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Molecular Genetics and Microbiology
William Navarre

MGY377 – Exam Study Note! Page 1 October 29 – Biofilm II Biofilms and antimicrobial resistance  A common theme of biofilm research involves study of antimicrobial resistance  Biofilms can survive exposure to high levels of antimicrobials that kill planktonic cells of the same strain o Antibiotics (biofilms up to 1000x more resistant to some antibiotics!) o Disinfectants e.g. chlorination  Dispersed biofilm cells are sensitive; it is not caused by a ‗selection of mutants‘ phenomenon Biofilms and antimicrobial resistance - Biofilm resistance to antibiotic chemotherapy not the same as conventional antibiotic Paper disk on top of biofilm and membrane cannot resistance o Often referred to as ―Antimicrobial pass though small bacteria can, antibiotic, diffusion Tolerance‖ on paper disk, how much the paper biotic transfer to disk, platonic, look at zone of clearance, some do not - Antibiotic Resistance involves genetically get through but some do, convective flow  encoded factors that prevent killing by antibiotics interaction with the matrix, not a simple model o Mechanisms will work for planktonic or Lack of Penetration/Diffusion biofilm bacteria - Some, but not all antibiotics have impaired - Tolerance involves resistance only in biofilms o Does not work for planktonic bacteria diffusion in biofilm, grow in different rate o Many mechanisms contribute to tolerance - Slow diffusion likely protects some of the bacteria in the biofilm  glow at different rate even at platonic ones, target cell wall, it already Antibiotic resistance - Antibiotic resistance genesconfer resistance for built PBP is already there, use penicillin, cell planktonicand biofilm growing bacteria… pop, actively grows - Protection is enhanced if bacteria also have - But bacteria that lack these genes are still tolerant of antibiotics,(less susceptible to their antibiotic resistance genes effects) when present in biofilms o Break down antibiotic as it diffuses o Group action, concentration of enzymes e.g. b-lactamase Horizontal gene transfer - Antibiotic resistance genes can be transferred - Biofilms may promote transfer Antibiotic Tolerance  lack of penetration/diffusion Lack of Penetration/Diffusion - First theory was diffusion limitation; based on the old ―grapes in jello‖ biofilm view - Thought that the cells at the surface would be killed by high concentrations of antimicrobial, while the cells at the bottom would be exposed to lower concentrations and survive - How can this be tested? (diffusion assay) MGY377 – Exam Study Note! Page 2 Slow Growth Effect o Polymer of glucose donunut, in cytoplasm – - Biofilms have slow growing bacteria sponge to soak up antibiotics o Reduced growth rate due to limiting oxygen, nutrients - Slow growing bacteria are more resistant to ―The proposed localization of the antibiotics NdvB protein to the inner o Planktonic membrane is shown to the right. o Biofilms NdvB is thought to catalyze the - Shigeta et al. (1997) and Tanaka et al. (1999) synthesis of circular glucose used leucine-requiring auxotroph of P. molecules known as cyclic aeruginosa to control growth rate; showed that glucans. These molecules are transported to the effect of beta-lactams is dependent on growth periplasm and are also exported outside the cell, rate (target cell wall synthesis), while effect of possibly via the action of the the NdvA protein.‖ fluoroquinolones (target gyrA/parC) is not Phenotypic tolerance - Changes in gene expression induced by growth in a biofilm that result in a tolerant sub- population: not permanent mutations - Includes many stress response genes o E.g. rpoS (encodes sigma-factor s ) Experimental approach - Made transposon library of Pseudomonas aeruginosa PA14 o Random library of 4000 mutations Expression of NdvB in biofilms - Screened mutants for loss of antibiotic resistance in biofilm o But, also selected for normal planktonic growth - Also wanted mutants that made normal looking biofilms - Identified one clone, 45E7: o More sensitive to several antibiotics in biofilm o Normal biofilm architecture in absence of antibiotics NdvB NdvB - Mutated NdvB gene in two other P. aeruginosa - Clone 45E7 had insertion in NdvB gene strains - Encodes glucosyltransferase - B. japonicum strain with mutation in NdvB o Less effect on antibiotic resistance - Speculate other factors are involved in resistance more antibiotic sensitive in biofilms - Makes cyclic-b-(1,3)-glucans: o Circular polymers of glucose o Thought to play a role in osmoadaptation, flagella-based motility MGY377 – Exam Study Note! Page 3 Intracellular Biofilms? ―Shown below is our current model for the role of cyclic glucans in biofilm resistance. The ndvB gene is preferentially expressed in biofilm cells likely resulting in an increase in the NdvB protein (yellow). NdvB catalyzes the synthesis of cyclic glucans in the periplasm (yellow circles). The red stars represent the fact that the antibiotic Tb can penetrate the biofilm. However, based on a demonstrated interaction between the periplasmic glucans Urinary Tract Infections and Tb, we propose that the diffusion of Tb into the Infection of urinary tract and bladder cytoplasm may be slowed by the glucans. The decreased Very common, estimated cost $1.6B/yr in USA to diffusion of Tb may allow the biofilm cells additional time to adapt to and resist the action of the antibiotic.