Progress 01/01/16 to 12/31/18
Outputs
Target Audience: Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? How have the results been disseminated to communities of interest?We have communicated information on the project at a number of scientific meetings What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Comparison of transcriptomic responses between naïve and adapted cultures Comparison of the transcriptional profiles between the naïve and reintroduced cultures to monensin concentrations to which they had previously shown adaptation was conducted using the RNA sequencing analysis as described above. In comparative transcriptomes of S. bovis JB1 between the naïve isolates exposed to monensin and the reverted ones to 75 μg/ml, up-regulation of the genes involved in the energy metabolism and the acid stress responses were observed, which was the same as the previous data, but at a moderated level. Uniquely, in the reverted transcriptomes, genes encoding drug resistance transporters, transmembrane secretion effector, modification of cell wall proteins, and bile acid:sodium symporters were actively expressed. A shift in the transcriptional profile was also observed in the reverted R. albus 7 culture. While most of the previously up-regulated genes in the MIC test of the naïve cultures were moderately overexpressed, new sets of genes encoding DDE transposase (RUMAL_RS12365) and protein translocase subunit SecD and SecF (RUMAL_RS01840 and RUMAL_RS01845) were strongly expressed in the reverted cultures. Among the resistant strains, the reverted P. bryantii B14 culture highly expressed genes encoding conjugative transposon proteins, which was a similar profile to that of the naïve culture exposed to monensin. The rest of the resistant strains showed relatively unique transcriptional profiles compared to those of the naïve culture. For instance, the reverted culture of F. succinogenes S85 overexpressed genes encoding biopolymer and MFS transporters (FSU_RS01105, FSU_RS03330, FSU_RS10620, FSU_RS10945, and FSU_RS12565) together with membrane ion channels (FSU_RS10615 and FSU_RS00080) and molecular chaperone proteins (FSU_RS00885, FSU_RS02170, and FSU_RS13415). The transcriptomic results of the reverted M. elsdenii T81 and S. ruminantium HD4 commonly overexpressed the genes encoding various sodium symporters as well as genes encoding a sulfate-transport system and cell wall modifying proteins. Effect of monensin on primary fermentation products Comparison of end product metabolites between naïve and adapted cultures of S. bovis JB1 and P. bryantii B14 was conducted to investigate how adaptation to monensin affected alteration of the primary fermentation products. For S. bovis JB1, the supernatant samples were collected from the naïve cultures grown at 0.0 μg/ml and 4.0 μg/ml, respectively, and the adapted cultures grown at 25 μg/ml over the growth. For P. bryantii B14, the samples were collected from the naïve cultures grown 0.0 μg/ml and 4.0 μg/ml, respectively, and the adapted cultures grown at 75 μg/ml. The naïve S. bovis JB1 culture at 0.0 μg/ml consumed fourg glucose/L in 4 hours and produced 2.96 g lactate/L while ODMAX reached to 1.16 with growth rate 1.02 hr-1, and the other naïve cultures at 4.0 μg/ml consumed all glucose in eighthours and produced 2.43 g lactate/L. Their growth only reached ODMAX 0.48 with a growth rate of0.31 hr-1. On the other hand, the adapted cultures grown at 25 μg/ml indicated a similar consumption and production pattern to the naïve culture grown at 4.0 μg/ml. The adapted cultures consumed the glucose completely in sevenhours and produced 2.54 g lactate/L while ODMAX reached 0.54 with a growth rate, 0.35 hr-1. The naïve P. bryantii B14 cultures at 0.0 μg/ml completely consumed 4 g glucose/L in tenhours and produced 1.40 g succinate/L while ODMAX reached to 1.4 with growth rate 0.37 hr-1. The naïve cultures grown at 4.0 μg/ml and the adapted cultures at 75 μg/ml consumed the glucose in sixteenand twentyhours, respectively, and the amount of succinate productions and the ODMAX were the same as those of the naïve culture grown at 0.0 μg/ml. These results suggested that the alteration effect on the metabolites by increasing monensin concentration in the media was alleviated in the adapted cultures of both strains. Effect of monensin on energy transduction To determine whether monensin adaptation could affect the formation of proton gradient in the sensitive and resistant strains, the membrane potential (ΔΨ), the transmembrane pH gradient (ΔpH), and the proton motive force (ΔP) were measured in the naïve and adapted cultures of S. bovis JB1 and P. bryantii