**7. Shape variation**

152 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

(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

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).

PG (De Pedro *et al*., 1997). Thus, the caps would restrict the width of the bacterium

**6. How the shape remain constant** 

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 phase of growth.

Morphological Characterization of *Mycobacterium tuberculosis* 155

suggested that V-forms resulted from "germ tube extrusions" from each of a pair of attached arthrospores and were not due to postfission movements. More studies have demonstrated that snapping division or V-forms could arise by any of three methods: (I) germination of adjacent coccoid elements, (ii) subpolar germination (budding) of rods, and (iii) snapping postfission movements (Starr and Khan, 1962). In mycobacterium, during septum formation the plasma membrane and inner cell wall grow inward but the outer cell wall layer remains intact. Upon completion of septum formation with a cross-wall, the inner layer may continue to grow and thus exert pressure upon the outer cell wall layer. The outer layer eventually ruptures first on one side of the cell, and the two daughter cells bend in on the side where the outer layer is still intact forming a "V-form (Dahl,2004; Farnia *et al*,2010;

Mycobacterium is known to form a "Y-shaped "cells with branches more interior to the cells and of greater length figure 3. Brieger *et al* in 1954, was among the first scientist who demonstrate the branching in the reproductive cycle of *M. avium*. He showed that young culture of bacilli when first transplanted to fresh medium it consists mainly of short coccoid rods. These elongate into filaments (8-10µ) which continue to divide and grow during a phase of filamentous proliferation. The filaments usually have two fully

Fig. 2. Atomic force microscopy shows the V-shape *M. tuberculosis* during exponential phase

Malhotra *et al*., 2010)

of growth

stress conditions (Velayati *et al*., 2009,2011; Farnia *et al*., 2010). Briefly, the reported morphological variation in *M. tuberculosis* are classified into two categories; those which frequently seen at exponential phase of growth that is rod, V, Y-shape, branched or buds, and those that are seen occasionally under stress or environmental conditions which are round, oval , ultra-virus, spore like, and cell wall defiant or L-forms.
