Priority effects in microbiome assembly.

Advances in next-generation sequencing have enabled the widespread measurement of microbiome composition across systems and over the course of microbiome assembly. Despite substantial progress in understanding the deterministic drivers of community composition, the role of historical contingency remains poorly understood. The establishment of new species in a community can depend on the order and/or timing of their arrival, a phenomenon known as a priority effect. Here, we review the mechanisms of priority effects and evidence for their importance in microbial communities inhabiting a range of environments, including the mammalian gut, the plant phyllosphere and rhizosphere, soil, freshwaters and oceans. We describe approaches for the direct testing and prediction of priority effects in complex microbial communities and illustrate these with re-analysis of publicly available plant and animal microbiome datasets. Finally, we discuss the shared principles that emerge across study systems, focusing on eco-evolutionary dynamics and the importance of scale. Overall, we argue that predicting when and how current community state impacts the success of newly arriving microbial taxa is crucial for the management of microbiomes to sustain ecological function and host health. We conclude by discussing outstanding conceptual and practical challenges that are faced when measuring priority effects in microbiomes.

Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation Under Ionic Liquid Stress.

Corynebacterium glutamicum is an ideal microbial chassis for production of valuable bioproducts including amino acids and next generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50 mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of an C. glutamicum strain engineered to produce IP.

Engineering Pseudomonas putida for efficient aromatic conversion to bioproduct using high throughput screening in a bioreactor.

Pseudomonas putida KT2440 is an emerging biomanufacturing host amenable for use with renewable carbon streams including aromatics such as para-coumarate. We used a pooled transposon library disrupting nearly all (4,778) non-essential genes to characterize this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays. Assessing differential fitness values by monitoring changes in mutant strain abundance identified 33 gene mutants with improved fitness across multiple stirred-tank bioreactor formats. Twenty-one deletion strains from this subset were reconstructed, including GacA, a regulator, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Thirteen deletion strains with roles in varying cellular functions were evaluated for conversion of para-coumarate, to a heterologous bioproduct, indigoidine. Several mutants, such as the ΔgacA strain improved fitness in a bioreactor by 35 fold and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g, 23% of maximum theoretical yield) from para-coumarate as the carbon source.

Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale.

High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. We achieve 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose). These phenotypes are maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr®, and 2-L bioreactors).

Iron Supplementation Eliminates Antagonistic Interactions Between Root-Associated Bacteria.

The rhizosphere microbiome (rhizobiome) plays a critical role in plant health and development. However, the processes by which the constituent microbes interact to form and maintain a community are not well understood. To investigate these molecular processes, we examined pairwise interactions between 11 different microbial isolates under select nutrient-rich and nutrient-limited conditions. We observed that when grown with media supplemented with 56 mM glucose, two microbial isolates were able to inhibit the growth of six other microbes. The interaction between microbes persisted even after the antagonistic microbe was removed, upon exposure to spent media. To probe the genetic basis for these antagonistic interactions, we used a barcoded transposon library in a proxy bacterium, Pseudomonas putida, to identify genes which showed enhanced sensitivity to the antagonistic factor(s) secreted by Acinetobacter sp. 02. Iron metabolism-related gene clusters in P. putida were implicated by this systems-level analysis. The supplementation of iron prevented the antagonistic interaction in the original microbial pair, supporting the hypothesis that iron limitation drives antagonistic microbial interactions between rhizobionts. We conclude that rhizobiome community composition is influenced by competition for limiting nutrients, with implications for growth and development of the plant.

Production of tetra-methylpyrazine using engineered Corynebacterium glutamicum.

Corynebacterium glutamicum ATCC 13032 is an established and industrially-relevant microbial host that has been utilized for the expression of many desirable bioproducts. Tetra-methylpyrazine (TMP) is a naturally occurring alkylpyrazine with broad applications spanning fragrances to resins. We identified an engineered strain of C. glutamicum which produces 5 â€‹g/L TMP and separately, a strain which can co-produce both TMP and the biofuel compound isopentenol. Ionic liquids also stimulate TMP production in engineered strains. Using a fed batch-mode feeding strategy, ionic liquid stimulated strains produced 2.2 â€‹g/L of tetra-methylpyrazine. We show that feedback from a specific heterologous gene pathway on host physiology leads to acetoin accumulation and the production of TMP.

Complete Genome Sequence of Agrobacterium sp. Strain 33MFTa1.1, Isolated from Thlaspi arvense Roots.

