Bacterial Genetics 1 Genetic information Genetic elements Replication

Bacterial Genetics 1 • Genetic information • Genetic elements • Replication • Genetic information transfer • Regulation of gene expression • Mutation and recombination Ch. 7, 8, 10

Sugar backbone (ribose or deoxiribose) Antiparallel – complementary (phosphate diester bond) purine – pyrimidine A T G C Deoxi-nucleotide triphosphate units: d. ATP, d. TTP, d. GTP, d. CTP

The three key processes of macromolecular synthesis are: • DNA replication; • transcription (the synthesis of RNA from a DNA template); and • translation (the synthesis of proteins using messenger RNA as template).

DNA vs. RNA Deoxyribose Thymine T-A G-C Double stranded Ribose Uracil U-A G-C Single but Loop-forming

Types of RNA m. RNA – messenger t. RNA – transfer r. RNA – ribosomal -------------------------Roles: Genetic (informational) Functional: structure (ribosomes) function (ribozymes)

• Bacteria: promoters are recognized by the sigma subunit of RNA polymerase. These promoters have very similar sequences. • Eukarya: the major classes of RNA are transcribed by three different RNA polymerases, with RNA polymerase II producing most m. RNA. • Archaea: have a single RNA that resembles in structure and function the RNA polymerase II.

Although the basic processes are the same in both prokaryotes and eukaryotes, but more complex in eukaryotes. 1. Eukaryotic genes have both: • Exons (coding regions and • Introns (noncoding regions). 2. Both are transcribed to primary m. RNA 3. Introns are excised Exons ligated 4. A cap is added at 3’ end 5. A poly-A tail added at 5’ end is clipped and a 6. Mature m. RNA leaves the nucleus 7. Translation takes place in cytoplasm

Transformation: Free DNA transfer; Requires competence factor Transduction: Virus mediated transfer Generalized: Accidental DNA fragments packed in the virion (at random; lytic cycle) Specialized: Genes next to prophage are transducted (lysogenic cycle) Conjugation: Plasmid-stimulated transfer FF+ Hfr F’ Recipient w/o plasmid Plasmid only is transferred Plasmid is integrated in the chromosome both transferred Plasmid w. chromosomal genes

Bacterial Genetics 2 • Mutation • Recombination • In vivo – techniques, transformation transduction, conjugation • Plasmids, Transposalbe elements • In vitro – techniques • Bacterial genomics • Genetic engineering Ch. 10, 15, 31

Different types of mutations can occur at different frequencies. For a typical bacterium, mutation rates of 10– 7– 10– 11 per base pair are generally seen. Although RNA and DNA polymerases make errors at about the same rate, RNA genomes typically accumulate mutations at much higher frequencies than DNA genomes.

Genetic Recombination General: Rec. A Site-specific: Transposase Eukaryotes: mating + crossing over Prokaryotes: transformation transduction conjungation

Transformation: Free DNA transfer; Requires competence factor Transduction: Virus mediated transfer Generalized: Accidental DNA fragments packed in the virion (at random; lytic cycle) Specialized: Genes next to prophage are transducted (lysogenic cycle) Conjugation: Plasmid-stimulated transfer FF+ Hfr F’ Recipient w/o plasmid Plasmid only is transferred Plasmid is integrated in the chromosome both transferred Plasmid w. chromosomal genes

Recombination General: homologous: same sequence-different source --- complement partial heteroduplex (prokaryotes). --- crossing-over (eukaryotes) Plasmids: mobilize external receptors & pilus F- no plasmid (competent vs. incompetent) F+ complete transfer regular--- separate circular Hfr complete transfer rare --- integrated F' plasmid + chromosomal genes Interrupted mating Site-specific: Transposable elements: transposase, inverted sequences & repeats Conservative Replicative Integrons Inversions

PCR – Polymerase Chain Reaction 1. 2. 3. 4. 5. 6. 7. 8. Target DNA (organismal) Oligonucleotide primers flanking the target sequence, DNA polymerase of a hyperthermophile (Taq) Heat denaturation of the target ds. DNA Cooling – annealing of the Primers Primer extension by polymerase in both directions Repeat of the cycle Accumulated sequence 10 -6 – 10 -9 (Taq = Thermus aquaticus)

