IM-Microbial Physiology [UPDATED]-1 PDF

POLYTECHNIC UNIVERSITY OF THE PHILIPPINES COLLEGE OF SCIENCE Department of Biology INSTRUCTIONAL MATERIAL IN MICROBIAL PHYSIOLOGY (Lecture) 1st SEMESTER SY 2021-2022 Lourdes V. Alvarez, MSc, PhD, RMicro. ...

POLYTECHNIC UNIVERSITY OF THE PHILIPPINES COLLEGE OF SCIENCE Department of Biology INSTRUCTIONAL MATERIAL IN MICROBIAL PHYSIOLOGY (Lecture) 1st SEMESTER SY 2021-2022 Lourdes V. Alvarez, MSc, PhD, RMicro.  ABOUT THE COURSE Microbial Physiology deals with the study of physiological processes in microorganisms including cellular structure, growth, nutrition, energy production, macromolecular biosynthesis, and metabolism with emphasis on prokaryotic system. The microorganisms are diverse group that play very valuable role in the environment as producers, consumers and decomposers. They are the only group of organisms that take part in all the three important stages in the ecosystem. Different type of microorganisms has their own mode of physiological processes. Being minute in size, microorganisms produce several enzymes and metabolites that has industrial importance. These enzymes and metabolites are regulated by specific genes under the influence of several environmental factors. It is therefore necessary to understand microbial physiology to identify these factors and learn how to manipulate and regulate the enzymes and metabolites produced by microorganisms which has important application in the industry, medicine and agriculture. Microbes are special group of organisms. They have unique and diverse metabolic activity that are not observed to other groups of living organisms. They have the ability to degrade different carbon sources converting them both aerobically and anaerobically to metabolites of commercial value. The main objective of this course is to discuss the different physiological processes in different kinds of microbes with the main focus on prokaryotes. Discussion will begin with the overview of the structure and function of the microbial cell, how the cells divide, and the necessary nutrients needed by the microbes to survive. The main topic of the course will focus on the different microbes and how they produce energy and synthesize macromolecules for metabolism. At the end of the course, the students are expected to understand the microbes and the physiological processes in various kinds of microorganisms. It is hoped that the students will be acquainted with the information that they are supposed to gain with the course and that these knowledges can be used and applied in their future work in the field of microbiology 2  COURSE OUTCOMES A. Cognitive Aims 1. Identify the physiological processes in microorganisms; 2. Illustrate the cellular structure of microorganisms with emphasis on prokaryotic system; 3. Discuss the microbial growth and know the various physical and chemical growth requirements of microbes and get equipped with various methods of bacterial growth measurement; 4. Explain the energy production, macromolecular biosynthesis as well as the microbial transport systems and the modes and mechanisms of energy conservation in microbial metabolism – Autotrophy and heterotrophy; and 5. Design a simple microbiological research in order to fully understand and appreciate the physiological process in microorganisms in the living world. B. Value Aims 1. Respect for the diversity of microscopic organisms with regards to their distinct physiological processes in nature; 2. Realization of the importance of microorganisms in harnessing the natural microbial physiological processes for the well-being of man; 3. Deep appreciation for personal and community hygiene thus, committed to share in the prevention and control of infectious diseases; and 4. Nurture the values of discipline, commitment strong sense of responsibility, intellectual honesty and appreciation for hard work. 3  GENERAL DIRECTIONS The following are the instructions on how you will take the course BIOL 40065 (Microbial Physiology) at home. 1. Study the attached course syllabus of BIOL 40065 (Microbial Physiology) carefully. The different topics in the course syllabus are distributed in accordance to the week number on which said topic needs to be studied. 2. Read and study this instructional material (IM) to serve as guide on what to study for the week. Reference material (e-copy of Madigan MT, Bender KS, Buckley DH, Sattley WM, Stahl DA. (2019). Brock Biology of Microorganisms 15th edition. PearsonBenjamin Cummings: New York) will be sent to your email upon your request. You may send your request to Dr. Lourdes V. Alvarez at [email protected] point presentations for each of the topic may also be sent to your email upon your request, including the links of the online reference materials. Other reference materials are available at http://ils.pup.edu.ph/. 3. At the end of each module in this IM is a set of questions that you need to answer to assess whether you have learned from the assigned topic. Answer each of the question in the not more 50 words and write your answers in a yellow paper. Compile all your answers and place it an envelope for submission at the end of the 1st semester SY 2021-2022.The below rubric will serve as guide on how to earn points for every question in the assessment. Criteria Level 4 Level 3 Level 2 Level 1 2 points 1.5 points 1 point 0.5 point No mistakes in Grammar grammar or Isolated Few Multiple spelling mistakes mistakes mistakes 2 points 2.5 points 1.5 points 1 point Information is Information Information is Information Information is complete with complete with is not very no added added information complete light/effortless information 5.5 points 4.5 points 3.5 points Quality of Response is 2.5 points response/answer insightful, shows Response Response is Response to the questions thoughtful and shows though just enough shows effort effort, shows and effort optimal thought Over-all score 10 8 6 4 4. Also, attached to this IM are the quizzes, Midterm Examination and Final Examination. You may answer and detach those pages of quizzes and exams from the IM and may be submitted at the end of the semester together with the answers of your assessments. Each question is equivalent to 1 point. 5. The grading system for this course is indicated in the course syllabus. 6. For any further questions, please contact your professors: - Lourdes V. Alvarez, MSc, PhD, RMicro. – [email protected] 4  TABLE OF CONTENTS Page No. About the Course 2 Course Outcomes 3 General Directions 4 Week Module Title No. No. 1 1 Definition and Scope of Microbial Physiology 6 1 2 Diversity of Life: Bacteria, Archaea, and Eukarya 7 2 3 Cells of Bacteria and Archaea 13 2 4 The Cytoplasmic Membrane and Transport 17 3 5 Bacterial Cell Walls: Peptidoglycan 22 3 6 Other Cell Surface and Inclusions 29 4 7 Endospore and the Sporulation Cycle 34 4 8 Flagella, Motility, and Chemotaxis 39 5 9 Eukaryotic Microbial Cells 46 Quiz no. 1 54 Microbial Cell Growth and its Control: Cell Division and Population 6 10 56 Growth Microbial Cell Growth and its Control: Culturing Microbes and 6 11 63 Measuring Their Growth Microbial Cell Growth and its Control: Environmental Effects on 7 12 67 Growth (Temperature) Microbial Cell Growth and its Control: Environmental Effects on 7 13 71 Growth (pH, Osmolarity, Oxygen) 8 14 Microbial Cell Growth and its Control: Control of Microbial Growth 75 Quiz no. 2 79 9 Mid-Term Examination 81 10 15 Microbial Nutrients and Nutrient Uptake 90 10 16 Energetics, Enzymes and Redox 97 11-12 17 Catabolism: Fermentation and Respiration 106 13 18 Microbial Metabolism: Biosynthesis 118 14 19 Metabolic Diversity of Microorganisms: Phototrophy 124 Quiz No. 3 136 Metabolic Diversity of Microorganisms: Autotrophy and Nitrogen 14 20 138 Fixation Metabolic Diversity of Microorganisms: Respiratory Processes 15 21 146 Defined by Electron Donor Metabolic Diversity of Microorganisms: Respiratory Processes 16 22 150 Defined by Electron Acceptor 16 23 Metabolic Diversity of Microorganisms: One-Carbon (C1) Metabolism 153 17 24 Metabolic Diversity of Microorganisms: Fermentation 156 17 25 Metabolic Diversity of Microorganisms: Hydrocarbon Metabolism 163 Quin No. 4 167 Final Examination 169 5  Module 1 DEFINITION AND SCOPE OF MICROBIAL PHYSIOLOGY I. Learning Outcomes After successful completion of this module, you should be able to appreciate and understand the definition and scope of the Microbial Physiology course, which includes microbial: l Cell structure and function l Microbial growth and nutrition l Bioenergetics and metabolism II. Course Material Microbial physiology is defined as the study of physiological processes in microscopic organisms which include the cellular structure, growth, nutrition, energy production macromolecular biosynthesis, and metabolism with emphasis on prokaryotic system. It also covers the study of viruses, bacteria, fungi and parasites. The study of microbial cell functions includes the study of microbial growth, microbial metabolism and microbial cell structure. Microbial physiology is important in the field of metabolic engineering and also functional genomics. Fundamentally, microbial physiology is an enormous discipline encompassing the study of thousands of different microorganisms. III. Other Learning Resources Reference file: l Course Syllabus in Microbial Physiology ASSESSMENT Answer the following questions in not more than 100 words (10 pts. Each). 1. In your own words, please define the meaning of microbial physiology. 2. What do you think is the importance of microbial physiology in this present time? 3. Can you give the advantages of microbial physiology to other microbial fields? 