MKB 244 Study Guide 2023 Final PDF

MICROBIOLOGY 244 Study Guide by Henry Viljoen __________________________________________________________________________________ Note to the Reader Hey there! Thanks for using these notes. I hope you find them helpful. If there are any problems regarding unnecessary grammatical or sentence errors...

MICROBIOLOGY 244 Study Guide by Henry Viljoen __________________________________________________________________________________ Note to the Reader Hey there! Thanks for using these notes. I hope you find them helpful. If there are any problems regarding unnecessary grammatical or sentence errors, or incorrect content, please feel free to contact me on WhatsApp: 082 865 8139, or send me an email: [email protected] Disclaimer: These notes have been compiled using the lecture notes provided by Prof Alfred Botha as intended for the Microbiology 244 Module of 2022 at Stellenbosch University, South Africa. These notes also include information form the prescribed textbook (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) and various online resources to fill any gaps left on the slides, and to give a more thorough understanding of the concepts. Unless stated otherwise, all diagrams and images are from the lecture slides and the prescribed textbook. Where there is background knowledge given that will not be assessed, I have indicated in textboxes. It is thus, recommended to read the textboxes before studying a page. __________________________________________________________________________________ Note for Studying At first glance, Microbiology 244 may seem quite intimidating, and the number of facts can be overwhelming. My advice firstly, is to relax, make sure you are comfortable with your surroundings and not easily distracted. Make your favourite cup of tea and put a relaxing playlist on if that will calm you. Once you are set up and ready to go, remember that the point of assessments is to test your knowledge and not purely your parrot-skills. As you work through these notes and any additional content, make sure you understand what you are reading and test yourself consistently. Understanding metabolism and oxygen requirements is a major and hidden gem that will undoubtedly bring you success. Search for links between the environments the organisms find themselves in and ponder how they would interact if they were included in a single niche. Would one’s metabolic products be the other’s metabolic substrates? Would they even survive the new habitat, or would their oxygen requirements lead to their fatality? As important as understanding is, however, there are quite a few random facts that might be important yet difficult to remember in the bigger picture. Here, mnemonics and acronyms are a lifesaver! I have given a few examples throughout the notes showing ways to memorize lists. Lastly, I would like to remind you that you are more than your memory and more than your knowledge. Scientists dedicate years to their respective fields. Undergraduate is merely an introduction so do not stress if you do not grasp all the concepts immediately – you are only human. __________________________________________________________________________________ Happy Studying! CHAPTER 3, 4 & 5: Bacterial, Archaeal & Eukaryotic Cell Structure HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Bacterial, Archaeal and Eukaryotic Cell Structure Presented by Prof. A. Botha Bacterial Cell Structure The diagrams represent a basic annotated sketch of the bacterial cell structure. Recall the functions of the different components by reviewing your Microbiology 214 notes. Study tip: Understand the structure of prokaryotic cells to make studying the differences and similarities between the three domains easier. The diagram to the left represents a more specific cell of the species Paramecium caudatum. MCB 244 1 CHAPTER 3, 4 & 5: Bacterial, Archaeal & Eukaryotic Cell Structure HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Eukaryotic Cell Structure The diagram to the left represents various eukaryotic microbes. It is not important to remember each one specifically. Just note that there is lots variation between eukaryotic cells and that reality often differs from the classic textbook cell diagram. General Characteristics of Eukaryotic Cells Membrane-delimited nuclei. Membrane-bound organelles that perform specific functions. More structurally complex than prokaryotic cells. Generally larger than a prokaryotic cell. Study tip: Understand the structure of eukaryotic cells to make studying the differences and similarities between the three domains easier. EXTRA The images on this page are from the textbook and just show the variation in the eukaryotic cells and their structure. MCB 244 2 CHAPTER 3, 4 & 5: Bacterial, Archaeal & Eukaryotic Cell Structure HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Arhacaeal Cell Structure Archaea will be discussed in more depth later in the module. For now, focus on the cell structure and features that differentiate this microbe from bacteria and eukarya. NOTE ❖ At first glance, archaeal cells may look like bacterial cells. The major difference lies in the composition and presence/ absence of components from the cells. ❖ The similarities and difference are shown in the table below which is from the textbook. ❖ Archaeal cell wall structure will be discussed later in the module. MCB 244 3 CHAPTER 3, 4 & 5: Bacterial, Archaeal & Eukaryotic Cell Structure HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Comparison of Prokaryotic, Eukaryotic and Archaeal Cells The most obvious difference between eukaryotic and prokaryotic cells is that the first possess a variety of complex membrane organelles in the cytoplasmic matrix, and their genetic material is found inside membrane-included nuclei. Each organelle has a characteristic structure that is directly related to its specific function(s). In eukaryotes, genetic material is distributed between cells by the highly organized, complex processes that are called mitosis and meiosis. Note that archaea contain glycerol diethers and diglycerol tetraethers as their plasma membrane lipids. This distinguishes them from both bacteria and eukarya. MCB 244 4 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Taxonomy Presented by Prof. A. Botha Introduction to Taxonomy: Terminology All definitions are derived from the textbook. Taxonomy: The science of biological classification; it consists of three parts: classification, nomenclature, and identification. A taxonomic scheme is used to arrange organisms into groups called taxa (singular: taxon). Nomenclature: The branch of taxonomy concerned with the assignment of names to taxonomic groups in agreement with published rules. Identification: The process of determining if a particular isolate belongs to a recognized taxon and, if so, which one. Systematics: The scientific study of organisms with the ultimate objective of characterizing and arranging them in an orderly manner; often considered synonymous with taxonomy. o I.e., The study of organisms and using the results in the taxonomy of these organisms. o May include the following: ▪ Morphology – size, shape. ▪ Ecology – interactions with other organisms and the environment. ▪ Epidemiology – how does it spread to the host? What are the hosts? ▪ Biochemistry – characteristic enzymes or cell wall constituents. ▪ Molecular biology – characteristic genes. ▪ Physiology – anaerobe or aerobic? Maximum salt concentration? Polyphasic taxonomy: An approach in which taxonomic schemes are developed using a wide range of phenotypic and genotypic information. Natural classification: Arranges organisms into groups whose members share many characteristics. Natural Classification Aims to arrange organisms whose members share many characteristics in groups and to reflect the biological nature of these organisms as much as possible. In animals and plants, morphology may provide insight into evolutionary relatedness. However, morphology