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MICR335 > Mihnea's lectures > Flashcards

Mihnea's lectures Flashcards

1
Q

the bacterial chromosome is linearly compacted in what fashion?

A

compacted in an ordered and heirarchical fashion in lockstep with DNA replication

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2
Q

the bacterial chromosome is compacted into a functional 3D form to allow what?

A

replication, recombination, segregation and transcription

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3
Q

what controls the architecture of the bacterial chromosome?

A

specialised proteins

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4
Q

is the bacterial chromosome enclosed by a membrane?

A

nah

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5
Q

the organisation of the bacterial chromosome recapitulates the genetic map - what are some important positions?

A

oriC (origin of replication) and ter (replication terminus) - these are on opposite poles i.e. pole anchoring proteins

the left and right chromosomal arms

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6
Q

how do the pole-anchoring proteins coordinate replication?

A

during replication the new DNA moves to its relative position

this means that each daughter cell inherits a full copy of the genome with the same structure

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7
Q

how does chromosome configuration differ?

A

it differs between bacteria and can also differ based on the growth conditions

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8
Q

what are chromosome interaction domains (CIDs)?

A

well packaged domains of DNA with lots of DNA-DNA interactions and insulated from flanking regions

boundaries between CIDs are highly expressed genes (HEGs) which are less ordered (hence higher expression cause easier access)

HEGs are usually housekeeping genes

CIDs are around 10-10^2 kbp and ordered into loops by other proteins called NAPs

the number of CIDs differs based on the growth conditions (makes sense cause different conditions requires different genes)

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9
Q

how are e. coli macrodomains kept together?

A

ter protein (matP) keeps ter in a compacted form by binding matS sites which are exclusively present in the ter macrodomain

matP binds matS site as a dimer and can interact with membranes anchoring the domain in place

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10
Q

what does the isolated nucleoid contain by weight?

A

80% DNA, 10% RNA, 10% protein

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11
Q

are all bacterial chromsomes circular?

A

most are

some are linear tho

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12
Q

in eukaryotes, DNA is condensed by histones - how is DNA condensed in bacteria?

A

DNA is organised in plectonomic supercoils by specific proteins which bend the DNA

different conformations are governed by nuclear-associated proteins (NAPs)

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13
Q

what are structural maintenance of chromosome (SMC) complexes?

A

have a hinge dimerisation domain and an ATPase head domain (on this domain is kite) and arms extending between these

this allows it to wrap around a loop of DNA and excise it (from the previously highly ordered form) making it accessible for transcription

so the SMC complexes embrace a pair of DNA segments as a single ring and dimerise making that region of DNA accessible

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14
Q

what is histone-like nucleoid structuring protein (H-NS)

A

small polypeptide with AT rich segments which binds the DNA using a c-terminal arginine hook motif

they form head to head and tail to tail interactions to form a long thread of proteins which interact with domains on the DNA

this allows formation of rigid DNA filaments or it can also allow bridging of two DNA filaments

this can also obscure RNAP binding sites and/or transcription activators leading to gene expression

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15
Q

how are H-NS proteins an example of convergent evolution?

A

they all have an arginine which allows DNA binding and are present in many bacterial species

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16
Q

what proteins allow sharp DNA bending?

A

HU has an alpha and beta domain allowing formation of homodimers and heterodimers and also a flexible hinge allowing a range of different angles. Very abundant and can allow formation of octamers

IHF causes much tighter 160 degree hairpin bends

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17
Q

what protein allows DNA light bending?

A

Fis is highly expressed during rapid cell division and is conserved in most gram-negatives

bends DNA by 50-90 degrees (hence light) to form stable nucleoprotein complexes

can regulate transcription

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18
Q

what are some techniques for studying the bacterial chromosome?

A

Hi-C

fluorescent repressor operator system

chromatin immunoprecipitation

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19
Q

what is Hi-C?

A

high-throughput method for studying the bacterial chromosome

fixed chromatin undergoes restriction digestion and DNA overhangs filled in with biotin-labelled nucleotides. It then undergoes ligation and labels are removed from unligated ends

then undergoes reverse crosslinking and fragmentation and labeled fragments are enriched

this can then undergo adapter ligation and amplification and then high-throughput sequencing

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20
Q

what is fluorescent repressor operator system?

A

used to look at specific genes

genes are labelled and then changes can be induced? (e.g. pH, osmolarity, temp.) allowing visualisation of if/if not genes undergo transcription

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21
Q

what is chromatin immunoprecipitation?

A

fixed chromatin undergoes fragmentation, exonuclease treatment and immunoprecipitation. DNA can then be purified and undergo adapter ligation and amplification followed by high-throughput sequencing

this allows you to see what regions of DNA have been bound and assess the specificity of this

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22
Q

what occurs due to the dsDNA circular molecule being topologically constrained?

