Pneumococcal cell biology in a new light

Hoewel bacteriën bijna overal te vinden zijn, zijn ze met het blote oog niet te zien. Daarvoor kunnen we een microscoop gebruiken: om kleine dingen te vergroten en zo zichtbaar voor ons te maken. Bacteriën groeien via binaire deling, waarbij één cel zich deelt in twee identieke kopieën van zichzelf. De meeste bacteriën zijn belangrijk of zelfs onmisbaar voor ons en onze omgeving. Sommige kunnen echter juist ziektes veroorzaken. Een voorbeeld hiervan is Streptococcus pneumoniae, ook wel de pneumokok genoemd. Deze rugbybalvormige bacterie kan ernstige infecties in het ademhalingskanaal veroorzaken. Sinds Fleming in 1920 penicilline heeft ontdekt, worden antibiotica gebruikt om bacteriële infecties te genezen. De toename in antibioticaresistente ziekteverwekkers is in de loop der jaren een serieus probleem geworden. Voor het identificeren van doelwitten voor nieuwe geneesmiddelen is het van essentieel belang dat we de basis van bacteriële groei begrijpen. De deling van een cel is een zeer dynamisch proces, waarvoor een (nauwkeurig afgestemd) samenspel van factoren nodig is. Wij onderzochten deze processen door individuele cellen te bestuderen met behulp van microscopietechnieken. Met behulp van fluorescente eiwitten kunnen we andere eiwitten labellen en hun lokalisatie binnen de cel daarmee zichtbaar maken. Een deel van ons werk beschrijft geoptimaliseerde methodes en gereedschappen voor fluorescentiemicroscopie die van algemeen belang zijn voor het veld. We hebben ook licht kunnen werpen op de regulatie van celdelingsprocessen, maar er is nog meer werk nodig om een duidelijker beeld te krijgen en nieuwe antibiotica te identificeren die de pneumokok kunnen bestrijden

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy)

Red fluorescent proteins for gene expression and protein localization studies in Streptococcus pneumoniae and efficient transformation with Gibson assembled DNA

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram positive bacteria. In the present work we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and tagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson Assembly products (isothermal assembly), a method speeding up the strain construction process significantly. Our data show that mCherry is the fastest maturing RFP in S. pneumoniae and is best suited for studying gene expression while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multi-color labeling in S. pneumoniae and related organisms

Streptococcus pneumoniae PBP2x mid-cell localization requires the C-terminal PASTA domains and is essential for cell shape maintenance

The transpeptidase activity of the essential penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of N-terminal GFP-PBP2x fusions under the control of a zinc-inducible promoter. The ectopic fusion protein localized at mid-cell. Cells showed no growth defects even in the absence of the genomic pbp2x, demonstrating that GFP-PBP2x is functional. Depletion of GFP-PBP2x resulted in severe morphological alterations, confirming the essentiality of PBP2x and demonstrating that PBP2x is required for cell division and not for cell elongation. A genetically or antibiotic inactivated GFP-PBP2x still localized at septal sites. Remarkably, the same was true for a GFP-PBP2x derivative containing a deletion of the central transpeptidase domain, although only in the absence of the protease/chaperone HtrA. Thus localization is independent of the catalytic transpeptidase domain but requires the C-terminal PASTA domains, identifying HtrA as targeting GFP-PBP2x derivatives. Finally, PBP2x was positioned at the septum similar to PBP1a and the PASTA domain containing StkP protein, confirming that PBP2x is a key element of the divisome complex

Protocol to assess metabolic activity of Pseudomonas aeruginosa by measuring heat flow using isothermal calorimetry

Summary: Here, we present a protocol for assessing metabolic activity of bacterial populations by measuring heat flow using isothermal calorimetry. We outline the steps for preparing the different growth models of Pseudomonas aeruginosa and performing continuous metabolic activity measurements in the calScreener. We detail simple principal component analysis to differentiate between metabolic states of different populations and probabilistic logistic classification to assess resemblance to wild-type bacteria. This protocol for fine-scale metabolic measurement can aid in understanding microbial physiology.For complete details on the use and execution of this protocol, please refer to Lichtenberg et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Regulation of peptidoglycan synthesis by outer-membrane proteins

Growth of the meshlike peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function respectively of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/ LpoB and their PBP docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth