Comparative whole genome transcriptional responses of Ruminococcus albus strain 7 and 8: a case for specialization and niche differentiation

Kwon, Inhyuk

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https://hdl.handle.net/2142/90731 Copy

Description

Title

Comparative whole genome transcriptional responses of Ruminococcus albus strain 7 and 8: a case for specialization and niche differentiation

Author(s)

Kwon, Inhyuk

Issue Date

2016-03-28

Director of Research (if dissertation) or Advisor (if thesis)

Mackie, Roderick I.

Doctoral Committee Chair(s)

Mackie, Roderick I.

Committee Member(s)

• Cann, Isaac K. O.
• Vanderpool, Carin K.
• Ridlon, Jason

Department of Study

Animal Sciences

Discipline

Animal Sciences

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Ruminococcus albus
• whole genome transcriptomic analyses

Abstract

Ruminococcus albus strains are one of dominant fibrolytic bacteria in the rumen that contribute to plant biomass as well as vitamin utilization in host nutrition. To better understanding of host-microbe interactions, it is relevant to establish the model for fiber degradation and vitamin metabolism of the dominant fibrolytic bacteria and investigate their roles in the gut ecosystem. However, the fibrolytic mechanism and vitamin metabolism of Ruminococcus albus remain largely unknown. In the current study, comparative genomic and transcriptomic analyses of two different strains 7 and 8 of R. albus for plant fiber and folate utilization were used to investigate the conserved and differential mechanism between the two R. albus strains. Through comparative transcriptomic analyses of both strains grown on alkaline peroxide hydrogen treated corn stalk (AHPCS), phosphoric acid swollen cellulose (PASC) and wheat arabinoxylan (WAX), this research demonstrated that the top 5 highly expressed glycoside hydrolase (GH) families, including the versatile GH5, GH9 (Cel9B), GH10, GH11, and GH48 (Cel48A), are the primary GH enzymes employed by both strains of R. albus for the hydrolysis of plant cell wall. In addition, the co-expression of these endoglucanases and endoxylanases in response to cellulose and hemicellulose was observed. The previously known adhering mechanism of R. albus were transcriptionally analyzed and verified in this research. The genes encoding Pil-like protein or a family 37 carbohydrate binding module (CBM37) domain were highly expressed in both strains during growth on different polysaccharides. Especially, the significant role of CBM37 in the fiber utilization of R. albus was highlighted based on the prevalence of CBM37 domain on the highly expressed GH genes as well as hypothetical genes. It is notable that distinct strategies between two strains for plant cell wall utilization were proposed in this research. Based on phenotypic, genomic, and transcriptomic evidence, wild type of R. albus 8 in rumen appears to preferentially utilize hemicellulose rather than cellulose embedded in the plant cell wall, while R. albus 7 prefers to utilize cellulose over hemicellulose. To support this conclusion, R. albus 8 utilized more hemicellosic sugars derived from the hydrolysis of AHPCS than R. albus 7. More CAZyme genes of R. albus 8 responded to WAX than PASC, while those genes of R. albus 7 responded to more PASC than WAX. When hemicellulose in AHPCS started to decrease in the culture, R. albus 8 down-regulated the expression of genes for sugar transporters and intracellular GH. In contrast, R. albus 7 exhibited a sequential expression of sugar transporters and intracellular GH genes, as preferred cellulosic sugars were released from AHPCS after removal of hemicellulose. Notably, we found the putative genes belonging to c-di-GMP regulatory and the accessory gene regulator quorum sensing (Agr QS) systems in R. albus 7 and 8. The transcriptional pattern of these genes were in accordance with differential transcriptional pattern of GH genes between both strains and the preferred planktonic growth of strain 8 on AHPCS as opposed to the substrate adherent growth of R. albus 7. These results suggest that c-di-GMP and Agr QS systems are implicated not only in biofilm formation of pathogenic bacteria, but also in the fibrolytic systems of commensal bacteria. Supported by the fermentation profile and the growth rate on beechwood xylan together with genomic and transcriptomic evidence, R. albus 8 was found to possess a predicted unique phosphoketolase (PK) pathway, which likely enables R. albus 8 to catabolize pentose rapidly as well as conserve energy and costs for enzyme synthesis required for the lower glycolytic sequence. With our proposal for the differential strategies between strains, the co-culture experiment demonstrated that despite a similar fibrolytic mechanism, R. albus 7 and 8 could co-exist on complex substrate containing cellulose and hemicellulose. This research on folate metabolism in R. albus 7 and 8 provided genomic evidence for three folate utilization pathways (either de novo synthesis, salvage, or both pathways) conserved in the Firmicutes including R. albus strains. Through the growth experiments in the presence or absence of folate and para-aminobenzoate (pABA), it was shown that R. albus strains 7 and 8 rely on different folate metabolic pathways, de novo synthesis or salvage pathway, respectively. In addition, the results of transcriptomic analysis suggest that the folate autotrophic strain, R. albus 7, also has an alternative pathway for pABA synthesis and likewise other Ruminococcus species lacking the canonical pABA synthetic pathway are likely autotrophs and not auxotrophs. Notably, the potential long non-coding RNA (lncRNA) loci was identified in the genomes of R. albus strains. The putative lncRNA loci consisted of four sequence components; lncRNA, DUF1292 gene, putative 6S RNA, and alcohol dehydrogenase. Based on their transcriptional profiles assessed by RNA-seq and northern blot analyses, it seems likely that the lncRNA loci are involved in the regulatory system related to the stationary phase of cells. This study provides molecular insight in conserved and differentiated fibrolytic system and folate metabolism between R. albus 7 and 8. In addition, the presence of novel lncRNA loci was identified, providing more information on the regulatory mechanism in Gram-positive Firmicutes.

Graduation Semester

2016-05

Type of Resource

text

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Copyright 2016 INHYUK KWON

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