Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Inherited chitinases enable sustained growth and rapid dispersal of bacteria from chitin particles


Many biogeochemical functions involve bacteria utilizing solid substrates. However, little is known about the coordination of bacterial growth with the kinetics of attachment to and detachment from such substrates. In this quantitative study of Vibrio sp. 1A01 growing on chitin particles, we reveal the heterogeneous nature of the exponentially growing culture comprising two co-existing subpopulations: a minority replicating on chitin particles and a non-replicating majority which was planktonic. This partition resulted from a high rate of cell detachment from particles. Despite high detachment, sustained exponential growth of cells on particles was enabled by the enrichment of extracellular chitinases excreted and left behind by detached cells. The ‘inheritance’ of these chitinases sustains the colonizing subpopulation despite its reduced density. This simple mechanism helps to circumvent a trade-off between growth and dispersal, allowing particle-associated marine heterotrophs to explore new habitats without compromising their fitness on the habitat they have already colonized.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Steady-state growth of two subpopulations on chitin particles.
Fig. 2: Planktonic cells are not replicating.
Fig. 3: Chitinase activity and the resulting carbon flow.
Fig. 4: Chitinase expression and proteome allocation.
Fig. 5: Enzyme secretion is coupled to high dispersal.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium52 via the UCSD MassIVE partner repository with the dataset identifier PXD034003. Summary tables including both absolute and relative quantitation as well as functional grouping can be found in Supplementary Tables 2 and 3. All other data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.


  1. Monod, J. The growth of bacterial cultures. Annu. Rev. Microbiol. 3, 371–394 (1949).

    CAS  Google Scholar 

  2. Nguyen, T. T. H. et al. Microbes contribute to setting the ocean carbon flux by altering the fate of sinking particulates. Nat. Commun. 13, 1657 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Yawata, Y. et al. Competition–dispersal tradeoff ecologically differentiates recently speciated marine bacterioplankton populations. Proc. Natl Acad. Sci. USA 111, 5622–5627 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Yawata, Y., Carrara, F., Menolascina, F. & Stocker, R. Constrained optimal foraging by marine bacterioplankton on particulate organic matter. Proc. Natl Acad. Sci. USA 117, 25571–25579 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Fernandez, V. I., Yawata, Y. & Stocker, R. A foraging mandala for aquatic microorganisms. ISME J. 13, 563–575 (2019).

    PubMed  Google Scholar 

  6. Pomeroy, L. R. The ocean’s food web, a changing paradigm. BioScience 24, 499–504 (1974).

    Google Scholar 

  7. Alldredge, A. L. & Gotschalk, C. C. The relative contribution of marine snow of different origins to biological processes in coastal waters. Cont. Shelf Res. 10, 41–58 (1990).

    Google Scholar 

  8. Kiørboe, T. & Jackson, G. A. Marine snow, organic solute plumes, and optimal chemosensory behavior of bacteria. Limnol. Oceanogr. 46, 1309–1318 (2001).

    Google Scholar 

  9. Gasol, J. M. & Kirchman, D. L. Microbial Ecology of the Oceans (John Wiley & Sons, 2018).

  10. Azam, F. & Malfatti, F. Microbial structuring of marine ecosystems. Nat. Rev. Microbiol. 5, 782–791 (2007).

    CAS  PubMed  Google Scholar 

  11. Souza, C. P., Almeida, B. C., Colwell, R. R. & Rivera, I. N. G. The importance of chitin in the marine environment. Mar. Biotechnol. 13, 823–830 (2011).

    Google Scholar 

  12. Grimes, D. J. et al. What genomic sequence information has revealed about Vibrio ecology in the ocean—a review. Microb. Ecol. 58, 447–460 (2009).

    CAS  PubMed  Google Scholar 

  13. Jollès, P. & Muzzarelli, R. A. A. Chitin and Chitinases (Birkhäuser Verlag, 1999).

  14. Muzzarelli, R. A. A., Gooday, G. W. & Jeuniaux, C. Chitin in Nature and Technology (Plenum Press, 1986).

  15. Li, X. & Roseman, S. The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase. Proc. Natl Acad. Sci. USA 101, 627–631 (2004).

    CAS  PubMed  Google Scholar 

  16. Meibom, K. L. et al. The Vibrio cholerae chitin utilization program. Proc. Natl Acad. Sci. USA 101, 2524–2529 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Bassler, B. L., Yu, C., Lee, Y. C. & Roseman, S. Chitin utilization by marine bacteria. Degradation and catabolism of chitin oligosaccharides by Vibrio furnissii. J. Biol. Chem. 266, 24276–24286 (1991).

