scholarly journals Control of acetyl phosphate-dependent phosphorylation of the response regulator CiaR by acetate kinase in Streptococcus pneumoniae

Microbiology ◽  
2020 ◽  
Vol 166 (4) ◽  
pp. 411-421
Author(s):  
Sabrina Kaiser ◽  
Lisa Marie Hoppstädter ◽  
Kevser Bilici ◽  
Kevin Heieck ◽  
Reinhold Brückner
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transcriptional Activation ◽  
Type Species ◽  
Response Regulator ◽  
Acetate Kinase ◽  
Acetyl Phosphate ◽  
Content Type ◽  
Link Type ◽  
Cell Extracts

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a large variety of physiological processes including ß-lactam resistance, competence development, maintenance of cell integrity, bacteriocin production, but also host colonization and virulence. The response regulator CiaR is active under a wide variety of conditions and the cognate CiaH kinase is not always needed to maintain CiaR activity. Using tetracycline-controlled expression of ciaR and variants, acetyl phosphate was identified in vivo as the alternative source of CiaR phosphorylation in the absence of CiaH. Concomitant inactivation of ciaH and the acetate kinase gene ackA led to very high levels of CiaR-mediated promoter activation. Strong transcriptional activation was accompanied by a high phosphorylation status of CiaR as determined by Phos-tag gel electrophoresis of S. pneumoniae cell extracts. Furthermore, AckA acted negatively upon acetyl phosphate-dependent phosphorylation of CiaR. Experiments using the Escherichia coli two-hybrid system based on adenylate cyclase reconstitution indicated binding of AckA to CiaR and therefore direct regulation. Subsequent in vitro CiaR phosphorylation experiments confirmed in vivo observations. Purified AckA was able to inhibit acetyl phosphate-dependent phosphorylation. Inhibition required the presence of ADP. AckA-mediated regulation of CiaR phosphorylation is the first example for a regulatory connection of acetate kinase to a response regulator besides controlling acetyl phosphate levels. It will be interesting to see if this novel regulation applies to other response regulators in S. pneumoniae or even in other organisms.

scholarly journals Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti

Microbiology ◽  
2020 ◽  
Vol 166 (5) ◽  
pp. 484-497 ◽  
Author(s):  
Alejandra Arteaga Ide ◽  
Victor M. Hernández ◽  
Liliana Medina-Aparicio ◽  
Edson Carcamo-Noriega ◽  
Lourdes Girard ◽  
...  
Keyword(s):  
Type Species ◽  
Sinorhizobium Meliloti ◽  
Arginase Activity ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Link Type ◽  
Arginine Catabolism

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

scholarly journals SufT is required for growth of Mycobacterium smegmatis under iron limiting conditions

Microbiology ◽  
2020 ◽  
Vol 166 (3) ◽  
pp. 296-305 ◽  
Author(s):  
Tsaone Tamuhla ◽  
Lydia Joubert ◽  
Danicke Willemse ◽  
Monique J. Williams
Keyword(s):  
Mycobacterium Smegmatis ◽  
Type Species ◽  
Model Organism ◽  
Culture Conditions ◽  
Growth Defect ◽  
Content Type ◽  
Link Type ◽  
Targeted Gene Deletion ◽  
Accessory Factor

Iron-sulphur (FeS) clusters are versatile cofactors required for a range of biological processes within cells. Due to the reactive nature of the constituent molecules, assembly and delivery of these cofactors requires a multi-protein machinery in vivo. In prokaryotes, SufT homologues are proposed to function in the maturation and transfer of FeS clusters to apo-proteins. This study used targeted gene deletion to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. Deletion of the sufT gene in M. smegmatis had no impact on growth under standard culture conditions and did not significantly alter activity of the FeS cluster dependent enzymes succinate dehydrogenase (SDH) and aconitase (ACN). Furthermore, the ΔsufT mutant was no more sensitive than the wild-type strain to the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), or the anti-tuberculosis drugs isoniazid, clofazimine or rifampicin. In contrast, the ΔsufT mutant displayed a growth defect under iron limiting conditions, and an increased requirement for iron during biofilm formation. This data suggests that SufT is an accessory factor in FeS cluster biogenesis in mycobacteria which is required under conditions of iron limitation.

