BIOL 371 Class Notes - Microbiology (Concordia University) PDF

lOMoARcPSD|14412004 BIOL 371 Class Note Microbiology (Concordia University) Studocu is not sponsored or endorsed by any college or university Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 BIOL 371 | Class Notes Lecture 8 | Viruses Virus - Genetic element that can multi...

lOMoARcPSD|14412004 BIOL 371 Class Note Microbiology (Concordia University) Studocu is not sponsored or endorsed by any college or university Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 BIOL 371 | Class Notes Lecture 8 | Viruses Virus - Genetic element that can multiply only in a living (host) cell. - Not a living organism; has own nuclei acid genome. - Obligate intracellular parasite: need host cell for energy, metabolic intermediates, protein synthesis. - Virion (virus particle) extracellular form of a virus facilitates transmission. Components of naked and enveloped virus - Capsid: protein shell that surrounds the genome of a virus. - Naked viruses: have no other layers (most bacterial and plant viruses). - Enveloped viruses: has an outer layer made of phospholipid bilayer (from host) and viral proteins. - Nucleocapsid: nucleic acid +protein in enveloped viruses. - Virion surface proteins important for host cell attachment and may include enzymes involved in infection/replication. Viral activities: modes of infection - Virulent (lytic) infection: replicates and destroys host. o Host cell metabolism redirected to support multiplication and virion assembly. Lysogenic infection: host cell genetically altered because viral genome becomes part of host genome. Viruses can be classified by nature of genetic material. - DNA or RNA genomes. - Single or double-stranded. - Classification based on the hosts they infect. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 o o o o o Bacterial: bacteriophage or phage. Archaeal Animal Plant Protozoan Not all viruses are bad. - Disease-causing viruses are extensively studied. - Some viruses exert beneficial effects. o Arabidopsis infected with plus pox virus increases drought tolerance. o Hepatitis G infection of HIV patients decreases HIV replication and indectivity. Virion structure - Capsomere: individual protein molecules arranged in a precise and highly repetitive pattern around the nucleic acid making up the capsid. - Some viruses only have one capsid protein because small size of viral genomes restricts number of proteins. - Capsids can be put together through self-assembly (spontaneous) or may require host cell folding assistance. Basic virus symmetry - Two primary shapes: Rod and spherical. - Helical symmetry: o Rod-shaped o Length determined by length of nucleic acid. o Width determined by size and packaging of capsomeres. - Icosahedral symmetry: o Spherical o 20 triangular faces, 12 vertices; 5,3 or 2 identical segments. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 o Most efficient arrangement of subunits in a closed shell. o Requires fewest capsomeres. Complex Viral structure - Virion contains several parts, each with their own shapes and symmetry. Most complex are head-plus tail bacteriophages [ T4 phage ]. Enveloped Viruses - Nucleocapsid surrounded by lipoprotein membrane. Most use outer surface proteins to attach and infect. Relatively few enveloped plant or bacterial viruses because of cell walls surrounding cell membrane. Entire virion enters animal cell during infection. Enveloped viruses exit more easily. Enzymes inside virions - Lysozyme: makes holes in bacterial cell wall to allow entry of nucleic acid. o Lyses bacterial cells to release new virions. - Neuraminidases (influenza virus) o Destroys glycoproteins and glycolipids. o Allow liberation of viruses from cells. - Nucleic acid polymerases o RNA replicases: RNA-dependent RNA polymerases. o Reverse transcriptase: RNA-dependent DNA polymerase in retrovirus. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Culturing, detecting and counting of bacteriophage. - Plaque assay: plaques are clear zone of cell lysis that developed on lawns of host cells where successful viral infection occurs. o Titer: number of infectious virions per volume of fluid. o Calculate titer from the number of plaques. Animal cell culture and viral plaques - Similar to plaque assay. Plating efficiency: estimates of viral titer by plaque assay. o Number of plaque-forming units is always lower than direct count by electron microscopy. o Efficiency of infection usually much lower than 100%.  Defective virions or inappropriate conditions for infectivity. Steps in replication cycle of prokaryotic viruses - 5 steps: o Attachment (absorption of the virion) o Penetration (entry, injection) of the viral nucleic acid. o Synthesis of viral nucleic acid and proteins by host cell as redirected by virus. o Assembly of capsids and packaging of viral genomes into new virions. o Release of new virions from host cell. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Growth curve of viruses - One-step growth curve: virion numbers increase when cells burst. o Eclipse phase: genome replicated, and proteins synthesized. o Maturation: packaging of nucleic acid in capsids. o Latent period: eclipse+ maturation. o Release: cell lysis, budding, or excretion.  Burst size: number of virions released. Receptors for bacteriophages - Attachment and entry of bacteriophage requires complementary receptors on host cell surface- host specificity a major factor. Receptor on host cell carry out normal host functions for cell. Depending on the phage, receptors include proteins, carbohydrates, lipoproteins. For T4 phage, receptor is carbohydrates of lipopolysaccharide of the outer membrane. Attachment and penetration of bacteriophage - Virion attaches to cell via tail fibers that interact with polysaccharides on E. coli lipopolysaccharide layer. Tail fibers retract, and tail pins contact cell wall. T4 lysozyme forms a small hole in peptidoglycan. Tail sheath contracts, resulting in injection of viral DNA into cytoplasm. Capsid stays outside. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Invasion of viral genome does not ensure infection. - Prokaryotes possess mechanisms to diminish viral infections. o Toxin-antitoxin molecules. o Antiviral CRISPR (clustered Regularly short palindromic repeats). o Restriction endonucleases: enzymes that cleave foreign DNA at specific site.  Some have modified genome that is unaffected by restriction enzymes. Virions synthesis - Within one minute if entry: host-specific protein synthesis ends and phage-specific protein synthesis starts. T4 genome encodes 3 major sets of proteins. o Early proteins: enzyme needed for DNA replication and proteins that modify host enzymes to transcribe viral genes. o Middle and late proteins: head and tail proteins and enzymes required to liberate mature phage particles. Packaging and Assembly - Packaging in 3 stages: o Empty proheads (phage head precursors) assembled. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Fiber Protec , motor iglosh ↓ En pump ijs were o Packaging motor assembled at prohead opening. o Genome pumped into prohead using ATP. o Motor discarded and capsid head sealed. - capi After head is filled, T4 tails, tail fibers, and other components are assembled. Late enzymes break membrane and peptidoglycan. Lysis occurs, 100+ virions released. Temperate bacteriophages and lysogeny - Temperate: viruses established long-term, stable relationship but are capable of virulence. Lysogen: host cell that harbours temperature virus. In lysogeny, viral genome is integrated into bacterial genome forming prophage (viral DNA). Lysogeny maintained by phage-encoded repressor protein. o Inactivation of repressor protein induces lytic stage (induction).  Viral DNA excised and starts lytic cycle. o Cell stress (DNA damage) induces lytic pathway. Animal Viruses - Processes universal to all viral infections o Capsids and DNA/RNA genome. o Infection and takeover of host. o Assembly and release. - Differences between animal and bacterial viruses. o Entire virion enters the animal cell. o Eukaryotic cells contain a nucleus, the site of replication for many animal viruses. Downloaded by Raghad Abushahin ([email protected]) sicht lOMoARcPSD|14412004 o Viroplasms (membrane-bound viral factories) form in some eukaryotic cells to increase virion assembly rate and protect from host defense. Viral infection of animal cells. - Bind specific host cell receptors, typically used for cell-cell contact or immune function. Often infect only certain tissue because different cell surface proteins expressed by different tissues/organs. Host cell entry by fusion with cytoplasmic membrane or by endocytosis. Uncoating occurs at cytoplasm or cytoplasmic membrane. Viral DNA genomes enter nucleus, most viral RNA genomes are replicated or converted to DNA within nucleocapsid. After genome packaging, many animal viruses are enveloped, during lysis or budding. Outcomes of virulent infection infection - - - Virulent infection: lysis of host cell, most common. Latent infection: Viral DNA exists in hots genome as provirus (similar to lysogeny) and virions are not produced; host cell is unharmed unless/until virulent pathway is triggered. Persistent infection: slow release of virions from host cell by budding does not result in cell lysis. o Infected cell remains alive and continues to produce virus. Transformation: conversion of normal cell into tumor cell-rare event. Retrovirus-integration into the host genome - Includes HIV-1 (human immunodeficiency virus-1), the causative agent of AIDS (acquired immunodeficiency syndrome) in humans. Enveloped virion contains two copies of the ss (+) RNA genome. Retroviral genome is not used as mRNA. Virion contains several enzymes and specific viral tRNA. o Reverse transcription: synthesize DNA form RNA template. o Ribonuclease activity degrades RNA strand of RNA: DNA hybrid. o DNA polymerase to make dsDNA from ssDNA using viral tRNA as primer. o dsDNA integration into genome by integrase. Influenza Virus - Segmented genome: influenza A genome has eight linear RNA molecules. Surface proteins interact with host cell surface. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - o Hemagglutinin highly immunogenic (stimulates immune system); antihemagglutinin antibodies prevent infection (immunization). o Neuraminidase breaks down sialic acid component of host cytoplasmic membrane, functions in virus assembly. Nucleocapsid goes to nucleus for transcription. Enveloped virion forms by budding. Antigenic shift: RNA genomes from two different strains of the virus infecting the same cell, leading to re-assorted genome. o Surface antigen of hybrid influenza virus not recognized by the immune system. o Cause of major outbreaks of influenza. Coronaviruses - Enveloped virus with glycoprotein spikes on surfaces giving “crown” (corona) appearance. Cause respiratory infections in humans and other animals. Include sever Acute Respiratory syndrome Coronavirus-2 virus, the causative agent of COVID-19. Genomic RNA used as template to produce-strand from which mRNA is produced and translated. o Virions assembled in Golgi complex. o Released from cell surface. Lecture 9 | Mechanisms of microbial evolution Origin of genetic diversity - Evolution: change in allele (gene version) frequencies in a population over time. Origins of genetic diversity o Mutation: random changes in DNA sequence over time.  Drive evolutionary process. o Most mutations are neutral or deleterious some beneficial. o Forms: Substitutions, deletions, insertions, duplications. o Recombination  break & rejoins DNA segments to make new combinations of genetic material.  Can re-assort genetic material already present.  Required for integration of acquired DNA.  Can be homologous (require short flanking segments of similar sequence) or non-homologous (doesn’t require high similarity). Evolutionary selection Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Defined by fitness which is the ability of an organism to produce progeny and contribute to genetic makeup of future generations. Most mutation neutral (no effect) and accumulate over time. Deleterious mutations decrease fitness and or removed by NS over time. Beneficial mutations increase fitness and are favoured by NS. o E.g.,  antibiotic resistance during therapy. Mutations occur by chance; environment selects for advantageous mutations. Genetic drift - Random process that can cause gene frequencies to change over time, resulting in evolution in the absence of NS. o Some pop. more offspring by chance. o Most powerful in small populations and those experiencing frequent “bottleneck” events.  Bottleneck severe reduction in pop. followed by regrowth from remaining cells, such as pathogens. Origins of microbial species - Sequence changes can be used as molecular clock to estimate time since a species diverged. Major assumption is that nucleotide changes accumulate in proportion to time, are generally neutral and do not interfere with function, and are random. o E.g.,  E. coli harmless K-12 and O157:H7 pathogen diverged ~4.5 mya.  E. coli and Salmonella enterica diverged ~100-140 mya. Experimental evolution - Uses experiments with microbes to investigate evolutionary processes. o Possible because of rapid growth and preservation through freezing. o Evolutionary events can be observed relatively quickly. - Loss-of-function mutations- relatively easy to obtain. o Deleterious mutations in any region of gene may lead to loss of function. - Gain-of-function mutations-rare o Requires precise mutations that lead to functional (structural) change without losing function. Selection in a population of phototrophic purple bacteria - Rhodobacter capsulatus: phototrophic purple bacterium, photopigments are required for harvesting light. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Random mutations can result in reduced levels of photopigments. o Deleterious mutation in the light, loss of mutant. o Beneficial mutation due to energy savings (Wild-type cells continue to make bacteriochlorophyll and carotenoids), takes over the population due to fitness. Long term experimental evolution in E. coli - Running since 1988, tracing evolution of 12 parallel lines for >60,000 generations. Aerobically grown on defined media with glucose as sole carbon source and citrate as buffering agent. Ancestral and evolved strains marked with neutral marker to distinguish them. Dramatic increase in fitness over the first 500 generations, then slowed down. Gain-of-function mutation after 31,500 generations - E. coli can’t utilize citrate. Citrate was present in long term evolution experiment as pH buffer. Random accumulation of mutations allowed for the evolution of the ability to use citrate. This occurred in only one of the 12 evolving population (a chance event) and provided a selective advantage once it occurred. Gene duplication in evolution Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - After gene duplication, one copy is free to evolve new function while the other copy continues with the original function. Genome analysis suggests the genome of the budding yeast saccharomyces cerevisiae had undergone whole genome duplication followed by extensive deletion. Genome evolution: gene families - Homologs: gene descended from a common ancestral sequence. Orthologs: sequence that have diverged due to a speciation event. Paralogs: homologous sequences that share a common ancestor due to one or more gene duplication events. Gene families: groups of gene homologs. Gene deletions in microbial genome dynamics - Gene deletions plays an important role in microbial genome dynamics. o Occur more often than insertions/duplications. o Nonessential/non-functional genes deleted over time.  Maintain small size of microbial genomes. - Deletions drive tiny genomes in obligate intracellular symbionts and intracellular pathogens. o Metabolites available in host cytoplasm. o Deletions removing biosynthetic genes might have little effect on fitness. o Also eliminate redundant functions (mitochondria, chloroplasts). Deletions and the evolution of interdependence Microorganisms grow in communities, so deletion preventing production of essential nutrient may not be lethal if nutrient is available elsewhere. - Can increase fitness but promote interdependency. - Microbes that grow in pure culture without growth factor additions preserve all essential functions but are at a disadvantage compared to a community. Horizontal gene transfer - Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Impact microbial evolution. Allows transfer of DNA between distant branches of evolutionary tree. If no fitness benefit, deleted over time. Mechanisms: transformation, transduction, conjugation. Involvement of mobile genetic elements; transposons, insertion sequences, plasmids. Comparative genomics reveal diversity within species. - Microbial genomes are highly diverse and dynamic. Microbial species consists of individual strains differing in composition. The Pan and Core genome concept for species - Core genome: genes shared by all strains of a species. Pan genome: core genome plus genes not shared by all strains. Species can have major differences in genome size, gene content, functional traits. Chromosomal and pathogenicity islands of E. coli Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Chromosomal islands: entire genetic pathways can be acquired as blocks via horizontal gene transfer. o Often flanked by repeats, implying transposition o Base composition and codon bias differ from genome. - Core genome of E. coli is 1976 genes. o Average genome is 4721 genes. o E. coli K-12 strain, and two pathogenic strains share 39% of genes. - Pathogenicity islands: genes associated with pathogenicity are clustered in blocks. Systematics - The study of the diversity of organisms and their relationships o Link phylogeny with taxonomy, in which organisms are characterized, named and grouped based in natural (evolutionary) relationships. - Species are fundamental units of diversity. o For plants and animals, species have a special status. - Biological species concept: species is an interbreeding population that is reproductively isolated from other such populations. o Problematic when describing bacteria and archaea. - Microbial species is a taxonomic category that defines a population of individual that. o Is monophyletic (descending from common ancestor) o Genomically coherent. o Phenotypically coherent o Can be clearly distinguished from other species. The polyphasic approach to taxonomy - Objective: reach a consensus classification by integrating multiple forms of fate and information into a classification that presents a minimum number of contradictions. - The polyphasic approach to taxonomy uses three methods: o Phenotypic (morphological, metabolic, physiological, chemical characteristics) analysis. o Genotypic (genome) analysis. o Phylogenetic (evolutionary) analysis. Phenotypic analysis Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Morphology, mobility, metabolism, physiology, cell lipid chemistry, cell wall chemistry, other traits. Molecular phylogeny - Phylogenetic trees: diagrams depicting evolutionary history. o Difference in nucleotide sequence between two homologous genes is a function of number of mutations accumulated since they shared a common ancestor. o Difference in DNA sequences can be used to infer relationships. - SSU (small subunits) ribosomal RNA (rRNA) genes highly conserved and easily sequenced and analyzed. Can amplify SSU rRNA genes from environmental samples or to sequence or using metagenomics. Sequence the SSU rRNA gene and align the sequence to SSU rRNA genes of other species. o Strains that exhibit >97% SSU rRNA sequence identity are considered to belong to the same species. - Multilocus sequence analysis - DNA sequences of protein-encoding gene accumulate mutations faster than rRNA genes, thus distinguishing species that cannot be resolved by rRNA sequences. Multilocus sequence analysis can distinguish at species level. o Use highly conserved genes. o Use single-copy gene: compare orthologs, not paralogs. o Use as many genes as available. Genome analysis - Use of entire genomes increasingly common as sequencing improves and cost declines. Analyses of content: present/absence of genes. Synteny: order of genes. GC content Can reconstruct metabolic/physiological characteristics with genome data. Average nucleotide identity: most used metric for estimating overall relatedness by aligning ~1000 bp fragments and calculating average nucleotide identity. o >96% average nucleotide identity- same species o <93% average nucleotide identity-different species. Lecture 10 | Fermentation Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Fermentations - Energy conservation depends primarily on substrate-level phosphorylation. o Defined by lack of external electron acceptor. o Achieve redox balance by donating electrons to metabolic intermediates excreted as fermentation products. - Two challenges o Conserve much less energy than respiratory organisms. o Difficult to achieve redox balance. - Tremendous reaction diversity o Many only ferments when lacking external electron acceptors anoxically. o Many more exclusively fermentative. Energy-rich compounds and substrate-level phosphorylation - Energy-rich compounds contain an energy-rich phosphate bond or coenzyme A. These compounds are formed during fermentation. o Allows microbes to make ATP by transferring the phosphate bond to ADP by substrate-level phosphorylation. o Production of fatty acids is common in fermentations, gives opportunity to do substrate-level phosphorylation by producing fatty acid coenzyme-A derivative. Achieving redox balance - Total number of each type of atom and electrons in reactants/substrates and products must balance. Redox balance achieved