    CAS  PubMed  Google Scholar 

  18. Pruzzo, C., Crippa, A., Bertone, S., Pane, L. & Carli, A. Attachment of Vibrio alginolyticus to chitin mediated by chitin-binding proteins. Microbiology 142, 2181–2186 (1996).

    CAS  PubMed  Google Scholar 

  19. Svitil, A. L., Chadhain, S., Moore, J. A. & Kirchman, D. L. Chitin degradation proteins produced by the marine bacterium Vibrio harveyi growing on different forms of chitin. Appl. Environ. Microbiol. 63, 408–413 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Suzuki, K. et al. Chitinases A, B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation. Biosci. Biotechnol. Biochem. 66, 1075–1083 (2002).

    CAS  PubMed  Google Scholar 

  21. Chuang, H.-H. & Lin, F.-P. New role of C-terminal 30 amino acids on the insoluble chitin hydrolysis in actively engineered chitinase from Vibrio parahaemolyticus. Appl. Microbiol. Biotechnol. 76, 123–133 (2007).

    CAS  PubMed  Google Scholar 

  22. Svitil, A. L. & Kirchman, D. L. Y. A chitin-binding domain in a marine bacterial chitinase and other microbial chitinases: implications for the ecology and evolution of 1,4-β-glycanases. Microbiology 144, 1299–1308 (1998).

    CAS  PubMed  Google Scholar 

  23. Davidi, D. et al. Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro kcat measurements. Proc. Natl Acad. Sci. USA 113, 3401–3406 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Belas, M. R. & Colwell, R. R. Adsorption kinetics of laterally and polarly flagellated Vibrio. J. Bacteriol. 151, 1568–1580 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Nalin, D. R., Daya, V., Reid, A., Levine, M. M. & Cisneros, L. Adsorption and growth of Vibrio cholerae on chitin. Infect. Immun. 25, 768–770 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Kaneko, T. & Colwell, R. R. Adsorption of Vibrio parahaemolyticus onto chitin and copepods. Appl. Microbiol. 29, 269–274 (1975).

    PubMed  PubMed Central  Google Scholar 

  27. Kiørboe, T., Tang, K., Grossart, H.-P. & Ploug, H. Dynamics of microbial communities on marine snow aggregates: colonization, growth, detachment, and grazing mortality of attached bacteria. Appl. Environ. Microbiol. 69, 3036–3047 (2003).

    PubMed  PubMed Central  Google Scholar 

  28. Bassler, B., Gibbons, P. & Roseman, S. Chemotaxis to chitin oligosaccharides by Vibrio furnissii, a chitinivorous marine bacterium. Biochem. Biophys. Res. Commun. 161, 1172–1176 (1989).

    CAS  PubMed  Google Scholar 

  29. Bassler, B. L., Gibbons, P. J., Yu, C. & Roseman, S. Chitin utilization by marine bacteria. Chemotaxis to chitin oligosaccharides by Vibrio furnissii. J. Biol. Chem. 266, 24268–24275 (1991).

    CAS  PubMed  Google Scholar 

  30. Datta, M. S., Sliwerska, E., Gore, J., Polz, M. F. & Cordero, O. X. Microbial interactions lead to rapid micro-scale successions on model marine particles. Nat. Commun. 7, 11965 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Karp, P. D. et al. The BioCyc collection of microbial genomes and metabolic pathways. Brief. Bioinform. 20, 1085–1093 (2019).

    CAS  PubMed  Google Scholar 

  32. Hayes, C. A., Dalia, T. N. & Dalia, A. B. Systematic genetic dissection of chitin degradation and uptake in Vibrio cholerae: genetic dissection of chitinases in V. cholerae. Environ. Microbiol. 19, 4154–4163 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Leventhal, G. E., Ackermann, M. & Schiessl, K. T. Why microbes secrete molecules to modify their environment: the case of iron-chelating siderophores. J. R. Soc. Interface 16, 20180674 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Aunkham, A. et al. Structural basis for chitin acquisition by marine Vibrio species. Nat. Commun. 9, 220 (2018).

    PubMed  PubMed Central  Google Scholar 

  35. Maaløe, O. in Biological Regulation and Development: Gene Expression (ed. Goldberger, R. F.) 487–542 (Springer, 1979).

  36. Scott, M., Gunderson, C. W., Mateescu, E. M., Zhang, Z. & Hwa, T. Interdependence of cell growth and gene expression: origins and consequences. Science 330, 1099–1102 (2010).

