Evolution of longitudinal division in multicellular bacteria of the Neisseriaceae family.

Rod-shaped bacteria typically elongate and divide by transverse fission. However, several bacterial species can form rod-shaped cells that divide longitudinally. Here, we study the evolution of cell shape and division mode within the family Neisseriaceae, which includes Gram-negative coccoid and rod-shaped species. In particular, bacteria of the genera Alysiella, Simonsiella and Conchiformibius, which can be found in the oral cavity of mammals, are multicellular and divide longitudinally. We use comparative genomics and ultrastructural microscopy to infer that longitudinal division within Neisseriaceae evolved from a rod-shaped ancestor. In multicellular longitudinally-dividing species, neighbouring cells within multicellular filaments are attached by their lateral peptidoglycan. In these bacteria, peptidoglycan insertion does not appear concentric, i.e. from the cell periphery to its centre, but as a medial sheet guillotining each cell. Finally, we identify genes and alleles associated with multicellularity and longitudinal division, including the acquisition of amidase-encoding gene amiC2, and amino acid changes in proteins including MreB and FtsA. Introduction of amiC2 and allelic substitution of mreB in a rod-shaped species that divides by transverse fission results in shorter cells with longer septa. Our work sheds light on the evolution of multicellularity and longitudinal division in bacteria, and suggests that members of the Neisseriaceae family may be good models to study these processes due to their morphological plasticity and genetic tractability.

Visualization of SpoVAEa Protein Dynamics in Dormant Spores of Bacillus cereus and Dynamic Changes in Their Germinosomes and SpoVAEa during Germination.

Bacillus cereus spores, like most Bacillus spores, can survive for years and germinate when their surroundings become suitable, and germination proteins play an important role in the initiation of germination. Because germinated spores lose the extreme resistance of dormant spores, information on the function of germination proteins could be useful in developing new strategies to control B. cereus spores. Prior work has shown that (i) the channel protein SpoVAEa exhibits high-frequency movement in the outer leaflet of the inner membrane (IM) in dormant B. subtilis spores and (ii) the formation of the foci termed germinosomes between two germination proteins, the germinant receptor GerR and the scaffold protein GerD, in developing B. cereus spores is slower than foci formation by GerR and GerD individually. However, the movement dynamics of SpoVAEa in B. cereus spores, and the behavior of the germinosome upon B. cereus spore germination, are not known. In this study, we found that SpoVAEa fluorescent foci in dormant B. cereus spores move on the IM, but slower than in B. subtilis spores, and they likely co-localize transiently with GerD-mScarlet-I in the germinosome. Our results further indicate that (i) the expression of GerR-SGFP2 and SpoVAEa-SGFP2 with GerD-mScarlet-I from a plasmid leads to more heterogeneity and lower efficiency of spore germination in B. cereus, and (ii) germinosome foci observed by Fluorescence resonance energy transfer (FRET) between GerR-SGFP2 and GerD-mScarlet-I can be lost soon after the spore-phase transition. However, this is not always the case, as some GerR-SGFP2 and GerD-mScarlet-I foci continued to exist, co-localize, and even show a weak FRET signal. These data highlight the heterogeneous behavior of spore germination protein complexes and indicate that some complexes may persist beyond the initiation of germination. IMPORTANCE Bacillus cereus is commonly present in soil and infects humans via contaminated food. In this study, we used B. cereus spores to investigate the movement of the spore-specific inner membrane (IM) channel protein SpoVAEa, the interaction between SpoVAEa and the germinosome scaffold protein GerD, and the dynamics of germinosomes with GerR and GerD in spore germination. Our results expand upon observations of interactions between specific B. cereus spore germination proteins, in particular the GerR germinant receptor A, B, and C subunits and GerD, as well as those between SpoVAEa and GerD. The approaches used in this work could also be used to examine the interactions between GerD and SpoVAEa and other germination proteins in spores of other Bacillus species.

Organization and dynamics of the SpoVAEa protein and its surrounding inner membrane lipids, upon germination of Bacillus subtilis spores.

