PHYSIOLOGY: THE BIOLOGICAL STUDY OF THE FUNCTIONS OF LIVING ORGANISMS AND THEIR PARTS.

RELATIONSHIP OF STRUCTURE TO FUNCTION

However, difficult for brain to function to without the heart or correspondingly, difficult for genome to function without ribosomes. Studying the parts doesn't tell the complete story.

Physiology can be regarded as the study of the life processes of an organism, mediated by its structures operating together, to accomplish the requirements of life for that organism.

Bacteria must accomplish in a few cubic micrometers all of the same processes that we perform in the context of life: nutrient acquisition and waste secretion; energy production; biosynthesis; replication; cell division; repair processes; sensing of and adaptation to environment.

Bacteria are very successful at growth and survival, yet they exhibit greater physiological and metabolic diversity than larger life forms (animals, plants, etc.). Microbial physiology can allow detailed studies of processes that can not be studied in larger life forms.

A fundamental difference between microbial physiology and plant or animal physiology:

the organism IS a cell, whereas large life forms are collections of cells

Consider the difference between an average bacterium (measured in mm) and an elephant (measured in meters).

V = 4/3 p r³ Volume of sphere/ coccus

V= p r² h Volume of cylinder/ bacillus

smallest bacteria (e.g., mycoplasmas) 0.2 mm in diameter---V = 4.18 x 10^-15 cm³

largest bacterium known (60 mm x 600 mm---V = 1.74 x 10^-6 cm³

The ratio of these two numbers is 4.2 x 10⁸ !!!

Eucaryotes span an even larger range of about 10¹⁸!!

However, it is not size per se that is important from a physiological perspective. It is surface to volume ratio that is critical.

Surface of cylinder S = 2 p r h, hence S/V = 2/r

Surface of sphere S = 4 p r h, hence S/V = 3/r

As r gets smaller and smaller, S/V gets larger and larger--difference between sphere and cylinder becomes insignificant. If elephant is approximated by a sphere of 3 m, then 3/3 = 1.0 m^-1 Correspondingly, a small mycoplasm would have S/V = 3/1 x 10^-7 = 3 x 10⁷ m^-1

Physiological consequence: microorganism can take up nutrients from, and secrete wastes into, the medium very rapidly. Microbial enzymes often more efficient as well. Rapid metabolism translates into rapid growth. Typical "generation times" for bacteria are 20 minutes to 3 hours. Elephants do not communicate with their environment at a biochemical level as rapidly--they have to find and eat food; they have elaborate respiratory systems, digestive systems, etc. Consequence: the generation time for an elephant is about 3 years.

Elephant: structures are composed of differentiated cells, all of which share many related properties.

Microbes: made up of macromolecular aggregates

Again, size is an issue. You can easily isolate the elephants liver and study it, even if you suffer a 99% loss during isolation. It is not possible to isolate an individual organelle from an individual cell for bacteria. With bacteria, one must study populations of organisms and populations of structures isolated from that population of organisms.

Many individual measurements can be made on a single elephant to determine the variability of that individual, and that individual can then be compared to other individuals. With microorganisms, one most always measure the average response of a population or the properties of the average organelle or molecule isolated from the population. Cells of the microbial population generally speaking will not be identical and will not respond identically; mutation, natural fluctuations, etc. insure this will be the case. In microbial physiology, we MUST study the population and make assumptions about individuals from those responses. However, it is easy to obtain large populations. Studying the genetics of microbe is easy; obtaining billions of elephants for study is not a possibility in any time frame.

Considered collectively, microorganisms exhibit unparalleled metabolic diversity and adaptability. They survive in hostile environments where large life forms cannot survive. Adaptability exceeds that of eucaryotes by many orders of magnitude. Hence, although microbes are morphologically simple, the are metabolically and physiologically complex. In contrast, large life forms are collectively complex in structure but physiologically simple.