    CAS  PubMed  Google Scholar 

  37. Neidhardt, F. C. & Magasanik, B. Studies on the role of ribonucleic acid in the growth of bacteria. Biochim. Biophys. Acta 42, 99–116 (1960).

    CAS  PubMed  Google Scholar 

  38. Hui, S. et al. Quantitative proteomic analysis reveals a simple strategy of global resource allocation in bacteria. Mol. Syst. Biol. 11, 784 (2015).

    PubMed  PubMed Central  Google Scholar 

  39. Mori, M. et al. From coarse to fine: the absolute Escherichia coli proteome under diverse growth conditions. Mol. Syst. Biol. 17, e9536 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Itoh, T. et al. Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7. Appl. Environ. Microbiol. 79, 7482–7490 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Itoh, T. et al. Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains. Biosci. Biotechnol. Biochem. 78, 624–634 (2014).

    CAS  PubMed  Google Scholar 

  42. Itoh, T. et al. Crystal structure of chitinase ChiW from Paenibacillus sp. str. FPU-7 reveals a novel type of bacterial cell-surface-expressed multi-modular enzyme machinery. PLoS ONE 11, e0167310 (2016).

    PubMed  PubMed Central  Google Scholar 

  43. Amarnath, K. et al. Stress-induced metabolic exchanges between complementary bacterial types underly a dynamic mechanism of inter-species stress resistance. Nat. Commun. 14, 3165 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Jansson, M. Phosphate uptake and utilization by bacteria and algae. Hydrobiologia 170, 177–189 (1988).

    CAS  Google Scholar 

  45. Herbert, D., Phipps, P. J. & Strange, R. E. in Methods in Microbiology Vol. 5 (eds Norris, J. R. & Ribbons, D. W.) 209–344 (Academic Press, 1971).

  46. You, C. et al. Coordination of bacterial proteome with metabolism by cyclic AMP signalling. Nature 500, 301–306 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Benthin, S., Nielsen, J. & Villadsen, J. A simple and reliable method for the determination of cellular RNA content. Biotechnol. Tech. 5, 39–42 (1991).

    CAS  Google Scholar 

  48. Balakrishnan, R. et al. Principles of gene regulation quantitatively connect DNA to RNA and proteins in bacteria. Science 378, eabk2066 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Cremer, J., Arnoldini, M. & Hwa, T. Effect of water flow and chemical environment on microbiota growth and composition in the human colon. Proc. Nat Acad. Sci. 114, 6438–6443 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Dai, X. et al. Reduction of translating ribosomes enables Escherichia coli to maintain elongation rates during slow growth. Nat. Microbiol. 2, 16231 (2017).

    CAS  Google Scholar 

  51. Lam, H. et al. Building consensus spectral libraries for peptide identification in proteomics. Nat. Methods 5, 873–875 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Vizcaíno, J. A. et al. ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat. Biotechnol. 32, 223–2266 (2014).

    PubMed  PubMed Central  Google Scholar 

Download references


We thank J. Schwartzman and O. Cordero for providing strains and helpful discussions, F. Azam for extremely informative general discussions and members of the Hwa research group for helpful input and feedback. We also commemorate Alma Dal Co, the review of this work being one of the last scientific activities of her short but brilliant career. This work was supported by the Simons Foundation through the Principles of Microbial Ecosystems (PriME) collaboration (Grant no. 542387 to T.H. and 542395 to J.R.W.). Illustrations were created using

Author information

Authors and Affiliations



T.H. and G.G. conceived the study, designed experiments and wrote the manuscript. G.G. conducted all experiments and performed modelling work. R.B. designed, optimized protocols, conducted radioactivity experiments and analysed data. V.P. processed proteomics samples, designed data processing pipelines and analysed data with guidance from J.R.W. T.C. contributed to modelling work. All authors contributed to editing the manuscript.

Corresponding author

Correspondence to Terence Hwa.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Microbiology thanks Alvaro Sanchez, Vaibhhav Sinha, Alma Dal Co and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Quantifying the RNA abundance of cells grown in different conditions.

