Anatomy of the pneumococcal nucleoid:Visualizing replication, chromosome segregation and chromosome condensation dynamics in Streptococcus pneumoniae

The pneumococcus is a bacterium that lives in the upper part of the throat behind the nose of many children and adults. In most cases it lives there harmlessly, but sometimes, it can move further into the body and cause serious illnesses as pneumonia or meningitis. Understanding how this bacterium grows could give us starting points for antibacterial drugs: if you can stop growth, you can stop the bacterium. Bacteria grow by cell division. The pneumococcus is no different: it grows in two directions, after which a wall is formed in the middle and the bacterium splits into two daughter cells. During this division cycle, the internal components of the cell need to be copied and brought to the two cell halves. In this thesis, we use several microscopy techniques to map how the DNA of the pneumococcus is copied and split into two so-called 'nucleoids' during the cell cycle. First, we benchmarked Red Fluorescent Proteins (RFPs) in the pneumococcus and developed analysis software to be able to create a map of the internal organization of the pneumococcus. Then, we followed different parts of the chromosome and proteins important for the cell cycle. We found that the correct splitting of the DNA in two nucleoids is also important for the correct placement of the septum in the middle of the cell. This shows that also in the pneumococcus, cell division and chromosome organization are connected processes

Computational tools for the synthetic design of biochemical pathways

As the field of synthetic biology is developing, the prospects for de novo design of biosynthetic pathways are becoming more and more realistic. Hence, there is an increasing need for computational tools that can support these efforts. A range of algorithms has been developed that can be used to identify all possible metabolic pathways and their corresponding enzymatic parts. These can then be ranked according to various properties and modelled in an organism-specific context. Finally, design software can aid the biologist in the integration of a selected pathway into smartly regulated transcriptional units. Here, we review key existing tools and offer suggestions for how informatic scan help to shape the future of synthetic microbiology.</p

Noise and Stochasticity in Gene Expression:A Pathogenic Fate Determinant

Not all cells in a bacterial population exhibit exactly the same phenotype, even though they grow in the same environment and are genetically identical. This phenomenon is known as phenotypic variation. The major source of phenotypic variation is noise or stochasticity in gene expression networks, which can directly promote the formation of population heterogeneity. Bistability, or the existence of two stable subpopulations, is a direct outcome of gene expression noise. In this chapter, we will discuss the origin of noise in gene expression and how researchers measure, quantify and engineer noise in gene circuits. Furthermore, we will describe how pathogenic organisms utilize noisy gene circuits for bistable gene expression of virulence factors. This serves to highlight the importance of noise in molecular decision making. We discuss how two pathogenic bacteria employ heterogeneous gene expression when invading a host: firstly, the heterogeneously expression of pilus formation, which are thought to aid the colonization the Gram-positive bacterium Streptococcus pneumoniae by facilitating attachment to host epithelium, and, secondly, the complex gene circuit with positive and double-negative feedback loops characteristic for noise-driven expression, used by the Gram-negative bacterium Salmonella enterica serovar Typhimurium to regulate flagella formation and the expression of its type 3 secretion system.

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

Modulation of prey size reveals adaptability and robustness in the cell cycle of an intracellular predator.

Despite a remarkable diversity of lifestyles, bacterial replication has only been investigated in a few model species. In bacteria that do not rely on canonical binary division for proliferation, the coordination of major cellular processes is still largely mysterious. Moreover, the dynamics of bacterial growth and division remain unexplored within spatially confined niches where nutrients are limited. This includes the life cycle of the model endobiotic predatory bacterium Bdellovibrio bacteriovorus, which grows by filamentation within its prey and produces a variable number of daughter cells. Here, we examined the impact of the micro-compartment in which predators replicate (i.e., the prey bacterium) on their cell-cycle progression at the single-cell level. Using Escherichia coli with genetically encoded size differences, we show that the duration of the predator cell cycle scales with prey size. Consequently, prey size determines predator offspring numbers. We found that individual predators elongate exponentially, with a growth rate determined by the nutritional quality of the prey, irrespective of prey size. However, the size of newborn predator cells is remarkably stable across prey nutritional content and size variations. Tuning the predatory cell cycle by modulating prey dimensions also allowed us to reveal invariable temporal connections between key cellular processes. Altogether, our data imply adaptability and robustness shaping the enclosed cell-cycle progression of B. bacteriovorus, which might contribute to optimal exploitation of the finite resources and space in their prey. This study extends the characterization of cell cycle control strategies and growth patterns beyond canonical models and lifestyles

Noise and Stochasticity in Gene Expression: A Pathogenic Fate Determinant

Not all cells in a bacterial population exhibit exactly the same phenotype, even though they grow in the same environment and are genetically identical. This phenomenon is known as phenotypic variation. The major source of phenotypic variation is noise or stochasticity in gene expression networks, which can directly promote the formation of population heterogeneity. Bistability, or the existence of two stable subpopulations, is a direct outcome of gene expression noise. In this chapter, we will discuss the origin of noise in gene expression and how researchers measure, quantify and engineer noise in gene circuits. Furthermore, we will describe how pathogenic organisms utilize noisy gene circuits for bistable gene expression of virulence factors. This serves to highlight the importance of noise in molecular decision making. We discuss how two pathogenic bacteria employ heterogeneous gene expression when invading a host: firstly, the heterogeneously expression of pilus formation, which are thought to aid the colonization the Gram-positive bacterium Streptococcus pneumoniae by facilitating attachment to host epithelium, and, secondly, the complex gene circuit with positive and double-negative feedback loops characteristic for noise-driven expression, used by the Gram-negative bacterium Salmonella enterica serovar Typhimurium to regulate flagella formation and the expression of its type 3 secretion system