MICROBIAL PHYSIOLOGY
INTRODUCTION
Objectives
• Knowledge of the cellular and molecular basis of the interactions between microorganisms and their
environment, and between microbes and higher organisms and between microbes and microbes
• Insight in the structure and function of bio-macromolecules and complexes involved in processes occurring
at microbial membranes
• Insight in the cellular physiology of microbial cells, differentiation processes, and the behaviour of
microorganisms in the context of complex communities
• Experimental approach
• Project work and discussions
Context
• Relevance of Microbial Physiology in the Master of Bioscience Engineering: Cellular and Genetic Engineering
Study material
• Presentations on Toledo, no compulsory handbook
• Optional:–‘Molecular Genetics of Bacteria’, L. Snyder and W. Champness, ASM Press, ISBN 155581-204-X
Guidance
• Questions or problems regarding the course material
Exam
• Knowledge questions (global and specific)
• Insight in molecular mechanisms of regulatory systems, transport systems and microorganism-host
interactions
• Exercises
Content
• Chapter 1. Regulatory systems (J. Michiels)
Global regulatory networks in micro-organisms in connection with nutrition and stress (stringent response,
catabolite repression, heat shock response, pH-stress, osmotic stress, protein folding and proteolysis, ...
• Chapter 2. Microbe-host and Microbe-microbe interactions
Molecular mechanisms, ecology and evolution of (i) beneficiary and pathogenic microbe-host interactions
(H. Steenackers), (ii) cooperative and competitive microbe-microbe interactions (H. Steenackers) and (iii)
interactions with viruses (J. Michiels)
• Chapter3. Transport systems (H. Steenackers)
Structure and functioning of microbial transport systems of bio-molecules (protein secretion systems,
receptors, ABC-transporters, cellular casing, ...)
• Chapter 4. Exercises (H. Steenackers)
Applications related to microbe-host and microbe-microbe interactions, including microbiome-based
medicinal products, anti-pathogenic drugs, AMR and resistance evolution to anti-pathogenic drugs :
exercises, project work, discussions, and experimental approach
1
,CHAPTER 1: REGULATORY SYSTEMS
Model: Escherichia coli : best know model system, important bacteria it lives in symbioses with our cells and is
the most important pathogen worldwide.
TRANSCRIPTION/ TRANSLATION IN BACTERIA
1. transcription
RNA polymerase
• Limited; 2000 molecules/cell for 4000-5000 genes and~2000 promoters (genome of E.Coli)
- this means that there is a huge competition of RNA polymerase enzymes for this promoters. The total
number of RNA polymerases is limited, it depends on how active the promotors are. Some other genes
has less RNA polymerases, important for gene regulation.
• produces all RNAs(rRNA, tRNA, mRNA, miRNAs) except Okazaki fragments(primase)
- Single RNA polymerase that is responsible for transcription of all genes. There is a primase that is
responsible to make the okazaki fragments.
• 5 subunits: α, β, β’, ω, σ(mw> 400.000)
• sigma factor for initiation of transcription(recycling) (also limited), there are several.
- Different from eukaryotes systems, the regulation is coordinated by sigma factor. Needed for initiate
not for elongation. They are involved in very specific responses. All bacteria has one house keeping
sigma factor, important for transcription of housekeeping genes.
• α2ββ’ω= core enzyme
• α2ββ’ωσ= holoenzyme: need a sigma factor to bind
• Active site =ββ’
• αNTD: dimerizes and is responsible for the assembly of ββ’
• αCTD: DNA binding or protein-protein interactions, important at some promoters
• ω: no direct role in transcription, foldingβ’
2
,Transcription - initiation
• no existing primers required
- no proofreading, can just start from scratch
• specific DNA regions: promoter regions-promoter recognition is dependent on σfactor (e.g. σ70, 70 kDa))
- promoter recognition –positioning RNA pol
- unwinding DNA near start site
• consensus sequences: most common sequence and often also the most strongest sequence. But you have
little variation to it.
- -10 hexamer (σ domain 2) TATAAT
- -35 hexamer (σ domain 4) sequences TTGACA
- σ70 binds both sequences: one of the house hold sigma factor in E.Coli. It involves in fast growth of the
cells.