Nutritional requirements of large life forms are generally speaking more complex than those of microorganisms. Nutritional diversity is reflected in the much more varied metabolism found in bacteria.

Microorganisms provide model systems for understanding how larger life forms function and respond to their environment. Example: studying photosynthesis in cyanobacteria is a good model system for understanding how this process occurs in complex seaweed or a tree. The continuity of life, established through evolution, makes this so.

How did diversity arise? Organisms evolved to fill all niches on Earth. In the last edition (4 volumes) of Bergey's Manual of Determinative Bacteriology, about 3200 species of bacteria are described. This is estimated to represent a maximum of about 1% of all microbial species on Earth--i.e., the real number of bacterial species could be as large as 300,000 to more than 1,000,000!!

4. 6 Billion Years Ago Formation of our solar system

4.0 BYA Earth cools and H20 condenses

4.0-3.5 BYA Primordial Soup develops "Chemical evolution occurs by photochemical synthesis of organic chemicals

3.5 BYA Origin of life--fossil evidence of life exists in rocks younger than this age--Stromatolites (fossilized microbial mats)

3.5-1.9 BYA Microbial diversification occurs

2.5 BYA Origin of oxygenic phototrophs (ancestors of modern of cyanobacteria

2 BYA Atmosphere becomes oxygenic (~1%); Fe deposits form

1.9-1.2 BYA Development of ozone shield; oxygen rises to ~10%

1.2 BYA Origin of Modern Eukaryotes

1.0 BYA Origin of Sexes

800 MYA Origin of Metazoans; oxygen content of atmosphere reachs ~20%

500 MYA Plants and Animals

400 MYA Animals develop on land

250 MYA Dinosaurs

190 MYA Mammals

65 MYA Dinosaurs extinct

5 MYA Humans Evolve

Chemical evolution: The primitive Earth had no oxygen, and the atmosphere was thus reducing. CH4, CO2, N2 NH3 were likely prerent, in addition to some CO and H2. There was probably lots of FeS and some HCN produced from discharge reactions of CH4 and NH3. The primitive Earth was probably 100°C, and early life was likely thermophilic by necessity. Geochemical and electrical discharges (lightning) produced simple then more complex organic molecules. Many biochemically important molecules can be produced in this manner in the laboratory from similar gas mixtures. These include: simple sugars, amino acids, purines, pyrimidines, nucleotides, and fatty acids. In the absence of bacteria, these molecules would have become quite concentrated in some environments. Polymerization reactions took place by dehydration reactions, possibly catalyzed by exposed surfaces of clays, pyrite, or basaltic glasses.

Primitive organisms: 1. must have had metabolism: ability to accumulate, convert and transform nutrients and energy; 2. hereditary mechanism--the ability to replicate and transfer its properties to its offsping. Both require the development of a cellular structure. Such structures probably developed spontaneously from lipids and possibly simple proteins that trapped polynucleotides, polypeptides, and other small organic molecules. It now seems likely that before DNA and proteins, much of catalytic and hereditary features of primitive organisms was performed by RNA--hence the term the "RNA world." A recent Science paper showed that a simple RNA molecule can faithfully make precise copies of itself--a prerequisite for the development of heredity as we know it today.

Metabolism in these primitive organisms was certainly anaerobic, but it is difficult to say what type of reactions occurred. One likely possibility would have been a primitive respiration based on oxidation of hydrogen with reduction of S0 to H2S. The resulting proton gradient could have been coupled to ATP formation through a primitive ATPase. Alternatively, organic compounds could have been fermented to produce energy and metabolites for biosynthesis. A third possibility, believed to be somewhat less likely as an original mechanism, would have been photosynthesis.

Further chemical evolution and the formation of tetrapyrroles would have led to cytochromes, thus respiratory electron transport chains, and more importantly photosynthesis. The first phototrophs were again anoxygenic and may have resembled purple or green photosynthetic bacteria of today. Photoreactions would have generated ATP while reductants would have come from H2S and other reduced compounds in the environment. The origins of oxygenic photosynthesis--the second photosystem--greatly accelerated evolution of microbes. An endless supply of electrons, through water, was now available.