Agrobacterium sp. strain 33MFTa1.1 was isolated for functional host-microbe interaction studies from the Thlaspi arvense root-associated microbiome. The complete genome is comprised of a circular chromosome of 2,771,937 bp, a linear chromosome of 2,068,443 bp, and a plasmid of 496,948 bp, with G+C contents of 59%, 59%, and 58%, respectively.

Rhizobacteria Mediate the Phytotoxicity of a Range of Biorefinery-Relevant Compounds.

Advances in engineering biology have expanded the list of renewable compounds that can be produced at scale via biological routes from plant biomass. In most cases, these chemical products have not been evaluated for effects on biological systems, defined in the present study as bioactivity, that may be relevant to their manufacture. For sustainable chemical and fuel production, the industry needs to transition from fossil to renewable carbon sources, resulting in unprecedented expansion in the production and environmental distribution of chemicals used in biomanufacturing. Further, although some chemicals have been assessed for mammalian toxicity, environmental and agricultural hazards are largely unknown. We assessed 6 compounds that are representative of the emerging biofuel and bioproduct manufacturing process for their effect on model plants (Arabidopsis thaliana, Sorghum bicolor) and show that several alter plant seedling physiology at submillimolar concentrations. However, these responses change in the presence of individual bacterial species from the A. thaliana root microbiome. We identified 2 individual microbes that change the effect of chemical treatment on root architecture and a pooled microbial community with different effects relative to its constituents individually. The present study indicates that screening industrial chemicals for bioactivity on model organisms in the presence of their microbiomes is important for biologically and ecologically relevant risk analyses. Environ Toxicol Chem 2019;38:1911-1922. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Engineering Corynebacterium glutamicum to produce the biogasoline isopentenol from plant biomass hydrolysates.

BACKGROUND: Many microbes used for the rapid discovery and development of metabolic pathways have sensitivities to final products and process reagents. Isopentenol (3-methyl-3-buten-1-ol), a biogasoline candidate, has an established heterologous gene pathway but is toxic to several microbial hosts. Reagents used in the pretreatment of plant biomass, such as ionic liquids, also inhibit growth of many host strains. We explored the use of Corynebacterium glutamicum as an alternative host to address these constraints. RESULTS: We found C. glutamicum ATCC 13032 to be tolerant to both the final product, isopentenol, as well to three classes of ionic liquids. A heterologous mevalonate-based isopentenol pathway was engineered in C. glutamicum. Targeted proteomics for the heterologous pathway proteins indicated that the 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein, HmgR, is a potential rate-limiting enzyme in this synthetic pathway. Isopentenol titers were improved from undetectable to 1.25 g/L by combining three approaches: media optimization; substitution of an NADH-dependent HmgR homolog from Silicibacter pomeroyi; and development of a C. glutamicum ∆poxB ∆ldhA host chassis. CONCLUSIONS: We describe the successful expression of a heterologous mevalonate-based pathway in the Gram-positive industrial microorganism, C. glutamicum, for the production of the biogasoline candidate, isopentenol. We identified critical genetic factors to harness the isopentenol pathway in C. glutamicum. Further media and cultivation optimization enabled isopentenol production from sorghum biomass hydrolysates.

Restoration of biofuel production levels and increased tolerance under ionic liquid stress is enabled by a mutation in the essential Escherichia coli gene cydC.

BACKGROUND: Microbial production of chemicals from renewable carbon sources enables a sustainable route to many bioproducts. Sugar streams, such as those derived from biomass pretreated with ionic liquids (IL), provide efficiently derived and cost-competitive starting materials. A limitation to this approach is that residual ILs in the pretreated sugar source can be inhibitory to microbial growth and impair expression of the desired biosynthetic pathway. RESULTS: We utilized laboratory evolution to select Escherichia coli strains capable of robust growth in the presence of the IL, 1-ethyl-3-methyl-imidizolium acetate ([EMIM]OAc). Whole genome sequencing of the evolved strain identified a point mutation in an essential gene, cydC, which confers tolerance to two different classes of ILs at concentrations that are otherwise growth inhibitory. This mutation, cydC-D86G, fully restores the specific production of the bio-jet fuel candidate D-limonene, as well as the biogasoline and platform chemical isopentenol, in growth medium containing ILs. Similar amino acids at this position in cydC, such as cydC-D86V, also confer tolerance to [EMIM]OAc. We show that this [EMIM]OAc tolerance phenotype of cydC-D86G strains is independent of its wild-type function in activating the cytochrome bd-I respiratory complex. Using shotgun proteomics, we characterized the underlying differential cellular responses altered in this mutant. While wild-type E. coli cannot produce detectable amounts of either product in the presence of ILs at levels expected to be residual in sugars from pretreated biomass, the engineered cydC-D86G strains produce over 200 mg/L D-limonene and 350 mg/L isopentenol, which are among the highest reported titers in the presence of [EMIM]OAc. CONCLUSIONS: The optimized strains in this study produce high titers of two candidate biofuels and bioproducts under IL stress. Both sets of production strains surpass production titers from other IL tolerant mutants in the literature. Our application of laboratory evolution identified a gain of function mutation in an essential gene, which is unusual in comparison to other published IL tolerant mutants.

