Spatial organisation of expanding bacterial colonies is affected by contact-dependent growth inhibition

Identifying how microbes are able to manipulate, survive and thrive in complex multispecies communities has expanded our understanding of how microbial ecosystems impact human health and the environment. The ability of bacteria to negatively affect neighbours, through explicit toxin delivery systems, provides them with an opportunity to manipulate the composition of growing microbial communities. Contact-dependent inhibition (CDI) systems (a Type Vb secretion system) are a distinct subset of competition systems whose contribution to shaping the development of spatially-structured bacterial communities are yet to be fully understood. Here we compare the impact of different CDI systems, at both the single cell and population level, to determine the key drivers of CDI-mediated competition within spatially-structured bacterial populations. Through an iterative approach using both an Escherichia coli experimental system and computational modelling, we show that CDI systems have subtle and system-specific effects at the single cell level, generating single cell wide boundaries between CDI-expressing inhibitor cells and their neighbouring targets. Despite the subtle effects of CDI at a single cell level, CDI systems greatly diminished the ability of susceptible targets to expand their range during colony growth. The inoculum density of the population, together with the CDI system-specific variables of the speed of inhibition after contact and biological cost of CDI, strongly affects CDI-mediated competition. In contrast, the magnitude of the toxin-induced growth retardation of target cells only weakly impacts the composition of the population. Our work reveals how distinct CDI systems can differentially affect the composition and spatial arrangement of bacterial populations

Design and implementation of tools to study cellular heterogeneity in clonal populations of Salmonella enterica

In this thesis genetic tools were designed and implemented to study cellular heterogeneity in clonal populations of Salmonella enterica. Salmonella is an intestinal, food-borne pathogen and was chosen as a model organism due to its large impact on human health and the global economy. The aim of this work was to develop customized transposons to study Salmonella behavior on the single cell level using time-lapse fluorescence microscopy (TLFM). Studying isogenic bacterial populations with single cell resolution can yield unprecedented insights in protein expression and function, often impossible to obtain with population level studies. The transposons constructed in this work harbor a gene encoding a fluorescent protein and after transposon mutagenesis, Salmonella mutants can be screened for unusual protein expression patterns (i.e. heterogeneous) and/or intracellular protein localization (i.e. non-diffuse localizations) using TLFM. The different transposons each serve slightly different purposes and were all validated in this work. Moreover, two major screens were set up with the developed transposons, using Salmonella Typhimurium as model organism, and atypical mutants regarding protein localization and expression could be isolated and were further characterized. Our first screen aimed for the retrieval of endogenous nucleoid reporters, which are important when studying the live impact of specific stresses on the bacterial chromosome, and uncovered the IscR protein as a valuable candidate. Although the DNA binding properties of this protein were already known, this is, to the best of our knowledge, the first time the IscR protein was studied using TLFM and further validated as a nucleoid reporter. The second screen performed in this work targeted the large virulence plasmid (pSLT) of Salmonella Typhimurium and this mobile genetic element is important for Salmonella to achieve full virulence. Different fluorescent mutants could be retrieved of which one particular mutant was found to have a bistable expression pattern, and this mutant could be linked to an important virulence operon. In depth analysis of this operon led to a more profound understanding of its regulation and more generally in how Salmonella regulates and coordinates its virulence factors. Together, these results demonstrate that our transposon mutagenesis approach, using customized transposons, is useful for screening large libraries for aberrant protein localizations and expression patterns, and can thus be the starting point in unraveling unknown protein functions and biological mechanisms in general. In turn, this will increase our knowledge regarding Salmonella behavior and may eventually lead to new approaches in combatting this pathogen.nrpages: 227status: publishe

Population heterogeneity tactics as driving force in Salmonella virulence and survival

Salmonella enterica comprises many pathogenic serovars that are able to colonize a variety of animal hosts and therefore constitute an important source of zoonotic food-borne illness. Their pathogenicity can range from gastroenteritis to typhoid fever, and depends on a series of virulence factors that are regularly located on laterally acquired genetic elements. The regulation of these virulence factors often also includes their differential expression within clonal populations. Moreover, exploitation of the resulting population heterogeneity appears to be an integral aspect of Salmonella virulence that could also affect its survival outside the host. This review therefore addresses how the regulation and heterogeneous expression of various virulence factors supports Salmonella's success as a food-borne pathogen.status: publishe

Construction and validation of the Tn5-PLtetO-1-msfGFP transposon as a tool to probe protein expression and localization

In this study we report the design, construction and validation of a novel transposon aimed to systematically screen for protein localization and expression patterns in prokaryotes using fluorescence microscopy. Upon random insertion in an open reading frame in the proper frame and orientation, the transposon creates an N-terminal fluorescent protein fusion to the msfGFP reporter. Moreover, in order to examine the localization of fusion proteins whose native expression might be too low or absent, the transposon was fitted with a PLtetO-1 promoter that makes the expression of the generated fluorescent protein fusions controllable by anhydrotetracycline. Importantly, upon flipping out the PLtetO-1 promoter and neighboring antibiotic resistance marker, an in-frame "sandwich" msfGFP fusion is created in which the N- and C-terminal portions of the targeted protein are again controlled by its native promoter.status: publishe

Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in <i>Salmonella</i> Typhimurium

<div><p>In this study we adapted a Mu<i>d</i>-based delivery system to construct a random <i>yfp</i> reporter gene (encoding the yellow fluorescent protein) insertion library in the genome of <i>Salmonella</i> Typhimurium LT2, and used fluorescence activated cell sorting and fluorescence microscopy to screen for translational fusions that were able to clearly and specifically label the bacterial nucleoid. Two such fusions were obtained, corresponding to a translational <i>yfp</i> insertion in <i>iscR</i> and <i>iolR</i>, respectively. Both fusions were further validated, and the IscR::YFP fluorescent nucleoid reporter together with time-lapse fluorescence microscopy was subsequently used to monitor nucleoid dynamics in response to the filamentation imposed by growth of LT2 at high hydrostatic pressure (40–45 MPa). As such, we were able to reveal that upon decompression the apparently entangled LT2 chromosomes in filamentous cells rapidly and efficiently segregate, after which septation of the filament occurs. In the course of the latter process, however, cells with a “trilobed” nucleoid were regularly observed, indicative for an imbalance between septum formation and chromosome segregation.</p></div

Digital Microfluidics for Single Bacteria Capture and Selective Retrieval Using Optical Tweezers

When screening microbial populations or consortia for interesting cells, their selective retrieval for further study can be of great interest. To this end, traditional fluorescence activated cell sorting (FACS) and optical tweezers (OT) enabled methods have typically been used. However, the former, although allowing cell sorting, fails to track dynamic cell behavior, while the latter has been limited to complex channel-based microfluidic platforms. In this study, digital microfluidics (DMF) was integrated with OT for selective trapping, relocation, and further proliferation of single bacterial cells, while offering continuous imaging of cells to evaluate dynamic cell behavior. To enable this, magnetic beads coated with Salmonella Typhimurium-targeting antibodies were seeded in the microwell array of the DMF platform, and used to capture single cells of a fluorescent S. Typhimurium population. Next, OT were used to select a bead with a bacterium of interest, based on its fluorescent expression, and to relocate this bead to a different microwell on the same or different array. Using an agar patch affixed on top, the relocated bacterium was subsequently allowed to proliferate. Our OT-integrated DMF platform thus successfully enabled selective trapping, retrieval, relocation, and proliferation of bacteria of interest at single-cell level, thereby enabling their downstream analysis

Growth and nucleoid dynamics of HP stressed LT2.

<p>(A) Representative images showing cell growth and nucleoid dynamics of HP stressed LT2 <i>iscR</i>::<i>yfp</i> cells (grown overnight at 40–45 MPa and 37°C) at the indicated time points after pressure release. Merged phase contrast and YFP images are shown. (B) Representative images showing cell growth and nucleoid dynamics of a HP stressed LT2 <i>iscR</i>::<i>yfp</i> cell (grown overnight at 40–45 MPa and 37°C) at the indicated time points after pressure release in the presence of DAPI and intermittent UV excitation. Phase contrast (top panels), DAPI (middle panels) and YFP (lower panels) images are shown. Scale bars correspond to 5 μm.</p

Construction and use of the MudY transposon.

<p>(A) During construction of the Mu<i>d</i>Y transposon, the <i>lacZYA</i>-<i>npt</i> of Mu<i>d</i>K was replaced by an <i>yfp</i>-<i>frt</i>-<i>cat</i>-<i>frt</i> module through recombineering (blue and green crosses indicate the homologous regions involved in recombination). (B) The <i>frt</i>-<i>cat</i>-<i>frt</i> cassette can be readily flipped out using the Flp recombinase, thereby reducing possible polar effects of the <i>cat</i> marker. (C) Exact location and genomic context of two Mu<i>d</i>Y insertions (<i>iscR</i>::Mu<i>d</i>Y and <i>iolR</i>::Mu<i>d</i>Y) in LT2 yielding endogeneous nucleoid reporters.</p

Representative images showing the colocalization of the IscR144::YFP and IolR159::YFP proteins with the DAPI stained nucleoid in LT2 <i>iscR</i>::Mu<i>d</i>Y (A–D) and LT2 and <i>iolR</i>::Mu<i>d</i>Y (E–H), respectively.

<p>Phase-contrast (A,E), DAPI (B,F), YFP (C,G), and merged (D,H) images are shown. Inset in panel C includes a larger image of an LT2 <i>iscR</i>::Mu<i>d</i>Y cell showing the apparent helical shape of the nucleoid highlighted by IscR144::YFP. Scale bars correspond to 5 μm.</p