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.

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.

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.

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.

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.

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.

Transcriptional regulation of the vanillate utilization genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-like repressor.

Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo, ferulate, vanillin, and vanillate induced PvanABK in C. glutamicum, while only vanillate induced the activity of PvanABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the PvanABK DNA fragment and the VanR protein had an equilibrium dissociation constant (KD) of 15.1 ± 1.7 nM. The VanR-DNA complex had a dissociation rate constant (Kd) of (267 ± 23) × 10(-6) s(-1), with a half-life of 43.5 ± 3.6 min. DNase I footprinting localized the VanR binding site at PvanABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered PvanABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N4)TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator.

Development of a markerless gene deletion system for Bacillus subtilis based on the mannose phosphoenolpyruvate-dependent phosphotransferase system.

To optimize Bacillus subtilis as a production strain for proteins and low molecular substances by genome engineering, we developed a markerless gene deletion system. We took advantage of a general property of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular the mannose PTS. Mannose is phosphorylated during uptake by its specific transporter (ManP) to mannose 6-phosphate, which is further converted to fructose 6-phosphate by the mannose-6-phosphate isomerase (ManA). When ManA is missing, accumulation of the phosphorylated mannose inhibits cell growth. This system was constructed by deletion of manP and manA in B. subtilis Δ6, a 168 derivative strain with six large deletions of prophages and antibiotic biosynthesis genes. The manP gene was inserted into an Escherichia coli plasmid together with a spectinomycin resistance gene for selection in B. subtilis. To delete a specific region, its up- and downstream flanking sites (each of approximately 700 bp) were inserted into the vector. After transformation, integration of the plasmid into the chromosome of B. subtilis by single cross-over was selected by spectinomycin. In the second step, excision of the plasmid was selected by growth on mannose. Finally, excision and concomitant deletion of the target region were verified by colony PCR. In this way, all nine prophages, seven antibiotic biosynthesis gene clusters and two sigma factors for sporulation were deleted and the B. subtilis genome was reduced from 4215 to 3640 kb. Despite these extensive deletions, growth rate and cell morphology remained similar to the B. subtilis 168 parental strain.

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.

The Bacillus subtilis mannose regulator, ManR, a DNA-binding protein regulated by HPr and its cognate PTS transporter ManP.

The transcriptional activator ManR of the Bacillus subtilis mannose utilization operon is composed of an N-terminal DNA-binding domain, two phosphotransferase system (PTS) regulation domains (PRDs), an EIIB(Bgl) - and an EIIA(Fru) -like domain. Site-specific mutagenesis of ManR revealed the role of conserved amino acids representing potential phosphorylation sites. This was investigated by ß-galactosidase activity tests and by mobility shift assays after incubation with the PTS components HPr and EI. In analogy to other PRD-containing regulators we propose stimulation of ManR activity by phosphorylation. Mutations in PRD1 lowered ManR activity, whereas mutations in PRD2 abolished ManR activity completely. The Cys415Ala (EIIB(Bgl)) and the His570Ala mutations (EIIA(Fru)) provoked constitutive activities to different degrees, whereas the latter had the greater influence. Addition of EIIBA(Man) reduced the binding capability significantly in a wild-type and a Cys415Ala background, but had no effect on a His570Ala mutant. The different expression levels originating from the two promoters PmanR and PmanP could be ascribed to different 5'-untranslated mRNA regions. Sequences of 44 bp were identified and confirmed as the ManR binding sites by DNase I footprinting. The binding properties of ManR, in particular the equilibrium dissociation constant KD and the dissociation rate kdiss, were determined for both promoter regions.

Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants.

Expression of mannitol utilization genes in Bacillus subtilis is directed by PmtlA, the promoter of the mtlAFD operon, and PmtlR, the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIB(Gat)-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates PmtlA in vivo. In the mtlR-H342D C419A mutant, PmtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled PmtlA and PmtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of PmtlA-PlicB hybrid promoters, as well as increasing the distance between the MtlR operator and the -35 box of PmtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at PmtlA and PmtlR during transcription initiation.

Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid.

Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficient to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86% within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.

Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.

The Gram-positive bacterium Bacillus subtilis is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced B. subtilis strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced B. subtilis strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD+ and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.

The tyrosine recombinase MrpA and its target sequence: a mutational analysis of the recombination site mrpS resulting in a new left element/right element (LE/RE) deletion system.

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to be a site-specific tyrosine recombinase that acts with the 36-bp recombination site mrpS. The present report gives a comprehensive characterization of the composition as well as the position of the spacer and MrpA binding sites within mrpS. Experiments revealed a spacer consisting of 6 remarkably variable nucleotides in the middle of the mrpS-site. A reduction in the spacer to 5 nucleotides abolished recombination. Investigation of the MrpA binding sites showed the importance of its 15 nucleotides on an effective recombination. Among almost randomly exchangeable nucleotides, two nucleotides were identified as essential for MrpA binding. Alteration of either of these nucleotides led to a reduction in MrpA binding down to 2 % or even to no binding. Based on these results, a new left element/right element (LE/RE) deletion system was developed. The constructed heteromeric mrpS-sites are efficiently resolved by MrpA. The resulting double mutated (LE/RE) site can no longer be used as a recombination site by MrpA. The system has been successfully applied for the generation of multiple-targeted deletions in the genome of E. coli.

Functional characterization and application of a tightly regulated MekR/P mekA expression system in Escherichia coli and Pseudomonas putida.

A methyl ethyl ketone (MEK)-inducible system based on the broad-host-range plasmid pBBR1MCS2 and on the P mekA promoter region of the MEK degradation operon of Pseudomonas veronii MEK700 was characterized in Escherichia coli JM109 and Pseudomonas putida KT2440. For validation, ß-galactosidase (lacZ) was used as a reporter. The novel system, which is positively regulated by MekR, a member of the AraC/XylS family of regulators, was shown to be subject to carbon catabolite repression by glucose, which, however, could not be attributed to the single action of the global regulators Crc and PtsN. An advantage is its extremely tight regulation accompanied with three magnitudes of fold increase of gene expression after treatment with MEK. The transcriptional start site of P mekA was identified by primer extension, thereby revealing a potential stem-loop structure at the 5' end of the mRNA. Since MekR was highly insoluble, its putative binding site was identified through sequence analysis. The operator seems to be composed of a 15-bp tandem repeat (CACCN5CTTCAA) separated by a 6-bp spacer region, which resembles known binding patterns of other members of the AraC/XylS family. Subsequent mutational modifications of the putative operator region confirmed its importance for transcriptional activation. As the -35 promoter element seems to be overlapped by the putative operator, a class II activation mechanism is assumed.

A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites.

A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.