• N17 spacer: important spacer if you increase of decrease it the gene will not express anymore. If you will
variate this spacer than it means you will end up with two helical turns. These sequences are normally at the
same site of the DNA. (DNA is a helix and these sequences rotated).
• start site (+1): A or G (T or C in template)
- purine base that is the initiation nucleotide of transcription. This is also linked to energy status.
• no helicase required (in contrast to DNA pol)
- they have their own helicase activity
Polymerization
• coding strand is identical to the mRNA except the
presence of Uracil in mRNA. The template strand
is used by the RNA polymerase, it is in the active
site.
• 5 promoter elements for binding of RNA
polymerase: interaction
- -10 (σ domain 2) and -35 (σ domain 4)
hexamers
- Discriminator: between +1 and -10 (σ domain
1; GGG)
- Extended -10 element: 3-4 bp motif 5’ of -10
element (recognized by σ domain 3)
- UP element: ~20 bp sequence 5’ of -35 hexamer (αCTD)
• No promoters with 5 perfect elements: would result in too strong binding. Too strong to remove it from the
promotor sequences. So all sigma 70 dependent genes have variation of the sequences of these different
elements.
3
,• Domains 3 and 4: important for initial positioning
• Domains 1 and 2: important for formation of open complex, place where the two strands will separated.
• The DNA is bended, this an important aspect. If there is no bended possible this can repressed transcription.
Closed complex
a) Closed complex moves along DNA (no σ
factor present)
b) Binding to promoter(σ factor). DNA
strands are melted at a promoter region to generate
an open complex (-10 to +2)
This means the DNA strands are separated. The
open complex overlaps with the transcriptional
start site. It is not initiation yet. Some genes need an
activator.
transfer of ss DNA strand to active site; transcription
is initiated
Rifampicin (antibiotic) inhibits initiation: binds to
active site and stops RNA synthesis after 2-3
nucleotides (targeted transcription)
c) Formation of transcription bubble
(approximately 17 bases long),
σ factor is released: Sigma factor only needed for
initiation not for elongation.
RNA polymerase is processive to continue, it will not
fall of the DNA. This interaction is stabilize by only -
9 bp DNA-RNA hybrid. That is not a lot, if there are
factor which destabilized than RNA polymerase will
released and transcription will stop.
d) RNA polymerase leaves DNA at a
transcription terminator; transcription is
terminated
4
,The transcription cycle. You
have the sigma binding then the
RNA polymerase can bind to the
DNA promoter. There is
isomerization from a closed
complex to an open complex.
The template strand is in the
active site, this is the main
channel. Initiation of
transcription: a few nucleotides
can be formed. But this is not
always a stable formation,
transcription can abort and then
released their nucleotides.
Initiation is not an easy process.
After initiation the sigma factor
is released, you enter in the
elongation phase.
Elongation complex
• The template strength is inside the active site and the
active side channel, so in the beta subunit. By the RNA
exit channel it goes out. The 8-9 bp are being formed
between RNA and DNA. The secondary channel allows
actually access from the outside to the active side and
so NTP obviously can enter, but also proteins as well
and some proteins have these large arms or extensions
that can regulate transcription as well
• Elongation: 30-100 nucleotides/sec
- A typically bacteria gene is thousand bases That mean you will need 10-30 seconds to synthesis a RN
molecule. Responses are fast, but they are not very stable. The most are degraded in 2-10 minutes.
• Number of RNA polymerases is limited
• Many are used by rRNA genes
• Number of σ is limited
• A lot of competition
Transcription does not occur continuously
• Pause hairpin: it can interact backwards with the RNA
polymerse, and will pause this. retroactive interaction
with β-subunit as RNA exits the exit channel, often at
5’end of mRNA (leads to attenuation of transcription,
hairpin broken by ribosomes) transcription and
translation are coupled.
