From the outer space to the inner cell: deconvoluting the complexity of Bacillus subtilis disulfide stress responses by redox state and absolute abundance quantification of extracellular, membrane, and cytosolic proteins.

Understanding cellular mechanisms of stress management relies on omics data as a valuable resource. However, the lack of absolute quantitative data on protein abundances remains a significant limitation, particularly when comparing protein abundances across different cell compartments. In this study, we aimed to gain deeper insights into the proteomic responses of the Gram-positive model bacterium Bacillus subtilis to disulfide stress. We determined proteome-wide absolute abundances, focusing on different sub-cellular locations (cytosol and membrane) as well as the extracellular medium, and combined these data with redox state determination. To quantify secreted proteins in the culture medium, we developed a simple and straightforward protocol for the absolute quantification of extracellular proteins in bacteria. We concentrated extracellular proteins, which are highly diluted in the medium, using StrataClean beads along with a set of standard proteins to determine the extent of the concentration step. The resulting data set provides new insights into protein abundances in different sub-cellular compartments and the extracellular medium, along with a comprehensive proteome-wide redox state determination. Our study offers a quantitative understanding of disulfide stress management, protein production, and secretion in B. subtilis. IMPORTANCE: Stress responses play a crucial role in bacterial survival and adaptation. The ability to quantitatively measure protein abundances and redox states in different cellular compartments and the extracellular environment is essential for understanding stress management mechanisms. In this study, we addressed the knowledge gap regarding absolute quantification of extracellular proteins and compared protein concentrations in various sub-cellular locations and in the extracellular medium under disulfide stress conditions. Our findings provide valuable insights into the protein production and secretion dynamics of B. subtilis, shedding light on its stress response strategies. Furthermore, the developed protocol for absolute quantification of extracellular proteins in bacteria presents a practical and efficient approach for future studies in the field. Overall, this research contributes to the quantitative understanding of stress management mechanisms and protein dynamics in B. subtilis, which can be used to enhance bacterial stress tolerance and protein-based biotechnological applications.

Let There Be Light: Genome Reduction Enables Bacillus subtilis to Produce Disulfide-Bonded Gaussia Luciferase.

Bacillus subtilis is a major workhorse for enzyme production in industrially relevant quantities. Compared to mammalian-based expression systems, B. subtilis presents intrinsic advantages, such as high growth rates, high space-time yield, unique protein secretion capabilities, and low maintenance costs. However, B. subtilis shows clear limitations in the production of biopharmaceuticals, especially proteins from eukaryotic origin that contain multiple disulfide bonds. In the present study, we deployed genome minimization, signal peptide screening, and coexpression of recombinant thiol oxidases as strategies to improve the ability of B. subtilis to secrete proteins with multiple disulfide bonds. Different genome-reduced strains served as the chassis for expressing the model protein Gaussia Luciferase (GLuc), which contains five disulfide bonds. These chassis lack extracellular proteases, prophages, and key sporulation genes. Importantly, compared to the reference strain with a full-size genome, the best-performing genome-minimized strain achieved over 3000-fold increased secretion of active GLuc while growing to lower cell densities. Our results show that high-level GLuc secretion relates, at least in part, to the absence of major extracellular proteases. In addition, we show that the thiol-disulfide oxidoreductase requirements for disulfide bonding have changed upon genome reduction. Altogether, our results highlight genome-engineered Bacillus strains as promising expression platforms for proteins with multiple disulfide bonds.

Enhancing bacterial fitness and recombinant enzyme yield by engineering the quality control protease HtrA of Bacillus subtilis.

IMPORTANCE: In the expanding market of recombinant proteins, microbial cell factories such as Bacillus subtilis are key players. Microbial cell factories experience secretion stress during high-level production of secreted proteins, which can negatively impact product yield and cell viability. The CssRS two-component system and CssRS-regulated quality control proteases HtrA and HtrB play critical roles in the secretion stress response. HtrA has a presumptive dual function in protein quality control by exerting both chaperone-like and protease activities. However, its potential role as a chaperone has not been explored in B. subtilis. Here, we describe for the first time the beneficial effects of proteolytically inactive HtrA on α-amylase yields and overall bacterial fitness.

