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ABSTRACT Gas vesicles (GVs) are microbial protein organelles that support cellular buoyancy. GV engineering has multiple applications, including reporter gene imaging, acoustic control and

payload delivery. GVs often cluster into a honeycomb pattern to minimize occupancy of the cytosol. The underlying molecular mechanism and the influence on cellular physiology remain unknown.

Using genetic, biochemical and imaging approaches, here we identify GvpU from _Priestia megaterium_ as a protein that regulates GV clustering in vitro and upon expression in _Escherichia

coli_. GvpU binds to the C-terminal tail of the core GV shell protein and undergoes a phase transition to form clusters in subsaturated solution. These properties of GvpU tune GV clustering

and directly modulate bacterial fitness. GV variants can be designed with controllable sensitivity to GvpU-mediated clustering, enabling design of genetically tunable biosensors. Our

findings elucidate the molecular mechanisms and functional roles of GV clustering, enabling its programmability for biomedical applications. Access through your institution Buy or subscribe

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ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A CATALOG OF THE DIVERSITY AND

UBIQUITY OF BACTERIAL MICROCOMPARTMENTS Article Open access 21 June 2021 ACTIVE PARTICLES INDUCE LARGE SHAPE DEFORMATIONS IN GIANT LIPID VESICLES Article 30 September 2020 COMPOSITION AND

FUNCTIONS OF BACTERIAL MEMBRANE VESICLES Article 17 March 2023 DATA AVAILABILITY The supporting data for findings of this study are available as source data. Models of docking are available

at https://github.com/lab-of-george-lu/Li_2024_NMICROBIOL67. Raw data of disorder profile of GvpU and structure modelling of GvpU are available at

https://github.com/holehouse-lab/supportingdata/tree/master/2024/Li_202468. Macromolecular structural data are available from the RCSB Protein Data Bank (PDB). GvpAAna: PDB 8GBS. GvpA2Mega:

PDB 7R1C. Source data are provided with this paper. CODE AVAILABILITY The computational script used for generating disorder profile of GvpU is available at

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Authority at Rice University for the access to core facilities and instruments and W. Guo for the training. We thank the Thyer Lab at Rice University for providing template plasmids encoding

the spectinomycin resistance gene and the p15A and CloDF13 origins of replication. This work was supported by the Cancer Prevention and Research Institute of Texas, the National Institutes

of Health (R00 EB024600 and R21 EB033607), the Welch Foundation, G. Harold and Leila Y. Mathers Foundation, Hearing Health Foundation and John S. Dunn Foundation. M.I. acknowledges support

from German Research Foundation (DFG) Postdoctoral Fellowship; E.T.U. is supported by a W.M. Keck Fellowship, and Z.L. acknowledges support from Edgar O’Rear and Mary F.D. Morse Travel Award

from the Institute of Biosciences and Bioengineering at Rice University. This work was supported by the Air Force Office of Scientific Research (FA9550-20-1-0241 to L.Y. and A.C). AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Department of Bioengineering, Rice University, Houston, TX, USA Zongru Li, Qionghua Shen, Andrew P. Anderson, Manuel Iburg, Richard Lin, Brandon Zimmer

 & George J. Lu * Department of Biomedical Engineering, Center for Biomolecular Condensates, Washington University in St. Louis, Saint Louis, MO, USA Emery T. Usher, Alex S. Holehouse 

& Yifan Dai * Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, Saint Louis, MO, USA Emery T. Usher & Alex S. Holehouse * Shared Equipment

Authority, Rice University, Houston, TX, USA Matthew D. Meyer * Department of Biomedical Engineering, Duke University, Durham, NC, USA Lingchong You, Ashutosh Chilkoti & Yifan Dai *

Center for Quantitative BioDesign, Duke University, Durham, NC, USA Lingchong You Authors * Zongru Li View author publications You can also search for this author inPubMed Google Scholar *

Qionghua Shen View author publications You can also search for this author inPubMed Google Scholar * Emery T. Usher View author publications You can also search for this author inPubMed 

Google Scholar * Andrew P. Anderson View author publications You can also search for this author inPubMed Google Scholar * Manuel Iburg View author publications You can also search for this

author inPubMed Google Scholar * Richard Lin View author publications You can also search for this author inPubMed Google Scholar * Brandon Zimmer View author publications You can also

search for this author inPubMed Google Scholar * Matthew D. Meyer View author publications You can also search for this author inPubMed Google Scholar * Alex S. Holehouse View author

publications You can also search for this author inPubMed Google Scholar * Lingchong You View author publications You can also search for this author inPubMed Google Scholar * Ashutosh

Chilkoti View author publications You can also search for this author inPubMed Google Scholar * Yifan Dai View author publications You can also search for this author inPubMed Google Scholar

* George J. Lu View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conceptualization, G.J.L., A.C., L.Y., Z.L., A.S.H and Y.D.; methodology,

G.J.L., A.C., L.Y., Z.L., Q.S., E.T.U., A.S.H. and Y.D.; investigation, Z.L., Q.S., Y.D., A.S.H., E.T.U., A.P.A., M.I., R.L., B.Z. and M.D.M.; formal analysis, Z.L., Q.S., Y.D., A.S.H.,

E.T.U., A.P.A., M.I., R.L., B.Z. and M.D.M.; writing—original draft, G.J.L., A.C., L.Y., Z.L., Q.S., A.S.H. and Y.D.; supervision and funding acquisition, G.J.L., Y.D., A.C. and L.Y.

