Biology subjects

Poehlein, A.

Publications and source records attributed to Poehlein, A..

At least 19 recordsLinked to original sources

bioRxiv2025

Spore type-specific gene expression profiles underlying development and leaf infection processes of Colletotrichum graminicola

Colletotrichum graminicola causes significant losses of the staple crop maize worldwide. The fungus produces two distinct asexual spore types, oval and falcate conidia, which show unique processes in development and plant interaction. Based on genome resequencing of our laboratory strain (CgM2/ M1.001), we investigated the gene expression profiles of oval and falcate conidia during development and early leaf infection using RNA-seq. Our results reveal specific gene expression profiles between the two spore types, indicating fundamental differences in their developmental programs that reflect different modes of infection. We identified expression patterns discriminating both conidia types from mycelium and spore type-specific ones for genes encoding transcription factors, conserved fungal developmental genes, transporters, genes of secondary metabolite clusters, and pathogenicity-related functions, including effectors and carbohydrate-active enzymes (CAZymes). Our study shows that despite the identical genomic basis, oval and falcate conidia show unique transcriptomes across vegetative development and early plant interaction. Taking together, these results provide new insights into the molecular mechanisms determining the biology of C. graminicola and its interaction with the plant host.

microbiology↗

bioRxiv2025

Time-resolved effects of cold atmospheric plasma on E. coli GW-AmxH19 transcriptome and proteome in an emulated wastewater environment

Cold atmospheric plasma (CAP) has been shown to be effective against a variety of microorganisms. In this study, we described effects on an Escherichia coli strain isolated from hospital wastewater caused by a treatment with physical plasma. E. coli GW-AmxH19 was incubated in artificial wastewater and treated for 15 minutes with CAP. Transcriptomes and proteomes were monitored at different timepoints within a 24 h period to differentiate between immediate physiological responses and adaptations in the recovery phase. Reduction of viable cells was on average at 90%. The short-term response of the surviving cells to physical plasma aims at repairing and protecting cellular structures from plasma-induced damages, mainly provoked by reactive oxygen and nitrogen species. Notably, CAP induced a temporary transcription of genes from a conjugative plasmid carrying antibiotic resistance determinants. The late response during recovery phase is dominated by a massive activation of two prophages turning cold plasma treatment into a novel possible strategy to induce the lytic cycle of prophages. This study is the first report on the combined analysis of transcriptional and translational effects of CAP on an environmental bacterial isolate in a time-resolved manner. Chances and risks of considering CAP as an additional purification step in wastewater treatment plants are depicted and discussed.

microbiology↗

bioRxiv2025

Polyethylene terephthalate (PET) primary degradation products affect c-di-GMP-, cAMP-signaling and quorum sensing (QS) in Vibrio gazogenes DSM 21264

Global plastic pollution in oceans and estuaries is increasing rapidly and its well known that bacteria colonize plastic particles of all sizes. Vibrio spp. are frequently found as part of the plastisphere. We recently showed that Vibrio gazogenes DSM 21264 harbors a promiscuous esterase designated PET6. We now provide evidence that the pet6 gene is expressed under a wide range of environmental conditions in its native host. However, in PET- and PE-grown biofilms the pet6 gene expression was not affected by the type of surface. The pet6 transcription was sufficient to allow enzyme production and release of {micro}M amounts of mono-(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA) already after 24 hours of incubation on PET foil. Notably, the highest pet6 gene transcription was observed in planktonic lifestyle in the presence of bis(2-hydroxyethyl) terephthalate (BHET) one of the primary degradation products of PET. BHET was further hydrolyzed by PET6 and UlaG, a lactonase that had not been known to be involved in BHET degradation. Elevated concentrations of BHET affected the major signaling circuits involved in bacterial quorum sensing (QS), c-di-GMP and cAMP-CRP signaling. This resulted in failure to form biofilms, synthesis of the red pigment prodigiosin and altered colony morphologies. While BHET had a very wide impact, TPA interfered mainly with the bacterial QS by attenuating the expression of the CAI-I autoinducer synthase gene. These observations imply a potential role of BHET and TPA as nutritional signals in Vibrio gazogenes and that may affect its growth and survival in the plastisphere. IMPORTANCEThis study provides first evidence that Vibrio gazogenes DSM 21264 secretes an active PET hydrolase and degrades the polymer using PET6 when growing in biofilms on foils and microplastic particles. The study further provides evidence that the primary PET degradation products BHET and TPA may have a profound impact on the global QS, c-di-GMP and cAMP-CRP signaling of V. gazogenes and its capability to colonize plastic particles in the marine environment.