B14. Among the S. bovis JB1 cultures, the naïve cultures at 0.0 μg/ml of monensin media maintained an internal pH of 7.64 while an external pH was 6.63, which yielded the ZΔpH of 62.01 mV. The difference in internal and external pH in the naïve culture at 4.0 μg/ml yielded the ZΔpH of 35.01 mV. This indicated that the intracellular alkalinity decreased when the cultures were exposed to monensin. However, in the adapted culture, the difference between the internal and external pHs was similar to that of the naïve cultures grown at 0.0 μg/ml. The ΔΨs in the naïve cultures at 0.0 and 4.0 μg/ml and in the adapted cultures at 25.0 μg/ml were -115.21, -100.14, and -96.50 mV, respectively. Taken together, the ΔPs in the naïve cultures at 0.0 and 4.0 μg/ml and in the adapted cultures at 25.0 μg/ml were -177.22, -135.60, and -157.54 mV, respectively. This indicated that effect of the monensin exposure to the adapted cultures, even grown at the higher monensin concentration, was reduced when it was compared to the naïve cultures exposed to the monensin. On the other hand, the difference between ΔpH in the P. bryantii B14 naïve cultures grown in the presence and absence of monensin was not significant, neither was shown an increased ΔpH in the adapted culture at 75.0 μg/ml. The total ΔPs in the naïve cultures at 0.0 and 4.0 μg/ml and in the adapted cultures at 75.0 μg/ml were -144.21, -149.24, and -140.75 mV, respectively, which were not significantly different each other. Summary In summary, we examined the growth inhibitory effect of monensin to the seven ruminal bacteria, each of which represented different cell wall types and monensin sensitivity. The susceptibility of naïve cultures of the bacteria to monensin correspond to their cell wall type, but varied over a range of monensin concentrations. Different from the susceptibility of the naïve cultures, the adaptability to monensin was not specifically related to the bacterial cell wall types as both of the sensitive strain, S. bovis JB1 and the resistant strain, P. bryantii B14 enabled growth at the highest monensin concentration tested in this study after adaptation. The whole genome transcriptomic analysis showed that the transcriptional responses of strains sensitive to monensin commonly upregulated genes encoding energy metabolism, ABC transporters at the first monensin exposure, and protein damage repair, whereas the expression of those genes was not significantly changed in the resistant strains. The transcriptional profiles of the monensin-adapted cultures were unique, as shifted toward overexpression of genes related to transmembrane secretion, modification of cell wall proteins, and response tochemiosmotic gradients. Comparison of end product metabolites between naïve and adapted cultures of S. bovis JB1 and P. bryantii B14 indicated that the alteration of the primary fermentation products caused by monensin was alleviated in the adapted cultures. Energy transduction effect by monensin was also reduced in the adapted cultures. The findings enhance our understanding of population based ecological studies and long-term efficacy of adding ionophores to ruminant diets.
Publications
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Progress 01/01/17 to 12/31/17
Outputs
Target Audience:Members of the target audience included graduate students, postdoctoral research associates, and associated professors at the university level. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided support for two Postdoctoral Research Associates (Dr Celia Mendez-Garcia left in May 2017,and Dr Na Kyung Kim)as well as for one PhD student (Mr. Michael Iakiviak who has since completed his dissertation) during work reported on this project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the next research period, based on the transcriptomic data, we will determine the genes involved in regulatory responses to monensin to make ruminal bacterial cells sensitive/resistant. By integrating this information with measurement of ion fluxes, energy transduction and metabolites, cellular and molecular mechanisms responding to monensin will expect to be interpreted. The determined genes and regulatory networks will be studied for mutational analyses and gene inactivation studies using model bacterial species (e.i., E. coli K12 and B. subtilis 168). In addition, to investigate whether the genetic regulatory responses to monensin are valid to other types of ionophores, a transcriptomic response of S. bovis JB1 to lasalocid, an ionophore preferentially affiliating divalent cations, will also be analyzed.
Impacts
What was accomplished under these goals?