CORRESPONDING AUTHORS Correspondence to Lingchong You, Ashutosh Chilkoti, Yifan Dai or George J. Lu. ETHICS DECLARATIONS COMPETING INTERESTS Z.L., Q.S, and G.J.L. are co-inventors on a US

provisional patent application that incorporates discoveries described in this paper. Their interests are reviewed and managed by Rice University in accordance with their conflict of

interest policies. The other authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Microbiology_ thanks Jon Marles-Wright 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 MASS SPECTRUM OF GVPU::HIS6-TAG OBTAINED FROM MALDI TOF MS. The x-axis displays the mass-to-charge ratio (m/z) of the ionized

species, while the Y-axis represents the ion intensity. The presence of the peak with 15681.311 m/z in the spectrum confirms the identity of GvpU::His6-tag. Source data EXTENDED DATA FIG. 2

ALIGNMENT OF CRYO-EM STRUCTURES OF MAJOR SHELL PROTEIN GVPAANA AND GVPA2MEGA. GvpAAna is depicted in yellow and GvpA2Mega in grey. The secondary structures, adapted from cryo-EM structures

for GvpAAna (PDB: 8GBS)49 and GvpA2Mega (PDB: 7R1C)30, are segmented into the N-terminal (Nt) and C-terminal (Ct) regions, two α-helices (α1 and α2), and two β-strands (β1 and β2). Notably,

both cryo-EM structures did not resolve the C-terminal tail due to its disordered conformations, and we labeled the last residue visible from the cryo-EM structures on the figure. EXTENDED

DATA FIG. 3 PHASE DIAGRAM OF PURIFIED GVPU PROTEINS, WHICH SHOWS A TYPICAL UCST PHASE TRANSITION BEHAVIOR. Source data EXTENDED DATA FIG. 4 MCHERRY DOES NOT FORM TIME-DEPENDENT PUNCTA IN _E.

COLI_. _E. coli_ induced with 0.05 mM IPTG grown for a) 5 and b) 8 hours from inoculation (2 and 5 hours after induction). Scale bar = 5 µm. EXTENDED DATA FIG. 5 GVPU IS PREDICTED TO

ASSEMBLE INTO DEFINED HOMO-OLIGOMERS OF 5-7 UNITS. A) The average overall pLDDT70 score for all chains in different oligomeric states. In general, regardless of oligomeric state, the

monomeric protein fold is predicted with relatively high confidence and is essentially identical, although trimer, tetramer, and octamers show lower pLDDT than pentamer, hexamer, and

heptamers. The data are boxplots of the pLDDT scores for all residues in the various oligomeric complexes; thus, there is one pLDDT score for each residue in the complex. n = 138 (monomer),

276 (dimer), 414 (trimer), 552 (tetramer), 690 (pentamer), 828 (hexamer), 966 (heptamer), and 1104 (octamer). The minima is the lower adjacent value; the maxima is the upper adjacent value;

the box is drawn from the 25th and 75th percentiles with the median as the horizontal line in center. Whiskers are truncated at min/max of data if they would overstep the data. B) The

predicted 3D structure obtained from AlphaFold2 for monomeric GvpU. The structure is colored according to the pLDDT score (blue = more confident, red = less confident). C–I) Predicted 3D

structure of dimers-thru-octamers and the Predicted Alignment Error (PAE). These data do not enable us to confidently assess which of pentamer, hexamer, and heptamer is most likely, although

naively, the pentamer is the most confident overall prediction in terms of pLDDT and PAE. All models predict the same overall topology regardless of assembly start with a short (8-residue)

hydrophobic low-confidence N-terminal region extending upwards (with respect to the right-hand-side representation) and a second short (8-residue) positively-charged low-confidence

C-terminal region extending downwards. Both these regions are predicted to be disordered (Fig. 4a). In both cases, these short extensions are positioned adjacent to the equivalent region

from the next protomer. Taken together, our structural model suggests these short disordered regions form a meshwork on the top and bottom of the oligomer. EXTENDED DATA FIG. 6

CONCENTRATION-DEPENDENT ASSEMBLY OF GVPU YIELDS ANOMALOUS SPECTROSCOPIC FEATURES. A) Far-UV circular dichroism (CD) spectra of three concentrations of GvpU. At the lowest concentration (1.27

 μM = 0.02 mg/mL, lightest purple) of GvpU, noise below 220 nm precludes certainty in secondary structure assignment. Qualitatively, the local minimum at ~222 nm (denoted by vertical dashed

line) may reflect some α-helical character at 1.27 μM. Notably, the spectra for the two higher concentrations do not trend as expected for a system of non-interacting chromophores71,72.