The SpoVA proteins make up a channel in the inner membrane (IM) of Bacillus subtilis spores. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca2+-DPA from the spore core during germination. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores. Notably, the SpoVAEa-SGFP2 proteins were present in a single spot in spores, similar to the IM complex formed by all GRs termed the germinosome. However, while the GRs' spot remains in one location, the SpoVAEa-SGFP2 spot in the IM moved randomly with high frequency. It seems possible that this movement may be a means of communicating germination signals from the germinosome to the IM SpoVA channel, thus stimulating CaDPA release in germination. The dynamics of the SpoVAEa-SGFP2 and its surrounding IM region as stained by fluorescent dyes were also tracked during spore germination, as the dormant spore IM appeared to have an immobile germination related functional microdomain. This microdomain disappeared around the time of appearance of a germinated spore, and the loss of fluorescence of the IM with fluorescent dyes, as well as the appearance of peak SpoVAEa-SGFP2 fluorescent intensity occurred in parallel. These observed events were highly related to spores' rapid phase darkening, which is considered as due to rapid Ca2+DPA release. We also tested the response of SpoVAEa and the IM to thermal treatments at 40-80 °C. Heat treatment triggered an increase of green autofluorescence, which is speculated to be due to coat protein denaturation, and 80 °C treatments induce the appearance of phase-grey-like spores. These spores presumably have a similar intracellular physical state as the phase grey spores detected in the germination but lack the functional proteins for further germination events.

Heat Activation and Inactivation of Bacterial Spores: Is There an Overlap?

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has the potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination and then inactivating the less-heat-resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single-spore levels. The heat treatments used were 40 to 80°C and for 0 to 300 min. The results were as follows. (i) Heat activation at 40 to 70°C promoted l-valine- and l-asparagine-glucose-fructose-potassium (AGFK)-induced germination in a time-dependent manner. (ii) The optimal heat activation temperatures for AGFK and l-valine germination via the GerB plus GerK or GerA germinant receptors were 65°C and 50 to 65°C, respectively. (iii) Heat inactivation of dormant spores appeared at 70°C, and the heat damage of molecules essential for germination and growth began at 70 and 65°C, respectively. (iv) Heat treatment at 75°C resulted in both activation of germination and damage to the germination apparatus, and 80°C treatment caused more pronounced heat damage. (v) For the spores that should withstand adverse environmental temperatures in nature, heat activation seemed functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth. IMPORTANCE Bacterial spores are thermal-stress-resistant structures that can thus survive food preservation strategies and revive through the process of spore germination. The more heat resistant spores are, the more heterogeneous their germination upon the addition of germinants. Upon germination, spores can cause food spoilage and food intoxication. Here, we provide new information on both heat activation and inactivation regimes and their effects on the (heterogeneity of) spore germination.

Dynamics of Germinosome Formation and FRET-Based Analysis of Interactions between GerD and Germinant Receptor Subunits in Bacillus cereus Spores.

Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores' extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. In this study, we confirmed that the GerR germinant receptor's (GR) A, B, and C subunits and GerD co-localize in B. cereus spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)-SGFP2 and GerD-mScarlet-I, but there was no FRET between GerD-mScarlet-I and either GerRA-SGFP2 or GerRC-SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)-SGFP2 and GerD-mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)-SGFP2 and GerD-mScarlet-I; and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous.

Investigating Synthesis of the MalS Malic Enzyme during Bacillus subtilis Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions.

Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore's inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:695-707, 2015, https://doi.org/10.1016/j.molcel.2014.12.019). However, in contrast to the earlier report, both Western blotting and SILAC (stable isotopic labeling of amino acids in cell culture) analysis showed there was no increase in MalS-GFP levels during the 15 min after the addition of germinants and that MalS synthesis did not begin until more than 90 min after germinant addition. Thus, the increase in MalS-GFP fluorescence early in germination is not due to new protein synthesis but is perhaps due to a change in the physical environment of the spore cores. Our findings also show that different sporulation conditions and spore maturation times affect expression of MalS-GFP and the germination behavior of the spores, albeit to a minor extent, but still result in no changes in MalS-GFP levels early in spore germination.IMPORTANCE The spores formed by Bacillus subtilis remain in a quiescent state for extended periods due to their dormancy and resistance features. Dormancy is linked to a very low level of core water content and a phase-bright state of spores. The present report, focusing on proteins MalS and PdhD (pyruvate dehydrogenase subunit D) and complementary to our companion report published in this issue, aims to shed light on a major dilemma in the field, i.e., whether protein synthesis, in particular that of MalS, takes place in phase-bright spores. Clustered MalS-GFP in dormant spores diffuses throughout the spore as germination proceeds. However, fluorescence intensity measurements, supported by Western blot analysis and SILAC proteomics, confirm that there is no new MalS protein synthesis in bright-phase dormant spores.