microbiology↗

bioRxiv2025

A single pathogen-secreted protein reprograms plants for drought resilience

Climate change-enforced drought stress conditions and diseases caused by pathogens often co-occur and represent one of the greatest challenges in plant science1-3. Wilt pathogens that colonize water-conducting plant tissues can aggravate the problem and affect a wide range of agricultural crops4,5. However, whilst fungal infections with the vascular pathogen Verticillium dahliae are typically associated with wilt symptoms due to occlusion of xylem tissues, the related V. longisporum induces de novo formation of tracheary elements6,7. This promotes not only its virulence but also enables elevated water storage capacity of the infected host plant and resilience against drought stress conditions6,7. Here, we identified a secreted Verticillium protein, TRANSDIFFERENTIATION EFFECTOR (TRADE), which triggers cell identity switches of bundle sheath cells into tracheary elements. We show that TRADE interacts with the intracellular plant protein VARICOSE (VCS), a conserved component of the mRNA turnover machinery and ortholog of the metazoan protein ENHANCER OF DECAPPING 4 (EDC4/HEDLS/Ge-1)8. The TRADE-VCS interaction induces SUCROSE NON-FERMENTING 1 (SNF1)-related protein kinase (SRK)-dependent phosphorylation and thus dysfunction of VCS. This affects the abundance of mRNAs encoding master regulators of xylem differentiation and demonstrates how a single pathogen effector protein triggers complex tissue-specific developmental reprogramming and thus promotes abiotic stress resilience.

plant biology↗

bioRxiv2024

Isolation and characterization of Sporomusa carbonis sp. nov., a carboxydotrophic hydrogenogen in the genus of Sporomusa isolated from a charcoal burning pile

A Gram-negative bacterial strain, designated ACPtT, was isolated from the covering top soil of an active charcoal burning pile. Cells of ACPtT were strictly anaerobic, rod-shaped and grew optimally at 40{degrees}C and pH 7. The substrates ribose, glucose, sucrose, raffinose, melezitose, pyruvate, vanillate, syringate, methanol and CO were utilized for growth. Phylogenomic analysis of the 4.1 Mb genome showed that strain ACPt represented a novel species of the genus Sporomusa. The most closely related species to ACPtT was Sporomusa malonica with an average amino acid identity of 80.1%. The genome of ACPtT encoded cytochromes, ubiquinones, the Wood-Ljungdahl gene cluster and an Rnf complex, which were identified as common features all Sporomusa type strains. However, strain ACPt did not ferment H2 + CO2 via acetogenesis as other Sporomusa species, but employed the metabolism of a carboxydotrophic hydrogenogen converting CO to H2 + CO2. Based on the genomic, morphological and physiological features presented in this study, strain ACPtT is proposed as a novel species in the genus Sporomusa with the name Sporomusa carbonis sp. nov. (DSM 116159T and CCOS 2105T).

microbiology↗

bioRxiv2024

Knock-down of genes essential for homoacetogenic growth using sugar inducible promoters in the thermophile Thermoanaerobacter kivui