Presented as molar ellipticity73,74, which is normalized to sample concentration and size, the spectra differ in shape and intensity. Specifically, as GvpU concentration increases, the

minima of the spectra decrease in negative intensity and undergo a modest red shift (higher wavelength), despite a linear increase in absorbance signal with increasing concentration. B)

Considering evidence from dynamic light scattering for the concentration-dependent formation of soluble oligomers of GvpU, the emergent CD spectral features at 12.7 and 22.9 μM likely

reflect interactions between chromophores in separate GvpU subunits. In particular, the AF2 model for the GvpU homomeric assembly implicates hydrophobic packing at the subunit interface;

drawing from exciton coupling theory, we speculate that the interactions between Phe and Tyr at the hydrophobic interface could be responsible for the observed hypochromic shift in the CD

signal at concentrations that support self-assembly. Source data EXTENDED DATA FIG. 7 REPRESENTATIVE TEM IMAGES OF GV VARIANTS. A) wildtype GvpA2Mega -based GVs (WT), B) WT GVs with _gvpU_

middle region Phe to Ser mutation (F → S), C) _gvpU_ middle region Glu to Ser/Gly mutations (E → S, G), D) and _gvpU_ middle region Phe/Glu to Ser/Gly mutations (F, E → S, G). GV clustering

in WT and E → S, G variants are zoomed in. Scale bar = 500 nm. Source data EXTENDED DATA FIG. 8 INTER-SUBUNIT INTERFACE OF THE PREDICTED GVPU PENTAMER. The predicted structure of the

pentameric oligomer with the hydrophobic interface between two protomers highlighted. A network of aromatic and aliphatic residues (with one threonine) line the interior of the interface,

with F71 and F57 forming a central pair of residues to connect the two protomers via hydrophobic interactions with the adjacent F26, L28, and F17 of the next protomers. EXTENDED DATA FIG. 9

GVPC DOES NOT INTERFERE WITH GVPU-MEDIATED GV CLUSTERING. A) Representative DLS measurement of A2Mega,2C_∆gvpU_ constructs (n = 3 for biologically independent samples). B) Representative TEM

images of A2Mega,2C_∆gvpU_ constructs (scale bar, 500 nm). Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Table 1 and Methods (sequences of oligonucleotides

and reagent or kit catalogue number). REPORTING SUMMARY SUPPLEMENTARY TABLE 1 Sequences of oligonucleotides and reagent or kit catalogue number. SOURCE DATA SOURCE DATA FIG. 1 Statistical

source data for Fig. 1c,d,h,j. SOURCE DATA FIG. 1 Unprocessed SDS–PAGE gel. SOURCE DATA FIG. 1 Unprocessed TEM micrographs. SOURCE DATA FIG. 2 Statistical source data for Fig. 2c,d,g,j,l.

SOURCE DATA FIG. 2 Unprocessed SDS–PAGE gel and western blot. SOURCE DATA FIG. 2 Unprocessed TEM micrographs. SOURCE DATA FIG. 3 Statistical source data for Fig. 3d,e,f,g,h,i. SOURCE DATA

FIG. 3 Unprocessed micrographs. SOURCE DATA FIG. 4 Statistical source data for Fig. 4d,f. SOURCE DATA FIG. 4 Unprocessed TEM micrographs. SOURCE DATA FIG. 5 Statistical source data for Fig.

5d,e,g,i,k. SOURCE DATA FIG. 5 Unprocessed SDS–PAGE gel (labelled). SOURCE DATA FIG. 5 Unprocessed TEM micrographs. SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data for Extended Data

Fig. 1. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data for Extended Data Fig. 3. SOURCE DATA EXTENDED DATA FIG. 6 Statistical source data for Extended Data Fig. 6. SOURCE DATA

EXTENDED DATA FIG. 7 Unprocessed TEM micrographs. SOURCE DATA EXTENDED DATA FIG. 9 Statistical source data for Extended Data Fig. 9a. SOURCE DATA EXTENDED DATA FIG. 9 Unprocessed TEM

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Li, Z., Shen, Q., Usher, E.T. _et al._ Phase transition of GvpU regulates gas vesicle clustering in bacteria. _Nat Microbiol_ 9, 1021–1035

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