William C. Fuqua - US grants

The process of Agrobacterium tumefaciens of plants is a well studied phenomenon, and is the only bonafide example of natural genetic engineering. The infecting bacterium transfers a fragment of its DNA from a tumor inducing (Ti) plasmid, into the host plant genome. Genes encoded on this fragment direct tumor formation and the synthesis of unusual sugars and amino acids termed opines, which serve as Agrobacterium-specific carbon/nitrogen sources. In turn, the opines stimulate the expression of A. tumefaciens genes involved in their uptake and utilization, conjugal transfer of the Ti plasmid, and a number of other functions that may be involved in tumor colonization. In octopine-type plasmids this induction is mediated at least in part by the LysR-type transcriptional activator OccR, which induces the transcription of these genes in response to the opine octopine and which also autoregulates its own synthesis. One of the targets for OccR regulation is the overlapping, divergently transcribed promoter region between the occ (octopine catabolism) operon and the occR gene itself. This unusual genetic organization is typical of LysR-type systems and probably allows simultaneous control of both operons. In the proposed study, we shall examine the interaction of OccR with this transcriptional control region. Evidence is presented that OccR binds to this region in the presence or absence of octopine, and that octopine induces conformational changes in Occr and/or its target site. We hypothesize that; i) OccR bends its target site, that octopine incites modulation of this bending, and that these changes result in the induction of transcription, or ii) that octopine-induced conformational changes in OccR itself result in transcriptional activation. The proposed investigation will employ biochemical, molecular biological, and genetic techniques to test these hypotheses.

Many species of bacteria employ signaling mechanisms to monitor their own population density and respond by altering physiological and developmental pathways. This process usually entails the release of a soluble pheromone, the local concentration of which provides a measure of cell density. Release and perception of the signaling molecule has been termed quorum sensing. Quorum sensing in a number of gram-negative bacteria is facilitated by production of acylated homoserine lactones (acyl HSL). Agrobacterium tumefaciens, the causative agent of plant crown gall neoplasia, utilizes the acyl HSL N-3-(oxooctanoyl) homoserine lactone to regulate the conjugal dissemination of its primary virulence factor, the Ti (tumor-inducing) plasmid, to other bacteria in the rhizosphere. The current proposal focuses on examination of a unique and essential inhibitory component of the A. tumefaciens acyl HSL regulatory pathway. For most acyl HSL-type quorum sensors, a pheromone synthase and a receptor protein that also serves as a transcriptional activator are sufficient to impart cell-density-dependent gene expression. However, A. tumefaciens requires a third component, a regulator protein called TraM, to facilitate quorum sensing. The hypothesis is that TraM forms a noncovalent complex with the acyl HSL receptor, TraR, which prevents the transcriptional activator from binding DNA. The overall goal of this research is to dissect the interaction between TraM and TraR and to understand how this interaction modulates the activity of TraR. Biochemical analysis will be employed to examine the inhibitory complex and to determine the effect of inhibition on the activity of TraM and TraR in the presence of acyl HSL. Mutant TraR and TraM proteins that exhibit altered regulatory phenotypes will be
isolated and biochemically analyzed to correlate in vitro and in vivo activities. Additional studies will assess the effects of regulation of traM expression and pool size. This research will not only elucidate a unique aspect of acyl HSL quorum sensing in the important model pathogen A. tumefaciens, but will also contribute to the understanding of cell-cell communication in other species of bacteria.