a) Growth curves of 1A01 grown in minimal media with different carbon sources. Cultures were inoculated from exponentially growing pre-cultures with the same carbon source. The replication rates (r) were obtained from the exponential fits and ranged from 1.15 h−1 to 0.26 h−1; see Supplementary Table 2 for values. b) RNA amounts per OD\•mL of culture, R/ρ, was obtained for exponentially growing cultures in different carbon sources and plotted against the respective replication rates, r (same symbols as Panel a). The RNA content exhibits a linear dependence on the replication rate r as indicated by the line of best-fit given in the plot. In the main text, we determined that the replication rate of particle-associated cells and planktonic cells are respectively rs ≈ 0.26 h−1 and rb ≈ 0. The best-fit line then allows us to deduce \({R}_{s}:{\rho }_{s}\approx 60\,\mu g/(OD\cdot mL)\) and \({R}_{b}:{\rho }_{b}\approx 50\,\mu g/(OD\cdot mL)\) for these two subpopulations of cells. Together with the result Rb: Rtotal ≈ 0.72 from Fig. 1d, we obtain ρb: ρtotal ≈ 0.75 ± 0.06. c) Direct measurement of RNA content of the different subpopulations of the chitin culture. RNA concentration was measured for samples of the full culture, planktonic cells and on particles only using perchloric acid precipitation. We observe that the planktonic samples (blue diamonds) were consistently below the full samples (black circles) and similarly to Fig. 1c, we interpret this difference as the biomass accumulating on the surface of the particles. Moreover, the linear increase of RNA concentration with OD in the three samples indicates again that the culture has reached a steady-state. The data points represent the mean value measured and the error bars correspond to the standard deviation across three biological replicates. d) To determine the planktonic fraction of the culture (blue diamonds), we took the ratio of the blue diamonds to the black circles in Panel c. The error bars are propagated from the standard deviations of the measurements obtained with three biological replicates in panel c. This fraction remained constant throughout growth, further establishing that the chitin culture was in a steady-state. The data provides another estimate of the fraction of RNA in the planktonic subpopulation, with Rb: Rtotal ≈ 0.82 ± 0.1, which is comparable to the estimate obtained in Fig. 1d using radio-labeling. Using the RNA content described in Panel b, the fraction of the planktonic subpopulation is estimated to be ρb: ρtotal ≈ 0.8 ± 0.1, consistent with the estimate of ρb: ρtotal ≈ 0.75 ± 0.16, given above.

Source data

Extended Data Fig. 2 Detecting the dynamics of GlcNAc in the media.

a) Standard curve showing the detection limit of GlcNAc using HPLC. Several high dilutions of GlcNAc, ranging from 10−2 μM were prepared in the minimal media. The samples were passed through a hydrophobic column equipped with a refractive index detector (see HPLC in Methods). Peaks corresponding to GlcNAc eluted between 19.8 and 20.5 minutes, and the total signal area under the curve was determined. The signal area scaled linearly with GlcNAc concentration at the input all the way down to 2 μM, below which peaks were not detected. Analyzing the supernatant of a growing chitin culture with the same method, yielded an absence of measurable peak, indicated the GlcNAc concentration in the supernatant is below 2 μM, the detection limit of the HPLC. b) Illustration of the dialysis setup in Fig. 2c-d. Chitin particles were separated from planktonic cells in the middle of exponential growth. Chitin particles were resuspended in the same volume of fresh media without particles and placed inside a dialysis bag with a 14kDa molecular weight cutoff. This pore size means that small molecules such as GlcNAc can exchange but not enzymes, cells nor chitin particles. The dialysis bag was inserted into a flask and the planktonic cells were transferred to the flask, to the exterior of the bag, with equal volumes inside and outside of the dialysis bag. Outside of the dialysis bag, the OD was tracked regularly over a time period of 30 hours, while inside of the bag only the initial and final ODs were recorded due to the difficulty of handling the dialysis tubing. c) 10mL of cell-less media with 20 μM of GlcNAc was placed in a dialysis bag and incubated with the same volume of media without GlcNAc outside of the bag. The concentrations start equilibrating immediately after the start of the experiment, and reach their equilibrium value by 1h. This indicates that with the full chitin culture in Fig. 2c-d, a low concentration of GlcNAc generated inside the bag can freely exchange between the interior and exterior of the bag if not taken up by cells inside the bag.

Extended Data Fig. 3 Instantaneous RNA synthesis rate measurements in the chitin culture.

From an exponentially increasing chitin culture at \(O{D}_{600}^{plank}\approx 0.3\), samples of the full culture (including chitin particles and planktonic cells) were compared to samples containing planktonic cells only. The instantaneous RNA synthesis rate, a proxy for the cells’ metabolic rate, was measured in both samples by pulsing 3H-Uridine and immediately tracking its incorporation into the total culture and planktonic culture (see RNA synthesis rate in Methods). The plot shows the radioactivity reading as a function of time. The solid lines are linear best fits to the data and their slope represents the metabolic rate in each sample. Specifically let Atotal = rsρs + rbρb be the metabolic rate of the full culture and Ab = rbρb be the metabolic rate of the planktonic fraction. The result Atotal/Ab ≈ 12 indicates that the particle-associated cells were the main contributor to RNA synthesis (and hence cell replication) even though they comprised a minority of the biomass as was determined in Fig. 1 and Extended Data Fig. 1. The replication rates on and off the particles can be compared by taking the ratio Atotal/Ab and expressing rb given its contribution to the total biomass: \({r}_{b}=({r}_{s}\,({\rho }_{total}/{\rho }_{b}\,-1))/({A}_{total}/{A}_{b}\,-1)={r}_{s}/33\). Given the estimate of rs = 0.26 h−1 (main text), this indicates that \({r}_{b}\approx 0.008\,{h}^{-1}\ll \lambda\), making rb practically negligible.