6  Module 2 DIVERSITY OF LIFE: BACTERIA, ARCHAEA AND EUKARYA I. Learning Outcomes After successful completion of this module, you should be able to: l Understand the diversity of living organisms based on molecular basis of life; and l Be familiar with the microbial life: Bacteria, Archaea, Eukarya and Viruses II. Course Material Molecular Basis of Life Experiments with bacterial cultures in the twentieth century were critical in describing the foundations of molecular biology, molecular genetics, and biochemistry. Microbiologists came to realize that while microorganisms were incredibly diverse, all cells operated on similar principles. Albert Jan Kluyver (1888–1956) was Beijerinck’s successor at what was then called the Delft Institute of Technology. Kluyver recognized that though microbial diversity was tremendous, microorganisms used many of the same biochemical pathways and their metabolic processes faced similar thermodynamic constraints. Kluyver promoted the study of comparative biochemistry to identify the unifying features of all cells. He famously proclaimed, “From elephant to butyric acid bacterium—it is all the same!” This was later reformulated by Jacques Monod (1910–1976) into the expression, “What is true for E. coli is also true for the elephant,” a statement that proclaimed the importance of working with bacteria to understand the fundamental principles that govern all living things.The use of microbes as metabolic model systems led to the discovery that certain macromolecules and biochemical reactions are universal, and that to understand their function in one cell is to understand their function in all cells. These discoveries were of central importance to understanding microbial evolution and none were as important as the discovery of DNA as the molecular basis of heredity, a discovery that is less than 80 years old. In the early twentieth century, it was clear that some molecule carried the hereditary information from parent to offspring, but the molecular basis of heredity remained a mystery. Most biologists thought that proteins carried this hereditary information. DNA had been discovered but it was thought to be merely a structural molecule, and too simple in its composition to encode cellular functions. The hunt for the molecular basis of heredity began in earnest with an experiment by Frederick Griffith (1879–1941). Griffith worked with a virulent strain of Streptococcus pneumoniae, a cause of bacterial pneumonia in both humans and mice. This strain, strain S, produced a polysaccharide coat that caused cells to form smooth colonies and conferred the ability to kill infected mice (Figure 2.1a). A related strain, strain R, lacked this polysaccharide and produced “rough” colonies that did not cause disease. However, Griffith observed that strain R could be transformed to type S, forming smooth colonies and causing disease, when it was mixed with the dead remains of cells of strain S. He reasoned that some molecule that contained genetic information must have been transferred from strain R to strain S in this process, and this experiment showed that genetic transfer could be studied in bacteria. 7 Figure 2.1 Early evidence that DNA is the molecular basis of heredity. Later, the Avery–MacLeod–McCarty experiment (1944), named for three scientists at the Rockefeller University, would show that this “transforming principle” is DNA. They treated the dead remains of cells of strain S with chemicals and enzymes that destroyed protein and left behind only DNA. They then repeated Griffith’s experiment with the pure DNA of strain S and showed that this DNA was sufficient to cause transformation, causing strain R cells to become S-type cells and virulent (Figure 2.1b). They also demonstrated that transformation failed if the DNA from strain S was degraded. These experiments proved that DNA is the genetic material of cells. The discovery that DNA is the basis of heredity was followed by intense effort to understand how this molecule stores genetic information. The structure of DNA was ultimately solved by James D. Watson (1928–) and Francis Crick (1916–2004) using X-ray diffraction images of DNA taken by their colleague Rosalind Franklin (1920–1958). They revealed that DNA is composed of a double helix that contains four nitrogenous bases: guanine, cytosine, adenine, and thymine. Later research would reveal how the genetic code is read from DNA and translated into a protein alphabet, and these principles are covered in Chapter 4. Once again, however, this research to crack the code of life was enabled by a microbial model system, in this case, the bacterium Escherichia coli (commonly called E. coli). Not long after the discovery that genetic information is encoded in the sequence of biological molecules, Emile Zuckerkandl (1922–2013) and Linus Pauling (1901–1994) proposed that molecular sequences could be used to reconstruct evolutionary relationships. They recognized that evolution, as described by Darwin, required variation in offspring and that these variations must be caused by changes in molecular sequences. They predicted that these sequence differences occur randomly in a clock-like fashion over time. This led to the conclusion that the evolutionary history of organisms is inscribed in the sequence of molecules such as DNA. Carl Woese seized upon these insights to pursue the ambitious goal of reconstructing the evolutionary history of all cells. 8 Woose and the Tree of Life The ribosomal RNA (rRNA) genes, present in all cells, revolutionized the understanding of microbial evolution and made it possible to construct the first universal tree of life. Woese recognized that genes encoding rRNAs are excellent candidates for phylogenetic analysis because they are: (1) universally distributed, (2) functionally constant, (3) highly conserved (that is, slowly changing), and (4) of adequate length to provide a deep view of evolutionary relationships. Woose found out that the rRNA sequences from the methanogens were distinct from those of Bacteria and Eukarya, the only two domains recognized at that time. He named this new group of prokaryotic cells the Archaea (originally Archaebacteria) and recognized them as the third domain of life alongside the Bacteria and the Eukarya (Figure 2.2). More importantly, Woese demonstrated that the analysis of rRNA gene sequences could be used to reveal evolutionary relationships between all cells, providing the first effective tool for the evolutionary classification of microorganisms. Figure 2.2 Evolutionary relationships and the phylogenetic tree of life. An Introduction to Microbial Life All cells are unified by the facts that their genetic blueprints are encoded in DNA and that evolution is the process by which their blueprints change over time. Microorganisms vary dramatically in size, shape, and structure. Moreover, the identification focuses on cellular forms of life, not all microbes form cells. In this section let’s study about Bacteria, Archaea, Eukarya, and viruses—the four groups into which all known microorganisms can be classified. 9  Figure 2.3 Microorganisms vary greatly in size and shape. Bacteria Bacteria have a prokaryotic cell structure and often thought of as undifferentiated single cells with a length that ranges from 1 to 10 μm. While bacteria that fit this description are common, the Bacteria are actually tremendously diverse in appearance and function. The smallest bacteria are no more than 0.15–0.2 μm in diameter and the largest can be as much as 700 μm long. Some bacteria can differentiate to form multiple cell types and others are even multicellular (for example, Magnetoglobus). Among the Bacteria, 30 major phylogenetic lineages (called phyla) have been described, and some of these phyla contain thousands of identified species while others contain only a few. More than 90% of bacteria in cultivation belong to one of only four phyla: Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes. The analysis of rRNA gene sequences and even entire genome sequences from environmental samples reveals that at least 80 bacterial phyla likely exist. Although species in some bacterial phyla are characterized by unique phenotypic traits, most bacterial phyla contain a wide diversity of species and show tremendous physiological diversity. The Proteobacteria illustrate this concept well as they include organisms with a diverse array of physiological traits including respiration (both with and without oxygen), fermentation of various types, diverse forms of phototrophy, and chemolithotrophic metabolisms using H2, sulfur or nitrogen compounds, or even metals. Species of Proteobacteria also possess a wide range of ecological strategies and can be found in all but the hottest and most salty environments on Earth. It is important to remember that while most phyla of plants and animals originated within the last 600 million years, bacterial phyla are billions of years old and this time has allowed for extensive experimentation and diversification. Archaea Like Bacteria, Archaea also have a prokaryotic cell structure and they are quite diverse in their physiology, cultured isolates have less morphological diversity than Bacteria, and most described Archaea exist as undifferentiated cells that are 1 to 10 μm in length. The domain 10 Archaea consists of five welldescribed phyla: Euryarchaeota, Crenarchaeota, Thaumarchaeota, Nanoarchaeota, and Korarchaeota. As for the Bacteria, many lineages of Archaea are known only from rRNA genes or genome sequences recovered from the environment. Analysis of these environmental DNA sequences indicate more than 12 archaeal phyla likely exist. Archaea have historically been associated with extreme environments; the first