BACKGROUND: Mycobacterium tuberculosis causes tuberculosis, one of the world’s deadliest diseases. It is an acid-fast bacillus that is 2-4 um in length and 0.2-0.5 um in width. How many weeks, days,& hours would it take to see a M.tuberculosis colony on solid media? Its generation time is listed in the reading (above). Assume that bacterial colonies are not visible to the naked eye unless they contain ≥ 2 millioncells. This species forms aggregates of 5 cells on average. Show your work. The Acid-Fast Solution of Tubercle Bacilli and Their lIk Some bacteria, notably the tubercle bacillus (Mycobacterium tu nd its relatives, have developed yet another way to protect their cell mem. branes from environmental challenges, the so-called acid-fast cell envel 2.13). The cell walls of these organisms contain large amounts ot (Fig. waxes (yes, you heard right!) called mycolic acids. These wax cules are made of fatty acids, as are the phospholipids of the cell m brane, but these fatty acids are longer (60 to 90 carbons), branched, complex. The mycolic acids orient themselves as a highly ordered lipid layer membrane to keep their hydrophobic tails in the middle and awa from water. Proteins are embedded in this layer, where they water-filled pores through which nutrients and certain drugs can pass slowly. The mycolic acid bilayer is thick and covalently attached to the mu rein cell wall via layers of complex sugars. y mole- em and With such a robust hydrophobic cover over their murein cell wall, the acid-fast bacteria are impervious to many harsh chem icals, including disin- fectants and strong acids. You can test this with the acid-fast stain (Table 2.3 any bacteria, including those with the waxy cell wall, can be stained red with a dye such as hot fuchsin by brief heating or transitory treatment with detergents. The dye can then be removed from most bacteria by dilute hydrochloric acid treatment, but not from mycobacteria, whose waxy cell walls make them resistant to the acid. Hence their name "acid fast"; they retain the red stain and remain red after the preparation is counterstained with a blue dye. You can use this procedure to easily differentiate cells with A Acyl glycolipids Free lipids Porin s 3 Mycolic acid Arabinogalactan Peptidoglycan Cytoplasmic membrane FIGURE 2.13 (A) Structure of the acid-fast cell envelope, with the outer mycolic acid bilayer and its attachment to the murein cel wall via an underlying layer of complex sugars. (B) Light micrograph showing the differential staining of acid-fast bacilli of Myco bacterium smegmatis (red) compared with the Gram-positive cocci of Staphylococcus aureus (blue). The waxy mycolic bilayer of acid-fast cells prevents the release of the hot fuchsin red dye after acid treatment. (MicrobeWorld O Tasha Sturm.) TABLE 2.3 The acid-fast stain 1. Stain with hot fuchsin (red); wash. 2. Decolorize with acid alcohol; wash. Only acid-fast bacteria remain red 3. Counterstain with methylene blue; wash. All other material becomes blue. a waxy envelope (red) from other bacteria (blue) (Fig. 2.13). The waxy envelope of acid-fast bacteria also makes them resistant to common soap- based disinfectants. For this reason, surfaces and materials contaminated with tubercle bacilli require something stronger than just soap and water for disinfection, typically certain quaternary detergents. In clinics, you will see such detergents labeled as mycobactericidal. The mycobacteria re sist not only strong chemicals but also white blood cells. All this protection comes at a cost: the waxy armor reduces the rate of nutrient uptake. For mple, the permeability for hydrophilic molecules is 100- to 1,000-fold lower than for the Gram-negative E. coli. Perhaps for this reason, these or- ganisms grow very slowly. Some acid-fast bacteria, such as the human tubercle bacillus, divide only once every 24 hours. Yet do not underesti mate these slow growers: millions of people are diagnosed with tuberculo- sis every year worldwide, with immunocompromised patients being particularly susceptible. Remember Aesop's fable about the tortoise an the hare? Well, think about acid-fast bacteria as the slow tortoise that ends up winning the race. Chapter 24 will discuss how the waxy mycobacterial envelope contributes to tuberculosis exa Bacteria without Cell Walls: The Mycoplasmas After our singing the praises of the peptidoglycan and acid-fast cell walls, you may be surprised to learn that some bacteria called mycoplasmas do not have a cell wall of any type. Some of these wall-less bacteria are human pathogens and difficult to treat in clinical settings. Why? Because com- mon antibiotics that target cell wall biosynthesis are ineffective against these organisms. Thus, mycoplasmas are resistant to penicillins and their relatives the cephalosporins. So how do these wall-less bacteria protect their cell membrane? Some, like Mycoplasma pneumoniae, the agent of "walking pneumonia," contain sterols in their membranes, the same strat- egy used by eukaryotic cells to make their cell membranes more rigid and preserve their integrity. Although delicate in culture, mycoplasmas are re markably persistent in the human body In addition to being insensitive to some commonly used antibiotics, mycoplasmas are very small and spread fast. They can attach to the sur- face of many types of host cells, including cells of the immune system, and some can even get inside host cells. And so mycoplasma bacteria grow either outside or inside the host cells, sometimes evading the host immune sys tem for long periods of time. Interestingly, phylogenetic analysis places the mycoplasmas close to the Gram-positive bacteria, from which they may well have been derived. Clearly, losing the peptidoglycan cell wall in the course