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Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3′- and 6′-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5′-monophospho- N -acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6′-sialyllactose synthesis but low levels of 3′-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmST WT under optimal conditions for 6′-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3′-sialyllactose over 6′-sialyllactose and the double mutant (PmST E271F/R313Y ) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmST E271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation. [ABSTRACT FROM AUTHOR]

Abstract: Recent advances in mass spectrometry allowed the charting of bacterial serine/threonine/tyrosine phosphoproteomes with unprecedented accuracy, including the acquisition of a large number of phosphorylation sites. Phosphorylated bacterial proteins are involved in some key housekeeping processes, and their phosphorylation is expected to play an important regulatory role. When coupled to stable isotope labeling by amino acids in cell culture (SILAC), high-resolution mass spectrometry allows the detection of changes in the occupancy of phosphorylation sites in response to various stimuli. This and similar approaches promise to lead bacterial phosphoproteomics into the era of systems biology, where the entire phosphorylation-based regulatory networks will be charted, modelled, and ultimately engineered to obtain desired properties. [Copyright &y& Elsevier]

Enterococci comprise a group of lactic acid bacteria (LAB) with considerable potential to serve as food fermentation microorganisms. Unfortunately, enterococci have received a lot of negative attention, due to the occurrence of pathogenic and multidrug resistant strains. In this study, we used genomics to select safe candidates among the forty-four studied enterococcal isolates. The genomes of the forty-four strains were fully sequenced and assessed for presence of virulence and antibiotic resistance genes. Nineteen isolates belonging to the species Enterococcus lactis, Enterococcus faecium, Enterococcus durans , and Enterococcus thailandicus , were deemed safe from the genome analysis. The presence of secondary metabolite gene clusters for bacteriocins was assessed, and twelve candidates were found to secrete antimicrobial compounds effective against Listeria monocytogenes isolated from cheese and Staphylococcus aureus. Physiological characterization revealed nineteen industrial potentials; all strains grew well at 42 °C and acidified 1.5 hours faster than their mesophilic counterpart Lactococcus lactis , with which they share metabolism and flavor forming ability. We conclude that a large fraction of the examined enterococci were safe and could serve as excellent food fermentation microorganisms with inherent bioprotective abilities. [ABSTRACT FROM AUTHOR]

Highlights: [•] A mutated sialidase from Trypanosoma rangeli was expressed in Pichia pastoris. [•] The sialidase mutant was tuned for trans-sialidase activity by optimizing reaction conditions. [•] 3′-Sialyllactose and other sialylated HMO molecules were produced via trans-sialidase biocatalysis. [•] Casein glycomacropeptide was used as substrate for the trans-sialidase mutant biocatalysis. [ABSTRACT FROM AUTHOR]