A

theres no rotation of free ends so the genome organises into plectonemic supercoils constrained by NAPs

RNAP induces -/+ supercoiling where overwinding occurs at one end (+) and underwinding at the other (-)

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23
Q

what is the solution for supercoiling?

A

topoisomerases bind and cut DNA - they cut the plectonemic domains so that just that domain will unwind

type I topoisomerases cut one DNA strand and type II topoisomerases cut two

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24
Q

outline bacterial chromosome segregation?

A

separation of newly replicated origins

condensation mediated by supercoiling

origin segregation facilitated by a highly conserved partitioning system

bulk chromosome segregation reverses? the orderly compaction of replicated sisters along adjacent DNA segments

condensation is mediated by the concerted action of supercoiling and NAPs and topoisomerases enriched ahead of replication forks and transcription bubbles

transport of replication termini at the division septum

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25
Q

outline how environmental conditions affect DNA organisation?

A

the chromosome structure changes with environmental conditions

conformational change is induced by ligands e.g. Mg stabilises H-NS helix conformation

temperature increases reduces H-NS oligomerisation and dissociation from DNA

when starving e. coli compacts its DNA to almost crystalline form, NAPs usually present in lower levels in starved cells (cause DNA organised well so dont need as many)

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26
Q

what is transcription?

A

the process by which DNA is copied into RNA

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27
Q

briefly outline RNA and RNA polymerases in eukaryotes?

A

we have three types of RNA made by three polymerases

rRNA made by polymerase I
mRNA made by polymerase II
tRNA made by polymerase III

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28
Q

how many RNA polymerases do bacteria have and what structure?

A

one - it is made of five conserved subunits:

beta prime
beta
alpha I
alpha II
omega

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29
Q

compare the structure of RNApol in eukaryotes and bacteria?

A

RNAP general architecture and catalytic function is conserved across the three domains of life

eukaryotes have a few extra subunits tho

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30
Q

discuss the structure of bacterial RNAP and how this interacts with dsDNA?

A

has a crab claw appearance that opens and closes during transcription

core of RNAP is parts of beta and beta prime and the two alpha subunits, and parts of beta subunit form the jaw-lobe

dsDNA goes into the cleft and gets unwound - one strand copied into mRNA and other not read - read strand goes into core where there is Mg2+ ion important for elongation

mRNA exits perpendicular to jaw and dsDNA rewound back into double helix

RNAP has lots of interaction partners

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31
Q

what is the bacterial RNAP holoenzyme?

A

all the RNAP subunits PLUS sigma factor

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32
Q

what does the sigma factor do?

A

participates in recognising the promoter of a gene that needs to be transcribed so initiates transcription

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33
Q

what are the three main steps of transcription?

A

initiation - RNAP binds promoter and makes transcription bubble

elongation - RNAP reads the template strand one base at a time and builds a complementary RNA molecule

termination - terminators at end of gene signal RNA transcript is complete and RNAP is released

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34
Q

what are the elements on the bacterial promoter and what is their position relative to the gene?

A

UP element (-37 to -58)

-35 element (-35 to -30)

extended -10 element (ext) (-17 to -14)

-10 element (-12 to -7)

the discriminator element (dis) (-6 to -4)

exact positions of each element may vary

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35
Q

where does transcription of the gene start?

A

+1

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36
Q

what parts of the bacterial promoter are recognised by what parts of the holoenzyme, and what does this position the promoter for?

A

UP element bound by alpha subunit C terminal domains

this much further upstream from gene so this bit getting recognised by polymerase

then -35, ext -10, -10 and discriminator recognised by sigma factor domains 1, 2, 3 and 4

sigma factor domains binding promoter and complexing with polymerase aligns promoter regions specifically to position the gene for transcription

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37
Q

outline the bacterial transcription cycle?

A

holoenzyme recognising promoter makes closed complex and dsDNA put in polymerase with help of sigma factor

this is followed open complex where transcription bubble opened in dsDNA - this has to happen in promoter region cause transcription needs to start at +1

then initiating complex where it starts making RNA transcripts and scrunching occurs

when DNA aligns well in catalytic core sigma factor released and elongation begins

now dsDNA being pulled through, read and mRNA released perpendicular to this

then termination

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38
Q

what do the domains of sigma factor do?

A

3 and 4 domains recognise -35 and ext; position promoter in precise orientation

1 and 2 domains recognise dsr and -10; open the promoter complex

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39
Q

outline the initiation step of bacterial transcription?