Gram-negative bacteria within the Proteobacteria group commonly use acylated homoserine lactones (acyl-HSLs) as molecular signals in the process of quorum sensing (QS). Quorum-sensing bacteria release diffusible signal molecules that accumulate with increasing cell number, eventually triggering adaptive responses. Regulatory proteins of the LuxI and LuxR families are usually required for synthesis of, and response to acyl-HSLs, respectively. Diffuse populations of cells generally produce a constant, low level of acyl-HSLs, and these rapidly diffuse out of cells down their concentration gradient. Elevated population density increases the relative acyl-HSL concentration, eventually fostering interaction of the signals with LuxR-type proteins, which in turn, control the transcription of target genes. Although this basic mechanism is well conserved, the context of QS regulation and the cellular functions under its control are highly variable among different bacteria. This project focuses on the regulatory context of a LuxI-LuxR-type QS system employed by the nitrogen-fixing plant symbiont Rhizobium sp. NGR234, its mechanism of action, and its effect on cellular growth rate. NGR234 incites the formation of nitrogen-fixing, symbiotic nodules on the roots of a wide range of leguminous plants. The molecular basis of this promiscuous host interaction has been extensively studied. Many of the functions that orchestrate the plant interaction are carried on the 536 kb pNGR234a plasmid. The pNGR234a plasmid also carries a large cluster of genes homologous to plasmid replication (rep) and conjugal transfer (trb/tra) genes from other bacteria. The rep/trb/tra cluster includes a LuxI-LuxR-type regulatory pair, TraI and TraR, and the additional QS regulator TraM. TraI synthesizes 3-oxo-octanoyl-L-homoserine lactone and TraR interacts with this acyl HSL to regulate tra/trb and rep operon expression. TraM acts to inhibit TraR through formation of an anti-activation complex. A molecular genetic approach is being employed to study the QS mechanism in NGR234. The regulatory signals and pathways that control traR expression will be investigated to determine the conditions that foster QS. Based on analogous systems, the host plant is likely to play a role in this regulation. The QS-regulated pNGR234a genes under TraR control will be identified and the mechanism by which TraR controls their expression elucidated. Lastly, control of cellular growth rate by QS, a common QS regulatory target among several Rhizobium species, will be examined. Findings generated from the research project will provide fundamental information on cell-to-cell communication in an important plant symbiont, and the role of this communication in its highly plastic host interactions. More generally, these studies will add to the understanding of how cell-to-cell communication directly and indirectly influences interactions with host organisms, and enables quorum-sensing microbes to balance their physiological activity with the host environment.

The bacterium Rhizobium sp. NGR234 uses cell-to-cell communication during its symbiotic relationships with higher plants. The research project examines the biochemical and genetic mechanisms underlying this intercellular communication, and its influence on the interaction of this bacterium and host plants. The findings generated from this work will provide fundamental knowledge required to take advantage of these bacterial communication systems, for combating infectious disease in plants and animals, as well as promoting beneficial microbial interactions.

The goal of this project is to investigate microbial interactions within ticks and how those interactions affect the prevalence of human pathogens. Ticks are the primary source of infectious disease transmitted by arthropod vectors in the United States, including Lyme Disease, Rocky Mountain Spotted Fever and many other newly-recognized and emerging human diseases. Ticks also carry a variety of non-pathogenic symbiotic bacteria. Most of this microbial diversity has only become apparent with the advent of powerful molecular tools. Preliminary evidence suggests that these symbionts can exclude pathogens, reducing human disease risk. Using molecular genetic methods, the microbial communities of medically-important tick species will be quantified and statistical tests will be applied to test the strength and direction of microbial interactions. In addition, microbial interactions will be incorporated into epidemiological models to determine conditions where microorganisms within ticks interfere with or amplify one another.

The results of this research will contribute to a general understanding of how microbial interactions affect pathogen prevalence, disease epidemiology, and human disease risk. This research will also explore new techniques for quantifying pathogen populations and microbial communities, and contribute significantly to scientific education at the undergraduate, graduate and post-doctoral levels. Microbial interactions within other arthropod vectors (e.g., mosquitoes, fleas, lice, bugs) are likely to be common, including vectors of an ever-growing list of emerging human diseases and pathogens identified as bioterrorism threats.

