Environmental & Pollution MicrobiologySpring 2010

Environmental Regulation of Microbial Metabolism

Organized as follows:

(I.) Metabolism and Energy Transduction: How bacteria gain fuel (catabolism), and how they make more cells (biosynthesis) and the link between fueling reactions (catabolism) and the generation of cellular energy to keep the bacterial “machine” working

(II.) Enzymes: the catalysts that do all of the work

(III.) Transcriptional organization and control: How the metabolic machine is regulated

(IV.) Catabolic pathways: Diverse strategies bacteria use to occupy almost every conceivable niche

I. Metabolism

(1.) Anabolism (= biosynthesis)

(A.) metabolism = anabolism + catabolism

(B.) anabolism = biosynthetic pathways that lead from the 12 precursor intermediates to cellular building blocks

(C.) catabolism = fueling reactions that lead from ingredients of the external medium to the metabolic needs (precursor metabolites, reduced pyridine nucleotides, energy, nitrogen, sulfur) of the biosynthetic pathways

(2.) How do we make sense out of biochemical complexity?

(A.) Employ a unit process approach

(B.) All of the 75-100 known building blocks, coenzymes, and prosthetic groups are synthesized from only 12 precursor metabolites by reactions that employ energy (high energy phosphate bonds from ATP), reducing power, and sources of nitrogen, sulfur, and single carbon units.

(C.) 12 precursor metabolites

(D.) Role of the 12 precursors as a “pool” linking catabolism and anabolism. ATP, reduced pyridine nucleotide, and C1 units are also provided from catabolism to build the precursor pool

(3.) intermediates formed during catabolism are used for biosynthesis during anabolism by heterotrophs as well as autotrophs

(A.) Consider the resources needed to produce the building blocks to make 1 gram of cells. Treat each pathway as a unit function. Make a list of components (number of enzymes)

and metabolic costs (consumption of energy [as high energy phosphate bonds from ATP], reducing power, nitrogen sulfur, and one-carbon units)

(B.) Detailed material balance sheet approach to biosynthesis.

(4.) Nitrogen assimilation

(A.) Precursor metabolites do not contain nitrogen. What is its source?

(i.) Entry into cell

(ii.) organic forms in soil and sediment habitats are often complexed with polyphenols and tannins

(iii.) Always enters biosynthetic pathways in inorganic form, as ammonium ion [NH4

+]

(B.) Common inorganic sources

(C.) Assimilative uptake of nitrate -- Importance of ammonia repression.

(D.) Ammonia ultimately is taken into biosynthetic pathways via 2 key enzymatic reactions: glutamine synthetase and glutamate synthase

(5.) Nitrogen fixation

(A.) only found in bacteria and archaea

(B.) mediated by nitrogenase

(C.) sequential electron transfer

(D.) 6 electron needed to convert nitrogen to ammonia, but 8 electrons are actually transferred

(6.) Precursor metabolites do not contain sulfur. What is its source?

(A.) Major assimilative route is via O-acetylserine sulfohydrolase H2S + O-acetyl-L-serine ---> L-cysteine + acetate + H2O

(B.) exogenous sulfur sources

(C.) sulfur source in oxic environments?

(D.) sulfate can be assimilated via ATP sulfurylase to make APS which is phosphorylated further to PAPS

Other components that must be obtained by the cell from its environment

(A.) K – via symport and antiport

(B.) Ca – via symport and antiport

(C.) Fe – via chelation

(D.) Mg – via symport and antiport

(E.) Trace elements (e.g., Mo, Cu, Zn, etc.) – various mechanisms

END – 2/17

RECAPITULATE ----> The important constituents are the 12 precursor metabolites, energy (high energy phosphate bonds from ATP), reducing power, and sources of nitrogen, sulfur, and single carbon units.

(7.) Catabolism (= fueling reactions)

(A.) Biosynthetic reactions, as discussed above, are remarkably similar among all microbes. In fueling reactions (= catabolism) microbes demonstrate incredible diversity.