isolates came from hot, salty, or acidic sites. But not all Archaea are extremophiles. Archaea are indeed common in the most extreme environments that support life, such as those associated with volcanic systems, and species of Archaea hold many of the records that define the chemical and physical limits of life. But in addition to these, Archaea are found widely in nature. For example, methanogens are common in wetlands and in the guts of animals (including humans). Methanogenic Archaea produce methane and have a major impact on the greenhouse gas composition of our atmosphere. In addition, species of Thaumarchaeota inhabit soils and oceans worldwide and are important contributors to the global nitrogen cycle. Archaea are also notable in that this domain lacks any known pathogens or parasites of plants or animals. Most described species of Archaea fall within the phyla Crenarchaeota and Euryarchaeota while only a handful of species have been described for the Nanoarchaeota, Korarchaeota, and Thaumarchaeota. Eukarya Plants, animals, and fungi are the most well-characterized groups of Eukarya. These groups are relatively young in relation to Bacteria and Archaea, originating during a burst of evolutionary radiation called the Cambrian explosion, which began about 600 million years ago. The first eukaryotes, however, were unicellular microbes. Microbial eukaryotes, which include diverse algae and protozoa, may have first appeared as early as 2 billion years ago, well before the origin of plants, animals, and fungi. The major lineages of Eukarya are traditionally called kingdoms instead of phyla. There are at least six kingdoms of Eukarya, and this diverse domain contains microorganisms as well as the plants and animals. Microbial eukaryotes vary dramatically in size, shape, and physiology. Among the smallest are the nanoflagellates, which are microbial predators that can be as small as 2 μm long. In addition, Ostreococcus, a genus of green algae that contains species that are 0.8 μm in diameter, is smaller than many bacteria. The largest single-celled organisms are eukaryotes, but they are hardly microbial. Xenophyophores are amoeba-like, single-celled organisms that live exclusively in the deep ocean. Exploration of the Mariana Trench has revealed xenophyophores up to 10 cm in length. In addition, plasmodial slime molds consisting of a single cytoplasmic compartment can be up to 30 cm in diameter. Microbial eukaryotes include diverse phototrophic organisms, microbial predators, symbionts and parasites, along with a range of other physiological types. VIruses Viruses are not found on the tree of life. Indeed, it can be argued that they are not truly alive. Viruses are obligate parasites that can only replicate within the cytoplasm of a host cell. Viruses are not cells, and they lack the cytoplasmic membrane, cytoplasm, and ribosomes found in all forms of cellular life. Viruses cannot conserve energy and they do not carry out metabolic processes; instead, they take over the metabolic systems of infected cells and turn them into vessels for producing more viruses. Unlike cells, which all have genomes composed of double-stranded DNA, viruses have genomes composed of DNA or RNA that can be either double- or single-stranded. Viral genomes are often quite small, with the smallest having only 11 three genes. The small size of most viral genomes means that no genes are conserved among all viruses, or between all viruses and all cells; hence it may be impossible to ever place viruses into the tree of life or build a universal viral phylogenetic tree that includes all viruses. Viruses are as diverse as the cells they infect, and viruses are known to infect cells from all three domains of life. Viruses are often classified on the basis of structure, genome composition, and host specificity. Viruses that infect bacteria are called bacteriophages (or phages, for short). Bacteriophages have been used as model systems to explore many aspects of viral biology. While most viruses are considerably smaller than bacterial cells, there are also unusually large viruses such as the Pandoraviruses, which can be more than 1 micrometer long and have a genome of as many as 2500 genes, larger than that of many bacteria. ASSESSMENT Answer the following questions in not more than 50 words (10 pts. Each). 1. Explain the strain S and R in the experiment of Griffith. 2. What experiment proved the DNA was the transforming principle? 3. What is the ribosomal RNA (rRNA) genes and its importance? 4. What are the 4 reasons Woese recognized that genes encoding rRNAs are excellent candidates for phylogenetic analysis? 5. What is the essence of phylogenetic tree in microbial physiology? 6. What are the main differences of Bacteria, Archaea, Eukarya and Viruses? 7. What are the five well-described phyla of Archaea? 12  Module 3 CELLS OF BACTERIA AND ARCHAEA I. Learning Outcomes After successful completion of this module, you should be familiar with the basic morphological features of microbial cell in accordance to the following: l Shapes and arrangements of the cell; and l Microbial cell sizes and its significance II. Course Material Cell Morphology In microbiology, the term morphology means cell shape. Several morphologies are found among Bacteria and Archaea, and the most common ones are described by terms that are part of the essential lexicon of the microbiologist. Common morphologies of prokaryotic cells are shown in Figure 3.1. A cell that is spherical or ovoid in morphology is called a coccus (plural, cocci). A cylindrically shaped cell is called a rod or a bacillus. Some cells form curved or loose spiral shapes and are called spirilla. The cells of some Bacteria and Archaea remain together in groups or clusters after cell division, and the arrangements are often characteristic. For instance, some cocci form long chains (for example, the bacterium Streptococcus), others occur in three- dimensional cubes (Sarcina), and still others in grape-like clusters (Staphylococcus). Some morphological groups are immediately recognizable by the unusual shapes of their individual cells. Examples include the spirochetes, which are tightly coiled Bacteria; bacteria that form extensions of their cells as long tubes or stalks (appendaged forms); and filamentous bacteria, which form long, thin cells or chains of cells. The cell morphologies described here are representative but certainly not exhaustive; many variations of these morphologies are known. For example, there can be fat rods, thin rods, short rods, and long rods, a rod simply being a cell—roughly in the shape of a cylinder—that is longer in one dimension than in the other. Cell morphologies thus form a continuum, with some shapes, such as rods and cocci, being very common, whereas others, such as spiral, budding, and filamentous shapes, are less common. 13  Figure 3.1 Cell morphologies, beside each drawing is a phase-contrast photomicrograph of cells showing that morphology. Morphology and Biology Although cell morphology is easily determined, it is a poor predictor of other properties of a cell. For example, under the microscope many rod-shaped Archaea are indistinguishable from rod-shaped Bacteria, yet we know they are of different phylogenetic domains. With rare exceptions, it is impossible to predict the physiology, ecology, phylogeny, pathogenic (disease- causing) potential, or virtually any other major property of a prokaryotic cell by simply knowing its morphology. Nevertheless, cell morphology is an important characteristic that is always noted when describing a particular species of Bacteria or Archaea. Although we know quite a bit about how cell shape is controlled, we know relatively little about why a particular cell displays the morphology it does. The morphology of a given microbe is undoubtedly the result of the selective forces that have shaped its evolution to maximize fitness for competitive success in its habitat. Some examples of these might include evolving an optimal cell shape to maximize nutrient uptake for survival in nutrient-limiting environments (small cells and others with high surface-to-volume ratios, such as appendaged cells), evolving a morphology to exploit swimming motility in viscous environments (helical- or spiral-shaped cells), or evolving a morphology that facilitates gliding motility along a surface (filamentous bacteria). The Small World Cells of Bacteria and Archaea vary in size from as small as about 0.2 micrometer (μm) in diameter to those more than 700 μm in diameter (Table 3.1). The vast majority of rod-shaped species that have been cultured are between 0.5 and 4 μm wide and less than 15 μm long. A few very large Bacteria are known, such as Epulopiscium fishelsoni, whose cells exceed 600 μm (0.6 millimeter) in length. This bacterium, phylogenetically related to the endospore-forming bacterium Clostridium and found in the gut of the surgeonfish, contains multiple copies of its genome. The many copies are apparently necessary because the volume of an Epulopiscium cell is so large that a single copy of its genome would be insufficient to support its transcriptional and translational demands. 