Bacterial and Archaeal Growth MCB3020 PDF

7 Bacterial and Archaeal Growth How Low Can You Go? T wenty million years ago, in a forest in what is now the western Pacific Ocean, organic debris accumulated to form peat. It supported a diverse microbial ecosystem that thrived on degrading plant matter. Over the millennia, peat was compressed i...

7 Bacterial and Archaeal Growth How Low Can You Go? T wenty million years ago, in a forest in what is now the western Pacific Ocean, organic debris accumulated to form peat. It supported a diverse microbial ecosystem that thrived on degrading plant matter. Over the millennia, peat was compressed into sedimentary rock and ultimately lignite coal, and the site receded into the ocean. The remains of the forest is now about 2 km below the sea floor. And that microbial ecosystem? It’s still there. We know the microbial ecosystem still exists because in the summer of 2012, scientists in the International Ocean Discovery Program drilled into this Miocene era coal bed. Core samples were crushed and set up in culture vessels incubated under conditions that mimic the original site: no oxygen, warm temperature (45°C), and simple carbon and nitrogen compounds. The scientific team patiently incubated these cultures for 2.5 years before breaking them open to examine their contents. They then made two notable discoveries. First, 16S rRNA analysis revealed what looked like a contemporary peat microbial community, not a marine sediment community. And while incorporation of isotopes like 2H2O and 15NH4+ indicated that life processes were occurring, scientists calculated that their generation times ranged from several months to over 100 years. In other words, in the time it takes for an average human to be born, have children, grow very old, and die, many of these bacteria didn’t divide a single time. Only microbes can live at such a slow pace. Amazingly, these cells seemingly counteract chemical degradation and maintain cellular integrity. They remain intact and capable of reviving. Although we are only beginning to study microbes with such long generation times, these observations confirm that growth in nature can be challenging, not to mention difficult for microbiologists to observe and quantify. Defining “growth” and “alive” confounds microbiologists. From our anthropomorphic ideas about what constitutes a habitable environment to our expectation of microbial cell division in a brief time frame, only in the last century have we realized that life exists in ©Arthur Dorety/Stocktrek Images/Getty Images extreme environments, and it is almost exclusively microbial. In this chapter, we present bacterial and archaeal growth patterns, how they are measured, and the environmental conditions that affect growth. Readiness Check: Based on what you have learned previously, you should be able to: ✓ Describe the functions of eukaryotic cytoskeletal proteins (section 5.3) ✓ Define and list examples of essential nutrients, and describe how they are used by cells ✓ Distinguish macroelements (macronutrients) from trace elements ✓ ✓ ✓ (micronutrients), list examples of each, and describe how they are used (section 3.3) Provide examples of growth factors needed by some microorganisms (section 3.3) Describe the eukaryotic cell cycle Describe the major events of mitosis and meiosis 7.1 Most Bacteria and Archaea Reproduce by Binary Fission After reading this section, you should be able to: a. Describe binary fission as observed in bacteria and archaea b. Compare binary fission with other bacterial reproductive strategies Most bacterial and archaeal cells reproduce by binary fission (figure 7.1). Binary fission is a relatively simple type of cell division. The cell elongates as new cell envelope material is synthesized, and subcellular structures like ribosomes and inclusions are abundant enough to be evenly distributed in the cytoplasm. Only the nucleoid is present as a single entity, and its replication and partitioning into each half of the elongated cell is a critical 128 wil11886_ch07_128-169.indd 128 23/10/18 9:18 am 7.2 Bacterial Cell Cycles Can Be Divided into Three Phases 129 Cell wall Cell membrane (a) A cell at early phase of cycle Chromosome 1 Chromosome 2 Ribosomes (b) A cell prepares for division by enlarging its cell wall, plasma membrane, and overall volume. DNA replication then starts. (c) The septum begins to grow inward as the chromosomes move toward opposite ends of the cell. Other cytoplasmic components are distributed to the two developing cells. (d) The septum is synthesized completely through the cell center, creating two separate cell chambers. (e) At this point, the daughter cells are divided. Some species separate completely as shown here, while others remain attached, forming chains, doublets, or other cellular arrangements. Figure 7.1 Binary Fission. MICRO INQUIRY In addition to chromosomes, what other cytoplasmic contents must be equally distributed between daughter cells? event in binary fission. Finally, a septum (cross wall) is formed at midcell, dividing the parent cell into two progeny cells, each having its own nucleoid and a complement of other cellular constituents. Several other reproductive strategies have been identified in bacteria (figure 7.2). Some bacteria reproduce by forming a bud. Certain cyanobacteria undergo multiple fission. The progeny cells, called baeocytes, are held within the cell wall of the parent cell until they mature. Other bacteria, such as members of the genus Streptomyces, form multinucleoid filaments that eventually divide to form uninucleoid spores. These spores are readily dispersed, much like the dispersal spores formed by filamentous fungi. Order Streptomycetales: an important source of antibiotics (section 23.1) Despite the diversity of bacterial reproductive strategies, certain features are shared. In all cases, the genome of the cell must be replicated and segregated to form distinct nucleoids. At some point during reproduction, each nucleoid and its surrounding cytoplasm becomes enclosed within its own plasma membrane. These processes are the major steps of the cell cycle. In section 7.2, we examine the bacterial cell cycle in more detail. wil11886_ch07_128-169.indd 129 7.2 Bacterial Cell Cycles Can Be Divided into Three Phases After reading this section, you should be able to: a. Summarize the three phases in a typical bacterial cell cycle b. Summarize current models for chromosome partitioning c. State the functions of cytoskeletal proteins during cytokinesis and in determining cell shape The cell cycle is the complete sequence of events extending from formation of a new cell through the next division. It is of intrinsic interest as a fundamental biological process. However, understanding the cell cycle has practical importance as well. For instance, synthesis of peptidoglycan during the cell cycle is the target of numerous antibiotics used to treat bacterial infections. Inhibitors of cell wall synthesis (section 9.4) The cell cycles of several bacteria—Escherichia coli, Bacillus subtilis, and the aquatic bacterium Caulobacter crescentus—have been examined extensively, and our understanding of the bacterial cell cycle is based largely on 23/10/18 9:18 am 130 CHAPTER 7 | Bacterial and Archaeal Growth (a) L. monocytogenes mother cell and bud (b) Dermocarpa cell undergoing multiple fission Spore Aerial hypha Figure 7.2 Some Bacteria Reproduce by Spore Germ tube Branch Vegetative hypha (c) Streptomyces spore formation these studies. The bacterial cell cycle consists of three phases: (1) a period of growth after the cell is born, which is similar to the G1 phase of the eukaryotic cell cycle; (2) chromosome replication and partitioning period, which functionally corresponds to the S and mitosis events of the M phase of the eukaryotic cycle; and (3) cytokinesis, during which a septum and daughter cells are formed (figure 7.3). Recall that in the eukaryotic cell cycle, the S phase is separated from the M phase by another period called G2. In G2, chromosome replication is completed and some time passes before chromosome segregation occurs. This is not the case for bacteria. As you will see in the discussion that follows, chromosome replication and partitioning occur concurrently. Furthermore, the initial events of cytokinesis actually occur before chromosome replication and partitioning are complete. Finally, some bacteria are able to initiate new rounds of replication before the first round of replication and cytokinesis is finished. wil11886_ch07_128-169.indd 130 Methods Other than Binary Fission. (a) Transmission electron micrograph (TEM) of a budding Listeria monocytogenes cell. (b) Baeocytes produced by the cyanobacterium Dermocarpa. (c) Spore formation by Streptomyces spp. Branched filaments, aerial hyphae, and chains of spores are visible. (a) ©Dr. Kari Lounatmaa/Science Source; (b) ©Dennis Kunkel Microscopy/Science Source (c) ©Smith Collection/Gado/Getty Images Although chromosome replication and partitioning overlaps with cytokinesis, we consider them separately. Chromosome Replication and Partitioning Most bacteria have a single circular chromosome. Each circular chromosome has a single site at which replication starts called the origin of replication, or simply the origin (fig ure 7.3). Replication is completed at the terminus, located directly opposite the origin. In a newly formed E. coli cell, the chromosome is compacted and oriented so that the origin and terminus are in opposite halves of the cell. Early in the cell cycle, the origin and terminus move to midcell, and proteins needed for chromosome replication assemble at the origin. This DNA synthesizing machinery is called the replisome, and DNA replication proceeds in both directions from the origin. As the progeny chromosome is synthesized, the two 23/10/18 9:18 am 7.2 Bacterial Cell Cycles Can Be Divided into Three Phases 131 1 Initiation mass reached Origin of replication Bacterium Cells divide. Chromosome Terminus Replisome 2 Initiation of replication Chromosomes separate as septation continues. 3 Septum formation begins when Z ring forms Origins separate. Cell elongates as chromosome replication and partitioning continue. Figure 7.3 Cell Cycle of E. coli. An increase in cell mass results in cell cycle initiation and accumulation of the DnaA protein, which initiates DNA replication. As the cell readies for DNA replication, the origin of replication migrates to the center of the cell and proteins that make up the replisome assemble. In this illustration, one round of DNA replication is completed before the cell divides. In rapidly growing cultures (generation times of about 20 minutes), second and third rounds of DNA replication are initiated before division of the original cell is completed. Thus the daughter cells inherit partially replicated DNA. MICRO INQUIRY Why is it important that the origin of replication migrate to the center of the cell prior to replication? origins move toward opposite ends of the cell, and the rest of close together and near the origin of replication, and this region each chromosome follows in an orderly fashion. functions like a eukaryotic centromere. Following replication iniAlthough the process of DNA synthesis and movement tiation, ParB binds to the parS sites on each chromosome. seems rather straightforward, the mechanism by which chromosomes are partitioned to each daughter cell varies somewhat among bacterial species. StudParA Stalk ParB ies of C. crescentus have provided the most comReplisome plete understanding of the partitioning process. This bacterium has an interesting life cycle in Replicated DNA which a sessile stalked cell divides to give rise to parS sites a slightly smaller, flagellated swarmer cell; the oriC flagellum forms at the pole opposite the stalk (see figure 22.8). The partitioning system for bacterial chromosomes has three components: ParA and ParB proteins Figure 7.4 The ParAB/parS Partitioning System of Caulobacter crescentus. ParB binds and a parS region on the chromosome (figure 7.4). each parS site on the two daughter chromosomes. A gradient of ParA proteins directs the The C. crescentus chromosome has two parS sites partitioning complex of ParB/parS to the opposite pole of the cell in a relay mechanism. wil11886_ch07_128-169.indd 131 23/10/18 9:18 am 132 CHAPTER 7 | Bacterial and Archaeal Growth Additional molecules of ParB bind nearby and ultimately cover a large chromosomal region. This protein-DNA assembly is termed the partitioning complex. One partitioning complex remains at the stalk pole of the dividing cell, while the other is guided by ParA to the opposite pole. The ParA-mediated movement of the second partitioning complex is described as a relay model. In a relay race, the baton is passed from one runner to the next; by the end of the race, the baton has traveled the full distance, but each runner has traveled only part of the distance. Similarly, movement of the newly replicated chromosome occurs by passing the partitioning complex to a series of ParA proteins. The ParA protein is distributed in a gradient in the cell and is most abundant at the opposite pole. ParA molecules in complex with ATP are bound loosely and nonspecifically to the chromosome. When a partitioning complex contacts ParA-ATP, ATP is hydrolyzed and the partitioning complex is moved in the direction of the opposite pole. Therefore, the abundance of ParA-ATP at the opposite pole directs chromosome movement from one pole to the other. It is still unclear how the ParA gradient forms and is maintained in the cell. Caulobacteraceae and Hyphomicrobiaceae bacteria reproduce in unusual ways (section 22.1) Binary Fission; Bidirectional DNA Replication Cytokinesis Septation is the process of forming a cross wall between two daughter cells. Cytokinesis, a term that was once used to describe the formation of two eukaryotic daughter cells, is now used to describe this process in all cells. In bacteria and archaea, septation is divided into several steps: (1) selection of the site where the septum will be formed; (2) assembly of the Z ring, which is composed of the cytoskeletal protein FtsZ; (3) assembly of the cell wall–synthesizing machinery (i.e., for synthesis of peptidoglycan and other cell wall constituents); and (4) constriction of the cell and septum formation. Synthesis of peptidoglycan occurs in the cytoplasm, at the plasma membrane, and in the periplasmic space (section 12.4) Correct septation requires that the Z ring form at the proper place at the proper time. In E. coli, the MinCDE system limits Z-ring formation to the center of the cell. Three proteins compose the system (MinC, MinD, and MinE). These proteins oscillate between the cell poles (figure 7.5). This creates high concentrations of MinC at the poles, where it prevents formation of the Z ring; thus Z-ring formation can occur only at midcell, which lacks MinCDE. A mechanism called nucleoid occlusion helps ensure that the Z ring forms only after most of the daughter chromosomes have separated from each other. This is important because the septum might otherwise guillotine the chromosome. In E. coli, a protein called SlmA is critical to this process. SlmA binds to specific sites distributed around the chromosome except near the terminus of replication. DNA-bound Slm inhibits FtsZ polymerization, thereby preventing the FtsZ ring from forming while the chromosome is at midcell. Once enough of the wil11886_ch07_128-169.indd 132 MinE FtsZ Figure 7.5 MinCDE Proteins Help Establish the Site of Septum Formation. MinC (not shown) blocks septum formation. It oscillates with MinD. MinD’s oscillations are controlled by MinE, which also follows MinD movements. As shown, MinD-ATP interacts with the plasma membrane and forms filaments. MinE binding to MinD-ATP causes hydrolysis of ATP, yielding MinD-ADP. MinD-ADP is released from the filament into the cytosol. There MinD-ADP is converted back to MinD-ATP, which then forms filaments at the opposite end of the cell. MinC and MinE follow and the process begins again. As a result of these oscillations, MinC concentrations are highest at the poles forcing septum formation at the center of the cell. MICRO INQUIRY What would be the outcome if FtsZ formed a Z ring that was not in the center of the cell? Consider both the morphology of the daughter cells and the partitioning of chromosomes. 23/10/18 9:18 am 7.2 Bacterial Cell Cycles Can Be Divided into Three Phases 133 Plasma membrane Peptidoglycan synthesizing enzymes FtsA minirings FtsZ filament Treadmilling FtsZ Figure 7.6 The E. coli Divisome. The cell division apparatus is composed of numerous proteins. The first step in divisome formation is FtsZ polymerization to form the Z ring. FtsA anchors the Z ring to the plasma membrane through insertion of a helix. Cytoplasmic FtsZ polymerizes in a treadmilling process, where it forms the platform for septal peptidoglycan synthesis. daughter chromosomes and SlmA molecules have moved away from midcell, the Z ring can form. Assembly of the Z ring is the critical early step in septation, as it forms the scaffold for the complete cell division machinery. The FtsZ protein is a homologue of eukaryotic tubulin, and like tubulin, it polymerizes, and its filaments create the meshwork that constitutes the Z ring. The Z ring is dynamic, with portions being exchanged constantly with newly formed, short FtsZ polymers from the cytosol. Although the detailed architecture of the Z ring has not yet been defined, FtsA and ZipA proteins are known to promote its attachment to the plasma membrane. Once the Z ring forms, the late division proteins assemble into the divisome, as illustrated in figure 7.6. The cell wall– synthesizing machinery is assembled using the Z ring as a scaffold (table 7.1). Short polymers of FtsZ, termed protofilaments, Table 7.1 Some E. coli Divisome Proteins and Their Functions move around the inner circumference of the cell near the Z ring in a motion called treadmilling. This motion occurs by the removal of individual FtsZ molecules from one end of the protofilament and the repolymerization at the other end. These treadmilling protofilaments associate with enzyme complexes that synthesize peptidoglycan to build the septum between the daughter cells. The Z ring constricts—that is, it is reduced in circumference as the septum grows inward. The plasma membrane invaginates, and the cytoplasms of the daughter cells are separate before the septal wall is complete. A final step involves amidases, enzymes that function to split the newly synthesized wall so that the daughter cells can separate. So far, our discussion of the cell cycle describes what occurs in slowly growing E. coli cells. In these cells, the cell cycle takes approximately 60 minutes to complete. However, E. coli can reproduce at a much more rapid rate, completing the entire cell cycle in about 20 minutes, despite the fact that DNA replication always requires at least 40 minutes. E. coli accomplishes this by beginning a second round of DNA replication (and sometimes even a third or fourth round) before the first round of replication is completed. Thus the progeny cells receive a chromosome with two or more replication forks, and replication is continuous because the cells are always copying their DNA. Bacterial Cell Cycle Cellular Growth and Determination of Cell Shape As we have seen, bacterial and archaeal cells have speciesspecific defined shapes. These shapes are neither accidental nor random, as demonstrated by the faithful propagation of shape from one generation to the next. In addition, some microbes change their shape under certain circumstances. For instance, Sinorhizobium meliloti switches from rods to Y-shaped cells when living symbiotically with plants. Likewise, Helicobacter pylori, the causative agent of gastric ulcers and stomach cancer, changes from its characteristic wil11886_ch07_128-169.indd 133 23/10/18 9:18 am 134 CHAPTER 7 | Bacterial and Archaeal Growth helical shape to a sphere in stomach infections and in prolonged culture. To consider the shape of a cell, we must first review cell wall function. The cell wall constrains the turgor pressure exerted by the cytoplasm, preventing the cell from swelling and bursting. Turgor pressure describes the force pushing against the cell wall because of the osmolarity of the cytoplasmic contents. Peptidoglycan is a rigid but elastic structure that protects the cell from lysis. The strength of the existing peptidoglycan in the cell wall must be maintained as new peptidoglycan subunits are added. Thus understanding peptidoglycan synthesis is critical to understanding the determination of cell shape. Peptidoglycan structure (section 3.4) Peptidoglycan synthesis involves many proteins, including a group of enzymes called penicillin-binding proteins (PBPs) so named because they were first noted for their capacity to bind penicillin. While this property is important, their function is to link strands of peptidoglycan together or hydrolyze bonds in existing strands so that new units can be inserted during cell growth. The PBP enzymes that hydrolyze bonds of peptidoglycan are also called autolysins; other autolysins besides PBPs also help sculpt the peptidoglycan sacculus during cell growth and cell division. Figure 7.7 illustrates some of the components of the peptidoglycan-synthesizing machinery and outlines a general scheme of peptidoglycan synthesis. Synthesis of the NAG-NAM-pentapeptide building block is completed while it is attached to a lipid carrier, bactoprenol, located in the plasma membrane. The carrierbound building block is then flipped across the membrane by MurJ. Upon release of the NAG-NAM-pentapeptide into 1. Peptidoglycan synthesis starts in the cytoplasm with the attachment of uridine diphosphate (UDP) to the sugar N-acetylglucosamine (NAG). Some of the UDP-NAG molecules are converted to UDPNAM. Amino acid addition to NAM is not shown for simplicity. 