Juan E. González

Rhizobium meliloti exopolysaccharides: Synthesis and symbiotic function ☆

Bacterial exopolysaccharide (EPS) is required for establishment of the nitrogen-fixing symbiosis between Rhizobium meliloti and its host plant, Medicago sativa (alfalfa), but the precise role of EPS in this interaction is not well defined. Bacterial mutants which fail to produce EPS induce nodules on the roots of the host plant, but fail to invade these root nodules. Research conducted in our lab and others suggests that EPS plays a specific role in the R. meliloti-M. sativa symbiosis. A common theme emerging from these studies is that small quantities of low-molecular-weight (LMW) EPS are sufficient to mediate successful invasion by R. meliloti mutants which fail to produce EPS, implying that LMW EPS may act as a signaling molecule during this process.

Orphan LuxR regulators of quorum sensing

Bacteria can modulate their behavior by releasing and responding to the accumulation of signal molecules. This population co-ordination, referred to as quorum sensing, is prevalent in Gram-negative and Gram-positive bacteria. The essential constituents of quorum-sensing systems include a signal producer, or synthase, and a cognate transcriptional regulator that responds to the accumulated signal molecules. With the availability of bacterial genome sequences and an increased elucidation of quorum-sensing circuits, genes that code for additional transcriptional regulators, usually in excess of the synthase, have been identified. These additional regulators are referred to as orphan regulators, because they are not directly associated with a synthase. Here, we review orphan regulators characterized in various Gram-negative bacteria and their role in expanding the bacterial regulatory network.

Chemical sensing in mammalian host–bacterial commensal associations

The mammalian gastrointestinal (GI) tract is colonized by a complex consortium of bacterial species. Bacteria engage in chemical signaling to coordinate population-wide behavior. However, it is unclear if chemical sensing plays a role in establishing mammalian host–bacterial commensal relationships. Enterohemorrhagic Escherichia coli (EHEC) is a deadly human pathogen but is a member of the GI flora in cattle, its main reservoir. EHEC harbors SdiA, a regulator that senses acyl-homoserine lactones (AHLs) produced by other bacteria. Here, we show that SdiA is necessary for EHEC colonization of cattle and that AHLs are prominent within the bovine rumen but absent in other areas of the GI tract. We also assessed the rumen metagenome of heifers, and we show that it is dominated by Clostridia and/or Bacilli but also harbors Bacteroidetes. Of note, some members of the Bacteroidetes phyla have been previously reported to produce AHLs. SdiA-AHL chemical signaling aids EHEC in gauging these GI environments, and promotes adaptation to a commensal lifestyle. We show that chemical sensing in the mammalian GI tract determines the niche specificity for colonization by a commensal bacterium of its natural animal reservoir. Chemical sensing may be a general mechanism used by commensal bacteria to sense and adapt to their mammalian hosts. Additionally, because EHEC is largely prevalent in cattle herds, interference with SdiA-mediated cattle colonization is an exciting alternative to diminish contamination of meat products and cross-contamination of produce crops because of cattle shedding of this human pathogen.

Complex Regulation of Symbiotic Functions Is Coordinated by MucR and Quorum Sensing in Sinorhizobium meliloti

In Sinorhizobium meliloti, the production of exopolysaccharides such as succinoglycan and exopolysaccharide II (EPS II) enables the bacterium to invade root nodules on Medicago sativa and establish a nitrogen-fixing symbiosis. While extensive research has focused on succinoglycan, less is known concerning the regulation of EPS II or the mechanism by which it mediates entrance into the host plant. Previously, we reported that the ExpR/Sin quorum-sensing system is required to produce the symbiotically active low-molecular-weight fraction of this exopolysaccharide. Here, we show that this system induces EPS II production by increasing expression of the expG-expC operon, encoding both a transcriptional regulator (ExpG) and a glycosyl transferase (ExpC). ExpG derepresses EPS II production at the transcriptional level from MucR, a RosR homolog, while concurrently elevating expression of expC, resulting in the synthesis of the low-molecular-weight form. While the ExpR/Sin system abolishes the role of MucR on EPS II production, it preserves a multitude of other quorum-sensing-independent regulatory functions which promote the establishment of symbiosis. In planktonic S. meliloti, MucR properly coordinates a diverse set of bacterial behaviors by repressing a variety of genes intended for expression during symbiosis and enhancing the bacterial ability to induce root nodule formation. Quorum sensing precisely modulates the functions of MucR to take advantage of both the production of symbiotically active EPS II as well as the proper coordination of bacterial behavior required to promote symbiosis.

Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas

Some members of the moderately halophilic genus Halomonas, such as H. eurihalina, H. maura, H. ventosae and H. anticariensis, produce exopolysaccharides with applications in many industrial fields. We report here that these four species also produce autoinducer molecules that are involved in the cell-to-cell signaling process known as quorum sensing. By using the N-acyl homoserine lactone (AHL) indicator strains Agrobacterium tumefaciens NTL4 (pZRL4) and Chromobacterium violaceum CV026, we discovered that all the Halomonas strains examined synthesize detectable AHL signal molecules. The synthesis of these compounds was growth-phase dependent and maximal activity was reached during the late exponential to stationary phases. One of these AHLs seems to be synthesized only in the stationary phase. Some of the AHLs produced by H. anticariens FP35T were identified by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry as N-butanoyl homoserine lactone (C4-HL), N-hexanoyl homoserine lactone (C6-HL), N-octanoyl homoserine lactone (C8-HL) and N-dodecanoyl homoserine lactone (C12-HL). This study suggests that quorum sensing may also play an important role in extreme environments.

Exopolysaccharides and Rhizobium Meliloti-Alfalfa Interactions

Rhizobium meliloti strain Rm 1021 excretes two exopolysaccharides. The first of these, succinoglycan (EPS I), is a high molecular weight polymer (Gravanis et al., 1987) composed of polymerized octasaccharide subunits. Each octasaccharide consists of a backbone of three glucoses and one galactose residue, a side chain of four glucose residues, and 1-carboxyethylidene (pyruvate), acetyl and succinyl modifications in a ratio of approximately 1:1:1. The structure of succinoglycan was determined through studies in a variety of labs (Bjorndal et al., 1971; Hisamatsu et al., 1980; Jansson et al., 1977; Aman et al., 1981). Recently in a collaboration with V. Reinhold’s lab the positions of the acetyl and succinyl modifications were determined (see figure) (B. Reinhold et al., in preparation). Before polymerization, the subunits are assembled on a lipid carrier, and in vitro labeling studies show that the sequence of assembly is, first galactose and β- 1,3 glucose, then the rest of the glucose residues and the other substituents (Tolmasky et al., 1980, 1982). R. meliloti strain Rm1021 also has the capacity to make a second exopolysaccharide, EPS II, whose structure is also shown (Glazebrook and Walker, 1989; Zhan et al., 1989).

A perspective on promoting diversity in the biomedical research workforce: The national heart, lung, and blood institute's pri de program

Aspiring junior investigators from groups underrepresented in the biomedical sciences face various challenges as they pursue research independence. However, the biomedical research enterprise needs their participation to effectively address critical research issues such as health disparities and health inequities. In this article, we share a research education and mentoring initiative that seeks to address this challenge: Programs to Increase Diversity among Individuals Engaged in Health Related Research (PRIDE), funded by the National Heart, Lung, and Blood Institute (NHLBI). This longitudinal research-education and mentoring program occurs through summer institute programs located at US-based academic institutions. Recruited participants are exposed to didactic and lab-based research-skill enhancement experiences, with year-round mentoring over the course of two years. Mentor-mentee matching is based on shared research interests to promote congruence and to enhance skill acquisition. Program descriptions and sample narratives of participants perceptions of PRIDEs impact on their career progress are showcased. Additionally, we highlight the overall program design and structure of four of seven funded summer institutes that focus on cardiovascular disease, related conditions, and health disparities. Mentees testimonials about the value of the PRIDE mentoring approach in facilitating career development are also noted. Meeting the clinical and research needs of an increasingly diverse US population is an issue of national concern. The PRIDE initiative, which focuses on increasing research preparedness and professional development of groups underrepresented in the biomedical research workforce, with an emphasis on mentoring as the critical approach, provides a robust model that is impacting the careers of future investigators.