This project will support the International Workshop Biocomplexity VI: Complex
Behavior in Unicellular Organisms, jointly organized by the Biocomplexity
Institute at Indiana University and the Interdisciplinary Center for the Study of
Biocomplexity at the University of Notre Dame. The collective behavior of
bacteria is as a problem in basic science and is significant in bioengineering, agriculture, medicine, naval architecture and geosciences.... Biofouling is a major problem in cases ranging from surgical implants to submarines, and biofilms are a major cause of antibiotic-resistant infections. The regulatory mechanisms for collective behaviors in bacteria differ from those in more advanced organisms and present an opportunity to compare different design solutions to similar biological problems related to embryonic development. This workshop will develop a more comprehensive view of our current understanding of these issues and include a panel to discuss and report on new directions and collaborations. It will bring together researchers in many disciplines who would not normally attend the same conference (including experimental and theoretical biology, biophysics, engineering, mathematics, and computer science). Approaches will range from single molecule interactions and genetics to systems biology and ecology. About fifty speakers will attend from the USA, Canada, Europe, Israel and Japan, with a total participation of about 100, including many non-specialists. The workshop will include tutorials for graduate students and junior scientists, especially those not currently in the field, but interested in learning about open problems, methodologies and opportunities. As part of the workshop's outreach effort, Dr. Eshel Ben-Jacob will present a public lecture for nonscientists and interested members of the community-at-large.

Bacteria can exhibit social behaviors, one of which is the process known as quorum sensing. Quorum sensing regulates a range of functions, often those involved with host interactions such as pathogenesis and symbiosis, in response to bacterial population density via signal molecules. Once the signal molecule (e.g. acylated homoserine lactones or AHLs for the Proteobacteria) reaches a threshold concentration, it effectively binds and activates a transcription factor(s), which regulates expression of genes responsible for collective bacterial activities. Although the role of AHL quorum sensing in microbial systems is relatively well accepted, mechanistic studies of the regulatory circuitry, including production and diffusion of AHLs, activation and inhibition of AHL-responsive transcription factors, and control of gene expression, are still at an early stage. The aim of this project is to examine the structural and biochemical basis of quorum sensing in Agrobacterium tumefaciens, a model plant pathogen and a premier system for mechanistic studies of quorum sensing, using a range of genetic, biochemical and structural methods particularly protein X-ray crystallography. In A. tumefaciens quorum sensing requires the AHL-responsive TraR transcription factor, and under non-inducing conditions this protein is inhibited by the TraM anti-activator through formation of a highly stable heterocomplex. The objectives of this research are as follows. First, structural and functional studies of TraM, based on the PIs' recently solved crystal structure of TraM, will be continued. These efforts will dissect the biochemical and functional complexity of TraM activity. Second, the TraM inhibitory mechanism will be studied by determining the structure of the anti-activator heterocomplex (TraM-TraR-AHL) using X-ray crystallography, complemented with biochemical analysis of this complex using biophysical and kinetic techniques. Third, functional studies will be performed to test the predictions revealed through the co-crystal structure on TraM inhibition of TraR function. These studies will represent the first structural studies on mechanistic aspects of transcription factor inhibition in a quorum sensing pathway, and the first kinetic characterizations on the molecular events of TraM antagonism on TraR. Results from this work will provide a comprehensive structure-function understanding of TraM-TraR interaction. These findings could lead to novel approaches for limiting the spread of infective A. tumefaciens in agricultural situations by the deliberate intervention into their quorum sensing. These studies will also have significant impact in the area of microbial cell-to-cell communication, and may help develop novel approaches such as the design of chemical compounds that interfere with bacterial communication, to control and combat microbial invasion. This work will also provide abundant opportunities for the involvement of undergraduates and the training of graduate students, integrating with the rich educational environment at Indiana University.

A bacterial pathogen's ability to infect hosts is frequently conferred by genes on mobile genetic elements called plasmids. These genetic elements sometimes spread through bacterial populations because of the competitive advantages they confer to the bacteria that harbor them. Plasmids can also decline in populations when their net effects are negative. A main goal of this project is to understand how ecological factors, such as resource levels, influence the maintenance of a disease-causing plasmid. The focal plasmid employs intercellular communication to coordinate a component of its spread.

A second goal of this project is to provide insight into the adaptive reasons many bacteria (and many pathogens) utilize communication. This project will combine experimental approaches with mathematical modeling to examine these issues.

Plasmids carry genes involved in many important functions, including pathogenesis and antibiotic resistance. However, because of the unique biology of plasmids, biologists do not fully understand their population dynamics. This project focuses on understanding the costs and benefits associated with maintaining a virulence plasmid in a common plant pathogen that is a frequent agricultural pest. As similar population dynamics occur in outbreaks of antibiotic resistant bacteria, this project could also inform a general understanding of the biology underlying this major public health problem.