2. NAM is transferred from UDP to bactoprenol, a carrier embedded in the plasma membrane. NAG is then attached to bactoprenol-NAM generating bactoprenol-NAM-NAG, called lipid II. The divisome protein MurJ (not shown) “flips” lipid II across the plasma membrane so that the NAMNAG units are available for insertion into the sacculus. the periplasmic space, it is inserted into a peptidoglycan strand. The cellular location of autolysin activity and peptidoglycan export is not random and plays an important role in determining cell shape. We begin our discussion with the simplest shape, the coccus. Although it has been stated that this is the “default” cellular shape, the growth of a spherical cell is more complicated than once thought. Studies of model cocci (e.g., Enterococcus faecalis and Staphylococcus aureus) show that new peptidoglycan forms only at the central septum (figure 7.8a). When daughter cells separate, each has one new and one old hemisphere. As in most bacteria, proper placement of the septum depends on FtsZ localization. In mutant cells without this tubulin homologue, peptidoglycan synthesis occurs in a random pattern around the cell, leading to bloated cells that lyse. Thus FtsZ placement determines the site of cell wall growth by recruiting enzymes needed for peptidoglycan synthesis to the divisome. The peptidoglycan of rod-shaped cells is synthesized by two molecular machines that share many proteins but differ in terms of placement and time of function. The first is responsible for elongation of the cell that occurs prior to septum formation. It is sometimes called the elongasome. The second molecular machine is the divisome, which synthesizes peptidoglycan during cytokinesis. During elongation, proteins in the actin homologue MreB family play an essential role. In a manner similar to that of FtsZ and the Z ring in cytokinesis, MreB proteins polymerize, creating patches of filaments along the cytoplasmic face of the plasma membrane (figure 7.8b). Cell wall growth occurs in numerous bands around the circumference of the cell. The NAG Cytoplasm UDP UDP NAM –P P– Bactoprenol Peptidoglycan A NAG NAM NAG Plasma membrane A NAM NAG NAM NAG NAM NAG NAM NAG NAM A 3. Autolysins (blue balls labeled “A”) located at the divisome degrade bonds in the existing peptidoglycan sacculus. This permits the insertion of new NAM-NAG units into the sacculus. NAG NAM NAG NAM Figure 7.7 Components of the Peptidoglycan Synthesizing Machinery. See figures 9.6 and 12.9 for detailed diagrams of peptidoglycan synthesis. wil11886_ch07_128-169.indd 134 23/10/18 9:18 am 7.2 Bacterial Cell Cycles Can Be Divided into Three Phases 135 AI. Spherical cells build new peptidoglycan only at midcell, where the septum will form during division. This leads to daughter cells that have one old and one new cell wall hemisphere. A Spherical/Coccoid I: Division Hemisphere formation BI. During growth, prior to division, new cell wall is made along the side of the cell but not at the poles. This placement is thought to be determined by the position of MreB homologues. BII. As division begins, FtsZ polymerization forms a Z ring and new cell wall growth is confined to the midcell. BIII. Rod-shaped daughter cells are formed with one new pole and one old pole. B Rod (common) I: Sidewall elongation II: Preseptal elongation III: Division Pole formation Figure 7.8 Cell Wall Biosynthesis and Determination of Cell Shape in Spherical and Rod-Shaped Cells. MICRO INQUIRY Which step in the development of rod-shaped cells is essential in determining cell morphology? bands are positioned along the length of the cell but not at the poles (see figure 3.30b). MreB functions as a scaffold upon which the cell-wall synthesizing machinery assembles. As with FtsZ, MreB filaments also undergo treadmilling, but in this case, the activity of the peptidoglycan synthesizing enzymes is required for filament movement. As the FtsZ ring forms at the midcell in cytokinesis, MreB proteins and other elongasome proteins redeploy from the sidewalls to the midcell, where they contribute to cell wall synthesis during cytokinesis. Thus, cell wall growth switches from the side wall to the septum at this time. The importance of MreB proteins in determining cell shape is demonstrated by two observations. First, rod-shaped cells in which MreB has been depleted assume a spherical shape. In addition, while almost all rod-shaped bacteria and archaea synthesize at least one MreB homologue, coccoid-shaped cells lack proteins in the MreB family. The last cell shape we consider is that of comma-shaped cells, as studied in the aquatic bacterium Caulobacter crescentus. In addition to the actin homologue MreB and the tubulin-like protein FtsZ, these cells (and other vibroid-shaped cells) produce a cytoskeletal protein called crescentin, a homologue of eukaryotic intermediate filaments. This protein localizes to one side of the cell, where it slows the insertion of new peptidoglycan units into the peptidoglycan sacculus wil11886_ch07_128-169.indd 135 (figure 7.9). The resulting asymmetric cell wall growth gives rise to the inner curvature that characterizes the comma shape. It is clear from these examples that common microbial cell shapes require cytoskeletal proteins that bear startling similarity with those of eukaryotes. However, some bacterial shapes appear to be determined by other mechanisms. For MreB FtsZ Crescentin Vibroid C. crescentus Figure 7.9 Vibroid Shape Is Determined by Crescentin in Caulobacter crescentus. The intermediate filament homologue crescentin polymerizes along the length of the inner curvature of the cell. Cells depleted of crescentin are rod-shaped. Note that C. crescentus has all three types of cytoskeleton protein homologues. 23/10/18 9:19 am 136 CHAPTER 7 | Bacterial and Archaeal Growth example, the spirochete Borrelia burgdorferi, which causes Lyme disease, relies on its periplasmic flagella to confer its spiral shape. As another example, Spiroplasma, a citrus pathogen, has no cell wall or flagella. It relies on contractile cytoplasmic fibrils to give its spiral shape. Phylum Spirochaetes (section 21.6); Class Mollicutes, Phylum Tenericutes: bacteria that lack cell walls (section 21.3) The study of bacterial cytoskeletal elements and cell shape has important practical aspects. Bacterial cytoskeletal homologues provide a tractable model for the study of more complicated eukaryotic cytoskeletal proteins and their assembly. In addition, these proteins may prove to be valuable targets for drug development. Indeed, molecules that block FtsZ function inhibit growth of the pathogen Staphylococcus aureus and might be effective treatments for staphylococcal diseases. Antimicrobial chemotherapy (chapter 9) Comprehension Check 1. Describe the three phases of a bacterial cell cycle. The overlapping of cytokinesis and chromosome partitioning could potentially create problems for a cell during the cell cycle. What mechanisms does the cell use to prevent any problems? 2. How does the bacterial cell cycle compare with the eukaryotic cell cycle? List two ways they are similar and two ways they differ. 3. Do you think MinCDE functions in coccoid-shaped cells? Explain your answer. 4. Do you think Spiroplasma produces FtsZ? What about MreB? Explain your reasoning. 