Given recent interest in hydrogen (H2) as a medium for storing and transporting renewable energy, the ability of acetogenic bacteria to use H2 to directly fix carbon dioxide (CO2) shows great promise. Thermophilic bacteria have long been of interest as industrial strains due to their resistance to contamination. Thermoanaerobacter kivui is among the most thermophilic acetogens. It can be genetically manipulated, but as yet only in an all-or-nothing manner, either complete gene knockouts or very strong constitutive promoters leading to protein overexpression. The sugar inducible promoters characterized here provide the finer control necessary to close remaining gaps in understanding of T. kivuis energy metabolism and for controlled expression of recombinant genes of industrial relevance. Acetogenic bacteria are capable of growing on mixtures of the gasses H2, CO2, and CO using the Wood-Ljungdahl pathway, the most energy efficient CO2 fixation pathway. T. kivui is a thermophilic (Topt = 66{degrees}C) acetogen also capable of growth on sugars including glucose, mannitol, and fructose. RNA-sequencing indicated an operon of phosphotransferase import genes up-regulated on mannitol, and growth on a mixture of glucose and mannitol was diauxic. Given this evidence for sugar responsive transcriptional regulation, the promoter region from the mannitol operon, and a similar operon involved in fructose uptake were used to control expression of a thermostable {beta}-galactosidase reporter gene. Both promoters resulted in higher reporter gene activity when grown on their inducing sugar relative to glucose. The inducible promoters were used to create "knock-down" mutants of the proton-pumping Ech1 hydrogenase. Ech1 is believed to be essential because repeated attempts to knock-out the gene operon have failed. The resulting sugar-inducible Ech1 expression strains grew better on their inducing sugar than on glucose. The mannitol inducible promoter was also used to control expression of an essential gene of the Wood-Ljungdahl pathway, formyl-THF synthetase (fhs), involved in consuming the metabolic intermediate formate. When fhs repressed by growth on glucose significantly more formate accumulated in the culture medium than during growth on mannitol, clear evidence of a metabolic bottleneck under the knock-down condition. The promoters characterized here provide finer control of recombinant or native gene expression in T. kivui, while the thermostable reporter gene allows for rapid characterization of new promoters.

microbiology↗

bioRxiv2023

Novel insights into phage biology of the pathogen Clostridioides difficile based on the active virome

The global pathogen Clostridioides difficile is a well-studied organism, and researchers work on unravelling its fundamental virulence mechanisms and biology. Prophages have been demonstrated to influence C. difficile toxin expression and contribute to the distribution of advantageous genes. All this underlines the importance of prophages in C. difficile virulence. Although several C. difficile prophages were sequenced and characterized, investigations on the entire active virome of a strain are still missing. Phages were mainly isolated after mitomycin C-induction, which does not resemble natural stressor for C. difficile. We examined active prophages from different C. difficile strains after cultivation in the absence of mitomycin C by sequencing and characterization of particle-protected DNA. Phage particles were collected after standard cultivation, or after cultivation in the presence of the secondary bile salt deoxycholate (DCA). DCA is a natural stressor for C. difficile and a potential prophage-inducing agent. We also investigated differences in prophage activity between clinical and non-clinical C. difficile strains. Our experiments demonstrated that spontaneous prophage release is common in C. difficile, and that DCA presence induces prophages. Fourteen different, active phages were identified by this experimental procedure. We could not identify a definitive connection between clinical background and phage activity. However, one phage exhibited distinctively higher activity upon DCA-induction in the clinical strain than in the corresponding non-clinical strain, although the phage is identical in both strains. We recorded that enveloped DNA mapped to genome regions with characteristics of mobile genetic elements other than prophages. This pointed to mechanisms of DNA mobility that are not well-studied in C. difficile so far. We also detected phage-mediated lateral transduction of bacterial DNA, which is the first described case in C. difficile. This study significantly contributes to our knowledge on prophage activity in C. difficile and revealed novel aspects on C. difficile (phage) biology.

microbiology↗

bioRxiv2023

Genome-based metabolic and phylogenomic analysis of the type strains of Terrisporobacter mayombei, Terrisporobacter petrolearius and Terrisporobacter glycolicus.

To date, three validly published type strains of the genus Terrisporobacter are covered by draft genome sequences, and the genes and pathway responsible for acetogenesis have not been analyzed. Here, we report complete genome sequences of the bacterial type strains Terrisporobacter petrolearius JCM 19845T, Terrisporobacter mayombei DSM 6539T and Terrisporobacter glycolicus DSM 1288T. Functional annotation, KEGG pathway module reconstructions and screening for virulence factors were performed. Various species-specific vitamin, cofactor and amino acid auxotrophies were identified and a model for acetogenesis of Terrisporobacter was constructed. The complete genomes harbored a gene cluster for the reductive proline-dependent branch of the Stickland reaction located on an approximately 21 kb plasmid, which is exclusively found in the Terrisporobacter genus. Phylogenomic analysis of available Terrisporobacter genomes suggested a reclassification of most isolates as T. glycolicus into T. petrolearius.