‖ treat infections Uropathogenic E. Coli (UPEC) accounts for 70-95% of UTI UPEC Infections - Bacteria adhere to epithelial cells in bladder, urinary tract - Invade epithelial cells, induce inflammatory responses - Bacteria can replicate in cytosol of epithelial cells o Perpetuate infection, and spread by shedding in urine - Thought to form persistant reservoir in epithelial Persisters cells - Some bacteria refuse to die with antibiotics, even - Very hard to treat! Resistant to antibiotics when severe cellular damage has occurred - Apparently lost ability for programmed cell Experimental approach death - Infected mice lacking normal immune response - Called Persisters, thought to be spore-like state with UPEC - Not clear how this is regulated o To prevent immune cell influx - Apparently not from genetic mutation o Better able to study infection - More persisters are formed in biofilms - Measured 100 fold increase in # of bacteria in - E.G. chronic UTI/Bladder infection bladder over 24h - Looked at bladder epithelial cells Antimicrobial tolerance summary microscopically - Diffusion limitations, enhanced by presence of enzymes that degrade antimicrobials - Slower growth rates that reduce susceptibility to UPEC-infected bladder epithelial cells - See pods, bladder cell infected by it, inside antibiotics that kill rapidly growing cells bacteria cluster together with a ECM round, - Phenotypic tolerance due to changes in gene colonies like biofilm with ECM around expression o Stress responses - Model of UPEC infection o NdvB, cyclic glucans - Generation of persister cells that refuse to die Biofilms The Black Death Until recently, thought that biofilms formed only - The Black Death is the name given to a massive pandemic of outside host cells i.e. Extracellular event bubonic plague that struck Asia and Europe in the mid- fourteenth century. MGY377 – Exam Study Note! Page 4 - The Black Death spread westward along the ―Silk Road‖ trading routes from Asia (started in China). By the time the pandemic was finished, in just five years, over 1/3 of Asia and Europe was dead and the Mongol Empire was on the brink of collapse. The Black Death – “Bubonic Plague” - After the incubation period of 2-6 days, - Yersinia pseudotuberculosis causes a symptoms of the plague appear including severe comparatively mild and self limiting disease in malaise, headache, shaking chills, fever, and humans. As far as we know it has no capacity to pain and swelling, or adenopathy, in the affected live in fleas. regional lymph nodes, also known as buboes. - Plague is caused by the bacteria Yersinia pestis. How do their genomes differ It is a Gram-negative rod closely related to - Genome sizes are similar Salmonella and E. coli. There are different forms - The pestis strains had an explosion of IS of plague depending on the route of infection. elements Bubonic plague is typically acquired through - The pestis strains have far more pseudogenes the skin. Pneumonic plague, which is caught - There are some regions of new DNA through the air (by inhalation) is the most deadly and results in death within two days. It Positions of the regions acquired and lost by has high mortality even when treated. Y. pestis on a schematic map of the CO92 - Gangrene in the extremities of infected victims chromosome. - A more recent analysis of several pestis and may have given the disease its name. pseudotuberculosis chromosomes revealed that all strains of Bubonic Plague is Spread by Fleas Y. pestis have picked up a phage genome (Ypff), six large - In 1914 Bacot and Martin discovered a solid culture of gene regions (R1-R6), and two small genes (CDS1 and CDS1) bacteria in the proventriculus, the valve-like organ between during their evolution away from pseudotuberculosis. They have also lost several regions (light gray arrows). the esophagus and midgut. - However deleting these newly acquired genes appears to do - They noted that when plague infected fleas attempt to feed, nothing to the ability of lab strains to infect fleas or mice. blood does not get past the proventriculus to the midgut. The We don‘t know what role they may play, if any, in plague. fleas starve, and do what any starving animal does – keep trying to eat. The fleas bite repeatedly, but are able to pump blood only to the esophagus. - When the infected fleas relax their pharyngeal sucking Y. pestis acquired two plasmids, pMT1 and muscles blood flows back to the mammalian host, taking pPCP1. some bacteria with it. It does not require many – the infective dose of Y. pestis is as few as ten cells. (a) Scanning electron micrograph of an uninfected flea, showing elongated spines in the pro- ventriculus. (b) A