A

sigma factor comes in with dsDNA and puts it in precise position on top of polymerase

binding to the promoter mediated by initiation factor (which i am 90% sure is just sigma factor)

dsDNA melted to unwind strand; one goes to catalytic core and one unread

sigma D1 domain is ejected to allow for bigger loop (from more melting) - begin to make RNA and scrunching occurs; this is the transcribing complex

once sigma released elongation occurs (cant happen while sigma there)

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40
Q

why can initiation factor and elongation factor not be bound at the same time during RNA synthesis?

A

they bind the same spot on RNAP as initiation factor

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41
Q

what is the name of the initiation factor and the elongation factor in e coli?

A

initiation factor - sigma 70

elongation factor - NusG

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42
Q

outline the process of elongation during transcription?

A

DNA-RNA hybrid of 8 to 9bp formed in transcription bubble - the downstream DNA is not yet transcribed and the upstream DNA has regained duplex form

backtracking can occur here? where incorrectly incorporated nucleotides excised

during elongation the rudder loop separates DNA and a series of loops maintains the transcription bubble, with active site located at the floor of the cleft

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43
Q

what are the two ways of terminating bacterial transcription?

A

intrinsic termination: DNA and RNA sequence sends termination signal to polymerase

other way is using helicase which engages with elongation factors and RNAP during elongation - helicase associates with C-rich RNA sequences expelling RNAP from DNA

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44
Q

why is it useful to know all the stages of bacterial transcription in detail?

A

it allows us to build more effective and more precise drugs which target components involved in the process

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45
Q

what does the RNAP holoenzyme use to act on DNA?

A

activators and repressors

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46
Q

is RNAP capable of random transcription with no promoter?

A

yes

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47
Q

is sigma factor necessary for recognising a promoter?

A

yes

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48
Q

what are lineage specific inserts?

A

well bacterial RNAP is highly conserved, different lineages have different inserts interacting with them

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49
Q

what is the RNAP holoenzyme?

A

RNAP with sigma factor

this recognises the promoter in a closed complex

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50
Q

what part of the promoter is recognised by the core RNAP enzyme itself?

A

the UP element; is recognised/bound by c-terminus of alpha subunits

rest of promoter elements are recognised by sigma domains

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51
Q

summarise the steps of bacterial transcription from initiation to elongation?

A

RNAP holoenzyme recognises the promoter in closed complex; covers from ~ -55bp to +15bp

transcription bubble

open complex

RNA synthesis

elongation (sigma not required so fucks off)

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52
Q

what do sigma factors do and can what do they recognise?

also how many are there

A

lead RNAP to the transcription start site (TSS)

initiate strand separation of dsDNA for the transcription bubble

incapable of recognising DNA alone, only with RNAP

multiple types of sigma factors which are evolutionarily related - some have less than four domains, most minimal one just domain 2 and 4

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53
Q

what is the downside of sigma factors having less domains?

A

it becomes more difficult to recognise the promoter which has five domains

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54
Q

what is sigma 70?

A

house keeping factor - responsible for recognising most promoters

each domain recognises specific promoter elements

sigma domain 2 can remain attached to elongating RNAP; continues recognising -10 and -35 like sequences allowing for transcriptional pausing

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55
Q

what is transcriptional pausing caused by sigma 2 domain remaining attached to elongating RNAP?

A

can bring in other parts of DNA on top of continuous impeding on function of polymerase

this important in some bacteria when u don’t want to express some genes

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56
Q

what is sigma ECF?

A

extra cytoplasmic function sigma factors; pretty much only used in response to stress or sudden changes which requires fast response - essential for transcribing genes/proteins required for stress

minimal size sigma factor - only domains 2 and 4

a bacteria on average has six sigma ECF

recognised by anti-sigma factors

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57
Q

what are anti-sigma factors and what are they important for?

A

sigma factors (e.g. sigma ECF) are arrested by anti-sigma factors in conformation where they can’t associate with RNAP

when stress occurs u just drop the anti-sigma factor from sigma ECF and it will become available to recognise promoters important for genes/proteins required to cope with the stress

there are also anti-anti-sigma factors which sequester anti-sigma factors i.e. this is a complex regulatory network

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58
Q

what is sigma factor 54?

A

recognises -24 and -12 promoter sites on a completely different promoter to sigma 70

small; only recognises two elements thus cannot allow formation of open complex and transcription bubble alone - requires an ATP-dependent activator which is regulated by stress-related signals

activator binds far upstream so in order to make contact with holoenzyme DNA looping is required - once it gets their initiation can start

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59
Q

outline the structure of sigma factor 54?

A

region I - interacts with activator

region II - implicated in DNA melting so necessary for initiation

region III - RNAP binding and recognises the -12 and -24 promoter elements

sigma 54 family structurally and evolutionary distinct from the sigma 70 family of sigma factors

60
Q

while there are five domains in the promoter, why can we start transcription with less than five being bound?

A

because not all five elements in the promoter are essential

61
Q

can different sigma factors recognise the same transcription start site (TSS)?

