Background:The molecular mechanism underlying the regulation of cellulase production by T. reesei is unclear. Results: The absence of sugar transporter Stp1 enhanced cellulase gene induction whereas the absence of Crt1 abolished cellulase gene expression. Conclusion: Crt1 is essential in cellulase gene induction independent of intracellular sugar delivery. Significance: These data shed light on the mechanism by which T. reesei senses and transmits cellulose signal.

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular ␤-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple ␤-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular ␤-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular ␤-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three ␤-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three ␤-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals.

BackgroundThe pure culture of prokaryotes remains essential to elucidating the role of these organisms. Scientists have reasoned that hard to cultivate microorganisms might grow in pure culture if provided with the chemical components of their natural environment. However, most microbial species in the biosphere that would otherwise be “culturable” may fail to grow because of their growth state in nature, such as dormancy. That means even if scientist would provide microorganisms with the natural environment, such dormant microorganisms probably still remain in a dormant state.ResultsWe constructed an enrichment culture system for high-efficiency isolation of uncultured strains from marine sediment. Degree of enrichment analysis, dormant and active taxa calculation, viable but non-culturable bacteria resuscitation analysis, combined with metatranscriptomic and comparative genomic analyses of the interactions between microbial communications during enrichment culture showed that the so-called enrichment method could culture the “uncultured” not only through enriching the abundance of “uncultured,” but also through the resuscitation mechanism. In addition, the enrichment culture was a complicated mixed culture system, which contains the competition, cooperation, or coordination among bacterial communities, compared with pure cultures.ConclusionsConsidering that cultivation techniques must evolve further—from axenic to mixed cultures—for us to fully understand the microbial world, we should redevelop an understanding of the classic enrichment culture method. Enrichment culture methods can be developed and used to construct a model for analyzing mixed cultures and exploring microbial dark matter. This study provides a new train of thought to mining marine microbial dark matter based on mixed cultures.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0613-2) contains supplementary material, which is available to authorized users.

S U M M A R YStable single-domain (SD) magnetite formed intracellularly by magnetotactic bacteria is of fundamental interest in sedimentary and environmental magnetism. In this study, we studied the time course of magnetosome growth and magnetosome chain formation (0-96 hr) in Magnetospirillum magneticum AMB-1 by transmission electron microscopy (TEM) observation and rock magnetism. The initial non-magnetic cells were microaerobically batch cultured at 26 • C in a modified magnetic spirillum growth medium. TEM observations indicated that between 20 and 24 hr magnetosome crystals began to mineralize simultaneously at multiple sites within the cell body, followed by a phase of rapid growth lasting up to 48 hr cultivation. The synthesized magnetosomes were found to be assembled into 3-5 subchains, which were linearly aligned along the long axis of the cell, supporting the idea that magnetosome vesicles were linearly anchored to the inner membrane of cell. By 96 hr cultivation, 14 cubo-octahedral magnetosome crystals in average with a mean grain size of ∼44.5 nm were formed in a cell. Low-temperature (10-300 K) thermal demagnetization, room-temperature hysteresis loops and first-order reversal curves (FORCs) were conducted on whole cell samples. Both coercivity (4.7-18.1 mT) and Verwey transition temperature (100-106 K) increase with increasing cultivation time length, which can be explained by increasing grain size and decreasing nonstoichiometry of magnetite, respectively. Shapes of hysteresis loops and FORCs indicated each subchain behaving as an 'ideal' uniaxial SD particle and extremely weak magnetostatic interaction fields between subchains. Low-temperature thermal demagnetization of remanence demonstrated that the Moskowitz test is valid for such linear subchain configurations (e.g. δ FC /δ ZFC > 2.0), implying that the test is applicable to ancient sediments where magnetosome chains might have been broken up into short chains due to disintegration of the organic scaffold structures after cell death. These findings provide new insights into magnetosome biomineralization of magnetotactic bacteria and contribute to better understanding the magnetism of magnetofossils in natural environments.

BackgroundTrichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The large-scale production is usually performed in a substrate-inducing manner achieved by a fine-tuned cooperation of a suite of transcription factors. Their production and subsequent analysis are, however, often either difficult to manipulate or complicated by the concomitant production of other inducible proteins. Alternatives to control gene expression independent of the nutritional state are thus preferred in some cases to facilitate not only biochemical studies of proteins but also genetic engineering of the producer.ResultsWe identified a copper transporter encoding gene tcu1 (jgi:Trire2:52315) in T. reesei, the transcription of which was highly responsive to copper availability. Whereas excess copper repressed the expression of tcu1 from T. reesei, eliminating copper addition in the medium resulted in a high-level transcription of tcu1. The usefulness of the system was further illustrated by the high-level expression of specific cellulases driven by the tcu1 promoter in T. reesei when cultivated on D-glucose or glycerol as the sole carbon source. A recombinant T. reesei strain, which overexpressed the main transcription activator of hydrolases (xylanase regulator 1) under the control of tcu1 promoter, was found to be relieved from the carbon catabolite repression and thus displayed a constitutive cellulase expression. Moreover, the amount and activities of cellulases produced by this strain on glycerol or glucose fully recapitulated those of the parental strain produced on Avicel.ConclusionExpression of T. reesei tcu1 gene was tightly controlled by copper availability, and a homologous protein expression system was developed based on this promoter. Deregulation of XYR1 (xylanase regulator 1) mediated by the tcu1 promoter not only overcame the carbon catabolite repression of cellulases but also resulted in their full expression even on the non-inducing carbon sources.