7.3 Archaeal Cell Cycles Are Unique After reading this section, you should be able to: a. Compare and contrast the Sulfolobus spp. cell cycle and the typical eukaryotic cell cycle b. Compare and contrast the Sulfolobus spp. cell cycle and a bacterial cell cycle As is true with many other aspects of archaeal biology, understanding of archaeal cell cycles lags behind that of bacterial cell cycles. However, studies of model archaea such as Sulfolobus spp. have yielded intriguing results. Sulfolobus spp. are members of the phylum Crenarchaeota and grow best in environments that are hot (80°C) and acidic (pH 3). Their cell cycle is reminiscent of a mitotic cell cycle. After a growth phase (G1), their DNA is replicated (S) using replicative machinery similar to that of eukaryotes. Of note is the fact that unlike bacteria, the circular chromosomes of Sulfolobus spp. have three origins of replication. Furthermore, once replicated, the daughter chromosomes remain unsegregated for some time (G2); the Sulfolobus G2 phase consumes more than 50% of the cell cycle. G2 is wil11886_ch07_128-169.indd 136 followed by segregation of the chromosome and then cytokinesis occurs. Archaea use homologues of eukaryotic replisome proteins (section 15.2) Advances have been made in elucidating how daughter chromosomes are segregated. Two proteins have been identified: SegA and SegB. SegA is similar to ParA proteins found in bacteria, and SegB is unique to archaea, but is thought to be the functional equivalent of bacterial ParB. The genes encoding the two proteins are located near one of the origins of replication, just as the genes for ParA and ParB are located near the bacterial origin. SegA polymerizes and is thought to exert the force, either pulling or pushing, that segregates the chromosomes. The role of SegB is unclear, although it is known to bind DNA and interact with SegA, enhancing SegA polymerization. However, parS-like sequences have not been clearly identified in Sulfolobus spp. Unlike chromosome segregation, which appears to be bacteria-like, cytokinesis in Sulfolobus spp. is most similar to eukaryotic processes. These archaea undergo cytokinesis using proteins related to the ESCRT proteins of eukaryotes. ESCRT proteins are so named because of their roles in endocytosis: endosomal sorting complex required for transport. ESCRT proteins form four distinct complexes that are involved in formation of multivesicular bodies (MVB), a type of endosome (see figure 5.9). During MVB formation, some ESCRT proteins cause the endosome membrane to invaginate. Then a complex of ESCRT proteins called ESCRT-III circles the neck formed by the invaginating membrane and brings about membrane scission, releasing a small vesicle into the developing MVB. Three division proteins have been identified in Sulfolobus spp. CdvA binds the plasma membrane and forms a ring at midcell. ESCRT-III and Vps4 are recruited to this CdvA belt and have been proposed to form an hourglass structure that constricts the membranes until they separate. Archaea belonging to the phylum Euryarchaeota have cell cycles that differ in several substantive ways from the Sulfolobus model just described. The euryarchaeal cell cycle is comparable to the bacterial cell cycle, featuring growth, DNA replication, and cytokinesis. FtsZ is present in most euryarchaea, often in two related forms. Recall that archaea lack peptidoglycan, so unlike the bacterial model where the Z ring is physically associated with the peptidoglycan synthesizing enzymes, the archaeal Z ring associates with new S-layer proteins and possibly other cell wall components. How this is accomplished awaits discovery. As we note in chapter 4, euryarchaea are polyploid, often with a dozen or more chromosome copies. It is possible that a genome segregation system does not exist, and the large number of chromosomes may compensate for this deficiency. Only Halobacterium salinarum, a halophile, has been demonstrated to actively segregate daughter chromosomes. Archaeal cytoplasm is similar to bacterial cytoplasm (section 4.3) 23/10/18 9:19 am 7.4 Growth Curves Consist of Five Phases 137 Stationary phase Comprehension Check Death phase Log10 number viable cells 1. What elements of the Sulfolobus spp. cell cycle are similar to that of Caulobacter crescentus? What elements are similar to the eukaryotic cell cycle? 2. Many archaea have genes encoding an FtsZ homologue. Describe how FtsZ might function in an archaeal cell cycle. Exponential phase Long-term stationary phase Lag phase Time 7.4 Growth Curves Consist of Five Phases After reading this section, you should be able to: a. Describe the five phases of a microbial growth curve observed when microbes are grown in a batch culture b. Describe three hypotheses proposed to account for the decline in cell numbers during the death phase of a growth curve c. Correlate changes in nutrient concentrations in natural environments with the five phases of a microbial growth curve d. Relate growth rate constant to generation (doubling) time and suggest how these values might be used by microbiologists doing basic research or working in industrial settings In section 7.2, we commented on the changes in bacterial cell size that accompany its preparation for division. This is one type of growth of concern to microbiologists. However, microbiologists are more frequently concerned with the increase in population size that follows cell division. Therefore the term growth is more commonly used to refer to growth in the size of a population. Population growth is often studied by analyzing the growth of microbes in liquid (broth) culture. When microorganisms are cultivated in broth, they usually are grown in a batch culture; that is, they are incubated in a closed culture vessel like a test tube or a flask with a single batch of medium. Fresh medium is not provided during incubation, so as nutrients are consumed, their concentrations decline, and wastes accumulate. Population growth of microbes reproducing by binary fission in a batch culture can be plotted as the logarithm of the number of viable cells versus the incubation time. The resulting curve has five distinct phases (figure 7.10), which we examine in this section. Although this is “life in the lab,” microbes do encounter conditions in their natural environments that mimic what occurs in a batch culture. Furthermore, humans routinely create artificial environments for microbes (e.g., the fermentation vessel in a pharmaceutical plant) that are batch cultures. Therefore understanding the growth curve is of paramount importance. Lag Phase When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs. wil11886_ch07_128-169.indd 137 Figure 7.10 Microbial Growth Curve in a Closed System. The five phases of the growth curve are identified. The dotted lines shown during the long-term stationary phase represent successive waves of genetic variants that evolve during this phase of the growth curve. MICRO INQUIRY Identify the regions of the growth curve in which (1) nutrients are rapidly declining and (2) wastes accumulate. This period is called the lag phase. It is not a time of inactivity; rather cells are synthesizing new components. This can be necessary for a variety of reasons. The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized before growth can begin. The medium may be different from the one the microorganism was growing in previously. In this case, new enzymes are needed to use different nutrients. Possibly the microorganisms have been injured and require time to recover. Eventually, however, the cells begin to replicate their DNA, increase in mass, and divide. As a result, the number of cells in the population begins to increase. Exponential Phase During the exponential phase, microorganisms grow and divide at the maximal rate possible given their genetic potential, the nature of the medium, and the environmental