flea infected with wild-type Y. pestis. Note that the spines (asterisks) are covered with and obscured by the biofilm. The shapes of bacterial In summary, the plasmids appear to encode most of cells are indistinct because they are encased in the genes necessary for the pathogenesis and spread the biofilm extracellular matrix. of plague. We don‘t yet know what role that gene (c) A flea infected with biofilm mutant. Bars, 5 μm. loss may play in pestis biology although this (d) The location of the proventriculus (circled). bacterium seems to have lost its ability to live Areas filled with black show typical extent of Y. independently in the soil. pestis in a blocked flea. Plague infected male Xenopsylla cheopis 28 days after feeding on an inoculated mouse. Studies have Was Y. pestis the Cause of Black Death? East Smithfield Gravesite shown that blockage of the proventriculus is not (1348-1349, excavated in 1986) necessary for infection of a bite victim but that it enhances the rate of biting. The biofilm also appears to help the Yersinia prevent from being digested One challenge of trying to sequence very old DNA from samples like these is that the human DNA and DNA from microbes in the and/or expelled from the flea. burial soil vastly exceed the trace amounts of DNA from Y. pestis. This would prove to be a difficult problem to solve even if the Y. Pestis is closely related to Yersinia sample were not old. On top of this, ancient DNA tends to be pseudotuberculosis. highly broken down with an average fragment length of only 50- 100 nucleotides (very small sample) MGY377 – Exam Study Note! Page 5 Molecular Archaeology Study of Plague (2011) • This is a way of telling how many bacteria (and - To enrich for sequences that are specific to the bacteria you what types) are present in the immediate want to study from a sample a ―targeted capture‖ technique environment: ―quorum sensing‖ can be used. [Target Capture Array] - A slide (shown) is spotted with short oligonucleotides that • When enough bacteria are present, the signal correspond in sequence to the bacterial genome being molecule concentration is high studied. Thousands of spots are required to give complete • Regulatory proteins bind the signal molecule, coverage of a bacterial genome. Beads with oligos attached would work similarly. and activate transcription (directly or indirectly) - Here an array is spotted with numerous oligos (attached to the slide by a chemical linkage). Enlarged is a section of the slide containing oligos for regions (A through E) of the genome. There would be thousands of such unique regions on a slide corresponding to the Y. pestis genome. - This is a highly biased approach. You will only find what you are looking for. - By applying the mixed sample DNA to the capture array, fragments specific to Yersinia pestis were captured by hybridization. The unbound DNA was washed away, leaving Low density, no signaling a sample that is greatly enriched for Y. pestis DNA. Large volume, not enough to produce a threshold - After enrichment the ―captured‖ Y. pestis DNA can be High density, signal activation washed off at high temperature and processed for next gen Cross [] threshold and the bacteria starts to act on it, sequencing. Note that gene E was missing from the strain change in behavior being tested in this example. - A problem with this technique is that if the test strain (old plague in this case) has a unique genomic island (labeled U) Quorum sensing that is not present in the molecular capture array it will be Quorum sensing allows bacteria to switch between lost from the sample. two distinct gene expression programs: one that is - It is possible that the plague strain that infected London in 1348-49 had an extra pathogenicity island that we do not favored at low-cell-density for individual, asocial know about that could account for its high degree of behaviors, and another that is favored at high-cell- transmission or virulence. density for social, group behaviors - Using this method the authors (Bos et al.) were able to complete a draft genome of the medieval plague strain. Importance of Quorum Sensing Interestingly surprisingly few changes were observed. • Quorum = ―the fixed minimum # of members Compared to the modern strain CO92, the Smithfield strain differed by only 97 single nucleotide changes and a few that must be present to make the proceedings of subtle chromosomal rearrangements. an assembly or society valid‖ - The Smithfield strain has even fewer differences when • Many important functions (some exceptions) compared to other modern strains that are believed to be more ―ancestral‖. – Biofilm formation - The authors conclude that Y. pestis was the cause of the – Virulence gene expression Black Death and that the Smithfield strain is not significantly – Sporulation different than modern plague strains. – Mating - The severity of the Black Death may have been due – Motility – Antibiotic production to other factors including nutrition and general – Metabolic control population health as well as frequent exposure