Biology subjects

Roseboom, W.

Publications and source records attributed to Roseboom, W..

18 recordsLinked to original sources

bioRxiv2024

Non-mechanical disruption of Bacillus subtilis spores allows sensitive and deep characterization of the minimal proteome for resuming a cellular lifestyle.

In response to extreme conditions, Bacillus subtilis generates highly resilient spores characterized by a unique multilayered structure. This confers resistance against various chemicals and enzymes yet adding complexity to the analysis of the spore proteome. As the first step in bottom-up proteomics, sample preparation poses a significant challenge. We assessed how an optimized protocol for sample preparation by easy extraction and digestion (SPEED) performed compared to previously established methods "One-pot" (OP) and single-pot, solid phase-enhanced sample-preparation (SP3) for the proteomic analysis of B. subtilis cell and spore samples. We found that SPEED outperformed both OP and SP3 in terms of peptides and proteins identified, moreover SPEED highly reproducibly quantified over 1000 proteins in limited input samples as low as 1 OD600 of B. subtilis cells and spores. SPEED was applied to analyze spore samples of different purity by applying sequential purification following harvesting of spores. Comparison of the differential abundance of proteins revealed clusters likely partially stemming from remaining vegetative cells in less purified spore samples. We show that ranking of absolute protein abundance in cellular and spore samples further enables us to rationally differentiate integral spore proteins from vegetative remnants. This is of importance in applications and organisms where highly homogenous spore samples are difficult to obtain. A deep proteomic analysis of spore and vegetative cell samples with the new approach led to the identification of 2447 proteins, 2273 of which were further quantified and compared between B. subtilis spores and cells. Our findings indicate that pathways related to peptidoglycan biosynthesis, glycolysis, carbon metabolism, and biosynthesis of secondary metabolites are shared between cells and spores. This corroborates and extends earlier work stressing that despite marked differences in their physiological states, spores preserve vegetative cell (core) proteins, essential for revival under conditions conducive to growth. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/584050v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@9ac7b1org.highwire.dtl.DTLVardef@1ac35c6org.highwire.dtl.DTLVardef@fe73a4org.highwire.dtl.DTLVardef@15a24e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Multi-omics analysis of Bacillus. subtilis spores formed at different environmental temperatures reveal differences at the morphological and molecular level.

Spore-forming bacteria play an essential role in the food industry and public health. Through sporulation, bacteria can withstand extreme environmental conditions that vegetative cells cannot survive. Although it is well established that the same environmental factors that affect the growth of vegetative cells also profoundly influence sporulation, the mechanisms of how growth conditions affect spore structure and function remain unknown. Prior research has shown that spores prepared at higher temperatures are more heat resistant than those prepared at lower temperatures. The present study examines, both at metabolomic and proteomic levels, the effect of different sporulation temperatures (25, 37 and 42{degrees}C) on the small molecule and protein composition of spores (strain PY79) of the model organism B. subtilis. Through differential harvesting times, spores of the same developmental stage were obtained for each temperature regime. The heat resistance, dipicolinic acid content, germination kinetics and spore morphology were assayed to compare spore properties. Metabolome and proteome analysis yielded unparalleled broad molecular detail of the spores formed at different environmental temperatures. Our findings indicate that peptidoglycan biosynthesis and 28 outer-layer proteins play a crucial role in the functional diversity of spores produced by B. subtilis under varying temperatures.

microbiology↗

bioRxiv2023

Integrative metabolomics and proteomics allow the global intracellular characterization of Bacillus subtilis cells and spores.

Reliable and comprehensive multi-omics analysis is essential for researchers to understand and explore complex biological systems more completely. Bacillus subtilis is a model organism for Gram-positive spore-forming bacteria, and in-depth insight into the physiology and molecular basis of spore formation and germination in this organism requires advanced multilayer molecular datasets generated from the same sample. In this study, we evaluated two monophasic methods for polar and nonpolar compound extraction (acetonitrile/methanol/water; isopropanol/water, and 60% ethanol), and two biphasic methods (chloroform/methanol/water, and methyl tert-butyl ether/methanol/water) on coefficients of variation of analytes, identified metabolite composition and the quality of proteomics profiles. The analytical workflow comprises (1) parallel metabolite and protein extraction, (2) monophasic or biphasic sample extraction, (3) proteomics comparison and (4) multi-omics-based data visualization. The 60% EtOH protocol proved to be the easiest in sample processing and more amenable to automation. Collectively, we annotated 505 and 484 metabolites and identified 1665 and 1562 proteins in B. subtilis vegetative cells and spores, respectively. We also show differences between vegetative cells and spores from a multi-omics perspective and demonstrated that an integrative multi-omics analysis can be implemented from one sample using the 60% EtOH protocol. Correlation analysis further demonstrated that the metabolome and proteome datasets were highly correlated in content for components annotated to be part of the pyrimidine metabolism pathway. The results obtained by the 60% EtOH protocol provide a comprehensive insight into differences in the metabolic and protein makeup of B. subtilis vegetative cells and spores. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/545067v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@18b0206org.highwire.dtl.DTLVardef@1321433org.highwire.dtl.DTLVardef@cc5e6eorg.highwire.dtl.DTLVardef@14bac12_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Whitefly effector G4 interacts with tomato proteins of which MIPDB141 affects whitefly performance