by excretion of fermentation products: acids, alcohols Redox balance often facilitated by hydrogen production. o H2 is a powerful electron donor for respiration. Mechanisms of reducing protons to hydrogen during fermentation Common fermentations and example organisms Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Fermentations are classified by either the substrate fermented or the products formed (e.g., alcohol) Homofermentative lactic acid bacteria - Lactic acid bacteria are Gram-positive nonsporulating bacteria that produce lactic acid as sole or major fermentation product. Homofermentative yields only lactic acid and two ATP/glucose. Difference is the presence of aldolase from glycolysis. Heterofermentative lactic acid bacteria - Heterofermentation yields lactic acid, ethanol, CO2, and one ATP/glucose. Absence of aldolase from glycolysis. Observation of CO2, in culture distinguishes heterofermenters from homofermenters. Mixed-acid fermentations and butanediol production Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Characteristics of enteric bacteria o Generate acetic, lactic and succinic acids. o Typically formed are ethanol, carbon dioxide and hydrogen. - Glycolysis used. Some produce neutral products such as butanediol. Obligate anaerobic fermenters - Grow under highly reducing conditions. Cannot tolerate oxygen, often produce hydrogen from fermentation. Clostridium species are obligately fermentative anaerobes. o Ferment sugars, aa, purines and pyrimidines o Most ATP synthesis from substrate-level phosphorylation. o Some generate proton motive force. Sugar fermentation by Clostridium - Saccharolytic (sugar fermenting) Produce butyric acid and hydrogen as major products. 1.5 glucose converted to 3 pyruvates and 3 NADH via glycolysis. Pyruvate from glycolysis split acetyl-CoA, carbon dioxide and ferredoxin. Cytoplasmic hydrogenase reduces H+ to H2 to balance redox. Amino Acid fermentation and the Stickland reaction Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - AA fermentation by Clostridia o Proteolytic clostridia degrade proteins released from dead organisms and ferment aa. o Some strictly proteolytic, some both saccharolytic and proteolytic. o Some ferment individual aa to yield fatty acid-CoA derivative, then produce ATP via substrate-level phosphorylation with ammonia and carbon dioxide. - Stickland reaction: ferment aa pairs with one aa as electron donor and the other acceptor. o Products are ammonia and carbon dioxide, and a carboxylic acid with one fewer carbon than the oxidized aa. o Purine and pyrimidines from nucleic acid degradation lead to many of the same fermentation products and ATP through fatty acid-CoA derivatives. Use of energy-converting hydrogenases - Uses modified glycolysis forming 3-phosphoglycertae instead of 1,3-biphosphoglyceric acid. Forms ferredoxin and ATP from conversion of pyruvate to acetate. Uses energy-converting hydrogenases to achieve redox balance. Translocate Na+ across membrane, sodium motive force synthesizes ATP. Generates 4 ATP from substrate-level and oxidative phosphorylation, better than lactic acid bacteria. Primary vs Secondary fermentations Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Primary: carried out by organism that break down and ferment carbohydrate, protein, fat polymers and monomers to reduced products, H2 and CO2. Secondary fermentation: use fermentation products as substrates for additional fermentation reactions. o Products are mainly volatile fatty acids, H2 and CO2. o Propionibacterium, an important agent in the ripening of Emmental (Swiss) cheese, probably uses lactic acid, a fermentation product of lactic acid bacteria, as a major substrate to produce propionic acid (nutty taste of the cheese) and carbon dioxide (holes in cheese). Fermentation that lacks substrate-level phosphorylation - Fermentations of certain compounds do not yield sufficient energy to synthesize ATP by substrate-level phosphorylation but support anaerobic growth. In these cases, catabolism is linked to ion pumps that establish a membrane gradient. (Succinate fermentation by propionigenium modestum, oxalate fermentation by oxalobacter formignes.) Succinate fermentation: lacking substrate-level phosphorylation - Require NaCl for growth and succinate catabolism under strict anoxic conditions. o An unusual decarboxylase, sodium-extruding decarboxylase, is involved in the fermentation. o Succinate is converted to propionate through the decarboxylation of the intermediate (s)-methylmalonyl-CoA. o Energy released by the decarboxylation reaction is used to translocate two NA+ across the membrane. o Energy is then conserved by using a NA+- linked ATPase. Syntrophy Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Two different microbes cooperate to perform a metabolic reaction neither can do alone. Most syntrophic reactions are secondary fermentations. o Major compounds are fatty acids and alcohols. - Interspecies electron transfer is the core of syntrophic reactions. o One species serves as the electron acceptor for another species that is the electron donor. o Electron transfer can be direct: through contact between cells. o Mediated through diffusion of metabolic products: H Interspecies H2 transfer - The ethanol fermentation carried out by the syntroph (Pelotamaculum) has a positive free energy; thus, it cannot grow in pure culture. o The H2 produced can be used as an electron donor by a methanogen to produce methane. o The sum of the two reactions is exergonic; hence when cultured together,both organisms grow. Oxygenase’s in aliphatic hydrocarbon - Oxygenase’s catalyze the incorporation of oxygen into organic and some inorganic compounds. o Dioxygenases: incorporate both oxygen atoms. o Monooxygenases: incorporate one oxygen atom with the second reduced to water.  Usually dependent on NADH or NADPH. - Oxygen atom is incorporated initially at a terminal carbon. o End-product is a fatty acid of the same carbon length as the original hydrocarbon. o Fatty acid is oxidized by beta-oxidation: removing two carbons at a time, forming NADH for electron transport, acetyl Co-A and a fatty acid two carbons shorter. o Acetyl-CoA is oxidized through the citric acid cycle or to make new cellular material. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Multiple oxygenase’s in aromatics catabolism - Typically starts with formation of catechol or structurally related compound formation via oxygenase’s. o Benzene to catechol using a hydroxylating monooxygenase. o Toluene to methyl catechol by a hydroxylating dioxygenase. - Catechol and related aromatics are cleaved by ring-cleavage dioxygenases. o Degraded into compounds that can enter the citric acid cycle. Lecture 11 | Environmental microbiology Enrichment culture microbiology - Inoculum: sample from which organisms will be isolated. Isolation: separation of individual pop. from the mixed community. Enrichment cultures: used to isolate bacteria from diverse and densely populated samples. o Select for desired organisms through manipulation of medium and incubation conditions. o Favouring growth of target organisms while inhibiting growth of non-target organisms. o Isolation of the aerobic nitrogen-fixing bacterium Azotobacter. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Enrichment culture outcomes - - - Successful enrichment cultures are this with appropriate resources (nutrients) and conditions (pH, temperature, osmotic pressure) that are needed for target organisms to grow. Enrichment culture can show that the presence of an organism in a habitat. o Cannot rule out that an organism does not inhabit an environment. o Say nothing about the relative abundance and ecological importance of the target organism. Enrichment bias: microorganisms cultured in the lab are often minor components of the microbial ecosystem. o Quantity of nutrients available in the lab are typically much higher than encountered in nature. o Dilution of inoculum maybe used to eliminate rapidly growing, but quantitively insignificant species. Classical procedures for isolating microorganisms - Most probable number techniques: o Serial 10X dilutions of inoculum in a liquid medium. o Often used to estimate number of microorganisms in food, wastewater, and other samples. - Pure (axenic) culture needs to be verified. o Colony characteristics. o Microscopy on cell shape, single morphological type, uniform staining. o Tests multiple culture conditions for expected growth and no growth. Laser tweezers for isolating single cells. - Laser beams move through the objective lens of a microscopy. o Focus on the cell and separate it from other cells. o Isolate slow-growing bacteria from mixed cultures Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Flow cytometry - Method of counting and examining a mixture of cells. Stream of suspended cells passing through an electronic detector in single file. Cell sorted by cell shape, size or fluorescent properties. High-throughput cultivation of previously uncultured microorganisms - Separates individual cells for culture in microtiter plate, one cell per well. Each cell can grow without competition from other species. o Important in isolating slow growing species that thrive in nutrient poor environment. Microfluidic platform for cultivation - Microfabrication to construct tiny wells for cultivation, one cell per well. General staining to examine microbial communities. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - - Culture-independent microscopic method of examining microbial communities. Commonly used nonspecific fluorescent dyes- fluoresce under UV. o DAPI (4,6- diamidino-2-phenylindole)-blue o Acridine orange. o SYBR Green. Nonspecific stains for nucleic acids, cannot distinguish live from dead cells. Viability stains - Two dyes o Green dye enters both dead or live cells. o Red dye (propidium iodide) can enter only dead cells (defective cytoplasmic membrane). - Provides information on abundance and viability. Fluorescent protein reporters - Genes encoding fluorescent proteins controlled by bacterial gene promoters. o Introduced into bacteria to track live bacteria and bacterial processes. General principle of nucleic acid hybridization - - Nucleic acids with complementary sequence can form hybrid (DNA:DNA, DNA:RNA or RNA:RNA) The probe should be single-stranded nucleic acid. o Fluorescently labelled for mots experiments. o Following hybridization, the unbound probes are removed by washing with buffer. Multiple probes, each labelled with a different colour fluorescent dye and complementary to a specific organism or group of organisms can be used to examine pop. of microorganisms. Fluorescence in situ hybridization (FISH) - Situ means in place: used to see microorganism in the context of their communities. Fluorescence in situ hybridization (FISH): the probing nucleic acid is tagged with a fluorescent dye and used it hybridize with nucleic acids of microorganisms in the community. Phylogenetics of microbial populations with FISH. - Fluorescently labelled oligonucleotides complementary to rRNA. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 o Can be highly specific to species, or multiple species or genera. o Used in microbial ecology food industry and clinical diagnostics. - Can be modified to measure gene expression or translational activity. PCR methods of microbial community analysis - Specific genes can be used as a measure of diversity. Isolate DNA from environmental samples. o PCR (polymerase chain reaction) amplification of specific genes; typically, rRNA genes. o Analysis of the amplified genes  Molecular cloning  Electrophoresis  Restriction enzyme digestion  Sequencing Phylogenetic analysis: massively parallel DNA sequencing - - Multiple sequencing technologies available o Do not require molecular cloning. o Generates billions of sequences reads. o Allows the detection of minor phylotypes. Results of phylogenetic analysis o rRNA sequences differ from those of all known laboratory cultures. o New phylogenetic distinct prokaryotes. o Fewer than 0.1% of bacteria have been cultured, enrichment bias a real problem in environmental microbiology. Geochip-functional gene microarray - DNA microarray containing gene probes that encompass most major biogeochemical processes. Fluorescently labelled environmental DNA and hybridize to Geochip. o Relatively fast and easy analyze. o Provides functional information to correlate with phylogenetic analysis. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Environmental multi-omics - - More complete understanding of how a microorganism functions requires and integrated accounting of all central cellular processes. These include integrative knowledge on: o Genomics – genes, gene function and gene regulation. o Transcriptomics- global gene expression under different conditions. o Proteomics- accumulation of proteins under different conditions. o Metabolomics- dynamics of accumulation of metabolites. Expand the analysis to community level: metagenomics, meta transcriptomics, metaproteomic, and metametabolomics. Environmental genomic (metagenomics) - DNA extracted from environmental samples. - Sequence, assemble and annotate – mostly partial genomes assembled. - Identify as many genes as possible. o Detect genes not amplified by available PCR primers. o Powerful tool for assessing the phylogenetic and metabolic diversity of an environment. Meta transcriptomics Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Analysis of community mRNA o Remove rRNA before sequencing (>90% of total RNA are rRNA) o Reveals genes in a community that are active. o Reveals level of gene expression Metaproteomic and metabolomics - Metaproteomic: measures the diversity and abundance of different proteins in a community. o Current state of technology can only detect the most abundant proteins - Metabolomics: the comprehensive analysis of cellular and extracellular metabolites of a microbial community Direct chemical measurements of metabolites - For many studies, direct chemical measurements are sufficient, e.g., lactate and H S can 2 be measured with high sensitivity by chemical assay - For some processes, higher sensitivity can be achieved with radioisotopes. Microsensors - Microsensors available to measure a wide range of activity. o Small glass electrodes that can be inserted into the habitat. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Isotopic fractionation - Stable isotopes: an element can have multiple nonradioactive stable isotopes, e.g., 13 and C. - Isotopic fractionation: biological reactions prefer lighter isotopes; hence cellular 12 13 materials are enriched in C and depleted in C relative to inorganic carbon. 