Transcript Environmental & Pollution Microbiology Spring 2010 Environmental Regulation of Microbial Metabolism Organized as follows: (I.) Metabolism and Energy Transduction: How bacteria gain fuel (catabolism), and.

Environmental & Pollution Microbiology
Spring 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
DG0’ = -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
Ramos, Marqués, & Timmis
3-methylbenzoate
-
+
Pu xyl UWCMABN
Pm xyl XYZLTEGFJQKIH
+
xyl S Ps2 Ps1 Pr xyl R
+ -
IHF
σ54
σ70/ σS
HU
σ70
σ54
σ70
xylene
(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
Ramos, Marqués, & Timmis
3-methylbenzoate
-
+
Pu xyl UWCMABN
Pm xyl XYZLTEGFJQKIH
+
xyl S Ps2 Ps1 Pr xyl R
+ -
IHF
σ54
σ70/ σS
HU
σ70
σ54
σ70
xylene
(2.) above is example of positively controlled operon
(A.) operon -- complete unit of gene expression involving genes
for several polypeptides on a polycistronic mRNA
coding
(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, heattransfer 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 2,4,5Trichloropehoxyacetic 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. 2,4D 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