AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase.

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/ß-fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature Tm 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.

Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus.

Alginate (alginic acid) is a linear polysaccharide, wherein (1→4)-linked ß-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1→4) glycosidic linkages between the monomers by a ß-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.

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.

A Timed Off-Switch for Dynamic Control of Gene Expression in Corynebacterium Glutamicum.

Dynamic control of gene expression mainly relies on inducible systems, which require supplementation of (costly) inducer molecules. In contrast, synthetic regulatory circuits, which allow the timed shutdown of gene expression, are rarely available and therefore represent highly attractive tools for metabolic engineering. To achieve this, we utilized the VanR/P vanABK * regulatory system of Corynebacterium glutamicum, which consists of the transcriptional repressor VanR and a modified promoter of the vanABK operon (P vanABK *). VanR activity is modulated by one of the phenolic compounds ferulic acid, vanillin or vanillic acid, which are co-metabolized with d-glucose. Thus, gene expression in the presence of d-glucose is turned off if one of the effector molecules is depleted from the medium. To dynamically control the expression of the aceE gene, encoding the E1 subunit of the pyruvate dehydrogenase complex that is essential for growth on d-glucose, we replaced the native promoter by vanR/P vanABK * yielding C. glutamicum ΔP aceE ::vanR-P vanABK *. The biomass yield of this strain increased linearly with the supplemented amount of effector. After consumption of the phenolic compounds growth ceased, however, C. glutamicumΔP aceE ::vanR-P vanABK * continued to utilize the residual d-glucose to produce significant amounts of pyruvate, l-alanine, and l-valine. Interestingly, equimolar concentrations of the three phenolic compounds resulted in different biomass yields; and with increasing effector concentration, the product spectrum shifted from pyruvate over l-alanine to l-valine. To further test the suitability of the VanR/P vanABK * system, we overexpressed the l-valine biosynthesis genes ilvBNCE in C. glutamicum ΔP aceE ::vanR-P vanABK *, which resulted in efficient l-valine production with a yield of about 0.36 mol l-valine per mol d-glucose. These results demonstrate that the VanR/P vanABK * system is a valuable tool to control gene expression in C. glutamicum in a timed manner by the cheap and abundant phenolic compounds ferulic acid, vanillin, and vanillic acid.

Crystal structures of the Bacillus subtilis prophage lytic cassette proteins XepA and YomS.

As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPß were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel ß-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each ß-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.

Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced ß-galactosidase production using the CRISPR/Cas9 system.

The ganA gene from Bacillus subtilis encoding a ß-galactosidase for degradation of the galactomannan was integrated in different loci of the B. subtilis chromosome employing the CRISPR/Cas9 system. Hereby a total of five copies of ganA cassettes in which the ganA gene was fused with the glucitol-promoter were inserted in the recipient chromosome wherein hypothetical, sporulation and protease genes were deleted. The strain with five copies of ganA expression cassette showed a ß-galactosidase activity similar to the one with the same gene on a pUB110 derived multi-copy plasmid and under the same regulatory control of the glucitol promoter and GutR activator. The production of ß-galactosidase in the strain with the multi-copy plasmid decreased rapidly when growth was performed under induced conditions and without antibiotic selection. In contrast, the strain with the five copies of ganA in the chromosome produced ß-galactosidase for at least 40 generations. This demonstrates that the CRISPR/Cas9 system is a valuable and easy tool for constructing stable producer strains. The bigger efforts that are needed for the multiple target gene integration into the chromosome compared to cloning in expression vectors were justified by the higher stability of the target genes and the lack of antibiotic resistance genes.

Evaluation of surfactin synthesis in a genome reduced Bacillus subtilis strain.