cannot be used with the same ease to classify microbes. o Polyphasic taxonomy is often used during Natural Classification to classify microbes. o Such approaches include phenotypic, phylogenetic, and genotypic features. Phenotypic (“Phenetic”) Classification Groups organisms together based on their phenotypic characteristics. As many as possible attributes are compared and organisms sharing many characteristics make up a single group or taxon. MCB 244 1 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Phylogenetic Classification Seeks to compare organisms based on evolutionary relationships. Phylogeny: Evolutionary development of a species. Today rRNA nucleotide sequence (16S (prokaryotic) and 18S (eukaryotic) rRNA sequences) analysis is used to determine evolutionary relationships among microbes. IMPORTANT Genotypic Classification Seeks to compare the genetic similarity between organisms. Individual genes or whole genomes can be compared. It was found that genomes with 70% or more shared homology, belong to the same species. Taxonomic Ranks Taxonomic ranks provide an organizational framework. The T indicates that it’s the Strain “Type strain”. Strains: o Biovars: Strains differ slightly based on their biochemical and physiological characteristics. o Morphovars: Strains differ in their morphology. o Serovars: Strains differ in their reaction with antibodies that are generated against these strains by the immune systems of laboratory organisms. NOTE Type strain: The first strain of a species that was described based on distinguishing characteristics and to which all other strains are compared. First name = Species are named according to the Binomial System of Linnaeus. Genus name Second name = o First time writing in a document: Write both names – do not abbreviate. Species name Written on paper: Underline both names. Typed: italicise both names. o Second time & beyond: Abbreviate the first name and write out the second. MCB 244 2 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Characteristics used Microbial Taxonomy Two approaches: Classical and Molecular Previously, microbial taxonomy and phylogeny consisted of several “classical” characteristics. These characteristics are still used today. However, modern microbial taxonomy and phylogeny are largely based on molecular characterization. The most durable identifications are those that are based on a combination of both classical and molecular approaches. Classical Characteristics Morphological characteristics. Physiological & Metabolic characteristics. Biochemical characteristics. Ecological characteristics. Study tip: Shape Size Stain – SSS Making acronyms and mnemonics Morphological Characteristics are useful for memorizing lists. Major characteristics: Cell shape, size and staining behaviour. Why stains? 1. Stained to visualize and group microbes according to their staining behaviour. 2. E.g., purple gram-positive and pink gram-negative bacteria. 1 2 4 3 MCB 244 3 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) The examples in the diagram on the previous page are visualised using stains and using a light microscope. Light microscopes are commonly used to analyse larger structures such as cell morphology. For the diagram on the previous page: Practice: Can you compare their 1. Escherichia coli and Staphylococcus aureus morphology and what colour(s) each ▪ E. coli: would stain? Gram-negative. Bacillus shaped. ▪ S. aureus: Gram-positive. Coccus shaped. ▪ Both are well-known to microbiologists since some strains belonging to these two species are used as experimental subjects in laboratories across the globe. 2. Bacillus subtilis ▪ Gram-positive. ▪ Staining: Green-stained spores (dormant cells). Red-stained vegetative cells (cells that are actively growing). 3. Bacillus anthracis ▪ Gram-positive. ▪ Rod-shaped. ▪ Risk: Causes anthrax. ▪ Historic role: Was developed as a biological weapon many years ago. 4. Corynebacterium diphtheria ▪ Gram-positive. ▪ Club-shaped. ▪ Risk: Causes diphtheria. Transmission electron microscopy (TEM) is used to study the ultrastructure of microbes (morphology of organelles in the cells). The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. MCB 244 4 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) NOTE ❖ This table is EXTRA information and not for assessment in MCB 244. ❖ It is purely included to give background knowledge regarding the different types of microscopies and which type can be used to visualise and analyse the various components of microbial cells. MCB 244 5 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) More examples of morphology visuals: NOTE The specialized way in which some bacteria move – some glide or wiggle through slimy substrates, and some cannot move at all. Type of cellular inclusions in cells of some microbial species – some have starch-like inclusions, while others may have lipid droplets inside their cells. Note: This table can be used to distinguish between organisms. E.g., The bacterium, E. coli, can be distinguished from the Archaea by looking at the chemical composition of the cell wall. MCB 244 6 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Physiological Characteristics Carbon Source Utilization 1. The ability to ferment certain carbohydrates. a. Test a microbe to ferment a series of different sugars. b. Traditionally used in the brewing industry, to identify yeast to be used in breweries. c. The microbe must ferment a carbohydrate that is dissolved in a liquid medium, within a test tube containing an inverted Durham tube. d. The inoculated tubes (each containing a Durham tube) are then incubated in an upright position at (generally) 25 oC for up to 3 weeks. e. The tubes are periodically inspected and if the yeast can ferment the sugar, a gas bubble will appear in the Durham tube. These tubes are inspected to see whether the organisms ferment and produce gas. f. Sometimes, a pH indicator is also added to the medium which changes colour as acids are created by the yeast. In such cases, the yeast is tested for both gas and acids during the fermentation of carbohydrates. 2. The ability to utilize certain carbon compounds aerobically. a. A series of different carbon sources (in liquid media) may be used to investigate what carbon source the yeast utilizes the best. The composition of the liquid media is not important – just take note of the process: i. The yeast is transferred from an agar slant/medium to sterile, distilled water. This water is used to wash off all the extra nutrients that occur on the surface of the yeast cells. ii. Then, a few drops of this watery suspension of the yeast, are used to inoculate a series of test tubes each containing synthetic media and a different carbon source. iii. The synthetic media are incubated further at 25 oC for up to 3 weeks. During the incubation period, the test tubes can be shaken to aerate them. The test tubes are inspected for milky yeast growth – which indicates that the yeast is growing and utilizing the carbon source. A Spectro- photometer can also be used to analyse yeast growth. EXTRA (Optional read): Examples of some carbon source compounds i. Hexoses: galactose, L-sorbose, L-rhamnose. ii. Saccharides: 1. Disaccharides: sucrose, maltose, cellobiose, trehalose, lactose, melibiose. 2. Trisaccharides: raffinose, melizitose. 3. Polysaccharides: soluble starch, inulin. 4. Pentoses: D-xylose, D-ribose, L-arabinose, D-arabinose iii. Alcohols: Erythritol, ribitol, D-mannitol, inositol, methanol, ethanol, glycerol, galactitol, D-glucitol. iv. Organic acids: succinate, citrate, lactate, malate, D-gluconate, 2-ketogluconate, 5-ketogluconate. v. Glucosides: α-methyl-D-glucoside, arbutin, salicin. MCB 244 7 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) 3. The concentration of the carbon sources is chosen in such a manner that there are just as many carbon atoms in the medium as there would have been in 0.5% glucose. 