand for binding (Swinger and Rice, 2004). These beta ribbons have a highly conserved proline which intercalates between adjacent base pairs and induces DNA bending.

HU is a homo dimer that binds to DNA in a non-sequence specific manner through intercalation of conserved proline residues to bring about DNA bending (Swinger and Rice, 2004)HU not only bends DNA but at higher concentrations can also form a rigid superhelical filament in which HU and DNA spiral around each other (van Noort et al., 2004).

Nucleoid associated proteins in M. tuberculosis

Histone-like nucleoid-associated proteins of eubacteria play important roles not only in compaction of DNA but also in regulation of various cellular processes of DNA replication, recombination, transcription etc. These proteins are involved in regulation of genes that respond to cellular stress or environmental changes in several eubacteria (Dorman and Deighan, 2003). Nucleoid-associated proteins in mycobacteria, however, have been discovered only recently owing to poor sequence similarities with other histone-like proteins. Of the 12 eubacterial NAPs, homologues of only HU and LRP are present in M. tuberculosis, while a few others identified so far appear to be specific to some mycobacteria or to M. tuberculosis only (Table 1). Little is understood about the changes in gene expression brought about by these proteins during mycobacterial infection to cause extremely slow growth rates of mycobacteria in response to environmental stress. Dissecting the interactions of these unique histone-like DNA-binding proteins of mycobacteria would help understand their roles in gene regulation in greater details.

Table I: Comparison of NAPs of Escherichia coli and Mycobacterium tuberculosis

In Mycobacterium tuberculosis, only five NAPs are reported till date, namely, HU, lrp, lsr-2, H-NS and IHF. Recently, another protein, GroEL has been postulated to possess unique nucleoid-associated properties in M. tuberculosis (Basu et al., 2009). HU and LRP of M. tuberculosis share sequence similarity with other eubacterial counterparts. However, H-NS, Lsr-2 and IHF do not share sequence similarity with any other eubacterial proteins (except a few other mycobacteria) and appear to be unique NAPs. The NAPs identified in M. tuberculosis and their unique characteristics are discussed in greater detail below.

Lsr-2: Lsr-2 (Rv3597c) is a small 12 KDa protein, found in all mycobacterial genomes. It is an essential gene for the survival of M. tuberculosis as described by Sassetti et al, 2003. Lsr-2 exhibits no homology with any other known NAPs in other eubacteria. Lsr-2 exhibits no conserved domain or sequence repeats as checked in pfam. AFM studies have shown that Lsr-2 is a nucleoid associated protein and achieves its function of compaction and global gene regulation by a DNA bridging mechanism. (Chen et al., 2008). Recent reports suggest its physical interaction in oligomeric form with DNA as well as protection of DNA from ROS damage (Colangeli et al., 2009). It is also involved in the transcriptional regulation of antibiotic resistance operon (Colangeli et al., 2007)and may be involved in regulatory function during multiple cellular events. Lsr-2 complements E. coli H-NS mutant (Gordon et al., 2008), confirming its role as a NAP with a similar function in M. tuberculosis. Further insights into the DNA binding mechanisms of this protein would help understand the regulatory effects of this unique NAP of M. tuberculosis in greater details.

H-NS : H-NS in M. tuberculosis (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 aid its interaction with DNA. While H-NS of other eubacteria has a preference for AT rich sequences as well as curved DNA, the preference of H-NS-Mtb for curved DNA has been demonstrated (Werlang et al., 2009)but a sequence preference, if any remains to be determined. Mtb-H-NS regulates proU Operon (Werlang et al., 2009). An E. coli H-NS mutant, however, could not be complemented with Mtb-H-NS, so it is speculated that the ORF annotated as putative "H-NS" of Mtb does not correspond to this family of proteins and may have unique properties. Further investigation in this aspect is necessary to understand the molecular events involved in DNA binding, compaction and regulation by this putative NAP of M. tuberculosis.

m-IHF:. A novel integration host factor of M. smegmatis and M. tuberculosis (Rv1388) that stimulates the integration of a mycobacteriophage has been reported earlier (Pedulla et al., 1996). The mycobacterial IHF (m-IHF) has several unique properties. It bears no sequence similarity to other eubacterial integration host factors (IHF) and shows no preference for integration at the attP site. m-IHF of both M. tuberculosis and M. smegmatis are essential for survival of the respective mycobacteria (Pedulla and Hatfull, 1998; Sassetti and Rubin, 2003). The role of m-IHF in regulatory roles during various physiological processes remains a largely unexplored field. IHF of other eubacteria have important roles in gene regulation and one would speculate similar roles for m-IHF. Interestingly, the IHF proteins of Salmonella typhimurium (Mangan et al., 2006)and Vibrio cholerae (Stonehouse et al., 2008)regulate the protein expression of stationary phase and virulence genes. A similar role for m-IHF has not yet been demonstrated. In addition, in the lack of a sequence preference for binding in M. tuberculosis it is intriguing how a specific mycobacteriophage integrates into its genome. Clearly, sequence preferences and molecular details involved in this event need to be investigated in much details to implicate this protein in its nucleoid associated and potential gene regulatory functions.

