microLife最新文献

SliP4 is a small, 37 amino acids protein that is strongly induced when the cyanobacterium Synechocystis sp. PCC 6803 is exposed to high-light (HL) conditions. Deletion mutants manifest a light-sensitive phenotype due to impaired cyclic electron flow and state transitions. In this study, we aimed to investigate the consequences of SliP4 deficiency on the process of high-light acclimation on systems level. Transcriptomic data revealed that the deletion mutant ΔsliP4 exhibited a wild-type-like gene regulatory response 30 minutes after the light intensity was increased from 50 to 250 μmol photons m^-2 s^-1, a process that is controlled by the RpaB-PsrR1 system. Proteome analysis showed consistent expression changes of many HL-regulated proteins. Metabolome analysis provided hints for a changed N and C metabolism in mutant cells compared to wild type. In addition, the mutant increased the production of extracellular polysaccharides causing the mutant cells to aggregate after the shift to HL. This effect corresponds to the upregulated expression of xssA-E and xssN-P genes for the production of the sulfated exopolysaccharide synechan. We interpret these observations as a response that counteracts the potential light stress effects caused by the impaired capacity for cyclic electron flow and state transitions in the ΔsliP4 mutant. Our results demonstrate that the unicellular cyanobacterium Synechocystis compensates for the loss of SliP4 and its crucial role by activating a genetic program for a population-level response that helps the cells to cope with HL conditions.

CRISPR and their associated Cas proteins provide adaptive immunity in prokaryotes, protecting against invading genetic elements. These systems are categorized into types and are highly diverse. Genomes often harbor multiple CRISPR arrays varying in length and distance from Cas loci. However, the ecological roles of multiple CRISPR arrays and their interactions with multiple Cas loci remain poorly understood. We present a comprehensive analysis of CRISPR systems that uncovers variation between diverse Cas types regarding the occurrence of multiple arrays, the distribution of their lengths and positions relative to Cas loci, and the diversity of their repeat sequences. Some types tend to occur as the sole Cas locus present in the genome, but typically have two or more associated arrays, especially for types I-E and I-F. Multiple Cas types are also common, with some systems showing a preference for specific co-occurrence. Distinct array distributions and orientations around Cas loci indicate substantial differences in functionality and transcriptional behavior among Cas types. Our analysis suggests that arrays with identical repeats in the same genome acquire new spacers at comparable rates, irrespective of their proximity to the Cas locus. Furthermore, repeat similarities indicate that arrays of systems that often co-occur with other systems tend to have more diverse repeats than those mostly appearing alongside solitary systems. Our results indicate that co-occurring Cas-type pairs might not only collaborate in spacer acquisition but also maintain independent and complementary functions and that CRISPR systems distribute their defensive spacer repertoire equally across multiple CRISPR arrays.

Salmonella enterica, a prominent enteric pathogen, employs sophisticated iron acquisition mechanisms to overcome host-imposed iron limitation, notably through the production and uptake of siderophores-small, high-affinity iron-chelating compounds that scavenge iron from the host environment. In this study, we investigate how environmental pH influences Salmonella's preference for its endogenous siderophores versus exogenous siderophores within the physiological range of the gastrointestinal tract. Through competition assays, gene expression analysis, and siderophore quantification, we demonstrate that Salmonella increasingly relies on exogenous siderophores under acidic conditions. This shift is attributed to reduced production of its endogenous siderophores, enterobactin and salmochelin. Deletion of the sigma factor RpoS enhances iron acquisition through increased endogenous siderophore production at low pH, suggesting a role in iron homeostasis regulation. Our findings reveal a pH-dependent difference in Salmonella's iron acquisition strategy, highlighting the pathogen's versatility in nutrient acquisition across varying gastrointestinal conditions. This research provides insights into Salmonella's pathogenicity and may inform the development of targeted interventions for Salmonella infections.