Comparison of whole genome transcriptomes of the strains grown in the presence and absence of monensin was conducted using RNA sequencing analysis. Given the results of the MICs, triplicate cultures of each strain grown at 0%, 25%, and 50% MIC (additional 12.5% MIC for P. bryantii B14 and S. bovis JB1) were cultivated. RNA samples isolated at a mid-log phase were subjected to RNA-Seq library construction using an Illumina 4000 sequencing platform. Resulting sequence data were continued to a differential gene expression analysis using the edgeR package in R. Genes that indicated greater than 4 fold-change to the control at an FDR of 5% were screened from the global transcriptional analysis. In a comparative transcriptome analysis of S. bovis JB1 with monensin added as compared to control (absence of monensin), the most up-regulated genes, among numerous overexpressed ones, are likely involved in membrane transporter systems, such as IE46DRAFT_00768, 00769, 01845, 01846, and 01847 (IMG accession: Gs0033970), encoding ABC transport systems: ATP-binding proteins and permease proteins. Another group of the substantially overexpressed genes are related to energy metabolism, such as IE46DRAFT_01179-01180, 01361, 01366-01368, and 01371-01375, encoding phosphotransferase systems: membrane-bound permeases that transport extracellular sugar molecules to the cell. The other up-regulated genes were involved in stress responses of low pH: potassium/sodium efflux ATPases, dehydrogenases, deaminases, chaperones protecting denatured proteins, and proteases removing remove damaged proteins. In the transcriptomic responses of the other sensitive strain to monensin, B. fibrisolvens D1, we observed that genes encoding similar functions were highly expressed. Besides ABC transports, efflux ATPases, and genes for repairing damage to proteins (SAMN02745229_00025, 00027, and 02958), to cope with de-energization due to maintaining ionic homoeostasis, B. fibrisolvens D1 greatly expressed genes (SAMN02745229_00661-00663) encoding substrate binding proteins and permeases together with beta-glucosidase (NCBI accession: SAMN02745229). The comparative transcriptomic results of the monensin resistant strains, P. bryantii B14, F. succinogenes S85, S. ruminantium HD4, and M. elsdenii T81, indicated fewerdifferences in gene expression between monensin treated and the controlcompared to those of the sensitive strains. P. bryantii B14 actively expressed genes encoding conjugative transposon proteins, TraE, TraG, and TraM. F. succinogenes S85 up-regulated genes involved in multidrug efflux transporters and membrane proteins functioning proton channels. In the transcriptomic results of S. ruminantium HD4, genes encoding bile acid:sodium symporters and membrane modifying proteins were overexpressed. To determine the persistence and genomic stability of the monensin adaptation, the seven selected rumen bacterial strains were grown in media containing 50% of their respective MIC, and then every six consecutive subcultures were performed at stepwise increasing monensin concentrations. At the end of each phase of the subculturing, the growth rate was determined. For the strains showing adaptability at more than 7xMIC, the subculture procedure was discontinued at 75 μg/ml because of the solubility of monensin in the media. Adaptation to higher concentrations of MIC was observed in all tested strains. P. bryantii B14 and S. bovis JB1 showed monensin adaptation enabling growth at 75 μg/ml, which was more than ten times the MIC for the naïve cultures. The difference was that the growth rate and the ODmax of P. bryantii B14 were maintained during the adaptation whereas those of S. bovis JB1 gradually decreased as the monensin concentration increased. The highest adapted monensin concentrations of R. albus 7 and B. fibrisolvens D1were 0.6 μg/ml and 0.2 μg/ml, respectively. These strains were not only sensitive to monensin but incapable of adaptation under our growth conditions. S. ruminantium HD4 and M. elsdenii T81, the Gram-intermediates greatly resistant to monensin, showed increasing growth rates and constant ODmax during the adaptation. The adaptation process of F. succinogenes S85 is ongoing. Putative reversibility of the monensin-adapted strains was tested. The isolates at the last adaptation phase were repeatedly transferred more than five times in monensin free media and then were re-introduced to media with the highest adapted monensin concentration. Five strains among the seven targeted strains, R. albus 7, S. bovis JB1, S. ruminantium HD4, M. elsdenii T81, and B. fibrisolvens D1were able to grow at the respective highest adapted monensin concentrations with decreased growth rates. When the growth rate of the reverted S. bovis JB1 culture, which was transferred from the 6th subculture in the monensin free media after the adaptation, was compared with that from the 26th subculture, a decrease in the growth rate was observed as the duration for the adapted culture to be transferred in monensin free media got longer. This indicates that the acquired adaptability of S. bovis JB1 to monensin may not be consistently conserved through generations. For P. bryantii B14, a procedure transferring in monensin free media after the adaptation is currently underway. Comparison of the transcriptional profiles between the naïve and reverted cultures after monensin-adaptation was conducted using the RNA sequencing analysis as described above. In comparative transcriptomes of S. bovis JB1 between the naïve isolates exposed to monensin and the reverted ones to 75 μg/ml, up-regulation of the genes involved in the energy metabolism and the acid stress responses were observed, which was the same as the previous data, but at a moderate level of transcription. Uniquely, in the reverted transcriptomes, genes encoding drug resistance transporters, transmembrane secretion effector, and bile acid:sodium symporters were actively expressed. The transcriptomic results of the reverted M. elsdenii T81 and S. ruminantium HD4 commonly overexpressed the genes encoding various sodium symporters together with genes encoding a sulfate-transport system and cell wall modifying proteins, respectively. The RNA-seq analysis for the remainder of the reverted strains is currently ongoing.