to – Growth fleas and no population immunity. Nov. 1 Quorum Sensing (10.8 + special section) It began in the ocean… • Bacteria such as Vibrio harveyi, Vibrio fisheri make light in specialized organs of fish and squid • Study of these bacteria led to discovery of quorum sensing, which is a general bacterial phenomenon Bioluminescence in marine animals Bacterial cell-cell communication The bacterial luminescence reaction, which is • Bacteria produce and can detect small freely catalyzed by luciferase, involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin diffusible signal molecules (―autoinducers‖ – self induced) – can trigger a change genetic behavior mononucleotide (FMNH2) with the liberation of of the (genetically similar neighbor) + self- excess free energy in the form of a blue-green light at 490nm: looping FMNH2 + RCHO + O2  FMN + RCOOH + H2O + light (490nm) MGY377 – Exam Study Note! Page 6 - Juvenile bobtail squid are colonized in the light Al-2 autoinducer  only organ by Vibrio fisherii. Bobtail squid biomolecule with boron colonization is specific to only this They are small hydrophobic bacterial species - no other bacteria carbon tail successfully accumulate in the light organ despite the presence of high numbers of other Autoinducers of Gram-positive bacteria in ocean water. bacteria are typically derived - This symbiotic relationship is thought to protect from peptides the squid by making it difficult for predators to - They are usually cyclic spot from below against the starry sky. Glow at peptide which are night, see star look up, food = star blocked, this synthesised in the ribosome makes it harder to understand it and moditifed - In the morning the numbers of bacteria in the light organ are low. No light is produced by The key experinment the Vibrio bacteria. It is important to note - Using absorbance to measure that the lack of light isn‘t due to the fact that growth of bacteria there are too few bacteria to make enough light - CRM  no luciferase made, to detect. It‘s because the bacteria have shut off then a lot of it made the genes necessary to make light. - Adding autoinducers - Throughout the day the bacteria are dividing in exogeniously can make them the light organ, fed by amino acids provided expressed by the squid. Eventually they will reach a concentration of approximately Quorum Sensing in Gram –ve 100,000,000,000 per milliliter (yes, 10 per involves LuxI –like and Lux-R like enzyme ml). That is a high []! LuxI makes AI-2 that go in or out … - At this high concentration of bacteria enough autoinducer has accumulated in the light organ to trigger bacterial synthesis of luciferase/luciferin. The bacterial luminescence reaction, which is catalyzed by luciferase, involves the oxidation of a long-chain aliphatic aldehyde (luciferin) and reduced flavin mononucleotide (FMNH2) with the liberation of excess free energy in the form of a blue-green light at 490nm. The squid uses its light organ to control the amount and the direction of light released (usually shining downwards). - It binds to a DNA binding protein which they are - In the morning a large quantity of bacteria are paired, so they always makes the AI-1 molecule expelled from the light organ (approximately that it is made by LuxR 95% of the culture). The process begins again… - In light or no light?! - Threshold [] and getting upshift is the +Ve The concept of quorum feedback loop sensing Autoinducer, get Positive-feedback loops occur in many AI-1 induced luciferase expression systems - In many systems one of the targets of LuxR is Autoinducers the LuxI gene. This leads to the synthesis of more inducer. of Gram- negative - The rapid amplification of autoinducer bacteria are production leads to a quick change from very low small organic to very high concentrations of autoinducer. compounds - This ensures all cells Autoinducers nearby trigger dervided from at nearly the acyl same time. MGY377 – Exam Study Note! Page 7 Quorum Sensing in – Important for many pathogens V. harveyi is more • In V. harveyi: complex than – Made by LuxM enzyme typical ―IR‖ – Recognized by LuxN, cell surface receptor systems and involves three Combinations of different autoinducers can give different each bacterial species its own ―scent‖ autoinducers (phosphor- Autoinducer-2 (AI-2)  cross- relay system) inducing molecule • Furanosyl borate diester - Makes 3 different autoinducing agent, AI-1, Ai-2 – (do not cross‘) and GAI-1 Membrane impermeant – Conserved structure • Except Salmonella – Made by more than 40 species of Gram -ves and +ves  Thought to be universal interspecies chemical signal • In V. harveyi and other bacteria: – Made by LuxS enzyme – Recognized by LuxQ, cell surface receptor • Note: Requires periplasmic binding - At low cell densities the sensor kinases protein LuxP for signaling (do not cross autophosphorylate ultimately shutting down membrane) LuxR and light production Formation of AHL and AI-2 - Intrinsically linked to metabolism  with SAM a important mehtylator which affects the [] of their production  decide if