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes.

Bacillus cereus can survive in the form of spores for prolonged periods posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. Our study aims at increasing knowledge of B. cereus spores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of B. cereus spores are a core with the genetic material encased by multiple protective layers, an important one being the spores' inner membrane (IM), the location of many important germination proteins. To study mechanisms involved in germination of B. cereus spores, we have examined the organization of germinant receptors (GRs) in spores' IM. Previous studies have indicated that in spores of B. cereus ATCC 14579 the L-alanine responsive GR, GerR, plays a major role in the germination process. In our study, the location of the GerR GR subunit, GerRB, in spores was examined as a C-terminal SGFP2 fusion protein expressed under the control of the gerR operon's promoter. Our results showed that: i) the fluorescence maxima and integrated intensity in spores with plasmid-borne expression of GerRB-SGFP2 were significantly higher than in wild-type spores; ii) western blot analysis confirmed the expression of the GerRB-SGFP2 fusion protein in spores; and iii) fluorescence microscopy visualized GerRB-SGFP2 specific bright foci in ~30% of individual dormant spores if only GerRB-SGFP2 was expressed, but, noticeably, in ~85% of spores upon co-expression with GerRA and GerRC. Our data corroborates the notion that co-expression of GR subunits improves their stability. Finally, all spores displayed bright fluorescent foci upon expression of GerD-mScarlet-I under the control of the gerD promoter. We termed all fluorescent foci observed germinosomes, the term used for the IM foci of GRs in Bacillus subtilis spores. Our data are the first evidence for the existence of germinosomes in B. cereus spores.

Distinct Requirements for Tail-Anchored Membrane Protein Biogenesis in Escherichia coli.

Tail-anchored membrane proteins (TAMPs) are a distinct subset of inner membrane proteins (IMPs) characterized by a single C-terminal transmembrane domain (TMD) that is responsible for both targeting and anchoring. Little is known about the routing of TAMPs in bacteria. Here, we have investigated the role of TMD hydrophobicity in tail-anchor function in Escherichia coli and its influence on the choice of targeting/insertion pathway. We created a set of synthetic, fluorescent TAMPs that vary in the hydrophobicity of their TMDs and corresponding control polypeptides that are extended at their C terminus to create regular type II IMPs. Surprisingly, we observed that TAMPs have a much lower TMD hydrophobicity threshold for efficient targeting and membrane insertion than their type II counterparts. Using strains conditional for the expression of known membrane-targeting and insertion factors, we show that TAMPs with strongly hydrophobic TMDs require the signal recognition particle (SRP) for targeting. Neither the SecYEG translocon nor YidC appears to be essential for the membrane insertion of any of the TAMPs studied. In contrast, corresponding type II IMPs with a TMD of sufficient hydrophobicity to promote membrane insertion followed an SRP- and SecYEG translocon-dependent pathway. Together, these data indicate that the capacity of a TMD to promote the biogenesis of E. coli IMPs is strongly dependent upon the polypeptide context in which it is presented.IMPORTANCE A subset of membrane proteins is targeted to and inserted into the membrane via a hydrophobic transmembrane domain (TMD) that is positioned at the very C terminus of the protein. The biogenesis of these so-called tail-anchored proteins (TAMPs) has been studied in detail in eukaryotic cells. Various partly redundant pathways were identified, the choice for which depends in part on the hydrophobicity of the TMD. Much less is known about bacterial TAMPs. The significance of our research is in identifying the role of TMD hydrophobicity in the routing of E. coli TAMPs. Our data suggest that both the nature of the TMD and its role in routing can be very different for TAMPs versus "regular" membrane proteins. Elucidating these position-specific effects of TMDs will increase our understanding of how prokaryotic cells face the challenge of producing a wide variety of membrane proteins.

A Bidimensional Segregation Mode Maintains Symbiont Chromosome Orientation toward Its Host.