• ‘Backtracks’: pushes RNA into secondary channel
(degradation of additional RNA in rNTP pore by GreA and
GreB (RNases))
• Anti terminators: reduce pauses, rRNA genes. They avoid the problem of premature termination of
transcription
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,Transcription termination
Factor independent (intrinsic)
• Intrinsic to the DNA sequence
• A. GC-rich inverted repeat followed by polyA sequence
• B. Formation of hairpin loop
• Formed RNA-RNA complex is more stable than DNA-RNA
hybrid; weaker A:U interactions; RNA polymerase
dissociates and transcription is terminated
Factor dependent
• E. coli: 3 transcription terminators: Rho, Tau and Nus A
(proteins)
• Ρ factor:
- Probably universal
- RNA dependent ATPase
- Active on ribosome-free RNA
- RNA-DNA helicase
• Mechanism
- Ρ binds to naked RNA, at free rut sites (rho utilization),
only when RNA is not translated
- Moves along RNA (rotates) in 5’-3’ direction (60nucl/sec)
(RNA pol: 100nucl/sec) only when RNA pol slows down,
the rho protein can catch up with RNA polymerase
- When RNA pol pauses at a Rho-dependent termination
site, RNA-DNA hybrid is separated
- RNA polymerase dissociates and transcription is
terminated
• Termination of transcription when translation is terminated
(end of gene (stop codon), nonsense codon)
• mRNA molecule free of ribosome: can occur if you have an
early stop codon (mutation or not corrected transcription).
The mRNA will be free of ribosome
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,Coupling of transcription and translation
• The figure is not in scale: Polymerases are 4-5 proteins and
the ribosomes is maybe 60protein. mRNA is in principle
never naked. It's immediately covered by the by the
ribosomes and transcription translation is coupled. This is
not the case in eukaryotes. Eukaryotes: the mRNA's are
produced in the in the nucleus and are then transported
through the cytoplasm and in cytoplasm that can be
translated. So there it is UN coupled.
• The coupling makes that bacteria have additional regulatory systems, like the ribosomes can actually collide
with the RNA polymerase and push the RNA polymerase forward.
rRNAs and tRNAs
• Play an important role in protein synthesis
• >95% of total RNA in bacterial cell are ribosomal RNA
(>50% of all RNA synthesis, stable RNAs) they do not have
the polyA tails, you can’t fish them easily out (there are
other tricks to use primers)
• Are synthesized as one precursor RNA
• rRNA: 5S, 16S, and 23S (7-10 copies in the genome:
extremely active transcript because translation is the
limited factor in growth in the cell)
• 23S: peptidyl transferase enzyme (ribozyme). It makes the bounds between the amino acids
• 16S: initiation and translation termination
degradation of RNA
• Sometimes polyadenylation
by poly(A) polymerase for
degradation at 3’ (binding
site for RNases, e.g. PNPase)
• Often co localisation of
poly(A) polymerase with
PNPase and other RNases in
a degradome
• Half-life mRNA: 1-3 min
They are degraded by
ribonucleases: You have
endoribonucleases that will cut
inside. One type will degrade RNA
from the five prime site. This is
the end so obviously it will be
created in the five prime to three prime direction. The other type will degrade it from the three prime end. RNA's
are very often protected as well by this stem loop structures at the end. The ribonuclease cannot initiate the
degradation of the RNA. So for that you have other RNA's is going either from the five prime site or cutting inside.
You have a special enzyme as well, that it's called Poly adenylation. It will adenylate the RNA. This polyA tail can
serve as a landing platform for this extra nucleases. Then they can start degrading. So often you have a
colocalization of the RNA’S within a structure, it's called a degradosome.
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,tmRNA and trans-translation
what if you would have premature termination of
transcription. Ribosome will bind as well, but they would stuck
to it. Ribosomes are very important to cells so they need to be
recycled. There is no stop codon so the ribosomes cannot be
released. The cell has a system tmRNA with a double function
to solve this problem.
• Double role(tRNA and mRNA)
• Ribosome functioning inhibited in the absence of stop
codon(release factor cannot bind) (as a result of mRNA
degradation, premature transcription termination)
• These tmRNA will enter the E site. Alanine attach to it and
will be transfer to the P site. the Alanine is coupled to the
C terminus of the protein. The rest of the tmRNA can be
used as an mRNA. So an alanine will be attached again
and finally you will get a stop codon.
• Encodes C-terminal tag (approximately10 aa) recognized
by Clp protease and degrade the protein.
• Biotech application: destabilise proteins.