Extended Data Fig. 4 Microscopy images of chitin particles from growing cultures.

Samples were incubated with two fluorescent dyes: FITC-WGA, here shown in the green channel and FM 4-64, a cell membrane dye, shown in the red channel, and fixed with glutaraldehyde. Imaging of the samples was done in custom-built chambers (see microscopy in Methods) under a confocal microscope with 40x magnification. Sample representative images were chosen but microscopy sessions were performed to image 10-50 particles per sample, which all showed similar colonization trends. a) Sterile chitin flake incubated with both dyes. FITC-WGA specifically binds to chitin particles, though some FM 4-64 can also be seen in the background. b) Chitin particles were isolated from an exponentially increasing chitin culture at \(O{D}_{600}^{plank}=0.04\) and resuspended in fresh media. Red (membrane dyed) cells can be seen to bind to chitin and form microcolonies. c) A sample including both planktonic cells and chitin particles from an exponentially increasing chitin culture at \(O{D}_{600}^{plank}=0.4\). Consistent with bulk measurements, more planktonic cells are observed than surface-associated cells. d) From the same growing culture as in Panel c, chitin particles were isolated and resuspended in fresh media to remove planktonic cells. In both c) and d), the surface of particles is not saturated with cells even at this moderately high OD. This suggests that cell detachment from particles is not a result of a ‘space limitation’ on the particle surface.

Extended Data Fig. 5 1A01 has a dispersal lifestyle.

a) Chitin particles pre-colonized with cells (green cells) were isolated at various time (τ) from an exponentially increasing chitin culture, and resuspended in the same volume of fresh media without chitin particles. The amount of particle-associated cells was estimated using the planktonic OD at the time of particle isolation ρb(τ) and the constant planktonic fraction ρb: ρtotal obtained in Fig. 1, that is, \({\rho }_{s}(\tau )=(\frac{{\rho }_{total}}{{\rho }_{b}}-1){\rho }_{b}(\tau )\). This is taken as the initial cell density \({\rho }_{s}^{init}\) of the resuspended culture. For each resuspended culture, the planktonic OD (Δρb, giving the density of blue cells) was measured at regular time intervals Δt. The increase in planktonic OD is expected to be linear in time with a rate kd for some time after resuspension. b) Inset: the planktonic OD for one such resuspensions increases linearly starting from a background reading ρ0, due to the turbidity caused by small chitin particles. The dashed line is the line of best fit to the data with a slope \({k}_{d}{\rho }_{s}^{init}\). Main: Traces of the planktonic OD (Δρb) were normalized to the initial amount of cells on the particles (\({\rho }_{s}^{init}\)) and plotted as a function of time. The normalized traces collapse on top of one another. The slope of the line of best fit (dashed line) allows an estimate of the detachment rate, with kd ≈ 0.18 ± 0.02h−1, with the error given by the 95% confidence interval of the fit. c) Similarly to Panel a, pre-colonized chitin particles from a 10mL exponentially increasing chitin culture were separated and re-suspended into the same volume of fresh media without chitin particles. After a time interval of Δt = 2h, the planktonic OD increased by an amount Δρb as a result of cell detachment and with the rate observed Panel b. At this point, the particles were sedimented, and resuspended in the same volume of fresh media without chitin particles. This process, which effectively removed all the planktonic cells and thereby prevented reattachment (effectively setting the attachment rate ka ≈ 0), was repeated 8 times, for a total of 16 hours. d) Traces of the planktonic OD as a function of time for successive resuspension cycles. From an initial OD measurement ρ0, which is the background OD due to the residual turbidity of small chitin particles, we observed an increase in the planktonic OD, Δρb, after the 2h time cycle. The background OD exhibits a time dependence ρ0(t) due to the successive removal of small particles in the fractionation process. The data shows that in the absence of planktonic cells, the particle-associated cells alone were able to sustain replication and shedding of new planktonic cells in the duration of our experiments, demonstrating that the planktonic culture is not necessary for the replication of cells on the particles. e) From the traces in Panel b, we plot the planktonic increase relative to the background OD as a function of time. The normalization by the dynamic background OD allows to adjust for the loss of small particles and hence of surface-associated cells from the culture during the sedimentation process. The dashed line is obtained by fitting an exponential model to the data, with the growth exponent being 0.065 h−1. Since the cell detachment rate is proportional to the number of cells on particles, the relative increase in planktonic OD, Δρb(t)/ρ0(t), is thus a proxy for the increase in cell density on the particles. We see that this relative increase in planktonic OD is exponential, and that its rate matches the exponential rate measured in an undisturbed chitin culture, λ ≈ 0.06 h−1 (Fig. 1). Since the rate of exponential increase in the planktonic population is hardly affected by the removal of planktonic cells from the chitin culture, we conclude from the data that the attachment rate \({k}_{a}\ll \lambda {\rho }_{s}/{\rho }_{b}=0.02{h}^{-1}\) is thus negligible compared to other rates in our culture.