[unreadable] DESCRIPTION: ASM Conference on Cell-Cell Communication in Bacteria, Clay Fuqua and Heidi B. Kaplan, Co-Organizers. Bacteria are the paradigm for unicellular life, yet they also exhibit elaborate coordinated behaviors that often defy unicellularity. Single bacterial cells respond to their immediate physical and chemical environment, adapting in response to changing conditions. In the 1960s and 1970s several reports suggested that bacteria might communicate with each other via chemical signals, specifically in regulating genetic competence in Streptococcus pneumoniae and in controlling bioluminescence in Vibrio fischeri, respectively. Research over the past decade has begun to reveal that a wide range of bacteria can communicate by diverse mechanisms. In most cases these microbial conversations occur through the exchange of diffusible signals, although there are also clear examples of cell contact-dependent communication. Many bacteria use these signaling mechanisms to monitor and respond to population density, a process often described as quorum sensing. Interbacterial communication is not however restricted to quorum sensing mechanisms and there is mounting evidence that signaling can function in a range of different capacities. Communication between microorganisms can have profound impacts on human health, as pathogens and commensals often regulate important aspects of their host interactions using signal production and perception. Target functions include, but are not restricted to virulence factors, adhesins, biofilm formation, horizontal gene transfer and the secretion of exoenzymes. Well established systems such as the cyclic oligopeptide signals that regulate Staphylococcus aureus virulence and acyl homoserine lactones (AHLs) that control of pathogenesis in Pseudomonas aeruginosa are now joined by a number of more recently identified signaling systems. The range and diversity of these systems continues to grow explosively. Due to the rapid pace of discovery in this area of microbiology and the excitement it has generated, the ASM has hosted two previous Cell-Cell Communication in Bacteria (CCCB) conferences, in 2001 and in 2004. Strong community support for a third conference led the ASM to commit to the current conference, to be held in Austin, Texas 2007. The goal of this conference is to act as a conduit for the exchange and synthesis and new ideas among leading US and international scientists working on communication. The past two conferences greatly stimulated the cell- cell communication community, led to outstanding discourse and productive new collaborations. A large number of the participants that attend this conference are funded through NIH, and the NIGMS has been generously supportive of the two previous two meetings. We are therefore hopeful that this trend continues for this important microbiology conference. Public Health Relevance Statement: The process of cell-cell communication in bacteria has tremendous impact on human health at several different levels. Pathogenic microorganisms that cause infectious disease in humans represent some of the most intensively studied examples of bacterial signaling. Coordination in the production of colonization factors, toxins, and tissue-damaging enzymes is often mediated through bacterial communication, whereby the infectious populations dictate the time, location and intensity of host damage, to best cope with the immune response. Advances in the understanding of how, why and when microbes employ signaling has great potential to improve our ability to combat infectious agents. New therapies directed towards microbial communication networks promise to augment and improve our current reliance on antibiotics, expanding our ability to treat and prevent disease, and manipulate bacterial behavior for beneficial purposes. The Cell-Cell Communication in Bacteria 2007 conference will gather together the best US and international scientists working in this area to exchange new information, ideas and strategies for targeting and harnessing the recently discovered communication mechanisms of microorganisms. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of the proposed research is to offer predoctoral training in the area of genetics, genomics, development, cellular biology, molecular biology, and/or biochemistry. Forty training faculty will participate in this program. The wide range of interests and expertise of the training faculty allows students to select among research programs that include molecular evolution, molecular biology, molecular genetics, developmental genetics, evolution of development, cell biology, microbiology, virology, structural biology, and biochemistry. A number of faculty have been hired in the past few years and add both breadth and depth in these areas. This interdisciplinary nature of our training program represents one of the major strengths of the program. The core curriculum is designed to give first year students a strong foundation in genetics, cell, developmental, and molecular biology, and biochemistry. Advance courses and other requirements are designed for the students to gain strength in their area of specialty, develop strong communication skills, and gain awareness of proper research conduct. Students are engaged in research training from the first year and are encourage to apply techniques and approaches derived from all of our areas of research strength. The majority of students supported by the training grant will be drawn from the Molecular Biology and Genetics (MBG) Program which awards Ph.D. degrees in Genetics, Molecular, Cellular and Developmental Biology, Microbiology, or Plant Sciences. It is also possible for students in other Departments or Programs to be supported by the training grant if their thesis mentor is a member of the Training Grant Faculty and the student fulfills the academic and thesis requirements that are related to the training mission of this program. We have had a long-standing tradition in the training of predoctoral students who have been productive, and established successful careers in biomedical related research. The training faculty remains dedicated to this mission. We are requesting continued funding of the current 16 training grant positions so that we can continue in this tradition.