The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate defense responses. Here, we present a set of proteins that was identified in artificial diet on which B. tabaci was salivating. We studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of the flg22-induced ROS response when transiently expressed in Nicotiana benthamiana. In addition, G4 was able to supress ROS in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). Fused to a fluorescence tag, G4 localized in the cytoplasm in N. benthamiana. A yeast two-hybrid screen combined with a luciferase bimolecular complementation and co-localization assays resulted in the identification of two target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato, using virus-induced gene silencing, reduced whitefly fecundity up to 40% demonstrating that the protein is involved in susceptibility to B. tabaci. Together our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with the ROS production and via a direct interaction with tomato susceptibility protein MIPDB141.

plant biology↗

bioRxiv2022

Cross-link scrambling in peptide pairs

Identification of peptides and their linked amino acids from chemically cross-linked protein complexes with bifunctional N-hydroxysuccinimidyl (NHS) esters can reveal interacting proteins and their spatial arrangements. With NHS esters both amide- and ester cross-links can be formed. Retention time prediction for strong cation exchange chromatography (SCXC) of cross-linked peptides at pH 3 can distinguish between ester and amide cross-links based on their charge differences. By this approach we show that about 98 % of cross-links are formed by two amide bonds. However MS/MS analysis revealed the presence of an ester linkage in more than 5% of peptide pairs predicted by SCXC to be linked by amide bonds. This discrepancy can be explained by intra-peptide amide-ester rearrangement in the gas phase during MS/MS analysis. So, SCXC retention time prediction can be used to distinguish amide-amide, amide-ester and ester-ester linkages actually formed in the cross-linking reaction and to detect scrambling of cross-linked sites. This information is important for studies aimed to understand the spatial arrangement of protein complexes by cross-linking at the highest possible resolution.

biochemistry↗

bioRxiv2021

High resolution analysis of proteome dynamics during Bacillus subtilis sporulation

Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (blue, brown, green and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue, is negatively correlated with modules brown and green, and contained ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green. We speculate that they may also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation. We speculate on their possible role in guiding or helping assembly of other coat proteins, after which they can be disposed of, but such a hypothesis remains to be experimentally addressed.

microbiology↗

bioRxiv2020

A composite filter for low FDR of protein-protein interactions detected by in vivo cross-linking

In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at a proteome wide-scale and at the peptide level. In vivo CX-MS requires a membrane permeable and cleavable cross-linker that enables isolation of target peptides and a fast and sensitive search engine to identify the linked peptides. Here we explore the use of the search engine pLink 2 for analysis of a previously obtained LCMSMS dataset from exponentially growing Bacillus subtilis treated in culture with the cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). Cross-linked peptide pairs were identified by pLink 2 in very short time at an overall FDR of < 5%. To also obtain a FDR < 5% for inter-protein cross-linked peptide pairs additional thresholds values were applied for matched fragment intensity and for the numbers of unambiguous y and b ions to be assigned for both composite peptides. Threshold values were based on a set of decoy sequences from yeast and human sequence databases. Also the mass- and charge-dependent retention times of target peptides purified by diagonal strong cation exchange chromatography were used as a criterion to distinguish true from false positives. After this filtering, pLink 2 identified more than 80% of previously reported protein-protein interactions. In addition the use of pLink 2 revealed interesting new inter-protein cross-linked peptide pairs, among others showing interactions between the global transcriptional repressor AbrB and elongation factor Tu and between the essential protein YlaN of unknown function and the ferric uptake repressor Fur. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/097956v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@974dd1org.highwire.dtl.DTLVardef@1760d10org.highwire.dtl.DTLVardef@1539d5eorg.highwire.dtl.DTLVardef@808e2e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIImproved protocol for identification of PPIs at low FDR by in vivo cross-linking with BAMG C_LIO_LIThe use of all intra-protein cross-linked peptide pairs as true positives C_LIO_LIThe cytosolic aminopeptidase (AMPA_BACSU) interacts with the 50S ribosomal protein L17 C_LIO_LIThe transition state regulator AbrB interacts with elongation factor Tu C_LIO_LIThe essential protein YlaN of unknown function interacts with the iron uptake repressor Fur C_LI SignificanceImportant for reliable identification of PPIs by chemical cross-linking in vivo is a low FDR of non-redundant inter-protein peptide pairs. Here we describe how to recognize the presence of spurious interactions in a dataset of cross-linked peptide pairs enriched by 2D strong cation exchange chromatography and identified by LCMSMS by taking into account chromatographic behavior of cross-linked peptide pairs and protein abundance of corresponding peptides. Based on these criteria we assessed that the FDR of the fraction of non-redundant inter-protein cross-linked peptide pairs was approx. 20-25% by interrogating an entire species specific database at an overall FDR of 5% or 0.1% with a search engine that otherwise scores best in sensitivity among other search engines. We have defined a composite filter to decrease this high FDR of inter-protein cross-linked peptide pairs to only about 2%.