A

yes

RNAP holoenzyme can use different recognition elements, so different sigma factors can initiate at the same TSS

62
Q

why do some promoters need additional factors?

A

the PRE promoter does not have capability to bind RNAP holoenzyme alone as -10 and -35 elements that differ from those in consensus sequence

u can have additional proteins which modify DNA structure so that domains fit in the holoenzyme; in this case CII protein binding site overlaps w -35 element allowing PRE to be bound by sigma factor and transcription to begin

63
Q

why is CII not an activator?

A

it doesn’t start transcription, it helps sigma factor recognise the promoter

64
Q

when/why/how might bacteria sequester RNAP?

A

during transitions between exponential and stationary growth phases bacteria may undergo transcriptional reprogramming by sequestering RNAP

don’t want to destroy RNAP cause will need later so rather just arrest it in inactive conformation

bacterial 6S RNA is a ncRNA that binds sigma70 holoenzymes (not other sigma families) promoting increased activity from other sigma factors

6S RNA has central bubble which mimics promoter structure

65
Q

what are transcription factors, what do they regulate and why is it complex?

A

proteins which bind DNA and help or inhibit transcription

most transcription factors regulate many promoters, and most promoters are regulated by multiple transcription factors

transcription factors may be expressed from promoters that are regulated by other transcription factors

its is a complex regulatory network

66
Q

when might transcription factors not be able to access promoters?

hint - its not that deep

A

when DNA is compacted

67
Q

what are activators?

A

something that helps transcription (different ways of doing this)

a type of transcription factor

68
Q

what are the two types of activators and how do they differ?

A

class I - binds upstream operator which recruits RNAP; RNAP recruited by protein-protein interaction

class II - binds to operator at the -35 element leading to interaction with sigma 4 domain

some activators bind operators between the -35 and -10 elements realigning the DNA (promoter conformation change) to bind RNAP

can also have combinations of activators recognising different promoters or combinations of class I and II activators

69
Q

what are repressors and how do they work?

A

repress gene expression by three mechanisms:

steric hindrance: block RNAP access to promoter; promoters may have multiple operators which can increase the strength of repression

looping: operators binding outside of the promoter i.e. not blocking it just making DNA unrecognisable to holoenzyme

anti-activators: prevent the recruitment of RNAP

a type of transcription factor

70
Q

what is the ligand dependence of target binding?

A

the fraction of transcription factors binding to the target site

71
Q

why are less abundant transcription factors more commonly bound to DNA than more common transcription factors?

A

if you don’t have many copies of something it might be hard to find them when you need them but if you have lots then its easy to find one when needed

hence why binding affinity (of activators and repressors in particular) is dependent on abundance

72
Q

what is the lac repressor and why is bound to DNA 90% of the time?

A

lac repressor is a transcription factor which binds specific region before three lactose-metabolising proteins (lacZ, Y, A)

when it binds lactose it changes shape and can no longer bind DNA; low lactose LacI repressor blocks RNAP, high lactose repressor is released allowing expression of genes which promote glucose import into cell

low glucose leads to high cAMP production which binds CAP protein and acts as promoter for same genes

so if both glucose and lactose absent the lac operon gets repressed

very few copies of lac repressor per cell (about 10) hence why usually bound

73
Q

outline the structure of the lac repressor?

A

has a C-terminal tetramerisation domain and a core domain that binds LacI sugar ligands

binds operator DNA via a helix-turn-helix motif and hinge

it can bind DNA and form a DNA loop so that -35 and -10 elements cannot be recognised by holoenzyme

74
Q

outline the coupling of transcription and translation?

like why are theyt coupled both structurally and practically

A

transcription and translation are functionally coupled; bacteria lack a membrane enclosed nucleus and nutrient availability and growth effect both transcription and translation rates

advantages of this is that it prevent accumulation of unused mRNA (saves energy), avoids formation of DNA-RNA loops, allows regulation of transcription by translation (gene regulatory mechanism) and contributes to genome-wide synchronisation

75
Q

outline the structure of the bacterial ribosome?

A

has a large multi-protein complex (70s) and two subunits (30s/small subunit and 50s/large subunit)

made of RNA and protein

76
Q

outline bacterial translation?

A

like transcription it has initiation, elongation and termination after which is recycling of ribosomes

key proteins: initiation factors (not sigma factors) which position mRNA in small subunit for 50s recognition, elongation factors which brings in tRNA w aa’s, release factors which release peptide, recycling factor which helps ribosome dissociate into small/large subunit again

occurs much faster in bacteria (17-20 residues/sec) than eukaryotes (6-9)

76
Q

what methods are used to solve macromolecular structures?