Mentored training to increase diversity among faculty in the biomedical sciences: The NHL BI Summer Institute Programs to Increase Diversity (SI PID ) and the Programs to Increase Diversity among Individuals Engaged in Health-related Research (PRIDE )

OBJECTIVEnTo report baseline characteristics of junior-level faculty participants in the Summer Institute Programs to Increase Diversity (SIPID) and the Programs to Increase Diversity among individuals engaged in Health-Related Research (PRIDE), which aim to facilitate participants career development as independent investigators in heart, lung, blood, and sleep research.nnnDESIGN AND SETTINGnJunior faculty from groups underrepresented in the biomedical-research workforce attended two, 2-3 week, annual summer research-education programs at one of six sites. Programs provided didactic and/or laboratory courses, workshops to develop research, writing and career-development skills, as well as a mentoring component, with regular contact maintained via phone, email and webinar conferences. Between summer institutes, trainees participated in a short mid-year meeting and an annual scientific meeting. Participants were surveyed during and after SIPID/PRIDE to evaluate program components.nnnPARTICIPANTSnJunior faculty from underrepresented populations across the United States and Puerto Rico participated in one of three SIPID (2007-2010) or six PRIDE programs (2011-2014).nnnRESULTSnOf 204 SIPID/PRIDE participants, 68% were female; 67% African American and 27% Hispanic/Latino; at enrollment, 75% were assistant professors and 15% instructors, with most (96%) on non-tenure track. Fifty-eight percent had research doctorates (PhD, ScD) and 42% had medical (MD, DO) degrees. Mentees feedback about the program indicated skills development (eg, manuscript and grant writing), access to networking, and mentoring were the most beneficial elements of SIPID and PRIDE programs. Grant awards shifted from primarily mentored research mechanisms to primarily independent investigator awards after training.nnnCONCLUSIONSnMentees reported their career development benefited from SIPID and PRIDE participation.

A Novel Denitrification Regulator, Adr, Mediates Denitrification Under Aerobic Conditions in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil dwelling bacteria capable of forming a symbiotic relationship with several legume hosts. Once symbiosis is established, S. meliloti fixes atmospheric nitrogen into nitrogenated compounds, thus carrying out an important step in the nitrogen cycle. S. meliloti is also capable of the reverse process, denitrification, the reduction of nitrate and nitrite to nitrogen gas. In this study we have identified a novel regulator of denitrification in S. meliloti, Adr, which affects the expression of the denitrification genes in aerobically grown cultures. Analysis of the Adr sequence reveals a LuxR-like quorum sensing regulator, however, it does not respond to the known quorum sensing signals produced by S. meliloti. Additionally, we show that FixJ, the major regulator of denitrification and microaerobic respiration in S. meliloti, is active under our growth conditions. Comparison of the FixJ microarray to our Adr microarray shows a significant overlap between the two regulons. We also show that while Adr is not necessary for symbiotic nitrogen fixation, a functional copy of this regulator confers a competitive advantage to S. meliloti during host invasion. Our findings suggest that Adr is a new type of denitrification regulator and that it acts at the same regulatory level as FixJ. Importance Rhizobia contribute to the nitrogen cycle by fixing atmospheric nitrogen to nitrogenated compounds and by denitrification, the reduction nitrate and nitrite to nitrogen gas. Denitrification enhances the survival of Sinorhizobium meliloti in the various environments it may encounter, such as free-living conditions in the rhizosphere, during invasion of the plant host, and after a symbiotic relationship has been established. Oxygen concentration is the typical signal for denitrification gene expression. Recent studies of low oxygen cultures of S. meliloti have outlined the regulation structure for denitrification. In this study, we examine the regulation of denitrification in aerobically grown S. meliloti cultures. Understanding how S. meliloti responds to various oxygen concentrations will result in a more complete picture of denitrification regulation in this agriculturally important organism and the impact of denitrification on the soil microbiome as a whole.

