Microbial growth. Steve Zinder MBL June 11, 2013

Transcription

1 Microbial growth Steve Zinder MBL June 11, 2013

2 The obvious goal of any bacterium is to become bacteria. In signature of: Lizzie Wilbanks UC Davis Course student 10, TA 11 Queen of the berries

3 Plos Biol. 3:295, 2005 YFC-labeled cells grown in slide culture on Luria broth at 30 o C 505 cells (~9 doublings) in 315 min ~34 minute T d E. coli dividing Cells with initial poles (old) grow slightly slower (1%/ gen) Old cells more likely to die than newer ones More protein aggregation at poles? (EMBO 29: )

4 E. coli dividing E. coli dividing optimally doubles every 20 minutes Makes up to 20,000 new ribosomes, > 1,000,000 molecules of >1000 different proteins >22,000,000 lipid molecules 2 simultaneous chromosome replications (each takes 40 minutes) If growth continued unimpeded, a culture s weight would be > than the Earth s after 48 hours

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6 Growth in batch culture dn/dt = µn N = number of cells present µ = specific growth rate (a.k.a. k ) = 0.69/T d

7 Measuring culture biomass by turbidity Beer s law: A(λ) = e(λ) l c A = absorption at wavelength λ c = concentration of solute l = length of light path e(λ) = (molar) absorptivity constant What are we measuring? From: Neidhardt et al, Physiology of the Bacterial Cell, Sinauer, 1990

8 Other ways to measure growth Metabolic product: CH 4 Thermophilic Methanosaeta Arch. Microbiol. 146:315, 1987 Cell protein and microscopic counts Metabolic product: vinyl chloride Dehalococcoides culture Science 276:1578, 1997

9 Measurement by qpcr Assumptions? Measurement of Dhc in PCB utilizing mixed culture AEM 73:2513, 2007

10 Life in the slow lane: Pelagibacter growth in seawater 10x increase (3.3 doublings) in 9 days 2.7 day Td

11 Colonies from soil dilute medium From AEM 68:2391 (2002)

12 Organisms carrying out biogeochemical processes often grow on a geological time scale Gas production from hexadecane by a methanogenic consortium. 4C 16 H H 2 O --> 49CH HCO H + G = kj/rxn or kj/ch 4 From: Nature 401:266, 1999

13 Growth ain t just binary fission: Epulopiscium From: Angert, Nature Rev. Microbiol 5:214, 2005 Metabacterium

14 Multiple cells and budding

15 Rhodomicrobium vannielii 4 buds/hyphum

16 Actinobacterial filamentous growth

17 Growth curve revisited Problems?

18 Lag phase Inoculum with 10% viability From: Neidhardt et al, Physiology of the Bacterial Cell, Sinauer, 1990

19 Diauxic (biphasic) growth of E. coli on glucose and lactate

20 Effect of substrate concentration on growth Reaction rate Vmax Growth rate µmax Km [substrate] Ks [substrate] Michaelis Menten enzyme kinetics V = Vmax * [S] [S] + Km Monod growth kinetics µ = µmax *[S] [S] + Ks Liebig s law of the minimum Only one thing limits growth at any one time. (bottom up control)

21 The chemostat D = flow/volume = 1/Retention time e.g. if volume is 10 L and flow is 1 L/h, D = 0.1 h -1 At steady state µ = D

22 Chemostat kinetics J. Gen Micro 14:601, 1956 Add Pirt plot S = Ks at 0.5 µ max Uses: Constant growth rate - omic studies Feeding toxic substrates at low concentration Competition experiments

23 Competition D = 0.32 h -1 S/P D = 0.05 h -1 P/S D = 0.24 h -1 P/S Pseudomonas: µmax = 0.64 h -1, Ks = 91 µm Spirillum: µmax = 0.35 h -1, Ks = 23 µm R vs K selection Physiological basis of the selective advantage of a Spirillum sp. in a carbon-limited environment. Matin A., Veldkamp H. (1978) J Gen Microbiol. 105:187

24 Growth physiology trna/total RNA Neidhardt book

25 Growth physiology maintenance energy Pirt double reciprocal plots: 1/yield vs 1/dilution rate Butyrivibrio fibrisolvens Maint coeff = Bacteroides ruminicola Maint coeff = Selenomonas ruminantium Shifts from acetate and propionate to lactate (less ATP) at high growth rates Russell and Baldwin AEM 37:537 (1979)

26 Other continuous feed methods Turbidistat dilute when culture reaches certain turbidity ph auxostat: substrate neutralizes ph change by organism s growth Conversion of formic or acetic acid to CO 2 Gradient cultures e.g. SOB

27 Wall growth = Biofilms

28 Growth on slides at 90 o C Suspending cover slips in Boulder Spring a boiling water spring extensively studied by Brock and coworkers Microbial colonization of cover slips incubated in boiling water springs - J. Bacteriol. 107:303 (1971)

29 Growth on slides EM pictures of some of the Boulder Spring "bacteria" which were clearly Archaea. Uptake of radiolabeled lactate, acetate, or thymidine by Boulder Spring bacteria attached to slides