biochemistry↗

bioRxiv2020

The Ice Nucleating Protein InaZ is Activated by Low Temperature

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Utilizing specialized ice-nucleating proteins (INPros), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds and may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INPro-induced freezing have remained largely elusive. In the present study, we investigated the folding and the structural basis for interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae strain R10.79. Using vibrational sum-frequency generation and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a {beta}-helical structure in solution and at water surfaces. In this configuration, hydrogen bonding between INPros and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with spectral calculations and molecular-dynamics simulations shows that the INPro reorients at lower temperatures. We suggest that the reorientation can enhance order-inducing water interactions and, thereby, the effectiveness of ice nucleation by InaZ.

biophysics↗

bioRxiv2020

A predictive processing model of episodic memory and time perception

Human perception and experience of time is strongly influenced by ongoing stimulation, memory of past experiences, and required task context. When paying attention to time, time experience seems to expand; when distracted, it seems to contract. When considering time based on memory, the experience may be different than in the moment, exemplified by sayings like "time flies when youre having fun". Experience of time also depends on the content of perceptual experience - rapidly changing or complex perceptual scenes seem longer in duration than less dynamic ones. The complexity of interactions between attention, memory, and perceptual stimulation is a likely reason that an overarching theory of time perception has been difficult to achieve. Here, we introduce a model of perceptual processing and episodic memory that makes use of hierarchical predictive coding, short-term plasticity, spatio-temporal attention, and episodic memory formation and recall, and apply this model to the problem of human time perception. In an experiment with ~ 13, 000 human participants we investigated the effects of memory, cognitive load, and stimulus content on duration reports of dynamic natural scenes up to ~ 1 minute long. Using our model to generate duration estimates, we compared human and model performance. Model-based estimates replicated key qualitative biases, including differences by cognitive load (attention), scene type (stimulation), and whether the judgement was made based on current or remembered experience (memory). Our work provides a comprehensive model of human time perception and a foundation for exploring the computational basis of episodic memory within a hierarchical predictive coding framework. Author summaryExperience of the duration of present or past events is a central aspect of human experience, the underlying mechanisms of which are not yet fully understood. In this work, we combine insights from machine learning and neuroscience to propose a combination of mathematical models that replicate human perceptual processing, long-term memory, attention, and duration perception. Our computational implementation of this framework can process information from video clips of ordinary life scenes, record and recall important events, and report the duration of these clips. To assess the validity of our proposal, we conducted an experiment with ~ 13, 000 human participants. Each was shown a video between 1-64 seconds long and reported how long they believed it was. Reports of duration by our computational model qualitatively matched these human reports, made about the exact same videos. This was true regardless of the video content, whether time was actively judged or based on memory of the video, or whether the participants focused on a single task or were distracted - all factors known to influence human time perception. Our work provides the first model of human duration perception to incorporate these diverse and complex factors and provides a basis to probe the deep links between memory and time in human experience.

neuroscience↗

bioRxiv2020

Accumulation of salient events in sensory cortex activity predicts subjective time

Human experience of time exhibits systematic, context-dependent deviations from veridical clock time; for example, time is experienced differently at work than on holiday. Here we test the proposal that differences from clock time in subjective experience of time arise because time estimates are constructed by accumulating the same quantity that guides perception: salient events. Healthy human participants watched naturalistic, silent videos of up to [~]1 minute in duration and estimated their duration while fMRI was acquired. We were able to reconstruct trial-by-trial biases in participants duration reports, which reflect subjective experience of time (rather than veridical clock time), purely from salient events in their visual cortex BOLD activity. This was not the case for control regions in auditory and somatosensory cortex, despite being able to predict clock time from all three brain areas. Our results reveal that the information arising during sensory processing of our dynamic environment provides a sufficient basis for reconstructing human subjective time estimates.

