A Survey of Pyridoxal 5′-Phosphate-Dependent Proteins in the Gram-Positive Model Bacterium Bacillus subtilis

The B6 vitamer pyridoxal 5′-phosphate (PLP) is a co-factor for proteins and enzymes that are involved in diverse cellular processes. Therefore, PLP is essential for organisms from all kingdoms of life. Here we provide an overview about the PLP-dependent proteins from the Gram-positive soil bacterium Bacillus subtilis. Since B. subtilis serves as a model system in basic research and as a production host in industry, knowledge about the PLP-dependent proteins could facilitate engineering the bacteria for biotechnological applications. The survey revealed that the majority of the PLP-dependent proteins are involved in metabolic pathways like amino acid biosynthesis and degradation, biosynthesis of antibacterial compounds, utilization of nucleotides as well as in iron and carbon metabolism. Many PLP-dependent proteins participate in de novo synthesis of the co-factors biotin, folate, heme, and NAD+ as well as in cell wall metabolism, tRNA modification, regulation of gene expression, sporulation, and biofilm formation. A surprisingly large group of PLP-dependent proteins (29%) belong to the group of poorly characterized proteins. This review underpins the need to characterize the PLP-dependent proteins of unknown function to fully understand the “PLP-ome” of B. subtilis

Visualisierung von Mutationen auf Einzelzellebene

Täuber S, Dormeyer M, Commichau FM, Grünberger A. Visualisierung von Mutationen auf Einzelzellebene. BIOspektrum. 2020;26(4):388-390.Bacterial mutations have been investigated since many years, but they remain difficult to observe directly in single cells, which limits the analysis of the underlying molecular mechanism. However, for the investigation of mutations at the level of single cells, precise analytical tools are currently developed. This article describes a workflow for visualizing mutations in single cells and lays the foundation for the quantification of bacterial mutation rates in the future

Adaptation of Listeria monocytogenes to perturbation of c-di-AMP metabolism underpins its role in osmoadaptation and identifies a fosfomycin uptake system

The human pathogen Listeria monocytogenes synthesizes and degrades c-di-AMP using the diadenylate cyclase CdaA and the phosphodiesterases PdeA and PgpH respectively. c-di-AMP is essential because it prevents the uncontrolled uptake of osmolytes. Here, we studied the phenotypes of cdaA, pdeA, pgpH and pdeA pgpH mutants with defects in c-di-AMP metabolism and characterized suppressor mutants restoring their growth defects. The characterization of the pdeA pgpH mutant revealed that the bacteria show growth defects in defined medium, a phenotype that is invariably suppressed by mutations in cdaA. The previously reported growth defect of the cdaA mutant in rich medium is suppressed by mutations that osmotically stabilize the c-di-AMP-free strain. We also found that the cdaA mutant has an increased sensitivity against isoleucine. The isoleucine-dependent growth inhibition of the cdaA mutant is suppressed by codY mutations that likely reduce the DNA-binding activity of encoded CodY variants. Moreover, the characterization of the cdaA suppressor mutants revealed that the Opp oligopeptide transport system is involved in the uptake of the antibiotic fosfomycin. In conclusion, the suppressor analysis corroborates a key function of c-di-AMP in controlling osmolyte homeostasis in L. monocytogenes.Peer Reviewe

The ␥-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

b PutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-DL-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the ␥-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHPresistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates

Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

Kampf J, Gerwig J, Kruse K, et al. Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability. mBio. 2018;9(5): e01464-18

Bacillus subtilis Spore Resistance to Simulated Mars Surface Conditions

In a Mars exploration scenario, knowing if and how highly resistant Bacillus subtilis spores would survive on the Martian surface is crucial to design planetary protection measures and avoid false positives in life-detection experiments. Therefore, in this study a systematic screening was performed to determine whether B. subtilis spores could survive an average day on Mars. For that, spores from two comprehensive sets of isogenic B. subtilis mutant strains, defective in DNA protection or repair genes, were exposed to 24 h of simulated Martian atmospheric environment with or without 8 h of Martian UV radiation [M(+)UV and M(-)UV, respectively]. When exposed to M(+)UV, spore survival was dependent on: (1) core dehydration maintenance, (2) protection of DNA by α/β-type small acid soluble proteins (SASP), and (3) removal and repair of the major UV photoproduct (SP) in spore DNA. In turn, when exposed to M(-)UV, spore survival was mainly dependent on protection by the multilayered spore coat, and DNA double-strand breaks represent the main lesion accumulated. Bacillus subtilis spores were able to survive for at least a limited time in a simulated Martian environment, both with or without solar UV radiation. Moreover, M(-)UV-treated spores exhibited survival rates significantly higher than the M(+)UV-treated spores. This suggests that on a real Martian surface, radiation shielding of spores (e.g., by dust, rocks, or spacecraft surface irregularities) might significantly extend survival rates. Mutagenesis were strongly dependent on the functionality of all structural components with small acid-soluble spore proteins, coat layers and dipicolinic acid as key protectants and efficiency DNA damage removal by AP endonucleases (ExoA and Nfo), non-homologous end joining (NHEJ), mismatch repair (MMR) and error-prone translesion synthesis (TLS). Thus, future efforts should focus on: (1) determining the DNA damage in wild-type spores exposed to M(+/-)UV and (2) assessing spore survival and viability with shielding of spores via Mars regolith and other relevant materials

The Bacillus subtilis Minimal Genome Compendium

To better understand cellular life, it is essential to decipher the contribution of individual components and their interactions. Minimal genomes are an important tool to investigate these interactions. Here, we provide a database of 105 fully annotated genomes of a series of strains with sequential deletion steps of the industrially relevant model bacterium Bacillus subtilis starting with the laboratory wild type strain B. subtilis 168 and ending with B. subtilis PG38, which lacks approximately 40% of the original genome. The annotation is supported by sequencing of key intermediate strains as well as integration of literature knowledge for the annotation of the deletion scars and their potential effects. The strain compendium presented here represents a comprehensive genome library of the entire MiniBacillus project. This resource will facilitate the more effective application of the different strains in basic science as well as in biotechnology

Essential genes in Bacillus subtilis: a re-evaluation after ten years

In 2003, an initial study on essential genes in the Gram-positive model bacterium described 271 genes as essential. In the past decade, the functions of many unknown genes and their encoded proteins have been elucidated. Moreover, detailed analyses have revealed that 31 genes that were thought to be essential are in fact non-essential whereas 20 novel essential genes have been described. Thus, 261 genes coding for 259 proteins and two functional RNAs are regarded essential as of January 2013. Among the essential proteins, the largest group is involved in protein synthesis, secretion and protein quality control. Other large sets of essential proteins are involved in lipid biosynthesis, cell wall metabolism and cell division, and DNA replication. Another interesting group of essential proteins protects the cell against endogenous toxic proteins, metabolites, or other intermediates. There are only six essential proteins in B. subtilis, for which no function is known. The functional analysis of these important proteins is predicted to be a key issue in the research on this model organism in the coming years