Microbial Evolution and Systematics

EARLY EARTH, THE ORIGIN OF LIFE, AND MICROBIAL DIVERSIFICATION Origin of Earth, Evidence for Microbial Life on Early Earth, Conditions on Early Earth: Hot and Anoxic. Origin of Life Catalysis and the Importance of Montmorillonite Primitive Life: The RNA World and Molecular Coding RNA Life The Modern Cell: DNA —> RNA —> Protein

Cambrian stromatolite, ca. 500 My. Old, South Australia Scale is in inches, Courtesy of Stanley M. Awramik

Great Slave Lake, Canada Ancient ca. 2 • 109 y. Stromatolites dominated 5/6 of the Recent etire history of Earth Cryptozoon Kalkowsky, V. H. 1908: Oolith und Stromatolith im Norddeutschen Buntsandstein Hamelin Pool, Shark Bay, W. Australia

Early Proterozoic stromatolite ca. 3000 My. old, South Africa Scale bar is 10 cm long

Modern subtidal stromatolites Shark Bay, Australia

Modern Conophyton equivalent

Old Faithful Geyser Microbially guided silica deposition Yellowstone National Park

Microbial reefs with silica deposition Yellowstone National Park

Tom Brock in Yellowstone 1975

Modern subtidal stromatolites, Lee Stocking Island, Bahamas

Sediment accumulation Microbial mat (red) – Stromatolite (yellow)

Fossil 2000 My old stromatolite Modern marine stromatolite Belcher Island Formation, Canada Shark bay, Western Australia Eoentophysalis belcherensis Entophysalis major Hofmann Ercegovic

Entophysalis major Baja California, Mexico Eoentophysalis belcherensis Mesoproterozoic, ca. 1400 Ma. Gauyuzhuang Formation, China

Planet Earth is approximately 4. 6 billion years old. The first evidence for microbial life can be found in rocks about 3. 86 billion years old. Early Earth was anoxic and much hotter than the present. The first biochemical compounds were made by abiotic syntheses that set the stage for the origin of life.

Condition on Early Earth: Hot and Anoxic Organic synthesis and stability Catalysis on clay and pyrite surfaces RNA and molecular coding

The Modern Cell: DNA —> RNA —> Protein

The first life forms may have been selfreplicating RNAs. These were both catalytic and informational. Eventually, DNA became the genetic repository of cells and the three part system, DNA, RNA, and protein, became universal among cells.

Primitive Life: Energy and Carbon Metabolism

Molecular Oxygen: Banded Iron Formations

Oxygenation of the Atmosphere: New Metabolisms and the Ozone Shield

• Primitive metabolism was anaerobic and likely chemolithotrophic, exploiting the abundant sources of Fe. S and H 2 S present. • Carbon metabolism may have included autotrophy. • Oxygenic photosynthesis led to development of banded iron formations, an oxic environment, and great bursts of biological evolution.

Phanerozoic 0 -0. 54 Neoproterozoic 0. 54 -1. 0 Mesoproterozoic 1. 0 -1. 6 Paleoproterozoic 1. 6 -2. 5 Late Archaean 2. 5 -3. 0 Early Archaean 3. 0 -3. 5 Hadean 3. 5 -4. 6

• Origin of the Nucleus • Origin of organelles as organisms • Endosymbiosis • Lateral flow of genetic information • Reduction of redundancies

Evoluntionary history of endosymbiosis: Mitochondria and Chloroplasts

Evoluntionary history of chloroplast endosymbiosis 2 3

1. Cyanobacteria 6. Euglenophyta 2. Glaucophyta 7. Chlorachniophyta 3. Cryptomonads 8. Dinoflagellata (green). 4. Rhodophyta 9. Dinoflagellata (brown) 5. Chlorophyta 10. Chrysophyta, Heterocontae, Diatoms

Evoluntionary history of chloroplast endosymbiosis: The Hosts

Evidence of Endosymbiosis • • • Size of ribosomes 80 S vs. 70 S Organellar DNA present Organellar DNA is circular Multiple membranes Sensitivity to antibiotics • Models of Symbioses

• The eukaryotic nucleus and mitotic apparatus probably arose as a necessity for ensuring the orderly partitioning of DNA in large-genome organisms. • Mitochondria and chloroplasts, the principal energy-producing organelles of eukaryotes, arose from symbiotic association of prokaryotes of the domain Bacteria within eukaryotic cells, • The process is called endosymbiosis. • Assuming that an RNA world existed, selfreplicating entities have populated Earth for over 4 billion years.

