The molecular microbiology and physics of bacterial flotation

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

Some aquatic bacteria can make intracellular chambers (made entirely of protein) that are permeable only to environmental gasses. The structures are called gas vesicles (GVs) and they form conglomerates (gas vacuoles) identifiable by phase contrast microscopy. The aquatic bacteria that make GVs may use them for the phenomenon of buoyancy, allowing upward flotation in a static water column. This ability can be useful for some photosynthetic bacteria (e.g. cyanobacteria) that need to rise in a stratified aquatic niche to access light of a specific wavelength, or to acquire nutrients or oxygen at the air-liquid interface, or perhaps to escape predators or competitors. The GVs usually comprise a major protein (GvpA) and a minor protein (GvpC) and form cylindrical structures with apical poles.

We recently discovered gas vesicles in strain ATCC39006 of the enterobacterium, Serratia ("related" to E. coli). The existence of GVs in this strain was a unique, and totally unexpected, observation. In addition to production of GV organelles and the capacity to float, this strain has other interesting traits. It makes two antibiotics. One antibiotic is antibacterial (a carbapenem) and another (prodigiosin) can kill protozoans and other microbes. ATCC39006 can swim via flagella (motility) and can swarm on solid surfaces and make surface detergent molecules (biosurfactants) enabling spreading and colonisation of new niches or host surfaces. The strain also rots plants (potato) by secreting plant cell wall degrading enzymes and it kills microscopic worms (Caenorhabditis elegans) and so it is also nematicidal.

We identified the cluster of 19 GV genes in strain ATCC39006 and we engineered E. coli strains that expressed the Serratia GV genes and allowed E. coli to float up to the air-liquid interface in static culture. Production of the GVs in Serratia was bacterial cell density-dependent in a process called "Quorum Sensing" controlled by a diffusible chemical signal that moves between cells. The signalling molecule is essential for production of the GVs in Serratia; quorum-sensing mutants don't float. Therefore, in this bacterium, an intercellular chemical signal controls the development of intracellular organelles and thus the chemical communication signal is also a morphogen. Quorum sensing also controls the production of the antibiotics in this strain and so these toxic molecules are made at the same time as the GVs are assembled. We showed that GV production was also up-regulated by oxygen limitation, implying that GVs may allow flotation to the liquid surface to acquire oxygen.

In collaboration with Professor Raymond Goldstein in the Department of Applied Mathematics and Theoretical Physics (DAMTP) in Cambridge, we have been investigating the phenomenon of buoyancy in this bacterium. Based on our knowledge of the genetics, physiology and physics of mobility in this bacterium we have developed a testable working hypothesis as to why GV production, and flotation, is under quorum sensing control; responsive to oxygen levels, and developmentally preferred to flagellar motility. Our model also predicts why the production of the two antibiotics has evolved to be co-incident with the development of the GVs, and flotation. We will now investigate the fascinating connections between bacterial cell population density, motility, bioconvection, GV development, buoyancy and antibiotic production. This study has wide ramifications. It impinges on areas such as ecological adaptation to environmental stress cues in microbes; intercellular chemical communication in bacteria; bacterial organelle morphogenesis; and the fitness value of microbial conflict and niche defence. Our understanding of the evolution of some of these biological processes will be significantly enhanced by an appreciation of the underlying mathematical physics that describes their behaviour; an exciting interdisciplinary study where microbiology meets mathematics!

Technical Summary

Serratia sp ATCC39006 (S39006) makes gas vesicles (GVs)- the only example of intracellular organelles existing naturally in a member of the Enterobacteriaceae. GVs form conglomerate light-refractive vacuoles under phase contrast microscopy. GVs are gas-permeable organelles providing buoyancy in bacteria. The ability to float upwards in a static water column can allow bacteria to gain access to nutrients, or access light, or air, or evade competitors. GVs were originally associated with marine and aquatic organisms such as cyanobacteria and halophilic Archea.