scholarly journals Azithromycin resistance mutations in Streptococcus pneumoniae as revealed by a chemogenomic screen

2020 ◽  
Vol 6 (11) ◽  
Author(s):  
Hélène Gingras ◽  
Kévin Patron ◽  
Philippe Leprohon ◽  
Marc Ouellette
Keyword(s):  
Streptococcus Pneumoniae ◽  
Ribosomal Proteins ◽  
Type Species ◽  
Chemical Mutagenesis ◽  
Resistance Mutations ◽  
Content Type ◽  
Link Type ◽  
Chromosomal Changes ◽  
23S Ribosomal Rna ◽  
Ribosomal Binding Site

We report on the combination of chemical mutagenesis, azithromycin selection and next-generation sequencing (Mut-Seq) for the identification of small nucleotide variants that decrease the susceptibility of Streptococcus pneumoniae to the macrolide antibiotic azithromycin. Mutations in the 23S ribosomal RNA or in ribosomal proteins can confer resistance to macrolides and these were detected by Mut-Seq. By concentrating on recurrent variants, we could associate mutations in genes implicated in the metabolism of glutamine with decreased azithromycin susceptibility among S. pneumoniae mutants. Glutamine synthetase catalyses the transformation of glutamate and ammonium into glutamine and its chemical inhibition is shown to sensitize S. pneumoniae to antibiotics. A mutation affecting the ribosomal-binding site of a putative ribonuclease J2 is also shown to confer low-level resistance. Mut-Seq has the potential to reveal chromosomal changes enabling high resistance as well as novel events conferring more subtle phenotypes.

scholarly journals Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization

2016 ◽  
Vol 84 (10) ◽  
pp. 2922-2932 ◽  
Author(s):  
Krystle A. Blanchette ◽  
Anukul T. Shenoy ◽  
Jeffrey Milner ◽  
Ryan P. Gilley ◽  
Erin McClure ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Sialic Acid ◽  
Metabolic Pathways ◽  
Biofilm Formation ◽  
Fatty Acid Biosynthesis ◽  
Growth Conditions ◽  
Acetyl Phosphate ◽  
Content Type ◽  
Carbon Availability

Streptococcus pneumoniaeis an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed thatS. pneumoniaemutants deficient in NanA and β-galactosidase A (BgaA) failed to form biofilmsin vivodespite normal biofilm-forming abilitiesin vitro. Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formationin vivo. Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role.

scholarly journals Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica

2012 ◽  
Vol 11 (10) ◽  
pp. 1249-1256 ◽  
Author(s):  
Matthew L. Fowler ◽  
Cheryl Ingram-Smith ◽  
Kerry S. Smith
Keyword(s):  
Entamoeba Histolytica ◽  
Acetate Kinase ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Acetyl Phosphate ◽  
Phosphate Binding ◽  
Methanosarcina Thermophila ◽  
Inorganic Pyrophosphate ◽  
Content Type ◽  
Cell Extracts

ABSTRACTAcetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protistEntamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PPi)/inorganic phosphate (Pi) as the sole phosphoryl donor/acceptor. Detection of ACK activity inE. histolyticacell extracts in the direction of acetate/PPiformation but not in the direction of acetyl phosphate/Piformation suggests that the physiological direction of the reaction is toward acetate/PPiproduction. Kinetic parameters determined for each direction of the reaction are consistent with this observation. TheE. histolyticaPPi-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterizedMethanosarcina thermophilaATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for theM. thermophilaACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway inEntamoeba.

scholarly journals Streptococcus pneumoniae, S. pyogenes and S. agalactiae membrane phospholipid remodelling in response to human serum