(B.) Goal is to produce reducing power in the form of NAD(P)H + H+ (or FADH + H+) and ATP (or Coenzyme A compounds such as acetyl-CoA)

(C.) Two catabolic strategies: fermentation and respiration

(8.) Fermentation

(A.) internally balanced oxidation-reduction reactions with energy conservation

C6H12O6 ---> 2 C3H4O3- + 2 H+

glucose ---> lactate

(B.) energy is conserved via substrate level phosphorylation

(C.) not all of the potential energy is gained

(D.) importance of excretion of fermentation products

(E.) Diversity of fermentations

(F.) importance of hydrogenases

(9.) Respiration

(A.) oxidation of organic compounds coupled to transfer of electron to an external electron acceptor; starting compounds are completely oxidized; potential difference between reactants and electron acceptor is very large.

(B.) glycolysis

(C.) overview of TCA = citric acid = Krebs cycle

(i.) pyruvate decarboxylated to acetyl moiety which combines with coA; this is added to oxaloacetate to yield citrate; series of dehydrations, decarboxylations, and oxidations regenerates oxaloacetate with CO2 released

(ii.) generation of 4 NADH + FADH + GTP

(D.) link between catabolism and anabolism

10. Oxidation-Reduction

(A.) fermentations -- redox cycle at substrate level

(B.) respiration -- NADH+H+ ---> membrane-bound carriers(i.) carriers embedded in membrane

(ii.) separate movement of protons and electrons

(iii.) NADH dehydrogenase

(iv.) flavoproteins

(v.) iron-sulfur proteins

(vi.) quinone pool (coenzyme Q)

(vii.) cytochromes

(viii.) terminal cytochrome and electron acceptor

(ix.) the quinone cycle (proton translocation)

(C.) protonmotive force

(i.) charge difference used by cells for:

(a.) ion transport

(b.) motility; rotation of flagellum

(c.) generation of ATP

(ii.) F0F1 ATPase

(iii.) ATP formation

(iv.) 3-4 protons per 1 ATP

(v.) proton translocation can be used for multiple cellular events

(vi.) inhibitors and uncouplers

(a.) inhibitors -- bind to and inactivate cytochromes

(b.) uncouplers -- leakage of protons across membrane

(D.) various electron acceptors can be used in respiration

(i.) denitrification: nitrate reductase, nitrite reductase, nitric oxide reductase, nitrous oxide reductase

(ii.) iron and manganese respiration

(iii.) sulfate reduction

(iv.) methanogenesis

(v.) halogenated organic compounds

(11.) 4 general modes of respiration in microbes

(A.) aerobic respiration -- oxygen used as electron acceptor

(B.) anaerobic respiration -- nitrate, ferric iron, manganese, sulfate, or carbonate used as electron acceptors

[Note that (A.) and (B.) really shouldn’t be separated]

(C.) chemolithotrophic metabolism -- inorganic sources used to generate a protonmotive force; H2S, NH3 or H2 can be used

(D.) phototrophic metabolism -- light energy used to generate protonmotive force.

END – 2/22

RECAPITULATE ---> In respiration, unlike fermentation, reducing equivalents are not used to conserve energy at substrate level; rather, a separation of electron flow and proton movement leads to the establishment of polarized membrane generating a protonmotive force. This proton gradient is used to generate ATP as well as for ion transport across the membrane, and for motility

II. Enzymes

(1.) metabolic transformations don’t happen spontaneously!

C6H12O6 + 6O2 ----> 6CO2 + 6H2O G0’ = -2872.2 kJ/mol

Energetics of above reaction are quite favorable but mixing oxygen with glucose won’t result in carbon dioxide and water (in my lifetime or yours!!)

(A.) thermodynamics vs. kinetics

(B.) activation energy

(C.) catalyst lowers activation energy needed for a reaction to occur; it increases the rate of the reaction and it is itself not consumed or transformed in the reaction; catalysts for reactions in metabolic pathways are enzymes -- proteins specific for the reactions that they catalyze

(D.) E + S <====> E-S <====> E + P

(i.) active site

(E.) enzymes increase rate of chemical reactions by 108 to 1020 times the rate at which reaction would occur spontaneously

REALITY CHECK ----> bring theoretical information into the context of real-world enzymes of importance to environmental scientists and engineers.

(i.) example of toluene as a model contaminant.

(ii.) microbial population adaptation.

III. Operon Structure and Transcriptional Control

(1.) organization of xyl regulon from pWW0

(A.) physical map

(i.) gene order

(ii.) relative placement

(iii.) restriction fragments

Pu xyl UWCMABN Pm xyl XYZLTEGFJQKIH xyl S Ps2 Ps1 Pr xyl R

+

3-methylbenzoate

--

σ70

σ54

σ54

HU

IHF

+

σ70σ70/ σS

+

xylene

-

Ramos, Marqués, & Timmis

(B.) pathway and enzymes

(i.) oxygen is a reactant

(ii.) link the pathway to the gene organization

(C.) regulatory paradigm

(i.) role of XylR at Pu and Ps1

(ii.) role of XylS at Pm

(iii.) role of effectors (= inducers)

(iv.) modular organization of some regulatory proteins

Pu xyl UWCMABN Pm xyl XYZLTEGFJQKIH xyl S Ps2 Ps1 Pr xyl R

+

3-methylbenzoate

--

σ70

σ54

σ54

HU

IHF

+

σ70σ70/ σS

+

xylene

-

Ramos, Marqués, & Timmis

(2.) above is example of positively controlled operon

(A.) operon -- complete unit of gene expression involving genes coding for several polypeptides on a polycistronic mRNA

(B.) positive control -- regulatory protein promotes binding of RNA polymerase and thus increases mRNA synthesis

(C.) positive control is widely used for catabolic operons of importance in biodegradation of chemical of environmental significance

(3.) Negatively controlled pathways

(A.) lactose operon as an example

(4.) Summary

(A.) Regulatory modes just described are a part of the “adaptation” process.

IV. Catabolic Pathways

NOTE ----> Material covered here is not in the text.

RESOURCE FOR THIS MATERIAL IS AT <umbbd.msi.umn.edu>

(1.) peripheral pathways that feed into the central pathways (EMP and TCA)

(A.) funnel -- diverse compounds feeding into a few conserved peripheral pathways that feed intermediates into TCA cycle

(B.) starting materials that can feed into a few central, key intermediates: catechol and protocatechuate

(2.) Intradiol (ortho) cleavage route for oxidation of catechol

(3.) Extradiol (meta) cleavage route for oxidation of substituted catechols

END – 2/24

OVERVIEW ----> will cover a variety of classes of environmental contaminants and show how their degradation fits into the paradigm of “diverse compounds feeding into a few conserved peripheral pathways that feed intermediates into TCA cycle”

(4.) toluene

(A.) not a biosynthetic product; diagenic origin; petroleum spills

(B.) initial oxidation routes

(i.) alkyl oxidation

(ii.) arene oxidation

(iii.) extradiol (meta) cleavage of catechols

Toluene (methylbenzene) is an aromatic hydrocarbon natural product of diagenic origin and an important commercial chemical. It is, for example, commonly used as a paint thinning agent and in other solvent applications. The BTEX mixtures referred to in bioremediation applications contain benzene, toluene, ethylbenzene and xylenes. The biodegradation of toluene has been well-studied at the molecular level and it, thus, serves as one of the principal models for understanding the mechanisms of bacterial benzene ring metabolism.

(5.) Naphthalene as a model polycyclic aromatic hydrocarbon (PAH)

(A.) PAHs also not biosynthetic products; diagenic origin or from combustion; contaminants as a result of manufactured gas plant (MGP) processes, creosote works, gas condensates, petroleum fractionation

(B.) pathway to catechol

(C.) extradiol (meta) cleavage

Naphthalene is a fused ring bicyclic aromatic hydrocarbon and thus serves as a model for understanding the properties of a large class of environmentally prevalent polycylic aromatic hydrocarbons (PAHs). Naphthalene and its substituted derivatives are commonly found in crude oil and oil products. Certain PAHs are strong human carcinogens leading to widespread interest in the microbial metabolism of these compounds.

(6.) Nitroaromatics

(A.) explosives

(B.) nitro group can be reduced leading to aminophenol which is degraded by extradiol (meta) cleavage

(C.) nitro group can be eliminated leading to catechol which is degraded by extradiol (meta) cleavage

(7.) Sulfonated aromatics

(A.) xenobiotics synthesized as dyestuffs or as starting materials in pharmaceutical industry

(B.) example of aminobenzenesulfonate, in which oxygenase attacks amino group (electron donating)

(C.) sulfonate group must also be eliminated by a dioxygenase

(D.) extradiol (meta) cleavage

Many aromatic sulfonates are produced on a multi-ton scale and can be detected in the environment. Hundreds of thousands of kilograms of 2-aminobenzenesulfonic acid are produced for use in the U.S. annually. It is used in organic synthesis and in the manufacture of various dyes and medicines.

(8.) Chlorobenzene

(A.) xenobiotic used as a starting material in chemical synthesis, as a solvent, and as a heat transfer agent

(B.) Cl on aromatic nucleus presents special problems in chemistry -- strong electron withdrawing group

(C.) modified intradiol (ortho) pathway for oxidation of chloroaromatics

(D.) Cl as a leaving group during lactone formation

(E.) oxoadipate is metabolized further to succinyl CoA and acetyl CoA

Although chlorobenzene is not formed in any significant quantity naturally, the United States chemical industry synthesized 231 million pounds of chlorobenzene in 1992 alone. Chlorobenzene is commonly used in the manufacture of nitrochlorobenzenes, phenol, aniline, and other industrial chemicals. It also functions as a paint solvent, heat-transfer medium, and an intermediate compound in the manufacture of some pesticides. Most chlorobenzene that is discharged into the environment quickly evaporates and is subsequently degraded atmospherically via reactions with photochemically-generated hydroxyl radicals. Enzymes involved in the microbial degradation of chlorobenzene are believed to have evolved from simliar enzymes catalyzing the degradation of benzene and toluene.

The Japanese Database for Environmental Fate of Chemicals has information on the rates and pathways of Biodegradation of Chlorophenols and Chlorobenzenes in Sediments.

(9.) 2,4-Dichlorophenoxyacetic acid (2,4-D)

(A.) broad leaf herbicide; often used together with 2,4,5-Trichloropehoxyacetic acid (2,4,5-T)

(B.) problem when 2 chlorines present on aromatic nucleus

(C.) variation of the modified intradiol (ortho) pathway

(D.) oxoadipate is metabolized further to succinyl CoA and acetyl CoA

2,4-Dichlorophenoxyacetic acid (2,4-D), a chlorinated phenoxy compound, functions as a systemic herbicide and is used to control many types of broadleaf weeds. It is used in cultivated agriculture and in pasture and rangeland applications, forest management, home and garden situations and for the control of aquatic vegetation. The wide use of this compound has prompted interest in its biodegradation. 2,4-D biodegradation may produce a byproduct antibiotic protoanemonin, which can be degraded to cis-acetylacrylate by dienelactone hydrolase of Pseudomonas sp. strain B13.

(10.) Alkanes

(A.) from petroleum; major constituent in crude oil spills

(B.) degradability is function of chain length and degree of branching

(C.) pathway proceeds by terminal oxidation to an acid which is converted to a CoA ester; metabolism by beta-oxidation

n-Octane is used in organic syntheses, calibrations, and azeotropic distillations and is a common component of gasoline and other petroleum products. The engine fuel antiknocking properties of an isomer of n-octane (2,2,4-trimethylpentane or isooctane) are used as a comparative standard in the Octane Rating System.