it goes social or not - At high cell densities the kinases are inactivated by AI-1, AI-2 and CAI-1  all those feed to lasI mutant  forms flat indifferent biofilm that LuxU, trigger some small RNA that looks LuxR unlike wrt and is sensitive to detergent which turn on the expression of LuxABCDE gene - Some will turn on their type III when there is Quorum sensing controls expression of the type III lots of autoinducers around secretion gene transcription and protein secretion in enterohemorrhagic and enterpopathogenic E Coli Autoinducer-1 (AI-1) - Hard to express virulent factor  quantum • N-acyl homoserine lactone sensing as they need to see point to express structures (AHL) those  Show luxS (makes AI-2) required for – Membrane permeant type III secretion by EPEC & EHEC – Specific to each pathogen – Some make >1 • e.g. P. aeruginosa makes two and secretes these in hierarchcal manner (one before the other) • Made by more than 50 Gram - ves MGY377 – Exam Study Note! Page 8 Quorum ‗interception‘ in Salmonella “Typical” Gram-positive quorum sensing • Salmonella lack luxI gene, can‘t make AHL, but can detect multiple AHL‘s from other species using luxR homologue SdiA  activates several genes e.g. rck (resistance to complement killing – immune response) • Also recognize modified AI-2 via LuxP Pepide precursor to ribosome, make larger peptide homologue LsrB  activates expression of AI-2 and make a derivate of the pepide, trasporter, some system transporter…clears AI-2 from medium  role of AI-2 signaling in Salmonella unclear Anti-QS strategies: bacteria Can Sdi A sense those molecule in in vivo situation - May allow certain bacteria to outcompete others from other bacteria  took Salmonella  go in the environment, or during infection of a host through animal, was quorum sensing ever occurring - Several mechanisms observed: when passing through them  it was activated • Metabolism of autoinducers e.g. V. paradoxus through Turtle and found that it was sensing some • B. subtilis degrades AHL using aiiA gene (many those homologues present in other bacteria) • S. aureus autoinducing peptides activate own virulence genes but inhibit those of other S. aureus groups Modified AI-2 in Salmonella Host responses to QS signals - QS signals similar to mammalian signaling molecules: • Hormones • Prostaglandins - Many host cells can respond to bacterial QS molecules: • IL-8 production by lung epithelial cells • IFNg production by T cells • Cox-2 expression by fibroblasts (makes prostaglandins) • Acceleration of apoptosis by macrophages and neutrophils LuxP in V,harveyi  do not make Beronease ester, Lsr B, Salmonella specifc, see LuxP Anti-QS strategies: host - Mammalian cells appear to have mechanisms to Quorum Sensing in Gram +ves eliminate QS signaling molecules • Autoinducers are short, usually - Present in multiple cell types, multiple modified peptides processed mechanisms from precursors Anti-QS strategies: host • Actively exported from cell • Cell impermeant Inactivation of bacteria virulence pheromone by • Interact with membrane- phagocyte-derived oxidant – new role for the bound receptors NADPH oxidase in host defense • Highly specific for peptide ligands • Signal transduction by phosphorylation cascade • Activate transcription factor AntiQS strategies MGY377 – Exam Study Note! Page 9 Inactivation of Pseudomona aeruginosa quorum sensing signal by human airway epithelia - Degraduation activity of different R group, 3OC6 - When those combine with NO2  something,  get eliminated  2 carbon shorter harder  ROS  many target of autoinducer incidates is an enzymatic process - Airway epithelia AntiQS strategies Experimental strategy - Different cell have different activity  some randomly sens it AntiQS - Chun et al also showed that 3OC12-HSL degrading activity is associated with cell membranes • Not a secreted factor - Pseudomonas aeruginosa makes two AHL: • 3OC12-HSL and C4-HSL - Boiling cell lysate inactivated QS degrading activity, demonstrated heat sensitive factor was - Grew polarized human lung epithelial cells on involved permeable support - Mechanism unclear, speculate mammalian - Added AHL‘s from P.aeruginosa to apical side and measured breakdown over time enzyme equivalent to aiiA in B. subtilis or other bacteria - Well with liquid media, grow cells on, hit certain density, make tight junction, like epithelial cell monolayer Anti-QS strategies: therapeutic? - Different side  apical and basolateral have - Quorum sensing intrinsic to virulence by pathogens different effects  going into the cell - Can QS signaling be - See how much go into the cell, like the GI - Adding AHL and see break down impaired to treat infection? - Nu- mber of avenues being pursued Anti-QS strategies: host - Different reaction and inhibition and whatever - Atypical  no basal lateral, start getting equalirium  More cell cell signal molecules are being discovered soak up by cell - 3OC12  atypical, never in basolateral, and then it stops  soaking it out and doing soming • Eliminated rapidly with no BL accumulation • Specificity  Graph MGY377 – Exam Study Note! Page 10 Nov, 4 – Competence I: Bacterial Genetic - Bacteria that can get DNA from environment, a (transformation) - Chapter 9 gummy looking colony, those can be grown and can kill the mouse, when passed, it can loses How do bacteria acquire gene needed to make the capsid and the colony new genes looks rough, the variant can generate an immune responds - What is killing the mouse Genetic exchange in prokaryotes Transformation in the history of molecular biology - Can transform the cells from rough to smooth by adding things to the structure How cells can take up DNA in environment and - E Coli not compentant  cannot pick up DNA incorporated in the genome - DNA not protein Transduction, phage get inside of cell and make multiple copies, then the DNA need to be package in Transformation and competence the head and jump on to the next bacteria • Genetic Transformation: a process by which Once in a while, get some the host DNA and when free DNA is incorporated into a recipient cell and pops out, get phage head which can infect other cells brings about genetic change Conjugation – makes a pump on the membrane of • Competent: a cell that is able to take up a the host cell and make contact to other cells, and molecule of DNA and be transformed infect other cells, a selfish DNA, bringing along DNA • Natural Competence: genetically from one cell to the programmed physiological state permitting the (1) Host efficient uptake of macromolecular DNA. Well- (2) PHAGE and another is the DNA element studied examples include: itself – Gram +: Streptococcus pneumoniae, Bacillus subtilis Transformation in the history of Molecular Biology – Gram -: Haemophilus influenzae, Neisseria gonorrhoeae Artificially-Induced Competence: a state of competence achieved by chemical or physical treatment of the cell Many bacteria are naturally competent Do not mean they are competent all the time, it undergoes some regulation Temporal Expression of Competence Phenotype in S. - Structure was solved but the mechanism of its pneumoniae and action was unknown, method of inheritance and B. subtilis genetic was unknown MGY377 – Exam Study Note! Page 11 Quorum Sensing-based The Gram-positive competence system is Control of Competence in composed of: S. pneumonia • The pheromone precursor (pre-CSP) is encoded by comC and is processed and secreted by the action of the ComAB proteins. • A ―pseudopilus‖ encoded by the ―ComG‖ operon • The pheromone is detected by the that is related but not identical to the Type-IV histidine kinase ComD, pilus (or Type-II secretion system – how protein leading to the can get out of bacteria) – rod that stick out of cell • A nuclease (NucA) phosphorylation of ComE. • A DNA binding protein (ComEA) • ComE~P drives the transcription of the comCDE • An ATPase helicase/motor protein (ComFA) • A transmembrane pore (ComEC) and comAB operons as well as that of comX. ComX is a minor sigma factor that appears to activate the transcription of the late competence proteins, which includes proteins involved in taking up DNA, RecA, and a negative regulatory protein that eventually turns off expression of the com operons. (competes with housekeeping, turns on, lead to expression of competence gene, and can feedback) Perhaps we‘ve only touched the surface of how bacteria take up DNA. The DNA gains access to the ComEA protein by way Chitin induce neutral competence in cholera  of the pseudopilus. The exact mechanism by which though to be incompetent but it does when grown this occurs is unclear but it is clear that this structure with other thing, could be competent in the wild is required. ComEA mutant are not compentent + - Gram vs Gram (diagram) ComEA is a DNA binding protein that is required for competence. The DNA must have a free end to enter the cell. In the Gram- positive Bacillus subtilis, the NucA nuclease does the trick by introducing double stranded breaks. Once bound to ComEA, the DNA is passed to the channel protein ComEC. The ComFA protein is an ATPase that is required for DNA import and may act as a helicase to unwind the DNA although this remains unclear. The DNA that enters the cell is single stranded, entering by the 3‘ end. How to get it a large –ve charged molecule into the The 5‘ end of the incoming DNA appears to cell be chopped into nucleotides although the Other side, gram –ve  there is a specific sequence, nuclease activity is yet to be defined. Dust?! Which must be recognized Gram + many binding site and the sequence can be anything MGY377 – Exam Study Note! Page 12 Once inside specific single-stranded DNA binding DNA import is proteins bind the incoming DNA. Thereafter it can an active be degraded (used as food) or recombine into the process that chromosome or plasmid by RecA (recombinase) generates significant force. catalyzed DNA transport into Bacillus subtilis requires protein homologous motive force to generate large molecular forces recombination. Force generated to slurp the DNA into the cell, it is so hard that over the 2 min it goes right to the bead that the DNA is