genomics↗

bioRxiv2023

A patchwork pathway of apparently recent origin enables degradation of the synthetic buffer compound TRIS in bacteria

The widely used synthetic buffer compound TRIS was long considered to be biologically inert. Herein, we describe the discovery of a complete bacterial degradation pathway for TRIS. By serendipity, a Pseudomonas strain was isolated from sewage sludge that was able to grow with TRIS as only carbon and nitrogen source. Genome and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be involved in TRIS degradation. Conjugational transfer of this plasmid into P. putida KT2440 enabled this strain to grow with TRIS, demonstrating that the complete TRIS degradation pathway can be transmitted by horizontal gene transfer. Heterologous gene expression revealed cluster I to encode a TRIS uptake protein, a TRIS alcohol dehydrogenase, and a TRIS aldehyde dehydrogenase, catalyzing the oxidation of TRIS into 2-hydroxymethylserine. Gene cluster II encodes a methylserine hydroxymethyltransferase and a D-serine dehydratase which plausibly catalyze the conversion of 2-hydroxymethylserine into pyruvate. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from the Pseudomonas and Shinella genera carrying highly similar TRIS degradation gene clusters. Our data indicate that TRIS degradation evolved recently via gene recruitment and enzyme adaptation from multiple independent metabolic pathways and database searches suggest that the TRIS degradation pathway is now globally distributed. Our study illustrates how engineered environments can enhance the emergence of new microbial metabolic pathways in short evolutionary time scales. This knowledge is valuable for developing new water purification processes in times of increasing water scarcity.

microbiology↗

bioRxiv2023

The previously uncharacterized RnpM (YlxR) protein modulates the activity of ribonuclease P in Bacillus subtilis

Even though Bacillus subtilis is one of the most studied organisms, no function has been identified for about 20% of its proteins. Among these unknown proteins are several RNA- and ribosome-binding proteins suggesting that they exert functions in cellular information processing. In this work, we have investigated the RNA-binding protein YlxR. This protein is widely conserved in bacteria and strongly constitutively expressed in B. subtilis suggesting an important function. We have identified the RNA subunit of the essential RNase P as the binding partner of YlxR. The main activity of RNase P is the processing of 5 ends of pre-tRNAs. In vitro processing assays demonstrated that the presence of YlxR results in reduced RNase P activity. Chemical cross-linking studies followed by in silico docking analysis and experiments with site-directed mutant proteins suggest that YlxR binds to the region of the RNase P RNA that is important for binding and cleavage of the pre-tRNA substrate. We conclude that the YlxR protein is a novel interaction partner of the RNA subunit of RNase P that serves to finetune RNase P activity to ensure appropriate amounts of mature tRNAs for translation. We rename the YlxR protein RnpM for RNase P modulator.

microbiology↗

bioRxiv2023

Direct and culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions emplyoing antibiotics. This suggests a significant bias among analysed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA operon and thereby identifies low abundances of C. difficile in environmental samples. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessing C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to detection PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research. ImportanceClostridioides difficile is mainly responsible for hospital-acquired infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence focused on bacterial isolation from clinical specimen under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies however require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoiding antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using NGS.

microbiology↗

bioRxiv2023

Pan-genome analysis of six complete Paracoccus type strain genomes from hybrid next generation sequencing

The genus Paracoccus capable of inhabiting a variety of different ecological niches both, marine and terrestrial is globally distributed. In addition, Paracoccus is taxonomically, metabolically and regarding lifestyle highly diverse. Until now, little is known on how Paracoccus can adapt to such a range of different ecological niches and lifestyles. In the present study, the genus Paracoccus was phylogenomically analyzed (n = 160) and revisited, allowing species level classification of 16 so far unclassified Paracoccus sp. strains and detection of five misclassifications. Moreover, we performed pan-genome analysis of Paracoccus-type strains, isolated from a variety of ecological niches, including different soils, tidal flat sediment, host association such as the bluespotted cornetfish, Bugula plumosa, and the reef-building coral Stylophora pistillata to elucidate either i) the importance of lifestyle and adaptation potential, and ii) the role of the genomic equipment and niche adaptation potential. Six complete genomes were de novo hybrid assembled using a combination of short and long-read technologies. These Paracoccus genomes increase the number of completely closed high-quality genomes of type strains from 15 to 21. Pan-genome analysis revealed an open pan-genome composed of 13,819 genes with a minimal chromosomal core (8.84 %) highlighting the genomic adaptation potential and the huge impact of extra-chromosomal elements. All genomes are shaped by the acquisition of various mobile genetic elements including genomic islands, prophages, transposases, and insertion sequences emphasizing their genomic plasticity. In terms of lifestyle, each mobile genetic elements should be evaluated separately with respect to the ecological context. Free- living genomes, in contrast to host-associated, tend to comprise (1) larger genomes, or the highest number of extra-chromosomal elements, (2) higher number of genomic islands and insertion sequence elements, and (3) a lower number of intact prophage regions. Due to the vast number of adaptive genes, Paracoccus can quickly adapt to changing environmental conditions.

