The filter feeding oligotrich ciliate Strobilidium lacustris, the raptorial prostome ciliate Balanion planctonicu~n and the diffusion feeding scuticociliate Histiobalantium bodamicurn could be cultivated for months/years on a sole &et of Cryptomonas sp., whereas the diatom Stephanodiscus hantzschii did not support their growth. With Cryptornonas sp. as food, numerical responses of all ciliates followed a modified Michaelis-Menten model, which at lS°C yielded maximum growth rates of 0.96. 1.87 and 0.33 d-' and threshold concentrations of 61, 7 8 and 290 ng C ml-' for S. lacustris, B. planctonicum and H. bodamicum, respectively. Functional response patterns differed between species. In all investigated ciliates, growth rates reached a maximum earlier than ingestion rates, and there were no threshold concentrations for zero ingestion. Food selectivity depended on feeding mode. H. bodamicum was not able to ingest the non-motile diatoms. Both S. lacustris and B. planctonicum selectively preferred cryptophytes when offered a mixed diet. This effect was more pronounced in the raptorial feeder compared to the filter feeder. Our results indicate that during the phytoplankton spring bloom in Lake Constance prostome and oligotrich ciliates mainly exploit cryptophytes, and that the scuticociliate H. bodamicum, due to its slow growth, is an inferior competitor during this season. The observed threshold concentrations suggest that during the rest of the year prostomes and oligotrichs must rely on small-scale patches of this food, whereas H. bodamicum, with maximum development in late summer and autumn, presumably consumes a much larger variety of prey.

The maltose system of Escherichia coli consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins. The system is positively regulated by MalT, its transcriptional activator. MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins. MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity. By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK. This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain. Mutants in MalT causing elevated mal gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors. The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them. Some of them became nearly completely resistant to Aes while still being sensitive to MalY. These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT.The Escherichia coli maltose system consists of 10 genes encoding proteins dedicated to the uptake and the metabolism of maltose and maltodextrins (4). These genes are under the control of MalT, a specific transcriptional activator of 901 amino acids (aa). MalT belongs to a class of bacterial transactivators, the MalT or LAL family (11, 42). They are large proteins (Ͼ90 kDa), possess an ATP binding site near their N terminus, and share homology with LuxR near their C terminus. MalT binds and activates its target promoters (29) only in the presence of ATP (34) and the inducer maltotriose (28). MalT consists of four structural domains (11): domain 1 (DT1, aa 1 to 241) binds ATP, domains 2 (DT2, aa 250 to 436) and 3 (DT3, aa 437 to 806) bind the inducer, and domain 4 (DT4, aa 807 to 901) harbors the DNA binding site (11,43). MalT exists in an equilibrium between a monomeric (inactive) and a monomeric (active) form and is prone to multimerize. This equilibrium is shifted to the active form by the inducer maltotriose, which triggers a conformational change involving DT1, -2, and -3 and the linkers in between. The conformational change, which also requires ATP, is a step towards the formation of a high-order oligomer, the transcriptionally competent form of the protein (11, 36). Point mutations in malT (malT c ) have been isolated that confer a constitutive expression of the maltose regulon when maltodextrin is not present in the growth medium (12, 13) The in vitro analysis of two corresponding MalT c proteins revealed that, in contrast to the wild-type MalT, they could activate transcription from a MalT-dependent promoter in the absence of maltotriose but could st...

Globally accessible preventive and therapeutic molecules against SARS-CoV-2 are urgently needed. DARPin molecules are an emerging class of novel therapeutics based on naturally occurring repeat proteins (∼15 kDa in size) and can be rapidly produced in bacteria in large quantities. Here, we report the identification of 380 DARPin molecules specifically targeting the SARS-CoV-2 spike protein selected from a naïve library of 1012 DARPin molecules. Using extensive biophysical and biochemical characterization, (pseudo)virus neutralization assays and cryo-EM analysis, 11 mono-DARPin molecules targeting either the receptor binding domain (RBD), the S1 N-terminal-domain (NTD) or the S2 domain of the SARS-CoV-2 spike protein were chosen. Based on these 11 mono-DARPin molecules, 31 anti-SARS-CoV-2 multi-DARPin molecules were constructed which can broadly be grouped into 2 types; multi-paratopic RBD-neutralizing DARPin molecules and multi-mode DARPin molecules targeting simultaneously RBD, NTD and the S2 domain. Each of these multi-DARPin molecules acts by binding with 3 DARPin modules to the SARS-CoV-2 spike protein, leading to potent inhibition of SARS-CoV-2 infection down to 1 ng/ml (12 pM) and potentially providing protection against viral escape mutations. Additionally, 2 DARPin modules binding serum albumin, conferring an expected half-life of about 3 weeks in humans, were included in the multi-DARPin molecules. The protective efficacy of one multi-DARPin molecule was studied in a Golden Syrian hamster SARS-CoV-2 infection model, resulting in a significant reduction in viral load and pathogenesis. In conclusion, the multi-DARPin molecules reported here display very high antiviral potency, high-production yield, and a long systemic half-life, and thereby have the potential for single-dose use for prevention and treatment of COVID-19.

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