Source data

Extended Data Fig. 6 Chitinase activity and properties.

a, b) SDS-PAGE gels were run to separate and identify differences in the proteomes of the three fractions of the chitin culture (planktonic cells, particles, and medium after filtering out planktonic cells). To identify the proteins in each band, the bands were excised, solubilized and analyzed using mass spectrometry for identification; see SDS-PAGE in Methods. Bands corresponding to chitinases (with molecular weight ~100kDa) are indicated on the images. Gel image: light intensity and contrast were adjusted. Original gel images are available upon request. Representative gel images were shown but at least three biological replicates were performed for each fraction. a) Samples were collected from a steady-state chitin culture at \(O{D}_{600}^{plank}=0.5,\,0.6\) (from left to right). 100 mL of culture was passed through a 0.22 μm filter, and the filtrate was concentrated 100-fold using 3kDa concentrators (see fractionation in Methods). The resulting protein mixture was loaded onto the gel. Chitinases are found in the supernatant. See Extended Data Table 1 for the amount quantified. Other unrelated lanes on the same gel were cropped out of the image shown. b) Throughout a steady-state chitin culture at multiple planktonic ODs, (at \(O{D}_{600}^{plank}=0.18,\,0.51\,{\rm{and}}\,0.6\) respectively from the left to the right lane), samples were collected and particles separated from planktonic cells. Particles were concentrated 4-fold compared to the volume of planktonic cells to make sure loading amounts were in the same range. After boiling and reducing the proteins (see SDS-PAGE in Methods), they were loaded onto the gel. From the band intensities, it is clear visually that the chitinases are enriched on the particles. This observation is quantitatively confirmed by mass spectrometry, which yields a 5-fold enrichment of chitinases per cell on the particles compared to planktonic cells (see Extended Data Table 1). Gel image: three unrelated lanes between the ladder and the particle samples shown were cropped out from the original image. c-d) Proteins from the supernatant of a steady-state chitin culture were collected at \(O{D}_{600}^{plank}\approx 0.5\) and concentrated using the method described above. They were then incubated with fresh chitin flakes and the activity of the enzymes was determined by measuring the GlcNAc concentration in the supernatant at different times using HPLC. c) To test the stability of the enzymes, they were resuspended in the same buffer used in our chitin culture. The activity of the enzymes was assayed for similar amounts immediately after their collection (circles) and after being incubated in the same shaking conditions as our culture at 27 °C for 24 hours (squares). This procedure resulted in the same rate of GlcNAc accumulation indicating that the enzymes were stable for the duration assayed. d) Because we used chloroform as a way of disabling nutrient uptake in our in situ measurements of enzymatic activity (Fig. 4 and Panel e), we independently tested the effect of chloroform on the absolute activity of the chitinases by incubating similar amounts of supernatant enzymes with chitin particles in our culture buffer with and without the addition of chloroform. For each case, we measured the concentration of GlcNAc in the media at regular time intervals (filled and open circles, respectively). The dashed lines represent the linear curves of best fit for the data and the best-fit parameters are indicated on the plots. The ratio of the slopes of the two lines was about 3.3, indicating that the activity of the chitinases enzymes was reduced ~3.3-fold due to chloroform treatment. e) Similarly to Fig. 3c, we tracked the accumulation of GlcNAc after treating exponentially growing chitin cultures with chloroform to inhibit carbon uptake by cells in the culture. In this case, GlcNAc accumulation was followed over a longer timescale (3 days) to assess possible changes in chitinase activity, through observing changes in the rate of GlcNAc accumulation. Samples were collected at planktonic ODs of 0.16 (open triangles) and 0.63 (filled circles). The resulting GlcNAc accumulation traces were normalized to the planktonic OD at sampling and we found that the traces collapsed onto each other after normalizing by the initial OD. The reaction was kept at 27 °C and shaking throughout the course of our measurement. The longer timescale of this experiment showed that after 15 hours, there was a change of rate in the accumulation of the GlcNAc concentration (compare dashed to dotted green lines). The second rate was thereafter maintained for ~3 days. This is incompatible with a gradual degradation of chitinases but rather suggests that the system finds a new equilibrium due to the exchange dynamics (attachment and detachment) of the enzymes. d) To interpret the data in Panel e) and extract the value of the relevant parameters, we formulate the following model governing the GlcNAc accumulation rate. Surface-attached enzymes (εs) which produce GlcNAc with a catalytic rate kE as in Fig. 3 are exchanged with the bulk enzymes (εb) with attachment and detachment rates respectively \({k}_{a}^{{\prime} }\) and \({k}_{d}^{{\prime} }\). We plot the best-fit solution for the traces as the solid black line. The full solution of this model can be found in Supplementary Note II. Here we briefly summarize the results: Initially, as explained in Fig. 4b–d, the rate of GlcNAc accumulation is proportional to the enzyme’s catalytic rate \({s}_{1}={\kappa }_{E}{m}_{E}{\varepsilon }_{s}\). At longer times the slope decreases and corresponds to the equilibration of attached and detached enzymes, with \({s}_{2}={{\rm{\kappa }}}_{{\rm{E}}}{m}_{E}({\varepsilon }_{s}+{\varepsilon }_{b})\,{k}_{a}^{{\prime} }/({k}_{a}^{{\prime} }+{k}_{d}^{{\prime} })\). The ratio of the two slopes is therefore: \({s}_{2}:{s}_{1}=({\varepsilon }_{s}+{\varepsilon }_{b}){k}_{a}^{{\prime} }/[{\varepsilon }_{s}({k}_{a}^{{\prime} }+{k}_{d}^{{\prime} })]\). The change from one regime to the other occurs at a point \(\tau ={({k}_{a}^{{\prime} }+{k}_{d}^{{\prime} })}^{-1}\). Thus, with the empirical values of the quantities s2: s1 and τ obtained from the data in panel a, as well as the ratio εb: εs obtained from Extended Data Table 1 (ratio of the third to the first entry in the last row), we can determine the attachment and detachment rates \({k}_{a}^{{\prime} }\) and \({k}_{d}^{{\prime} }\), with \({k}_{a}^{{\prime} }=0.02\,{h}^{-1}\) and \({k}_{d}^{{\prime} }=0.04\,{h}^{-1}\).