Enhancing diversity in the hematology biomedical research workforce: A mentoring program to improve the odds of career success for early stage investigators

The necessity for greater racial and ethnic diversity in the US biomedical research workforce is evident, however many challenges must be overcome to achieve this formidable goal. Historically, underrepresented minority (URM) groups are the most rapidly growing segment of the US population and there is an urgent need to ensure that scientific talent among these groups is recognized, mentored and actively supported. For example, in 2010, Hispanics/Latinos, Blacks/African Americans, and American Indians/Alaskan Natives represented 29.8% of the US population, yet only 4.8% of National Institutes of Health (NIH) research project grants (RPG) were awarded to URM principal investigators. A study by Ginther et al. revealed that PhD-trained African American applicants are 13.2% less likely than White applicants to be awarded RPG. While the NIH is the largest research funding agency in the world, it has not achieved proportional representation of URM investigators in the US biomedical research workforce. Likewise, the imperative to increase diversity is justified by inequities in access to health care and health outcomes. Improving these statistics will require interventions such as the introduction of innovative training models involving dedicated mentoring by established NIH-funded investigators, which are tested by rigorous evaluations. Analysis of the results from these training models will demonstrate the extent to which current interventions increase representation of URM groups in the biomedical research enterprise. Recently, the NIH established the National Research Mentoring Network (NRMN) to improve the success of URM investigators with the goal of diversifying our nation’s biomedical research workforce. There is a paucity of published data demonstrating that structured research mentoring programs promote grant funding, and professional development of early stage investigators (ESI). To provide expanded mentoring support for URM investigators, in 2006 the National Heart, Lung, and Blood Institute (NHLBI) established the Summer Institute Program to Increase Diversity (SIPID), and subsequently the PRIDE (Program to Increase Diversity Among Individuals Engaged in HealthRelated Research) Program. The scope of the PRIDE Program consists of seven academic sites, each focused on a specific research area. The objective of all programs is to provide intense research and career development mentorship coordinated through a central PRIDE Coordination Core (PCC) described recently. The PRIDE Program at Augusta University is focused on “Functional and Translational Genomics of Blood Disorders” (FTG-PRIDE), and has been funded by NHLBI since 2006. During each funding period, 3 cohorts of 6–10 mentees were recruited after the FTG-PRIDE Admissions Committee reviewed and ranked applications. Top candidates were interviewed to ensure the program requirements were fulfilled and a suitable mentor-mentee dyad could be established. While many approaches can be taken to address the inequity of URM representation in the US biomedical research workforce, the objectives of the PRIDE Program has principally focused on training ESI in grant writing skills to achieve extramural funding and expanded research-related technical skills. To evaluate program effectiveness, the PCC developed and administered a series of evaluation questionnaires during the 2-year training period and for 8 years after training completion. Mentee demographics and career development-related outcomes have been collected since 2006. To assess research self-efficacy, a 19item Clinical Research Appraisal Inventory (CRAI-19) previously validated in the PRIDE Program, is completed annually. To accomplish these objectives, the FTG-PRIDE Program leadership organizes two consecutive Summer Institutes at Augusta University, each lasting 2 to 3 weeks. In addition, a mid-year face-to-face meeting is attended by each mentee with their primary mentor to review research progress, and to update skills and adopt new technologies. Mentees are also required to attend the National PRIDE Meeting convened annually in Bethesda, MD. The purpose of this meeting is to provide opportunities for trainees to interact with NHLBI program staff, present their research to other trainees, mentors and teaching faculty from all PRIDE programs, and establish research collaborations. During the period 2006–2017, we trained 76 mentees in the FTGPRIDE Program (Supporting Information Table S1) under Institutional Review Board approval and informed consent for data collection by the PCC. Since the last cohort of participants in PRIDE 2 has not completed its second year of training, the data presented here are limited to the 48 mentees trained in SIPID and PRIDE 1. Of this group, 6 mentees were excluded from the analysis due to withdrawal from the program, matriculation into a second PRIDE Program, or noncompliance with program evaluations. As a result, the outcomes of 42 evaluable mentees are described in this report. The design of the 2-year FTG-PRIDE Program is summarized in Supporting Information Figure S1. The first Summer Institute commences with a Welcome Ceremony attended by mentees, mentors, teaching faculty, and program leadership along with high-level administrators from Augusta University. After orientation to review program requirements, one-on-one mentee/mentor meetings are held to initiate the