Microbiology ◽  
2021 ◽  
Vol 167 (5) ◽  
Author(s):  
Luke R. Joyce ◽  
Ziqiang Guan ◽  
Kelli L. Palmer
Keyword(s):  
Streptococcus Pneumoniae ◽  
Human Serum ◽  
Type Species ◽  
Cellular Membrane ◽  
Defined Medium ◽  
Content Type ◽  
Link Type ◽  
Streptococcal Species ◽  
Group A ◽  
Group B

Streptococcus pneumoniae , S. pyogenes (Group A Streptococcus ; GAS) and S. agalactiae (Group B Streptococcus ; GBS) are major aetiological agents of diseases in humans. The cellular membrane, a crucial site in host–pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization MS, we characterized the phospholipids and glycolipids of S. pneumoniae , GAS and GBS in routine undefined laboratory medium, streptococcal defined medium and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodelling by the major pathogenic streptococci in response to metabolites present in human serum.

scholarly journals Co-infection in critically ill patients with COVID-19: an observational cohort study from England

2021 ◽  
Vol 70 (4) ◽  
Author(s):  
Vadsala Baskaran ◽  
Hannah Lawrence ◽  
Louise E. Lansbury ◽  
Karmel Webb ◽  
Shahideh Safavi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cohort Study ◽  
Streptococcus Pneumoniae ◽  
Critically Ill ◽  
Type Species ◽  
Severe Illness ◽  
Gram Negative ◽  
Content Type ◽  
Link Type ◽  
Icu Stay

Introduction. During previous viral pandemics, reported co-infection rates and implicated pathogens have varied. In the 1918 influenza pandemic, a large proportion of severe illness and death was complicated by bacterial co-infection, predominantly Streptococcus pneumoniae and Staphylococcus aureus . Gap statement. A better understanding of the incidence of co-infection in patients with COVID-19 infection and the pathogens involved is necessary for effective antimicrobial stewardship. Aim. To describe the incidence and nature of co-infection in critically ill adults with COVID-19 infection in England. Methodology. A retrospective cohort study of adults with COVID-19 admitted to seven intensive care units (ICUs) in England up to 18 May 2020, was performed. Patients with completed ICU stays were included. The proportion and type of organisms were determined at <48 and >48 h following hospital admission, corresponding to community and hospital-acquired co-infections. Results. Of 254 patients studied (median age 59 years (IQR 49–69); 64.6 % male), 139 clinically significant organisms were identified from 83 (32.7 %) patients. Bacterial co-infections/ co-colonisation were identified within 48 h of admission in 14 (5.5 %) patients; the commonest pathogens were Staphylococcus aureus (four patients) and Streptococcus pneumoniae (two patients). The proportion of pathogens detected increased with duration of ICU stay, consisting largely of Gram-negative bacteria, particularly Klebsiella pneumoniae and Escherichia coli . The co-infection/ co-colonisation rate >48 h after admission was 27/1000 person-days (95 % CI 21.3–34.1). Patients with co-infections/ co-colonisation were more likely to die in ICU (crude OR 1.78,95 % CI 1.03–3.08, P=0.04) compared to those without co-infections/ co-colonisation. Conclusion. We found limited evidence for community-acquired bacterial co-infection in hospitalised adults with COVID-19, but a high rate of Gram-negative infection acquired during ICU stay.

scholarly journals Delivery of antibacterial silver nanoclusters to Pseudomonas aeruginosa using species-specific DNA aptamers

2020 ◽  
Vol 69 (4) ◽  
pp. 640-652 ◽  
Author(s):  
Jennifer Soundy ◽  
Darren Day
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Activity ◽  
Type Species ◽  
Galleria Mellonella ◽  
Host Cells ◽  
Silver Nanoclusters ◽  
Bacterial Cells ◽  
Content Type ◽  
Link Type