Source data

Extended Data Fig. 7 Analysis of proteomic data.

a) Specific examples of proteins whose abundances increased strongly on carbon sources resulting in poor growth. The mass fraction is obtained as the fractional spectral count of these individual proteins in each sample. The x-axis represents the growth rate of the culture under carbon sources of varying quality: Glucose (0.81 h−1), GlcNAc (0.8 h−1), Glucosamine (0.57 h−1), Succinate (0.34 h−1) and Galactose (0.25 h−1). The data in the shaded bar show the mass fraction of these proteins on chitin particles (excluding the chitinases). The abundance of these proteins is more comparable to that on slow growth in poor carbon sources than on fast growth. b) Analysis of individual ribosomal proteins. For each ribosomal protein, a linear curve was fitted for its abundance as a function of growth rate using the series described in panel A with varying carbon quality. This linear fit was used as a predictor of the growth rate of surface-associated cells depending on the abundance of individual ribosomal proteins on the particles subtracted of chitinases. The histogram (blue bars) shows the result of the predicted growth rates of surface-associated cells based on each of the 53 ribosomal proteins detected. They yield an average predicted growth rate of 0.23h−1 (dashed vertical line) with the median being even lower, 0.1 h−1, (dotted vertical line). c-d) Scatter plots of the relative abundances of proteins from surface-associated cells and cells grown on c) glucose and d) galactose. Each data point represents the abundance of a protein relative to our 15N labelled standard in both conditions examined. Our standard was composed of a mixture of cells extracted from a fast (glucose) and a medium (succinate) growing condition and injected in equal amount into all of our samples to allow for comparisons between them (see media recipe in Methods). Stronger correlation (0.23) is seen between the proteome of surface-associated cells and cells grown on galactose (poor carbon source giving growth rate of 0.25 h−1), while weaker correlation (0.007) is seen between surface-associated cells and cells grown on glucose (good carbon source giving growth rate of 0.81 h−1). e) The correlation coefficient is calculated between the proteome of cells grown in each of the carbon sources studied and that of particle-associated cells as done in panels c and d except for GlcNAc to avoid biases due to the substrate’s nature. Plotting the correlation coefficient with the growth rate of the corresponding carbon source, we see that higher correlation is progressively obtained for slower carbon sources, suggesting that particle-associated cells are carbon-limited.

