Georges Rapoport

Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg;

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety‐two open reading frames were predicted to encode putative proteins among which only forty‐two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.

Antagonistic effects of dual PTS-catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR

LevR, which controls the expression of the lev operon of Bacillus subtilis, is a regulatory protein containing an N‐terminal domain similar to the NifA/NtrC transcriptional activator family and a C‐terminal domain similar to the regulatory part of bacterial anti‐terminators, such as BglG and LicT. Here, we demonstrate that the activity of LevR is regulated by two phosphoenolpyruvate (PEP)‐dependent phosphorylation reactions catalysed by the phosphotransferase system (PTS), a transport system for sugars, polyols and other sugar derivatives. The two general components of the PTS, enzyme I and HPr, and the two soluble, sugar‐specific proteins of the lev‐PTS, LevD and LevE, form a signal transduction chain allowing the PEP‐dependent phosphorylation of LevR, presumably at His‐869. This phosphorylation seems to inhibit LevR activity and probably regulates the induction of the lev operon. Mutants in which His‐869 of LevR has been replaced with a non‐phosphorylatable alanine residue exhibited constitutive expression from the lev promoter, as do levD or levE mutants. In contrast, PEP‐dependent phosphorylation of LevR in the presence of only the general components of the PTS, enzyme I and HPr, regulates LevR activity positively. This phosphorylation most probably occurs at His‐585. Mutants in which His‐585 has been replaced with an alanine had lost stimulation of LevR activity and PEP‐dependent phosphorylation by enzyme I and HPr. This second phosphorylation of LevR at His‐585 is presumed to play a role in carbon catabolite repression.

Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase

SummaryThe structural gene for the enzyme levanase of Bacillus subtilis (SacC) was cloned in Escherichia coli. The cloned gene was mapped by PBS1 transduction near the sacL locus on the B. subtilis chromosome, between leu4 and aroD. Expression of the enzyme was demonstrated both in B. subtilis and in E. coli. The presence of sacC allowed E. coli to grow on sucrose as the sole carbon source. The complete nucleotide sequence of sacC was determined. It includes an open reading frame of 2,031 bp, coding for a protein with calculated molecular weight of 75,866 Da, including a putative signal peptide similar to precursors of secreted proteins found in Bacilli. The apparent molecular weight of purified levanase is 73 kDa. The sacC gene product was characterized in an in vitro system and in a minicellproducing strain of E. coli, confirming the existence of a precursor form of levanase of about 75 kDa. Comparison of the predicted aminoacid sequence of levanase with those of the two other known β-D-fructofuranosidases of B. subtilis indicated a homology with sucrase, but not with levansucrase. A stronger homology was detected with the N-terminal region of yeast invertase, suggesting the existence of a common ancestor.

Transcriptional Regulation of the phoPR Operon in Bacillus subtilis

When Bacillus subtilis is subjected to phosphate starvation, the Pho regulon is activated by the PhoP-PhoR two-component signal transduction system to elicit specific responses to this nutrient limitation. The response regulator, PhoP, and its cognate histidine sensor kinase, PhoR, are encoded by the phoPR operon that is transcribed as a 2.7-kb bicistronic mRNA. The phoPR operon is transcribed from two sigma(A)-dependent promoters, P(1) and P(2). Under conditions where the Pho regulon was not induced (i.e., phosphate-replete conditions or phoR-null mutant), a low level of phoPR transcription was detected only from promoter P(1). During phosphate starvation-induced transition from exponential to stationary phase, the expression of the phoPR operon was up-regulated in a phosphorylated PhoP (PhoP approximately P)-dependent manner; in addition to P(1), the P(2) promoter becomes active. In vitro gel shift assays and DNase I footprinting experiments showed that both PhoP and PhoP approximately P could bind to the control region of the phoPR operon. The data indicate that while low-level constitutive expression of phoPR is required under phosphate-replete conditions for signal perception and transduction, autoinduction is required to provide sufficient PhoP approximately P to induce other members of the Pho regulon. The extent to which promoters P(1) and P(2) are activated appears to be influenced by the presence of other sigma factors, possibly the result of sigma factor competition. For example, phoPR is hyperinduced in a sigB mutant and, later in stationary phase, in sigH, sigF, and sigE mutants. The data point to a complex regulatory network in which other stress responses and post-exponential-phase processes influence the expression of phoPR and, thereby, the magnitude of the Pho regulon response.

Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis.

SummaryThe crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichiacoli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 230 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.