4. An internationally standardized synthetic medium is used to provide the rest of the nutrients. A carbon source test can also be miniaturized. o Each tube contains a differential medium. o These strips test for a range of physiological characteristics, such as carbon source utilization, the formation of fermentation products (acid formation) and enzyme production. Oxygen Relations Another physiological characteristic used to classify microbes, is their oxygen relations. This can be done by growing them in a semi-solid medium containing a little bit of agar within a test tube that is incubated in an upright position. The nature of a bacterial strain’s relationship with oxygen can be determined by observing where the bacterial growth occurs in the test tube. IMPORTANT MCB 244 8 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Other Physiological & Metabolic Characteristics Study Tip: Try to find 5 easy ones to 1. Carbon and nitrogen source remember by heart and use an acronym 2. Energy source or mnemonic. However, take note that 3. General nutritional type the exam might require one to know which examples are physiological or 4. Growth temperature – optimum and range biochemical. It is thus, important to 5. Oxygen relationships recognise these examples. 6. pH optimum and growth range 7. Salt requirements and tolerance 8. Secondary metabolites 9. Sensitivity to metabolic inhibitors & antibodies 10. Storage inclusions 11. Cell wall constituents 12. Fermentation products Question: Is osmotic tolerance 13. Luminescence morphological, physiological or 14. Motility metabolic? 15. Osmotic tolerance 16. Photosynthetic pigments MCB 244 9 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) While it is important to understand and Molecular Characteristics appreciate classical taxonomic approaches, modern-day genetics and microbiology rely Nucleic Acid Base Composition (Whole Genome Comparisons) profoundly on molecular classification methods. Determining G+C content: o The G+C content of a microbe’s genome can be determined from the melting temperature (Tm) value of its DNA. Tm is the temperature of DNA where 50% of the DNA is melted and 50% is still normal/intact. o This is usually determined in a specialised spectrophotometer that gradually heats the cuvette containing the DNA solution. o As the cuvette is heated, the absorbance of the DNA solution increases due to the breaking of hydrogen bonds and the DNA undergoes thermal denaturation (“melts”) to form single-stranded DNA. o The Tm of DNA is directly proportional to the relative quantity of G and C. Analyses of G+C content: o When the G+C content of two organisms differs by more than 10% = totally different genomes are involved. o When the G+C content is the same – no conclusions are possible because it might just be due to the chance that the concentrations are the same while the actual DNA sequences might differ. Notes on G+C content: o Prokaryote G+C ranges between 25 – 80%. o G+C content is conserved within a species. o Many bacterial genera are characterized by their G+C content. MCB 244 10 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Nucleic Acid Hybridization (Whole Genome Comparisons) Definition: Level of hybridization between the genome of an unknown microbe and that of a reference organism in vitro (in a cuvette in a spectrophotometer). When dsDNA is heated – ssDNA is formed. When ssDNA is cooled to 25 oC below Tm (melting temperature), complementary strands will re-associate and stable dsDNA forms again. If all the DNA in the cuvette is from the same organism, the re-association of DNA will occur relatively quickly, when compared to when two different species’ genomes are mixed in a cuvette. The relative quantity of complementary ssDNA to a particular strand of ssDNA is now much less than when all the ssDNA in the cuvette was from the same organism. The result is that the complementary strands of DNA will now take much longer to re-associate and form dsDNA again. The more stable the dsDNA (as a result of more complimentary pieces of ssDNA), the higher the temp. needed to obtain ssDNA again or the more rapidly the ssDNA will re-associate at a given temp. The percentage homology can therefore be calculated. The closer the two organisms are related in the cuvette, the faster the complementary strands of DNA re-associate to form dsDNA. This re-association of DNA in the cuvette can be monitored by measuring the absorbance of the DNA solution at 260 nm. The rate at which the re-association occurs is used to calculate the percentage homology between the genomes of the two different microbial strains. Two strains of which the DNAs show at least 70% homology and differ with less than 5% in Tm may be considered members of the same species. Normally, relationships between closely related types are investigated with DNA-DNA hybridizations and more distantly related types are investigated with (ribosomal) rDNA sequencing. MCB 244 11 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Nucleic Acid Sequencing (Signature Sequences) Taxonomic informative gene sequences such as those encoding for the 16S ribosomal subunit of prokaryotes; and for the 18S ribosomal subunit of eukaryotes are often used to determine relationships among microbes. To initiate the taxonomic identification process, target gene sequences, within the organism’s ribosomal gene cluster, first need to be amplified using the polymerase chain reaction (PCR). Polymerase Chain Reaction (PCR) Amplifies Targeted DNA 1. PCR enables the synthesis of millions of copies of a target DNA sequence, e.g., a taxonomic informative gene sequence that occurs somewhere within the complex mixture of DNA molecules (such as those bacteria’s genome). 2. PCR enables the amplification of specific target DNA sequences. The amplified target sequence can be used for taxonomic purposes but first, need to be purified whereafter the sequences of the PCR products are determined using a genetic sequencer. The bacterium can then be identified by first comparing its sequence with known sequences available on the internet, for example, via the GenBank website, using a BLAST search. 3. After the BLAST search, the bacterium’s identity can be confirmed by phylogenetic analysis of the 16S ribosomal gene sequence, using appropriate computer software that results in the construction of a phylogenetic tree. This process entails determining the phylogenetic distance between a particular bacterium and the reference strains of closely related species. MCB 244 12 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Phylogentic Trees NOTE ❖ Phylogenetic trees (also called dendrograms) show inferred evolutionary relationships in the form of multiple branching lineages connected by nodes. ❖ Organisms whose nucleotide sequences have been analyzed are identified at the tip of each branch. ❖ Each node represents a divergence event. ❖ The length of the branch represents the number of molecular changes that have taken place between the nodes. ❖ Corrected Evolutionary distance refers to the normalization of phylogenetic data since computers may differ slightly in their analysis of the trees. It gives a generalised/ average/ standardized tree. Tree C: Rooted tree is rooted by providing data from an outgroup (a species known to be very distantly related to all the species in the tree). The root is then determined by the point of the tree where the outgroup joins. The node closest to the outgroup is viewed as the oldest on the tree. Tree A: An unrooted tree that doesn’t show the common ancestor of the four species or the direction of evolutionary change. Tree B: Rooted tree gives a node that serves as the common ancestor and shows the development of the four species from the root. MCB 244 13 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Evolutionary Processes and the Concept of Microbial species Most commonly accepted model is that of Carl Woese, which divides life into three domains: o Bacteria (Eubacteria) o Archaea (previously called archaeobacteria) o