Publications
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided support for two Postdoctoral Research Associates (Dr. Celia Mendez Garcia and Dr. Na Kyung Kim) and one PhD student (Mr. Michael Iakiviak) during the work reported on this project. How have the results been disseminated to communities of interest?One presentaion has been given at The Congress on Gastrointestinal Function (CGIF) 2015 by Dr. Amy Biddle entitled "Gene expression of Streptococcus bovis JB1 in response to monensin". What do you plan to do during the next reporting period to accomplish the goals?As reported in the proposal.
Impacts
What was accomplished under these goals?
Global genomic and transcriptomic analysis of S. bovis JB1 in response to monensin A chemostat experiment was carried out where S. bovis JB1 was grown at two different dilution rates (2X and 4X turnovers per day) to simulate a slow and fast ruminal turnover and bacterial growth rate. After completion of this sampling protocol, monensin (5 ppm) was added to the medium influent and samples collected at intervals for the following 48 hours and before washout of S. bovis. Dramatic transcriptional changes were observed after 1.5 as well as 47 hours after introduction of monensin. After monensin challenge, lactate fermentation increased and the corresponding transcript (RPKM) ratio of Pyruvate-formate lyase to Lactate dehydrogenase changed. Key changes occurred in many of the proton pump associated genes i.e. F0F1 ATP synthase subunits and associated membrane proteins. From this initial experiment we concluded that many of the transcriptional responses of S. bovis to monensin were similar to the protective mechanism from acid stress in acid tolerant Gram-positive bacteria, including ATPase, deaminases, and antioxidants, but S. bovis did not contain the common amino acid decarboxylase/antiporter system for combating acid stress. Unlike other monensin resistant Gram positive bacteria, S. bovis seems to mostly depend on ATPase to expel or remove intracellular H+ and Na+, consequently causing a depletion of total intracellular ATP. Thus, the decreased growth rate or growth cessation effect of monensin would result from insufficient ATP to meet biological demands for growth. However, in other Gram positive bacteria the presence of an amino acid decarboxylase/antiporter system would enable the G+ bacteria to save intracellular ATP for the removal of protons. Thus, an amino acid decarboxylase/ antiporter system would be another potential resistant protective mechanism of bacteria against the inhibitory effects of monensin. Monensin Minimum Inhibitory Concentrations (MIC's) In order to investigate the growth inhibitory effects of monensin on ruminal bacteria with different cell wall types, pure cultures of Gram-negative (P. bryantii B14 and F. succinogenes S85), Gram-positive (R .albus 7 and S. bovis JB1), and Gram-intermediate (S. ruminantium HD4, M. elsdenii T81 and B. fibrisolvens D1) were grown in batch culture over a range of monensin concentrations. Minimum inhibitory concentrations (MICs) of the strains were determined; P. bryantii B14 and F. succinogenes S85 showed growth inhibition at 7.0 μg/ml and 3.2 μg/ml, respectively. In the Gram-positive strains, R. albus 7 was sensitive to 0.2 μg/ml whereas growth of S. bovis JB1 was resistant to 4.0 μg/ml and showed delayed growth at a concentration of 7.0-13.0 μg/ml with prolonged incubation of up to 12 hours and at 16.0-20.0 μg/ml with up to18 hours of incubation. Among the Gram-intermediates, S. ruminantium HD4 and M. elsdenii T81 were insensitive up to 50.0 μg/ml and 25.0 μg/ml, respectively, whereas growth of B. fibrisolvens D1 was inhibited by 0.2 μg/ml. Most of the strains showed susceptibility to monensin that was related to their cell wall type except for S. bovis JB1. Among the strains that exhibited resistance to monensin, while P. bryantii B14, S. ruminantium HD4, and M. elsdenii T81 grew to a similar MaxOD at the late-exponential phase of growth, the MaxOD of S. bovis JB1 at 4.0 μg/ml barely reached t46% of the control by decreasing with respect to the increasing monensin concentrations. To compare genetic regulatory responses between monensin sensitive and resistant bacteria, transcriptional profiles of the seven strains are under analysis using RNA-seq. Triplicate cultures of each strain grown at 0%, 25%, and 50% MIC (additional 12.5% MIC for P.bryantii B14 and S. bovis JB1) were cultivated and are currently being subjected to RNA sequencing analysis for quantification of their full transcriptional repertoire.
Publications
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