HU: HU or the histone-like protein (Hlp) of M. tuberculosis (Rv2986c), is present in all mycobacterial species investigated till date. Transposon-based gene knock-out experiments have shown Hlp to be essential for the survival of M. tuberculosis (Sassetti and Rubin, 2003). Although hlp knockout of M. smegmatis grows well in anaerobic cultures at 37°C, these cells could not grow at 10°C (Shires and Steyn, 2001), suggesting that mycobacterial Hlp proteins play an important role in stress response. It is interesting to note that the highly reduced M. leprae genome also encodes the hlp gene (Cole et al., 2001)underlining its importance. Using an antisense approach, (Lewin et al., 2008)have recently shown that M. bovis cells harboring antisense DNA to hlp grow poorly confirming the importance of this protein in maintaining proper growth of mycobacterial cells.

In comparison to other eubacteria, mycobacterial Hlps are unique proteins as these consist of an additional C-terminal domain rich in lysine and proline residues with weak homology to the eukaryotic histone H1 proteins downstream of the N-terminal HU-like domain. The N-terminal domain shares homology with other bacterial HU proteins (Figure 3a) and also contains a similar overall three-dimensional fold (Fig 3b)(Ramagopal et al., 2009.,unpublished data)



Fig. 3:HU of M.tuberculosis. (A) Domain organization of M. tuberculosis Hlp-protein (obtained from Pfam). The C-terminal domain is characteristic of Mycobacterial proteins only. (B) Crystal structure of N-terminal Domain of M.tuberculosis (PDB ID : ) Structure is very similar to Anabena HU. (also see figure 2e)

Like other HU-like sequences, M. tuberculosis Hlp has DNA binding properties (Prabhakar et al., 1998). This DNA binding property, however, has been shown to be present in both N- and C-terminal regions in at least the M. smegmatis ortholog. Both domains appear to bind DNA with different affinities (Mukherjee et al., 2008). Although the C-terminus domains of mycobacterial sequences are variable, it is not clear whether these differences are responsible for differential stress response in different mycobacteria.

M. tuberculosis Hlp has recently been shown to mediate transfer of mycolic acids during cell wall biosynthesis (Katsube et al., 2007). Cell wall of mycobacteria is unique and its correct assembly is essential for its persistence in the extreme host cell environment. Understanding the role of mycobacterial HU, and its unique C-terminal domain hence assumes an even important role as a potential drug target.

Lrp: Lrp (Rv3291c) of M. tuberculosis (Lrp-Mtb) is a 150 amino acid protein and shares homology with E. coli Lrp. The structure of Lrp-Mtb is available (Reddy et al., 2008; Shrivastava et al., 2009) at high resolution and is very similar to that of E. coli (Fig. 2c) Lrp-Mtb binds to DNA with high specificity and is expected to be a general regulator transcription as its E. coli counterpart.

Conclusions and Perspectives

DNA organization and compaction serves for the accommodation of long DNA into cell compartment in addition to playing an important role in global regulation in eubacteria. Several eubacterial NAPs have been studied in detail and their structural information has revealed the nature of DNA compaction and their differing mechanisms of DNA binding. Apart from the architectural role, eubacterial NAPs play an important role in global gene regulation. Temporal expression pattern of bacterial NAPs reveals the differential expression levels of various NAPs at different stages of growth. In M. tuberculosis, only five NAPs have been reported compared to twelve of E. coli and other eubacterial counterparts. Of these, only two, HU and LRP share sequence similarity with other eubacteria. It is hence expected that there may be structural and mechanistic differences in DNA binding, compaction and regulation by M. tuberculosis NAPs. Further investigation for dissecting the structural mechanism of DNA binding and interaction is necessary to understand these events at a molecular level. It is intriguing to note that Lsr 2 , m-IHF, H-NS of M. tuberculosis have been implicated in gene regulation of several important operons, including the multidrug resistance operon, cell wall biosynthesis, etc. Moreover, the G:C content of M. tuberculosis is higher compared to many other eubacteria. While some eubacterial NAPs exhibit sequence preferences for binding to DNA, sequence preferences of mycobacterial NAPs remains to be investigated. It is plausible that some of the unique NAPs in M. tuberculosis may have evolved to bind to certain regions of the higher G:C rich genome of M. tuberculosis. Characterization of M. tuberculosis NAPs and their structural and mechanistic differences with their eubacterial counterparts hence form an important step to investigate some of these intriguing questions.