Improving membrane protein expression and function using genomic edits.

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. We hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression of five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.

Latent typologies of posttraumatic stress disorder in World Trade Center responders.

Posttraumatic stress disorder (PTSD) is a debilitating and often chronic psychiatric disorder. Following the 9/11/2001 World Trade Center (WTC) attacks, thousands of individuals were involved in rescue, recovery and clean-up efforts. While a growing body of literature has documented the prevalence and correlates of PTSD in WTC responders, no study has evaluated predominant typologies of PTSD in this population. Participants were 4352 WTC responders with probable WTC-related DSM-IV PTSD. Latent class analyses were conducted to identify predominant typologies of PTSD symptoms and associated correlates. A 3-class solution provided the optimal representation of latent PTSD symptom typologies. The first class, labeled "High-Symptom (n = 1,973, 45.3%)," was characterized by high probabilities of all PTSD symptoms. The second class, "Dysphoric (n = 1,371, 31.5%)," exhibited relatively high probabilities of emotional numbing and dysphoric arousal (e.g., sleep disturbance). The third class, "Threat (n = 1,008, 23.2%)," was characterized by high probabilities of re-experiencing, avoidance and anxious arousal (e.g., hypervigilance). Compared to the Threat class, the Dysphoric class reported a greater number of life stressors after 9/11/2001 (OR = 1.06). The High-Symptom class was more likely than the Threat class to have a positive psychiatric history before 9/11/2001 (OR = 1.7) and reported a greater number of life stressors after 9/11/2001 (OR = 1.1). The High-Symptom class was more likely than the Dysphoric class, which was more likely than the Threat class, to screen positive for depression (83% > 74% > 53%, respectively), and to report greater functional impairment (High-Symptom > Dysphoric [Cohen d = 0.19], Dysphoric > Threat [Cohen d = 0.24]). These results may help inform assessment, risk stratification, and treatment approaches for PTSD in WTC and disaster responders.

Risk, coping and PTSD symptom trajectories in World Trade Center responders.

Trajectories of disaster-related posttraumatic stress disorder (PTSD) symptoms are often heterogeneous, and associated with common and unique risk factors, yet little is known about potentially modifiable psychosocial characteristics associated with low-symptom and recovering trajectories in disaster responders. A total of 4487 rescue and recovery workers (1874 police and 2613 non-traditional responders) involved during and in the aftermath of the unprecedented World Trade Center (WTC) attacks, were assessed an average of 3, 6, 8, and 12 years post-9/11/2001. Among police responders, WTC-related PTSD symptoms were characterized by four trajectories, including no/low-symptom (76.1%), worsening (12.1%), improving (7.5%), and chronic (4.4%) trajectories. In non-traditional responders, a five-trajectory solution was optimal, with fewer responders in a no/low-symptom trajectory (55.5%), and the remainder in subtly worsening (19.3%), chronic (10.8%), improving (8.5%), and steeply worsening (5.9%) trajectories. Consistent factors associated with symptomatic PTSD trajectories across responder groups included Hispanic ethnicity, pre-9/11 psychiatric history, greater WTC exposure, greater medical illness burden, life stressors and post-9/11 traumas, and maladaptive coping (e.g., substance use, avoidance coping). Higher perceived preparedness, greater sense of purpose in life, and positive emotion-focused coping (e.g., positive reframing, acceptance) were negatively associated with symptomatic trajectories. Findings in this unique cohort indicate considerable heterogeneity in WTC-related PTSD symptom trajectories over 12 years post-9/11/2001, with lower rates of elevated PTSD symptoms in police than in non-traditional responders. They further provide a comprehensive risk prediction model of PTSD symptom trajectories, which can inform prevention, monitoring, and treatment efforts in WTC and other disaster responders.