Diamide-based screening method for the isolation of improved oxidative stress tolerance phenotypes in Bacillus mutant libraries.

IMPORTANCE: During their life cycle, bacteria are exposed to a range of different stresses that need to be managed appropriately in order to ensure their growth and viability. This applies not only to bacteria in their natural habitats but also to bacteria employed in biotechnological production processes. Oxidative stress is one of these stresses that may originate either from bacterial metabolism or external factors. In biotechnological settings, it is of critical importance that production strains are resistant to oxidative stresses. Accordingly, this also applies to the major industrial cell factory Bacillus subtilis. In the present study, we, therefore, developed a screen for B. subtilis strains with enhanced oxidative stress tolerance. The results show that our approach is feasible and time-, space-, and resource-efficient. We, therefore, anticipate that it will enhance the development of more robust industrial production strains with improved robustness under conditions of oxidative stress.

The high-light-induced protein SliP4 binds to NDH1 and photosystems facilitating cyclic electron transport and state transition in Synechocystis sp. PCC 6803.

An increasing number of small proteins has been identified in the genomes of well-annotated organisms, including the model cyanobacterium Synechocystis sp. PCC 6803. We describe a newly assigned protein comprising 37 amino acids that is encoded upstream of the superoxide dismutase SodB encoding gene. To clarify the role of SliP4, we analyzed a Synechocystis sliP4 mutant and a strain containing a fully active, Flag-tagged variant of SliP4 (SliP4.f). The initial hypothesis that this small protein might be functionally related to SodB could not be supported. Instead, we provide evidence that it fulfills important functions related to the organization of photosynthetic complexes. Therefore, we named it a small light-induced protein of 4 kDa, SliP4. This protein is strongly induced under high-light conditions. The lack of SliP4 causes a light-sensitive phenotype due to impaired cyclic electron flow and state transitions. Interestingly, SliP4.f was co-isolated with NDH1 complex and both photosystems. The interaction between SliP4.f and all three types of complexes was further confirmed by additional pulldowns and 2D-electrophoreses. We propose that the dimeric SliP4 serves as a molecular glue promoting the aggregation of thylakoid complexes, which contributes to different electron transfer modes and energy dissipation under stress conditions.

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila.

The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba Dictyostelium discoideum. Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δsey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.

Unraveling the small proteome of the plant symbiont Sinorhizobium meliloti by ribosome profiling and proteogenomics.

The soil-dwelling plant symbiont Sinorhizobium meliloti is a major model organism of Alphaproteobacteria. Despite numerous detailed OMICS studies, information about small open reading frame (sORF)-encoded proteins (SEPs) is largely missing, because sORFs are poorly annotated and SEPs are hard to detect experimentally. However, given that SEPs can fulfill important functions, identification of translated sORFs is critical for analyzing their roles in bacterial physiology. Ribosome profiling (Ribo-seq) can detect translated sORFs with high sensitivity, but is not yet routinely applied to bacteria because it must be adapted for each species. Here, we established a Ribo-seq procedure for S. meliloti 2011 based on RNase I digestion and detected translation for 60% of the annotated coding sequences during growth in minimal medium. Using ORF prediction tools based on Ribo-seq data, subsequent filtering, and manual curation, the translation of 37 non-annotated sORFs with ≤ 70 amino acids was predicted with confidence. The Ribo-seq data were supplemented by mass spectrometry (MS) analyses from three sample preparation approaches and two integrated proteogenomic search database (iPtgxDB) types. Searches against standard and 20-fold smaller Ribo-seq data-informed custom iPtgxDBs confirmed 47 annotated SEPs and identified 11 additional novel SEPs. Epitope tagging and Western blot analysis confirmed the translation of 15 out of 20 SEPs selected from the translatome map. Overall, by combining MS and Ribo-seq approaches, the small proteome of S. meliloti was substantially expanded by 48 novel SEPs. Several of them are part of predicted operons and/or are conserved from Rhizobiaceae to Bacteria, suggesting important physiological functions.

Revealing the small proteome of Haloferax volcanii by combining ribosome profiling and small-protein optimized mass spectrometry.