A

x-ray crystolography initially used to solve stuff like ribosome structure

now cryo-electron microscopy way better and easier cause u just freeze it so everything stuck in natural position

then u can do shit like put in antibiotics and understand how they effect translation and so on - essentially allows a high res snap shot of bacterial translation

77
Q

describe the movement of tRNA during bacterial translation?

A

three tRNA binding sites in ribosome; tRNAs translocate A to P to E

A - aminoacyl-tRNA enters ribosome

P - holds tRNA carrying nascent (peptide) chain

E - (exit) tRNA dissociates from ribosome

78
Q

how coordinated are the rates of transcription and translation?

A

transcription - 10-100nt/s

translation - 10-20aa/s

1 aa = 3 nt

i.e. they are pretty much the same

79
Q

what is indirect coupling?

A

regulation of transcription by translation as a result of amino acid starvation

aa starvation means ribosomes associated with uncharged tRNAs which gets detected by RelA

RelA gets activated and synthesises ppGpp which binds RNAP and slows transcription

this how cells know how to slow down when not enough materials to make protein

80
Q

what is direct coupling?

A

regulation of transcription by leader regions

charged tRNA is abundant cause lots of amino acids meaning translation occurring rapidly leading to formation of terminator loop –> decreased transcription and translation

charged tRNA is deficient (few amino acids) so translation slow leading to formation of anti-terminator loop –> increased transcription and translation

81
Q

what is the expressome?

A

RNAP + ribosome

82
Q

outline the expressome structure?

A

RNAP and ribosome bridged by dedicated transcription factors

NusG - has two domains for specific interactions linking RNAP with 30s

NusA - has five domains

NusA and NusG can be simultaneously present, they are molecular bridges which are elastic and allow ribosome and RNAP to undergo conformational changes

they align ribosome with polymerase so that it directs mRNA to ribosome for translation

83
Q

how is cryo-ET useful for analysing the expressome structure in situ?

A

can view the in-cell architecture of a transcribing and translating expressome

can also view the effect of drugs e.g. translation inhibitors or transcription inhibitors

84
Q

what is ribosome profiling?

A

deep sequencing of ribosome-protected mRNA fragments

allows genome-wide investigation of translation and monitoring of what proteins are translated in different conditions

85
Q

elaborate on the statement “gene order does not explain differential synthesis rates”?

A

genes are ordered one after the other, make a single mRNA which in principle is translated at same rate

many multiprotein complexes require different levels of gene products, however somehow cell doesn’t make more protein than needed

synthesis rates determined by frequency of initiation, and is tuned precisely to the stoichiometry of each molecular complex

this minimises wastage

86
Q

is tuning of synthesis rates to subunit stoichiometry of complexes achieved through transcriptional or translational control?

A

translational control

87
Q

what things are most of the proteins in the bacterial proteome dedicated to and what does this mean?

A

translation (41% in rich media)

transcription only 5%, and translation goes down to 21% in minimal media

so the reason all this shit so highly regulated is cause transcription and translation have a huge energy demand

88
Q

what different strategies do bacteria use for motility?

A

flagella driven

spirochetal mode

swarming

pili driven

gliding

each mode associated with special protein complexes which can be remodelled following changes in the environment

89
Q

what type of sensing determines the movement direction of bacteria?

A

bacteria use temporal sensing (not spatial sensing)

90
Q

outline bacterial taxis, specifically the receptors?

A

receptors bind ligands in the periplasm and send signals across inner membrane

these assemble into extended hexagonal arrays (trimers of dimers) at cell pole integration signals of 1000s of receptors - e. coli has 5 chemoreceptors of similar length and high sequence conservation packed into same array

hexagonal array structure common to all bacteria

91
Q

outline magnetotaxis?

A

pockets of inner membrane filled w crystals of magnetite organised by cytoskeletal proteins into chains

chains are inherited from parent cells and function unclear (mayb magentic orientation or vert movement)

92
Q

what is the bacterial flagellum?

A

semirigid helical filament which serves as a propellor allowing alternating run and tumble movements

rotates >100 times/sec

key flagellum modules include basal body, hook and filament

93
Q

how many flagella do bacteria usually have and where are they positioned?

A

bacteria can have any number of flagella in any position

94
Q

outline the architecture and assembly of the flagellum?

A

~50 genes expressing ~25 proteins that form the flagellum which is located in cytoplasm, IM, peptidoglycan layer, OM

three domains; basal body (incl. rotary machine; rotor, stator units), hook, filament

components have multiple symmetries and assembly occurs by polymerisation at the distal end

95
Q

what do flagella (and thus bacterial swimming is dependent on) use as the source of energy?

A

transmembrane proton gradient

96
Q

discuss the rotation mechanisms of the flagella?