Eukarya (Eukaryotes) Using sequence analyses of the small subunit RNA it was determined that all living organisms belong to one of three domains: General differences: o Eukarya – eukaryotic rRNA, membranes with glycerol-fatty acid-di-esters. o Bacteria (Eubacteria) – eubacteria rRNA, membranes with diacyl-glycerol- diesters. Sometimes called “true prokaryotes”. o Archaea – archaeal rRNA, membranes with isoprenoid-glycerol-diethers or diglycerol tetraether lipids. Ether bonds are more stable than ester bonds. MCB 244 14 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Polycistronic mRNA: RNA that has more than one coding region, formed when an operon is transcribed. This mRNA codes multiple proteins. Eukarya have mono-cistronic mRNA where each mRNA only codes for one specific protein. MCB 244 15 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Bergey’s Manual of Systematic Bacteriology Detailed work containing descriptions of all prokaryotic species currently identified. The First Edition of Bergey’s Manual of Systematic Bacteriology The First Edition of Bergey’s Manual of Systematic Bacteriology is a primarily focused phenetic classification of bacteria, including cell morphology and cell wall characteristics. Property Gram-Negative Bacteria Gram-Positive Bacteria Thin peptidoglycan inner layer Thick peptidoglycan layer Cell wall Outer membrane of lipid, protein No outer membrane & lipopolysaccharide Spheres Spheres Rods Rods Cell shape Filaments Filaments Sheaths or capsules True branching Binary fission Binary fission Reproduction Budding Tip extension of filamentous forms Chemo-organo-heterotroph Chemo-organo-heterotroph Metabolism Chemo-litho-autotroph No Phototroph Phototroph (some) Motile and non-motile Mostly non-motile Varied flagella placement Peritrichous flagella placement Motility Axial filaments (spirochetes) No axial filaments (spirochetes) Gliding No gliding Pili, fimbriae, prosthecae, stalks Lack appendages Appendages Endospores No Yes (some) The Second Edition of Bergey’s Manual of Systematic Bacteriology The Second Edition of Bergey’s Manual of Systematic Bacteriology explains bacterial classification mostly according to phylogenetic analysis of taxonomic informative gene sequences occurring in the 16S ribosomal subunit. MCB 244 16 CHAPTER 19: Microbial Taxonomy & the Evolution of Diversity HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) TO CONCLUDE: 1. To make sense of the diversity of organisms, it is necessary to group similar organisms and organize these groups in a non-overlapping hierarchical arrangement. Taxonomy is the science of biological classification. 2. A polyphasic approach is used to classify prokaryotes. This combines information that is based on the analysis of microbial phenotypic, genotypic, and phylogenetic features. The results of these analyses are often summarized in treelike diagrams called dendrograms. 3. Morphological, physiological, metabolic, ecological, genetic and molecular characteristics are all useful in taxonomy because they reflect the organization and activity of the genome. The structure of nucleic acid is probably the best indicator of relatedness, as nucleic acid is either the genetic material itself, or it is the product of gene transcription. Small subunit rRNAs and the genes that encode them display several features that make them useful in determining microbial phylogenies. 4. Bacterial taxonomy is rapidly changing due to the acquisition of new data, especially the use of molecular techniques such as the comparison of ribosomal RNA structures. This led to new phylogenetic classifications. 5. The evolutionary relationships among the three domains of life - Bacteria, Archaea, and Eukarya – are represented in the universal phylogenetic tree. The root, or origin, is placed early in the bacterial line of descent. This suggests that the Archaea and Eukarya share a common ancestry that is independent of the Bacteria. This long evolutionary history has generated a spectacular degree of microbial diversity. MCB 244 17 CHAPTER 18: Microbial Genomics HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Microbial Genomics Presented by Prof. A. Botha Introduction to Microbial Genomics Microbial genomics is an interdisciplinary research field, which focuses on the structure, function, evolution, mapping, and editing of the microbial genome, which includes all a microorganism’s genes. The aim is to collectively characterize and quantify all of a microorganism’s genes, their interrelations and their influence on the microorganism and its environment. Since its inception in the late 1970s, the field has expanded to the field of metagenomics, which is the study of microbial genomes directly from the environment. Metagenomics has emerged as a key technology across many biological disciplines including ecology, environmental microbiology, and immunology. Because metagenomics samples the entire pool of nucleic acids found in a particular ecosystem, it is most frequently used to determine the composition of the microbial community living in that ecosystem. In the past, such analyses were performed using PCR to amplify small subunit rRNA genes (16S for bacteria and archaea, 18S for eukaryotes) or other target genes. Although this approach is continued to be used, metagenomics enables the sequencing of large portions of the genomes in a particular habitat, which has led to the discovery of new microbial groups and novel genes in numerous microbiomes. Metagenomics is also a much faster way to identify organisms in an environmental sample than individual identification using PCR. It is now generally accepted that microbiomes play pivotal roles in the natural environment and are central to the well-being of humans. Human dietary changes and subsequent changes in the gut microbiome have, for example, been linked to colon cancer risk and obesity. The most striking findings, however, provide evidence for the existence of bidirectional brain-gut-microbiome interactions in humans. Changes in these interactions have not only been implicated in functional gastrointestinal disorders such as irritable bowel syndrome, but also in psychiatric and neurologic pathologies, including autism, Parkinson's disease, multiple sclerosis, and chronic pain. A Few Definitions Genome: The full set of genes present in a cell or virus; all the genetic material in an organism. Microbiome: The totality of microorganisms and microbial genomes that constitute the normal microbiota in a particular habitat. Metagenomics: The study of genomes recovered from environmental samples (including the human body) without first isolating members of the microbial community and growing them in cultures. MCB 244 1 CHAPTER 18: Microbial Genomics HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Effects of the Gut Microbiome on Human Health For the picture above. ❖ Some of the many known effects of the gut microbiome on diseases of various organ systems are illustrated. ❖ The disease and detrimental effects include autism Parkinson’s, depression, multiple sclerosis, drug metabolism resulting in toxic metabolites, non-alcoholic fatty liver disease, asthma, allergies, heart disease, Celiac disease, IBD, and colorectal cancer. ❖ Abbreviations: dsDNA = double-stranded DNA; GABA = gamma-aminobutyric acid; E. coli = Escherichia coli; IBD = inflammatory bowel disease (a collection of several intestinal disorders that includes Crohn’s disease and ulcerative colitis); PSA = capsular polysaccharide A from Bacteroides fragilis; TMA/TMAO = trimethylamine/ trimethylamine N-oxide. MCB 244 2  CHAPTER 18: Microbial Genomics HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Genome Shotgun Sequencing “A genomic library is a collection of overlapping Whole-genome shotgun cloning entails the construction of a segments of genomic DNA, genomic library. cloned into a backbone vector, The entire sequencing process can be subdivided into four stages: which statistically includes all o Library construction. regions of the genome of an o Random sequencing. organism. The resulting cloned o Fragment alignment and gap closure. DNA is then transformed into a o Editing. suitable host cell line.” Study Tip: Luna catches - source 2 Ron and Fred’s Eyes See the last page for the link to 1. Library Construction the website. The genome is randomly broken into fragments using Extra: You’ll ultrasonic waves or restriction enzymes, whereafter the fragments are purified. learn more These fragments are next inserted into plasmids or bacterial artificial chromosome about BACs in (BAC) vectors and isolated. Genetics 244. E. coli strains are next transformed with plasmids or (BACs) to produce a library of clones whose inserts represent the entire genome to be sequenced. 