Microbial evolution, phylogeny and classification Fossil record vs. Molecular view of evolution DNA composition GC to AT ratio DNA-hybridization: melting – reanealing DNA-sequence similarity and interrelatedness DNA-sequencing: in vivo vs. in vitro Synthetic DNA – PCR – Molecular cloning Fossil record, and endosymbiotic events Phylogenetic classification Bacterial Classification by phenotypes

Criteria for Molecular Chronometers • Universal distribution • • Functional homology Conserved sequences for alignment Slow rates of evolutionary change Lack of functional constraint • Ribosomal database Project (RDP) >100, 000 sequences.

• Ribosomal RNAs as Evolutionary Chronometers • Ribosomal RNA Sequences as a Tool of Molecular Evolution • Sequencing Methodology • Generating Phylogenetic Trees from RNA Sequences

• Comparisons of sequences of ribosomal RNA can be used to determine the evolutionary relationships between organisms. • Phylogenetic trees based on ribosomal RNA have now been prepared for all the major prokaryotic and eukaryotic groups.

• Comparative ribosomal RNA sequencing is now a routine procedure involving: • Amplification of the gene encoding 16 S r. RNA, • Sequencing it, and • Analyzing the sequence in reference to other sequences. • Two major treeing algorithms are: distance and parsimony.

Signature sequences, short oligonucleotides found within a ribosomal RNA molecule, can be highly diagnostic of a particular organism or group of related organisms. Signature sequences can be used to generate specific phylogenetic probes, useful for FISH or microbial community analyses.

Archaea Crenarchaeota Euryarchaeota

Life on Earth evolved along three major lines, called domains, all derived from a common ancestor. Each domain contains several phyla. Two of the domains, Bacteria and Archaea, remained prokaryotic, whereas the third line, Eukarya, evolved into the modern eukaryotic cell.

• Although the three domains of living organisms were originally defined by ribosomal RNA sequencing, subsequent studies have shown that they differ in many other ways. • In particular, the Bacteria and Archaea differ extensively in cell wall and lipid chemistry and in features of transcription and protein synthesis.

Conventional bacterial taxonomy places heavy emphasis on analyses of phenotypic properties of the organism. Determining the guanine plus cytosine base ratio of the DNA of the organism can be part of this process.

Chemotaxonomy DNA-DNA hybridization Ribotyping Multi. Locus Sequence Typing (MLST) Lipid profiling

The species concept applies to prokaryotes as well as eukaryotes, and a similar taxonomic hierarchy exists, with the domain as the highest level taxon. Bacterial speciation may occur from a combination of repeated periodic selection for a favorable trait within an ecotype and lateral gene flow.

Domain Bacteria Phylum Gamma Proteobacteria Class Zymobacteria Order Chromatiales Family Chromatiaceae Genus Allochromatium Species warmingii

Prokaryotes are given descriptive genus names and species epithets. Formal recognition of a new prokaryotic species requires depositing a sample of the organism in a culture collection and official publication of the new species name and description. Bergey’s Manual of Systematic Bacteriology is a major taxonomic compilation of Bacteria and Archaea. International Journal of Systematic and Evolutionary Microbiology IJSEM

Methods in Microbial Ecology

The enrichment culture technique is a means of obtaining microorganisms from natural samples. Specific reactions can be investigated by enrichment methods by choosing media and incubation conditions selective for particular organisms.

Once a successful enrichment culture has been established, a pure culture can be obtained by conventional microbiological procedures, including streak plates, agar shakes, and dilution methods. Laser tweezers allow one to “pick” a cell from a microscope field and literally move it away from contaminants.

DAPI is a general stain for identifying microorganisms in natural samples. Some stains can differentiate live versus dead cells, and fluorescent antibodies that are specific for one or a small group of related cells can be prepared. The green fluorescent protein makes cells autofluorescent and is a means for tracking cells introduced into the environment. Unlike pure cultures, in natural samples, morphologically similar cells may actually be quite different genetically.

The polymerase chain reaction (PCR) can be used to amplify specific target genes such as small subunit ribosomal RNA genes or key metabolic genes. Denaturing gradient gel electrophoresis (DGGE) can be used to resolve slightly to greatly different versions of these genes present in the different species inhabiting a natural sample.

The activity of microorganisms in natural samples can be assessed very sensitively using radioisotopes and/or microelectrodes. In most cases, measurements are of the net activity of a microbial community rather than of a population of a single species.