14 Table 3.1 Cell size and volume of some cells of Bacteria, from the largest to the smallest. Cells of the largest known bacterium, the sulfur-oxidizing chemolithotroph Thiomargarita, are even larger than those of Epulopiscium, about 750 μm in diameter; such cells are just visible to the naked eye. Why these cells are so large is not well understood, although for sulfur bacteria a large cell size may have evolved for storing inclusions of sulfur (used as an energy source). No species of Archaea are known that rival Epulopiscium or Thiomargarita in cell size, but that may simply be because they remain undiscovered. It is hypothesized that the upper size limit for prokaryotic cells results from the decreasing ability of larger and larger cells to transport nutrients (their surface-to-volume ratio is very small; see the next subsection). Since the metabolic rate of a cell varies inversely with the square of its size, for very large cells, nutrient uptake would eventually limit metabolism to the point that the cell would no longer be competitive with smaller cells. Figure 3.2 Two very large Bacteria. (a) Epulopiscium fishelsoni. The rod-shaped cell is about 600 μm (0.6 mm) long and 75 μm wide and is shown with four cells of the protist Paramecium (a microbial eukaryote), each of which is about 150 μm long. (b) Thiomargarita namibiensis, a large sulfur chemolithotroph and currently the largest known of all prokaryotic cells. Cell widths vary from 400 to 750 μm. 15  Very large cells are uncommon in the prokaryotic world. In contrast to Thiomargarita or Epulopiscium (Figure 3.2), the dimensions of an average rod-shaped bacterium, such as Escherichia coli, for example, are about 1–2 μm; these dimensions are typical of cells in the prokaryotic world. By contrast, eukaryotic cells can be as small as 2 to more than 600 μm in diameter, although very small microbial eukaryotes (cells less than about 6 μm in diameter) are uncommon. ASSESSMENT Answer the following questions in not more than 50 words (10 pts. Each). 1. Give at least five morphology shapes found in Bacteria and Arcahea. 2. Using a microscope, can you easily identify a pathogenic organism to a non-pathogenic organism? 3. What are the advantages of cell being small? 4. What is the ratio of a cell controls many of its properties, including its growth rate and evolution? 16  Module 4 THE CYTOPLASMIC MEMBRANE AND TRANSPORT I. Learning Outcomes After successful completion of this module, you should be able to: l Know the composition of the bacterial and archaeal cell membranes and their function as permeability barrier, protein anchor and for energy conservation l Understands the three classes of transport systems II. Course Material Bacterial Cytoplasmic Membrane The cytoplasmic membrane of all cells is a phospholipid bilayer containing embedded proteins. Phospholipids are composed of both hydrophobic (water-repelling) and hydrophilic (waterattracting) components (Figure 4.1). In Bacteria and Eukarya, the hydrophobic component consists of fatty acids and the hydrophilic component of a glycerol molecule containing phosphate and one of several other functional groups (such as sugars, ethanolamine, or choline) bonded to the phosphate. The fatty acids point inward toward each other to form a hydrophobic region, while the hydrophilic portion remains exposed to either the environment or the cytoplasm. That is, the outer surface of the cytoplasmic membrane faces the environment while the inner Figure 4.1 Phospholipid bilayer membrane. (a) Structure of the surface faces the cytoplasm and phospholipid phosphatidylethanolamine. The side chains are fatty acids and the ester linkage (characteristic of the lipids of Bacteria and Eukarya interacts with the cytoplasmic milieu. but not Archaea) is boxed with a red dashed line. (b) General architecture This type of membrane structure is of a bilayer membrane; the blue spheres depict glycerol with phosphate and/or other hydrophilic groups. (c) Transmission electron micrograph of a called a lipid bilayer, or a unit membrane. The light inner area is the hydrophobic region of the model membrane because each membrane shown in b. phospholipid “leaf” forms half of the unit (Figure 4.2). The cytoplasmic membrane is only 8–10 nanometers wide but can be resolved easily by transmission electron microscopy. In addition, although physically weak, the cytoplasmic membranes of some Bacteria are strengthened by sterol-like molecules called hopanoids. 17 Sterols are rigid and planar molecules that strengthen the membranes of eukaryotic cells, many of which lack a cell wall. A variety of proteins are attached to or integrated into the cytoplasmic membrane; membrane proteins typically have hydrophobic domains that span the membrane and hydrophilic domains that contact the environment or the cytoplasm. Proteins significantly embedded in the membrane are called integral membrane proteins. By contrast, peripheral membrane proteins are more loosely attached. Some peripheral membrane proteins are lipoproteins, proteins that contain a hydrophobic lipid tail that anchors the protein into the membrane. Peripheral membrane proteins typically interact with integral membrane proteins in important cellular processes such as energy metabolism and transport. Figure 4.2 Structure of the cytoplasmic membrane. The inner surface (In) faces the cytoplasm and the outer surface (Out) faces the environment. Phospholipids compose the matrix of the cytoplasmic membrane with proteins embedded (integral) or surface associated (peripheral). The general design of the cytoplasmic membrane is similar in both prokaryotic and eukaryotic cells, although there are chemical differences between different species. Archaeal Membrane The cytoplasmic membrane of Archaea is structurally similar to those of Bacteria and Eukarya, but the chemistry is somewhat different. In contrast to the lipids of Bacteria and Eukarya in which ester linkages bond fatty acids to glycerol (Figure 4.1), the lipids of Archaea contain ether bonds between glycerol and a hydrophobic side chain that is not a fatty acid (Figure 4.3). The hydrophobic region of archaeal membranes is formed from repeating units of the five-carbon hydrocarbon isoprene, rather than from fatty acids. The cytoplasmic membrane of Archaea is constructed from either phosphoglycerol diethers, which have C20 side chains (called a phytanyl group), or diphosphoglycerol tetraethers (C40 side chains, called a biphytanyl group). In the tetraether lipid structure, the ends of the inwardly pointing phytanyl groups are covalently linked at their termini to form a lipid monolayer instead of a lipid bilayer membrane. 18  Figure 4.3 Major lipids of Archaea and the architecture of archaeal membranes. (a, b) Note that the hydrocarbon of the lipid is bonded to the glycerol by an ether linkage (in dashed red box in a) in both cases. The hydrocarbon is phytanyl (C20) in a and biphytanyl (C40) in b; both are multiples of the parent structure, isoprene (in dashed red oval; detailed structure shown in black box). (c) A major lipid of Thaumarchaeota is crenarchaeol, a lipid containing 5- and 6-carbon rings. (d, e) The membrane structure in Archaea may form a lipid bilayer or a lipid monolayer (or a mix of both). Some archaeal lipids contain rings within the hydrocarbon side chains. For example, crenarchaeol, a common membrane lipid in cells of Crenarchaeota (a major phylum of Archaea) contains four C5 rings and one C6 ring. These rings affect the chemical properties of the lipids and thus influence membrane function. As in other organisms, the polar head groups in archaeal lipids can be sugars, ethanolamine, or a variety of other molecules. Hopanoids, present in the cytoplasmic membranes of many Bacteria, have not been found in the cytoplasmic membranes of Archaea.Despite differences in chemistry between the cytoplasmic membranes of Archaea and organisms in the other phylogenetic domains, the fundamental construction of the archaeal cytoplasmic membrane—inner and outer hydrophilic surfaces and a hydrophobic interior—is the same as that of membranes in all cells. Obviously, evolution has selected this fundamental design as the best solution to the major functions of the cytoplasmic membrane, an issue we turn to now. 19 Cytoplasmic Membrane Function The cytoplasmic membrane has at least three major functions (Figure 4.4). First, it is the cell’s permeability barrier, preventing the passive leakage of solutes into or out of the cell. Second, the cytoplasmic membrane anchors several proteins that catalyze a suite of key cell functions. And third, the cytoplasmic membrane of Bacteria and Archaea plays a major role in energy conservation and consumption. Figure 4.4 The major functions of the cytoplasmic membrane. The cytoplasmic membrane is a barrier to the diffusion of most substances, especially polar or charged molecules. Because the cytoplasmic membrane is so impermeable, most substances that enter or leave the cell must be carried in or out by transport proteins. These are not simply ferrying proteins but instead function to accumulate solutes against the concentration gradient, a process that diffusion alone cannot do (Figure 4.5). Transport, which requires energy, ensures that the cytoplasm has sufficient concentrations of the nutrients it needs to perform biochemical reactions efficiently. Transport proteins typically display high sensitivity and high specificity. If the concentration of a solute is high enough to saturate the transporter, which often occurs at the very low concentrations of nutrients found in nature, the rate of uptake can be near maximal. Some nutrients are transported by a low-affinity transporter when present at high external concentration and by a separate, typically higher- affinity, transporter for those present at low concentration. Moreover, many transport Figure 4.5 The importance of transport in membrane function. In transport, the proteins transport only a single uptake rate shows saturation at relatively low external concentrations. Both high- kind of molecule while others affinity and low-affinity transport systems are depicted. 