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

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.

Pentapeptide-rich peptidoglycan at the Bacillus subtilis cell-division site.

Peptidoglycan (PG), the major component of the bacterial cell wall, is one large macromolecule. To allow for the different curvatures of PG at cell poles and division sites, there must be local differences in PG architecture and eventually also chemistry. Here we report such local differences in the Gram-positive rod-shaped model organism Bacillus subtilis. Single-cell analysis after antibiotic treatment and labeling of the cell wall with a fluorescent analogue of vancomycin or the fluorescent D-amino acid analogue (FDAA) HCC-amino-D-alanine revealed that PG at the septum contains muropeptides with unprocessed stem peptides (pentapeptides). Whereas these pentapeptides are normally shortened after incorporation into PG, this activity is reduced at division sites indicating either a lower local degree of PG crosslinking or a difference in PG composition, which could be a topological marker for other proteins. The pentapeptides remain partially unprocessed after division when they form the new pole of a cell. The accumulation of unprocessed PG at the division site is not caused by the activity of the cell division specific penicillin-binding protein 2B. To our knowledge, this is the first indication of local differences in the chemical composition of PG in Gram-positive bacteria.

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method.

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 (isothermal assembly) products, a method that speeds 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 multicolor 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.

Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging.

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two "superfolder" GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

The localization of key Bacillus subtilis penicillin binding proteins during cell growth is determined by substrate availability.

The shape of bacteria is maintained by the cell wall. The main component of the cell wall is peptidoglycan (PG) that is synthesized by penicillin binding proteins (PBPs). The correct positioning of PBPs is essential for the maintenance of cell shape. In the literature, two different models for localization of PBPs have been proposed - localization through interaction with a cytoskeletal structure or localization through the presence of substrate. Here, we show that the localization of PBPs critical for the rod shape of Bacillus subtilis is altered when the substrate LipidII is delocalized by treatment of the cells with nisin. Alteration of this localization is only seen in a LipidII-dependent manner and is not influenced by dissipation of the membrane potential, a secondary effect of nisin treatment. Our results strongly suggest that the localization of PG synthesis at the periphery of the cell is substrate-driven, even in bacteria that contain actin-like MreB cytoskeletal structures.

A single amino acid substitution in the MurF UDP-MurNAc-pentapeptide synthetase renders Streptococcus pneumoniae dependent on CO2 and temperature.

The respiratory tract pathogen Streptococcus pneumoniae encounters different levels of environmental CO2 during transmission, host colonization and disease. About 8% of all pneumococcal isolates are capnophiles that require CO2 -enriched growth conditions. The underlying molecular mechanism for caphnophilic behaviour, as well as its biological function is unknown. Here, we found that capnophilic S. pneumoniae isolates from clonal complex (CC) 156 (i.e. Spain(9V) -3 ancestry) and CC344 (i.e. Norway(NT) -42 ancestry) have a valine at position 179 in the MurF UDP-MurNAc-pentapeptide synthetase. At ≤ 30°C, the growth characteristics of capnophilic and non-capnophilic CC156 strains were equal, but at > 30°C growth and survival of MurF(V) (179) strains was dependent on > 0.1% CO2 -enriched conditions. Expression of MurF(V) (179) in S. pneumoniae R6 and G54 rendered these, otherwise non-capnophilic strains, capnophilic. Time-lapse microscopy revealed that a capnophilic CC156 strain undergoes rapid autolysis upon exposure to CO2 -poor conditions at 37°C, and staining with fluorescently labelled vancomycin showed a defect in de novo cell wall synthesis. In summary, in capnophilic S. pneumoniae strains from CC156 and CC344 cell wall synthesis is placed under control of environmental CO2 levels and temperature. This mechanism might represent a novel strategy of the pneumococcus to rapidly adapt and colonize its host under changing environmental conditions.

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP.

How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.

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). Here, we provide a detailed description of a microscopy method used in several recent studies (4, 5, 6, 7), which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Regulation of peptidoglycan synthesis by outer-membrane proteins.

Growth of the mesh-like 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 of 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.