2. Random Sequencing The vectors carrying the cloned DNA are purified and thousands of DNA fragments are sequenced with automated sequencers, employing primers that recognize the plasmid DNA sequences adjacent to the cloned, chromosomal insert. For background information, on how this works, you can read the sections on Sanger sequencing in your textbook. Up to 96 samples can be sequenced simultaneously, making it possible to sequence up to a million bases per day, per sequencer. 3. Fragment Alignment and Gap Closure Using computer analysis, the DNA sequence information of each fragment is assembled into longer stretches of sequence. Two fragments are joined together to form a larger stretch of DNA if the sequences at their ends overlap and match. This comparison process results in a set of larger, contiguous nucleotide sequences called contigs. Contigs: DNA fragments with identical sequences at the ends and thus, the ends overlap in sequence. Sometimes an overlapping sequence is missing, generating gaps between contigs. There are several strategies to obtain the missing sequences. Ultimately, however, contigs are aligned in the proper order to form the complete genome sequence. The term scaffold is used to describe sequence data with gaps that persist between contigs. 4. Editing The sequence is then carefully proofread to resolve any ambiguities or frameshift mutations in the sequence. Proofreading is accomplished by ensuring that all reads of the same sequence are identical and that the sequences of the two DNA strands are complementary. MCB 244 3 CHAPTER 18: Microbial Genomics HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) FOR THE PICTURE ABOVE. Shotgun sequencing is a laboratory technique for determining the DNA sequence of an organism's genome. The method involves breaking the genome into a collection of small DNA fragments (1) that are sequenced individually (2). A computer program looks for overlaps in the DNA sequences and uses them to place the individual fragments in their correct order to reconstitute the genome (3). The sequence is then carefully proofread to resolve any ambiguities or frameshift mutations (4). As background also read about Sanger DNA sequencing in your textbook. Extra: You’ll learn more about Shotgun and Sanger sequencing in Genetics 244. DNA Sequencing Methods In 1977 the Nobel Laureate Fredrick Sanger and colleagues introduced the "dideoxy" chain- termination method for sequencing DNA molecules, also known as the Sanger method. This method later became the first automated sequencing platform in the late 20th century. The method involves the synthesis of a new strand of DNA using the DNA to be sequenced as a template. The reaction begins when single strands of template DNA are mixed with an oligonucleotide primer (a short piece of DNA complementary to the region to be sequenced), DNA polymerase, the four deoxynucleoside triphosphates (dNTPs), and dideoxynucleotide triphosphates (ddNTPs). o ddNTPs differ from dNTPs in that the 3’ carbon lacks a hydroxyl group. In such a reaction mixture, DNA synthesis will continue until a ddNTP, rather than a dNTP, is added to the growing chain. o Without a 3’-OH group to attack the 5’-PO4- of the next dNTP to be incorporated, synthesis stops. To obtain sequence information, four separate synthesis reactions must be prepared, one for each ddNTP. The ddNTP is mixed with all four normal dNTPs, and as DNA synthesis proceeds, sometimes the ddNTP will be incorporated into the growing DNA strand, rather than its dNTP cousin. This results in a collection of DNA fragments of varying lengths, each ending in the same ddNTP. MCB 244 4  CHAPTER 18: Microbial Genomics HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Source 2: https://www.sciencedirect.com/topics/neuroscience/genomic- library#:~:text=A%20genomic%20library%20is%20a%20collection%20of%20overla pping,then%20transformed%20into%20a%20suitable%20host%20cell%20line. MCB 244 5 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) The Archaea Presented by Prof. A. Botha Study Tip: First revise “Archaeal Cell Structure” Overview of the Archaea as discussed in Chapter 4. The Archaea is a diverse domain of organisms in terms of both metabolism and morphology. They are typically found in extreme environments, such as geothermically heated water (e.g., in Yellowstone National Park, USA) or salt pans. Archaea can, however, also be found in less extreme environments, such as in soil or ruminants (e.g., methanogens in the gut of cows). Nevertheless, only a small subset of the archaea can be grown in the laboratory. Examples of Archaea include methanogens (anaerobic – killed by oxygen), halophiles (require high salt concentrations), and thermoacidophiles (require high temperatures and low pH). Archaeal Cell Walls Although the archaea either stains as Gram-positive or Gram-negative, the cell wall differs considerably from that of bacteria in terms of chemical composition and ultrastructure. Thus, since Archaea stain both positive and negative, they can’t be differentiated using Gram-staining They are also resistant to lysozymes and penicillin – indicating that they do not contain peptidoglycan in their cell walls. Gram-positive archaea contain a diversity of polymers in their cell walls. Example: Methanobacterium and other methanogens possess: o Pseudomurein which consists of chains of N-acetyl-talosaminuronic acid (instead of N-acetylmuramic acid) bound with ß(1→3) glycosidic bonds to N-acetylglucosamine. o The peptide bridge consists of L-amino acids. MCB 244 1 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Other Gram-positive archaea, e.g., Methanosarcina and Halococcus, have complex polysaccharides related to chondroitin sulphate in their cell walls. Gram-negative-staining archaea have a layer of glycoprotein or protein (S) on the outside of the cell membrane. The layer may be as thick as 20 – 40 nm. Sometimes there are two or more layers. MCB 244 2 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Note: Some archaea do not contain a cell wall. E.g., Thermoplasma. Archaeal Cell Membranes Characteristic property: Composition of the cell membrane, contains branched carbohydrates that are bound to glycerol by ether bonds. Polar lipids in membranes: o Phospholipids, Sulpholipids and glycolipids. Non-polar lipids in membranes: o 7-30% of lipids in membranes are usually derivatives of squalene. Note: ❖ The “cell envelope” refers to the plasma membrane and everything external to it. ❖ Archaea have monolayers that act as bilayers. ❖ For many archaea, the S-layer is the major (or only) component of their cell wall. ❖ Slime layers and capsules are rare. MCB 244 3 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) NOTE ❖ Archaeal cell membranes often form monolayers instead of bilayers. ❖ The monolayer acts as a bilayer. ❖ This monolayer is due to the presence of unique lipids in archaeal plasma membranes. ❖ Two major archaeal lipids: Glycerol diether lipids – form a typical bilayer. Diglycerol tetraether lipids – form a monolayer. This structure is more stable and thus mostly occurs in thermophiles. MCB 244 4 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Archaeal Genetics and Molecular Biology In