**4. Control of peptidoglycan synthesis**

Enzymes involved in remodeling PG can be grouped as either biosynthetic or hydrolytic. Biosynthetic enzymes include transglycosylase and transpeptidase domains, often found on a single, bifuntional protein. Hydrolytic enzymes include muramidase, glucosaminidase, lytic transglycosylase, amidase, endopeptidase and carboxypeptidase (Young, 2003; Cabeen and Jacobs-Wagner, 2005). The reaction of these enzymes may be antagonistic, or they may physically interact to form complexes capable of breaking bonds to generate openings for new monomers, while also forming bonds necessary to unit PG strands. The production of these enzymes should be regulated, otherwise the bacterial cell wall would be degraded and the bacteria would be lyses. There are several ways to governorate these enzymes; one method is through formation of complexes with other proteins (Hett and Rubin, 2008).These proteins could suppress the activity of the enzyme, or they could be enzymes themselves with antagonistic reactions that join rather than degrade PG. Another possibility is that the enzymes are sequestered from their substrate until they needed. A third method could be that the appropriate substrate is not made available until cleavage of it is required.

### **5. The role of PG in cell shape regulation**

The PG –synthesizing enzyme organize into complexes that likely contributes to the resulting shape. Various models have been proposed which explain how this organization affects the bacterial shape. The two –competing sites" model (TCS) for peptidoglycan assembly advocates that, in bacterial rods, the shape depends on the activity of two biochemical reactions (sites) which occur in the terminal stages of peptidoglycan synthesis; one site is responsible for lateral wall elongation , and the other is responsible for septum formation (Lleo *et al*., 1990; Alaedini and Day, 1999). The two sites compete with each other in such a way that the lateral wall is not extended during septum formation and *vice versa*  (Lleo *et al*., 1990; Satta *et al* .,1994). The actual shape of the bacteria is thus determined by the balance between the two competing reactions, correct balance leading to normal rods; abnormal prevalence of the site for lateral wall elongation leads to long rods or filaments, whereas prevalence of the site for septum formation leads to formation of coccobacilli or cocci (Lleo *et al*., 1990). The other bacteria carry only one site for peptidoglycan assembly which can form only septa and can grow only as cocci. Another model is "three –for –one " predict the insertion of PG along a track, using an existing strand of PG as a template

Morphological Characterization of *Mycobacterium tuberculosis* 153

The tubercle bacillus is a prototrophic (i.e., it can build all its components from basic carbon and nitrogen sources) and heterotrophic (i.e., it uses already synthesized organic compounds as a source of carbon and energy), metabolically flexible bacterium( Edson, 1951; Ramakrishnan *et al*., 1972; Niederweis, 2008). The success of tubercle bacilli as a pathogen can be attributed to its extraordinary capacity to adapt to environmental changes throughout the course of infection. Generally, the nutritional quality and physical conditions will determine the temporary lifestyle of bacillus. These changes include: nutrient deprivation, hypoxia, temperature, PH, salinity and various exogenous stress conditions (Vera and Rettger, 1939; Smeulders *et al*., 1999; Honer *et al*., 2001; Young et *al*, 2005; Anuchin *et al*., 2009; Velayati *et al,* 2009; Farnia *et al*., 2010; Singh *et al*., 2010; Shleeva *et al.,* 2002, 2010). Unfortunately, in most of cases we do not know if shape *per se* is beneficial, because few experiments have addressed the question. Knowledge of the physiology of *M. tuberculosis* during this process has been limited by the slow growth of the bacterium in the laboratory and other technical problems such as cell aggregation. Recent advances in microscopy techniques have revealed adaptive changes in size and shape of bacilli under

Fig. 1. Scanning electron microscope shows shape variation in *M. tuberculosis* at exponential

**7. Shape variation** 

phase of growth.

(Holtje, 1998). This result in doubling the length in one direction, but since following a strand, no additional length is added in the direction perpendicular to the strand. Thus width would stay constant. Another theory as to how cells maintain a constant width posits that the poles are capped with a type of PG that prevents rapid turnover or insertion of new PG (De Pedro *et al*., 1997). Thus, the caps would restrict the width of the bacterium

### **6. How the shape remain constant**

Uniform cell shapes are favored by the need to segregate the chromosome and cytoplasmic material between daughter cells (Errington *et al*., 2003). The regular shape would seem to be the best way to ensure each daughter, because a symmetrical cell can be halved accurately by mechanisms that measure length or volume (Helmstetter *et al.*, 1990; Young, 2006). In an irregular cell, misplaced septation might leave one cell with both chromosomes or with more than its fair share of other components. Therefore, once a particular shape is adapted bacteria have a vested interest in keeping it (Stewart, 2005). The major incentive for doing so is to maintain a consistent relationship between cytoplasmic volume and surface area so that cell cycle events can be coordinated properly. This is visualized by considering the septation event that creates two daughter cells (Harry, 2001; Errington *et al*., 2003). The septum is formed through the in-ward growth of cytoplamic membrane and cell wall material that invaginates from opposing directions at the central plane of the cell. In such case, the concentration of essential division proteins will not change, but the surface area over which they must act will be greater in the sphere. The amounts of these proteins, if optimized for dimensions of a rod , might not be sufficient to initiate or complete normal septation and division in a coccus (Young, 2006). Thus limited concentrations of division proteins will dictate that the cell maintain a specific and constant diameter. To do this, bacteria must coordinate events spatially and temporally. Recently it was shown that the divisome will assemble at midcell, before chromosomes partitioned. The divisome consists of a set of 10 to 15 proteins that are required to the middle of the cell and are responsible for generating the septum that divides two daughter cells (Margolin, 2006; Buddelmeijer and Beckwith, 2002). This is accomplished by synthesizing septal PG, constricting the cell wall to eventually close off the cytoplasmic compartments of each daughter cell, and finally hydrolyzing part of the PG that holds two together in order to physically separate the cells. These divisome proteins (FtsA, FtsB, FtsE, FtsI, FtsK, FtsL, FtsN, FtsQ, FtsW, FtsX, FtsZ, Zip A, AmiC and EnvC) encoded in different bacterial genomes and have different function (Di Lallo *et al*., 2003; Karimova *et al*., 2005; Vicente and Rico, 2006). The FtsZ is the first protein to assemble at midcell (Bi and Lutkenhaus, 1992). Its formation of a ring around the cell, just under the plasma membrane, gives the assembled divisome the name Z ring. This sub cellular organelle, a functional analog of the contractile ring used in cytokinesis of many eukaryotic cells, is thought to form the scaffold for recruitment of the other key cell division proteins. In *E. coli*, successful cell division depends on a constant and critical concentration of Ftsz combined with proper proportions of Z-ring stabilizing and destabilizing proteins. Significantly, small changes in the concentrations of FtsZ or other essential division proteins disrupt cell growth. Thus, division is inhibited if FtsZ is under produced, extra divisions occur if the protein is overproduced and no division occurs if FtsZ levels are adequate but FtsZ/FtsA ratio is incorrect (Errington *et al*., 2003; Maki *et al*., 2000; Chauhan *et al*., 2006).