Mating between Bacillus subtilis and Bacillus thuringiensis and transfer of cloned crystal genes

SummaryWe took advantage of the recently discovered high frequency transfer of plasmids between strains of B. thuringiensis to obtain the heterospecific mating between a Bacillus subtilis strain, which contained the plasmid crystal gene from strain berliner 1715, and different B. thuringiensis strains. The plasmid-coded crystal gene was inserted in the plasmid pBT 42-1 and introduced into an acrystalliferous B. thuringiensis strain where it promoted the synthesis of the crystal protein. The plasmid was maintained stably and allowed the synthesis of a toxic parasporal body. The plasmid pBT 42-1 was also introduced into a wild-type strain israelensis and the transcipient strains produced both types of δ endotoxin, which are active on both lepidopteran and dipteran larvae. The transfers were also performed using the cloned crystal gene of chromosomal origin, but in this case, the gene was integrated into the chromosomal DNA of the transcipient strains and did not seem to be expressed.

Specificity of action on mosquito larvae of Bacillus thuringiensis israelensis toxins encoded by two different genes

SummaryA 135 kDa protein gene and two open reading frames (ORF1 and ORF2) have been cloned from a large plasmid of Bacillus thuringiensis israelensis (Bourgouin et al. 1986). The Escherichia coli recombinant clones containing these genes were highly toxic to larvae of Aedes aegypti, Anopheles stephensi and Culex pipiens. From subcloning experiments it was deduced that the 135 kDa polypeptide alone was responsible for the toxic activity on both A. aegypti and An. stephensi larvae. In contrast, the presence of two polypeptides, the 135 kDa protein and the ORF1 product was required for toxicity to C. pipiens larvae. The minimal toxic fragment of the 135 kDa polypeptide has been delineated. The results indicate that a polypeptide of about 65 kDa, corresponding to an amino-terminal part of the 135 kDa protein is sufficient for toxicity. Sequence comparisons indicate that the ORF1 product may correspond to an N-terminal part of a rearranged 130 kDa protein.

Nucleotide sequence of the sucrase gene of Bacillus subtilis

The sucrase gene (sacA) and part of the sacP locus, which corresponds to a membrane component of the phosphotransferase system (PTS) of sucrose transport of Bacillus subtilis, were previously cloned on a 2.1-kb EcoRI DNA fragment. Genes sacA and sacP were localized on this DNA fragment and the nucleotide sequence of the 2.1-kb DNA fragment was determined. A 1440-bp open reading frame (480 codons) was identified coding for a deduced polypeptide of Mr54827, which corresponds to that of purified sucrase. The amino acid sequence shares homology with that of yeast invertase (SUC2 gene product). The sacA gene and the preceding sacP gene seem to belong to the same operon.

Nucleotide sequence and characterization of a new insertion element, IS240, from Bacillus thuringiensis israelensis ☆

The nucleotide sequence of two repeated sequences (RS) in opposite orientations flanking the 125-kDa toxin gene of Bacillus thuringiensis israelensis (C. Bourgouin et al., J. Bacteriol. 170, 3575-3583, 1988) is reported in this paper. The analysis of these sequences indicates that these two RS display characteristic features of bacterial insertion sequences (IS) and are therefore referred to as IS240. IS240 B is 865 bp long and has two perfect terminal-inverted repeats of 16 bp; IS240 A is 99% identical to IS240 B. A long open reading frame encoding a polypeptide of 235 amino acids spans almost the entire sequence of both IS240 elements. Both the sequence of the inverted repeats and the putative transposases are homologous to IS26 of Proteus vulgaris, IS15-delta of Salmonella panama, IS431 of Staphylococcus aureus, and ISS1 of Streptococcus lactis.

The clpP multigenic family in Streptomyces lividans: conditional expression of the clpP3 clpP4 operon is controlled by PopR, a novel transcriptional activator

The clpP genes are widespread among living organisms and encode the proteolytic subunit of the Clp ATP‐dependent protease. These genes are present in a single copy in most eubacteria. However, five clpP genes were identified in Streptomyces coelicolor. The clpP1 clpP2 operon was studied: mutations affected the growth cycle in various Streptomyces. Here, we report studies of the expression of the clpP3 clpP4 operon in Streptomyces lividans. The clpP3 operon was induced in a clpP1 mutant strain, and the regulation of expression was investigated in detail. The product of the putative regulator gene, downstream from clpP4, was purified. Gel migration shift assays and DNase I footprinting showed that this protein binds to the clpP3 promoter and recognizes a tandem 6 bp palindromic repeat (TCTGCC‐3N‐GGCAGA). In vivo, this DNA‐binding protein, named PopR, acts as an activator of the clpP3 operon. Studies of popR expression indicate that the regulator is probably controlled at the post‐transcriptional level.