Isotopic fractionation can reveal the biological origin of various substances. Fractionation is a result of the activity of enzymes that discriminate against the heavier form of an element when binding their substrates.

1. Microbial impact on global nutrient cycling Carbon - Nitrogen - Sulfur - Phosphorus – Iron 2. Aquatic environments, nutrients & microbes Benthos – Plankton. Biofilms – Dee. P Sea – Hydrotermal vents – Coastal environments – Microbial endoliths – bioerosion – Freshwater eutrophication and pollution – Thermal springs. 3. Aquatic environments Soil microbiology – Rhizobium symbiosis - Mycorhzae

• Microbial communities consist of guilds of metabolically related organisms. • Microorganisms play major roles in energy transformations and biogeochemical processes, thus in • The recycling of elements essential to living systems.

• Microorganisms are very small and their habitats are likewise small. • The microenvironment is the place in which the microorganism actually lives. • Microorganisms in nature often live a feastor-famine existence such that only the bestadapted species thrive in a given niche. • Cooperation among microorganisms is also important in many microbial interrelationships.

• The soil is a complex habitat with numerous microenvironments and niches. • Microorganisms are present in the soil primarily attached to soil particles. • The most important factor influencing microbial activity in surface soil is the availability of water, whereas in deep soil (the subsurface environment) nutrient availability plays a major role.

• In aquatic ecosystems phototrophic microorganisms are usually the main primary producers. • Most of the organic matter produced is consumed by bacteria, which can lead to depletion of oxygen in the environment. • BOD is a measure of the oxygenconsuming properties of a water sample.

• Marine waters are more nutrient deficient than many freshwaters, yet substantial numbers of microorganisms exist there. • Many of these use light to drive ATP synthesis. • Among the prokaryotes, species of the domain Bacteria tend to predominate in oceanic surface waters whereas Archaea are more prevalent in deeper waters.

Energetic Base for Chemolithotrophy at the Deep Ocean Hydrothermal Vents Donors S - reduciers Methanogens H - oxidizers Nitrifyiers S - oxidizers Methylotrophs Fe - Mn oxidizers Acceptors H 2 S°, S 2 O 3 H 2 CO H 2 O 2 , NO 3 NH 4+, NO 2 HS, S°, S 2 O 3 O 2, NO 3 CH 4, CO O 2 Fe 2+, Mn 2+ O 2 Products H 2 S CH 4 H 2 O, NO 2, NO 3 S°, SO 4 CO 2 Fe 3+, Mn 4+

• Iron exists in nature primarily in two oxidation states, ferrous (Fe 2+) and ferric (Fe 3+), and bacterial and chemical transformation of these metals is of geological and ecological importance. • Bacterial ferric iron reduction occurs in anoxic environments and results in the mobilization of iron from swamps, bogs, and other iron-rich aquatic habitats. • Bacterial oxidation of ferrous iron occurs on a large scale at low p. H and is very common in coal-mining regions, where it results in a type of pollution called acid mine drainage.

• The principal form of nitrogen on Earth is nitrogen gas (N 2), which can be used as a nitrogen source only by the nitrogen-fixing bacteria. • Ammonia produced by nitrogen fixation or by ammonification from organic nitrogen compounds can be assimilated into organic matter or • it can be oxidized to nitrate by the nitrifying bacteria. • Losses of nitrogen from the biosphere occur as a result of denitrification, in which nitrate is converted back to N 2.

• Bacteria play major roles in both the oxidative and reductive sides of the sulfur cycle. • Sulfur- and sulfide-oxidizing bacteria produce sulfate, while sulfate-reducing bacteria consume sulfate as an electron acceptor in anaerobic respiration, producing hydrogen sulfide. Because sulfide is toxic and also reacts with various metals, sulfate reduction is an important biogeochemical process. • Dimethyl sulfide is the major organic sulfur compound of ecological significance

Proteobacteria: 1. - Phototrophes anoxygenic: a – Purple sulfur: Chromatium, Ectothiorhodospira, Thiocapsa b – Purple non-sulfur: Rhodospirillum, Rhodomicrobium 2. - Chemolithotrophs: a - Nitrosifyers & nitrifyers: Nitrosococcus, Nitrobacter b - Sulfur oxidizers: Thiobacillus, Beggiatoa, Thioploca c - Iron oxidizers: Leptothrix, Gallionella d - Hydrogen oxidizers e - Methane oxidizer 3. - Chemoorganotrophs: a - Aerobic respirers: Pseudomonads, Acetic A. , N-fixers: Azotobacter, Photobacteria b - Anaerobic respirers: S - reducers, Desulfovibrio c - Facultative aerobes: Enteric bacteria, E. coli d - Fermenters: Zymomonas e - Pathogens: Neisseria, Campylobacter, Salmonella Vibrios, Spirilla, Prostecate bacteria, Myxobacteria