Cancer incidence in world trade center rescue and recovery workers, 2001-2008.

BACKGROUND: World Trade Center (WTC) rescue and recovery workers were exposed to a complex mix of pollutants and carcinogens. OBJECTIVE: The purpose of this investigation was to evaluate cancer incidence in responders during the first 7 years after 11 September 2001. METHODS: Cancers among 20,984 consented participants in the WTC Health Program were identified through linkage to state tumor registries in New York, New Jersey, Connecticut, and Pennsylvania. Standardized incidence ratios (SIRs) were calculated to compare cancers diagnosed in responders to predicted numbers for the general population. Multivariate regression models were used to estimate associations with degree of exposure. RESULTS: A total of 575 cancers were diagnosed in 552 individuals. Increases above registry-based expectations were noted for all cancer sites combined (SIR = 1.15; 95% CI: 1.06, 1.25), thyroid cancer (SIR = 2.39; 95% CI: 1.70, 3.27), prostate cancer (SIR = 1.21; 95% CI: 1.01, 1.44), combined hematopoietic and lymphoid cancers (SIR = 1.36; 95% CI: 1.07, 1.71), and soft tissue cancers (SIR = 2.26; 95% CI: 1.13, 4.05). When restricted to 302 cancers diagnosed ≥ 6 months after enrollment, the SIR for all cancers decreased to 1.06 (95% CI: 0.94, 1.18), but thyroid and prostate cancer diagnoses remained greater than expected. All cancers combined were increased in very highly exposed responders and among those exposed to significant amounts of dust, compared with responders who reported lower levels of exposure. CONCLUSION: Estimates should be interpreted with caution given the short follow-up and long latency period for most cancers, the intensive medical surveillance of this cohort, and the small numbers of cancers at specific sites. However, our findings highlight the need for continued follow-up and surveillance of WTC responders.

The burden of full and subsyndromal posttraumatic stress disorder among police involved in the World Trade Center rescue and recovery effort.

BACKGROUND: This study examined the prevalence, correlates, and perceived mental healthcare needs associated with subsyndromal PTSD in police involved in the World Trade Center (WTC) rescue and recovery effort. METHODS: A total of 8466 police completed an interview/survey as part of the WTC Medical monitoring and Treatment Program an average of four years after 9/11/2001. RESULTS: The past month prevalence of full and subsyndromal WTC-related PTSD was 5.4% and 15.4%, respectively. Loss of someone or knowing someone injured on 9/11 (odds ratios [ORs]=1.56-1.86), pre-9/11 stressors (ORs=1.30-1.50), family support (ORs=0.83-0.94), and union membership (ORs=0.50-0.52) were associated with both full and subsyndromal PTSD. Exposure to the dust cloud (OR=1.36), performing search and rescue work (OR=1.29), and work support (OR=0.89) were additionally associated with subsyndromal PTSD. Rates of comorbid depression, panic disorder, and alcohol use problems (ORs=3.82-41.74), and somatic symptoms and functional difficulties (ORs=1.30-1.95) were highest among police with full PTSD, with intermediate rates among police with subsyndromal PTSD (ORs=2.93-7.02; and ORs=1.18-1.60, respectively). Police with full and subsyndromal PTSD were significantly more likely than controls to report needing mental healthcare (41.1% and 19.8%, respectively, versus 6.8% in trauma controls). CONCLUSIONS: These results underscore the importance of a more inclusive and dimensional conceptualization of PTSD, particularly in professions such as police, as operational definitions and conventional screening cut-points may underestimate the psychological burden for this population. Accordingly, psychiatric clinicians should assess for disaster-related subsyndromal PTSD symptoms in disaster response personnel.

Increased rates of asthma among World Trade Center disaster responders.