Marine sponges have proven to be a rich source for novel pharmaceuticals such as anticancer drugs and antibiotics. These simple animals also harbor a vast, yet stable population of symbiotic microorganisms that are often the source for the medicinally active compounds. In addition to generating useful metabolites for the host animal these microbes can also perform other important functions, including acquisition of limiting nutrients and protection against harmful disease agents. The microbial communities
within sponges are complex and diverse, representing many different types of microbes with a great range of capabilities. As with most complex microbial communities, there is virtually no understanding of the factors and mechanisms that establish and stabilize the microbiota in sponges. For marine sponges, it is unclear how specific microbes are introduced and maintained in the face of the huge volume of microbe-containing seawater surrounding them, and how the appropriate balance of different microbes is fostered within the sponge tissue. One mechanism by which bacteria coordinate their activities is through the exchange of chemical signals. Over recent years it has become clear that many bacteria can "converse" through different chemical languages, and that this microbial conversation can have a major impact on microbial community function. In a related NSF Microbial Observatory project, a large group of sponge-associated microbes has been found to produce a specific family of communication signals called acyl-homoserine lactones (AHLs), known to regulate diverse processes including antibiotic synthesis, nutrient acquisition, and microbial gene exchange in other bacteria. AHLs are prime candidates for factors that shape the sponge community and facilitate coordination of microbial activities. This study will test the pervasiveness and importance of these signaling mechanisms for the well-characterized microbial communities of several shallow-water, tropical sponges. Advanced chemical, biochemical, molecular, microscopic and microbiological analyses will be applied to determine the extent to which these indigenous microorganisms communicate within the sponge, the functions under control of the signaling, and the connection to important attributes of the sponge, including production of novel metabolites.

The study will provide broad insights into the ways that microbial communities proliferate and mature during host association, the regulation of microbial activity to promote symbiosis rather than disease, and the functional relationships between these microbes and the sponge. In a wider sense, this study will advance understanding of complex symbiotic processes and contribute to knowledge on signaling in interactions between bacteria and their hosts. This interdisciplinary project involves three different laboratories with different areas of expertise, and will train graduate students and postdoctoral researchers in ways that bridge the traditional areas of chemistry, ecology and molecular microbiology. Undergraduate student participation is also an important component of the study. In addition to undergraduate research assistants, the project also includes support for continuation of the successful Summer Microbiology and Research Training (SMaRT) course that offers a two week intensive laboratory research experience for minority students at the Center of Marine Biotechnology in Baltimore. This course provides students with a realistic research experience focused on sponge microbial communities, in close collaboration with the research scientists, postdoctoral researchers and graduate students.