2. activity of RNA polymerase
• Promoter sequences
• Sigma factors
• Small ligands
• Transcription factors
• Chromosome structure
promoter sequences
• 2000 promoters, differ in sequence (static regulation)
- If a gene has a promotor sequence it cannot change it, it will have a certain affinity
- Many of the strongest bacterial promoters have an almost-consensus sequence and UP elements
• Sub optimally-functioning promoters: are the most interesting ones
- possibility of upregulation (adaptive regulation) by trans-functioning factors. Increase or decrease their
activity
Different sigma factors (see further)
• E. coli: σ 70 and different alternative sigma factors (70 stands for the molecular mass, 70 killo Dalton)
- Accumulate at different stresses, competition between σ factors
- σ S: stationary phase, general stress response
- σ H: heat shock
- σ F: flagellar motility, chemotaxis
- σ E: periplasmic stress
- σ FecI: Fe-citrate transport
- σ 54: N-metabolism
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,• Mycoplasma genitalium (1 sigma factor); Streptomyces coelicor(63 sigma factors): diversity of
environments. Mycoplasma has a verry stable environment and Streptomyces is a soil bacteria more
variation of environment, more sigma factors.
• Complex regulation: transcriptional, translational and posttranslational control
• Anti-sigma factors (inhibit) and anti-anti-sigma factors (inhibit the Inhibiting)
Small ligands (see further)
• Guanosine 3’,5’ diphosphate (ppGpp) (control by aa availability)
• Destabilizes open complexes at promoters which form unstable open complexes (often promoters with GC-
rich sequence close to +1; often genes involved in translation) (control by starvation)
• These promoters often function poorly at low concentration of the initiator nucleotide ATP (control by
growth rate, energy levels)
Chromosome structure
• Chromosome is compact due to supercoiling and interaction with proteins
• In general: more compact implies less transcription, can be local
• Less important than eukaryotes, compaction is not very well understood in prokaryotes, not very important
• E. coli: a dozen proteins involved in compacting the chromosome
- Fis, IHF, H-NS (silences horizontally acquired genes: it is always a little bit danger if external DNA comes
in the cells), HU, StpA(HNS homologue) and DpS (DNA-protein from starved cells, protection)
- nucleoid proteins
- Clearly important but general regulation by this system is not well known
Transcription regulators (see further)
3. Translation
Translation initiation
• Translation initiation regions(TIRs)
• Important for in frame translation of mRNA
• Initiation codon: AUG, GUG (sometimes CUG, UUG) (‘Wobble
in reverse’, normally wobble is the 3 position, here in reverse
the first)
• 5’ untranslated region(5’UTR) or leader sequence: important
for the position of the ribosome to start translation
• Shine-Dalgarno sequence: complementary to 16S rRNA
• Other regions can also interact with16S rRNA
• First aa always meth(f-meth): it inserted first a the p site and not at the a site. The formyl site is cleaved of
or the whole amino acid is cleaved of.
- after protein synthesis, formyl groups and N terminal methionine are removed by peptide deformylase
(A) or methionine aminopeptidase (B)
- a protein does not always needs to start with a methionine
• Initiation similar in archae and eubacteria RBS, f-meth
• Resembles peptidyl-tRNA instead of normal aminoacyl-tRNA; binds at P site instead of A site
9
, Polycistronic mRNA
• Genes in bacteria are organized in operons,
multiple genes on a single mRNA molecule.
- Very often the proteins of these systems are
related, so they are co transcribed. The
ribosomes will recognize them
independently. Not the same ribosome that
will translate everything.
• Bacterial phenomenon (archae and eubacteria)
• A single mRNA encodes multiple proteins
• Simultaneous translation requires multiple TIRs
• Coding sequences can overlap
• Translational coupling
- TIR of second gene forms hairpin loop
- initiation codon is unrecognizable for ribosome
- unless translation of the first gene disrupts the secondary
structure of the second gene
Polar effects on gene expression
• Insertion of transposon (transcriptional terminators in Tn)
• Insertion Ab-R cassette with transcriptional terminator
• Nonsense mutation (deletion, insertion) upstream of
translationally coupled polypeptide
• Carefully by interpreting the phenotypes of the coupled
genes.
ρ-dependent polarity
• Nonsense mutation releases rut sequence, enabling
factor-dependent termination: inhibition of transcription
• The ribosome will be released earlier. The rho system can
leads to stop transcription.
• No transcription anymore of the second gene.
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