A

power for rotation provided by rotary machine; the C-ring

stator units form a ring around C-ring and spin clockwise driving C-ring rotation

change in C-ring interaction with stator ring results in directional switch of flagella motor

i.e. flagella motor is BIDIRECTIONAL

97
Q

which direction does flagella spin?

A

both cunt

98
Q

how does the flagellum adapt to different loads?

A

senses environmental conditions: load viscosity, surface contact, sodium conc., ion motive force, pH

stator units floating in IM can be recruited and released as needed (the more stator units around C-ring the more energy it has to rotate)

99
Q

what the fuck is a periplasmic flagella and what does it allow?

A

found in spirochetes - flagella wrapped around the body and assembled inside cell envelope; multiple copies pack together into helical ribbon allowing wave-like motion of cell

allows bacteria to penetrate between cells in host tissue

100
Q

how is the gene expression of the flagellum heirarcichal?

A

the extracellular components of the flagellum are secreted by a type III secretion apparatus

transcription of the genes encoding extracellular components occurs after basal body is assembled and functional

101
Q

how does gene regulation of flagellum assembly occur?

A

FliA (sigma28) required for transcription of genes encoding extracellular components of flagellum

FlgM (anti-sigma factor) interacts w FliA destabilising sigma28 holoenzyme

FlgM is substrate for type III secretion apparatus so once that part of flagella assembled FlgM is secreted leaving FliA free to carry out function (express flagella assembly genes)

102
Q

what is swarming?

A

flagella propelled group movement on surfaces

dependent on type of surface, nutrient, temperature, humidity, cell-cell signalling etc.

switching from swimming to swarming associated with morphological changes; more elongated and flagellated

103
Q

how do archael flagellum differ to that of bacteria?

A

smaller, no central channel, uses ATP for energy (not TM potential), more related to a pilus

archael flagella has no evolutionary relation to bacterial flagella

104
Q

what is a type IV pili (T4P)?

A

responsible for gliding motility, several copies per bacteria

dynamic structure with pilin (pilA) and adhesins at tip; pilA recruited from membrane (extension) and released (contraction)

ATPases (pilB and pilT) rotate at base polymerising pilA through channel (~5 pilA per turn)

allows for gliding movement

105
Q

what is gliding and what bacteria use this movement?

A

mycoplasma is an intracellular pathogen which uses internal organelles to glide across surfaces

gliding is inchworm movement - uses double spring mechanism

uses type IV pilus

106
Q

how do bacteria use gas vesicles to move?

A

gas vesicles are widespread in bacteria and archaea and involve ~14 genes

they are flotation devices allow bacteria an alternative form of movement which keeps them at suitable depth in aqueous environments - narrower vesicles resistant to greater pressure allowing greater depths

so bacteria can choose between motility and flotation

107
Q

why do bacteria secrete stuff and how do they do it?

A

to protect themselves

to manipulate their environment

to interact with other individuals

they do so using a series of specialised systems to translocate substrates

108
Q

why is secretion more complex for gram negative bacteria?

A

cause they got two membranes

109
Q

what are the two types of secretion systems?

A

sec (the general secretion)

tat (twin arginine translocation)

110
Q

outline secretion across the cytoplasmic membrane in bacteria?

A

secretion systems universal in bacteria

proteins remain inside the cell (periplasm or IM)

111
Q

what is sec?

A

general secretion system which secretes unfolded protein

secreted proteins have 20 residue hydrophobic N-terminus signal sequence

this recognised by secYEG complex which forms membrane channel through which translocation of peptide chain by the ATPase secA

112
Q

what is tat?

A

twin arginine translocation; translocates fully folded proteins through inner membrane through recognition of N-terminal signal

113
Q

how do bacteria transport virulence factors outside the cell?

A

using dedicated secretion systems and through recognising N or C terminal signals on proteins

a lot of diseases associated with toxins from secretion systems

114
Q

why are bacterial secretion systems such good drug targets?

A

cause so many diseases associated with toxins from secretion systems and bacteria can still live without so low chance of developing mutants

115
Q

outline type I secretion systems?

A

sec-independent (don’t need sec to secrete stuff from IM to periplasm)

contain C-terminal signal sequence

e.g. HlyA (toxin that lyses RBCs)

functions by ATPase transporter protein in IM which is bridged by channel to OM. OM factor is tolC which is multifunctional porin involved in transport

116
Q

outline type II secretion systems?

A

transports folded proteins from periplasmic space (already been transported by sec or tat)

found in many path/non-path gram negatives, 12-15 proteins and multiple substrates

bacteria have no energy source in OM so uses cytoplasmic ATPase to power assembly of periplasmic pseudopilus pushing protein through OM pore in piston-like motion

used by legionella to secrete virulence factors and proteins that facilitate growth allowing survival in many environments

117
Q

outline type III secretion systems?