microbiology↗

bioRxiv2022

SUB-OPTIMAL ENVIRONMENTAL CONDITIONS PROLONG PHAGE EPIDEMICS IN BACTERIAL POPULATIONS

Infections by filamentous phages influence bacterial fitness in various ways. While phage-encoded accessory genes, e.g., virulence genes, can be highly beneficial, the production of viral particles is energetically costly and often reduces bacterial growth. Consequently, if costs outweigh benefits, bacteria evolve resistance which can shorten phage epidemics. Abiotic conditions are known to influence the net-fitness effect for infected bacteria. Their impact on the dynamics and trajectories of host resistance evolution, however, remains yet unknown. To address this, we experimentally evolved the bacterium Vibrio alginolyticus in the presence of a filamentous phage at three different salinity levels, i.e., (1) ambient (2) 50% reduction, and (3) fluctuations between reduced and ambient. In all three salinities, bacteria rapidly acquired resistance through super infection exclusion (SIE), whereby phage-infected cells acquired immunity at the cost of reduced growth. Over time, SIE was gradually replaced by evolutionary fitter surface receptor mutants (SRM). This replacement was significantly faster at ambient and fluctuating conditions compared to the low saline environment. Our experimentally parameterized mathematical model explains that suboptimal environmental conditions, in which bacterial growth is slower, slow down phage resistance evolution ultimately prolonging phage epidemics. Our results imply that, if filamentous phages encode virulence genes, these may persist longer in bacterial populations at sub-optimal environmental conditions, which, in times of climate change, are becoming more frequent. Thus, our future ocean may favour the emergence of phage-born pathogenic bacteria, and impose a greater risk for disease outbreaks, impacting not only marine animals but also humans.

evolutionary biology↗

bioRxiv2022

New Microviridae isolated from Sulfitobacter reveals two cosmopolitan subfamilies of ssDNA phages infecting marine and terrestrial Alphaproteobacteria

The Microviridae family represents one of the major clades of ssDNA phages. Their cultivated members are lytic and infect Proteobacteria, Bacteroidetes, and Chlamydiae. Prophages have been predicted in genomes from Bacteroidales, Hyphomicrobiales, and Enterobacteraceae and cluster within the "Alpavirinae", "Amoyvirinae" and Gokushovirinae. We have isolated "Ascunsovirus oldenburgi" ICBM5, a novel phage distantly related to known Microviridae. It infects Sulfitobacter dubius SH24-1b and uses both a lytic and a carrier-state life strategy. Using ICBM5 proteins as a query, we uncovered in publicly available resources 65 new microviridae prophages and episomes in bacterial genomes and retrieved 47 environmental viral genomes (EVGs) from various viromes. Genome clustering based on protein content and phylogenetic analysis showed that ICBM5, together with Rhizobium phages, new prophages, episomes, and EVGs cluster within two new phylogenetic clades, here tentatively assigned the rank of subfamily and named "Tainavirinae" and "Occultatumvirinae". They both infect Rhodobacterales. Occultatumviruses also infect Hyphomicrobiales, including nitrogen-fixing endosymbionts from cosmopolitan legumes. A biogeographical assessment showed that tainaviruses and occultatumviruses are spread worldwide, in terrestrial and marine environments. The new phage isolated here shed light onto new and diverse branches of the Microviridae tree, suggesting that much of the ssDNA phage diversity remains in the dark.

microbiology↗

bioRxiv2021

The Bacillus phage SPβ and its relatives: A temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

