A method to isolate fragments of DNA that promote gene expression in Bacillus subtilis is described. The system is based on production ofcatechol 2,3-dioxygenase [CatO2ase; catechol:oxygen 2,3-oxidoreductase (decyclizing), EC 1. 13 Bacillus subtilis is an attractive alternative to Escherichia coli as a host for expression of cloned genes. The Gram-positive organism is nonpathogenic, free ofendotoxins, and an important producer of extracellular enzymes on a large industrial scale. Critical to the development ofthe microorganism as a host-vector system for recombinant DNA technology is the efficient expression of heterospecific genes. To express plasmid-borne genes in B. subtilis, transcriptional or translational signals that differ from those of E. coli are required (1). Plasmid vectors suitable for cloning fragments of DNA that carry transcriptional promoter or termination signals for Gram-negative bacteria into E. coli have been characterized (2-6). Detection in these systems is based on expression ofgenes that encode P-galactosidase (4-6) or confer antibiotic resistance to host cells (2, 3). An approach similar to the latter has been successful in B. subtilis using chloramphenicol acetyltransferase genes originating from Bacillus pumilus (7) or the transposable genetic element Tn9 (8). In the E. coli /-galactosidase system, selection ofDNA fragments that promote expression of the lacZ gene is based on an easily visualized color change ofbacterial colonies grown on indicator plates containing a chromogenic substrate (4). An analogous system that functions in B. subtilis would greatly facilitate the effort to decipher problems of heterospecific gene expression in Gram-positive bacteria.In this report, we present a method whereby fragments of DNA that promote expression of a foreign gene in B. subtilis are detected by a change ofcolor of bacterial colonies. The system is based on the cloning and expression, in B. subtilis, ofthe xylE gene, which originated from the TOL plasmid pWWO (9) of Pseudomonas putida mt-2. The assay is rapid and inexpensive, does not require special indicator plates but offers the advantages ofa genetic indicator test (10), and can be used for the development of efficient plasmid gene expression vectors. MATERIAL AND METHODSBacterial Strains and Plasmids. The B. subtilis strains used are derivatives of Marburg strain 168. Strains BZ2 cysB3 recE4 and TGB1 trpC2 recE4 spo331 were constructed by transformation (11). MI112 argl5 leuB thr5 r-mM recE4 was from T. Tanaka. Bacillus licheniformis 9945A and Bacillus pumilus BP1 were obtained from the Bacillus Genetic Stock Center (Ohio State University, Columbus). E. coli strain BZ18 was from W. Arber; C600 rj m' was from J. W. Little; Pseudomonas putida mt-2 was donated by K. Timmis. The bifunctional E. coli/B. subtilis plasmid pHV33 (12) was obtained from R. Dedonder. Plasmid DNA was prepared by an alkaline extraction procedure (13) or a cleared lysate method (14) followed by cesium chloride/ ethidium bromide density gradient centrifugati...

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BackgroundThe increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains.ResultsIn a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process.ConclusionsWe propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery.

Several variants of al-proteinase inhibitor (al-PJ) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) a,-Proteinase inhibitor (al-PI) is a member of the serine proteinase inhibitor (serpin) family [l -41. Inhibitors of this class play crucial roles in controlling proteolytic activities of major physiological importance, such as in coagulation, fibrinolysis and complement activation [5]. Several studies provided evidence that serpins are recognized by their target proteinases as substrates and that cleavage occurs between their P1 and PI' residues in the active site (positions 358 and 359 in al-PI) leading to a major conformational change in human al-P1 [6 -12al. More detailed information on how this conformational transition might take place was obtained from X-ray crystallographic analysis of the three-dimensional structure of al-PI cleaved at its active site by chymotrypsinogen. The most striking feature of this structure was that residues Met358 (Pl) and Ser359 (Pl') (see Fig. 1B) were separated by approximately 7 nm in the cleaved inhibitor [6]. It was hypothesized that insertion of residues 345 -358 of native a l -P1 into a pre-existing p-sheet in an antiparallel fashion was the key event of this conformational change. More recent studies on the structure of ovalbumin, a serpin with no inhibitory activity, and its cleaved form plakalbumin (see Fig. 1 A), which do not show this conformational change, confirmed this hypothesis and provided experimental evidence for the conformation of an uncleaved serpin [9, 131. In the follow- MATERIALS AND METHODS Production of a,-PI variantsOligonucleotide directed mutagenesis of the cDNA coding for [Met358 -+ Arg]al-PI with a deletion of its five N-termind amino acids was performed as previously described [15, 161. Expression of [Thr345 4 Arg, Met358 + ArgJal-Pi, [Met351 --$ Glu, Met358 -+Arg]a,-PI and [Met358 +Arg]al-PI was carried out in Escherichia coli (strain TGE 901) from plasmids pTG 7952, pTG 7955 and pTG 1943, respectively, under the control of the leftward promoter of phage 1 . in the presence of a temperature-sensitive repressor cl. For increased expression, a synthetic ribosome-binding site was employed [17]. Cultures were grown under selective conditions (200 mg/l ampicillin) at 30°C in a 20-1 fermenter (LSL Biolafitte, Saint-Germain en Laye, France) to an A600 of 10. Expression was induced by shifting the growth temperature to 42°C for 6 h. Isolation of the a,-PI variants was performed as previously described [18].

Genomic information about Clostridium tetani, the causative agent of the tetanus disease, is scarce. The genome of strain E88, a strain used in vaccine production, was sequenced about 10 years ago. One additional genome (strain 12124569) has recently been released. Here we report three new genomes of C. tetani and describe major differences among all five C. tetani genomes. They all harbor tetanus-toxin-encoding plasmids that contain highly conserved genes for TeNT (tetanus toxin), TetR (transcriptional regulator of TeNT) and ColT (collagenase), but substantially differ in other plasmid regions. The chromosomes share a large core genome that contains about 85% of all genes of a given chromosome. The non-core chromosome comprises mainly prophage-like genomic regions and genes encoding environmental interaction and defense functions (e.g. surface proteins, restriction-modification systems, toxin-antitoxin systems, CRISPR/Cas systems) and other fitness functions (e.g. transport systems, metabolic activities). This new genome information will help to assess the level of genome plasticity of the species C. tetani and provide the basis for detailed comparative studies.