How bacteria in colonies can survive by killing their brothers and sisters

Transcription

1 How bacteria in colonies can survive by killing their brothers and sisters Harry Swinney University of Texas at Austin bacterial colony 6 cm at U. Texas: Avraham Be er Hepeng Zhang Shelley Payne E.L. Florin G. Ariel with E. Ben-Jacob (Tel Aviv U)

2 Fractal growth in electrodeposition in 0.15 M zinc sulffate solution Argoul, Arneodo, & Swinney, Phys. Rev. Lett. 61 (1988) D = mm 12 V electrodes glass plates

3 Praud, Swinney, Phys. Rev. E (2005); Leshchiner, Thrasher, Mineev, Swinney Phys. Rev. E (2010) Pattern grown from an air bubble in oil layer dia = 288 mm oil layer old young pump air into center hole D = 1.70

4 Fractal pattern formed by growing bacterial colony Matsuyama, Harshey, Matsushita, Fractals (1993); Ben-Jacob et al, Nature (1995) 35 mm Bacillus subtilis

5 Bacterial colony pattern Paenibacillus dentritiformis (C morphotype) Eshel Ben-Jacob et al., Ann. Review Microbiology (1998) initial inoculation

6 TEM Inoculate agar gel with bacteria Petri dish 5 μl drop (~ 10 5 bacteria) agar gel 88 mm Paenibacillus dentritiformis Bacillus subtilis 1 μm flagella 7 μm

7 Paenibacillus subtilis colony (after six days growth on a gel) 40 mm

8 Motion and growth occurs only near the colony edge

9 Growth of nearby bacterial colonies Paenibacillus dendritiformis sibling colonies (from same culture)

10 Competing Bacterial Colonies Fujikawa & Matsushita --- Bacillus subtilis: J. Phys. Soc. Japan (1991) 10 μm 10 μm Explanation: food depletion

11 Competing Paenebacillus dendritiformis colonies: x final /d growth stops no matter how much food available MID-LINE d x final 25 mm MID-LINE

12 Change research direction from a study of bacterial colony patterns to address question: Why don t growing neighboring colonies collide?

13 Transition from growth to inhibition growth front position x (mm) single colony 2 colonies x final (mm) d final position x f sharp transition from unhibited to inhibited growth

14 Transition to inhibited growth occurs for colonies with any initial separation d x (mm) deceleration 1.1 μm/h 2

15 Inoculate gel with an annulus No growth inside even though there is sufficient nutrient there

16 Grow colonies from different initial gel inoculation patterns

17 Model: assume prespores secrete inhibitor u(r,t) take colony radius R c (t) from experiment: agar=1.5% peptone=2g/l prespore spore R ( t) = Vt c Vt lag x (mm) 10 D (mm) time (h) (h) 4 μm experiment also indicates R p = αr c (t) where α is constant

18 Model for inhibitor concentration u(r,t) u( r, t) t where = D 2 u( r, t) + A H ( R p r C limits inhibitor generation to prespore region i ) H ( u max u( r, t)) limits u to a maximum value D A H(x) C i u max diffusion coefficient (measured) an amplitude Heaviside step function center of the ith inoculation maximum u for inhibitor (for u > u max sporolation is complete and no inhibitor is generated) u T threshold: for u > u T, colony growth decelerates (as observed)

19 Model prediction for two competing colonies inhibitor concentration (mass/mm 2 ) u max threshold u T neighboring colonies

20 Inhibitor saturates below threshold u T in direction away from neighbor inhibitor concentration (mass/mm 2 ) outer edge of colony threshold u T

21 Initial inoculation: C-shaped

22 Calculate initial inhibitor concentration u(r,t=0) from model: u(r,t) [mass/mm 2 ] u threshold

23 Compare model prediction with experiment u(r,t) [mass/mm 2 ] u threshold Be er et al. PNAS (2009) 0.012

24 More than starvation or inhibition Bacteria from b grow in a rich nutrient broth, but bacteria from a don t grow at all they are dead. b a

25 What kills the bacteria?

26 Extract and analyze material from gel between two competing colonies

27 Gel electrophoresis of material extracted from gel between two growing colonies 2 colonies reference 1 reference 40 kda flagellin subtilisin 30 kda [Da (Dalton) is an atomic mass unit] 20 kda 15 kda XXX? (12 kda) 10 kda

28 Effect of subtilisin on a single colony Initially promotes growth, then growth is inhibited 0 h 10 h 20 h subtilisin (0.2% solution) introduced 1 day after bacterial inoculation 30 h 40 h extract gel for analysis

29 Analyze gel extract after introducing subtilisin near a single growing colony 1 colony flagellin subtilisin colony + subtilisin reference markers increasing molecular weight 17 kda XXX 14 kda 11 kda 8 kda XXX is present only when subtilisin concentration exceeds a threshold 6 kda

30 Isolate XXX and determine its effect on growth of a single colony lysed (ruptured) cells XXX 5 mm

31 Analyze XXX: It is a protein, but it is not listed in the Protein Data Bank. name it Slf --- Sibling lethal factor Be er et al., PNAS (2010)

32 The genome of Paenebacillus dentritiformis bacteria has previously been sequenced. Therefore, the gene that codes for our protein can be identified. However, the protein that the gene encodes has Molecular Weight 20.4 kda, not 12 kda (??) Name the gene dfsb, dendritiformis sibling Bacteriocin, and the 20.4 kda protein DfsB Surprisingly, DfsB has no effect on a P. dentriformis bacterial colony

33 Amino acid sequence of the 20.4 kda protein DfsB Large letters (positions 5-169): amino acid sequence associated with a conserved Pfam family domain, which occurs in many bacteria, but previously was of unknown function.

34 Amino acid sequence of the 20.4 kda protein DfsB subtilisin cleaves here Slf (12 kda) Large letters (positions 5-169): amino acid sequence associated with a conserved Pfam family domain, which occurs in many bacteria, but previously was of unknown function.

35 A better model Gil Ariel Scalar concentration fields: b(r,t): bacteria p(r,t): pre-spores n(r,t): nutrients s(r,t): subtilisin x(r,t): Slf

36 Model results: regulatory mechanism subtilisin and Slf concentration at the colony s moving front subtilisin threshold subtilisin: 2 colonies subtilisin: 1 colony Slf Bb Be er et al. PNAS (2010) front position (cm) 0.4 0

37 Look inside the colony dead rod-shaped bacterium TEM log 10 (# bacteria in 250x250 μm 2 ) RODS 1 μm

38 Transition: rods to spheres ( cocci ) a new Paenibacillus dentritiformis morphotype Be er et al. mbio (2011) coccus 0.8 μm log 10 (# bacteria in 250x250 μm 2 ) COCCI RODS TEM Conclude: at low Slf, rods switch to cocci, which are not affected by Slf zone

39 Cocci and rods are genetically the same colony of cocci colony of rods 5 μm

40 Cocci colonies grow even with Slf present, but growth is slower than growth of rods Cocci colonies are circular, not fractal. Slf

41 Cocci colony after 48 hours grown at high nutrient level colony edge agar 100 μm

42 10 μm rods agar at 54 hours: rods form at the edge of the colony Be er et al. mbio (2011) cocci colony

43 Later: most cocci have switched to rods 10 μm

44 Neighboring bacterial colonies Transition from growth to deadly sibling competition Deadliness due to protein Slf, a narrow spectrum bacteriocin (first in a new class of antibacterial proteins?) Slf Slf Survival by a transition a reversible switch to a new form (3 forms: WT rods, cocci, spores)