some cases, the chromosome is significantly smaller than that of eubacteria. o Example 1: E. coli = 2.5 × 10-9 daltons. o Example 2: Thermoplasma acidophilum = 0.8 × 10-9 daltons. G+C content varies between 21% and 68%. This table is Other distinctive properties include ribosomes, mRNA and RNA-polymerases, etc. from Chapter 19 Archaeal Metabolism CO2 Fixation Pathways: o Most known autotrophic archaea are either anaerobic or live in low-oxygen conditions. o They use ferredoxin (Fd) instead of NADPH as their electron carriers. o Three different pathways have been characterized: ▪ Reductive acetyl-CoA: Methanogens: Used for carbon fixation and energy production. Other archaea: used for carbon fixation. ▪ 3-hydroxyproprionate/4-hydroxybutyrate (HP/HB): Crenarchaeote (Sulfolobales and Thaumarchaeota). Extra (from the Pathway operates under aerobic conditions. textbook) ▪ Dicarboxylate/4-hydroxybutyrate (DC/HB): Crenarchaeote (Thermoproteales and Desulfurococcales). Include reductive TCA steps. Chemo-organotrophic Pathways: o Three different pathways have been characterized: ▪ Modified Embden-Meyerhoff ▪ Modified Phosphorylative Entner-Doudoroff ▪ Modified Non-Phosphorylative Entner-Doudoroff MCB 244 5 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Extreme Thermophiles Extreme Halophiles Methanogens Carbohydrate Most archaea Catabolism ✓ ✓ Glucose Catabolism Modified Entner-Doudoroff pathway (Phosphorylative pathway) ✓ ✓ Gluconeogenesis Reversed/Modified Embden-Meyerhof pathway Oxidation of pyruvate to acetyl CoA using pyruvate ferredoxin ✓ ✓ ✓ oxidoreductase (do not have pyruvate dehydrogenase) Functional tricarboxylic Use reductive acetyl acid cycle used for ✓ ✓ CoA pathway energy production Glycogen is used as a ✓ ✓ reserve material ✓ ✓ CO2 fixing via the CO2 fixing via the Autotrophic reductive tricarboxylic reductive acetyl CoA acid cycle pathway Reductive tricarboxylic acid cycle Reductive CoA pathway This image shows two different pathways used to fix CO2. MCB 244 6 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) EXTRA (Optional read) MCB 244 7 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) EXTRA (Optional read) Archaeal Taxonomy MCB 244 8 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Three Phyla of Archaea (Based on rRNA Data) Crenarchaeota o Temperature: All are thermophiles or hyperthermophiles. Thaumarchaeota o Where: Soils and different watery environments. o Metabolism: ammonia oxidizers. o Temperature: Mesophiles. Euryarchaeota o Where: Lower temperature niches. o Examples: Methanogens, extreme halophiles, sulphate utilisers and extreme thermophiles with sulfur-dependant metabolisms. Phylum: Crenarchaeota General characteristics: o Mostly strict anaerobes. o Temperature: Extreme thermophiles. o pH: Acidophiles (dependent on sulfur). o Metabolism: ▪ The sulfur serves as an electron acceptor during anaerobic respiration. ▪ The sulfur also serves as an electron donor in lithotrophs. o Where: Geothermally heated water or soil containing sulfur. o This phylum only has one class, Thermoprotei, which is divided into 4 orders. A) Order: Desulfurococcales Genus: Pyrodictium o Temperature: Extreme thermophiles (840 – 1100 oC). o Where: Hydrothermal vents in the seabed. B) Order: Sulfolabales Genus: Sulfolobus o Gram-negative-stain. o Aerobes. o Morphology: Irregularly lobed spherical. o Temperature: 70 – 80 oC. o pH: Thermoacidophiles (pH 2 – 3). o Metabolism: Grow lithotrophically on sulfur granules, which they oxidize to sulfuric acid. Oxygen is the normal electron acceptor, but ferric iron may also be used. Sugars and amino acids may also serve as carbon and energy sources. MCB 244 9 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) C) Order: Thermoproteales Genus: Thermoproteus o Strictly anaerobes. o Morphology: Long thin rod, which may be branched. o Temperature: 70 – 97oC. o pH: 2.5 – 6.5. o Metabolism: Organotrophs may oxidize glucose and other organic compounds, with sulfur as the final electron acceptor. Also grows chemolithotrophically - then using H2 and S0. CO and CO2 may serve as carbon sources. D) Order: Caldisphaerales Genus: Caldisphaera o Anaerobic. o pH: Acidophiles. o Metabolism: Chemotrophs. NOTE (Optional read) Phylum Thaumarchaeota General characteristics: o Capable of nitrification. o Ammonia oxidation is the metabolic feature that defines Thaumarchaeota. o Carbon source: Some fix CO2 via the HP/HB cycle. Others use organic carbon rather than CO2 as their carbon source. These are considered mixotrophic rather than heterotropic. Why? They capture energy through the oxidation of ammonia to nitrite using oxygen as the terminal electron acceptor while using organic carbon for anabolic purposes. The first step in ammonia oxidation is its conversion to hydroxylamine (NH2OH), a reaction catalyzed by ammonia monooxygenase (AMO). o Examples: Cenarchaeum symbiosum and Nitrosopumilus maritimus MCB 244 10 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Phylum Euryarchaeota Study tip: Methane = CH4 Methanogens o Very large and diverse group, with differences in 16S rRNA, cell morphology, cell wall and membrane composition. o Strict anaerobes. o Where: Anaerobic environments rich in organic compounds, e.g., sewage water or rumens of ruminants (cattle, sheep, and bovines). o Metabolism: Obtain energy through methanogenesis, involving the conversion of CO2, H2, muramic acid, methanol, and acetate to either methane or again CO2. Possess several unique coenzymes required for methanogenesis: Tetrahydromethanopterin (H4MPT). Methanofuran (MFR). Coenzyme M, Coenzyme F420 and Coenzyme F430 The rate of methane production may be high. Anaerobic decomposition of sewage sludge results in the production of H2, CO2 and acetate. “CO2-reducing methanogens” form methane from H2 and CO2. “Aceticlastic methanogens” cleave acetate to form methane and CO2. Methanogenesis Methane (product) NOTE: Halobacterium salinarium ❖ H. salinarium can produce a modified cell membrane Halobacteria (purple membrane) that contains the protein o Aerobes. archaerhodopsin. At low oxygen concentrations, ATP o Metabolism: Chemoheterotrophs is produced via a unique type of photosynthesis. ❖ It also contains halorhodopsin which uses light energy with respiratory metabolism. to transport chloride ions to the inside of the cell (to o Need complex nutrients. maintain intracellular concentrations of 4 – 5M KCl). o Motility: Motile (lofotrichous ❖ Two other rhodopsins serve as photoreceptors that flagella) or nonmotile. control the activity of the flagella so that the cell can o Optimal growth at 3 – 4 M NaCl. maintain a suitable depth in the water. MCB 244 11 CHAPTER 20: Archaea HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Thermoplasma o Temperature & pH: Thermoacidophiles without cell walls. o Where: Waste dumps at coal mines. The dumps become very hot and acidic. Thermoplasma grows in this habitat (55 – 590C; pH 1 – 2). o Morphology: At lower temperatures, coccus is formed. At higher temperatures, filamentous is formed. o Metabolism: Chemolithotrophs oxidize FeS to sulfuric acid. o Cell structure: Membranes are strengthened by diglycerol-tetra-ethers, lipopolysaccharides, and glycoproteins. DNA is stabilized by association with histone-like proteins. o Example: Picrophilus Aerobes. Temperature: 47 – 60 0C. pH: 0 – 3.7. Morphology: Irregular cocci. Contains an S-layer on the outside of the cell membrane. Extremely thermophilic S0-metabolisers o Temperature: Thermophiles. o Morphology: Cocci with cell walls. o Example: Thermococcales Strict anaerobes. Motility: Motile (possess flagella). Temperature: 88 – 100 oC. Sulfate-reducing Euryarchaeota o Archaeoglobales: Gram-negative-stain. Morphology: Irregular coccus-shaped cells,cell walls possess glycoprotein subunits. Temperature: Optimal growth at 83 oC. Metabolism: Possess methanogen co-enzymes, F420 and Methanopterin. Where: Hydrothermal vents in the ocean bed. MCB 244 12 CHAPTER 21: Nonproteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Non-proteobacterial Gram-Negative Bacteria Presented by Prof. A. Botha Introduction EXTRA (Optional read) Phylum: Deinococcus-Thermus ❖ It grows optimally at 70°C. Orders: Deinococcales and Thermales ❖ It contains the famous thermostable DNA polymerase, Taq, which is commonly used in Aerobes. PCR. Motility: Non-motile. Morphology: o Rods or cocci. o Cells are arranged in pairs or tetrads. Temperature: o Mesophiles. o Except Thermus aquaticus (thermophile). Identification: o They stain Gram-positive due to their thick cell walls, but the ultrastructure of the cell wall is like Gram-negative bacteria (contain an outer membrane). Metabolism: heterotrophs producing acid form only a few sugars. Unique features: o Resistant to oxidative stress, desiccation and irradiation. o Able to withstand radiation of 3 – 5 million rad. Panspermia Theory ▪ It can splice seriously damaged chromosomes after “Organisms such as bacteria, irradiation. complete with their DNA, ▪ This is done by enzymes that are protected against could be transported via radiation by high levels of manganese [Mn (II)]. comets through space to ▪ This provides support for the Panspermia Theory. planets including Earth.” MCB 244 1 CHAPTER 21: Nonproteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Photosynthetic Bacteria Phylum: Chlorobi (Green Sulfur Bacteria) EXTRA (Optional read) ❖ Morphology: Rods, cocci, or vibrios. ❖ Grass-green or chocolate brown in colour. Obligate anaerobes. Motility: Non-motile. Morphology: Rods. Where: o Flourish in anoxic, sulfide-rich zones of lakes and muddy ponds. o Can be isolated by taking a litre of muddy water from the muddy ponds, to prepare a Winogradsky column. After an incubation period, during which the column was exposed to light, different layers formed. o The green sulfur bacteria will accumulate and grow in the anaerobic zones near the bottom of the column. Metabolism: o Photolithoautotrophic bacteria that metabolize sulfur granules (elemental sulfur – S0), H2S or H2 (instead of H2O) as photosynthetic electron donors. o When sulfide is oxidized, elemental sulfur is deposited outside the cell. o No oxygen is generated during photosynthesis – anoxygenic photosynthesis. o Photosynthesis occurs in the chlorosomes. ▪ Chlorosomes are elongated, intramembranous, ellipsoidal vesicles that are attached to the plasma membrane by a proteinasceous baseplate. ▪ Chlorosomes contain light-harvesting pigments (bacteriochlorophylls). ▪ Chlorosome membranes are lipid monolayers, rather than bilayers. o The ATP produced by photophosphorylation is used to fuel CO2 fixation by the reductive citric acid cycle. MCB 244 2 CHAPTER 21: Nonproteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) EXTRA (Optional read) Phylum: Chloroflexi (Green Nonsulfur Bacteria) Note: Not all members of the Order: Chlorflexales Green Nonsulfur Bacteria are green, and some can use sulfide. A) Genus: Chloroflexus Facultative anaerobes or strict anaerobes. Temperature: Thermophilies. Where: o Neutral to alkaline hot springs. o Grow in the form of orange-reddish mats, usually in association with cyanobacteria. Motility: Gliding. Morphology: Filamentous. Metabolism: o Chemoorganotrophic or photoheterotrophic. o Anoxygenic photosynthesis. ▪ Possess chlorosomes with bacteriochlorophylls. o Sulfide or H2 as a source of electrons. o Heterotrophic growth: Supported by pyruvate, acetate, glycerol, and glucose oxidation. o Autotrophic growth: Fix CO2 using the 3-hydroxypropionate bi-cycle. Order: Herpetosiphonales A) Genus: Herpetosiphon Aerobes. Temperature: Thermophiles and mesophiles. Where: Freshwater or soil habitats. Motility: Gliding. Morphology: Rod-shaped or filamentous. Metabolism: o Chemoorganotrophs with respiratory metabolism. o Non-photosynthetic. Name: Chloroflexi Source: https://de-academic.com/dic.nsf/dewiki/255987 MCB 244 3 CHAPTER 21: Nonproteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Phylum: Cyanobacteria Aerobes. Where: o Oceans. o On land in polluted water bodies that contain excess phosphorus and nitrogen. o Muddy ponds. ▪ One can prepare a Winogradsky column by taking a sample of muddy water. ▪ Cyanobacteria would accumulate and grow in the aerobic zones at the top of the column. Role: Global carbon fixation via photosynthesis Metabolism: o Utilize H2O as a photosynthetic electron donor. o Oxygen is generated during photosynthesis – oxygenic photosynthesis. o Photosynthesis occurs in the phycobilisomes – particles, on the thylakoid membranes (lining the inside of the cell wall), that contain light-harvesting photosynthetic pigments (chlorophyll a and phycobiliproteins). Morphology: o Single-celled coccus-shaped species, e.g., Prochlorococcus marinus. o Filamentous species, e.g., Oscilatoria, Anabaena and Nostoc. These filamentous species produce specialized structures such as trichomes, hormogonia or akinetes. MCB 244 4 CHAPTER 21: Nonproteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) NOTE ❖ A heterocyst is a specialized cell that is a site where nitrogen fixation occurs. Their ability to fix atmospheric nitrogen increases the range of habitats in which these organisms can grow. E.g., can grow in environments with low nitrogen levels. ❖ As a result of their carbon fixation abilities, they act as major primary producers for marine ecosystems. Nonproteobacterial Proteobacterial Gram- Nonproteobacterial Gram-Negative Bacteria Negative Bacteria Gram-Negative Bacteria MCB 244 5 CHAPTER 21: Nonproteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Phylum: Planctomycetes Anaerobes. Morphologically unique bacteria having compartmentalized cells. The plasma membrane is closely surrounded by the cell wall, which lacks peptidoglycan. The largest internal compartment – the intracytoplasmic membrane – is separated from the plasma membrane by a peripheral, ribosome-free region called the paryphoplasm. The genera Brociadia, Kuenenia, Scalindua, and Anammoxoglobus contain an additional compartment called the anammoxosome that occupies much of the cell volume. The anammoxosome is the site of the energy-yielding anammox reaction. MCB 244 6 CHAPTER 21: Nonproteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) The Anammox Reaction NOTE The anammox reaction refers to anaerobic It was found that during growth, nitrite acts as both ammonia oxidation. an electron donor for biomass production, as well as Ammonium (NH4+) is used as an electron donor, an electron acceptor for ammonium oxidation and while nitrite (NO2-) is used as a terminal electron generation of the proton motive force that results in acceptor. Nitrite is reduced to nitrogen gas (N2). ATP production; the overall reaction is in two parts: Hydrazine (N2H4) is an important intermediate in Anabolic reaction: the anommox reaction. It donates electrons to ❖ 2NO2- + CO2 + H2O → CH2O + 2NO3- ferredoxin. Ferredoxin becomes reduced. The electron shutteling is used to simultaneously pump protons across the Catabolic reaction: anammoxosome membrane and creates a PMF ❖ NO2- + NH4+ → N2 + 2H2O (protomotoforce). The PMF drives ATP synthesis via ATP synthase Anammox may contribute as much as 70% of the which is embedded in the anammoxosome cycling of nitrogen in the world’s oceans. membrane. Globally, research is being conducted to use Reduced ferredoxin is also used to fix CO2. anammox bacteria to treat wastewater that Nitrite is also used as an electron donor for CO2 contains high concentrations of nitrite and fixation and biomass production. Thus, nitrite ammonium, using anaerobic microbiological acts in both anabolic and catabolic processes. reactors.. MCB 244 7 CHAPTER 21: Nonproteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Phylum Spirochaetes Faculative anaerobes. Motility: Motile. Morphology: Spiral -shaped. Metabolism: o Chemoheterotroph. o Carbohydrates, amino acids, long-chain fatty acids and long-chain fatty alcohols may serve as energy and carbon sources. They use a range of organic molecules as carbon sources. Characteristic structure and means of locomotion: o Long and slender (0.1 – 3.0 um × 5 - 250 um ) with a flexible helical shape. o With axial filament (periplasmic flagella), which makes movement through a very viscous (e.g., muddy) solution, as well as over solid surfaces possible. NOTE For image on the left: The terminally situated axial fibrils are twisted around a long, slender protoplasmic cylinder, resulting in their characteristic helical shape. Where: May occur in mud, fresh and seawater, even in the human mouth. Risk: A well-known human pathogen is Treponema pallidum – which resulted in thousands of deaths (syphilis). MCB 244 8 CHAPTER 22: Proteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) The Proteobacterial Gram-Negative Bacteria Presented by Prof. A. Botha Introduction The phylum, Proteobacteria is divided into 5 classes. All proteobacteria are Gram- negative. 