neuroscience↗

bioRxiv2019

Towards the minimal proteome of life: Proteome profiles of Bacillus subtilis vegetative cells and spores

The method of 15N metabolic labelling of Bacillus subtilis enabled mass spectrometric quantification of relative protein levels in the vegetative cells and the spores of this model organism. A total of 1501 proteins have been identified from the combined spore and vegetative cell samples. From these 1086 proteins have been relatively and reproducibly quantified between spores and vegetative cells. Of the quantified proteins, 60% are common to the vegetative cells and spores, indicating that spores host a minimal set proteins sufficient for the resumption of metabolism upon completion of germination. The shared proteins represent, the most basic survival kit for life on earth that is known thus far.

microbiology↗

bioRxiv2018

Multiple Duration Priors Within and Across the Senses

Perception can be understood as an active process in which sensory samples are combined with prior expectations to shape perceptual content. A prominent example of the influence of priors on perception is that manually reproduced temporal durations are biased towards the mean of previously experienced durations. However, little is known about how prior expectations are acquired and maintained in environments in which multiple competing cues may indicate whether a given prior should be applied in that specific context. We tested whether human participants could acquire and maintain multiple priors for duration, dependent on the sensory signal in which the duration was presented. Human participants were presented with visual flashes or auditory tones, high or low pitch tones, or white noise versus pure tone audio. In each case, the presented duration on a given trial was drawn from a distribution that was, on average, shorter for tones than for flashes, or vice versa. Our participants timing reports were consistent with having acquired distinct duration priors dependent on the sensory signal in which the duration was presented (e.g. auditory or visual). Moreover, this was true whether signals differed across, or within, sensory modality. We account for our findings within a Bayesian framework in which duration priors are iteratively updated depending on determination of a common or distinct origin between successive events. Overall, these results show that the human brain can acquire and maintain multiple perceptual priors based on differences in stimulus properties both within and across the senses.

animal behavior and cognition↗

bioRxiv2018

A Sensory Processing Hierarchy for Thermal Touch: Thermal Adaptation Occurs Prior to Thermal-Tactile Integration

The brain consistently faces a challenge of whether and how to combine the available information sources to estimate the properties of an object explored by hand. Thermal referral (TR) is a phenomenon that demonstrates how thermal and tactile modalities coordinate to resolve inconsistencies in spatial and thermal information. When the middle three fingers of one hand are thermally stimulated, but only the outer two fingers are heated (or cooled), thermal uniformity is perceived across three fingers. This illusory experience of thermal uniformity in TR compensates for the discontinuity in the thermal sensation across the sites in contact. The neural loci of TR is unclear. While TR reflects the diffuse nature of the thermoceptive system, its similarities to perceptual filling-in and its facilitative role in object perception also suggest that TR might involve inference processes associated with object perception. To clarify the positioning of this thermo-tactile interaction in the sensory processing hierarchy, we used perceptual adaptation and Bayesian decision modelling techniques. Our results indicate that TR adaptation takes place at a peripheral stage where information about temperature inputs are still preserved for each finger, and that the thermal-tactile interaction occurs after this stage. We also show that the temperature integration across three fingers in TR is consistent with precision weighted averaging effect - Bayesian cue combination. Altogether, our findings suggest that for the sensory processing hierarchy of thermal touch, thermal adaptation occurs prior to thermo-tactile integration, which combines thermal and tactile information to give a unified percept to facilitate object recognition.\n\nSignificance StatementThermal touch refers to the perception of temperature of objects in contact with the skin and is key to object recognition based on thermal cues. While object perception is an inference process involving multisensory inputs, thermal referral (TR) is an illusion demonstrating how the brains interpretation of object temperature can deviate from physical reality. Here we used TR to explore the processing hierarchy of thermal touch. We show that adaptation of thermal perception occurs prior to integration of thermal information across tactile locations. Further, we show that TR results from simple averaging of thermal sensation across locations. Our results illuminate the flexibility of the processing that underlies thermal-tactile interactions and facilitates object exploration and identification in our complicated natural environment.

neuroscience↗

bioRxiv2018

The illusion of uniformity does not depend on low-level vision: evidence from sensory adaptation