Extended Data Fig. 8 Possible environmental factors favoring or disfavoring dispersal.

Diverse scenarios are presented favoring either the colonizing or the dispersing lifestyle. Briefly, in the colonizing lifestyle, cells do not detach from particles until they are fully consumed, after which all cells are released in a ‘burst’. In this case, the ‘bursted’ population would all die unless one of the cells colonized a fresh particle during the cell lifetime following the burst. In the dispersing lifestyle, a fraction of the population is continuously shed as the particle is being degraded. This allows for cells to seed fresh particles at any time before the particle is fully consumed. For each scenario, the frames from left to right illustrate possible dynamics of the population on and surrounding the particle being degraded. Filled symbols represent colonized particles and the green shade represents planktonic cells. Open symbols represent fresh particles. Diamonds represent a different solid substrate supporting faster growth. a) The vertical direction represents depth from the water surface. High detachment rates allow the population to maintain itself along the water column and avoid inhospitable depths where oxygen and other factors may become limiting for growth. b) A high detachment rate allows the seeding of a particle made of a different substrate (red diamond) providing faster growth. This can be beneficial for certain Vibrio sp. species that favor, for example, alginate50,51. However, we remark that if detachment reduces the fitness of the population on the existing niche as commonly believed, then exploring alternative resource patches would be more costly. c) In situations where the particle density in the environment is low and thus the encounter rate is also low, a high dispersal rate increases the probability of encountering a fresh particle during the ‘lifetime’ of a colonized particle. If seeding itself is not difficult, this increases the probability of encounter for dispersing cells. On the other hand, if successful seeding is a low probability event while encountering fresh particles is not, then ‘bursting’ would be more advantageous given the larger number of cells released at burst. This may arise from the requirement on the absolute numbers of cells for the successful colonization of new particles2, or due to Allee effects arising from other mechanisms.

Extended Data Fig. 9 Cellular and environmental factors determining growth and dispersal.

For bacteria such as Vibrio sp. 1A01 which release their secreted chitinases, the rate of increase of the population colonized on chitin particles, λ, is decoupled from the rate of detachment from the particles, kd, circumventing the commonly held trade-off between growth and dispersal, illustrated by the dashed grey line. Instead, growth and dispersal can be separately set molecularly (red arrows), with the population increase rate λ set by φE, the allocation towards chitinase synthesis (equation 6). The two molecular parameters kd and φE are in turn dependent on environmental factors such that the population-level fitness, which involves both growth and successful colonization of many particles, increases over long timescales. We speculate on a number of such environmental factors, represented by the blue arrows. Examples of factors favoring detachment are illustrated in Extended Data Fig. 8. Factors setting the rate of increase of the colonized population may include the rate of particle influx and the rate of mortality on particles (due to grazing, phage killing…) since the survival of the population requires it to grow above the rate of mortality, and as much as allowed by the overall nutrient influx, but not above it to avoid long periods of starvation. We remark that even with growth and dispersal decoupled, an anti-correlation between these two traits may arise if environmental factors favoring one factor also disfavors another. for example, a low particle density resulting from low particle influx would favor slow growth and high detachment.

Extended Data Table 1 Chitinase abundance in different components of the chitin culture

Supplementary information

Supplementary Information

Supplementary Table 1 and Notes (Chapters I through III) including embedded figure in Chapter I.

Reporting Summary

Supplementary Tables 2 and 3

2. Proteomics data. Summary of labelled and unlabelled spectral counts of Vibrio sp. 1A01 proteome growing in different nutrient conditions. 3. Summary of proteomics data by functional group. Proteome of Vibrio sp. 1A01 growing in different nutrient conditions, with relevant proteins grouped by their functions.

Supplementary Video 1

1A01 cells near a chitin particle. Vibrio sp. 1A01 cells and chitin particles were trapped in a sealed capillary tube. Cells are seen to rapidly attach to and detach from chitin particles as well as rapidly swim in the vicinity of the particles.

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Guessous, G., Patsalo, V., Balakrishnan, R. et al. Inherited chitinases enable sustained growth and rapid dispersal of bacteria from chitin particles. Nat Microbiol 8, 1695–1705 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing