EloR interacts with the lytic transglycosylase MltG at midcell in Streptococcus pneumoniae R6

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A novel proteinaceous molecule produced by Lysinibacillus sp. OF-1 depends on the Ami oligopeptide transporter to kill Streptococcus pneumoniae.

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Sustainability of food side streams: a case study of fermented blends made with sour whey and sunflower press cake powder using the back-slopping technique

The exploitation of by-products is a key factor to increase the sustainability of the agri-food chain and fermentation is a simple and eco-friendly process for achieving safe and suitable food materials. In this study, we investigated the possibility to manage a spontaneous fermentation of blends made with different proportions of two food side streams (bovine acid whey and sunflower press cake powder) through the application of a back-slopping technique of the mixed material incubated at 26°C in static conditions. A full-factorial 2-factor 3-level design of experiment was applied to infer the effect of the percent (w/w) of press cake powder in the mixture (20, 25, and 30%) and the rate of back-slopping inoculum (15, 30, and 45%). The pH value, titratable acidity, content of sugars, organic acids, and phenolic acids, enumeration of lactic acid bacteria, yeasts and molds, bacterial contaminants, presumptive Bacillus cereus, and Escherichia coli were measured for each fermentation step at 0, 24, 48, and 72 h. On the same samples, a metataxonomics analysis, targeted on bacterial 16S rRNA gene and fungal ITS region, was performed by using the Illumina MiSeq platform. Acidification of the blends (on average, starting pH = 5.45 ± 011, final pH = 4.61 ± 0.11; starting acidity =13.68 ± 1.02 °SH/50 mL, final acidity = 28.17 ± 2.92°SH/50 mL) and high LAB counts (on average, 9.39 log CFU/g ± 0.25) were observed at the end of each refreshment. In all fermented mixtures, B. cereus, E. coli, and molds counts were lower than the detection limit (<2 log CFU/g), whereas bacterial contaminants, overall spore-formers, were always present (3.74 log CFU/g ± 0.27). After 72 h, the dropping of pH value was maximum, yielding significant differences compared to previous fermentation steps (p < 0.01); particularly, the lowest pH (4.45 ± 0.06) was achieved in the central points of DoE (25% of press cake powder and 30% of back-slopping rate), representing the most suitable condition. Results from both culture-dependent and -independent techniques were consistent; although Lactococcus lactis, continuously deriving from the acid whey, was the main LAB, Pediococcus pentosaceus appeared and, in some cases, became the dominant species. Finally, a long-term trial (about 1 month), using the best condition previously pointed out, was performed with an extension of the incubation time to 84 h for each refreshment. The increase in acidity forced the natural selection toward acid-tolerant microbial strains confirming the former results. Although preliminary, these findings can be useful for developing innovative operations to manage these two relevant side streams implementing the circularity of food resources

Regulering av celleform hos Streptococcus pneumoniae : EloR/KhpA, en ny reguleringsvei for celle-elongering

The oval shaped Streptococcus pneumoniae utilizes both septal and peripheral peptidoglycan (PG) synthesis in order to maintain its shape. The protein complexes responsible for synthesizing PG are called the elongasome and the divisome, and as the names imply the elongasome synthesizes peripheral PG while the divisome synthesizes the PG responsible for dividing the cell into two [1]. The core of the elongasome and the divisome is the essential class B penicillin binding proteins (PBPs) 2b and 2x, respectively. PBP2b and PBP2x are transpeptidases creating peptide cross-links in PG between glycan strands [1, 2]. They work alongside the glycosyltransferases RodA and FtsW, respectively, to incorporate new PG into the existing PG sacculus [3-5]. The elongasome and divisome activities must be precisely coordinated throughout the cell cycle, but detailed knowledge about the control systems the cells possess to manage these PG machineries are lacking. In the current work, I present a pathway for regulation of elongation that has emerged recently: the EloR/KhpA complex. EloR and KhpA both contain RNA binding domains commonly seen in proteins involved in transcriptional or post-transcriptional regulation [6-8]. We and others have found that in order to survive the loss of pbp2b, S. pneumoniae can create suppressor mutations in the genes encoding EloR, KhpA and MltG rendering the protein products inactive [9, 10]. Our results show that EloR and KhpA work as a complex controlling cell elongation, most likely in a pathway including StkP, a Ser/Thr kinase known to have a regulatory role in cell division [11-15], and the essential lytic transglycosylase MltG. In paper I we show that the loss of EloR resulted in shorter cells in the laboratory strain R6. We confirmed that EloR is phosphorylated by StkP and conclude that it is likely that phosphorylation of EloR leads to release of bound RNA, stimulating elongation. We speculate that the reason PBP2b and RodA are essential in a wild type background is that these proteins are required in cells where the muralytic activity of MltG has a normal function. The reason pbp2b and rodA can be deleted in an DeloR mutant may be that the MltG activity is reduced without EloR present. In paper II we show that EloR interacts directly with a small RNA binding protein called KhpA. Using 3D modelling and site directed mutagenesis we identified the interaction surface between the two proteins and two amino acid residues important for this interaction. We could use this information to investigate how cells reacted to the loss of complex formation between the two proteins. A study by Zheng et al., 2017 showed that a khpA deletion mutant phenocopies an eloR deletion mutant [9]. We demonstrate in paper II that EloR and KhpA is one functional unit, and if the direct interaction between EloR and KhpA is broken, the cells behave like a DeloR or DkhpA mutant, i.e. PBP2b/RodA become redundant. We also show that KhpA depends upon EloR interaction to reach its midcell localization. In addition to two RNA binding domains (KH-II and R3H) at the C-terminal end, EloR has a Jag domain with unknown function at its N-terminus. In the final manuscript, paper III, we set out to unravel the function of the Jag domain. We found that the Jag domain is critical for midcell localization of EloR. Furthermore, by screening for protein-protein interactions between EloR and other elongasome proteins, the Jag domain was found to interact with the cytoplasmic domain of the lytic transglycosylase MltG. We hypothesize that the EloR/KhpA complex is recruited to midcell through the Jag-MltG interaction where it somehow controls the muralytic activity of MltG, either through protein – protein interaction or by RNA binding.Den ovale bakterien Streptococcus pneumoniae benytter både septal og perifer peptidoglykansyntese for å opprettholde celleformen. Proteinkompleksene som er ansvarlige for å syntetisere peptidoglykan (PG) kalles elongasomet og divisomet, og som navnene tilsier syntetiserer elongasomet perifert PG mens divisomet syntetiserer PG som er ansvarlig for å dele cellen i to [1]. Kjernevirksomheten i elongasomet og divisomet utføres av de essensielle klasse B penicillinbindende proteinene (PBP) 2b og 2x. PBP2b og PBP2x er transpeptidaser som danner peptid-kryssbindinger mellom glykantrådene i PG [1, 2]. De jobber sammen med glykosyltransferasene RodA og FtsW for å inkorporere ny PG i det eksisterende PG nettverket [3-5]. Elongasom- og divisom-aktivitetene må være nøyaktig koordinerte gjennom hele cellesyklusen, men detaljert kunnskap om hvordan cellene kontrollerer disse PG-maskineriene mangler. I dette prosjektet presenterer jeg en vei for regulering av elongering som nylig har blitt oppdaget: EloR/KhpA-komplekset. EloR og KhpA inneholder begge RNA-bindende domener som ofte finnes hos proteiner involvert i transkripsjonell eller post-transkripsjonell regulering [6-8]. Vi og andre har oppdaget at for å overleve tapet av pbp2b kan S. pneumoniae skape suppressormutasjoner i genene som koder for EloR, KhpA og MltG, slik at proteinproduktene blir inaktive [9, 10]. Resultatene våre viser at EloR og KhpA fungerer som et kompleks som kontrollerer celle-elongering sammen med StkP, en Ser/Thr-kinase kjent for å ha en regulerende rolle i celledeling [11-15], og den essensielle lytiske transglykosylasen MltG

Fluorescent Biomarkers for Membrane Separation

The verification of the protein content in membrane fractions after separation of Gram-negative bacterial membranes is to date a tedious and demanding process. With this project, we wanted to create Escherichia coli (E. coli) strains with a stable expression of membrane bound fluorescent biomarkers. Labeling the membranes of E. coli BL21 Gold (DE3) with fluorescent proteins allows a membrane separation to be easily verified with simple fluorescence detection. Furthermore, we wanted to investigate and compare the grade of separation obtainable with the two membrane separation techniques selective detergent treatment and density gradient centrifugation. Six fluorescent membrane labels were produced by fusing the genes encoding a fluorescent protein and a membrane protein, or an artificial signal sequence. These fusion genes were expressed from the plasmid pACYCDuet-1 in E. coli BL21 Gold (DE3), and the membranes were separated with the selective detergent treatment. This method proved to be an outer membrane (OM) enrichment technique, where the OM fraction was relatively clean, while the inner membrane (IM) fraction contained contaminants in the form of OM proteins and lipopolysaccharides. Four membrane labels were chosen for further experiments based on the transport to the intended membrane and the grade of separation obtained with the selective detergent treatment. In the next part of the project, the transport and localization of the fluorescent biomarkers to the intended membrane was verified by utilizing the density gradient centrifugation technique. This proved to require more precision and time, but gave a more complete separation of the two membranes. The biggest issue with the fluorescent biomarkers was the loss of a major part of the total fluorescence after fluorescent protein expression and separation of the membranes. We found that biologically active inclusion bodies were part of the problem, and the addition of a centrifugation step of 10 000 x g before pelleting the membranes removed these from the membrane fractions. To reduce the accumulation of inclusion bodies in the cells, a lower expression level of the fluorescent biomarkers was required. In the final stage of the project, the two most suitable fluorescent markers were to be transferred into the E. coli BL21 Gold (DE3) genome using λ red recombination. Due to time limitations, this part of the project was not completed. More experimentation and optimization is required to obtain recombinant clones with this technique

Cell-shape regulation in Streptococcus pneumoniae : EloR/KhpA, a new regulatory pathway administering cellelongation

The oval shaped Streptococcus pneumoniae utilizes both septal and peripheral peptidoglycan (PG) synthesis in order to maintain its shape. The protein complexes responsible for synthesizing PG are called the elongasome and the divisome, and as the names imply the elongasome synthesizes peripheral PG while the divisome synthesizes the PG responsible for dividing the cell into two [1]. The core of the elongasome and the divisome is the essential class B penicillin binding proteins (PBPs) 2b and 2x, respectively. PBP2b and PBP2x are transpeptidases creating peptide cross-links in PG between glycan strands [1, 2]. They work alongside the glycosyltransferases RodA and FtsW, respectively, to incorporate new PG into the existing PG sacculus [3-5]. The elongasome and divisome activities must be precisely coordinated throughout the cell cycle, but detailed knowledge about the control systems the cells possess to manage these PG machineries are lacking. In the current work, I present a pathway for regulation of elongation that has emerged recently: the EloR/KhpA complex. EloR and KhpA both contain RNA binding domains commonly seen in proteins involved in transcriptional or post-transcriptional regulation [6-8]. We and others have found that in order to survive the loss of pbp2b, S. pneumoniae can create suppressor mutations in the genes encoding EloR, KhpA and MltG rendering the protein products inactive [9, 10]. Our results show that EloR and KhpA work as a complex controlling cell elongation, most likely in a pathway including StkP, a Ser/Thr kinase known to have a regulatory role in cell division [11-15], and the essential lytic transglycosylase MltG. In paper I we show that the loss of EloR resulted in shorter cells in the laboratory strain R6. We confirmed that EloR is phosphorylated by StkP and conclude that it is likely that phosphorylation of EloR leads to release of bound RNA, stimulating elongation. We speculate that the reason PBP2b and RodA are essential in a wild type background is that these proteins are required in cells where the muralytic activity of MltG has a normal function. The reason pbp2b and rodA can be deleted in an DeloR mutant may be that the MltG activity is reduced without EloR present. In paper II we show that EloR interacts directly with a small RNA binding protein called KhpA. Using 3D modelling and site directed mutagenesis we identified the interaction surface between the two proteins and two amino acid residues important for this interaction. We could use this information to investigate how cells reacted to the loss of complex formation between the two proteins. A study by Zheng et al., 2017 showed that a khpA deletion mutant phenocopies an eloR deletion mutant [9]. We demonstrate in paper II that EloR and KhpA is one functional unit, and if the direct interaction between EloR and KhpA is broken, the cells behave like a DeloR or DkhpA mutant, i.e. PBP2b/RodA become redundant. We also show that KhpA depends upon EloR interaction to reach its midcell localization. In addition to two RNA binding domains (KH-II and R3H) at the C-terminal end, EloR has a Jag domain with unknown function at its N-terminus. In the final manuscript, paper III, we set out to unravel the function of the Jag domain. We found that the Jag domain is critical for midcell localization of EloR. Furthermore, by screening for protein-protein interactions between EloR and other elongasome proteins, the Jag domain was found to interact with the cytoplasmic domain of the lytic transglycosylase MltG. We hypothesize that the EloR/KhpA complex is recruited to midcell through the Jag-MltG interaction where it somehow controls the muralytic activity of MltG, either through protein – protein interaction or by RNA binding

Prevention of EloR/KhpA heterodimerization by introduction of site-specific amino acid substitutions renders the essential elongasome protein PBP2b redundant in Streptococcus pneumoniae

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Metataxonomic analysis and host proteome response in dairy cows with high and low somatic cell count: a quarter level investigation

Abstract Host response to invasive microbes in the bovine udder has an important role on the animal health and is essential to the dairy industry to ensure production of high-quality milk and reduce the mastitis incidence. To better understand the biology behind these host-microbiome interactions, we investigated the somatic cell proteomes at quarter level for four cows (collected before and after milking) using a shotgun proteomics approach. Simultaneously, we identified the quarter microbiota by amplicon sequencing to detect presence of mastitis pathogens or other commensal taxa. In total, 32 quarter milk samples were analyzed divided in two groups depending on the somatic cell count (SCC). The high SCC group (>100,000 cell/mL) included 10 samples and significant different proteome profiles were detected. Differential abundance analysis uncovers a specific expression pattern in high SCC samples revealing pathways involved in immune responses such as inflammation, activation of the complement system, migration of immune cells, and tight junctions. Interestingly, different proteome profiles were also identified in quarter samples containing one of the two mastitis pathogens, Staphylococcus aureus and Streptococcus uberis, indicating a different response of the host depending on the pathogen. Weighted correlation network analysis identified three modules of co-expressed proteins which were correlated with the SCC in the quarters. These modules contained proteins assigned to different aspects of the immune response, but also amino sugar and nucleotide sugar metabolism, and biosynthesis of amino acids. The results of this study provide deeper insights on how the proteome expression changes at quarter level in naturally infected cows and pinpoint potential interactions and important biological functions during host-microbe interaction

Identification of EloR (Spr1851) as a regulator of cell elongation in Streptococcus pneumoniae.

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