bound to Artificially-Induced Competence A. Calcium-induced competence: Transformation in Gram-negative cells • E. coli treated with high concentrations of calcium ions and then stored in the cold become transformable at low efficiency (heat shock Several Gram-negative cells including Neisseria and helps!) • double-stranded DNA, including plasmids Haemophilus require that a specific sequence be present in the incoming DNA for efficient • no specific DNA sequences required (although transformation to occur. These sequences are called DNA methylation pattern will affect efficiency) ―DUS‖ for DNA-uptake sequences • simple reagents, no special equipment • <10% of cells typically become competent • DUS sequences have been identified for Neisseria (5′-GCCGTCTGAA-3′) and • mechanism uncertain Haemophilus (5′-AAGTCGGT-3′). These B. Electroporation (zip cells in metal plate) sequences are found frequencies much higher • pulsed electric fields produce small, transient pores in lipid membranes than would be expected by chance in their respective genomes. This fact is believed to • when DNA present outside the cells during the facilitate DNA transfer between similar species. electric pulse, they can enter through the pores • There is sequence specificity (also • requires sophisticated power supply which species-specific) allows pulse current, duration, and electrical • Ways to exchange DNA?! resistance to be carefully controlled • works with most species - conditions vary • single or double-stranded DNA DNA containing • ~95% of cells become competent (but high a DUS death) sequence is What Use is Competence? bound by an DNA as food? unknown • Why degrade half of food? receptor • Why complex machinery - B. subtilis also (DR) on secretes powerful nuclease and takes up free the cell nucleotides surface (binding event). From there it is passed DNA Repair? through the PilQ-secretin complex. • Stationary phase may correlate with increased DNA damage? (e.g. stress, B. subtilis oxidative ComE, a periplasmic protein delivers the DNA to metabolism) ComA, a channel similar to ComEC in Bacillus. The • Competence is not induced by UV stress DNA is degraded to ssDNA and passed through the To build a better bug? channel in a manner similar to that observed in • Sample genes that are in the environment (intra- Gram-positive and inter-species) to overcome stress - acquire bacteria new functions (enzymes) or generate new allelic combinations although the specific Biofilm formation (newer hypothesis)? nucleases • Is it a coincidence that DNA is found in biofilms have not been and that competence is regulated by quorum- identified. sensing or quorum-sensing like systems? MGY377 – Exam Study Note! Page 13 What Use is unaffected in signaling, showed reduced biofilm Competence? formation. During growth in the presence of DNase DNA as food? I, biofilm was reduced in the wild type to levels - E. coli is not similar to those found with the comGB mutant, naturally suggesting that DNA plays an important role in the competent. wild-type biofilm formation. We also showed that Here the authors growth in the presence of synthetic CSP promoted use a wild type strain significant release of DNA, with similar levels in and a mutant strain the wild type and in the comGB mutant… that lacks a (How can it get DNA out the cell and not kill the cell homologue of one of and pull together to make a rope) the Gram negative natural competence Biofilm formation in S. mutans is diminished proteins (previously without the pseudopilus had unknown function in E. coli) Wihtout SCSP compare to with, more robust bifilm - So, perhaps all bacteria can take up DNA, but ComGB, slight, but somewhat defective most use as food while a select few have learned to use it for natural competence. DNase treatment of S. mutans lowers biofilm production DNA Repair (or as a The fact that DNAse treatment doesn‘t lower the bug) biofilms of comGB mutants any further than their • An individual already low levels suggests that DNA is not involved nucleotide is in the small amount of biofilm made by comGB 50-80 – fold mutants. more likely to There is a DNA dependent/independent biofilm change by formation recombination The Dental floss, rope together, or rid it, not needed than by anmore and work together mutation in naturally Enterococcus facalis produce abundant extracellular competent structure containing DNA in the Absence of cell lysis species such as during early biofilmformation (reading) N. meningitidis & S. pneumoniae • When we compare genes of closely related (A and B) Long intercellular strands (yarn strains or species of a naturally competent structures) visible in early (4-h) E. faecalis biofilms bacteria, observe frequent exchange of genes or (arrows – see ropes) Bar, 5 µm. (C) Strong punctate gene fragments(mosaic gene) labeling of the strands visualized by using an anti- dsDNA monoclonal antibody conjugated to an Biofilm formation (newer hypothesis)? immunogold particle (asterisks – very electronic - In the ECM, in 2002 dense and can be seen in AB, all DNA rope after 