5 Classes as shown on the left. Class: Alpha-proteobacteria MCB 244 1 CHAPTER 22: Proteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Order: Purple Non-Sulfur Bacteria Colour: Red or orange. Where: Occurs in mud, dams as well as the ocean. Metabolism: Anoxygenic photosynthetic bacteria. Role: Used as probiotics in fish feed in aquaculture. E.g., Rhodospirillum. NOTE Revisit Chapter 21 to see how Order: Rickettsiales these bacteria differ from the other photosynthetic bacteria. Genus: Rickettsia Gram-negative. Motility: Non-motile (No flagella). Morphology: o Rod-shaped, coccus-shaped or pleomorphic. o Very small (0.3 – 0.5 μm × 0.8 – 2.0 μm). Reproduction: o Species are intracellular parasites of humans and animals. o Reproduce in blood- and endothelial cells. o Enter cells through phagocytosis. Rickettsia escapes from the phagosome and reproduces by binary fission in the cytoplasm. o Host cells eventually burst to release new bacteria. The host is also harmed by the toxic effect of the bacterium’s cell walls. Metabolism: o The bacterium has no glycolytic pathway. o It oxidises glutamate and intermediary compounds from the host’s tricarboxylic acid cycle. A) Species: Rickettsia prowazekii, Rickettsia typhi Risk: Typhoid fever. B) Species: Rickettsia rickettsii Risk: Rocky Mountain spotted fever. Distribution: Spread to humans via tick bites. MCB 244 2 CHAPTER 22: Proteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Order: Rhizobiales Genus: Rhizobium Morphology: o Favourable conditions give rise to rods. o Unfavourableable conditions give rise to pleomorphic cells. Motility: Motile. Metabolism: Contain poly--hydroxybutyrate. Where: Grows symbiotically within the root nodule cells of legumes. Role: Fix nitrogen as bacteroids – legumes are the most successful plant species on Earth. Genus: Agrobacterium Risk: Many pathogens, e.g., hairy root formation and Crown’s Gall Disease. Order: Hyphomicrobiales Genus: Nitrobacter Metabolism: o Chemoautotroph. o Nitrification: Oxidise nitrite (NO2-) to nitrate (NO3-). o NOX (nitrate oxidoreductase) catayses this oxidation process. Role: Nitrate is used by plants as a nitrogen source. Note: Nitrobacter (α-proteobacteria) and Nitrosomonas (β-proteobacteria) are commonly found together and have a symbiotic relationship where Nitrosomonas produces nitrite which is the electron donor (substrate) for Nitrobacter which oxidises this to nitrate. Study Tip: refer to Chapter 28 (Biogeochemical Cycling)’s Nitrogen Cycle to see how these processes tie together. MCB 244 3 CHAPTER 22: Proteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Class: Beta-proteobacteria Metabolically diverse group but tends to utilize substrates from the anaerobe zones in habitats. Order: Neisseriales Genus: Neisseria Gram-negative. Aerobe. Motility: Non-motile Morphology: Capsules and pili. Metabolism: o Chemo-organotrophs. o Oxidase and catalase. Where: Mucous membranes of mammals. A) Species: Neisseria meningitidis a. Risk: Meningitis. B) Species: Neisseria gonorrhoeae a. Risk: Gonorrhoea. MCB 244 4 CHAPTER 22: Proteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Order: Burkholderiales Genus: Burkholderia Gram-negative. Aerobes. Motility: Motile (single or cluster polar flagella). Temperature: Mesophiles. Morphology: Straight rods. Metabolism: Oxidase and catalase positive. A) Species: Burkholderia cepacia a. Role: Degrades and recycles organic compounds. b. Risk: Plant pathogen that may infect humans, especially in hospitals. Genus: Bordetella Gram-negative. Aerobe. Motility: Motile or Non-motile. Metabolism: o Chemo-organotrophs. o Require sulfur and nitrogen compounds for growth. Morphology: Coccobacilli. A) Species: Bordetella pertussis a. Motility: Non-motile. b. Morphology: Capsule. c. Risk: Pertussis – infects the epithelial cells of the upper respiratory tracts of humans. B) Species: Bordetella bronchiseptica a. Motility: Motile. b. Morphology: Coccobacillus. c. Risk: Causes kennel cough in dogs. MCB 244 5  CHAPTER 22: Proteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Order: Nitrosomonadales Genera: Nitrosomonas, Nitrosococcus and Nitrospira. Metabolism: NOTE o Chemoautotrophs. o Nitrification: Oxidise nitrate to nitrite The table is from the textbook and is the (AMO and HAO enzymes). same as the table in the lecture slides. When Nitrosomonas and Nitrobacter occur in the same niche in the soil, ammonium is converted to nitrate. This process is called nitrification: NH4+ → NO2- → NO3- However, the term co-anammox (Chapter 28) is used to describe the direct conversion from NH4+ to NO3-. This is not happening here since these are two separate reactions. Source: Lecture slides. Source: Textbook. MCB 244 6 CHAPTER 22: Proteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Order: Hydrogenophilales Genus: Thiobacillus Colour: Colourless (“colourless sulfur bacteria”). Metabolism: o Chemolithotrophs. o Not photosynthetic. o Electron donor: Reduced inorganic sulfur compounds. o Electron acceptor: O2. A) Species: Thiobacillus denitrificans a. Where: Acid mine drainage. b. Metabolism: i. Denitrification (reduces nitrate to nitrogen gas). ii. Electron donors: Hydrogen sulfide (H2S), thiosulfate (S2O32-), or ferrous iron (Fe2+). iii. Electron acceptor: Oxygen or nitrate. c. Role: Useful in biomining since these organisms are used to oxidise metals and solubilised sulfide ores to enable the leeching of precious metals from these ores. Class: Gamma-proteobacteria MCB 244 7 CHAPTER 22: Proteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Order: Purple Sulfur Bacteria Colour: Purple. Metabolism: o Anoxygenic photosynthesis. o Electron donors: Reduced sulfur compounds, such as hydrogen sulfide or H2. NOTE Revisit Chapter 21 to see how these bacteria differ from the other photosynthetic bacteria. Practice: Why do you think the purple sulfur bacteria is found in this region of the Winogradsky column? Where: Muddy ponds with lots of organic debris. Anaerobic conditions are preferred and fermentation products of anaerobic bacteria (e.g., H2S) can be used as electron donors for the anoxygenic photosynthesis of the purple sulfur bacteria. Order: Pseudomonadales Genus: Pseudomonas Gram-negative. Aerobe or anaerobe. Morphology: Motility: Motile (polar flagella). o Straight or slightly curved rods. o No prosthecae and sheaths. Metabolism: o Chemoheterotrophs. o Functional TCA-cycle – oxidizes substrate to CO2. o Electron acceptor: O2 (sometimes also nitrate during anaerobic metabolism). Where: Plants, human bladder, refrigerators. Role: Degrade large variety of organic compounds; important in the mineralization process where it breaks down organic compounds to inorganic compounds; important experimental subjects. Risk: Plant pathogen; opportunistic pathogens in humans (bladder infections); cause food decay in refrigerated food (milk, eggs, meat, seafood). MCB 244 8 CHAPTER 22: Proteobacterial Gram-Negative Bacteria HENRY VILJOEN (PRESCOTT’S MICROBIOLOGY, ELEVENTH EDITION) Order: Vibrionales Gram-negative. Morphology: Straight or curved rods. Motility: Motile (polar flagella). Metab