BACKGROUND: Studies have documented high rates of asthma symptoms among responders to the World Trade Center (WTC) disaster. However, whether there are increased rates of asthma among responders compared to the general population is unknown. METHODS: The study population consisted of a prospective cohort of 20,834 responders participating in the WTC Medical Monitoring and Treatment Program between July 2002 and December 2007. We calculated prevalence and standardized morbidity ratios (SMRs) of lifetime asthma and 12-month asthma (defined as ≥1 attacks in the prior 12 months) among WTC responders. The comparison population consisted of >200,000 adults who completed the National Health Interview Survey in 2000 (for pre-9/11 comparisons) and between 2002 and 2007 (for post-9/11 comparisons). RESULTS: WTC responders were on average 43 ± 9 years old, 86% male, 59% white, and 42% had an occupation in protective services. The lifetime prevalence of asthma in the general population was relatively constant at about 10% from 2000 to 2007. However, among WTC responders, lifetime prevalence increased from 3% in 2000, to 13% in 2002, and 19% in 2007. The age-adjusted overall SMR for lifetime asthma among WTC responders was 1.8 (95% CI: 1.8-1.9) for men and 2.0 (95% CI: 1.9-2.1) for women. Twelve-month asthma was also more frequent among WTC responders compared to the general population (SMR 2.4, 95% CI: 2.2-2.5) for men and 2.2 (95% CI: 2.0-2.5) for women. CONCLUSIONS: WTC responders are at an increased risk of asthma as measured by lifetime prevalence or active disease.

Persistence of multiple illnesses in World Trade Center rescue and recovery workers: a cohort study.

BACKGROUND: More than 50,000 people participated in the rescue and recovery work that followed the Sept 11, 2001 (9/11) attacks on the World Trade Center (WTC). Multiple health problems in these workers were reported in the early years after the disaster. We report incidence and prevalence rates of physical and mental health disorders during the 9 years since the attacks, examine their associations with occupational exposures, and quantify physical and mental health comorbidities. METHODS: In this longitudinal study of a large cohort of WTC rescue and recovery workers, we gathered data from 27,449 participants in the WTC Screening, Monitoring, and Treatment Program. The study population included police officers, firefighters, construction workers, and municipal workers. We used the Kaplan-Meier procedure to estimate cumulative and annual incidence of physical disorders (asthma, sinusitis, and gastro-oesophageal reflux disease), mental health disorders (depression, post-traumatic stress disorder [PTSD], and panic disorder), and spirometric abnormalities. Incidence rates were assessed also by level of exposure (days worked at the WTC site and exposure to the dust cloud). FINDINGS: 9-year cumulative incidence of asthma was 27·6% (number at risk: 7027), sinusitis 42·3% (5870), and gastro-oesophageal reflux disease 39·3% (5650). In police officers, cumulative incidence of depression was 7·0% (number at risk: 3648), PTSD 9·3% (3761), and panic disorder 8·4% (3780). In other rescue and recovery workers, cumulative incidence of depression was 27·5% (number at risk: 4200), PTSD 31·9% (4342), and panic disorder 21·2% (4953). 9-year cumulative incidence for spirometric abnormalities was 41·8% (number at risk: 5769); three-quarters of these abnormalities were low forced vital capacity. Incidence of most disorders was highest in workers with greatest WTC exposure. Extensive comorbidity was reported within and between physical and mental health disorders. INTERPRETATION: 9 years after the 9/11 WTC attacks, rescue and recovery workers continue to have a substantial burden of physical and mental health problems. These findings emphasise the need for continued monitoring and treatment of the WTC rescue and recovery population. FUNDING: Centers for Disease Control and Prevention and National Institute for Occupational Safety and Health.

"Sarcoid like" granulomatous pulmonary disease in World Trade Center disaster responders.

BACKGROUND: More than 20,000 responders have been examined through the World Trade Center (WTC) Medical Monitoring and Treatment Program since September 11, 2001. Studies on WTC firefighters have shown elevated rates of sarcoidosis. The main objective of this study was to report the incidence of "sarcoid like" granulomatous pulmonary disease in other WTC responders. METHODS: Cases of sarcoid like granulomatous pulmonary disease were identified by: patient self-report, physician report and ICD-9 codes. Each case was evaluated by three pulmonologists using the ACCESS criteria and only "definite" cases are reported. RESULTS: Thirty-eight patients were classified as "definite" cases. Six-year incidence was 192/100,000. The peak annual incidence of 54 per 100,000 person-years occurred between 9/11/2003 and 9/11/2004. Incidence in black responders was nearly double that of white responders. Low FVC was the most common spirometric abnormality. CONCLUSIONS: Sarcoid like granulomatous pulmonary disease is present among the WTC responders. While the incidence is lower than that reported among firefighters, it is higher than expected.