Prophages constitute a substantial portion of bacterial genomes, yet their effects on hosts remain poorly understood. We examine the abundance, distribution, and activity of prophages in Bacillus subtilis using computational and laboratory analyses. Genome sequences from the NCBI database and riverbank soil isolates reveal prophages primarily related to mobile genetic elements in laboratory strains. Distinct and previously unknown prophages in local isolates prompt an investigation into factors shaping prophage presence, with phylogenetic relatedness predicting the prophage repertoire slightly better than geographical origin. Data also show that prophages exhibit strong co-occurrence and exclusion patterns within genomes. Laboratory experiments indicate that most predicted prophages are cryptic, as they are not induced under DNA-damaging conditions. Importantly, stress responses increase with the number of predicted prophages, suggesting their influence on host physiology. This study highlights the diversity, integration patterns, and potential roles of prophages in B. subtilis, shedding light on bacterial genome evolution and phage-host dynamics.

Soon after the discovery of genetic competence in the Gram-positive bacterium lated. For instance, the lytic phage phi 29 became a model for studying processes such as viral DNA packaging, replication, and transcription. By contrast, only a handful of temperate B. subtilis phages have been comprehensively characterized. However, the discovery of a peptide-based quorum sensing (QS) system in 2017 has brought temperate B. subtilis phages, particularly those of the SP(3 family, back into the focus of research. The QS system is used by these phages to modulate lysis-lysogeny decisions. Meanwhile, many key components of the lysis-lysogeny management system have been identified. It turned out that a complex co-adaptation between the B. subtilis host cell and SP(3-like phages occurred during evolution and that a host-encoded toxin-antitoxin system plays a key role in controlling lysis-lysogeny decisions. There are many similarities and many important differences between the two well-studied model phages. Thus, a further comparative analysis of the lysis-lysogeny systems is essential to uncover the fundamental differences between phi 3T and SP(3. Moreover, we believe that it would be exciting to revive research on temperate B. subtilis phages that are not related to SP(3-family phages.

In lysogenic conversion, temperate phages can profoundly alter the phenotypes of their bacterial hosts. When studying the effects of SPbeta-like viruses on a Bacillus subtilis soil isolate, we observed that phage phi3T induced a stable and heritable acquisition of a spherical cell morphology, departing from the typical rod shape of its ancestor. Although time-lapse imaging revealed that the lysogen retained cell wall integrity and symmetric division, it exhibited reduced fitness and increased susceptibility to cell wall-targeting antimicrobials. Remarkably, when SPβ, a homologous SPbeta-like virus, was present, the host retained its rod-shaped morphology despite phi3T superinfection. This study uncovers a novel example of lysogenic conversion in which phage integration into the bacterial genome induces heritable changes in host biology. Additionally, we revealed intriguing phage-phage interactions during polylysogeny that may benefit the host. These findings offer insight into the persistence and absence of specific Spbetavirus variants in natural B. subtilis populations.

We present the complete genome of Schlesneria sp. DSM 10557, isolated from compost heap leakage (leachate). The genome consists of a single chromosome (7,067,087 bp) with a GC content of 56.48%. Genome-based phylogenetic analysis revealed that strain DSM 10557 is most closely related to members of the genus Schlesneria.

Prokaryotic antiviral defence systems are frequently toxic for host cells and stringent regulation is required to ensure survival and fitness. These systems must be readily available in case of infection but tightly controlled to prevent activation of an unnecessary cellular response. Here we investigate how the bacterial cyclic oligonucleotide-based antiphage signalling system (CBASS) uses its intrinsic protein modification system to regulate the nucleotide cyclase. By integrating a type II CBASS system from Bacillus cereus into the model organism Bacillus subtilis, we show that the protein-conjugating Cap2 (CBASS associated protein 2) enzyme links the cyclase exclusively to the conserved phage shock protein A (PspA) in the absence of phage. The cyclase-PspA conjugation is reversed by the deconjugating isopeptidase Cap3 (CBASS associated protein 3). We propose a model in which the cyclase is held in an inactive state by conjugation to PspA in the absence of phage, with conjugation released upon infection, priming the cyclase for activation.