DESCRIPTION (provided by applicant): A novel exopolysaccharide-containing structure has recently been discovered that localizes to a single pole of Agrobacterium tumefaciens cells and appears to promote cellular contact with surfaces, presumptively functioning as an adhesin. Production or extrusion of the exopolysaccharide is stimulated under growth conditions with limiting phosphorous and is regulated by the PhoR-PhoB two component system. A. tumefaciens is a pathogenic microbe that transfers DNA and proteins directly to plant host cells via a Type IV Secretion (T4S) system. There is virtually nothing known regarding the mechanism by which A. tumefaciens attaches to plant cells during the infection process. Unlike mammalian pathogens, there is limited understanding of host cell surface interactions in plant pathogens. The wide potential host range of A. tumefaciens, including fungi and mammalian cells, as well as its proclivity to form biofilms on diverse surfaces suggest an extremely versatile adhesion mechanism. It is well documented that A. tumefaciens tightly attaches to plant tissues and abiotic surfaces in a polar orientation. Recent evidence suggests that the A. tumefaciens T4S is also localized to a single pole of the cell, analogous to the polar exopolysaccharide. This study will determine whether the exopolysaccharide and the T4S machine can occupy the same pole of the cell, whether this occurs during surface colonization, and if the exopolysaccharide plays a role in productive infections. Co-localization of additional polar structures, flagella and pili, will also be tested. The chemical composition of the exopolysaccharide will be determined and the genes encoding its synthesis, elaboration and localization isolated. The mechanism of phosphorous-responsive regulation of the exopolysaccharide will be examined and the integration of this structure with other adhesion pathways during adaptation to the surface environment elucidated. The findings generated in this study will shed light on the process of surface attachment and biofilm formation during plant host interactions, including implementation of the T4S toxin delivery system. The properties of this polar polysaccharide are similar to the adhesive holdfast of Caulobacter species and suggest that these structures could be more common than anticipated, and may provide novel antimicrobial targets. The pathogenic microbe Agrobacterium tumefaciens produces a sticky, sugar-containing substance that concentrates on one end of cell. We are investigating the role of this structure in disease and bacterial survival using molecular genetics, high resolution microscopy and genomic analysis. This study promises to provide important insights into the relationship between adhesion and disease progression.

DESCRIPTION (provided by applicant): The goal of the proposed Genetics, Cellular and Molecular Sciences Training Grant (GCMSTG) is to provide comprehensive, relevant and engaging pre-doctoral training in the molecular sciences across a range of scales from atomic resolution, through macromolecules, to the cell and its structure, and to populations of cells and tissues. Fifty six Training Faculties from the Indiana University Biology, Biochemistry and Chemistry departments and the Medical Sciences Program, cover a broad range of expertise and research interests that encompass diverse disciplines including biochemistry, development, cell biology, microbiology, molecular genetics, structural biology, and molecular evolution. Trainees have the opportunity to engage in the highly collaborative and stimulating intellectual environment facilitated by a large, integrated faculty who span a tremendous breadth of life science research. Trainees enroll in a core curriculum designed to teach molecular science from a primary research perspective. Courses on literature analysis, grant writing, teaching and ethics, plus opportunities to develop presentation skills complement the core curriculum. Trainees cultivate their specific interests with advanced coursework. Throughout their studies, students are extensively mentored by the Training Faculty, the TG Committee and their thesis committees. Specific training grant activities including a yearly symposium, a monthly trainee meeting, travel support, and opportunities to meet with visiting seminar speakers, enhance their experience, and create a rich intellectual environment. Many students supported though the GCMSTG are from the Molecular, Biology and Genetics graduate program in the Biology Department, although a significant number of trainees with a molecular biological focus will also be supported from the Ecology, Evolution and Behavior program in Biology, and the Interdisciplinary Biochemistry and Medical Sciences graduate programs. The lU GCMSTG has a long and outstanding track record of training pre-doctoral students, who go on to develop successful and productive careers in academic and industrial life science sectors. This resubmitted proposal requests continuation of 16 training positions, through which we will contribute to and enhance this tradition. RELEVANCE: The lU GCMS TG enables talented pre-doctoral students to realize their potential and develop into outstanding life scientists, contributing to human health and society at many different levels. Comprehensive coursework, careful mentoring, advanced facilities, intensive scientific discourse and teaching, creates a program in which dedicated students thrive, and propel their careers forward into the biomedical sciences.