Strain engineering is often a method of choice towards increasing the yields of the biosurfactant surfactin which is naturally synthesized by many Bacillus spp., most notably Bacillus subtilis. In the current study, a genome reduced B. subtilis 168 strain lacking 10% of the genome was established and tested for its suitability to synthesize surfactin under aerobic and anaerobic conditions at 25 °C, 30 °C, 37 °C and 40 °C. This genome reduced strain was named IIG-Bs20-5-1 and lacks, amongst others, genes synthesizing the lipopeptide plipastatin, the antibiotic bacilysin, toxins and prophages, as well as genes involved in sporulation. Amongst all temperatures tested, 37 °C was overall superior. In comparison to the reference strain JABs24, a surfactin synthesizing variant of B. subtilis 168, strain IIG-Bs20-5-1 was both aerobically and anaerobically superior with respect to specific growth rates µ and yields YX/S. However, in terms of surfactin production, strain JABs24 reached higher absolute concentrations with up to 1147.03 mg/L and 296.37 mg/L under aerobic and anaerobic conditions, respectively. Concomitant, strain JABs24 reached higher YP/S and YP/X. Here, an outstanding YP/X of 1.541 g/g was obtained under anaerobic conditions at 37 °C. The current study indicates that the employed genome reduced strain IIG-Bs20-5-1 has several advantages over the strain JABs24 such as better conversion from glucose into biomass and higher growth rates. However, regarding surfactin synthesis and yields, the strain was overall inferior at the investigated temperatures and oxygen conditions. Further studies addressing process development and strain engineering should be performed combining the current observed advantages of the genome reduced strain to increase the surfactin yields and to construct a tailor-made genome reduced strain to realize the theoretically expected advantages of such genome reduced strains.

The melREDCA Operon Encodes a Utilization System for the Raffinose Family of Oligosaccharides in Bacillus subtilis.

Bacillus subtilis is a heterotrophic soil bacterium that hydrolyzes different polysaccharides mainly found in the decomposed plants. These carbohydrates are mainly cellulose, hemicellulose, and the raffinose family of oligosaccharides (RFOs). RFOs are soluble α-galactosides, such as raffinose, stachyose, and verbascose, that rank second only after sucrose in abundance. Genome sequencing and transcriptome analysis of B. subtilis indicated the presence of a putative α-galactosidase-encoding gene (melA) located in the msmRE-amyDC-melA operon. Characterization of the MelA protein showed that it is a strictly Mn2+- and NAD+-dependent α-galactosidase able to hydrolyze melibiose, raffinose, and stachyose. Transcription of the msmER-amyDC-melA operon is under control of a σA-type promoter located upstream of msmR (P msmR ), which is negatively regulated by MsmR. The activity of P msmR was induced in the presence of melibiose and raffinose. MsmR is a transcriptional repressor that binds to two binding sites at P msmR located upstream of the -35 box and downstream of the transcriptional start site. MsmEX-AmyCD forms an ATP-binding cassette (ABC) transporter that probably transports melibiose into the cell. Since msmRE-amyDC-melA is a melibiose utilization system, we renamed the operon melREDCAIMPORTANCEBacillus subtilis utilizes different polysaccharides produced by plants. These carbohydrates are primarily degraded by extracellular hydrolases, and the resulting oligo-, di-, and monosaccharides are transported into the cytosol via phosphoenolpyruvate-dependent phosphotransferase systems (PTS), major facilitator superfamily, and ATP-binding cassette (ABC) transporters. In this study, a new carbohydrate utilization system of B. subtilis responsible for the utilization of α-galactosides of the raffinose family of oligosaccharides (RFOs) was investigated. RFOs are synthesized from sucrose in plants and are mainly found in the storage organs of plant leaves. Our results revealed the modus operandi of a new carbohydrate utilization system in B. subtilis.

Phosphosugar Stress in Bacillus subtilis: Intracellular Accumulation of Mannose 6-Phosphate Derepressed the glcR-phoC Operon from Repression by GlcR.