Visual experience appears richly detailed despite the poor resolution of the majority of the visual field, thanks to foveal-peripheral integration. The recently described Uniformity Illusion (UI), in which peripheral elements of a pattern seem to take on the properties of foveal elements, may shed light on this integration. We examined the basis of UI by generating adaptation to a pattern of Gabors suitable for producing UI on orientation. After removing the pattern, participants reported the tilt of a single peripheral Gabor. The tilt after-effect (TAE) followed the physical adapting orientation rather than the global orientation perceived under UI, even when the illusion had been reported for a long time. Conversely, a control experiment replacing illusory for physical uniformity for the same durations did produce an after-effect to the global orientation. Our results indicate that the UI is not associated with changes in sensory encoding, but likely depends on high-level processes.

neuroscience↗

bioRxiv2017

The Hallucination Machine: A Deep-Dream VR platform for Studying the Phenomenology of Visual Hallucinations

Altered states of consciousness, such as psychotic or pharmacologically-induced hallucinations, provide a unique opportunity to examine the mechanisms underlying conscious perception. However, the phenomenological properties of these states are difficult to isolate experimentally from other, more general physiological and cognitive effects of psychoactive substances or psychopathological conditions. Thus, simulating phenomenological aspects of altered states in the absence of these other more general effects provides an important experimental tool for consciousness science and psychiatry. Here we describe such a tool, the Hallucination Machine. It comprises a novel combination of two powerful technologies: deep convolutional neural networks (DCNNs) and panoramic videos of natural scenes, viewed immersively through a head-mounted display (panoramic VR). By doing this, we are able to simulate visual hallucinatory experiences in a biologically plausible and ecologically valid way. Two experiments illustrate potential applications of the Hallucination Machine. First, we show that the system induces visual phenomenology qualitatively similar to classical psychedelics. In a second experiment, we find that simulated hallucinations do not evoke the temporal distortion commonly associated with altered states. Overall, the Hallucination Machine offers a valuable new technique for simulating altered phenomenology without directly altering the underlying neurophysiology.

neuroscience↗

bioRxiv2017

Neurophysiological signatures of duration and rhythm prediction across sensory modalities

Effective behaviour and cognition requires the ability to make predictions about the temporal properties of events, such as duration. It is well known that violations of temporal structure within sequences of stimuli lead to neurophysiological effects known as the (temporal) mismatch negativity (TMMN). However, previous studies investigating this phenomenon have typically presented successive stimulus intervals (i.e., durations) within a rhythmic structure, conflating the contributions of rhythmic temporal processing with those specific to duration. In a novel behavioural paradigm which extends the classic temporal oddball design, we examined the neurophysiological correlates of prediction violation under both rhythmically (isochronous) and arrhythmically (anisochronous) presented durations, in visual and auditory modalities. Using event-related potential (ERP), multivariate pattern analysis (MVPA), and temporal generalisation analysis (TGA) analyses, we found evidence for common, and distinct neurophysiological responses related to duration predictions and their violation, across isochronous and anisochronous conditions. Further, using TGA we could directly compare processes underlying duration prediction violation across different modalities, despite differences in processing latency of audition and vision. We discovered a common set of neurophysiological responses that are elicited whenever a duration prediction is violated, regardless of presentation modality, indicating the existence of a supramodal duration prediction mechanism. Altogether, our data show that the human brain encodes predictions specifically about duration, in addition to those from rhythmic structure, and that the neural underpinnings of these predictions generalize across modalities. These findings support the idea that time perception is based on similar principles of inference as characterize predictive processing theories of perception.

neuroscience↗

bioRxiv2017

A functioning model of human time perception

Despite being a fundamental dimension of experience, how the human brain generates the perception of time remains unknown. Here, we provide a novel explanation for how human time perception might be accomplished, based on non-temporal perceptual clas-sification processes. To demonstrate this proposal, we built an artificial neural system centred on a feed-forward image classification network, functionally similar to human visual processing. In this system, input videos of natural scenes drive changes in network activation, and accumulation of salient changes in activation are used to estimate duration. Estimates produced by this system match human reports made about the same videos, replicating key qualitative biases, including differentiating between scenes of walking around a busy city or sitting in a cafe or office. Our approach provides a working model of duration perception from stimulus to estimation and presents a new direction for examining the foundations of this central aspect of human experience.

neuroscience↗

bioRxiv2016

In culture cross-linking of bacterial cells reveals proteome scale dynamic protein-protein interactions at peptide level

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique inter-protein cross-linked peptides with less than a 1% false discovery rate by mass spectrometry and genome-wide data base searching. Nearly 60% of the inter-protein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the {delta} and {beta}' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how {delta} might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth.

biochemistry↗