PROJECT SUMMARY The establishment of productive, stable surface interactions is an important process for bacteria, that can lead to formation of the adherent communities known as biofilms. These assemblages are challenges in agricultural, industrial and medical settings, and are intrinsically tolerant to many antimicrobial therapies. For a number of bacteria in the large and diverse Alphaproteobacteria (APB) group, attachment to surfaces and to other cells requires production of a structure comprised of polysaccharide localized to a single cellular pole. In the model pathogen Agrobacterium tumefaciens this structure is called the unipolar polysaccharide (UPP). Polar adhesins similar to the UPP are widespread among the APB, including other pathogens and symbionts, and the A. tumefaciens UPP is therefore a representative model for these diverse bacteria. Among these, the stalked bacterium Caulobacter crescentus produces a similar structure called the holdfast at the stalk tip, and although it has been well studied, remains poorly understood and is less broadly representative than the UPP. These polar polysaccharides can drive stable surface attachment and host interactions. In A. tumefaciens the UPP is comprised of at least two distinct polysaccharide species, and the genes required for synthesis suggest that there may be overlapping biosynthesis pathways. We aim to determine how A. tumefaciens coordinates and regulates production of these polysaccharides during surface colonization, including dynamic localization of the biosynthetic complexes. Production of the A. tumefaciens UPP is strictly regulated by contact with the surface, and cells rarely if ever produce the UPP when free-swimming. The proposed studies will dramatically improve our current understanding of UPP properties and biosynthesis, and will elucidate its regulation via a network of intracellular signal cascades, its surface-dependent polar localization, and other environmental signals that affect its production, and thereby attachment. At the core of this control network is the ubiquitous bacterial second messenger cyclic diguanylate monophosphate, which regulates UPP production. Among the primary UPP regulatory mechanisms are a novel signaling pathway involving small metabolites called pterins, and the response to low pH. The project utilizes an extensive collection of genetic mutants and variants, quantitative microscopic imaging approaches, genomic technology, and sophisticated biochemical approaches to illuminate the cellular processes that promote attachment via the UPP. The findings generated will contribute to the understanding of the motile to sessile transition and initiation of biofilm formation. We will characterize the biosynthesis of a novel biological adhesive(s) and a potential antimicrobial target, and will reveal how bacterial cells control production of these important products to promote surface interactions that lead to biofilm formation. Our findings will provide fundamental information about these polar adhesins, identifying new targets for anti-bacterial approaches and facilitating development of new biomaterials.

Project description Microbial biofilms are among the most common forms in which microorganisms exist, and as such the topic of biofilms is relevant to virtually all of the microbial sciences. Biofilms create enormous challenges in the medical, agricultural and industrial sectors of society, but also can be harnessed for important beneficial applications. A tremendous number of research programs worldwide study biofilms spanning fundamental to applied science. In order to connect this large, active and diverse community the American Society for Microbiology (ASM) has convened the Conference on Biofilms, which has repeatedly catalyzed the development of new ideas and approaches in the field. The conference gathers the top researchers studying biofilms from both inside and outside of the United States, and from academia, government, and the private sector. The ASM has discontinued most of its small conferences, but given the vitality and community support for the Biofilm Conference, has retained this meeting. The 9th ASM Biofilm conference is scheduled for November 14-18, 2021 in Charlotte, North Carolina, an accessible yet affordable venue, chosen partly in response to participant concerns over high conference costs. The ASM provides all administrative support for this conference, but cannot cover all of the meeting expenditures and keep the conference fees manageable. Funds are requested in this proposal to defray the costs of participation by invited speakers, students and early career scientists. The conference format has been developed by the conference co-chairs, Karen Visick (Loyola University Chicago, USA) and Clay Fuqua (Indiana University Bloomington USA), along with the nine member Organizing Committee, representing a diversity of biofilm sub-specialties. The conference will be comprised of 11 topical sessions and 2 keynote presentations. Oral presentations will include 22 invited speakers, 33 selected abstract talks, and 12 short poster talks, and there will be four two-hour poster sessions. The afternoon of conference day two will have optional activities including a hands-on biofilm image analysis workshop, tours of local attractions, or networking time. Prior to the start of the full conference, there will be a single-day Early Career conference, that is being organized by a junior scientist committee in consultation with the co-chairs. The conference is specifically designed to convey the breadth and depth of biofilm research, and to actively engage the participation of scientists at all career stages. Special attention is focused to ensure diverse participation among conferees, including from inside the U.S. and internationally. Organizing committee membership also represents this diversity, helping to achieve this important goal. The conference proceedings will be published in a meeting review within a special biofilm-focused issue of the Journal of Bacteriology. Although the 9th ASM Biofilms Conference is intended to be in-person, contingency plans are in place with the ASM should public health concerns require an on-line only conference.