Chapter 3

Bacterial Metabolism and Genetics

Bacterial Metabolism
Metabolic requirements
Metabolism, Energy, and Biosynthesis Metabolism of Glucose Embden-Meyerhof-Parnas Pathway Tricarboxylic Acid Cycle Pentose Phosphate Pathway

Metabolic Requirements

Minimum requirement for growth

Oxygen requirements
Classification based on nutritional requirements

Minimum requirements for growth

C, O, H, N, S, P K, Na, Mg, Ca, Cl Fe, Zn, Mn, Mo, Se, Co, Cu, Ni Many bacteria secrete special proteins, siderophores, to concentrate iron from dilute solutions.

Oxygen requirements

Obligate anaerobes: Clostridium perfringens Obligate aerobes: Mycobacterium tuberculosis Facultative anaerobes: Escherichia coli

Classification based on nutritional requirements

autotrophs

lithotrophs
organotrophs

Metabolism, Energy, and Biosynthesis

catabolism: substrate breakdown and conversion anabolism: synthesis of cellular constituents The metabolites are converted via one or more pathways into pyruvate.

From pyruvate, carbons are channeled to energy production or the synthesis of new carbohydrates, amino acids, lipids and nucleic acids.

Metabolism of Glucose

production of energy of useful forms production of energy through fermentation, anaerobic respiration, or aerobic respiration

Embden-Meyerhof-Parnas Pathway (Glycolysis)

Converting glucose to pyruvate substrate level phosphorylation: generating ATP from ADP via kinases conversion of NAD to NADH produces ATP (via electron transport chain) Fermentation starts from pyruvate.

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Fermentation

Organic molecules, rather than oxygen, are used as electron acceptors to recycle NADH. Monosodium glutamate is the largest fermentation product in the world.

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Tricarboxylic Acid Cycle

Pyruvate is completely oxidized to water and CO2 in the presence of O2 via the TCA cycle. Oxaloacetate and acetyl-CoA condense to form citrate.

Citrate is then converted to oxaloacetate through a series of steps.

Produces 2 CO2, 3 NADH, 1 FADH2, and 1 GTP.

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Electron Transport Chain

The electrons carried by NADH are passed through a stepwise fashion through a series of donor-acceptor pairs and ultimately to O2 (aerobic respiration) or other terminal acceptors, including nitrate, sulfate, CO2, ferric ion (anaerobic respiration).

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Energy Generated through Aerobic Glucose Metabolism

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Importance of the TCA Cycle

the most efficient mechanism for generating ATP the final common pathway for the complete oxidation of amino acids, fatty acids, and carbohydrates supplying key intermediates, e.g. ketoglutarate, pyruvate, oxaloacetate for the ultimate synthesis of amino acids, lipids, purines, and pyrimidines

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Pentose Phosphate Pathway or Hexose monophosphate Shunt

Ribulose-5-phosphate is converted to ribose-5phosphate, a precursor of nucleotide biosynthesis, or xylulose-5-phosphate. The pathway uses transketolases and transaldolases to generate various sugars, which are used metabolically or shunt into the glycolysis pathway.

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The Bacterial Genes and Expression

Transcription

Translation
Control of Gene Expression

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Transcription

mRNA DNA-dependent RNA polymerase sigma factors promoters and operators

operons
polycistronic mRNA

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Template and Sense Strands

CAAGAAAAAT

AAATTAATTA ATACTATATT

CAAAAAAACT GTTATAAAAA

-51

TTTTATATTT

TTTATTGTAC TTTAAAAAAA

TTCGGTTA AT ATAA TGCATA

-1

-35 +1
TATGGATTAT ATAGTCATAT AATTTCTTTT

-10
TATTGTAATT ATTTCAGTTT +50

CTTATCCTCT

TATAAATTAG AATT GGAGGG AATTC GTTGA AAGAAAATAC RBS M K E N T

+100

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Translation -- tRNA and mRNA

tRNA codon anticodon

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Translation -- Reading Frames

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Translation -- Initiation and Termination Codons

initiation codons: AUG, GUG, UUG termination (nonsense) codons: UAA (ochre), UAG (amber), UGA (opal) nonsense mutations: a sense codon that is mutated to a nonsense codon nonsense suppressor mutation (a conditional lethal mutation) -- a tRNA mutation at the anti-codon loop that allows readthrough of a nonsense mutation.

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Translation -- Initiation
How do bacteria choose an AUG codon to initiate translation?
What is the function of ribosomalbinding site (RBS)? RBS is an A-G-rich sequence, 4n.t. long, located about 10 nucleotides upstream of an initiation codon. RBS complements the 3 region of 16S rRNA in the ribosome. What is the amino acid that starts the sequence of a protein when GUG (Val) or UUG (Leu) is used for initiation? Transpeptidation
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Control of Gene Expression

sigma factor and gene expression quorum sensing: N-acyl homoserine lactone (AHL) or small peptides

cAMP
pathogenicity island negative control: repressors operators positive control: apoinducer

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The lactose operon

E. coli does not use lactose when glucose is present. Allolactose is the inducer. RNA polymerase does not have a strong affinity to the lac promoter. LacI, the repressor, promotes the binding of RNA polymerase to the promoter. The CAP-cAMP complex promotes the binding of RNA polymerase, through the C-terminal domain of the -subunit.

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The Tryptophan Operon

Repression of the operon requires TrpR. Tryptophan is a corepressor. Binding of TrpR to the operator blocks the binding of RNA polymerase to the promoter. The expression of the operon depends on the translation of the leader region of the mRNA (posttranscriptional control; attenuation).

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DNA Replication

helicase, primase gyrase, DNA polymerases, DNA ligase DNA cannot be synthesized without a primer. semiconservative synthesis bidirectional synthesis

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Bacterial Growth

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Population Dynamics

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Mutations

transition, transversion silent mutation, missense mutation, nonsense mutation, frameshift mutations, null mutation, insertional mutation, deletion, inversion conditional lethal mutations: temperature-sensitive mutations, suppressor mutations reversion mutagens Ames test

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Carcinogenicity Test (Ames Test)

Do you know why a histidine mutant instead of a wild-type Salmonella strain is used in Ames test?

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DNA Repair

direct DNA repair excision repair recombinational or postreplicational repair the SOS response error-prone repair

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Gene Exchange in Prokaryotic cells

plasmids
bacteriophages fertility factor transposons

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Plasmids

circular or linear containing a replicon containing a constant copy number carrying genes that are necessary for plasmid replication may contain genes critical to bacterial survival F, the fertility factor, is involved in conjugation

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Bacteriophages

T 4

T 5

T 7

P 2

P2 2

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Transposons
IS10, Tn10 transposases: encoded by tnp or tnpA; enzyme necessary for transposition IS: insertion sequences; insertion sequences in a composite transcription are the same type nonreplicative (cut-and-paste) and replicative transposition tnpR, the resolvase gene; necessary for the resolution of the cointegrate form by the Tn3- (TnA) family of transposons; Res site: resolution site or binding site for TnpR, the resolvase Mu and HIV are actually transposons.
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Replicative Transposition (TnA, Tn3)

Resolvase

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Noneplicative Transposition

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Mechanisms of Genetic Transfer

transformation conjugation transduction

transposition

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Transformation

Not all the bacterial species can be transformed.

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Conjugation

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Transduction

generalized transduction specialized transduction

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Recombination

homologous (legitimate) recombination nonhomologous (illegitimate) recombination

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