All living organisms require accurate segregation of their genetic material. However, in microbes, chromosome segregation is less understood than replication and cell division, which makes its decipherment a compelling research frontier. Furthermore, it has only been studied in free-living microbes so far. Here, we investigated this fundamental process in a rod-shaped symbiont, Candidatus Thiosymbion oneisti. This gammaproteobacterium divides longitudinally as to form a columnar epithelium ensheathing its nematode host. We hypothesized that uninterrupted host attachment would affect bacterial chromosome dynamics and set out to localize specific chromosomal loci and putative DNA-segregating proteins by fluorescence in situ hybridization and immunostaining, respectively. First, DNA replication origins (ori) number per cell demonstrated symbiont monoploidy. Second, we showed that sister ori segregate diagonally prior to septation onset. Moreover, the localization pattern of the centromere-binding protein ParB recapitulates that of ori, and consistently, we showed recombinant ParB to specifically bind an ori-proximal site (parS) in vitro. Third, chromosome replication ends prior to cell fission, and as the poles start to invaginate, termination of replication (ter) sites localize medially, at the leading edges of the growing septum. They then migrate to midcell, concomitantly with septation progression and until this is completed. In conclusion, we propose that symbiont ParB might drive chromosome segregation along the short axis and that tethering of sister ter regions to the growing septum mediates their migration along the long axis. Crucially, active bidimensional segregation of the chromosome allows transgenerational maintenance of its configuration, and therefore, it may represent an adaptation to symbiosis. VIDEO ABSTRACT.

Evaluating novel synthetic compounds active against Bacillus subtilis and Bacillus cereus spores using Live imaging with SporeTrackerX.

An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.

Different Amounts of DNA in Newborn Cells of Escherichia coli Preclude a Role for the Chromosome in Size Control According to the "Adder" Model.

According to the recently-revived adder model for cell size control, newborn cells of Escherichia coli will grow and divide after having added a constant size or length, ΔL, irrespective of their size at birth. Assuming exponential elongation, this implies that large newborns will divide earlier than small ones. The molecular basis for the constant size increment is still unknown. As DNA replication and cell growth are coordinated, the constant ΔL could be based on duplication of an equal amount of DNA, ΔG, present in newborn cells. To test this idea, we measured amounts of DNA and lengths of nucleoids in DAPI-stained cells growing in batch culture at slow and fast rates. Deeply-constricted cells were divided in two subpopulations of longer and shorter lengths than average; these were considered to represent large and small prospective daughter cells, respectively. While at slow growth, large and small prospective daughter cells contained similar amounts of DNA, fast growing cells with multiforked replicating chromosomes, showed a significantly higher amount of DNA (20%) in the larger cells. This observation precludes the hypothesis that ΔL is based on the synthesis of a constant ΔG. Growth curves were constructed for siblings generated by asymmetric division and growing according to the adder model. Under the assumption that all cells at the same growth rate exhibit the same time between initiation of DNA replication and cell division (i.e., constant C+D-period), the constructions predict that initiation occurs at different sizes (Li) and that, at fast growth, large newborn cells transiently contain more DNA than small newborns, in accordance with the observations. Because the state of segregation, measured as the distance between separated nucleoids, was found to be more advanced in larger deeply-constricted cells, we propose that in larger newborns nucleoid separation occurs faster and at a shorter length, allowing them to divide earlier. We propose a composite model in which both differential initiation and segregation leads to an adder-like behavior of large and small newborn cells.

Assessing Membrane Fluidity and Visualizing Fluid Membrane Domains in Bacteria Using Fluorescent Membrane Dyes.

Membrane fluidity is a key parameter of bacterial membranes that undergoes quick adaptation in response to environmental challenges and has recently emerged as an important factor in the antibacterial mechanism of membrane-targeting antibiotics. The specific level of membrane fluidity is not uniform across the bacterial cell membrane. Rather, specialized microdomains associated with different cellular functions can exhibit fluidity values that significantly deviate from the average. Assessing changes in the overall membrane fluidity and formation of membrane microdomains is therefore pivotal to understand both the functional organization of the bacterial cell membrane as well as antibiotic mechanisms. Here we describe how two fluorescent membrane dyes, laurdan and DiIC12, can be employed to assess membrane fluidity in living bacteria. We focus on Bacillus subtilis, since this organism has been relatively well-studied with respect to membrane domains. However, we also describe how these assays can be adapted for other bacteria such as Staphylococcus aureus and Streptococcus pneumoniae.