We now know from comparative analyses of nucleic acid sequences that three main lines of descent were established relatively early in cellular evolution. Ancestral eucaryotic cells probably resembled modern procaryotic cells and lacked mitochondria, chloroplasts, and possibly even the membrane-bound nucleus. Modern eucaryotes arose late in evolution through endosymbiotic events, in which the primitive eucaryote acquired a respiratory aerobic bacterium to give rise to mitochondria and a cyanobacterium to give rise to the chloroplast. Development of the eucaryotic nucleus may have developed later as the complexity of the genome increased.

The majority of Earth's evolution has been dominated by microbes: nearly 80% of the total evolutionary time span. Until recently, we understood some aspects of metazoan evolution because of the fossil record, we knew little about the evolution of microorganisms. This has changed dramatically as molecular sequence analysis methods have developed in the past 25 years.

1. Molecule should be universally distributed across organisms to be studied.

2. Molecular should be functionally homologous in all organism.

3. It must be possible to align the molecules without ambiguity in order to identify regions of homology and heterogeneity clearly.

4. Sequence of molecule chosen should change at a rate commensurate with the evolutionary distance to be measured. The more similar two sequences, the more closely related the two organisms.

Ribosomal RNAs are ancient molecules, functionally constant, universally distributed, and moderately well conserved over broad phylogenetic distances.

Prokaryotic ribosomes contain 3 RNA molecules: 5S, 16S, and 23S rRNAs. 5S has been used but is too small (~120 nt). Both 16S rRNA (~1500 nt) and 23S (~3000 nt) rRNAs have been used; 16S is most common, and more than 2000 different organisms have now been sequenced. In eukaryotes, the equivalent molecule is the 18S rRNA of the small subunit of the 80S ribosome.

Ribosomal RNAs are easily sequenced these days. Polymerase Chain Reaction can be used to amplify even tiny amounts of RNA into DNA sequences which can then be sequenced. It is no longer necessary to grow a particular organism, since sequences can be obtained from field collected material. This offers hope of identifying the degree of diversity of microbial life on the Earth.

Other molecules, including a variety of proteins can be used to provide additional information. Examples include RecA protein and RNA polymerase subunits of eubacteria.

"There must be a prokaryote somewhere: Microbiology's search for itself." Carl R. Woese, Microbiological Reviews 58: 1-9 (1994).

"The winds of (evolutionary) change: breathing new life into microbiology. G. J. Olsen, C. R. Woese, and R. Overbeek. Journal of Bacteriology 176:1-6 (1994).

Most of you were taught that there are 5 kingdoms of organisms a la Whittaker (~1930): Animals, Plants, Fungi, Protists and Monera. Although convenient from some points of view for grouping and classifying organisms, this view of life doesn't represent the phylogenetic (genealogical or evolutionary) relationships among these organisms.

Most of us also once taught that life could be conveniently divided into two superkingdoms: eucaryotes and procaryotes, and this difference is a central dogma of biology and microbiology. This notion developed in the late 1950's and early 1960's.

1. Proteobacteria (Purple Bacteria). Largest, most physiologically diverse group of bacteria. At least five subdivisions (a, b, g, d , e). Three subdivisions have phototrophic members; many are heterotrophs or chemolitotrophs, however. Sulfur oxidizers, sulfur reducers, nitrate/nitrite oxidizers; etc. Very diverse, and most highly evolved group.

2. Cyanobacteria: Heterogeneous group. Characterized by oxygen-evolving photosynthesis, the presence of chlorophylls and phycobiliproteins

3. Gram-positive bacteria. Divided into Low G+C and High G+C groups. Contains one photosynthetic group (heliobacteria), a relative of Clostridium. Low G+C group includes Bacillus, Clostridium, heliobacteria, and lactic acid bacteria. High G+C group includes Corynebacterium, propionic acid bacteria, mycobacteria, Nocardia, Streptomyces, Micromonospora.

