Comparative Genome Analysis of the Lignocellulose Degrading Bacteria Citrobacter freundii so4 and Sphingobacterium multivorum w15.

Two bacterial strains, denoted so4 and w15, isolated from wheat straw (WS)-degrading microbial consortia, were found to grow synergistically in media containing WS as the single carbon and energy source. They were identified as Citrobacter freundii so4 and Sphingobacterium multivorum w15 based on 16S rRNA gene sequencing and comparison to the respective C. freundii and S. multivorum type strains. In order to identify the mechanisms driving the synergistic interactions, we analyzed the draft genomes of the two strains and further characterized their metabolic potential. The latter analyses revealed that the strains had largely complementary substrate utilization patterns, with only 22 out of 190 compounds shared. The analyses further indicated C. freundii so4 to primarily consume amino acids and simple sugars, with laminarin as a key exception. In contrast, S. multivorum w15 showed ample capacity to transform complex polysaccharides, including intermediates of starch degradation. Sequence analyses revealed C. freundii so4 to have a genome of 4,883,214 bp, with a G + C content of 52.5%, 4,554 protein-encoding genes and 86 RNA genes. S. multivorum w15 has a genome of 6,678,278 bp, with a G + C content of 39.7%, 5,999 protein-encoding genes and 76 RNA genes. Genes for motility apparatuses (flagella, chemotaxis) were present in the genome of C. freundii so4, but absent from that of S. multivorum w15. In the genome of S. multivorum w15, 348 genes had regions matching CAZy family enzymes and/or carbohydrate-binding modules (CBMs), with 193 glycosyl hydrolase (GH) and 50 CBM domains. Remarkably, 22 domains matched enzymes of glycoside hydrolase family GH43, suggesting a strong investment in the degradation of arabinoxylan. In contrast, 130 CAZy family genes were found in C. freundii so4, with 61 GH and 12 CBM domains identified. Collectively, our results, based on both metabolic potential and genome analyses, revealed the two strains to harbor complementary catabolic armories, with S. multivorum w15 primarily attacking the WS hemicellulose and C. freundii so4 the cellobiose derived from cellulose, next to emerging oligo- or monosaccharides. Finally, C. freundii so4 may secrete secondary metabolites that S. multivorum w15 can consume, and detoxify the system by reducing the levels of (toxic) by-products.

Cortes-Tolalpa L ，Wang Y ，Salles JF ，van Elsas JD ... -  《Frontiers in Microbiology》

Functioning of a tripartite lignocellulolytic microbial consortium cultivated under two shaking conditions: a metatranscriptomic study.

In a previous study, shaking speed was found to be an important factor affecting the population dynamics and lignocellulose-degrading activities of a synthetic lignocellulolytic microbial consortium composed of the bacteria Sphingobacterium paramultivorum w15, Citrobacter freundii so4, and the fungus Coniochaeta sp. 2T2.1. Here, the gene expression profiles of each strain in this consortium were examined after growth at two shaking speeds (180 and 60 rpm) at three time points (1, 5 and 13 days). The results indicated that, at 60 rpm, C. freundii so4 switched, to a large extent, from aerobic to flexible (aerobic/microaerophilic/anaerobic) metabolism, resulting in continued slow growth till late stage. In addition, Coniochaeta sp. 2T2.1 tended to occur to a larger extent in the hyphal form, with genes encoding adhesion proteins being highly expressed. Much like at 180 rpm, at 60 rpm, S. paramultivorum w15 and Coniochaeta sp. 2T2.1 were key players in hemicellulose degradation processes, as evidenced from the respective CAZy-specific transcripts. Coniochaeta sp. 2T2.1 exhibited expression of genes encoding arabinoxylan-degrading enzymes (i.e., of CAZy groups GH10, GH11, CE1, CE5 and GH43), whereas, at 180 rpm, some of these genes were suppressed at early stages of growth. Moreover, C. freundii so4 stably expressed genes that were predicted to encode proteins with (1) β-xylosidase/β-glucosidase and (2) peptidoglycan/chitinase activities, (3) stress response- and detoxification-related proteins. Finally, S. paramultivorum w15 showed involvement in vitamin B2 generation in the early stages across the two shaking speeds, while this role was taken over by C. freundii so4 at late stage at 60 rpm. We provide evidence that S. paramultivorum w15 is involved in the degradation of mainly hemicellulose and in vitamin B2 production, and C. freundii so4 in the degradation of oligosaccharides or sugar dimers, next to detoxification processes. Coniochaeta sp. 2T2.1 was held to be strongly involved in cellulose and xylan (at early stages), next to lignin modification processes (at later stages). The synergism and alternative functional roles presented in this study enhance the eco-enzymological understanding of the degradation of lignocellulose in this tripartite microbial consortium.

Wang Y ，Jiménez DJ ，Zhang Z ，van Elsas JD ... -  《-》

Bacterial Synergism in Lignocellulose Biomass Degradation - Complementary Roles of Degraders As Influenced by Complexity of the Carbon Source.

Cortes-Tolalpa L ，Salles JF ，van Elsas JD 《Frontiers in Microbiology》

Considerations on the Identity and Diversity of Organisms Affiliated with Sphingobacterium multivorum-Proposal for a New Species, Sphingobacterium paramultivorum.

Wang Y ，Brons JK ，van Elsas JD 《-》

Defining the eco-enzymological role of the fungal strain Coniochaeta sp. 2T2.1 in a tripartite lignocellulolytic microbial consortium.

Coniochaeta species are versatile ascomycetes that have great capacity to deconstruct lignocellulose. Here, we explore the transcriptome of Coniochaeta sp. strain 2T2.1 from wheat straw-driven cultures with the fungus growing alone or as a member of a synthetic microbial consortium with Sphingobacterium multivorum w15 and Citrobacter freundii so4. The differential expression profiles of carbohydrate-active enzymes indicated an onset of (hemi)cellulose degradation by 2T2.1 during the initial 24 hours of incubation. Within the tripartite consortium, 63 transcripts of strain 2T2.1 were differentially expressed at this time point. The presence of the two bacteria significantly upregulated the expression of one galactose oxidase, one GH79-like enzyme, one multidrug transporter, one laccase-like protein (AA1 family) and two bilirubin oxidases, suggesting that inter-kingdom interactions (e.g. amensalism) take place within this microbial consortium. Overexpression of multicopper oxidases indicated that strain 2T2.1 may be involved in lignin depolymerization (a trait of enzymatic synergism), while S. multivorum and C. freundii have the metabolic potential to deconstruct arabinoxylan. Under the conditions applied, 2T2.1 appears to be a better degrader of wheat straw when the two bacteria are absent. This conclusion is supported by the observed suppression of its (hemi)cellulolytic arsenal and lower degradation percentages within the microbial consortium.

Jiménez DJ ，Wang Y ，Chaib de Mares M ，Cortes-Tolalpa L ，Mertens JA ，Hector RE ，Lin J ，Johnson J ，Lipzen A ，Barry K ，Mondo SJ ，Grigoriev IV ，Nichols NN ，van Elsas JD ... -  《-》