Biol371 Lecture 6: Molecular Aspects of Microbial Growth PDF

Lecture 6 - Molecular aspects of microbial growth Presented by: Michael Sgro Materials covered:  Chapter 8.2-8.5, 8.10-8.12 Bacterial cell growth  Binary fission: a cell division following enlargement of a cell to twice its minimum size  Generation time: time required for microbial cells to do...

Lecture 6 - Molecular aspects of microbial growth Presented by: Michael Sgro Materials covered:  Chapter 8.2-8.5, 8.10-8.12 Bacterial cell growth  Binary fission: a cell division following enlargement of a cell to twice its minimum size  Generation time: time required for microbial cells to double in number  During cell division, each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell Overview of the bacterial cell cycle  Bacteria typically have a circular chromosome. The DnaA protein binds to the origin of replication (oriC) and initiates bidirectional DNA synthesis  SeqA binds to hemimethylated DNA and blocks DnaA from binding to the oriC region, preventing additional rounds of replication  Genomes segregate to poles of elongating cell  Septum formation and cell division Genome replication in fast-growing cells  E. coli has a generation time of 20 minutes but it takes 40 minutes to replicate its chromosome. How?  Cells growing at generation times less than the replication time contain multiple replication forks (a) Generation time, 1 h ; replication time, 40 min our utes  New rounds of DNA replication begin before the last round is complete (b) Generation time, 20 min ; replication time. 40 min utes utes Chromosome segregation  Required so daughter cells gets a copy of genome and for septum formation  Many bacteria use the Par (partitioning) system to distribute chromosomes and plasmids equally  PopZ localizes to old pole of the cell and associates with ParB, which binds to the parS sequence near oriC  The parS sequence on the daughter chromosome is bound by ParB and moved to the new pole by the activity of ParA Cell division and Fts proteins  The divisome is a cell division apparatus made up of Fts proteins and other proteins essential for binary fission  Involved in synthesis of new cytoplasmic membrane and cell wall material (septum)  FtsZ forms a ring around the center of the cell  ZipA anchors FtsZ to the cytoplasmic membrane  FtsA recruits other Fts proteins  FtsI helps synthesize new peptidoglycan Cell division and Min proteins  DNA replication occurs before divisome formation because nucleoids block formation of the FtsZ ring (nucleoid occlusion)  The Min proteins ensure the divisome forms at the center of the cell  MinD binds MinC and together they prevent the FtsZ ring from forming. These oscillate between the poles  MinE also oscillates, pushing MinCD aside toward the poles  MinCD spend most of the time at the poles, allowing the FtsZ ring to form only in the center of the cell Determinants of cell morphology  Like eukaryotes, bacteria contain a cytoskeleton to give cells their shape  Shape-determining proteins known as SEDS (shape, elongation, division, sporulation) proteins  MreB is the major shapedetermining factor in Bacteria  MreB recruits other proteins involved in cell wall growth to form the elongasome in rodshaped bacteria Determinants of cell morphology  Filaments of MreB move along the cell allowing growth at several points rather than a single plane (FtsZ ring) as in spherical bacteria MreB filaments in rod-shaped cell  Crescentin forms filaments which give vibrioshaped cells their curve  Crescentin localizes to the concave surface of the cell and is present in addition to MreB Crescentin in vibrio-shaped cells Diverse cell morphologies  Diverse morphologies exist in bacteria, especially in the gram-negative Alphaproteobacteria  Some synthesize peptidoglycan only at the poles, some grow by budding  Morphology and phylogeny are typically unrelated Peptidoglycan biosynthesis  Production of new cell wall is a major feature of cell division  In cocci, cell walls only grow in opposite directions outward from the FtsZ ring  Preexisting peptidoglycan needs to be severed to allow newly synthesized peptidoglycan to form  All bacterial cells must synthesize new peptidoglycan and export it outside the cytoplasmic membrane Peptidoglycan biosynthesis  Bactoprenol (orange section) is a highly hydrophobic molecule which carries peptidoglycan precursors through the cytoplasmic membrane  Lipid II is the entire molecule including bactoprenol bound to the peptidoglycan precursor consisting of N-acetylglucosamine (G), N-acetylmuramic acid (M), and a pentapeptide Pentapeptide Peptidoglycan biosynthesis Removal of extra D-alanine provides energy for transpeptidation Biofilm formation Four stages of biofilm formation  Random collision leads to initial attachment, facilitated by structures like flagella or by cell surface proteins  Attachment causes expression of biofilm-specific genes encoding proteins for intercellular communication and extracellular polysaccharides  Cyclic di-GMP is used as an intracellular signaling molecule. Accumulation triggers a transition from planktonic to biofilm growth Biofilm formation – cyclic di-GMP  Two molecules of GTP are converted to c-di-GMP by proteins with GCDEF domains  Phosphodiesterases degrade c-di-GMP  Many effects including modifications to extracellular polysaccharides and proteins, reduced motility and virulence Biofilm formation – Pseudomonas aeruginosa  Pseudomonas aeruginosa is an opportunistic pathogen that forms a tenacious biofilm containing polysaccharides that increase pathogenicity and prevent antibiotic penetration  Intercellular communication by quorum sensing is critical for biofilm development and maintenance  Accumulation of acyl homoserine lactones (AHLs) signal population growth and high levels trigger c-di-GMP synthesis and activate other biofilm-specific genes  DNA released by lysed cells can integrate into polysaccharides and promote biofilm formation  A cell lysis protein causes some cells to burst in response to stress Biofilm formation – Vibrio cholerae  In Vibrio cholerae, quorum sensing acts in the opposite way as it does in P. aeruginosa  Biofilm formation is triggered by low cell densities and repressed by high densities  Accumulation of signaling molecules represses biofilm-formation genes and activates flagellar genes  Biofilms are more likely to occur in natural marine environment compared to intestinal cells where nutrients are more plentiful.  