Aminophosphonates, like glyphosate (GS) or metal chelators such as ethylenediaminetetra(methylenephosphonic acid) (EDTMP), are released on a large scale worldwide. Here, we have characterized a bacterial strain capable of degrading synthetic aminophosphonates. The strain was isolated from LC/MS standard solution. Genome sequencing indicated that the strain belongs to the genus Ochrobactrum. Whole-genome classification using pyANI software to compute a pairwise ANI and other metrics between Brucella assemblies and Ochrobactrum contigs revealed that the bacterial strain is designated as Ochrobactrum sp. BTU1. Degradation batch tests with Ochrobactrum sp. BTU1 and the selected aminophosphonates GS, EDTMP, aminomethylphosphonic acid (AMPA), iminodi(methylene-phosphonic) (IDMP) and ethylaminobis(methylenephosphonic) acid (EABMP) showed that the strain can use all phosphonates as sole phosphorus source during phosphorus starvation. The highest growth rate was achieved with AMPA, while EDTMP and GS were least supportive for growth. Proteome analysis revealed that GS degradation is promoted by C-P lyase via the sarcosine pathway, i.e., initial cleavage at the C-P bond. We also identified C-P lyase to be responsible for degradation of EDTMP, EABMP, IDMP and AMPA. However, the identification of the metabolite ethylenediaminetri(methylenephosphonic acid) via LC/MS analysis in the test medium during EDTMP degradation indicates a different initial cleavage step as compared to GS. For EDTMP, it is evident that the initial cleavage occurs at the C-N bond. The detection of different key enzymes at regulated levels, form the bacterial proteoms during EDTMP exposure, further supports this finding. This study illustrates that widely used and structurally more complex aminophosphonates can be degraded by Ochrobactrum sp. BTU1 via the well-known degradation pathways but with different initial cleavage strategy compared to GS.

Antiviral defence systems build the prokaryotic immune system and their proper regulation is vital for survival and fitness. While it is important that they are readily available in case of infection, they need to be tightly controlled to prevent activation of an unnecessary cellular response. Here we describe how the bacterial cyclic oligonucleotide-based antiphage signalling system (CBASS) uses its intrinsic protein modification system to regulate the nucleotide cyclase. By integrating a Type II CBASS system from Bacillus cereus into the cognate host Bacillus subtilis , we show that the Cap2 protein conjugates the cyclase exclusively to the conserved phage shock protein A (PspA) in the absence of phage. This cyclase-PspA conjugation is reversed after infection by the isopeptidase Cap3. Finally, we propose a model in which the cyclase is held in an inactive state by conjugation to PspA in the absence of phage and that this conjugation is released upon infection, priming the cyclase for activation.

Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis.

Glyphosate (GS) specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that converts phosphoenolpyruvate (PEP) and shikimate-3-phosphate to EPSP in the shikimate pathway of bacteria and other organisms. The inhibition of the EPSP synthase depletes the cell of the EPSP-derived aromatic amino acids as well as of folate and quinones. A variety of mechanisms (e.g., EPSP synthase modification) has been described that confer GS resistance to bacteria. Here, we show that the Burkholderia anthina strain DSM 16086 quickly evolves GS resistance by the acquisition of mutations in the ppsR gene. ppsR codes for the pyruvate/ortho-Pi dikinase PpsR that physically interacts and regulates the activity of the PEP synthetase PpsA. The mutational inactivation of ppsR causes an increase in the cellular PEP concentration, thereby abolishing the inhibition of the EPSP synthase by GS that competes with PEP for binding to the enzyme. Since the overexpression of the Escherichia coli ppsA gene in Bacillus subtilis and E. coli did not increase GS resistance in these organisms, the mutational inactivation of the ppsR gene resulting in PpsA overactivity is a GS resistance mechanism that is probably unique to B. anthina.

Phages are viruses of bacteria and have been known for over a century. They do not have a metabolism or protein synthesis machinery and rely on host cells for replication. The model organism Bacillus subtilis has served as a host strain for decades and enabled the isolation of many unique viral strains. However, many viral species representatives remained orphans as no, or only a few, related phages were ever re-isolated.The presented protocol describes how a CRISPR-Cas9 system with an artificial CRISPR-array can be set up and used to discriminate abundant and well-known B. subtilis phage from a host-based metagenome enrichment. The obtained viral suspension can be used for metagenome sequencing and isolating new viral strains.