The GV locus has 19 genes, divisible into a left hand subcluster (L) encoding structural proteins and an R (right hand) subcluster that is regulatory. GV development is under quorum sensing (QS) control, via the signaling molecule (BHL) - a morphogen. Air-restricted cultures make more GVs and float better. The post-transcriptional regulator system, RsmAB also regulates GV morphogenesis. We will dissect the requirement for each of the 19 GV genes using non-polar mutants, assessing colonial impacts, GV architecture and protein composition, and flotation capacity. We will examine flotation functionality of apparently redundant structural genes encoding isoforms of GvpA. We will study the signal transduction pathways that sense QS (cell density) and oxygen levels and that use post-transcriptional control to link environmental stress to the developmental biology of GV biogenesis. We will test the hypothesis that the strain makes GVs as an adaptive response to avoid the consequences of flagellum-driven bioconvection processes seen during motility towards air-liquid interfaces. We will exploit various mutants of S39006 to describe mathematically the competing processes of motility and flotation and model the biophysical implications of GV production. Our aim is to compare the mathematical predictions with the experimental reality to test our view of the likely adaptive, evolutionary purpose of GV morphogenesis.

Planned Impact

Who will benefit from this research?
Potential beneficiaries include biotechnology industries using culture of mammalian cells or potentially generating hybrid vaccines using gas vesicles as scaffolds with antigen presentation features. GVs could have utility for such applications. Purified GVs have potential uses in mammalian cell culture systems because they represent a "gentle" mode of gas delivery to labile cells which can be susceptible to aeration damage.

We considered the fact that GVs engineered with hybrid structural proteins, including viral peptide antigens, are potentially immunologically protective and this could be a technology for the development of vaccines. As our system involves oxygen and QS-mediated control, this could be useful for possible translation in bioprocessing for control of gene expression in industrial microbiology. QS systems and GVs have clear synthetic biology applications as molecular bio-bricks for regulation control systems (and for flotation). Indeed our work has already been highlighted on an iGEM web blog site with this in mind.

How will they benefit from this research?
The biotechnological potential of GV research and QS research needs industrial collaboration and translation with financial investment that would extend beyond this project. The PDRA to be appointed will benefit enormously from acquiring the molecular microbiology skills involved plus his/her exposure and involvement in the biological physics and mathematics of modeling bacterial behavior. He/she will also gain skills and experience in teaching students and in science communication.

What will be done to ensure that they have the opportunity to benefit from this research?
The research outcomes will be disseminated to scientists (universities, institutes and commercial organisations) via international publications, lectures and posters at international symposia. Open access journals ensure global accessibility of research knowledge generated from this study. We have a track record of BBSRC-CASE studentships (two recently completed with UK companies) and collaboration with UK research institutes. We will try to extend such associations to generate enhanced funding for our research programmes and bring in added value by synergy of research interests with collaborators. This university encourages commercial liaisons and spin-out, after the establishment of solid IP positions. Our track record shows that we actively consider filing patent applications e.g. on carbapenem antibiotics and cryptic gene activation systems e.g. Salmond et al (US Patent 5821077 - issued 1998); Salmond et al (WO/1995/032294). We filed a patent application on antiviral abortive infection systems in 2008.

When we discovered GV production in Serratia and engineered the GV locus genes in E. coli, we considered filing a patent on the strains and technology because GVs have utility e.g. see above. However, after taking advice about IP from Cambridge Enterprise, we dropped the idea because pre-existing patents on GVs were sufficiently broadly written to make protection for our GV system very difficult. Furthermore, a very recent (December 2011) solid state NMR analysis of GVs from a group at MIT now suggests that GVs may have amyloid properties - perhaps not an ideal protein system for immunization! So our initial desire for IP protection was well intentioned, but pragmatically restricted. Nevertheless, we remain open to the possibility of securing IP on any aspects of our technology.

Finally, the PI and the Co-I are also involved in other impacts in national governance roles (e.g. Governing Boards of Research Institutes, Advisory Councils and Learned Societies). They are also involved in outreach activities. The Co-I has presented Science Week activities in Cambridge and school liaison presentations and the PI gives talks to student societies and to the University of the 3rd Age.

Publications

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Description This grant has very recently finished.

Bacteria have many modes of movement. This grant aims to explore and exploit the production of gas vesicles (GVs) in different Enterobacteria. Some aquatic organisms can make intracellular proteinaceous chambers that are permeable only to gasses, GVs. The bacteria that make these GVs can use them for buoyancy, allowing upward movement in a static water column. This is useful for photosynthetic bacteria that need to rise to the appropriate niche to access light of a specific wavelength. In this project we aimed to examine and exploit production of GVs in the Enterobacterium Serratia sp. ATCC39006. This strain swims, swarms and floats, utilises a well defined quorum sensing system (allowing for cell-density dependent transcriptional regulation) and produces two antimicrobial secondary metabolites.