of evolution was advantageous to them. To date, mycoplasmas ap- pear to be the only bacteria that lack peptidoglycan. Many archaea are also wall-less. But, unlike the mycoplasmas, wall-less archaea are protected by a protein S-layer (see below) The Acid-Fast Solution of Tubercle Bacilli and Their lIk Some bacteria, notably the tubercle bacillus (Mycobacterium tu nd its relatives, have developed yet another way to protect their cell mem. branes from environmental challenges, the so-called acid-fast cell envel 2.13). The cell walls of these organisms contain large amounts ot (Fig. waxes (yes, you heard right!) called mycolic acids. These wax cules are made of fatty acids, as are the phospholipids of the cell m brane, but these fatty acids are longer (60 to 90 carbons), branched, complex. The mycolic acids orient themselves as a highly ordered lipid layer membrane to keep their hydrophobic tails in the middle and awa from water. Proteins are embedded in this layer, where they water-filled pores through which nutrients and certain drugs can pass slowly. The mycolic acid bilayer is thick and covalently attached to the mu rein cell wall via layers of complex sugars. y mole- em and With such a robust hydrophobic cover over their murein cell wall, the acid-fast bacteria are impervious to many harsh chem icals, including disin- fectants and strong acids. You can test this with the acid-fast stain (Table 2.3 any bacteria, including those with the waxy cell wall, can be stained red with a dye such as hot fuchsin by brief heating or transitory treatment with detergents. The dye can then be removed from most bacteria by dilute hydrochloric acid treatment, but not from mycobacteria, whose waxy cell walls make them resistant to the acid. Hence their name "acid fast"; they retain the red stain and remain red after the preparation is counterstained with a blue dye. You can use this procedure to easily differentiate cells with A Acyl glycolipids Free lipids Porin s 3 Mycolic acid Arabinogalactan Peptidoglycan Cytoplasmic membrane FIGURE 2.13 (A) Structure of the acid-fast cell envelope, with the outer mycolic acid bilayer and its attachment to the murein cel wall via an underlying layer of complex sugars. (B) Light micrograph showing the differential staining of acid-fast bacilli of Myco bacterium smegmatis (red) compared with the Gram-positive cocci of Staphylococcus aureus (blue). The waxy mycolic bilayer of acid-fast cells prevents the release of the hot fuchsin red dye after acid treatment. (MicrobeWorld O Tasha Sturm.) TABLE 2.3 The acid-fast stain 1. Stain with hot fuchsin (red); wash. 2. Decolorize with acid alcohol; wash. Only acid-fast bacteria remain red 3. Counterstain with methylene blue; wash. All other material becomes blue. a waxy envelope (red) from other bacteria (blue) (Fig. 2.13). The waxy envelope of acid-fast bacteria also makes them resistant to common soap- based disinfectants. For this reason, surfaces and materials contaminated with tubercle bacilli require something stronger than just soap and water for disinfection, typically certain quaternary detergents. In clinics, you will see such detergents labeled as mycobactericidal. The mycobacteria re sist not only strong chemicals but also white blood cells. All this protection comes at a cost: the waxy armor reduces the rate of nutrient uptake. For mple, the permeability for hydrophilic molecules is 100- to 1,000-fold lower than for the Gram-negative E. coli. Perhaps for this reason, these or- ganisms grow very slowly. Some acid-fast bacteria, such as the human tubercle bacillus, divide only once every 24 hours. Yet do not underesti mate these slow growers: millions of people are diagnosed with tuberculo- sis every year worldwide, with immunocompromised patients being particularly susceptible. Remember Aesop's fable about the tortoise an the hare? Well, think about acid-fast bacteria as the slow tortoise that ends up winning the race. Chapter 24 will discuss how the waxy mycobacterial envelope contributes to tuberculosis exa Bacteria without Cell Walls: The Mycoplasmas After our singing the praises of the peptidoglycan and acid-fast cell walls, you may be surprised to learn that some bacteria called mycoplasmas do not have a cell wall of any type. Some of these wall-less bacteria are human pathogens and difficult to treat in clinical settings. Why? Because com- mon antibiotics that target cell wall biosynthesis are ineffective against these organisms. Thus, mycoplasmas are resistant to penicillins and their relatives the cephalosporins. So how do these wall-less bacteria protect their cell membrane? Some, like Mycoplasma pneumoniae, the agent of "walking pneumonia," contain sterols in their membranes, the same strat- egy used by eukaryotic cells to make their cell membranes more rigid and preserve their integrity. Although delicate in culture, mycoplasmas are re markably persistent in the human body In addition to being insensitive to some commonly used antibiotics, mycoplasmas are very small and spread fast. They can attach to the sur- face of many types of host cells, including cells of the immune system, and some can even get inside host cells. And so mycoplasma bacteria grow either outside or inside the host cells, sometimes evading the host immune sys tem for long periods of time. Interestingly, phylogenetic analysis places the mycoplasmas close to the Gram-positive bacteria, from which they may well have been derived. Clearly, losing the peptidoglycan cell wall in the course of evolution was advantageous to them. To date, mycoplasmas ap- pear to be the only bacteria that lack peptidoglycan. Many archaea are also wall-less. But, unlike the mycoplasmas, wall-less archaea are protected by a protein S-layer (see below)