The zebrafish embryo as a model to quantify early inflammatory cell responses to biomaterials.

To rapidly assess early inflammatory cell responses provoked by biomaterials in the full complexity of the living organism, we developed a zebrafish embryo model which allows real time analysis of these responses to biomaterial microspheres. Fluorescently labeled microspheres with different properties were injected into embryos of selected transgenic zebrafish lines expressing distinct fluorescent proteins in their neutrophils and macrophages. Recruitment of leukocytes and their interactions with microspheres were monitored using fluorescence microscopy. We developed a novel method using ImageJ and the plugin ObjectJ project file "Zebrafish-Immunotest" for rapid and semi-automated fluorescence quantification of the cellular responses. In the embryo model we observed an ordered inflammatory cell response to polystyrene and poly (ε-caprolactone) microspheres, similar to that described for mammalian animal models. The responses were characterized by an early infiltration of neutrophils followed by macrophages, and subsequent differentially timed migration of these cells away from the microspheres. The size of microspheres (10 and 15 µm) did not influence the cellular responses. Poly (ε-caprolactone) microspheres provoked a stronger infiltration of neutrophils and macrophages than polystyrene microspheres did. Our study shows the potential usefulness of zebrafish embryos for in vivo evaluation of biomaterial-associated inflammatory cell responses. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2522-2532, 2017.

Asynchronous division by non-ring FtsZ in the gammaproteobacterial symbiont of Robbea hypermnestra.

The reproduction mode of uncultivable microorganisms deserves investigation as it can largely diverge from conventional transverse binary fission. Here, we show that the rod-shaped gammaproteobacterium thriving on the surface of the Robbea hypermnestra nematode divides by FtsZ-based, non-synchronous invagination of its poles-that is, the host-attached and fimbriae-rich pole invaginates earlier than the distal one. We conclude that, in a naturally occurring animal symbiont, binary fission is host-oriented and does not require native FtsZ to polymerize into a ring at any septation stage.

Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells.

Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHi homeostasis. Unfortunately, accurate pHi quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pHi at single-cell levels in Bacillus subtilis Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pHi in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pHi and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pHi regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies. IMPORTANCE: This study shows how the physiological response to commonly used weak organic acid food preservatives, such as sorbic and acetic acids, can be measured at the single-cell level. These data are key to coupling often-observed single-cell heterogeneous growth behavior upon the addition of weak organic acid food preservatives. Generally, these data are gathered in the form of plate counting of samples incubated with the acids. Here, we visualize the underlying heterogeneity in cellular pH homeostasis, opening up avenues for mechanistic analyses of the heterogeneity in the weak acid stress response. Thus, microbial risk assessment can become more robust, widening the scope of use of these well-known weak organic acid food preservatives.

When Phase Contrast Fails: ChainTracer and NucTracer, Two ImageJ Methods for Semi-Automated Single Cell Analysis Using Membrane or DNA Staining.

Within bacterial populations, genetically identical cells often behave differently. Single-cell measurement methods are required to observe this heterogeneity. Flow cytometry and fluorescence light microscopy are the primary methods to do this. However, flow cytometry requires reasonably strong fluorescence signals and is impractical when bacteria grow in cell chains. Therefore fluorescence light microscopy is often used to measure population heterogeneity in bacteria. Automatic microscopy image analysis programs typically use phase contrast images to identify cells. However, many bacteria divide by forming a cross-wall that is not detectable by phase contrast. We have developed 'ChainTracer', a method based on the ImageJ plugin ObjectJ. It can automatically identify individual cells stained by fluorescent membrane dyes, and measure fluorescence intensity, chain length, cell length, and cell diameter. As a complementary analysis method we developed 'NucTracer', which uses DAPI stained nucleoids as a proxy for single cells. The latter method is especially useful when dealing with crowded images. The methods were tested with Bacillus subtilis and Lactococcus lactis cells expressing a GFP-reporter. In conclusion, ChainTracer and NucTracer are useful single cell measurement methods when bacterial cells are difficult to distinguish with phase contrast.