12 C 12 13 o The ratio of C and C can be used to trace the biological or geological origin of ancient environment. Stable isotope probing - Stable isotope probing: feed microorganisms with substrate labelled with stable heavy 13 isotope, e.g., C-benzoate. o Microorganisms that can utilize benzoate will incorporate 13 C into their DNA. o The heavier DNA can be separated by ultracentrifugation.  13 Compare the C-labelled DNA sequence with metagenomics data will identify microorganisms that can utilize benzoate in the microbial community. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Lecture 12 | Microbial Ecosystems General ecological concepts - Ecosystem: the sum total of all organisms and abiotic (physical rather than biological) factors in a particular environment - Habitat: portion of an ecosystem where a community could reside o An ecosystem contains many habitats. o Microbes account of ~50% of all biomasses on Earth, on the surface and deep within - Population: a group of microorganisms of the same species that reside in the same place at the same time o May be descendants of a single cell. - Community: consists of populations living in association with other populations Microbial diversity: richness vs abundance - Diversity of microbial species in an ecosystem is expressed in two ways o Species richness: total number of different species present o Species abundance: proportion of each species in an ecosystem - Microbial species richness and abundance are functions of the kinds and amounts of nutrients available in a given habitat. Resources and conditions that govern microbial growth in nature. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 Populations, guilds and communities - Guilds: metabolically related microbial populations o sets of guilds form microbial communities that interact with macroorganisms and abiotic factors in the ecosystem. - Niche: habitat shared by a guild o Supplies nutrients and conditions for growth Biogeochemistry and nutrient cycles - Biogeochemistry: the study of biologically mediated chemical transformations - A biogeochemical cycle defines the transformations of a key element by biological or chemical agents. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 o Microorganisms play essential role in cycling elements C, N, S, and Fe between their different chemical forms. o Typically proceed by oxidation–reduction reactions - Microbes play critical roles in energy transformations and biogeochemical processes that result in the recycling of elements to living systems. The microbial environment - Growth of microbes depends on resources and growth conditions. - Differences in the type and quantity of resources and the physiochemical conditions of a habitat define the niche for each microbe. - Fundamental niche: Full range of environmental conditions under which an organism can exist. - Realized niche or prime niche: for each organism, there exists at least one niche in which that organism is most successful. Microenvironment - Microenvironment: the immediate environmental surrounding of a microbial cell or groups of cells - Soil particles contain many microenvironments - Physiochemical conditions in a microenvironment are subject to rapid change, both spatially and temporally - Resources in natural environments are highly variable, and many microbes in nature face a feast-or-famine existence - Growth rates of microbes in nature are usually well below maximum growth rates defined in the laboratory Competition and cooperation - Competition and cooperation occur between microbes in natural systems. - Syntrophy: microbes work together to carry out transformations that neither can accomplish alone. o Microbial partnerships are particularly important for anoxic carbon cycling Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 o Metabolic cooperation can also be seen in the activities of organisms that carry out complementary metabolisms. Surfaces are important microbial habitats. - Surfaces: offer microbes greater access to nutrients and protection from predation and physicochemical disturbances o Nutrients adsorb to surfaces. o Attachment to a surface also offers cells a means to remain in a favorable habitat, modify the habitat by their own activities, and not be washed away Biofilms - Biofilms: assemblages of bacterial cells adhered to a surface and enclosed in an adhesive matrix excreted by the cells o The matrix is typically a mixture of polysaccharides. o Biofilms trap nutrients for microbial growth and help prevent detachment of cells in flowing systems. Implications of biofilms - - Why bacteria form biofilms? o Self-defense: Biofilms resist physical forces that sweep away unattached cells, phagocytosis by immune system cells, and penetration of toxins (e.g., antibiotics o Allows cells to remain in a favorable niche. o Allows bacterial cells to live in close association with one another. Biofilms have been implicated in several medical and dental conditions. o Periodontal disease, kidney stones, tuberculosis, Legionnaires’ disease, Staphylococcus infections - In industrial settings, biofilms can slow the flow of liquids through pipelines and accelerate corrosion. - Surface colonization and biodegradation of plastic alters the surface chemistry, density, and sinking rates of microplastics. Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Few highly effective antibiofilm agents are available Microbial mats - Microbial mats: thick biofilms o Built by phototrophic and/or chemolithotrophic bacteria. o Phototrophic mats contain filamentous cyanobacteria. o Chemolithotrophic mats contain filamentous sulfur-oxidizing bacteria. Soils-layers - Loose outer materials of Earth’s surface o Mineral soils: derived from rock weathering and other inorganic materials o Organic soils: derived from sedimentation in bogs and marshes Soil-composition and microenvironment - Composition of soils by soil volume o Air and water – 50%s o Inorganic mineral matter – 40% o Organic matter – 5% o Microorganisms and macroorganisms – 5% - Most microbial growth takes place on the surfaces of soil particles - Soil aggregates contain many different microenvironments supporting the growth of multiple types of microbes Soils-formation - Soils are formed by interdependent physical, chemical, and biological processes o Carbon dioxide is formed by respiring organisms that form carbonic acid that breaks down rock o Physical processes such as freezing and thawing break apart rocks, allowing plant roots to penetrate and form an expanded rhizosphere Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 o The rhizosphere, the area around plant roots where plants secrete sugars and other compounds, is rich in organic matter and microbial life - Water availability is variable and dependent on rainfall, plant coverage, drainage, and soil composition - Soil water has many dissolved materials and is called a soil solution - Well-drained soils have oxygen available, while waterlogged soils are typically anoxic, with the oxygen being consumed by soil microbiota Arid soils - Arid soils: dry, limited plant growth o Make up ~5% of the Earth’s land mass o Extreme environments: low water availability and variable temperatures (>60°C and below – 24°C) o Home to microbial communities specialized for extreme conditions o Arid soils are slow to form and are subject to desertification - Biological soil crusts made up of photosynthetic cyanobacteria and filamentous fungi that stabilize the soil o Damage to biological soil crusts leads to decrease soil fertility Soil bacterial and archaeal diversity - Phylogenetic sampling – pooled analysis from multiple studies of 16S rRNA gene sequencing - Microbial diversity varies with soil type and geographical location - Undisturbed, unpolluted soils support very high prokaryotic diversity - Soil perturbations and environmental changes trigger measurable shifts in community composition The terrestrial subsurface - Microbial life extends at least 3,000 metres below surface Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Microbial diversity of relatively shallow subsurface areas is similar to that of surface soils, but abundance is lower - Subsurface microbial life grows in an extremely nutrient- limited environment o Small microbial cells (<1 μM) are common - Few archaea are found in surface soils - Deep subsurface is home to the Asgard group of archaea that are most closely related to eukaryotes Freshwaters - Freshwater environments: highly variable in the resources and conditions available for microbial growth - The balance between photosynthesis and respiration controls the oxygen and carbon cycles - Oxygenic phototrophs, including algae and cyanobacteria, are the primary producers o Produce oxygen and organic material o They can be either planktonic (free floating) or benthic (attached to the bottom or sides of a lake or stream o Heterotrophic microbes in aquatic systems are highly dependent upon the activity of the primary producers - Oxygen has limited solubility in water o Concentrations are dependent on the amount of organic matter present and the physical mixing of the system o Deep layers of freshwater lakes can become anoxic once oxygen is consumed Stratification of water column in temperate lakes - In many temperate lakes, the water column becomes stratified during the summer o Epilimnion: warmer, less dense surface water o Hypolimnion: cooler, denser water at the bottom of a lake or pond o Thermocline: zone separating the epilimnion and the hypolimnion Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - These layers vary greatly in temperature, oxygen availability, and chemical composition Influx of organic-rich wastewaters into aquatic systems - Biochemical oxygen demand: the microbial oxygen-consuming capacity of a body of water o Increases with the influx of organic material (e.g., from sewage), then decreases over time Prokaryotic diversity of freshwater lakes - Phylogenetic sampling: 16S rRNA genes sequencing - High microbial diversity reflects dynamic character of lake o Seasonally variable inputs of endogenous and exogenous nutrients sustains a phylogenetically and metabolically complex community of bacteria and a few groups of archaea Differences between freshwater and marine environments - With the exception of oxygen, open ocean as compared to freshwater is: o Saline o Low in nutrients, especially nitrogen, phosphorus, and iron o Cooler 6 7 o Lower microbial activity (~10 /mL as compared to ~10 /mL for freshwater) - Because of the size of the oceans, the microbial activities taking place in them are major factors in Earth’s carbon balance Phototrophic microorganisms in near-shore waters Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Terrestrial runoff, retention of nutrients, and upwelling of nutrient-rich waters combine to support higher populations of phototrophic microorganisms in near- shore waters than in pelagic waters Diversity of marine systems all mute -excime - Eutrophication resulting from nutrient inputs can lead to the waters becoming intermittently anoxic from the removal of oxygen by respiration and the production of H S by sulfate-reducing bacteria 2 - Oxygen minimum zones: regions of oxygen-depleted waters at intermediate depths (1001,000 metres) and extend over large expanses of the ocean - Marine dead zone: example, an area of 6,000-8,000 square miles of seasonal oxygen depletion in the Gulf of Mexico associated with agricultural runoff of the Mississippi River o Excessive oxygen consumption by chemoorganotrophs and the formation oxygendepleted water Major marine phototroph- Prochlorococcus - Most of the primary productivity in the open oceans is due to photosynthesis by prochlorophytes o Prochlorococcus accounts for >40 percent of the biomass of marine phototrophs and ~50% of the net primary production o Abundance of different genotypes correlates with seasonal changes in temperature, light, nutrients, and predators (e.g., bacteriophage)  Each strain has about 2,000 genes with a core genome of 1,100 genes Distribution of bacteria and archaea in marine water - Abundant small planktonic heterotrophic prokaryotes o Pelagibacter: the most abundant marine heterotrophs  Contain proteorhodopsin, a form of rhodopsin that captures light energy to drive ATP synthesis - Prokaryote densities decrease with depth - Bacterial species dominate in surface waters Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Bacteria and archaea are roughly equal in deeper waters Marine Viruses - Viruses are typically 10X more abundant than microorganisms in the ocean - Highly diverse - Mostly affecting prokaryotic populations Deep sea - Deep sea: lying between 1000 and 6000 metres o >75% of all ocean water - Organisms that inhabit the deep sea must deal with: o Low temperature (psychrophilic or psychrotolerant) o High pressure (piezophilic or piezotolerant) o Low nutrient levels o Must be chemotrophic because deep-sea water is completely dark Deep-sea sediments - Deep-sea core samples are harvested by drilling the ocean floor - Archaeal and bacterial populations occur at depths >2000 metres - Fewer deep-sea microbes at greater depths than are found closer to the surface of the rock layer - Proteobacteria dominate - Novel phyla of archaea are widespread in deep subsurface Hydrothermal vents - Chemolithotrophic bacteria dominate at vent Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 o Utilize inorganic materials from vents o Thermophiles and hyperthermophiles present - More diversity in bacteria than in archaea Lecture 13 | Nutrient cycles Carbon reservoirs - Carbon is cycled through all of Earth’s major carbon reservoirs o Includes atmosphere, land, oceans, freshwater, sediments, rocks, and biomass o All nutrient cycles are linked to the carbon cycle, but the nitrogen (N) cycle links particularly strongly because, other than water, carbon and nitrogen make up the bulk of living organisms - Reservoir size and turnover time are important parameters in understanding the cycling of elements The carbon cycle – removal of carbon dioxide - While the sediments and rocks in the Earth’s crust are the largest reservoirs, CO in the 2 atmosphere is the most rapidly transferred carbon reservoir - Carbon dioxide is removed from the atmosphere by photosynthetic land plants and marine microbes, so a large amount of carbon is found there - More carbon is found in humus (dead organic material) than is found in