Ammonia and nitrite can be used as electron donors by the nitrifying bacteria. The ammonia -oxidizing bacteria produce nitrite, which is then oxidized by the nitrite-oxidizing bacteria to nitrate. Anoxic NH 3 oxidation is coupled to both N 2 and NO 3– production in the anammoxosome.

Thiocapsa roseopersicina - a sulfide oxidizing, non-oxygenic phototroph containing intracellular sulfur grains and bundled tubular pigment vesicles So

Purple bacteria are anoxygenic phototrophs that grow phototrophically, obtaining carbon from CO 2 + H 2 S (purple sulfur bacteria) or organic compounds (purple nonsulfur bacteria). Purple nonsulfur bacteria are physiologically diverse and most can grow as chemoorganotrophs in darkness. The purple bacteria reside in the alpha, beta, and gamma subdivisions of the Proteobacteria.

3 – Cyanobacteria • Gram-negative bacteria (formerly blue-green ‘algae’) • Evolutionary origins and paleoecology of: Oxygenic phototrophy (unique event in evolution) All chloroplasts in eukaryotes through endosymbiosis Atmospheric oxygen provided by Cyanobacteria Most of the global primary production Stromatolites, organo-sedimentery structures • Ecological significance today: Dinitrogen fixation, respond to P-load as ‘algal blooms’ in coastal and interior waters and enrichment of tropical ocean (Trichodesmium) Picoplankton contribution to open ocean (Synechococcus, Prochlorococcus). Sediment and soil stabilization Microbial endoliths and bioerosion

Microcystis flos aquae – a bloom. Forming, gas-vesicle loaded, Toxic coccoid cyanobacterium Petalonema alatum – a Heterocystous, N 2 -fixing, Filamentous cyanobacterium

hν Phycoerythrin Phycocyanin Allophycocyanin Thylakoid membrane Chlorophyll a Phycobilisome

Microbial Bioerosion Microbial bioerosion is carried by phototrophic cyanobacteria, green and red algae and organotrophic fungi. They may remove up to 50% of carbonate along the surfaces of substrates, such as shells, corals and limestone rocks.

Solentia achromatica Endolithic cyanobacterium responsible for destruction of limestone coasts at the intertidal zone.

Hyella racemus – a modern endolithic cyanobacterium and its Neoproterozoic counterpart Eohyella dichotoma

After microbial endoliths have Successfully colonized the rock…… Microbial euendoliths are integrated in the community of prokaryotes and eukaryotes. Consequently, the combined bioerosion of microbial endoliths (bio-corrosion) and their grazers becomes a progressive force that undercuts limestone coasts, and creates sharp and bizarre shapes called ‘biokarst’.

Biokarst & bioerosional notch are geologically significant Modifications of limestones caused by combined biocorrosion by microbial endoliths and bioabrasion by heir grazers detail

Ammonia and nitrite can be used as electron donors by the nitrifying bacteria. The ammonia -oxidizing bacteria produce nitrite, which is then oxidized by the nitrite-oxidizing bacteria to nitrate. Anoxic NH 3 oxidation is coupled to both N 2 and NO 3– production in the anammoxosome.

Iron Bacteria They are chemolithotrophs able to use ferrous iron (Fe 2+) as sole energy source. Most iron bacteria grow only at acid p. H and are often associated with acid pollution from mineral and coal mining. Some phototrophic purple bacteria can oxidize Fe 2+ to Fe 3+ anaerobically.

Methanotrophy & Methylotrophy Methane is oxidized by methanotrophic bacteria. Methane (CH 4 ) is converted to methanol (CH 3 OH) by the enzyme methane monooxygenase (MMO). The electrons needed to drive this first step come from cytochrome c, and no energy is conserved in this reaction. A proton motive force is established from electron flow in the membrane, and this fuels ATPase. Carbon for biosynthesis comes primarily from formaldehyde (CH 2 O), MMO is a membraneassociated enzyme.