(11.) Atrazine

(A.) most widely used herbicide; detected in almost all well water sampled in agricultural regions of New Jersey

(B.) degraded to urea, which is converted to carbon dioxide and ammonia

(C.) pH effects; metabolic regulation effects?

Atrazine is a broad-leaf, pre-emergence herbicide. Eighty million pounds are applied to soils annually in the United States, more than any other herbicide. Atrazine is the leading member of a class of triazine ring-containing herbicides that includes simazine and terbuthylazine. Atrazine has been found to be less biodegradable than other less substituted s-triazine ring compounds with a half-life ranging from 1 week to 1 year in different soils.

(12.) Degradation via anaerobic respiratory heterotrophs

(A.) less information; organisms more difficult to work with

(B.) toluene as a model

(C.) the importance of CoA ester

(D.) conversion of toluene via benzylsuccinate to benzoylCoA; benzoylCoA reductively converted to ketocyclohexane CoA carboxylate which is hydrolyzed; succesive dehydrogenations and decarboxylations yields acetylCoA

(13.) How are all of these pathways regulated?

(A.) For those that have been studied in detail, all use positive transcriptional control

(i.) toluene pathways -- XylR/S and TbuT as models

(ii.) naphthalene -- NahR

(iii.) nitroaromatics -- unknown

(iv.) sulfonated aromatics -- unknown

(v.) chlorobenzene -- unknown

(vi.) 2,4-D -- TfdR/S

(vii.) alkanes -- AlkT

(viii.) atrazine -- unknown

(ix.) anaerobic toluene -- TutT

(14.) Contaminants occur in mixtures

(A.) metabolic suicide from co-induction of extradiol (meta) pathway when chloroaromatics are also being degraded in the same organism.

(B.) Is suicide inactivation inevitable?

Table 1: Compound concentration (mg/Kg) in material over 9 weekly (WK) intervals

Parameter Start-up WK-1 WK-2 WK-3 WK-4 WK-5 WK-6 WK-7 WK-8 WK-9

phenol 90 19 50 33 34 21u 29 20 22 22u

2,4-dimethylphenol NR NR NR NR NR NR NR 38u 146 125

anthracene 110 94 208 124 108 114 88 89 59 74

benzo(a)anthracene 110 69 164 108 114 90 70 60 56 50

1,2,4-trichlorobenzene NR NR NR NR NR NR NR 11u 10u 4

1,2-dichlorobenzene 820 139 543 49 108 158 113 104 59 20

1,4-dichlorobenzene 100 22u 70 9 7 9u 9 10u 5 9u

acenaphthene 250 154 346 214 204 197 152 135 127 98

dibenzofuran 310 142 345 230 231 207 167 143 171 127

fluoranthene 110 43 95 64 53 48 38 35 27 27

chrysene NR NR NR 11 10 5u 8 6u 7 5u

fluorene 1600 20u 920 426 468 812 469 531 83 68

2-methylnaphthalene 1800 974 2420 1040 947 1170 1020 1040 977 591

nitrobenzene 600 190 722 248 240 449 266 176 122 72

naphthalene 43000 12100 208000 10000 9970 12100 15900 16400 13700 3360

N-nitrosodiphenylamine 1700 799 1970 1500 1870 1340 967 950 869 1050

phenanthrene 320 961 396 230 250 237 174 161 162 143

benzo(b)fluoranthene NR NR NR NR NR NR NR 10u 6 4

benzo(k)fluoranthene NR NR NR NR NR NR NR 20u 3 5

benzo(a)pyrene NR NR NR NR NR NR NR 9u 3 3

pyrene 31 24 62 53 10 35 37 29 21 18

Notes: NR; data not reported u; below detection limits

Source: O'Brien & Gere Bioremediation Pilot Test 1996