In any given organism, about 1/3 of all proteins have a yet unknown function. A widely distributed domain of unknown function is DUF1127. About 17,000 proteins with such an arginine-rich domain are found in 4,000 bacteria. Most of them are single domain proteins, and a large fraction qualifies as small proteins with less than 50 amino acids. We systematically identified and characterized the seven DUF1127 members of the plant pathogen Agrobacterium tumefaciens. They all give rise to authentic proteins and are differentially expressed as shown at the RNA and protein levels. The seven proteins fall into two sub-classes on the basis of their length, sequence and reciprocal regulation by the LysR-type transcription factor LsrB. The absence of all three short DUF1127 proteins caused a striking phenotype in later growth phases, and increased cell aggregation and biofilm formation. Protein profiling and RNA-seq analysis of the wild type and triple mutant revealed a large number of differentially regulated genes in late exponential and stationary growth. The most affected genes are involved in phosphate uptake, glycine/serine homeostasis and nitrate respiration. The results suggest a redundant function of the small DUF1127 paralogs in nutrient acquisition and central carbon metabolism of A. tumefaciens. They may be required for diauxic switching between carbon sources when sugar from the medium is depleted. We end by discussing how DUF1127 might confer such a global impact on cell physiology and gene expression. Importance Despite being prevalent in numerous ecologically and clinically relevant bacterial species, the biological role of proteins with a domain of unknown function DUF1127 is unclear. Experimental models are needed to approach their elusive function. We used the phytopathogen Agrobacterium tumefaciens, a natural genetic engineer that causes the crown-gall disease, and focused on its three small DUF1127 proteins. They have a redundant and pervasive role in nutrient acquisition, cellular metabolism and biofilm formation. The study shows that small proteins have important, previously missed biological functions. How small basic proteins can have such a broad impact is a fascinating prospect of future research.

The assembly of large, multi-cofactor membrane protein complexes like photosystem II (PSII) requires a high level of coordination. The process is facilitated by a large network of auxiliary proteins that bind transiently to unassembled subunits, preassembled modules or intermediate states of PSII, which are comprised of a subset of subunits. However, analysis of these immature, partially assembled PSII complexes is hampered by their low abundance and intrinsic instability. In this study, PSII was purified from the thermophilic cyanobacterium Thermosynechococcus elongatus via Twin-Strep-tagged CP43 and further separated by ion exchange chromatography into mature and immature complexes. Mass spectrometry analysis of the immature Psb27-PSII intermediate revealed six different Psb27 proteoforms with distinct lipid modifications. The maturation and functional role of thylakoid localized lipoproteins are discussed.

Oxygenic phototrophs frequently encounter environmental conditions that result in intracellular energy crises. Growth of the unicellular green alga Chlamydomonas reinhardtii in hypoxia in the light depends on acclimatory responses of which the induction of photosynthetic cyclic electron flow is essential. The microalga cannot grow in the absence of molecular oxygen (O ) in the dark, although it possesses an elaborate fermentation metabolism. Not much is known about how the microalga senses and signals the lack of O or about its survival strategies during energy crises. Recently, nitric oxide (NO) has emerged to be required for the acclimation of C. reinhardtii to hypoxia. In this study, we show that the soluble guanylate cyclase (sGC) CYG12, a homologue of animal NO sensors, is also involved in this response. CYG12 is an active sGC, and post-transcriptional down-regulation of the CYG12 gene impairs hypoxic growth and gene expression in C. reinhardtii. However, it also results in a disturbed photosynthetic apparatus under standard growth conditions and the inability to grow heterotrophically. Transcriptome profiles indicate that the mis-expression of CYG12 results in a perturbation of responses that, in the wild-type, maintain the cellular energy budget. We suggest that CYG12 is required for the proper operation of the photosynthetic apparatus which, in turn, is essential for survival in hypoxia and darkness.

The pharmaceutical industry has developed various highly effective semi-synthetic cephalosporins, which are generated by modifying the side chains of the core molecule 7-aminocephalosporanic acid (7-ACA). In industrial productions, the 7-ACA nucleus is obtained in vitro from cephalosporin C (CPC) by chemical or enzymatic processes, which are waste intensive and associated with high production costs. Here, we used a transgenic in vivo approach to express bacterial genes for cephalosporin C acylase (CCA) in the CPC producer Acremonium chrysogenum. Western blot and mass spectrometry analyses verified that the heterologous enzymes are processed into α- and β-subunits in the fungal cell. Extensive HPLC analysis detected substrates and products of CCAs in both fungal mycelia and culture supernatants, with the highest amount of 7-ACA found in the latter. Using different incubation times, temperatures, and pH values, we explored the optimal conditions for the active bacterial acylase to convert CPC into 7-ACA in the culture supernatant. We calculated that the best transgenic fungal strains exhibit a one-step conversion rate of the bacterial acylase of 30%. Our findings can be considered a remarkable contribution to supporting future pharmaceutical manufacturing processes with reduced production costs.

The synthesis of multi-span thylakoid membrane proteins initiates at ribosomes off the membrane. Subsequently, the ribosome nascent chain complexes (RNCs) are transferred to the translocase machinery in the thylakoid membrane for cotranslational protein insertion. These steps require finely tuned mechanisms for protein processing, quality control, and targeting to prevent misfolding or aggregation and to ensure efficient transfer of the nascent chain to the insertion machinery. However, little is known about the regulatory network underlying these processes. To identify factors specifically involved in the cotranslational biogenesis of the reaction center protein D1 of photosystem II we established a chloroplast-derived in vitro translation method that allows the production and affinity purification of stalled RNCs bearing nascent chains of D1 of different defined lengths. Stalled RNCs translating the soluble ribosomal subunit uS2c were affinity-purified for comparison. Quantitative tandem-mass spectrometry revealed a set of about 120 proteins specifically associated with D1 RNCs. The interactome includes proteins with broad functions in protein processing, biogenesis and metabolic pathways, such as chlorophyll biosynthesis. We identified STIC2 as a new factor specifically associated with D1 RNCs. Furthermore, our results demonstrated that the interaction of STIC2 with the thylakoid insertase Alb3 and its homologue Alb4 is mediated by the conserved motif III within the C-terminal regions of Alb3 and Alb4. Our data suggest that STIC2 is involved in cotranslational substrate delivery at the thylakoid membrane by coordinating the binding of the D1 RNCs to the insertase machinery.