The Bacillus phage SP{beta} has been known for about 50 years, but only a few strains are avalible. We isolated four new wild type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SP{beta}-like phages. We could also reveal the SP{beta}-like phage genome replication strategy, the genome packaging mode, and the phage genome opening point. We extracted 55 SP{beta}-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resembled four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SP{beta}-like prophages consists of 38 proteins. The integration cassette proved to be not conserved even though present in all strains. It consists of distinct integrases. Analysis of SP{beta} transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

microbiology↗

bioRxiv2021

HIGH VIRULENCE SELECTS FOR FAST HOST RESISTANCE EVOLUTION

Parasites and pathogens vary strikingly in their virulence and the resulting selection they impose on their hosts. While the evolution of different virulence levels is well studied, the evolution of host resistance in response to different virulence levels is less understood and as of now mainly based on observations and theoretical predictions with few experimental tests. Increased virulence can increase selection for host resistance evolution if resistance costs are outweighed by the benefits of avoiding infection. To test this, we experimentally evolved the bacterium Vibrio alginolyticus in the presence of two variants of the filamentous phage, VALG{Phi}8, that differ in their virulence. The bacterial host exhibited two alternative defence strategies against future phage infection: (1) super infection exclusion (SIE) whereby phage-infected cells were immune to subsequent infection at a cost of reduced growth, and (2) surface receptor mutations (SRM) in genes encoding the MSHA type-IV pilus providing resistance to infection by preventing phage attachment. While SIE emerged rapidly against both phages, SRM evolved faster against the high virulence compared to the low virulence phage. Using a mathematical model of our system we show that increasing virulence strengthens selection for SRM due to the higher costs of infection suffered by SIE immune hosts. In both the experiments and the model, higher levels of SRM in the host population drove more rapid phage extinction. Thus, by accelerating the evolution of host resistance, more virulent phages caused shorter epidemics.

evolutionary biology↗

bioRxiv2021

Nitrogen availability drives gene length of dominant prokaryotes and diversity of genes acquiring Nitrogen-species in oceanic systems

Nitrogen (N) is a key element for prokaryotes in the oceans and often limits phytoplankton primary production. An untested option to reduce prokaryotic N-demand under N-limitation is to reduce gene length. Here we show that in the sunlit Atlantic Ocean genes of the prokaryotic microbial communities in the permanently stratified N-limited (sub)tropics are up to 20% shorter than in N-replete regions further south and north. Average gene length (AGL) of major pelagic prokaryotic genera and two virus families correlated positively with nitrate concentrations. Further, the genomic G+C content of 60% of the taxa was lower and the gene repertoire to acquire inorganic and organic N-species higher in N-limited than in N-replete regions. A comparison of the N-demand by reducing gene length or G+C content showed that the former is much more efficient to save N. Our findings introduce a novel and most effective mode of evolutionary adaptation of prokaryotes to save resources including N and energy. They further show an enhanced diversification of genes acquiring N-species and -compounds in N-deplete relative to N-replete regions and thus add important information for a better understanding of the evolutionary adaptation of prokaryotes to N-availability in oceanic systems.

microbiology↗

bioRxiv2020

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) suggesting the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal mediu. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology. AUTHOR SUMMARYBacteria are exposed to constantly changing environmental conditions. In order to respond to these changes, they use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Among the repertoire of bacterial second messenger nucleotides, cyclic di-AMP (c-di-AMP) stands out as it is the only second messenger that is essential for the bacteria that produce it, including the Gram-positive model organism Bacillus subtilis. C-di-AMP plays a major role in the control of potassium homeostasis, and we found that glutamate is toxic to a B. subtilis strain lacking c-di-AMP to the same extent as potassium. These toxic conditions were the starting point for an extensive suppressor analysis, which led to the identification of a novel glutamate transporter (AimA). If the B. subtilis strain lacking c-di-AMP was cultivated on complex medium, the isolation of suppressor mutants was only possible under anaerobic conditions and if either of the two c-di-AMP-binding signal transduction proteins was absent. This suggests that these proteins are a major burden for the cell on complex medium in their c-di-AMP free state. Our result underline the complexity of c-di-AMP signaling and propose new directions for research.

microbiology↗