A

needle-like structure with more than 15 conserved components; goes from bacterial cytosol to host cell membrane (3 membranes)

related to flagellum

essential role in virulence making it attractive drug target as there is less evolutionary pressure to develop resistance (as opposed to targeting growth)

118
Q

what is the structure of the type III secretion system?

A

has four domains:

domain I - ATPase and export gate allowing recruitment of effectors and transport through apparatus

domain II - basal body spans both membranes and periplasmic space

domain III - extracellular side; needle is hollow filament capped at distal end by needle tip (size varies between bacterial species)

domain IV - translocon complex; forms pore in membrane of target cells onto which needle tip docks activating secretion of effectors and passage to cytosol of target cells

119
Q

outline assembly of t3ss?

A

five sctR assemble as helix with single sctT decorated by four sctS which template the assembly of 24-fold symettric sctDJ, a ring of nine sctV and correct formation of six C-ring pods

120
Q

outline the secretion of t3ss?

A

cytoplasmic substrates are delivered to export gate by ATPase and into export cage where solvent accessible channels in IM ring power delivery past small cytoplasmic loop of sctVN to sctRSTU for secretion through +vely charged axial lumen

121
Q

outline the sensory transduction of t3ss?

A

conformational changes in the needle propogate through sctDJ to sctV where they may influence substrate specificity

this allows the bacteria to be like aight we got the right host lets pump this cunt with toxins

122
Q

outline how t3ss are regulated?

A

RNA thermometer (RNAT) allows controlled translation of lcrF coding for virulence master regulator which activates transcription of t3ss regulon - RNAT basically recognises when it contacts host cell so it knows when to transcribe/translate t3ss

t3ss encoded on virulence plasmid and copy number increases in elevated temperatures

synth of Y. pseudotuberculosis t3ss components rely on host body temperature

regulatory elements are thermolabile hairpins which sequester SD domain (sequence at beginning of gene recognised by ribosomes)

123
Q

why has the structure of t3ss historically been difficult to study?

A

double membrane spanning protein complexes are difficult to purify so difficult to study

bacteria can also have multiple kinds of t3ss

structural info is important for development of antibacterials and gaining evolutionary insights

124
Q

discuss the evolution of t3ss and the flagellum?

A

minimal part of flagellum can be referred to as flagellar t3ss which exports flagellar proteins outside cell cutting costs of motility and protein export

over time loss of flagellum genes and motility left basic t3ss apparatus which could then acquire further genes (e.g. pore-forming secretins) so diff bacterial phylum evolved their own specific t3ss from this

these t3ss are then also frequently passed around via HGT and some bacteria encode multiple t3ss for multiple hosts

125
Q

outline type 4 secretion systems?

A

found in both bacteria and archaea

require cell-to-cell contact but some t4ss are employed to take up extracellular DNA

can act as conjugation system and disseminate MGEs spreading AMR

can also translocate effectors and deliver proteins to eukaryotic target cells

126
Q

discuss the structure of t4ss?

A

minimal core 12 subunits but can be expanded to 25 increasing efficiency

internal membrane complex consists of two concentric rings extending into cytoplasm

in IM there are six arches (6-fold symmetric) which stabilise IM complex and 5-fold symmetric stalk connecting this to OM

OM core complex has two layers

127
Q

outline t4ss biogenesis?

A

virB2 recruited from IM and pilus polymerises through channel with virB5 at tip

bacteria can encode multiple copies of virB5 or virB2 which bind diff cell receptors i.e. diff hosts

pilus architecture is conserved

128
Q

discuss type 5 secretion systems?

A

autotransporter (self-mediated transport) and the largest group of secreted proteins in gram negs; important virulence factor involved in adhesion, biofilm, AB deg etc.

sec gets unfolded protein to periplasmic space

contains signal peptide w 3 domains, translocator domain is beta-barrel and passenger folds after its translocated; passenger can remain uncleaved and is either released by being cleaved or via OM vesicles

129
Q

outline the function of type 6 secretion systems?

A

present in ~25% gram negs, dynamic (~one shot/min) and highly regulated

analogous to phage long contractile tail; allows translocation of proteins which bind spike

sheath and tube traverse periplasmic space and reach host membrane

only used on bacteria; directed against competitors - time lapse microscopy has allowed visualisation of cell duelling in P. aeruginosa

130
Q

outline the structure of t6ss?

A

base plate is pseudohexameric, attached to IM and starts assembly and sheath and tube are helical and hexameric

sheath monomers lost upon firing or unfolded and reassembled

membrane complex is pentameric

131
Q

discuss how bacterial war occurs with t6ss?