Introduction. The use of silver as an antimicrobial therapeutic is limited by its toxicity to host cells compared with that required to kill bacterial pathogens. Aim. To use aptamer targeting of DNA scaffolded silver nanoclusters as an antimicrobial agent for treating Pseudomonas aeruginosa infections. Methodology. Antimicrobial activity was assessed in planktonic cultures and in vivo using an invertebrate model of infection. Results. The aptamer conjugates that we call aptabiotics have potent antimicrobial activity. Targeted silver nanoclusters were more effective at killing P. aeruginosa than the equivalent quantity of untargeted silver nanoclusters. The aptabiotics have an IC50 of 1.3–2.6 µM against planktonically grown bacteria. Propidium iodide staining showed that they rapidly depolarize bacterial cells to kill approximately 50 % of the population within 10 min following treatment. In vivo testing in the Galleria mellonella model of infection prolonged survival from an otherwise lethal infection. Conclusion. Using P. aeruginosa as a model, we show that targeting of DNA-scaffolded silver nanoclusters with an aptamer has effective fast-acting antimicrobial activity in vitro and in an in vivo animal model.

Mining indigenous honeybee gut microbiota for Lactobacillus with probiotic potential

Microbiology ◽  
2021 ◽  
Author(s):  
Ghazal Aziz ◽  
Muhammad Tariq ◽  
Arsalan Haseeb Zaidi
Keyword(s):  
Type Species ◽  
Amplicon Sequencing ◽  
Culturable Bacteria ◽  
Human Pathogens ◽  
Carbohydrate Utilization ◽  
Potential Health ◽  
Content Type ◽  
Link Type

The present study was done to explore the diversity of lactic acid bacteria (LAB) associated with the gastrointestinal tract (GIT) of honeybee species endemic to northeastern Pakistan. Healthy worker bees belonging to Apis mellifera, A. dorsata, A. cerana and A. florea were collected from hives and the surroundings of a major apiary in the region. The 16S rRNA amplicon sequencing revealed a microbial community in A. florea that was distinct from the others in having an abundance of Lactobacillus and Bifidobacteria. However, this was not reflected in the culturable bacteria obtained from these species. The isolates were characterized for safety parameters, and 20 LAB strains deemed safe were evaluated for resistance to human GIT stresses like acid and bile, adhesion and adhesiveness, and anti-pathogenicity. The five most robust strains, Enterococcus saigonensis NPL780a, Lactobacillus rapi NPL782a, Lactobacillus kunkeei NPL783a, and NPL784, and Lactobacillus paracasei NPL783b, were identified through normalized Pearson (n) principal components analysis (PCA). These strains were checked for inhibition of human pathogens, antibiotic resistance, osmotic tolerance, metabolic and enzymatic functions, and carbohydrate utilization, along with antioxidative and cholesterol-removing potential. The findings suggest at least three strains (NPL 783a, 784 and 782a) as candidates for further in vitro and in vivo investigations of their potential health benefits and application as novel probiotic adjuncts.

scholarly journals The prevalence of pilus islets in Streptococcus pneumoniae isolates from healthy children in Indonesia

2020 ◽  
Author(s):  
Dodi Safari ◽  
Feby Valentiya ◽  
Korrie Salsabila ◽  
Wisiva Tofriska Paramaiswari ◽  
Wisnu Tafroji ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Type Species ◽  
Pneumococcal Vaccination ◽  
Healthy Children ◽  
Upper Respiratory Tract ◽  
Content Type ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Link Type ◽  
Islet 1

Streptococcus pneumoniae produces pili that function as adherence factors to bind to epithelial cells in the human upper respiratory tract. In this study, we investigated the prevalence of pilus islets (PIs) in S. pneumoniae strains carried by healthy children below 5 years of age prior to pneumococcal vaccination in 2012 in Lombok Island, Indonesia. In all, 347 archived S. pneumoniae isolates were screened using polymerase chain reactions for the presence of rrgC and pitB genes representing pilus islet 1 (PI-1) and pilus islet 2 (PI-2), respectively. We found that 40 isolates (11.5 %) contained the PI genes: 5.2% carried both PI-1 and PI-2, and 3.5 and 2.9% carried PI-1 and PI-2, respectively. Furthermore, we found that most of the strains carrying either of the PIs belonged to the vaccine serotypes 19F and 19A and were less susceptible to chloramphenicol and tetracycline.

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