Pleiotropic mutations affecting sporulation conditions and the syntheses of extracellular enzymes in Bacillus subtilis 168

Pleiotropic mutations of the chromosome of Bacillus subtilis 168 affecting simultaneously the levels of extracellular levansucrase and proteolytic activities are described. These mutations have been mapped at the sacU locus identified by PBS 1 mediated transduction. Several pleotropic hyperproducers and pleiotropic hypoproducers of these extracellular enzymatic activities, genotypically designated sacU^h and sacU⁻ respectively, have been isolated. sacU^h mutants are capable of sporulation in rich media or in mineral media containing amino acids in the presence of an excess of glucose in both cases; under these conditions the sporulation of the wild type strain 168 is inhibited. One pleiotropic mutation conferring hyperproduction of levansucrase and proteolytic activities was mapped at the sacQ locus distant from sacU.The sacU and sacQ mutants may be affected in a not yet identified regulation mechanism which controls simultaneously the production of several extracellular enzymatic activities and the sporulation conditions of B. subtilis 168.     

Distinct control sites located upstream from the levansucrase gene of Bacillus subtilis

The sacR regulatory region, which modulates the expression of sacB, the structural gene for levansucrase, was separated into two parts: an upstream region which carries a constitutive promoter and a downstream region which carries a palindromic structure. Three types of fusions were constructed in which the aphA3 gene coding for kanamycin resistance of Streptococcus faecalis was placed downstream from different deleted sacR regions. Other fusions were constructed by inserting a promoter from phage SPO1 upstream from the sacB gene and part of the sacA region. A third kind of fusion was constructed in which the palindromic structure was flanked by a heterologous promoter and a heterologous structural gene. After introduction of these fusions into the chromosomal DNA of mutants affected in sacB regulation, it was possible to reveal different targets for the regulatory genes sacU, sacQ and sacS: the sacU and sacQ genes act on a region located near or just upstream from the promoter, and the sacS gene, which is involved in the induction process, acts on the palindromic structure.     

Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis

Summary The sacU ^h , amyB and pap mutations are identical with respect to their pleiotropic phenotype and their genetic location. Strains bearing these mutations overproduce several exocellular enzymes: -amylase, lavansucrase and proteases, they are poorly or not at all transformable and most of them are devoid of flagella. These mutations are tightly linked to the sacU ^- mutations by transformation and therefore lie between the hisA1 and gtaB290 markers. It is possible that the sacU ^h , amyB and pap mutations on one hand and the sacU ^- mutations on the other are two different classes of alterations of the same regulatory gene controlling the synthesis of some exocellular enzymes and several other cellular functions. Furthermore an amy ^- mutation, leading to the lack of -amylase activity, was mapped between the lin2 and aroI906 markers which are not linked to the sacU locus.     

Cloning of the sacS gene encoding a positive regulator of the sucrose regulon in Bacillus subtilis

The sacS gene controls the expression of 2 saccharolytic enzymes in Bacillus subtilis (sucrase and levansucrase).This paper describes a recombinant plasmid containing a mutant allele, sacS^c. The plasmid was isolated from a B. subtilis DNA bank established in Escherichia coli. Moreover, it was shown that the sacS^c allele, placed on a high-copy plasmid, is dominant over the wild-type chromosomal sacS⁺ allele. This result strongly suggests that the sacS gene encodes a positive regulatory protein.     

The sacB and sacC genes encoding levansucrase and sucrase form a gene cluster in Zymomonas mobilis

Summary The Zymomonas mobilis gene sacB that encodes the extracellular levansucrase was cloned and expressed in Escherichia coli. The gene product exhibited both sucrose hydrolysis activity and levan forming capability. Sub-cellular fractionation of E. coli carrying pLSS41 revealed that about 95% of the total sucrase activity was detected in the cytoplasmic fraction. The levansucrase gene was overexpressed (about hundred fold) in E. coli under T7 polymerase expression system. Nucleotide sequence analysis of this gene revealed an open reading frame of 1269 bp long coding for a protein of 423 amino acids with a molecular mass of 46.7 KDa. The deduced amino acid sequence was identical to the N-terminal amino acids of protein A51 of Z. mobilis ZM4. Therefore, the product of sacB is levansucrase. This is the first extracellular enzyme of Z. mobilis sequenced which does not possess a signal sequence. This gene is located 198 bp upstream of sacC gene encoding for the extracellular sucrase forming a gene cluster     

Induction of saccharolytic enzymes by sucrose in Bacillus subtilis: evidence for two partially interchangeable regulatory pathways.

Sucrose induces two saccharolytic enzymes in Bacillus subtilis, an intracellular sucrase and an extracellular levansucrase, encoded by sacA and sacB, respectively. It was previously shown that the sacY gene encodes a positive regulator involved in a sucrose-dependent antitermination upstream from the sacB coding sequence. We show here that the sacY product is not absolutely required for sacB induction: a weak but significant induction can be observed in strains harboring a sacY deletion. The sacY-independent induction was altered by mutations located in the sacP and sacT loci but was observed in both sacU+ and sacU32 genetic backgrounds. These results suggest that B. subtilis has two alternative systems allowing sacB induction by sucrose. Both systems also seem to be involved in sacA induction.     

5'-noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis.