living organisms in a region Carbon turnover - CO is returned to the atmosphere by 2 o Respiration and decomposition o Microbial decomposition is the largest source of CO release to the atmosphere 2 - Since the Industrial Revolution, human (anthropogenic) activities have increased atmospheric carbon by 40% Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 o This rise in carbon dioxide has led to steadily increasing temperatures worldwide (global warming) because carbon dioxide is a greenhouse gas Photosynthesis and respiration - Phototrophic organisms produce organic or fixed carbon and reduce the level of carbon dioxide in the atmosphere - Oxygenic phototrophic organisms can be divided into two groups: plants and microorganisms o Plants dominate terrestrial environments o Microorganisms dominate aquatic environments - Photosynthesis and respiration are part of redox cycle - Photosynthesis: reduces inorganic carbon dioxide to organic carbohydrates CH O 2 o CO + H O (CH O) + O 2 2 2 2 - Respiration: oxidizes organic carbohydrates to inorganic carbon dioxide o (CH O) + O CO + H O 2 2 2 2 End products of decomposition - The two major products of decomposition are: o Methane (CH ) – a potent greenhouse gas and is produced in anoxic (oxygen4 free) environments o Carbon dioxide (CO ) – most methane is converted to carbon dioxide by 2 methanotrophs  Some CO enters the atmosphere 2 Methane hydrates - Methane hydrates: form when high levels of methane are under high pressure and low temperature Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Huge amounts of methane are trapped underground as methane hydrates; e.g., beneath the permafrost in the Arctic and in marine sediments - Methane hydrates can absorb and release methane - Methane hydrates fuel deep-sea ecosystems called cold seeps Couples’ cycles - All nutrient cycles are interconnected and feedback upon one another - Major changes in one cycle affect the functioning of other cycles - The carbon cycle and the nitrogen cycle are closely coupled - Example: the rate of carbon fixation and plant growth is often limited by the available nitrogen; adding nitrogen to farm fields increases yield Syntrophy and methanogenesis - Methanogenesis is central to carbon cycling in anoxic environments - Most methanogens use carbon dioxide as a terminal electron acceptor, reducing CO to 2 CH with H as an electron donor 4 2 o Some can reduce other substrates (e.g., acetate) to form CH - 4 Syntrophy: methanogens teaming up with partners that supply them with necessary substrates Acetogenesis - Acetogenesis: a H -consuming process that compete with methanogenesis in some 2 environments o Occurs in termite hindgut and rumen with the methanogens and protists Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Methanogenesis is energetically more favorable than acetogenesis - Acetogens can ferment glucose and methoxylated (methyl group bound to oxygen) aromatic compounds (like lignin, found in woody biomass), whereas methanogens cannot. This expands the diet of termites Nitrogen - N is the most stable form of nitrogen and is a major reservoir 2 o N comprises ~70% of the Earth’s air 2 - Most of the nitrogen recycled on Earth is already fixed; that is, in combination with other – elements such as ammonia (NH ) or nitrate (NO ) 3 3 The nitrogen cycle -nitrogen fixation, denitrification, ammonification - - Major nitrogen transformation by microorganisms Nitrogen fixation: N + 8H 2 NH +H 2 3 2 o Carried out by few free-living bacteria and archaea as well as symbiotic bacteria – Denitrification: reduction of nitrate (NO ) to gaseous nitrogen (N , NO (nitric oxide)) 3 2 Ammonification: decomposition of organic nitrogen compounds (such as amino acids and nucleotides) to ammonia Dissimilative nitrate reduction to ammonia: pathway used by nitrate-reducing bacteria under anoxic, nutrient-rich environments (e.g., human gastrointestinal tract) The nitrogen cycle- nitrification, anammox - – Nitrification: oxidation ammonia (NH ) to nitrate (NO ) 3 3 o Two-step process in many cases Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004  – Oxidation of ammonia (NH ) to nitrite (NO ) performed by many 3 2 bacteria and archaea  Oxidation of nitrite (NO – – ) to nitrate (NO ) by some other bacteria 2 3 o Comammox (complete ammonia oxidation) bacteria oxidize ammonia completely to nitrate - Anammox (anaerobic ammonia oxidation): ammonia is oxidized anaerobically with – nitrite (NO ) as the electron acceptor, forming N as the final product 2 2 o Beneficial in wastewater treatment by removing fixed nitrogen that could trigger algal and other microbial blooms Mercury and the environment - - - Mercury is not a microbial nutrient, but an ingredient in many pesticides, a pollutant from the chemical and mining industries, and a contaminant of aquatic systems and wetlands 0 Elemental mercury (Hg ) is the major form in the atmosphere, which is volatile and 2+ oxidized to mercuric ion (Hg ) photochemically Most mercury enters aquatic environments as Hg 2+ Mercury transformation - Mercury has a tendency to accumulate in living tissues and is highly toxic - Hg 2+ readily adsorbs to particulate matter where it can be modified by microorganisms o Primarily by sulfate-reducing and iron-reducing bacteria and methanogenic archaea o Specific enzymes are involved in the methylation of mercury to yield + methylmercury, CH Hg 3 o Methylmercury is extremely toxic and accumulates in the muscle tissues of fish  In addition to neurotoxicity, methylmercury can cause liver and kidney damage in humans Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 o Other microbial transformation of mercury include: 2+  H2S + Hg HgS by sulfate-reducing bacteria  + 0 CH Hg Ch4 + Hg by methanogens 3 Mercury resistance - Some bacteria can convert the toxic forms of mercury to nontoxic or less toxic forms + 2+ o The enzyme organomercury lyase cleaves CH Hg to Ch4 and Hg , which is 3 0 reduced by mercuric reductase to Hg o Plasmid- or transposon-borne operon of mercury-resistant genes 2+  MerP in the periplasm binds Hg  MerT interacts with the mercuric reductase MerA to reduce 2 0 Hg to Hg  and transfers it to MerT 0 Hg is volatile and nonpolar, exits through the cytoplasmic membrane Human impact on the carbon cycle - Since the start of the Industrial Revolution, CO levels have increased by >40% 2 - CO is a greenhouse gas that traps long-wave (heat waves) from the Earth’s surface, in 2 effect making the entire planet a large greenhouse. This phenomenon is called radiative forcing - Dissolved carbon dioxide decreases the pH of the ocean. This acidification endangers coral reefs, which release calcium carbonate as they die - Air and ocean water temperatures are increasing, which increases the oxygen minimum zones in the ocean Major sources of methane emissions Downloaded by Raghad Abushahin ([email protected]) lOMoARcPSD|14412004 - Increases in atmospheric methane account for ~20% of the increase in radiative forcing Human impact on the nitrogen cycle - Humans produce large amounts of nitrogenous fertilizers - Ecological effects of fertilizers are unknown, but the alteration of nitrogen cycles will also change iron availability and the carbon cycle - Nutrient cycles are coupled: change in either nitrogen or carbon cycle