Quantifying the effect of sorbic acid, heat and combination of both on germination and outgrowth of Bacillus subtilis spores at single cell resolution.

Bacillus subtilis spores are a problem for the food industry as they are able to survive preservation processes. The spores often reside in food products, where their inherent protection against various stress treatments causes food spoilage. Sorbic acid is widely used as a weak acid preservative in the food industry. Its effect on spore germination and outgrowth in a combined, 'hurdle', preservation setting has gained limited attention. Therefore, the effects of mild sorbic acid (3 mM), heat-treatment (85 °C for 10 min) and a combination of both mild stresses on germination and outgrowth of B. subtilis 1A700 spores were analysed at single spore level. The heat-treatment of the spore population resulted in a germination efficiency of 46.8% and an outgrowth efficiency of 32.9%. In the presence of sorbic acid (3 mM), the germination and outgrowth efficiency was 93.3% and 80.4% respectively whereas the combined heat and sorbic acid stress led to germination and outgrowth efficiencies of 52.7% and 27.0% respectively. The heat treatment clearly primarily affected the germination process, while sorbic acid affected the outgrowth and generation time. In addition a new 'burst' time-point was defined as the time-point at which the spore coat visibly breaks and/or is shed. The combined stresses had a synergistic effect on the time of the end of germination to the burst time-point, increasing both the mean and its variation more than either of the single stresses did.

Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.

The rod-shaped Gram-negative bacterium Escherichia coli multiplies by elongation followed by binary fission. Longitudinal growth of the cell envelope and synthesis of the new poles are organized by two protein complexes called elongasome and divisome, respectively. We have analyzed the spatio-temporal localization patterns of many of these morphogenetic proteins by immunolabeling the wild type strain MC4100 grown to steady state in minimal glucose medium at 28°C. This allowed the direct comparison of morphogenetic protein localization patterns as a function of cell age as imaged by phase contrast and fluorescence wide field microscopy. Under steady state conditions the age distribution of the cells is constant and is directly correlated to cell length. To quantify cell size and protein localization parameters in 1000s of labeled cells, we developed 'Coli-Inspector,' which is a project running under ImageJ with the plugin 'ObjectJ.' ObjectJ organizes image-analysis tasks using an integrated approach with the flexibility to produce different output formats from existing markers such as intensity data and geometrical parameters. ObjectJ supports the combination of automatic and interactive methods giving the user complete control over the method of image analysis and data collection, with visual inspection tools for quick elimination of artifacts. Coli-inspector was used to sort the cells according to division cycle cell age and to analyze the spatio-temporal localization pattern of each protein. A unique dataset has been created on the concentration and position of the proteins during the cell cycle. We show for the first time that a subset of morphogenetic proteins have a constant cellular concentration during the cell division cycle whereas another set exhibits a cell division cycle dependent concentration variation. Using the number of proteins present at midcell, the stoichiometry of the divisome is discussed.

Segregation of chromosome arms in growing and non-growing Escherichia coli cells.

In slow-growing Escherichia coli cells the chromosome is organized with its left (L) and right (R) arms lying separated in opposite halves of the nucleoid and with the origin (O) in-between, giving the pattern L-O-R. During replication one of the arms has to pass the other to obtain the same organization in the daughter cells: L-O-R L-O-R. To determine the movement of arms during segregation six strains were constructed carrying three colored loci: the left and right arms were labeled with red and cyan fluorescent-proteins, respectively, on loci symmetrically positioned at different distances from the central origin, which was labeled with green-fluorescent protein. In non-replicating cells with the predominant spot pattern L-O-R, initiation of replication first resulted in a L-O-O-R pattern, soon changing to O-L-R-O. After replication of the arms the predominant spot patterns were, L-O-R L-O-R, O-R-L R-O-L or O-L-R L-O-R indicating that one or both arms passed an origin and the other arm. To study the driving force for these movements cell growth was inhibited with rifampicin allowing run-off DNA synthesis. Similar spot patterns were obtained in growing and non-growing cells, indicating that the movement of arms is not a growth-sustained process, but may result from DNA synthesis itself. The distances between loci on different arms (LR-distances) and between duplicated loci (LL- or RR-distances) as a function of their distance from the origin, indicate that in slow-growing cells DNA is organized according to the so-called sausage model and not according to the doughnut model.