A

p. aeruginosa t6ss-dependent killing of v. cholerae

v cholerae hitting p aeruginose causes it to build its own t6ss at location of assault and fire and kill

baseplate can then be disassembled and reused

this assembly and counterattack is regulated by a signal that corresponds to attack of t6ss apparatus elaborated by second bacterial cell - p aeruginosa can differentiate between aggressors w t6ss and pacifists without (doesn’t kill)

132
Q

outline type 7 secretion systems and what bacteria use them?

A

central to virulence and crucial for nutrient and metabolite transport across mycobacterial cell envelope

secrete ~200 proteins

pathogenic mycobacterium have up to 5 different t7ss used in diff environments according to their needs - promising drug targets as less evolutionary pressure –> AMR

133
Q

outline the structure of t7ss?

A

located on inner mycobacterial membrane; five membrane components and two cytoplasmic components (chaperone EspG and ATPase EccA)

t7ss are in a 6:6:12:6:3 stoichiometry

protease MycP5 stabilises the entire complex, EccC5 is gating pore and has two conformations

134
Q

give an example of a structural strategy of recognition in bacterial secretion systems?

A

gating pore e.g. EccC5 in t7ss

135
Q

give several examples of how bacterial secretion systems work together?

A

in e. coli typeIII translocates tir to host cell where it acts as receptor; typeV autotransporter can then recognise this receptor

in plants typeII release proteins which damage cell wall and then typeIII can pierce and kill

typeIV pili get bacteria close and then typeVI can pierce

136
Q

why do bacteria have toxins?

A

inhibit growth of other microbes to give themselves a competitive advantage for resources

evade host immune response allowing proliferation within the host

break down host tissues and cells to release nutrients

137
Q

what are pore forming toxins?

A

largest class of bacterial toxin; widely distributed in gram neg/pos and also in archaea and eukaryotes

secretion can occur via sec or t1,2,3,5,10ss

PFT structures have two conformations based on either alpha-helices or ampipathic beta-strands

they perforate the membrane causing the PMF to dissipate (important source of energy for cells so fucks them up)

138
Q

discuss the mechanism of pore forming toxin?

A

alpha-PFT: penetrate membrane and start to oligomerise; more flexible and structurally heterogeneous

beta: bind receptors, assemble into pre-pore, major conformational change to insert in membrane and form beta-barrel pore

139
Q

describe the structure and function of anthrax toxin?

A

major virulence factor of B. anthracis; plays critical role in infection by suppressing innate immune response (early) and destroying host vasculature (late)

two components: PA (heptameric pre-pore confomration, major component of vaccine) and LF/EF of which there are three copies

PA binds host receptor, is cleaved and oligomerises forming pre-channel, LF binds this and toxin undergoes receptor-mediated endo, forms pore, delivers toxic enzymes (LF) to cytosol, powered only by pH gradient across membrane - not PMF or ATPase

140
Q

describe the structure of aerolysin?

A

toxins of aerolysin superfamily have mushroom-like shape in pre-pore conformation

pore formation has intermediate steps and similar mechanism to anthrax toxin

141
Q

discuss the function of Tc toxins?

A

toxin complexes present in bacterial pathogens, common and spread by HGT

large complex consisting of three subunits that perforate host membrane causing to deterioration and death of cel

pre-pore conf is bell-shape protecting unfolded toxic enzyme and alpha helical channel already formed, pH triggers insertion of channel via syringe-like mechanism

differs to beta-PFT cause toxin fully formed upon contacting cell

142
Q

how do Tc toxins enter the cell?

A

bind to receptors on cell triggering endocytosis

pH decreases and at certain level triggers conformational change opening channel and translocating toxin

toxin folds as it leaves channel into cytoplasm

143
Q

how is Tc toxin release regulated?

A

RoeA is usually present in low levels, howoever environmental signals (e.g. nutrient/thermo/quorum sensing) upregulate

RoeA activates many toxicity related genes; proteomic analysis has showed Tc can be enriched despite having no export signal sequence

microscopy has shown only a small percentage of bacterial population express the toxin (soldier cells) - shows how pathogen virulence can be determined by small number of specialised cells

144
Q

what are tailocins?

A

aka phage-tail like bacteriocins (PTLBs) are bacterial toxins which kill other bacteria or insects; makes channel in membrane decoupling membrane potential - a single particle can kill the cell

kill competing bacteria and narrow spectrum i.e. high specificity allowing targeting of diff strains within species

evolutionarily related to t6ss and cells resistant to bactericidal activity of their own PTLBs

145
Q

discuss the reprogrammability of tailocins?

A

many toxins have adopted phage tail-like syringe mechanism; called extracellular contractile injection systems (eCISs) - can inject protein payloads in eukaryotic cells

are highly specific due to tail fibres interacting with cell receptors - can modify RBD, load diverse payloads and engineer to target mammalian cells

this could serve as a delivery system of drugs due to high specificity

146
Q
A

MICR335 (4 decks)

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