Bacillus subtilis phosphorylates sugars during or after their transport into the cell. Perturbation in the conversion of intracellular phosphosugars to the central carbon metabolites and accumulation of phosphosugars can impose stress on the cells. In this study, we investigated the effect of phosphosugar stress on B. subtilis Preliminary experiments indicated that the nonmetabolizable analogs of glucose were unable to impose stress on B. subtilis In contrast, deletion of manA encoding mannose 6-phosphate isomerase (responsible for conversion of mannose 6-phosphate to fructose 6-phosphate) resulted in growth arrest and bulged cell shape in the medium containing mannose. Besides, an operon encoding a repressor (GlcR) and a haloic acid dehalogenase (HAD)-like phosphatase (PhoC; previously YwpJ) were upregulated. Integration of the P glcR-lacZ cassette into different mutational backgrounds indicated that P glcR is induced when (i) a manA-deficient strain is cultured with mannose or (ii) when glcR is deleted. GlcR repressed the transcription of glcR-phoC by binding to the σA-type core elements of P glcR An electrophoretic mobility shift assay showed no interaction between mannose 6-phosphate (or other phosphosugars) and the GlcR-P glcR DNA complex. PhoC was an acid phosphatase mainly able to dephosphorylate glycerol 3-phosphate and ribose 5-phosphate. Mannose 6-phosphate was only weakly dephosphorylated by PhoC. Since deletion of glcR and phoC alone or in combination had no effect on the cells during phosphosugar stress, it is assumed that the derepression of glcR-phoC is a side effect of phosphosugar stress in B. subtilisIMPORTANCEBacillus subtilis has different stress response systems to cope with external and internal stressors. Here, we investigated how B. subtilis deals with the high intracellular concentration of phosphosugars as an internal stressor. The results indicated the derepression of an operon consisting of a repressor (GlcR) and a phosphatase (PhoC). Further analysis revealed that this operon is not a phosphosugar stress response system. The substrate specificity of PhoC may indicate a connection between the glcR-phoC operon and pathways in which glycerol 3-phosphate and ribose 5-phosphate are utilized, such as membrane biosynthesis and teichoic acid elongation.

New CRISPR-Cas9 vectors for genetic modifications of Bacillus species.

Genetic manipulation is a fundamental procedure for the study of gene and operon functions and new characteristics acquisition. Modern CRISPR-Cas technology allows genome editing more precisely and increases the efficiency of transferring mutations in a variety of hard to manipulate organisms. Here, we describe new CRISPR-Cas vectors for genetic modifications in bacillary species. Our plasmids are single CRISPR-Cas plasmids comprising all components for genome editing and should be functional in a broad host range. They are highly efficient (up to 97%) and precise. The employment and delivery of these plasmids to bacillary strains can be easily achieved by conjugation from Escherichia coli. During our research we also demonstrated the absence of compatibility between CRISPR-Cas system and non-homologous end joining in Bacillus subtilis.

Cross Talk among Transporters of the Phosphoenolpyruvate-Dependent Phosphotransferase System in Bacillus subtilis.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) is the main carbohydrate uptake system in Bacillus subtilis A typical PTS consists of two general proteins, enzyme I (EI) and a histidine-containing protein (HPr), as well as a specific carbohydrate transporter (or enzyme II [EII]), all of which transfer the phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. The specific PTS transporters are formed by multidomain proteins or single-domain subunits. These domains are domain C (EIIC), the transmembrane channel for the carbohydrate transport; domain B (EIIB), the membrane-bound domain responsible for phosphorylation of the carbohydrate; and domain A (EIIA), the mediator between HPr(H15∼P) and EIIB. There are 16 PTS transporters in B. subtilis, 6 of which, i.e., NagP, MalP, MurP, TreP, SacP, and SacX, contain no EIIA domain. Deletion of the single-EIIA-containing transporters showed that there is cross talk between the noncognate EIIA and EIIB domains in PTS. By deletion of all EIIA-containing proteins, strain KM455 (ΔEIIA) was constructed, and the EIIA-containing proteins were individually introduced into the strain. In this way, the PTS transporters of the glucose family, namely, PtsG, GamP, and PtsA (also known as YpqE), enabled growth with maltose, N-acetylglucosamine, sucrose, or trehalose as the sole carbon source. Construction of TkmA-EIIA fusion proteins confirmed the probable interaction between the EIIAs of the glucose family of PTS transporters and the EIIA-deficient PTS transporters. Likewise, we have shown that SacX is mainly phosphorylated by PtsA and GamP. PtsG and GmuA were also able to phosphorylate SacX, albeit less well than GamP and PtsA.IMPORTANCE The phosphoenolpyruvate-dependent phosphotransferase system (PTS) not only is a carbohydrate uptake system in B. subtilis but also plays an important role in sensing the nutrient fluctuation in the medium. This sensing system enables the cells to respond to these fluctuations properly. The PTS transporters have a pivotal role in this sensing system since they are carbohydrate specific. In this study, we tried to understand the interactions among these transporters which revealed the cross talk among PTSs. Three PTS proteins, namely, PtsG (the specific transporter of glucose), GamP (the specific transporter of glucosamine), and PtsA (a cytoplasmic single-domain EIIA protein) were shown to play the major role in the interaction among the PTSs.