conditions. Their rate of growth is constant during the exponential phase; that is, they are completing the cell cycle and doubling in number at regular intervals (figure 7.10). The population is most uniform in terms of chemical and physiological properties during this phase; therefore exponential phase cultures are usually used in biochemical and physiological studies. The growth rate during exponential phase depends on several factors, including nutrient availability. When microbial growth is limited by the low concentration of a required nutrient, the final net growth or yield of cells increases with the initial amount of the limiting nutrient present (figure 7.11a). The rate of growth also increases with nutrient concentration (figure 7.11b) but it saturates, much like what is seen with many enzymes (see figure 10.16). The shape of the curve is thought to reflect the rate of nutrient uptake by microbial transport proteins. At sufficiently high nutrient levels, the transport systems are saturated, and the growth rate does not rise 23/10/18 9:19 am CHAPTER 7 | Bacterial and Archaeal Growth affect the growth of individual cells. DnaA, the protein that binds to the chromosome’s origin to initiate replication, becomes less active in stationary phase. Ongoing replication is completed, but no further initiation occurs. This is one of many ways the cell conserves energy by eliminating processes that are not essential to survival. Growth rate (hr–1) Yield (cells or mg/ml) 138 Nutrient concentration Nutrient concentration Death Phase Cells growing in batch culture cannot remain in stationary phase indefinitely. Eventually they enter a phase known as the death phase Figure 7.11 Nutrient Concentration and Growth. (a) The effect of changes in limiting nutrient (figure 7.10). During this phase, the number concentration on total microbial yield. At sufficiently high concentrations, total growth will plateau. of viable cells declines exponentially, with (b) The effect on growth rate. cells dying at a constant rate. Detrimental environmental changes such as nutrient deprivation and the buildup of toxic wastes cause irreparable harm further with increasing nutrient concentration. Bacteria to the cells. use many mechanisms to bring nutrients into the cell (section 3.3) (a) (b) Long-Term Stationary Phase Stationary Phase In a closed system such as a batch culture, population growth eventually ceases and the growth curve becomes horizontal (figure 7.10). This stationary phase is attained by some bacteria at a population level of around 109 cells per milliliter. Protist cultures often have maximum concentrations of about 10 6 cells per milliliter. Final population size depends on nutrient availability and other factors, as well as the type of microorganism. In stationary phase, the total number of viable microorganisms remains constant. This may result from a balance between cell division and cell death, or the population may simply cease to divide but remain metabolically active. Microbes enter the stationary phase for many reasons. One important reason is nutrient limitation; if an essential nutrient is severely depleted, population growth will slow and eventually stop. Aerobic organisms often are limited by O2 availability. Oxygen is not very soluble and may be depleted so quickly that only the surface of a culture will have an O2 concentration adequate for growth. Population growth also may cease due to the accumulation of toxic waste products. This seems to limit the growth of many cultures growing in the absence of O2. For example, streptococci can produce so much acid from sugar fermentation that growth is inhibited (see figure 11.20). Finally, some evidence exists that growth may cease when a critical population level is reached. Thus entrance into the stationary phase may result from several factors operating in concert. Information about the nutrient level that affects the growth of the population must be transmitted to the molecules that wil11886_ch07_128-169.indd 138 Long-term growth experiments reveal that after a period of exponential death some microbes have a long period where the population size remains more or less constant. This longterm stationary phase (also called extended stationary phase) can last months to years (figure 7.10). During this time, the bacterial population continually evolves so that actively reproducing cells are those best able to use the nutrients released by their dying brethren and best able to tolerate the accumulated toxins. This dynamic process is marked by successive waves of genetically distinct variants. Thus natural selection can be witnessed within a single culture vessel. Mathematics of Growth Knowledge of microbial growth rates during the exponential phase is indispensable to microbiologists. Growth rate studies contribute to basic physiological and ecological research, and are applied in industry. The quantitative aspects of exponential phase growth discussed here apply to microorganisms that divide by binary fission. During the exponential phase, each microorganism is dividing at constant intervals. Thus the population doubles in number during a specific length of time called the generation (doubling) time (g). This can be illustrated with a simple example. Suppose that a culture tube is inoculated with one cell that divides every 20 minutes (table 7.2). The population will be 2 cells after 20 minutes, 4 cells after 40 minutes, and so forth. Because the population is doubling every generation, the increase in population is always 2n where n is the number of 23/10/18 9:19 am 7.4 Growth Curves Consist of Five Phases 139 An Example of Exponential Growth1 60 ) Table 7.2 50 ) 1.000 1 Definitions of n, N0, Nt are as in figure 7.13. 2 The hypothetical culture begins with one cell having a 20-minute generation time. 3 Number of cells in the culture. 30 0.500 20 10 0 generations. The resulting population increase is exponential (figure 7.12). The mathematics of growth during the exponential phase is illustrated in figure 7.13, which shows the calculation of two important values. The growth rate constant (k) is the number of generations per unit time and is often expressed as generations per hour (hr−1). It can be used to calculate the generation time. As can be seen in figure 7.13, the generation time is simply the reciprocal of the growth rate constant. The generation time can also be determined directly from a semilogarithmic plot of growth curve data (figure 7.14). Once this is done, it can be used to calculate the growth rate constant. Calculation of the growth rate constant Let N0 = the initial population number For populations reproducing by binary fission Nt = N0 × 2n Solving for n, the number of generations, where all logarithms are to the base 10, log Nt = log N0 + n · log 2, and n= log Nt − log N0 log Nt − log N0 = log 2 0.301 The growth rate constant (k) is the number of generations n per unit time t . Thus () k= 0.000 0 20 40 60 80 Minutes of incubation Figure 7.12 Exponential Microbial Growth. Four generations of growth are plotted directly (•—•) and in the logarithmic form ( ˚—˚ ). The growth curve is exponential, as shown by the linearity of the log plot. Generation times vary markedly with the microbial species and environmental conditions. They range from less than 10 minutes (0.17 hours) to several days (table 7.3). Generation times in nature are usually much longer than in laboratory culture. Calculation of generation (doubling) time If a population doubles, then Nt = 2N0 Nt = the population at time t n = the number of generations in time t Log10 number of cells ( Number of cells ( 40 Substitute 2N0 into the growth rate constant equation and solve for k= log (2N0) − log N0 log 2 + log N0 − log N0 = 0.301g 0.301g k = g1 The generation time is the reciprocal of the growth rate constant. 