In contrast to extensively studied prokaryotic 'small' transcriptomes (encompassing all small noncoding RNAs), small proteomes (here defined as including proteins ≤70 aa) are only now entering the limelight. The absence of a complete small protein catalogue in most prokaryotes precludes our understanding of how these molecules affect physiology. So far, archaeal genomes have not yet been analyzed broadly with a dedicated focus on small proteins. Here, we present a combinatorial approach, integrating experimental data from small protein-optimized mass spectrometry (MS) and ribosome profiling (Ribo-seq), to generate a high confidence inventory of small proteins in the model archaeon Haloferax volcanii. We demonstrate by MS and Ribo-seq that 67% of the 317 annotated small open reading frames (sORFs) are translated under standard growth conditions. Furthermore, annotation-independent analysis of Ribo-seq data showed ribosomal engagement for 47 novel sORFs in intergenic regions. A total of seven of these were also detected by proteomics, in addition to an eighth novel small protein solely identified by MS. We also provide independent experimental evidence in vivo for the translation of 12 sORFs (annotated and novel) using epitope tagging and western blotting, underlining the validity of our identification scheme. Several novel sORFs are conserved in Haloferax species and might have important functions. Based on our findings, we conclude that the small proteome of H. volcanii is larger than previously appreciated, and that combining MS with Ribo-seq is a powerful approach for the discovery of novel small protein coding genes in archaea.

The Cellular Abundance of Chemoreceptors, Chemosensory Signaling Proteins, Sensor Histidine Kinases, and Solute Binding Proteins of Pseudomonas aeruginosa Provides Insight into Sensory Preferences and Signaling Mechanisms.

Chemosensory pathways and two-component systems are important bacterial signal transduction systems. In the human pathogen Pseudomonas aeruginosa, these systems control many virulence traits. Previous studies showed that inorganic phosphate (Pi) deficiency induces virulence. We report here the abundance of chemosensory and two-component signaling proteins of P. aeruginosa grown in Pi deficient and sufficient media. The cellular abundance of chemoreceptors differed greatly, since a 2400-fold difference between the most and least abundant receptors was observed. For many chemoreceptors, their amount varied with the growth condition. The amount of chemoreceptors did not correlate with the magnitude of chemotaxis to their cognate chemoeffectors. Of the four chemosensory pathways, proteins of the Che chemotaxis pathway were most abundant and showed little variation in different growth conditions. The abundance of chemoreceptors and solute binding proteins indicates a sensing preference for amino acids and polyamines. There was an excess of response regulators over sensor histidine kinases in two-component systems. In contrast, ratios of the response regulators CheY and CheB to the histidine kinase CheA of the Che pathway were all below 1, indicative of different signaling mechanisms. This study will serve as a reference for exploring sensing preferences and signaling mechanisms of other bacteria.

Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling.

Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purified LCVs indicate the presence of membrane contact sites (MCS) proteins possibly implicated in lipid exchange. Using dually fluorescence-labeled Dictyostelium discoideum amoeba, we reveal that VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L. pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L. pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation.

Differential Virulence of Aggregatibacter actinomycetemcomitans Serotypes Explained by Exoproteome Heterogeneity.

Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative bacterial pathogen associated with periodontitis and nonoral diseases like rheumatoid arthritis and Alzheimer´s disease. Aa isolates with the serotypes a, b, and c are globally most prevalent. Importantly, isolates displaying these serotypes have different clinical presentations. While serotype b isolates are predominant in severe periodontitis, serotypes a and c are generally encountered in mild periodontitis or healthy individuals. It is currently unknown how these differences are reflected in the overall secretion of virulence factors. Therefore, this study was aimed at a comparative analysis of exoproteomes from different clinical Aa isolates with serotypes a, b, or c by mass spectrometry, and a subsequent correlation of the recorded exoproteome profiles with virulence. Overall, we identified 425 extracellular proteins. Significant differences in the exoproteome composition of isolates with different serotypes were observed in terms of protein identification and abundance. In particular, serotype a isolates presented more extracellular proteins than serotype b or c isolates. These differences are mirrored in their virulence in infection models based on human salivary gland epithelial cells and neutrophils. Remarkably, serotype a isolates displayed stronger adhesive capabilities and induced more lysis of epithelial cells and neutrophils than serotype b or c isolates. Conversely, serotype c isolates showed relatively low leukotoxicity, while provoking NETosis to similar extents as serotype a and b isolates. Altogether, we conclude that the differential virulence presentation by Aa isolates with the dominant serotypes a, b, or c can be explained by their exoproteome heterogeneity. IMPORTANCE Periodontitis is an inflammatory disease that causes progressive destruction of alveolar bone and supporting tissues around the teeth, ultimately resulting in tooth loss. The bacterium Aggregatibacter actinomycetemcomitans (Aa) is a prevalent causative agent of periodontitis, but this oral pathogen is also associated with serious extraoral diseases like rheumatoid arthritis and Alzheimer's disease. Clinical Aa isolates are usually distinguished by serotyping, because of known serotype-specific differences in virulence. Aa with serotype b is associated with aggressive forms of periodontitis, while isolates with serotypes a or c are usually encountered in cases of mild periodontitis or healthy individuals. The molecular basis for these differences in virulence was so far unknown. In the present study, we pinpoint serotype-specific differences in virulence factor production by clinical Aa isolates. We consider these findings important, because they provide new leads for future preventive or therapeutic approaches to fight periodontitis and associated morbidities.

Sample Preparation for Mass Spectrometry-Based Absolute Quantification of Bacterial Proteins in Antibiotic Stress Research.

Absolute protein quantification is an essential tool for system biology approaches and elucidation of stoichiometry of multi-protein complexes. In this updated chapter, a universal protocol for gel-free absolute protein quantification in bacterial systems is described, which provides adapted methods for cytosolic and membrane proteins. This protocol can be used for sample preparation prior to miscellaneous mass spectrometry-based quantification workflows like AQUA, Hi3, and emPAI. In addition, a focus has been set to the specific challenges in antibiotic stress research.

Staphylococcus aureus populations from the gut and the blood are not distinguished by virulence traits-a critical role of host barrier integrity.

BACKGROUND: The opportunistic pathogen Staphylococcus aureus is an asymptomatically carried member of the microbiome of about one third of the human population at any given point in time. Body sites known to harbor S. aureus are the skin, nasopharynx, and gut. In particular, the mechanisms allowing S. aureus to pass the gut epithelial barrier and to invade the bloodstream were so far poorly understood. Therefore, the objective of our present study was to investigate the extent to which genetic differences between enteric S. aureus isolates and isolates that caused serious bloodstream infections contribute to the likelihood of invasive disease. RESULTS: Here, we present genome-wide association studies (GWAS) that compare the genome sequences of 69 S. aureus isolates from enteric carriage by healthy volunteers and 95 isolates from bloodstream infections. We complement our GWAS results with a detailed characterization of the cellular and extracellular proteomes of the representative gut and bloodstream isolates, and by assaying the virulence of these isolates with infection models based on human gut epithelial cells, human blood cells, and a small animal infection model. Intriguingly, our results show that enteric and bloodstream isolates with the same sequence type (ST1 or ST5) are very similar to each other at the genomic and proteomic levels. Nonetheless, bloodstream isolates are not necessarily associated with an invasive profile. Furthermore, we show that the main decisive factor preventing infection of gut epithelial cells in vitro is the presence of a tight barrier. CONCLUSIONS: Our data show that virulence is a highly variable trait, even within a single clone. Importantly, however, there is no evidence that blood stream isolates possess a higher virulence potential than those from the enteric carriage. In fact, some gut isolates from healthy carriers were more virulent than bloodstream isolates. Based on our present observations, we propose that the integrity of the gut epithelial layer, rather than the pathogenic potential of the investigated enteric S. aureus isolates, determines whether staphylococci from the gut microbiome will become invasive pathogens. Video Abstract.

Virulence Induction in Pseudomonas aeruginosa under Inorganic Phosphate Limitation: a Proteomics Perspective.