Inactivation of genes involved in base excision repair of Corynebacterium glutamicum and survival of the mutants in presence of various mutagens.

Base Excision Repair (BER) is considered as the most active DNA repair pathway in vivo, which is initiated by recognition of the nucleotide lesions and excision of the damaged DNA base. The genome of Corynebacterium glutamicum ATCC 13032 contains various DNA glycosylases encoding genes (ung, fpg/mutM, tagI, alkA, mutY), two AP-endonuclease encoding genes (nei and nth) and an exonuclease encoding gene xth. To investigate the role of these genes during DNA repair in C. glutamicum, mutants with deletions of one or more genes in BER pathway were created. After treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C (MMC), zeocin and UV-light, we characterised the function of the different BER genes by determination of the survival capability. DNA lesions caused by MNNG strongly reduced survival of the tagI, mutY and alkA mutants but had a negligible effect on the ung and mutM mutants. The endonucleases Nth and Nei turned out to be essential for the repair of base modifications caused by MMC while UV-light and zeocin did not seem to address the BER. So far, BER in C. glutamicum appears to be very similar to that in E. coli.

Large-scale reduction of the Bacillus subtilis genome: consequences for the transcriptional network, resource allocation, and metabolism.

Understanding cellular life requires a comprehensive knowledge of the essential cellular functions, the components involved, and their interactions. Minimized genomes are an important tool to gain this knowledge. We have constructed strains of the model bacterium, Bacillus subtilis, whose genomes have been reduced by ∼36%. These strains are fully viable, and their growth rates in complex medium are comparable to those of wild type strains. An in-depth multi-omics analysis of the genome reduced strains revealed how the deletions affect the transcription regulatory network of the cell, translation resource allocation, and metabolism. A comparison of gene counts and resource allocation demonstrates drastic differences in the two parameters, with 50% of the genes using as little as 10% of translation capacity, whereas the 6% essential genes require 57% of the translation resources. Taken together, the results are a valuable resource on gene dispensability in B. subtilis, and they suggest the roads to further genome reduction to approach the final aim of a minimal cell in which all functions are understood.

Role of the ganSPQAB Operon in Degradation of Galactan by Bacillus subtilis.

Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-ß-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a ß-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain ß-1,4-galacto-oligosaccharides as well as synthetic ß-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the -35 box of the σ(A)-type promoter of Pgan, which is located upstream of ganS IMPORTANCE: Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis Nevertheless, the B. subtilis utilization system of galactan, which is found as the side chain of the rhamnogalacturonan type I complex in pectin, has remained partially studied. Here, we investigated the galactan utilization system consisting of the ganSPQAB operon and its regulator ganR This study improves our knowledge of the carbohydrate degradation systems of B. subtilis, especially the pectin degradation systems. Moreover, the galactan-degrading enzymes may be exploited for the production of galacto-oligosaccharides, which are used as prebiotic substances in the food industry.

Modification of linear (ß1→3)-linked gluco-oligosaccharides with a novel recombinant ß-glucosyltransferase (trans-ß-glucosidase) enzyme from Bradyrhizobium diazoefficiens.

Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (ß1→3) linkages preferably and to a lesser extent (ß1→6) or (ß1→4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (ß1→3)(ß1→6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic ß-glucans in B. diazoefficiens.

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System.

UNLABELLED: The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temperature-sensitive replication origin of plasmid pE194(ts) for B. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries the cas9 gene under the control of the B. subtilis mannose-inducible promoter PmanP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B. subtilis chromosome and by repair of the trpC2 mutation of B. subtilis 168. IMPORTANCE: In prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing of B. subtilis overcomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

A novel esterase subfamily with α/ß-hydrolase fold suggested by structures of two bacterial enzymes homologous to L-homoserine O-acetyl transferases.

MekB from Pseudomonas veronii and CgHle from Corynebacteriumglutamicum belong to the superfamily of α/ß-hydrolase fold proteins. Based on sequence comparisons, they are annotated as homoserine transacetylases in popular databases like UNIPROT, PFAM or ESTHER. However, experimentally, MekB and CgHle were shown to be esterases that hydrolyse preferentially acetic acid esters. We describe the x-ray structures of these enzymes solved to high resolution. The overall structures confirm the close relatedness to experimentally validated homoserine acetyl transferases, but simultaneously the structures exclude the ability of MekB and CgHle to bind homoserine and acetyl-CoA. Insofar the MekB and CgHle structures suggest dividing the homoserine transacetylase family into subfamilies, namely genuine acetyl transferases and acetyl esterases with MekB and CgHle as constituting members of the latter.

Identification and characterization of the vanillin dehydrogenase YfmT in Bacillus subtilis 3NA.

With vanillin as one of the most important flavoring agents, many efforts have been made to optimize its biotechnological production from natural abundant substrates. However, its toxicity against the hosts results in rather low yields and product concentrations. Bacillus subtilis as a soil-dwelling bacterium is a possible lignin-derived compound-degrading microorganism. Therefore, its vanillin and ferulic acid metabolism was investigated. With a rather high tolerance for vanillin up to 20 mM, it is a promising candidate to produce natural vanillin. In this study, the well-studied phenolic acid decarboxylases PadC and BsdBCD could be ascribed to function as the only enzymes in B. subtilis 3NA converting ferulic acid to 4-vinylguaiacol and vanillic acid to guaiacol, respectively. As vanillin also becomes converted to guaiacol, a previous conversion to vanillic acid was assumed. Usage of bioinformatic tools revealed YfmT, which could be shown to function as the only vanillin dehydrogenase in B. subtilis 3NA. Thus, YfmT was further characterized regarding its temperature and pH optima as well as its substrate range. Vanillin and ferulic acid metabolic routes in the tested B. subtilis strain were revealed, a direct conversion of ferulic acid to vanillin, however, could not be found.

Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27.

We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a ß-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27.

Development of an anhydrotetracycline-inducible expression system for expression of a neopullulanase in B. subtilis.

Bacillus subtilis is a widely used bacterium for production of heterologous and homologous proteins. The primary challenge in the production of proteins in B. subtilis is choosing a relevant expression system. In this study, we developed a robust expression system based on optimized PtetR of transposon Tn1721, which is repressible by its specific repressor, TetR. The first step of this work was focused on the optimization of structure and core elements of Tn1721 anhydrotetracycline-inducible promoters, PtetA and PtetR. Both promoters were inserted upstream of eGFP on a pUB110-derivative with high copy number. Reduction of the 18 bp spacer region of both PtetA and PtetR to 17 bp significantly increased their strength in B. subtilis. Nevertheless, only the optimized PtetR with 17 bp spacer region (PtetR2) directed high level of eGFP expression. In the second step, regulation of the system was optimized by testing the expression of tetR using well-known promoters, such as PmtlA, PmtlR, PptsG and PpenP. Expression of tetR by PptsG resulted in a tight regulation of PtetR2-eGFP showing 44-fold induction. By using the final expression plasmid in B. subtilis, neopullulanase was produced up to 15% of the total soluble protein.