20 carry a related class of molecules, such as different sugars or different amino acids. This economizing reduces the need for separate transport proteins for each different sugar or amino acid. In addition to its permeability and transport functions, the cytoplasmic membrane of Bacteria and Archaea is a major site of both energy conservation and consumption. It have been discussed how the cytoplasmic membrane can be energized when protons (H+) are separated from hydroxyl ions (OH−) across the membrane surface. This charge separation creates an energized state of the membrane called the proton motive force, analogous to the potential energy present in a charged battery. Dissipation of the proton motive force can be coupled to several energy-requiring reactions, such as transport, cell locomotion, and the biosynthesis of ATP. In eukaryotic microbial cells, although transport across the cytoplasmic membrane is just as necessary as it is in prokaryotic cells, energy conservation takes place in the membrane systems of the cell’s key organelles, the mitochondrion (respiration) and chloroplast (photosynthesis). ASSESSMENT Answer the following questions in not more than 100 words (10 pts. Each). 1. Explain the structure and composition of cytoplasmic membrane. 2. What are the three main functions of cytoplasmic membrane? 3. What are the similarities and differences of cytoplasmic membrane in Archaea and Bacteria? 4. What is the importance of the fluid mosaic structure in cytoplasmic membrane?. 21  Module 5 BACTERIAL CELL WALLS: PEPTIDOGLYCAN I. Learning Outcomes After successful completion of this module, you should be able to: l Appreciate the uniqueness of bacterial and archaeal cell walls; l Identify the differences between Gram-positive and Gram-negative bacteria; and l Understand the relationship of the cell wall structure to the Gram-stain techniques II. Course Material Bacterial Cell Wall The cytoplasm of prokaryotic cells maintains a high concentration of dissolved solutes that creates significant osmotic pressure—about 2 atm (203 kPa); this is about the same as the pressure in an automobile tire. To withstand these pressures and prevent bursting—a process called cell lysis—most cells of Bacteria and Archaea have a layer outside the cytoplasmic membrane called the cell wall. Besides protecting against osmotic lysis, cell walls also confer shape and rigidity on the cell. Knowledge of cell wall structure and function is important not only for understanding the biology of microbial cells, but also because certain antibiotics, for example, the penicillins and cephalosporins, target bacterial cell wall synthesis, leaving the cell susceptible to osmotic lysis. Since human cells lack cell walls and are therefore not a target of such antibiotics, these drugs are of obvious benefit for treating bacterial infections. Cells of Bacteria can be divided into two major groups, gram-positive and gram- negative. The distinction between gram-positive and gram-negative bacteria is based on the Gram stain reaction, and differences in cell wall structure play a major role in the reaction. The surface of gram-positive and gram-negative cells as viewed in the electron microscope differs markedly, as shown in Figure 5.1. The gram-negative cell wall, or cell envelope as it is also called, consists of at least two layers, whereas the gram-positive cell wall is typically thicker and consists primarily of a single type of molecule. Gram-Positive Cell Wall As much as 90% of the cell wall of a gram-positive bacterium can consist of peptidoglycan. Although some bacteria have only a single layer of peptidoglycan, many gram- positive bacteria form several layers of peptidoglycan stacked one upon another. It is thought that peptidoglycan is synthesized by the cell in the form of “cables” about 50 nm wide, with each cable consisting of several glycan strands (Figure 5.2a). As peptidoglycan is synthesized, the cables themselves become cross-linked to form an even stronger cell wall structure. 22  Figure 5.1 Cell walls of Bacteria. (a, b) Schematic diagrams of gram-positive and gram-negative cell walls; the photo of Gram-stained bacteria in the center shows cells of Staphylococcus aureus (purple, gram-positive) and Escherichia coli (pink, gram-negative). (c, d) Transmission electron micrographs showing the cell wall of a gram-positive bacterium and a gram- negative bacterium, respectively. (e, f) Scanning electron micrographs of gram-positive and gram-negative bacteria, respectively. Note differences in surface texture. Each cell is about 1 μm wide. In addition to peptidoglycan, many gram-positive bacteria produce acidic molecules called teichoic acids embedded in their cell wall. Teichoic acids are composed of glycerol phosphate or ribitol phosphate with attached molecules of glucose or d-alanine (or both). Individual alcohol molecules are then connected through their phosphate groups to form long strands, and these are then covalently linked to peptidoglycan (Figure 5.2b). Teichoic acids also function to bind divalent metal ions, such as Ca2+and Mg2+, prior to their transport into the cell. Some teichoic acids are covalently bonded to membrane lipids rather than to peptidoglycan, and these are called lipoteichoic acids. Figure 5.2c summarizes the structure of the cell wall of gram-positive Bacteria and shows how teichoic acids and lipoteichoic acids are arranged in the overall wall structure. 23  A very few Bacteria and Archaea lack cell walls altogether. These include in particular the mycoplasmas, pathogenic Bacteria related to gram-positive bacteria that cause a variety of infectious diseases of humans and other animals, and Thermoplasma and some of its relatives (Archaea). Lacking a cell wall, these cells would be expected to contain unusually tough cytoplasmic membranes, and chemical analyses show that they do. For example, most mycoplasmas contain sterols in their cytoplasmic membranes; these molecules function to add strength and rigidity to the membrane as they do in the cytoplasmic membranes of eukaryotic cells. Thermoplasma membranes contain molecules called lipoglycans that serve a similar strengthening function. Gram-Negative Cell Wall In gram-negative bacteria, only a small amount of the total cell wall consists of peptidoglycan, as most of the wall is composed of the outer membrane. This layer is effectively a second lipid bilayer, but it is not constructed solely of phospholipid and protein, as is the cytoplasmic membrane. Instead, the outer membrane also contains polysaccharide, and the lipid and polysaccharide are linked to form a complex. Hence, the outer membrane is often called the lipopolysaccharide layer, or simply LPS for short. The outer membrane confers only modest structural strength on the gram-negative cell (peptidoglycan remains the major strengthening agent), but it acts as an effective barrier against many substances such as lipophilic antibiotics and other harmful agents that might otherwise penetrate the cytoplasmic membrane. Indeed, many antibiotics that are clinically useful against gram- positive bacterial pathogens show little to no activity against gram- Figure 5.2 Structure of the gram-positive bacterial cell wall. (a) Schematic of a gram-positive rod showing the internal architecture of negative pathogens because of their the peptidoglycan “cables.” (b) Structure of a ribitol teichoic acid. The outer membrane. teichoic acid is a polymer of the repeating ribitol unit shown here. (c) Summary diagram of the gram-positive bacterial cell wall. 24 Figure 5.3 The gram-negative bacterial cell wall. (a) Arrangement of lipopolysaccharide, lipid A, phospholipid, porins, and Braun lipoprotein in the outer membrane. (b) Transmission electron micrograph of a cell of Escherichia coli showing the cytoplasmic membrane and wall. (c) Molecular model of porin proteins. Note the four pores present, one within each of the proteins forming a porin molecule and a smaller central pore (circled) between the porin proteins. The view is perpendicular to the plane of the membrane. Differential Staining Stains that render different kinds of cells different colors are called differential stains. An important differential-staining procedure used in microbiology is the Gram stain (Figure 5.5). On the basis of their reaction in the Gram stain, bacteria can be divided into two major groups: gram-positive and gram-negative. After Gram staining, gram-positive bacteria appear purple- violet and gram-negative bacteria appear pink. The color difference in the Gram stain arises because of differences in the cell wall structure of gram-positive and gram-negative cells. Staining with a basic dye such as crystal violet renders cells purple in color. Cells are then treated with ethanol, which decolorizes gram-negative cells but not gram-positive cells. Finally, cells are counterstained with a different-colored stain, typically the red stain safranin. As a result, gram-positive and gram-negative cells can be distinguished microscopically by their different colors. 