Inorganic phosphate (Pi) is a central nutrient and signal molecule for bacteria. Pi limitation was shown to increase the virulence of several phylogenetically diverse pathogenic bacteria with different lifestyles. Hypophosphatemia enhances the risk of death in patients due to general bacteremia and was observed after surgical injury in humans. Phosphate therapy, or the reduction of bacterial virulence by the administration of Pi or phosphate-containing compounds, is a promising anti-infective therapy approach that will not cause cytotoxicity or the emergence of antibiotic-resistant strains. The proof of concept of phosphate therapy has been obtained using primarily Pseudomonas aeruginosa (PA). However, a detailed understanding of Pi-induced changes at protein levels is missing. Using pyocyanin production as proxy, we show that the Pi-mediated induction of virulence is a highly cooperative process that occurs between 0.2 to 0.6 mM Pi. We present a proteomics study of PA grown in minimal medium supplemented with either 0.2 mM or 1 mM Pi and rich medium. About half of the predicted PA proteins could be quantified. Among the 1,471 dysregulated proteins comparing growth in 0.2 mM to 1 mM Pi, 1,100 were depleted under Pi-deficient conditions. Most of these proteins are involved in general and energy metabolism, different biosynthetic and catabolic routes, or transport. Pi depletion caused accumulation of proteins that belong to all major families of virulence factors, including pyocyanin synthesis, secretion systems, quorum sensing, chemosensory signaling, and the secretion of proteases, phospholipases, and phosphatases, which correlated with an increase in exoenzyme production and antibacterial activity. IMPORTANCE Antibiotics are our main weapons to fight pathogenic bacteria, but the increase in antibiotic-resistant strains and their consequences represents a major global health challenge, revealing the necessity to develop alternative antimicrobial strategies that do not involve the bacterial killing or growth inhibition. P. aeruginosa has been placed second on the global priority list to guide research on the development of new antibiotics. One of the most promising alternative strategies is the phosphate therapy for which the proof of concept has been obtained for P. aeruginosa. This article reports the detailed changes at the protein levels comparing P. aeruginosa grown under Pi-abundant and Pi-depleted conditions. These data describe in detail the molecular mechanisms underlying phosphate therapy. Apart from Pi, several other phosphate-containing compounds have been used for phosphate therapy and this study will serve as a reference for comparative studies aimed at evaluating the effect of alternative compounds.

Connections between Exoproteome Heterogeneity and Virulence in the Oral Pathogen Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterial pathogen associated with severe periodontitis and nonoral diseases. Clinical isolates of A. actinomycetemcomitans display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, nonadherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on A. actinomycetemcomitans have been performed with S strains of A. actinomycetemcomitans, whereas the virulence of clinical R isolates has received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this subproteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of A. actinomycetemcomitans with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular A. actinomycetemcomitans proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Together, our findings provide novel clues for understanding the virulence of A. actinomycetemcomitans and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen. IMPORTANCE Periodontitis is one of the most common inflammatory diseases worldwide, causing high morbidity and decreasing the quality of life of millions of people. The bacterial pathogen Aggregatibacter actinomycetemcomitans is strongly associated with aggressive forms of periodontitis. Moreover, it has been implicated in serious nonoral infections, including endocarditis and brain abscesses. Therefore, it is important to investigate how A. actinomycetemcomitans can cause disease. In the present study, we applied a mass spectrometry approach to make an inventory of the virulence factors secreted by different clinical A. actinomycetemcomitans isolates and derivative strains that emerged upon culturing. We subsequently correlated the secreted virulence factors to the pathogenicity of the investigated bacteria in different infection models. The results show that a limited number of extracellular virulence factors of A. actinomycetemcomitans have central roles in pathogenesis, indicating that they could be druggable targets to prevent or treat oral disease.

Expression of the Cyanobacterial FoF1 ATP Synthase Regulator AtpΘ Depends on Small DNA-Binding Proteins and Differential mRNA Stability.

FoF1 ATP synthases produce ATP, the universal biological energy source. ATP synthase complexes on cyanobacterial thylakoid membranes use proton gradients generated either by photosynthesis or respiration. AtpΘ is an ATP synthase regulator in cyanobacteria which is encoded by the gene atpT. AtpΘ prevents the hydrolysis of ATP (reverse reaction) that otherwise would occur under unfavorable conditions. In the cyanobacterium Synechocystis sp. PCC 6803, AtpΘ is expressed maximum in darkness but at very low levels under optimum phototrophic growth conditions or in the presence of glucose. DNA coimmunoprecipitation experiments followed by mass spectrometry identified the binding of the two transcriptional regulators cyAbrB1 and cyAbrB2 to the promoter and the histone-like protein HU to the 5'UTR of atpT. Analyses of nucleotide substitutions in the promoter and GFP reporter assays identified a functionally relevant sequence motif resembling the HLR1 element bound by the RpaB transcription factor. Electrophoretic mobility shift assays confirmed interaction of cyAbrB1, cyAbrB2, and RpaB with the promoter DNA. However, overall the effect of transcriptional regulation was comparatively low. In contrast, atpT transcript stabilities differed dramatically, half-lives were 1.6 min in the light, 33 min in the dark and substantial changes were observed if glucose or DCMU were added. These findings show that transcriptional control of atpT involves nucleoid-associated DNA-binding proteins, positive regulation through RpaB, while the major effect on the condition-dependent regulation of atpT expression is mediated by controlling mRNA stability, which is related to the cellular redox and energy status. IMPORTANCE FoF1 ATP synthases produce ATP, the universal biological energy source. Under unfavorable conditions, ATP synthases can operate in a futile reverse reaction, pumping protons while ATP is used up. Cyanobacteria perform plant-like photosynthesis, but they cannot use the same mechanism as plant chloroplasts to inhibit ATP synthases during the night because respiratory and photosynthetic complexes are both located in the same membrane system. AtpΘ is a small protein encoded by the gene atpT in cyanobacteria that can prevent the ATP synthase reverse reaction (ATPase activity). Here we found that three transcription factors contribute to the regulation of atpT expression. However, the control of mRNA stability was identified as the major regulatory process governing atpT expression. Thus, it is the interplay between transcriptional and posttranscriptional regulation that position the AtpΘ-based regulatory mechanism within the context of the cellular redox and energy balance.

Migration of Acanthamoeba through Legionella biofilms is regulated by the bacterial Lqs-LvbR network, effector proteins and the flagellum.

The environmental bacterium Legionella pneumophila causes the pneumonia Legionnaires' disease. The opportunistic pathogen forms biofilms and employs the Icm/Dot type IV secretion system (T4SS) to replicate in amoebae and macrophages. A regulatory network comprising the Legionella quorum sensing (Lqs) system and the transcription factor LvbR controls bacterial motility, virulence and biofilm architecture. Here we show by comparative proteomics that in biofilms formed by the L. pneumophila ΔlqsR or ΔlvbR regulatory mutants the abundance of proteins encoded by a genomic 'fitness island', metabolic enzymes, effector proteins and flagellar components (e.g. FlaA) varies. ∆lqsR or ∆flaA mutants form 'patchy' biofilms like the parental strain JR32, while ∆lvbR forms a 'mat-like' biofilm. Acanthamoeba castellanii amoebae migrated more slowly through biofilms of L. pneumophila lacking lqsR, lvbR, flaA, a functional Icm/Dot T4SS (∆icmT), or secreted effector proteins. Clusters of bacteria decorated amoebae in JR32, ∆lvbR or ∆icmT biofilms but not in ∆lqsR or ∆flaA biofilms. The amoeba-adherent bacteria induced promoters implicated in motility (PflaA ) or virulence (PsidC , PralF ). Taken together, the Lqs-LvbR network (quorum sensing), FlaA (motility) and the Icm/Dot T4SS (virulence) regulate migration of A. castellanii through L. pneumophila biofilms, and - apart from the T4SS - govern bacterial cluster formation on the amoebae.

Streptococcus pneumoniae and Influenza A Virus Co-Infection Induces Altered Polyubiquitination in A549 Cells.