The regulation of the levansucrase gene sacB was studied in Bacillus subtilis strains. Fusions were constructed in which genes of cytoplasmic proteins such as lacZ were placed immediately downstream from sacR, the regulatory region located upstream from sacB. These fusions were introduced in mutants affected in sacB regulation. In all cases the marker gene was affected in the same way as sacB by the genetic context. This result is of particular interest for the sacU pleiotropic mutations, which affect sacB expression and other cellular functions such as the synthesis of several exocellular enzymes. We also showed that strains harboring sacU+ or sacU-hyperproducing alleles contained different amounts of sacB mRNA, which was proportional to their levansucrase secretion. We concluded that the sacU gene does not affect sacB expression at the level of secretion but acts on a target within sacR. We discuss the possibility that sacU acts on a part of sacR, a homologous copy of which was found upstream from the gene of another sacU-dependent secreted enzyme of B. subtilis, beta-glucanase.     

Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis

The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. the gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239 bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).     

Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors

The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene-replacement events into Mycobacterium smegmatis. A sucrose counter-selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrFKm) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one-step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic-exchange mutants. Two-step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.     

The regulation of urea amidolyase of Saccharomyces cerevisiae: mating type influence on a constitutivity mutation acting in cis.

Summary Constitutivity for the synthesis of the urea amidolyase bienzymatic complex is obtained by dur0^hmutations located in the regulatory genetic region adjacent to the dur1, dur2 gene cluster. The dur0^hmutations act only in cis and are a new case of cis effect strongly cancelled in /a diploid, similar to cargA ⁺0^hmutation shown previously to lead to arginase constitutivity. Illegitimate diploids do not show such a cancellation of constitutivity.The constitutivity produced by dur0^hmutation comprises the process of induction and the release of the glutamine effect. It does not impair the NH ₄ ⁺ effect.     

The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites

We present the sequence of a 2 kb fragment of the Bacillus subtilis Marburg genome containing sacB, the structural gene of levansucrase, a secreted enzyme inducible by sucrose. The peptide sequence deduced for the secreted enzyme is very similar to that directly determined by Delfour (1981) for levansucrase of the non-Marburg strain BS5. The peptide sequence is preceded by a 29 amino acid signal peptide. Codon usage in sacB is rather different from that in the sequenced genes of other secreted enzymes in B. subtilis, especially alpha-amylase. Genetic evidence has shown that the sacB promotor is rather far from the beginning of sacB (200 bp or more). The 200 bp region preceding sacB shows some of the features of an attenuator. A preliminary discussion of the putative workings and roles of this attenuator-like structure is proposed. sacRc mutations, which allow constitutive expression of levansucrase, have been located within the 450 bp upstream of sacB. It is shown that sacRc and sacR+ alleles control in cis the expression of the adjacent sacB gene.     

The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn 5501 and Tn 5502 , are cryptic transposons of the Tn 3 family

Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1. Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons). The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily. The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons. Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies. Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501. While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors. This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment. It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat). Transposition of phenol degradation genes could not be detected. Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon. Received: 21 July 1997 / Accepted: 7 July 1998     

Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli.

A clone bearing the structural gene sacB, coding for the exoenzyme levansucrase, was isolated from a library of Bacillus subtilis DNA that was cloned in phage lambda charon 4A on the basis of the transforming activity of the chimeric DNA. This lambda clone also was found to contain the sacR and smo loci. Subcloning the sacB-sacR region in plasmid pBR325 resulted in a clone which directed levansucrase synthesis in Escherichia coli. The nucleotide sequence coding for the secreted protein was localized on the physical map of the cloned DNA.     

Inducible Secretion of a Cellulase from Clostridium thermocellum in Bacillus subtilis

A host-vector system for inducible secretion during the logarithmic growth phase in Bacillus subtilis has been developed. The B. subtilis levansucrase gene promoter and the region encoding its signal sequence have been used. The endoglucanase A of Clostridium thermocellum was used as a model protein to test the efficiency of the system. Effective inducible secretion of the endoglucanase A was observed when either the levansucrase signal sequence or its own signal sequence was used. Expression of the endoglucanase A in different genetic backgrounds of B. subtilis showed that its regulation was similar to that of levansucrase, and high enzyme activity was recovered from the culture supernatant of a hyperproducing B. subtilis sacU(Hy) strain. The molecular weight of 46,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the secreted endoglucanase A is compatible with the calculated molecular weight of the mature polypeptide.     

Prevalence of germline mutations of hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in 75 French kindreds with nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is a syndrome characterized by familial predisposition to colorectal carcinoma and extracolonic cancers of the gastrointestinal, urological, and female reproductive tracts. This dominant disorder is caused by germline defects in one of at least five DNA mismatch repair (MMR) genes: hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 (GTBP). Germline mutations of hMSH2 and hMLH1 are also frequently identified in families not fulfilling all the Amsterdam criteria, thereby demonstrating that the involvement of these genes is not confined to typical HNPCC. To evaluate the respective involvement of the various MMR genes in typical and incomplete HNPCC syndromes, we have performed an analysis of the hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in a large series of French kindreds (n=75) with colorectal tumors and/or aggregation of extracolonic cancers belonging to the HNPCC spectrum. Mutational analysis has been performed in all families, without preselection for the tumor phenotype. We have detected 26 pathogenic germline mutations of the hMLH1 and hMSH2 genes and several novel variants of the hPMS1, hPMS2, and hMSH6 genes. Our data confirm that, regardless of the type of families and the tumor phenotype, hPMS1, hPMS2, and hMSH6 germline mutations are rare in familial aggregation of colorectal cancers. Furthermore, they suggest that the presence of multiple primary malignancies in a single individual and the observation of extracolonic tumors in relatives of a colorectal cancer patient should be included among the guidelines for referring patients for genetic testing. Electronic Publication