4. Chlamydia. Obligate intracellular parasites; lack peptidoglycan, but have outer membrane; agent of some sexually transmitted infections.

5. Planctomyces-Pirella. Budding organisms that lack peptidoglycan, have a proteinaceous cell wall. Obligate aerobes, prefer dilute nutrients.

6. Bacteriodes-Flavobacteria. Major line of gram-negative bacteria. Cytophaga/Flavobacteria is one lineage; Bacteroides a 2nd. Mixture of properties, including obligate fermentative anaerobes (Bacteroides), obligate aerobes (Sporocytophaga).

7. Green sulfur bacteria. All are strictly anaerobic phototrophs; contain chlorosomes, bacteriochlorophylls, and fix carbon by reverse TCA cycle

8. Sprirochetes. (Spirochaeta, Borrelia, Treponema). Free-living or parasitic; many are pathogens. Unique morphology is a valid taxonomix criterion.

9. Deinococcus-Thermus. Only two well-known genera, one gram-positive and one gram-negative. Deinococcus is highly radiation resistant; Thermus is a chemoorganotrophic thermophile.

10. Green nonsulfur bacteria. Confusing properties. Affinities to both cyanobacteria, purple bacteria and green bacteria. Appear to have evolved and diverged very early.

11. Thermotoga-Thermosipho. Only 2 genera. Anaerobic, fermentative, marine hyperthermophiles (55-90°C).

12. Aquifex-Hydrogenobacter. Extreme thermophiles (85-90°C). Aquifex is an aerobic chemolithotroph that oxidizes H₂ or reduced sulfur compounds. Hydrogenobacter oxidizes only H₂.

1. Extremely halophilic Archaea. Halobacterium sp. Unique phototrophy based on bacteriorhodopsin.

2. Thermoplasma acidophilum. Acidophilic thermophile (55°C). Aerobic chemoheterotroph.

3. Hyperthermophiles. Some grow above 100°C; most are strictly anaerobic and require elemental sulfur as an electron acceptor. Can oxidize H₂ or organic compounds. Sulfolobus oxidzes sulfur aerobically. Archaeglobus reduces sulfate and can produce methane as well.

4. Methanogens. Strictly anaerobic organisms that produce CH₄. CO₂ can be reduced with electrons from various sources (often H₂) or CH₄ can be produced from methyl substrates such as CH₃OH (methanol).

Bacteria have peptidoglycan in their walls (exceptions: Planctomyces-Pirella (proteins), chlamydia and mycoplasma--no walls in these two)

No Archaea have peptidoglycan in their walls. Some have glycoproteins; some have sulfated polysaccharides, but most have proteinaceous walls.

A few methanogens have a pseudopeptidoglycan, but this lacks muramic acid (unique to peptidoglycan).

Most useful of all non-genetic markers--very distinctive.

Bacteria and Eucarya have fatty acids linked to glycerol in ESTER linkages.

Archaea have branched chain alcohols linked to glyercol in ETHER linkages. Both Phytanyl and Biphytanyl forms are found.

All Bacteria have a single type of RNA polymerase (a2 b b') = Core Polymerase. To this is added a sigma subunit (s) that recognizes specific promoters to initiate transcription.

There are several types of Archaeal RNA polymerases. These have 8-10 subunits, more similar to Eucaryotic RNA polymerase.

Initiator is Formyl-methionine in Bacteria; Methionine in Archaea and Eucarya

Archaeal and Eucaryotic elongation factors sensitive to diptheria toxin; not so in Bacteria

Differential sensitivity to antibiotics (see Handout)

Functional hybrids of ribosomal subunits from Sulfolobus (Archaea) and Yeast (Eucarya) have been made. Sulfolobus 50S subunit + 40S subunit of Yeast = functional ribosome! No functional hybrids of Bacterial and Eucaryotic ribosomes have been made.