Epithelial cells are an important line of defense within the lung. Disruption of the epithelial barrier by pathogens enables the systemic dissemination of bacteria or viruses within the host leading to severe diseases with fatal outcomes. Thus, the lung epithelium can be damaged by seasonal and pandemic influenza A viruses. Influenza A virus infection induced dysregulation of the immune system is beneficial for the dissemination of bacteria to the lower respiratory tract, causing bacterial and viral co-infection. Host cells regulate protein homeostasis and the response to different perturbances, for instance provoked by infections, by post translational modification of proteins. Aside from protein phosphorylation, ubiquitination of proteins is an essential regulatory tool in virtually every cellular process such as protein homeostasis, host immune response, cell morphology, and in clearing of cytosolic pathogens. Here, we analyzed the proteome and ubiquitinome of A549 alveolar lung epithelial cells in response to infection by either Streptococcus pneumoniae D39Δcps or influenza A virus H1N1 as well as bacterial and viral co-infection. Pneumococcal infection induced alterations in the ubiquitination of proteins involved in the organization of the actin cytoskeleton and Rho GTPases, but had minor effects on the abundance of host proteins. H1N1 infection results in an anti-viral state of A549 cells. Finally, co-infection resembled the imprints of both infecting pathogens with a minor increase in the observed alterations in protein and ubiquitination abundance.

The global proteome and ubiquitinome of bacterial and viral co-infected bronchial epithelial cells.

Viral infections facilitate bacterial trafficking to the lower respiratory tract resulting in bacterial-viral co-infections. Bacterial dissemination to the lower respiratory tract is enhanced by influenza A virus induced epithelial cell damage and dysregulation of immune responses. Epithelial cells act as a line of defense and detect pathogens by a high variety of pattern recognition receptors. The post-translational modification ubiquitin is involved in almost every cellular process. Moreover, ubiquitination contributes to the regulation of host immune responses, influenza A virus uncoating and transport within host cells. We applied proteomics with a special focus on ubiquitination to assess the impact of single bacterial and viral as well as bacterial-viral co-infections on bronchial epithelial cells. We used Tandem Ubiquitin Binding Entities to enrich polyubiquitinated proteins and assess changes in the ubiquitinome. Infecting 16HBE cells with Streptococcus pyogenes led to an increased abundance of proteins related to mitochondrial translation and energy metabolism in proteome and ubiquitinome. In contrast, influenza A virus infection mainly altered the ubiquitinome. Co-infections had no additional impact on protein abundances or affected pathways. Changes in protein abundance and enriched pathways were assigned to imprints of both infecting pathogens. SIGNIFICANCE: Viral and bacterial co-infections of the lower respiratory tract are a burden for health systems worldwide. Therefore, it is necessary to elucidate the complex interplay between the host and the infecting pathogens. Thus, we analyzed the proteome and the ubiquitinome of co-infected bronchial epithelial cells to elaborate a potential synergism of the two infecting organisms. The results presented in this work can be used as a starting point for further analyses.

AtpΘ is an inhibitor of F0F1 ATP synthase to arrest ATP hydrolysis during low-energy conditions in cyanobacteria.

Biological processes in all living cells are powered by ATP, a nearly universal molecule of energy transfer. ATP synthases produce ATP utilizing proton gradients that are usually generated by either respiration or photosynthesis. However, cyanobacteria are unique in combining photosynthetic and respiratory electron transport chains in the same membrane system, the thylakoids. How cyanobacteria prevent the futile reverse operation of ATP synthase under unfavorable conditions pumping protons while hydrolyzing ATP is mostly unclear. Here, we provide evidence that the small protein AtpΘ, which is widely conserved in cyanobacteria, is mainly fulfilling this task. The expression of AtpΘ becomes induced under conditions such as darkness or heat shock, which can lead to a weakening of the proton gradient. Translational fusions of AtpΘ to the green fluorescent protein revealed targeting to the thylakoid membrane. Immunoprecipitation assays followed by mass spectrometry and far western blots identified subunits of ATP synthase as interacting partners of AtpΘ. ATP hydrolysis assays with isolated membrane fractions, as well as purified ATP synthase complexes, demonstrated that AtpΘ inhibits ATPase activity in a dose-dependent manner similar to the F0F1-ATP synthase inhibitor N,N-dicyclohexylcarbodimide. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration and indicate that ATP synthase activity can be controlled in surprisingly different ways.