Quantitative information on protein abundance is crucial to understand biological processes and is therefore frequently gathered in proteomic studies. However, the quality of a quantitative proteomic dataset is greatly affected by the number of missing values, which need to be minimized to produce robust and meaningful data. In this context, small proteins (≤100 amino acids) pose specific analytical challenges, which hinder their efficient identification and quantitative characterization in complex proteomes. In this study, methods for sample preparation and MS-data processing are systematically evaluated for their contribution to identification and quantification of small proteins of Clostridioides difficile 630 Δerm. Results show that small protein enrichment can enhance the number of identified and quantified proteins also for low abundant small proteins. Through application of spectral libraries for identification of MS spectra the number of robustly quantified proteins is increased and a lower limit of their detection is reached. Additionally, the dataset presented here is currently the most comprehensive protein repository for C. difficile covering 84.7% of the predicted proteome and 61.4% of all predicted small proteins of this important pathogen.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated protein (Cas) systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR-Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56 343 CRISPR arrays across 25 628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting higher spacer conservation. This observation corroborates recent findings indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations might play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) represents a family of important transcription factors in innate immunity. We have previously reported that the gastric pathogen Helicobacter pylori needs the actin-binding protein cortactin for efficient interleukin-8 (IL-8) secretion, which requires NF-κB activation. However, it remained unknown, which exact cortactin signaling mechanism contributes to IL-8 release. In fact, H. pylori profoundly activates NF-κB in wild-type AGS gastric epithelial cells by the effector molecule adenosine diphosphate (ADP)-β-d-manno-heptose (ADPH) in a type IV secretion system-dependent manner. However, the injected CagA protein might contribute to NF-κB activation. The ADPH-stimulated canonical NF-κB cascade involves alpha-kinase 1 and adapter protein TRAF-interacting protein with forkhead-associated domain (TIFA) to activate inhibitor of kappa B (IκB) kinases (IKKs), followed by phosphorylation-dependent degradation of IκBα and subsequent nuclear translocation of p65 NF-κB and IL-8 release. Here, we show that infection of cortactin knockout cells leads to reduced activation of focal adhesion kinase (FAK) and c-Sarcoma (Src) kinase resulting in diminished phosphorylation of IKKβ at tyrosine residue 199 and subsequently phosphorylation of p65 at serine residue 536, both of which are associated with downregulated NF-κB activity. Our results were further supported using FAK and TIFA knockout cells and treatments with purified ADPH and overexpression of CagA, showing cumulative effects in wild-type, but not in knockout cells. These data demonstrate that ADPH-dependent NF-κB activation and IL-8 secretion are enhanced by CagA. Together, we present here a novel CagA>cortactin>FAK>Src>IKKβ signaling cascade, contributing to proinflammatory responses by H. pylori.

In proteome studies, the application of alternative proteases, exclusively or in addition to trypsin, often increases protein sequence or proteome coverage. It has recently been shown that, in particular, the analysis of small proteins benefits from such multi-protease approaches. However, selecting the most optimal combination of proteases either requires laboursome experiments or the decision of an experienced user, which might be biased. In this manuscript, we present a protease score that enables the objective comparison of multiple-protease digestions and a Python-based tool named CoMPaseD (Comparison of Multiple Protease  Digestions), which utilizes Monte-Carlo simulations to predict this score for a user-defined set of proteases and any combination of these. By analysis of the small proteomes of the two model organisms Bacillus subtilis and Methanosarcina mazei with five proteases and different experimental setups, we demonstrate a good correlation between experimentally derived and predicted scores. This highlights the broad applicability of CoMPaseD, which can effectively guide the selection of proteases to enhance the characterization of specific subsets of the proteome, e.g. based on factors such as protein size, localization or isoelectric point. CoMPaseD is freely available at https://github.com/MicrobialProteomics/CoMPaseD.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated genes) systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii. Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing interspecies mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favor of the targeting strain (the strain containing the CRISPR-Cas system) during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the Mre11-Rad50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems in archaea play roles beyond their canonical immune function. They may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.

Guanosine tetra- and pentaphosphate ((p)ppGpp) are one of the key players in the stress response of bacteria. Accumulation of these alarmones activates the stringent response, usually triggered by different nutritional stresses. For Pseudomonas putida, there is only limited data available on the importance of the stringent response in stress situations. Also, in recent years, different specific phage defence systems have received much attention, but little is known about the involvement of stringent response in phage infection. Here, we show that P. putida PaW85 (p)ppGpp⁰ is prototrophic and tolerates chemical stress well. However, in the stationary phase P. putida cells deprived of (p)ppGpp have impaired membrane integrity. In addition, we conducted a large-scale screening of stringent response effects on phage infections using the CEPEST phage collection. We tested 67 phages of 22 different species and revealed that the lack of (p)ppGpp has opposing effects on phage infection with nearly half of the tested phages showing higher infection efficiency on the (p)ppGpp⁰ cells, whereas the other half shows reduced infection. We show that the differences in phage infection efficiency for phages Aura and Amme-3 are not caused by adsorption rate differences, but alterations in downstream steps of the infection cycle-prolonged latent period in the absence of (p)ppGpp or unproductive infection in the presence of (p)ppGpp. Altogether, results indicate that the role of stringent response in phage infection is highly diverse, and over half of the times the presence of (p)ppGpp facilitates phage infections rather than protects the cells.

The establishment of a replicative niche in the hostile environment of the host presents an enormous challenge for pathogens. Intracellular pathogens such as Brucella spp., the Gram-negative causative agents of Brucellosis, must subvert diverse host functions to ensure survival and replication. One of the key adaptations to achieve this is the translocation of effector proteins into host cells via its type 4 secretion system (T4SS). But effector identification in Brucella is particularly challenging, as previously identified effectors lack a conserved translocation signal, exhibit variable requirements for translocation, and in some cases appear to be translocated in a T4SS-independent manner. Here, we demonstrate that a subset of previously described T4SS effector proteins associates with outer membrane vesicles (OMVs) in different Brucella species. Most of these effector proteins encode predicted signal peptides for periplasmic export or transmembrane domains. Among them, BspC and VceA carry functional signal peptides that direct their export into the periplasm in a Sec-dependent manner. From the periplasm, these proteins are subsequently secreted into the extracellular milieu, likely via the formation and release of OMVs. Our findings provide new insights into protein secretion by Brucella, suggesting that OMVs may represent an alternative secretion pathway to the T4SS.