4 hr - Young P. aeruginosa biofilms are held together when cell is added, no real cell lysis). The image with DNA and will disperse if treated with shows the significant eDNA component (the central DNAse; older biofilms are DNAse resistant area is magnified in panel D). Bar, 250 nm. (E) Surface morphology can be appreciated in the DNA biding Uptake System corresponding secondary electron image. Bar, 1 µm. DNA has recently been described as a major structural component of the extracellular matrix in Thick enveloping mats of extracellular matrix biofilms. In streptococci, the competence- (sweater structure) make up a large fraction of the stimulating peptide (CSP) cell-to-cell signal is E. faecalis biofilm (A, secondary electron image) and involved in competence for genetic transformation, contain significant eDNA, as shown via immunogold biofilm formation, and autolysis. Among the genes labeling of the anti-dsDNA monoclonal antibody (B, regulated in response to the CSP are those involved backscatter image of the same field). Bars, 1 µm. (C) in binding and uptake of extracellular DNA. We False-color SEM image showing the relationship show in this study that a functional DNA between the surface of the ECM (red; secondary binding-uptake system is involved in biofilm electron detector) and the immunogold-labeled anti- formation. A comGB mutant of Streptococcus dsDNA antibody (green; backscatter detector). mutans deficient in DNA binding and uptake, but MGY377 – Exam Study Note! Page 14 eDNA appears to be encased within another ECM Nov. 7 - Bacterial Gene Transfer: component (arrows). Bar, 500 nm. Plasmids and Conjugation - The swater structure on B, zoom in cell rope of Recommended reading: Chapter 9 – S and F. DNAanda red layer  DNA is surrounded in a capsule of its own WHAT ABOUT PLASMIDS? Bacteria in the wild can have 0 to 21 plasmids (A and B) Immuno-SEM micrographs demonstrating localization of the eDNA probe near the E. faecalis septum (where daughter (Borrelia have 21) cell is dividing). (C) Endogenous lysis of cells in an older (48-h) Plasmids (there are exceptions to rules) biofilm display an entirely different morphology from that seen in early biofilms, as DNA (asterisks) is released from a ruptured cell • small (<30 genes; 0.2 - 4% size of genome), (arrow). Bars, 500 nm. (See popped cell and see that, distant from circular (usually), supe the early time point). Building up the case • rcoiled DNA molecules Correlative microscopy. • present in most species but not all strains Though imaged using different microscopic techniques, both the • may have ~1-1000 copies/cell SEM (A and B; bars, 500 nm) and immunofluorescent • use replication machinery of the host (some also micrographs (C) show similar patterns of eDNA labeling in early E. faecalis biofilms—long intercellular strands (yarn structures require plasmid-encoded proteins) [asterisks]) as well as a pericellular labeling component of the • have own replication origin, are autonomously replicated (replication using control signal sweater matrix (Δ) (C; bar, 25 µm). Monoclonal antibody labeling of extracellular dsDNA (green, DyLight 488; blue, Hoechst 33342 different from regular machinery), and stably highlighting individual E. faecalis cells) is shown. inherited (are a ‗replicon‘) Cell have it own DNA and also outside, case is that when it is still • are dispensable for growth under many alive conditions Biochemical assays. (detecting lysing) (A) Luciferase-based quantification of Plasmids stably maintain themselves in the extracellular ATP. No significant differences were found between biofilm cell through common strategies and planktonic levels at 2, 4, or 8 h • in selfish DNA element, makes sure it is passed postinoculation in matched E. faecalis on samples. Results are averages from four independent experiments. Only the values • autonomous replication and copy number for the 4-h OG1RF samples (planktonic control (plasmid dictate on) • providing a useful function for the cell such as and biofilm) are above the instrument background; neither rose to statistical drug resistance (often virulence genes) significance. (Not as much light produced, • partition systems that actively segregate the added ATP and see if it works – yellow is plasmid to daughter cells  make sure it passes control) (B) The cell-impermeant fluorescent dye on to each generation Sytox green (Invitrogen) was used to label • ―addiction systems‖  how they are parasitic nucleic acids outside functional cells. eDNA levels were >10 -fold and stay in own population of cell higher under biofilm conditions than in matched planktonic controls. Exogenous lysis of a small fraction of cells (~5% by microscopy) produced a luciferase signal that saturated the Examples of plasmids instrument detector under the displayed conditions (not shown). From different region, pUC was artificially Error bars indicate 1 SD. (more lysis, less extracellular DNA, as dye do not get into cell, platkontic, see more DNA in biofilm, not synthesi
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