We have used gene cloning and engineering techniques to create in-frame deletions of each of the 19 genes with the GV genetic cluster. From this, we determined that 14 genes were required for synthesis of full GVs. We demonstrated that a mutation in gvpC resulted in GVs with reduced strength and mutations in either gvpN or gvpV resulted in small bicone shaped vesicles, significantly smaller than GVs found in wild type cells. We have also engineered gas vesicle formation in strains of E. coli and these strains also float up to air-liquid interfaces. We have shown that the gas vesicle structures occupy around 30% of the bacterial cytoplasm in Serratia but around 50% in E. coli where their production may be deregulated. However, the apparent structural stability of the gas vesicles is equal in these different bacteria. We have done mathematical modelling of the buoyancy and "bioconvection" movement of Serratia strains carrying the gas vesicles but also mutants defective in vesicles, defective in quorum sensing through signalling molecules, or in production of flagella required for active swimming. Production is regulated by multiple physiological and environmental cues, including quorum sensing, oxygen levels, carbon source and other chemicals.
Exploitation Route Bacterial gas vesicles enable bacterial flotation towards air-liquid interfaces. Consequently, expression of these structures in other bacteria that do not naturally make them could have environmental applications (e.g. ecological biodegradation and "clean up") especially if done in a regulated fashion. Gas vesicles may be also structures/scaffolds that can be engineered as carriers of heterologous fusion proteins or peptides that could act as immunogens in synthetic vaccine development. They could also have synthetic biology and industrial biotechnology applications, for example, by regulated expression and morphogenesis in industrial E. coli strains cytoplasmic and exported proteins (and perhaps small bioactive molecules) may be influenced by alterations in local concentration and in the efficiency of cellular targeting - an industrial biotechnology aspiration.
Sectors Agriculture, Food and Drink,Chemicals,Education,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description We are considering synthetic biology/industrial biotechnology applications for gas vesicle development. The production and potential applications of gas vesicles is a story with wide appeal educationally; the concept of floating bacteria. Multiple invited talks and presentations have been given on this topic on the genetics and molecular biology of the biogenesis of the structures and their regulation by physiological and environmental cues. The work has been presented at international meetings in the UK, Europe and USA.
Sector Education
Impact Types Societal

 
Description Scottish Science Advisory Council
Geographic Reach National 
Policy Influence Type Citation in other policy documents
 
Description Cambridge International Student Scholarship (CISS)
Amount £109,947 (GBP)
Organisation University of Cambridge 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 09/2017
 
Title Development of plasposon mutagenesis system for generic use in Gram-negative bacteria 
Description Development of an engineered transposon mutagenesis system that allows mutagenesis of diverse bacteria for facile mutagenesis, cloning of insertion sites and sequencing. 
Type Of Material Biological samples 
Year Produced 2016 
Provided To Others? Yes  
Impact The system makes it easier to mutate genes, clone insertion sites and sequence them 
 
Title Genome sequence of gas vesicle-producing Serratia strain 
Description deposited in the ncbi database 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact This genome has been a resource for multiple projects in different labs. Scientific themes include secondary metabolite gene clusters (prodigiosin and a carbapenem antibiotic) plus the gas vesicle-producing cluster. 
 
Description Mathematical modelling of gas vesicle and flagellar functionality in bacterial movement 
Organisation University of Cambridge
Department Department of Applied Mathematics and Theoretical Physics (DAMTP)
Country United Kingdom 
Sector Academic/University 
PI Contribution The postdoctoral researcher on the grant collaborated with the the DAMPT group in the construction and use of bacterial mutants affected in motility, quorum sensing and gas vesicle production. The mutants and wild type were imaged in the DAMPT laboratories under conditions that allowed analysis of bacterial migration and bioconvection. The complex fluid dynamics of the outcomes are being mathematically modelled to assess impacts of flagellar-based motility and gas vesicle morphogenesis and signalling molecule production on bacterial movement.
Collaborator Contribution The setting up of the imaging microscopy and iterative analytical techniques and most of the mathematical skills were all provided through the DAMPT collaboration
Impact There are no modelling paper outputs so far but we expect to produce one, or perhaps two, later this year.
Start Year 2013
 