Dispersal aids in transmission to new hosts Biofilm formation – Vibrio cholerae Small regulatory RNA reduces expression of repressor Polysaccharide-binding matrix proteins Antibiotic targets  Antibiotics are antibacterial agents (generally) naturally produced by microbes  Kill or inhibit bacterial growth  Target essential molecular processes Antibiotic targets  Many antibiotics target DNA replication, RNA synthesis, or translation  Quinolones target DNA gyrase and topoisomerase IV, interfering with DNA unwinding and replication  Rifampin and actinomycin prevent RNA synthesis by blocking RNA polymerase active site or RNA elongation Antibiotic targets  Inhibition of protein synthesis  Ribosomes in bacteria are 70S; eukrayotic are 80S  Puromycin binds to A site in 70S ribosome, inducing chain termination  Aminoglycoside antibiotics (e.g. streptomycin) target 16S rRNA and 30S ribosome, leading to error-filled proteins that inhibit growth Antibiotic targets  Antibiotics targeting the cell membrane  Daptomycin specifically binds to phosphatidylglycerol residues of the bacterial cytoplasmic membrane, leading to pore formation, depolarization and death  Polymyxins are cyclic peptides whose long hydrophobic tails target the lipopolysaccharide layer, disrupting the membrane and causing leakage and death Antibiotic targets  Antibiotics targeting the cell wall  Β-lactams (penicillin, cephalosporin, derivatives) interfere with transpeptidation (formation of cross-links) between muramic acid residues  Vancomycin binds to pentapeptide precursor and prevents interbridge formation  Bacitracin binds to bactoprenol and prevents new peptidoglycan precursors from reaching site of synthesis Antibiotic resistance mechanisms  Antibiotic resistance mechanisms are genetically encoded in four classes: 1. Modification of drug target 2. Enzymatic inactivation 3. Removal via efflux pumps 4. Metabolic bypasses  Resistance can arise due to random chromosomal mutations  E.g. Spontaneous mutants resistant to rifampin can be obtained by exposing a large population to the antibiotic  Resistance can also exist on mobile genetic elements and be transferred by horizontal gene flow  Many mobile resistance genes encode enzymes that inactivate antibiotic (e.g. Βlactamase cleaves a ring structure, or an acetylating enzyme adds acetyl groups to chloramphenicol) Antibiotic resistance mechanisms  Penicillin is shown here as an example for different mechanisms of resistance  Mechanisms of resistance shown in green  PBP = penicillin-binding protein Antibiotic resistance – Efflux pumps  Efflux pumps are ubiquitous and transport various molecules, including antibiotics, out of the cell  Lowers intracellular concentration, allowing cell to survive at higher external concentrations  Many can act on a broad range of compounds and transport multiple classes of antibiotics, contributing to multidrug resistance  E.g. AcrAB-TolC in E. coli can pump out rifampin, chloramphenicol and fluoroquinolones Antibiotic resistance – Metabolic bypasses  A metabolic bypass makes the antibiotic target no longer essential  E.g. methicillin-resistant Staphylococcus aureus (MRSA) contains an alternative penicillinbinding protein that is not recognized by β-lactams  MRSA synthesize this protein only in the presence of β-lactams due to a repressor and a β-lactam sensor Persistence and dormancy  Persistence: When a population of antibiotic-sensitive bacteria produces rare cells that are transiently tolerant to multiple antibiotics  Persisters are genetically identical but dormant (viable but do not grow)  Dormancy prevents antibiotics from killing the cell  When treatment is stopped, cells emerge from dormancy and grow  Can cause recurring infections (e.g. cystic fibrosis from Pseudomonas aeruginosa) Toxin-Antitoxin (TA) modules  Toxin-Antitoxin (TA) modules encode two components: a toxin which inhibits cell growth and an antitoxin that binds and counteracts the toxin  Found in almost all bacteria  Toxic activity thought to promote cellular adaptation by slowing cell growth to ensure survival during stress Toxin-Antitoxin (TA) modules  The hipAB genes encode a TA module In E. coli  HipA is a toxin that inhibits translation  HipB is an antitoxin susceptible to Lon protease  Normally they form a stable, non-toxic complex  If Lon is activated by the signalling molecule PolyP, HipB is degraded and growth is arrested by HipA Stringent response  HipA phosphorylates glutamyl-tRNA synthetase (GltX), preventing the tRNA synthetase from charging amino acids. This leads to ribosome stalling, inhibiting translation  Uncharged tRNA entering the A site of the ribosome causes RelA to produce the alarmone (p)ppGpp, inducing the stringent response pathway  Stringent response leads to cell dormancy Exiting dormancy  Once antibiotic exposure ends, persisters exit the stringent response pathway and produce HipB (or other antitoxin) again, allowing cells to grow  HipA-induced persistence occurs in cells randomly producing higher amounts of the PolyP signal molecule (phenotypic heterogeneity)