1 g= k n log Nt − log N0 = t 0.301t Figure 7.13 Calculation of the Growth Rate Constant and Generation Time. The calculations are only valid for the exponential phase of growth, when the growth rate is constant. wil11886_ch07_128-169.indd 139 23/10/18 9:19 am 140 CHAPTER 7 | Bacterial and Archaeal Growth 3.00 Table 7.3 Examples of Generation Times1 Exponential phase 2.00 Number of cells (X107) 1.00 0.50 Lag phase 0.10 0 1 2 4 3 5 g Time (hours) Figure 7.14 Generation Time Determination. The generation time can be determined from a microbial growth curve. The population data are plotted with the logarithmic axis used for the number of cells. The time to double the population number is then read directly from the plot. The log of the population number can also be plotted against time on regular axes. 1 Generation times differ depending on the growth medium and environmental conditions used. Comprehension Check 1. Define microbial growth. 2. Describe the phases of the growth curve and discuss the causes of each. 3. Why would cells that are vigorously growing have a shorter lag phase than those that have been stored in a refrigerator when inoculated into fresh culture medium? 4. Calculate the growth rate constant and generation time of a culture that increases in the exponential phase from 5 × 102 to 1 × 108 in 12 hours. 5. Suppose the generation time of a bacterium is 90 minutes and the number of cells in a culture is 103 cells at the start of the exponential phase. How many bacteria will there be after 8 hours of exponential growth? wil11886_ch07_128-169.indd 140 7.5 Environmental Factors Affect Microbial Growth After reading this section, you should be able to: a. Use terms that describe a microbe’s growth range or requirement for each of the factors that influence microbial growth b. Summarize the adaptations of extremophiles to their habitats c. Summarize the strategies used by nonextremophiles to acclimate to changes in their environment d. Describe enzymes observed in microbes that protect them against toxic O2 products Almost every year at Yellowstone National Park, a visitor is seriously burned or killed after falling into one of the park’s hot 23/10/18 9:19 am 7.5 Environmental Factors Affect Microbial Growth 141 springs. Yet, a rich microbial community lives in these same springs, as well as in the hot pots, fumaroles, and other thermal features of the park. Clearly, the adaptations of some microorganisms to what humans perceive as inhospitable, extreme environments are truly remarkable. Indeed, microbes are thought to be present nearly everywhere on Earth. Bacteria such as Bacillus infernus are able to live over 2.4 km below Earth’s surface, without oxygen and at temperatures above 60°C. Other microbes live at great ocean depths or in lakes such as the Great Salt Lake in Utah (USA) that have high sodium chloride concentrations. Microorganisms that grow in such harsh conditions are called extremophiles. Whether an extremophile or not, all microbes must respond to changes in their environment. However, if the conditions exceed their ability to respond, they will not grow and eventually they may die. Thus for each environmental parameter, all microbes have a characteristic range at which growth occurs defined by high and low values beyond which the microbe cannot survive. Within the range is an optimal value at which growth is best. Microorganisms in marine ecosystems (section 30.2); The subsurface biosphere is vast (section 31.4) To study the ecological distribution of microbes, it is important to understand the strategies they use to survive. An understanding of the environmental influences on microbes and their activity also aids in the control of microbial growth. In this section, we briefly review the effects of the most important environmental factors on microbial growth. Major emphasis is given to solutes and water activity, pH, temperature, oxygen level, pressure, and radiation. The adaptations that microbes have evolved to live in their environments also are discussed. Table 7.4 summarizes how microorganisms are categorized in terms of their response to these factors. Solutes Affect Osmosis and Water Activity Water is critical to the survival of all organisms, but water can also be destructive. Solutes in an aqueous solution alter the behavior of water. One way this occurs is the phenomenon of osmosis, which is observed when two solutions are separated by a semipermeable membrane that allows movement of water but not solutes. If the solute concentration of one solution is higher than the other, water moves to equalize the concentrations. In other words, water moves from solutions with lower solute concentrations to those with higher solute concentrations. Because a selectively permeable plasma membrane separates a cell’s cytoplasm from its environment, microbes can be affected by changes in the solute concentration of their surroundings. If a microorganism is placed in a hypotonic solution (one with a lower solute concentration; solute concentration is also referred to as osmotic concentration or osmolarity), water will enter the cell and cause it to burst unless something prevents the influx of water or inhibits plasma membrane expansion. Conversely, if the microbe is placed in a hypertonic solution (one with a higher osmotic concentration), water will flow out of the cell. In microbes that have cell walls, the membrane shrinks away from the cell wall. wil11886_ch07_128-169.indd 141 Dehydration of the cell in hypertonic environments may damage the plasma membrane and cause the cell to become metabolically inactive. Because of the potential damaging effects of uncontrolled osmosis, it is important that microbes be able to respond to changes in the solute concentrations of their environment. Microbes in hypotonic environments are protected in part by their cell wall, which prevents overexpansion of the plasma membrane. However, not all microbes have cell walls. Wall-less microbes can be protected by reducing the osmotic concentration of their cytoplasm; this protective measure is also used by many walled microbes to provide protection in addition to their cell walls. Microbes use several mechanisms to lower the solute concentration of their cytoplasm. For example, some bacteria have mechanosensitive (MS) channels in their plasma membrane. In a hypotonic environment, the membrane stretches due to an increase in hydrostatic pressure and cellular swelling. MS channels then open and allow solutes to leave. Thus MS channels act as escape valves to protect cells from bursting. Many protists use contractile vacuoles to expel excess water. Some microbes are adapted to extreme hypertonic environments, and can be called osmophiles. By definition, osmophiles include halophiles, which require the presence of NaCl at a concentration above about 0.2 M (figure 7.15). However, usually osmophile refers to organisms that require high concentrations of sugars. Here we focus on halophiles. Extreme halophiles have adapted so completely to hypertonic, saline conditions that they require NaCl concentrations between about 3 M and saturation (about 6.2 M). Archaeal halophiles can be isolated from the Dead Sea (a salt lake between Israel and Jordan), the Great Salt Lake in Utah, and other aquatic habitats with salt concentrations approaching saturation. Halophiles generally are able to live in their high-salt habitats because they synthesize or obtain from their environment molecules called compatible solutes. Compatible solutes can be kept at high intracellular concentrations without interfering with metabolism and growth. Some compatible solutes are inorganic molecules such as potassium chloride (KCl). Others are organic molecules such as choline, betaines (neutral molecules having both negatively charged and positively charged functional groups), and amino acids such as proline and glutamic acid. The use of compatible solutes extends beyond halophiles. This is also a strategy utilized by many osmophiles. Furthermore, many microorganisms, whether in hypotonic or hypertonic environments, use compatible solutes to keep the osmotic concentration of their cytoplasm somewhat above that of the habitat so that the plasma membrane is always pressed firmly against the cell wall. For instance, fungi and photosynthetic protists employ the compatible solutes sucrose and polyols (e.g., arabitol, glycerol, and mannitol) for this purpose. Some of the best-studied halophiles belong to the archaeal order Halobacteriales. These archaea accumulate potassium and chloride ions to remain hypertonic to their environment; the internal potassium concentration may reach 4 to 7 M. Their 23/10/18 9:19 am 142 CHAPTER 7 Table 7.4 | Bacterial and Archaeal Growth Microbial Responses to Environmental Factors wil11886_ch07_128-169.indd 142 23/10/18 9:19 am Growth rate 7.5 Environmental Factors Affect Microbial Growth 143 0 2 1 3 4 water. These osmotolerant microorganisms grow over wide ranges of water activity but optimally at higher levels. Osmotolerant organisms can be found in all domains of life. For example, Staphylococcus aureus is halotolerant, can be cultured in media containing up to about 3 M sodium chloride, and is well adapted for growth on the skin. The yeast Zygosaccharomyces rouxii grows in sugar solutions with aw values as low as about 0.65. The photosynthetic protist Dunaliella viridis tolerates sodium chloride concentrations from 1.7 M to a saturated solution. In contrast, xerotolerant microbes can withstand either high solute concentrations or the effects of desiccation. These microbes exist in desert regions, household dust, and in preserved foods. However, most microorganisms only grow well at water activities around 0.98 (the approximate aw for seawater) or higher. This is why drying food or adding large quantities of salt and sugar effectively prevents food spoilage. Various methods are used to control food spoilage (section 41.2) NaCl concentration (M) Comprehension Check Nonhalophile Moderate halophile Halotolerant Extreme halophile Figure 7.15 The Effects of Sodium Chloride on Microbial Growth. Four different patterns of microbial dependence on NaCl concentration are depicted. The curves are only illustrative and are not meant to provide precise shapes or salt concentrations required for growth. MICRO INQUIRY What is the difference between halophilic and halotolerant? enzymes, ribosomes, and transport proteins are unusual because they require high potassium levels for stability and activity. In addition, their plasma membranes and cell walls are stabilized by high concentrations of sodium ion. If the sodium concentration decreases too much, the wall and plasma membrane disintegrate. Although extreme halophiles have successfully adapted to environmental conditions that would destroy most organisms, they have become so specialized that they lack ecological flexibility. Haloarchaea (section 20.3) Another way solutes change the behavior of water is by decreasing the availability of water to microbes. When solutes such as salts and sugars are present, the water is “tied up” by its interaction with the solutes. Microbiologists express quantitatively the degree of water availability by determining water activity (aw). The water activity of a solution is 1/100 the relative humidity of the solution (when expressed as a percent). It is also equivalent to the ratio of the solution’s vapor pressure (Psoln) to that of pure water (Pwater). Distilled water has an aw of 1, milk has an aw of 0.97, a saturated salt solution has an aw of 0.75, and the aw of dried fruits is only about 0.5. To survive in a habitat with a low aw value, microorganisms must maintain a high internal solute concentration to retain wil11886_ch07_128-169.indd 143 1. How do microorganisms adapt to hypotonic and hypertonic environments? 2. Define water activity and briefly describe how it can be determined. Why is it difficult for microorganisms to grow at low aw values? 3. What are halophiles and why do they require sodium and potassium ions? pH pH is a measure of the relative acidity of a solution and is defined as the negative logarithm of the hydrogen ion concentration (expressed in terms of molarity). pH = − log [H+] = log(1/[H+]) The pH scale extends from pH 0.0 (1.0 M H+) to pH 14.0 (1.0 × 10−14 M H+), and each pH unit represents a 10-fold change in hydrogen ion concentration. Figure 7.16 shows that microbial habitats vary widely in pH—from pH 0 to 2 at the acidic end to alkaline lakes and soil with pH values between 9 and 10. Each species has a definite pH growth range and pH growth optimum. Acidophiles have their growth optimum between pH 0 and 5.5; neutrophiles, between pH 5.5 and 8.0; and alkaliphiles (alkalophiles), between pH 8.0 and 11.5. In general, different microbial groups have characteristic pH preferences. Most known bacteria and protists are neutrophiles. Most fungi prefer more acidic surroundings, about pH 4 to 6; photosynthetic protists also seem to favor slight acidity. Many archaea are acidophiles. For example, the archaeon Sulfolobus acidocaldarius is a common inhabitant of acidic hot springs; it grows well from pH 1 to 3 and at high temperatures. The archaea Ferroplasma acidarmanus and Picrophilus oshimae can actually grow very close to pH 0. Alkaliphiles are distributed among all three domains of life. They include bacteria belonging to the genera Bacillus, Micrococcus, Pseudomonas, and Streptomyces; yeasts 23/10/18 9:19 am 144 CHAPTER 7 | Bacterial and Archaeal Growth 1M pH optima of some microbes pH [H+] ACIDIC 10–1M 0 Ferroplasma spp. (A) 1 Human stomach fluid Lemon juice Acid mine drainage Dunaliella acidophila (E) Cyanidium caldarium (E) Thiobacillus thiooxidans (B) Sulfolobus acidocaldarius (A) 10–2M 2 10–3M 3 Grapefruit juice Oranges 10–4M 4 Beer Tomato juice 10–5M 5 Cheese Physarum polycephalum (E) 10–6M 6 Beef Lactobacillus acidophilus (B) E. coli, Pseudomonas aeruginosa (B) 7 Milk Pure water Human blood 10–7M NEUTRAL 10–8M 8 –9 9 10 M 10–10M 10 10–11M 11 Seawater Staphylococcus aureus (B) Nitrosomonas spp. (B) Baking soda Soap Microcystis aeruginosa (B) Bacillus alcalophilus (B) Household ammonia 10–12M 12 10–13M 13 Bleach 10–14M ALKALINE 14 Figure 7.16 The pH Scale. The pH scale and examples of substances with different pH values. Several microorganisms are placed at their growth optima. A, archaeon; E, eukaryote; B, bacterium. and filamentous fungi; and numerous archaea. Bec