Description Gordon Conference in Massachusetts, USA, Invited talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a talk on gas vesicle production in Serratia to a large audience of environmental microbiologists, educators and students. Took part in a Q&A session and poster sessions, plus networking with attendees from very different backgrounds.
Year(s) Of Engagement Activity 2013
 
Description HE+ Program - Cambridge Outreach 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact February 21-22, 2014 - HE+ Program as a part of Cambridge University's Outreach Activities. PDRA taught enrichment classes for students in Swansea, Wales as part of a group of Cambridge University members (postgraduate students, postdocs and lectureres) for local students participating in the HE+ program. Also met with local teachers from state schools to explain the application process to Cambridge University and encourage them to bring students to Open Days in the University. Science presentations.
Year(s) Of Engagement Activity 2014
 
Description HE+ Program in Swansea 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact February 27-28, 2015 - PDRA took part in the HE+ Program as a part of Outreach Activities. She taught enrichment classes for students in Swansea, Wales as part of a group of Cambridge University members (postgraduate students, postdocs and lectureres) for local students participating in the HE+ program. She also met with local teachers from state schools to explain the application process to Cambridge University and encourage them to bring students to Open Days in the University.
Year(s) Of Engagement Activity 2015
 
Description Invited talk: 2nd Midlands Molecular Microbiology Meeting, Nottingham. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk on gas vesicles in bacteria, quorum sensing and co-regulation with antibiotic production, plus Q&A session. Attended other talks.
Year(s) Of Engagement Activity 2015
 
Description London Sixth Form Conference February 2015 - "The Variety of Living Organisms" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact February 11, 2015 - PDRA presented a session to students in London as part of a Sixth form 'conference' entitled 'The Variety of Living Organisms'.
Year(s) Of Engagement Activity 2015
 
Description Lumina Program in Harrow London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact June 29, 2015 - PDRA took part in the Lumina program in Harrow London, a program for local state school children for one week (her participation was on a single day) which attempts to widen appreciation of what University entails, how to apply for University, how to apply for student finance. She conducted a session describing the importance of Science and Maths A-level subjects as a part of applications to Universities. Afterwards, she spoke with local teachers about how to encourage students to apply for University courses in science.
Year(s) Of Engagement Activity 2015
 
Description Lumina Program in Harrow, London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact July 1st and 4th, 2014 - Lumina program in Harrow London, a program for local state school children for one week. PDRA contributed. The Program attempts to open state school children's appreciation of what University entails, how to apply for University, how to apply for student finance. This involved the PDRA in a half day session describing the importance of Science and Maths A-level subjects as a part of applications to Universities. The same students were then encouraged to come to Cambridge for an open day that Friday and the PDRA met them for lunch and walked around Cambridge with them that Friday.
Year(s) Of Engagement Activity 2014
 
Description Open Days 2014 - assorted 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact April 7th the PDRA gave a lecture as part of the Cambridge Admissions Office open day to students interested in medicine, biological natural sciences and veterinary sciences.
September 26th - - the PDRA hosted a lunch for students interested in attending the University as part of Open Days.
Year(s) Of Engagement Activity 2014
 
Description Open Days 2015 - assorted 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact April 9th - The PDRA gave a lecture as part of the Cambridge Admissions Office open day to students interested in medicine, biological natural sciences and veterinary sciences.
July 2nd, 3rd and September 25th - The PDRA hosted a lunch each day for students interested in attending the University as part of Open Days.
Year(s) Of Engagement Activity 2015
 
Description Talk to Young Microbiologists, Cork Ireland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An invited talk as one of a group of international scientists on a themed programme relevant to aspiring young microbiologists (mostly postgraduates and undergraduates). Talk plus Q&A plus networking a poster presentations.
Year(s) Of Engagement Activity 2012
 
Description Wisconsin meeting on Molecular Genetics of Bacteria and Phages 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a talk on gas vesicles in bacteria, followed by a Q&A session.
Year(s) Of Engagement Activity 2015