25 Figure 5.4 Staining cells for microscopic observation. Stains improve the contrast between cells and their background. The Gram stain is the most common staining procedure used in microbiology, and it is often performed to begin the characterization of a new bacterium. If a fluorescence microscope is available, the Gram stain can be reduced to a one-step procedure; grampositive and gram-negative cells fluoresce different colors when treated with a special chemical. Figure 5.5 The Gram stain. (a) Steps in the procedure. (b) Microscopic observation of gram- Archaeal Cell Walls positive (purple) and gram-negative (pink) bacteria. The organisms are Staphylococcus aureus and Escherichia coli, respectively. (c) Cells of Pseudomonas aeruginosa A variety of cell wall structures are (gram-negative, green) and Bacillus cereus (gram- positive, orange) stained with a one-step fluorescent found in Archaea, including walls containing staining method. This method allows for differentiating polysaccharides, proteins, or glycoproteins or gram-positive from gram-negative cells in a single some mixture of these macromolecules. staining step. Pseudomurein and Other Polysaccharide Cell Walls The cell walls of certain methane-producing Archaea (methanogens) contain a molecule that is remarkably similar to peptidoglycan, a polysaccharide called pseudomurein (the term “murein” is from the Latin word for wall and was an old term for peptidoglycan) (Figure 5.6). The backbone of pseudomurein is formed from alternating repeats of N- acetylglucosamine (also present in peptidoglycan) and N-acetyltalosaminuronic acid; the latter 26 replaces the N-acetylmuramic acid of peptidoglycan. Pseudomurein also differs from peptidoglycan in that the glycosidic bonds between the sugar derivatives are β-1,3 instead of β-1,4, and the amino acids are all of the l stereoisomer (Figure 5.6). Because in many respects they are so similar, it is likely that peptidoglycan and pseudomurein are variants of a cell wall polysaccharide originally present in the common ancestor of Bacteria and Archaea. However, although they are structurally very similar, they differ sufficiently that pseudomurein is immune from destruction by both lysozyme and penicillin, molecules that destroy peptidoglycan. Figure 5.6 Pseudomurein. Structure of pseudomurein, the Cell walls of some other Archaea lack cell wall polymer of Methanobacterium species. pseudomurein and instead contain other polysaccharides. For example, Methanosarcina species have thick polysaccharide walls composed of polymers of glucose, glucuronic acid, galactosamine uronic acid, and acetate. Extremely halophilic (salt-loving) Archaea such as Halococcus, which are related to Methanosarcina, have similar cell walls that contain large amounts of sulfate. The negative charges on the sulfate ion (SO42–) bind the abundant Na+ present in the habitats of Halococcus. S-Layers The most common type of cell wall in Archaea is the paracrystalline surface layer, or S-layer as it is called. S- layers consist of interlocking molecules of protein or glycoprotein (Figure 5.7). The paracrystalline structure of S-layers can form various symmetries, including hexagonal, tetragonal, or trimeric, depending upon the number and structure of the subunits of which it is composed. S- layers have been found in representatives of all major lineages of Archaea and also in some species of Bacteria (Figure 2.16). The cell walls of some Archaea, for example the methanogen Methanocaldococcus jannaschii, consist only of an S-layer. Thus, S-layers are sufficiently strong to withstand osmotic pressures without any other wall components. However, in many organisms S-layers are present in addition to other cell wall components, Figure 5.7The S-layer. Transmission electron micrograph of an S- usually polysaccharides. When an S-layer layer fragment showing its paracrystalline nature. Shown is the S- layer from Aquaspirillum (Bacteria); this S-layer shows hexagonal accompanies other wall components, the symmetry common in S-layers of Archaea. S-layer is always the outermost wall layer; 27 that is, the layer that is in direct contact with the environment. Besides serving as protection from osmotic lysis, S-layers undoubtedly have other functions. For example, as the interface between the cell and its environment, it is likely that the S-layer functions as a selective sieve, allowing the passage of low-molecular-weight solutes while excluding large molecules or structures (such as viruses or lytic enzymes). The S-layer may also function to retain proteins near the cell surface that must function outside the cytoplasmic membrane, much as the outer membrane retains periplasmic proteins and prevents their drifting away in gram-negative Bacteria. ASSESSMENT Answer the following questions in not more than 100 words (10 pts. Each). 1. What is the main composition of peptidoglycan? 2. What are the main differences of cell wall in gram-positive and gram-negative bacteria? 3. Differentiate the results of differential staining method in gram-positive and gram-negative bacteria? 28  Module 6 OTHER CELL SURFACE AND INCLUSIONS I. Learning Outcomes After successful completion of this module, you should be able to: l Identify other bacterial cell surface structures such as capsules, slime layers, fimbriae, pili, cell inclusions, and gas vesicles. l Familiar with the important function of each structure in Bacteria and Archaea. II. Course Material Bacteria and Archaea may have other layers or structures in addition to the cytoplasmic membrane and cell wall. Cells in contact with the environment often contain one or more types of cellular inclusions. Cell Surface Structures Many Bacteria and Archaea secrete sticky or slimy materials on their cell surface that consist of either polysaccharide or protein. However, these are not considered part of the cell wall because they do not confer significant structural strength on the cell. The terms “capsule” and “slime layer” are used to describe these layers. Capsules and Slime Layers The terms capsule and slime layer are often used interchangeably, but the two terms do not refer to the same thing. If the layer is organized in a tight matrix that excludes small particles and is tightly attached, it is called a capsule. Capsules are readily Figure 7.1 Bacterial capsules and slime formation. (a) A viscid colony of the bacterium Leuconostoc mesenteroides (lifted up by an inoculating loop) contains a thick dextran (glucose polymer) slime layer formed by the cells. (b) Capsules of Acinetobacter species observed by phase-contrast microscopy after negative staining with India ink. (c) Transmission electron micrograph of a thin section of cells of Rhodobacter capsulatus with capsules (arrows) clearly evident; cells are about 0.9 μm wide. (d) Transmission electron micrograph of Rhizobium trifolii stained with ruthenium red to reveal the capsule. The cell is about 0.7 μm wide. 29 visible by light microscopy if cells are treated with India ink, which stains the background but not the capsule, and can also be seen in the electron microscope (Figure 6.1b-d). By contrast, if the layer is more easily deformed and loosely attached, it will not exclude particles and is more difficult to see microscopically. This form is called a slime layer and is easily recognized in colonies of slime-forming species such as the lactic acid bacterium Leuconostoc (Figure 6.1a). Outer surface layers have several functions. Surface polysaccharides assist in the attachment of microorganisms to solid surfaces. As we will see later, pathogenic microorganisms that enter the body by specific routes usually do so by first binding specifically to surface components of host tissues; this binding is often facilitated by bacterial cell surface polysaccharides. When the opportunity arises, many bacteria will bind to solid surfaces, often forming a thick layer of cells called a biofilm. Extracellular polysaccharides play a key role in the development and maintenance of biofilms as well. Besides attachment, outer surface layers have other functions.These include acting as virulence factors (molecules that contribute to the pathogenicity of a bacterial pathogen) and preventing dehydration. For example, the causative agent of the diseases anthrax and bacterial pneumonia—Bacillus anthracis and Streptococcus pneumoniae, respectively—each contain a thick capsule of either protein (B. anthracis) or polysaccharide (S. pneumoniae). Encapsulated cells of these bacteria avoid destruction by the host’s immune system because the immune cells that would otherwise recognize these pathogens as foreign and destroy them are blocked from doing so by the bacterial capsule. In addition to this role in disease, outer surface layers of virtually any type bind water and because of this likely protect the cell from desiccation in periods of dryness. Fimbriae, Pili, and Hami Fimbriae and pili are thin (2–10 nm in diameter) filamentous structures made of protein that extend from the surface of a cell and can have many functions. Fimbriae (Figure 7.2) enable cells to stick to surfaces, including animal tissues in the case of pathogenic bacteria, or to form pellicles (thin sheets of cells on a liquid surface) or biofilms on solid surfaces. Pili are similar to fimbriae, but are typically longer and only one or a few pili are present on the surface of a cell. All gram-negative bacteria produce pili of one Figure 7.2 Fimbriae. Electron micrograph of a dividing sort or another, and many gram-positive cell of Salmonella enterica (typhi), showing flagella and fimbriae. A single cell is about 0.9 μm wide. bacteria also contain these structures. Because pili can be receptors for certain types of viruses, they can be easily seen under the electron microscope when they become coated with virus particles (Figure 7.3). Many classes of pili are known, distinguished by their structure and function. Two very important functions of pili include facilitating genetic exchange between cells in a process called conjugation (conjugative or Figure 7.3 Pili. The pilus on an Escherichia coli cell that sex pili) and enabling the adhesion of is undergoing conjugation with a second cell is better pathogens to specific host tissues that they resolved because viruses have adhered to it. The cells are about 0.8 μm wide. 30  subsequently invade (type IV and other pili). Type IV pili not only facilitate specific adhesion but also support an unusual form of cell movement called twitching motility in certain bacterial species. On rod-shaped cells that move by twitching, type IV pili are present only at the poles. Twitching motility is a type of gliding motility, movement along a solid surface. In twitching motility, extension of pili followed by their retraction drags the cell along a solid surface, and ATP supplies the energy necessary for this movement. The motility of certain species of Pseudomonas and Moraxella are the best-known examples of twitching motility. Cell Inclusions Prokaryotic cells often contain inclusions of one sort or another. Inclusions function as energy reserves and/or carbon reservoirs or have special functions. Inclusions can often be seen in cells with the light microscope and are enclosed by a thin membrane that partitions off the inclusion in the cytoplasm. Storing carbon or other substances in an insoluble form is advantageous because it reduces the osmotic stress that the cell would encounter should the same amount be dissolved in the cytoplasm. Carbon Storage Polymers One of the most common inclusion bodies in prokaryotic organisms is poly-b-hydroxybutyric acid (PHB), a lipid that is formed from β- hydroxybutyric acid units. The monomers of PHB polymerize by ester linkage and then the polymer aggregates into granules; the granules can be seen by either light or electron microscopy (Figure 7.4). Another storage inclusion is glycogen, which is a polymer of glucose; like PHA, glycogen is a reservoir of both carbon and energy and is produced when carbon is in excess. Glycogen resembles starch, the major storage reserve of plants, but differs slightly from starch in the manner in which the glucose units are linked together. Polyphosphate, Sulfur, and Carbonate Minerals Many prokaryotic and eukaryotic microbes accumulate inorganic phosphate (PO43−) in the form of polyphosphate granules. These granules are formed when phosphate is in excess and can Figure 7.4 Pili. Poly-b-hydroxyalkanoates (PHAs). (a) Chemistry of poly- β-hydroxybutyrate, a common PHA. A be drawn upon as a source of phosphate for monomeric unit is shown in color. Other PHAs are made by nucleic acid and phospholipid biosynthesis when substituting longer-chain hydrocarbons for the –CH3 group on the β-carbon. (b) Electron micrograph of a thin section of phosphate is limiting. In addition, in some cells of a bacterium containing granules of PHB. Color organisms, polyphosphate can be broken down to photo: Nile red–stained cells of a PHA-containing synthesize the energy-rich compound ATP from bacterium. ADP. Many gram-negative Bacteria and Archaea oxidize reduced sulfur compounds, such as hydrogen sulfide (H2S); these organisms are the 31 “sulfur bacteria,” discovered by the great Russian microbiologist Sergei Winogradsky. The oxidation of sulfide generates electrons for use in energy metabolism (chemolithotrophy) or CO2 fixation (autotrophy). In either case, elemental sulfur(S0) from the oxidation of sulfide may accumulate in the cell in microscopically visible granules. This sulfur remains as long as the source of reduced sulfur from which it was derived is still present. However, as the reduced sulfur source becomes limiting, the S0 in the granules is oxidized to sulfate (SO42−), and the granules slowly disappear. Interestingly, although sulfur globules appear to reside in the cytoplasm, they are actually present in the periplasm. In these cells the periplasm expands outward to accommodate the growing globules as H2S is oxidized to S0 and then contracts inward as S0 is oxidized to SO42−. Filamentous cyanobacteria have long been known to form carbonate minerals on the external surface of their cells. However, some cyanobacteria also form carbonate minerals inside the cell, as cell inclusions. For example, the unicellular cyanobacterium Gloeomargarita forms intracellular granules of benstonite, a carbonate mineral that contains barium, strontium, and magnesium. The microbiological process of forming minerals is called biomineralization. Magnetic Storage Inclusions Some bacteria can orient themselves within a magnetic field because they contain magnetosomes. These structures are biomineralized particles of the magnetic iron oxides magnetite [Fe(II)Fe(III)2O4] or greigite [Fe(II)Fe(III)2S4] (Figure 7.5). Magnetosomes impart a magnetic dipole on a cell, allowing it to orient itself in a magnetic field. This allows the cell to undergo magnetotaxis, the process of migrating along Earth’s magnetic field lines. Magnetosomes have been found in several aquatic organisms that grow best at low O2 concentrations or are anaerobic. It has thus been hypothesized that one function of magnetosomes may be to guide these aquatic cells downward (the direction of Earth’s magnetic field) toward the sediments where O2 is low or absent. Gas Vesicles Figure 7.5 Magnetotactic bacteria and magnetosomes. (a) Differential interference contrast micrograph of coccoid Some Bacteria and Archaea are magnetotactic bacteria; note chains of magnetosomes planktonic, meaning that they inhabit the (arrows). A cell is 2.2 μm wide. (b) Magnetosomes isolated from the magnetotactic bacterium Magnetospirillum water column of lakes and the oceans. magnetotacticum; each particle is about 50 nm wide. (c) Most planktonic organisms move up and Transmission electron micrograph of magnetosomes from an unnamed magnetic coccus. The arrow points to the membrane down with changes in currents, but some that surrounds each magnetosome. A single magnetosome is can float because they contain gas about 90 nm wide. vesicles, structures that confer buoyancy 32 and allow the cells to position themselves in regions of the water column that best suit their metabolisms. Gas vesicles are conical-shaped structures made of protein; they are hollow yet rigid and of variable length and diameter (Figure 7.6). Gas vesicles in different species vary in length from about 300 to more than 1000 nm and in width from 45 to 120 nm, but the vesicles of a given species are of constant size. Gas vesicles may number from a few to hundreds per cell and are impermeable to water and solutes but permeable to Figure 7.6 Gas vesicles. Phase-contrast photomicrograph of gases. The presence of gas vesicles in Anabaena. cells can be detected either by light microscopy, where clusters of vesicles, called gas vacuoles, appear as irregular bright inclusions (Figure 7.6), or by transmission electron microscopy of cell thin sections. ASSESSMENT Answer the following questions in not more than 100 words (10 pts. Each). 1. Explain the difference between a capsule and slime layer? 2. Give at least one function of fimbriae, pili, and hami. 3. How are the two proteins that make up the gas vesicle, GvpA and GvpC, arranged to form such a water-impermeable structure? 33  Module 7 Endospore and Sporulation Cycle I. Learning Outcomes After successful completion of this module, you should be able to: l Understand the structure and functions of endospres; l Learn how they are form during sporulation II. Course Material Endospores: An Overview Certain species of Bacteria produce structures called endospores (Figure 7.1) during a process called endosporulation or sporulation. Endospores (endo- means “within”) are highly differentiated cells that are extremely resistant to heat, harsh chemicals and radiations. It function as survival structures and enable the organisms to endure favorable growth condition, including but not limited to extremes of temperature, drying or nutrient depletion. Thus, endospores can be thought of as the dormant stage of a bacterial life cycle. Moreover, endospores are ideal for dispersal through wind, water, or animal gut. It is only present in some gram-negative bacteria. Some bacteria that forms endospores are considered as serious pathogens of humans and other animals because of its ability to survive outside the host during endospore stage until a favorable condition within the host support the disease. (a) Terminal (b) Subterminal (c) Central endospores endospores endospores Figure 7.1 The bacterial endospore. Phase-contrast photomicrographs showing different intracellular locations of endospores in different species of bacteria. Endospore Formation and Germination During endospore formation, a vegetative cell is converted ito a nongrowing, heat- resistant, and light-refractive structure (Figure 7.2). Cells do not sporulate when they are actively growing but only when growth ceases owing to the exhaustion of an essential nutrient. 34  Endospores can remain dormant for years but can convert back to a vegetative cell rapidly. This process occurs in three steps (Figure 7.3): l Activaion. It occurs when endospores are heated for several minutes at an elevated but sublethal temperature. l Germination. It is typically a rapid process which occurs in a matter of minutes and is signaled by the loss of reflectility of the endospore and loss of resistance to heat and chemicals. l Outgrowth. It involves visible swelling due to water uptake and synthesis RNA, proteins, and DNA. The vegetative cell emerges from the broken endospore and begins to grow, remaining in vegetative growth until environmentals signals once again trigger sporulation. Figure 7.2 The life cycle of an endospore-forming bacterium. The phase-contrast photomicrographs are of cells of Clostridium pascui. Endospore Structure and Features l Endospores are visible by light microscopy s strongly refractile structure; l Endospores are impermeable to most dyes, so occasionally they are seen as unstained regions within cells that have been stained with (a) (b) (c) (d) basic dyes such as methylene Figure 7.3 Endospore germination in Bacillus. (a) a highly refractile free endospore. (b) Activation: Refractility is blue. diminishin. (c, d) Outgrowth: The new vegetative cell is l Special stains and procedures emerging. are used to stain endospores. n Malachite green is used in the classical endospore-staining protocol. It is infused into the spore with steam. l Endospore contains many layers absent from vegetative cell (Figure 7.4). n The outermost layer of the endospore is called exosporium, a thin protein covering. n Moving inward, there are several spore coats, composed of layers of spore- specific proteins (Figure 7.4b). n Below the spore coat is the cortex, which consists of loosely cross-linked peptidoglycan. n Inside the cortex is the core, which contains the core wall, cytoplasmic membrane, cytoplasm, nucleoid, ribososmes, and other cellular essentials. 35  l One substance found in endospores but not in vegetative cells is dipicolinic acid (Figure 7.5a), which accumulates in the core.Endospores contain large amounts of calcium (Ca2+), most of which is complexed with dipicolinic acid. l The calcium-dipicolinic acid (DPA) complex forms about 10% of the dry weight of the endospore and functions to bind free water within the endospore, helping to dehydrate the developing endospore. It inserts between bases in DNA, which helps stabilize DNA against heat denaturation. l The ensospore core cotains high levels of Figure 7.4 Structure of the bacterial small acid-soluble spore proteins (SAPs), which are endospore. (a)Transmission electron only made during sporulation process and have at least micrograph of a thin section through an endospore of Bacillus megaterium. (b) two functions: Fluorescent photomicrograph of a cell of n SAPs bind tightly to DNA in the core and Bacillus subtilis undergoing protect it from potential damage from ultraviolet sporuulation. The green color is a dye that specifically stains a sporulation radiation, desiccation, and dry heat. n SAPs function as a carbon and energy source for the outgrowth of a new vegetative cell from the endospore during germination. Figure 7.5 Dipicolnic acid. (a) Table 7.1 Differences between endospores and Structure of DPA. (b) How Ca2+ cross-links DOA molecules to form a vegetative cells. complex. Characteristic Vegetative Endospore cell Microscopic Nonrefractile Refractile appearance Calcium content Low High Dipicolinic acid Absent Present Enzymatic activity High Low Respiration rate High Low or Absent Macromolecular Present Absent sythesis Heat resistance Low High Radiation Low High resistance Resistance to Low High chemicals Lysozome Sensitive Resistant Water content High, 80-90% Low, 10-25% in core 36  Small acid-soluble spore Absent Pres proteins The Sporulation Cycle Sporulation is a form of cellular differentiation and many genetically directed changes in the cell occur during the conversion from vegetative growth to sporulation (Table 7.1). The structural chainges in sporulating cells of Bacillus are illustrated in Figure 7.6. Sporulation can be divided into several stages and in certain species such as Bacillus subtilis, the conversion of a vegetative cell into a endospore takes about 8 hours and begin with asymmetric cell division. Key events such as asymmetric cell division, cortex formation, and SASP production take place in defined sequence and at specific time in the sporulation cycle. Figure 7.6 Stages in endospore formation. The stages are defined from genetic and microscopic analyses of sporulation in Edosporulation requires Bacillus subtilis, the model organism for studies of sporulation. differential protein synthesis which SAPs, small acid-soluble proteins.. occurs by the sequential activation of several families of endospore-specific genes ad the turning off of many vegetative cell functions. The proteins encoded by sporulation-specific genes catalyze the series of events leading from the moist, metabolizing, vegetative cell to the relatively dry, metabolically inert, but extremely resistant endospores. III. Other Learning Resources Reference video l Sporulation:https://youtu.be/NAcowliknPs and https://youtu.be/VbDHV7j5-PQ ASSESSMENT Answer the following questions in not more than 100 words (10 pts. Each). 1. Is an endospore still the same bacteria cell? 2. Enumerate and describe the different types of endospores as to their location in the cell. 3. Illustrate and describe the structure of endospores. 4. Briefly explain the importance of SASPs in the sporulation process. 5. In your own words, explain how endospores are form during sporulation. 37  Module 8 Flagella, Motility, Chemotaxis I. Learning Objectives After successful completion of this module, you should be able to: l Appreciate microbial locomotion through bacterial and archaeal flagella; l Know flagellar structure and its synthesis and how are they related to cell’s speed and motion; and l Learn how bacterial cell respond to environmental attractant through chemotaxis. II. Course Material Many microbial cells can move under their own power. Motility allows cells to reach different parts of their environment, and in nature, a new location may offer additional resources for a cell and spell the difference between life and death. Flagella, Archaella, and Swimming Motility Many Bacteria are motile by swimming due to a structure called the flagellum (plural, flagella) (Figure 8.1); an analagous structure called the archaellum is present in many Archaea. The flagella and archaella are tiny (a) (b) (c) rotating machines that function to push or Figure 8.1 Bacterial flagella. (a) Peritrichous (b) Polar (c) pull the cell through a liquid. Lophotrichous. Flagella and Flagellation Bacterial flagella are long thin appendages, usually 15 to 20 nm wide, depending on the species), free at one end and anchored into the cell at the other end. It can be stained and observed by light microscopy (Figure 8.1) or electron microscopy (Figure 8.2). Flagella can be anchored to a cell in different location (Figure 8.3): l Polar flagellation. In this flagellation, flagella are attached at one or both ends of a cell. n Lophotrichus is a type of polar flagellation where a group of flagella called tuft, arise at one end of the cell. n Amphitrichous is a type of polar flagellation where a tuft of flagella emerges from both poles of the cell. l Petrichous flagellation. In this flagellation, flagella are inserted around the cell surface. The swimming motions of polarity and lophotrichously flagellated organisms differ from those of peritrichously flagellated organisms, and this can be distinguished microscopically. Peritrichously flagellated organisms typically move slowly in a straight line. By contrast, polarly flagellated organisms move more rapidly, often spinning around and seemingly dashing from place to place. 38 (a) (b) Figure 8.2 Bacterial flagella as observed Figure 8.3 Flagellation in bacteria. Illustration of polar and by negative stain in the transmission peritrichoud flagella. electron microscope. (a) Single polar flagellum (b) Peritrichous flagella. Flagella Structure Activity Flagella are not straight structures but are helical. The main part of the flagellum, called the filament, is composed of many copies of protein called flagellin. The amino acis sequences of flagellin is highly conserved in Bacteria, suggesting that flagellar motility evolved early and has deep roots within this domain. In addition to the filament, a flagellum consists of several other components. A wider region at the base of the filament called the hook consists of a single type of protein and connects the filament to the flagellum motor in the base (Figure 8.4). The flagellum motor is a reversible rotating machine composed of several proteins and is anchored in the cytoplasmic membrane and cell wall. The motor consists of a central rod that passes through a series of rings: l L-ring is an outer ring anchored in the outer membrane in gram-negative bacteria. 39  l P-ring is the second ring anchored in the peptidoglycan layer. l MS and C rings are the third set of rings located within the cytoplasmic membrane and cytoplasm, resectively (Figure 8.4a). In gram-positive bacteria, which lack outer membrane, only the inner pair of rings is present. Surrounding the inner ring and anchored in the cytoplasmic membrane and peptidoglycan are a series of proteins called Mot proteins. Another set of proteins, called Fili proteins, function as the Figure 8.4 Structure and function of the motor switch , reversing the direction of rotation of flagellum in gram-enegative bacteria. (a) the flagella in response to intracellular signals. Structure (b) Proton Turine model. The flagellum motor contains two main components: n Rotor. It is consists of the central rod and the L, P, C, and MS rings. Collectively, these structures make up the flagellar basal body. n Stator. It is consists of the Mot proteins that surround the rotor and function to generate the torque. Rotation of the flagellum occurs at the expense of the proton motive force, and it is thought that rotation is imparted to the flagellum by a type of “proton turbine” process. In this model, potons that are flowing through the Mot proteins, exert forces on charges present on the C and MS rings, thereby spinning the rotor. Flagellar Synthesis Several genes encode the motility apparatus of Bacteria. In Escherichia and Salmonella species, in which motility studies have been extensive, over 50 genes are linked to motility in one way or another. These genes encode the structural proteins of the flagellum and motor apparatus, of course, but also encode proteins that export the structural proteins through the cytoplasmic membrane to the outside of the cell and proteins that r