Focusing on visible plaques for phage isolation leaves the question if we miss the diversity of non-plaque forming phages. We addressed this question through direct plaque-based isolation by employing the new hosts Brevundimonas pondensis LVF1 and Serratia marcescens LVF3 dsDNA, ssDNA, dsRNA, and ssRNA host-associated metavirome analysis. Of the 25 distinctive dsDNA phage isolates, 14 were associated with Brevundimonas and 11 with Serratia. TEM analysis revealed that 6 were myoviruses, 18 siphoviruses and 1 podovirus, while phages infecting Brevundimonas belonged all to siphoviruses. The associated viromes suggested a higher phage diversity in summer than in winter, and dsDNA phages were the dominant group. Isolation of vB_SmaP-Kaonashi was possible after investigating the viromes associated with Serratia, demonstrating the great potential of accompanying host-associated metavirome analysis. The ssDNA virome analysis showed that the B. pondensis LVF1 host is associated with Microviridae and Inoviridae phages, although none of them were isolated. The results demonstrated that the classical isolation technique is not exhausted, leading to the isolation of new dsDNA phages. It can be further improved by combination with metavirome techniques, which revealed further diversity.

A new Vibrio strain, K08M4T, was isolated from the broad-nosed pipefish Syngnathus typhle in the Kiel Fjord. Infection experiments revealed that K08M4T was highly virulent for juvenile pipefish. Cells of strain K08M4T were Gram-stain-negative, curved rod-shaped and motile by means of a single polar flagellum. The strain grew aerobically at 9-40° C, at pH 4-10.5 and it tolerated up to 12 % (w/v) NaCl. The most prevalent (>10 %) cellular fatty acids of K08M4T were C16 : 1 ω7c and C16 : 0. Whole-genome comparisons revealed that K08M4T represents a separate evolutionary lineage that is distinct from other Vibrio species and falls within the Splendidus clade. The genome is 4,886,292 bp in size, consists of two circular chromosomes (3,298,328 and 1, 587,964 bp) and comprises 4,178 protein-coding genes and 175 RNA genes. In this study, we describe the phenotypic features of the new isolate and present the annotation and analysis of its complete genome sequence. Based on these data, the new isolate represents a new species for which we propose the name Vibrio syngnathi sp. nov. The type strain is K08M4T (=DSM 109818T=CECT 30086T).

Luteibacter is a genus of the Rhodanobacteraceae family. The present study describes a novel species within the genus Luteibacter (EIF3T). The strain was analyzed genomically, morphologically and physiologically. Average nucleotide identity analysis revealed that it is a new species of Luteibacter. In silico analysis indicated two putative prophages (one incomplete, one intact). EIF3T cells form an elliptical morphotype with an average length of 2.0 µm and width of 0.7 µm and multiple flagella at one end. The bacterial strain is an aerobic Gram-negative with optimal growth at 30 °C. EIF3T is resistant towards erythromycin, tetracycline and vancomycin. We propose the name Luteibacter flocculans sp. nov. with EIF3T (=DSM 112537T = LMG 32416T) as type strain. Further, we describe the first known Luteibacter-associated bacteriophage called vB_LflM-Pluto.

A new Vibrio strain K08M4T was isolated from the broad-nosed pipefish Syngnathus typhle in the Kiel Fjord. Infection experiments revealed that K08M4T is highly virulent for juvenile pipefish. Cells of strain K08M4T are Gram-stain-negative, curved rod-shaped and motile by means of a single polar flagellum. The strain can grow aerobically at 9 to 40°C, at pH 4 to 10.5 and tolerates up to 12% (w/v) NaCl. The most prevalent (> 10%) cellular fatty acids of K08M4T were C16:1 ω 7 c and C16:0. Whole-genome comparisons revealed that K08M4T represents a separate evolutionary lineage which is distinct from other Vibrio species and falls within the Vibrio Splendidus clade. The genome is 4,886,292 bp in size, consists of two circular chromosomes (3,298,328 bp, 1,587,964 bp), and comprises 4,178 protein-coding genes and 175 RNA genes. In this study, we describe the phenotypic features of the new isolate and present the annotation and analysis of its complete genome sequence. Based on these data, the new isolate represents a new species for which we propose the name Vibrio syngnathi . The type strain is K08M4T (=DSM 109818T).Supplementary material One supplementary figure and six supplementary table are available with the online version of the Manuscript.### Competing Interest StatementThe authors have declared no competing interest.* TCBS : thiosulfate-citrate-bile-sucrose; TYGS : Type Strain Genome Server, dDDH : digital DNA-DNA hybridization

Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPβ prophage whose lysogenic state depends on the MrpR (YopR) protein, a key component of the lysis-lysogeny decision system. Using a historic B. subtilis strain harboring the heat-sensitive SPβ c2 mutant, we demonstrate that the lytic cycle of SPβ c2 can be induced by heat due to a single nucleotide exchange in the mrpR gene, rendering the encoded MrpRG136E protein temperature-sensitive. Structural characterization revealed that MrpR is a DNA-binding protein resembling the overall fold of tyrosine recombinases. MrpR has lost its recombinase function and the G136E exchange impairs its higher-order structure and DNA binding activity. Genome-wide profiling of MrpR binding revealed its association with the previously identified SPbeta repeated element (SPBRE) in the SPβ genome. MrpR functions as a master repressor of SPβ that binds to this conserved element to maintain lysogeny. The heat-inducible excision of the SPβ c2 mutant remains reliant on the serine recombinase SprA. A suppressor mutant analysis identified a previously unknown component of the lysis-lysogeny management system that is crucial for the induction of the lytic cycle of SPβ.

Bacteriophages, also called phages, are viruses of bacteria. They are the most common and diverse biological entities on this planet. For metagenomic investigation, their diversity is also their biggest obstacle. The direct metagenomic sequence of environmental phage communities often leads to short genomic fragments limiting the investigation to a few individual aspects of phage biology and diversity.The presented protocol for generating a host-associated metagenome reduces the phage diversity to a concise and accessible size. Metagenome sequencing often leads to complete genomes, and the availability of a suitable host system ensures further experimental investigation.

ABSTRACTProphages need to tightly control their lifestyle to either be maintained within the host genome or enter the lytic cycle. The SPβ prophage present in the genome ofBacillus subtilis168 was recently shown to possess anarbitriumsystem defining its replication stage. Using an historicB. subtilisstrain harboring the heat-sensitive SPβ c2 mutant, we analyzed a key component of the lysis-lysogeny decision system called YopR, which is critical for maintenance of lysogeny. Here, we demonstrate that the heat-sensitive SPβ c2 phenotype is due to a single nucleotide exchange in theyopRgene, rendering the encoded YopRG136Eprotein temperature sensitive. Structural characterization of YopR revealed that the protein is a DNA-binding protein with an overall fold like tyrosine recombinases. Biochemical and functional analyses indicate that YopR has lost the recombinase function and the G136E exchange impairs its higher order structure and DNA binding activity. We further show that the heat-inducible SPβ excision of the c2 mutant still depends on the serine recombinase SprA. Finally, an evolution experiment identified the YosL protein of unknown function as a novel component of the lysis-lysogeny management system, as the presence ofyosLis crucial for the induction of the lytic cycle of SPβ.

The functional relevance of microbial necromass in terrestrial biogeochemical cycles remains one of the unre-solved mysteries of element cycling in ecosystems, especially considering the high microbial abundance and turnover in soil. We therefore established a protocol to manufacture multi-isotope (C-14, N-15 and P-33) labeled microbial necromass to comprehensively track the turnover of microbial necromass elements within element cycles. This protocol encompasses the i) microbial cultivation of Pseudomonas kilonensis ACN4 (Gram-negative) and Bacillus licheniformis DSM13 (Gram-positive) on labeled minimal medium as well as fungal cultivation of Hypsizygus tessulatus on a complex yeast medium, ii) quantification of radio-(C-14, P-33) and stable (N-15) isotope incorporation as well their cellular pool partitioning, and iii) determination of element and tracer isotope uptake efficiency. We achieved 1 g of bacterial biomass per liter minimum medium within 24 h and 2.9 g l(-1) fungal biomass in complex medium within 18 d. This production rate enabled us to produce more than 100 g of nec-romass within only one half-life time of P-33, including post-harvest processing. Isotope uptake and incorporation for P-33 ranged from 10 to 73%, for N-15 from 24 to 52%, and for C-14 from 12 to 23%. Each of the cultivated species showed individual patterns of tracer element uptake. The nutritional value of the carbon-(C), nitrogen -(N) and phosphorus-(P) labeled microbial necromass was characterized by a water-based, necromass species-specific partitioning scheme with subsequent elemental analysis of the pools. We separated Gram-negative, Gram-positive and fungi's cellular pools to characterize element and tracer partitioning among dissolved versus particulate fractions. That is essential because these properties subsequently affect the respective pool's availability for ecosystem nutrition. Our procedure allows a defined production of microorganism-based nec-romass, enabling versatile use to determine necromass-related nutrient fluxes in terrestrial ecosystem studies.