CcpC, a novel regulator of the LysR family required for glucose repression of the citB gene in Bacillus subtilis.

Synergistic carbon catabolite repression of the Bacillus subtilis aconitase (citB) gene by glucose and a source of 2-ketoglutarate is dependent on DNA sequences located upstream of the gene. Mutations in a dyad symmetry element centered at position -66 and in a repeat of the downstream arm of the dyad symmetry at position -27 cause derepressed citB expression. In this work, a protein able to bind to a DNA fragment containing these elements was purified and identified. This protein, named CcpC (Catabolite control protein C), shares sequence similarity with members of the LysR family of transcriptional regulators. In addition to binding to the citB promoter, CcpC bound to the promoter of the citZ gene, which encodes the cell's major citrate synthase and is subject to carbon catabolite repression. In a ccpC null mutant, expression of both citB and citZ was derepressed in glucose-glutamine minimal medium, indicating that CcpC is a negative regulator of citB and citZ gene expression. DNase I footprinting experiments showed that CcpC binds to two sites within the citB promoter region, corresponding to the dyad symmetry and -27 elements. In the presence of citrate, a putative inducer, only the dyad symmetry element was fully protected by CcpC. When the dyad symmetry element was mutated, CcpC was no longer able to bind to either the dyad symmetry or -27 elements. Repression of citB and citZ gene expression during anaerobiosis also proved to be mediated by CcpC.

Adaptation of Bacillus subtilis to oxygen limitation.

Bacillus subtilis grows anaerobically by at least two different pathways, respiration using nitrate as an electron acceptor and fermentation in the absence of electron acceptors. Regulatory mechanisms have evolved allowing cells to shift to these metabolic capabilities in response to changes in oxygen availability. These include transcriptional activation of fnr upon oxygen limitation, a process requiring the ResD-ResE two-component signal transduction system that also regulates aerobic respiration. FNR then activates transcription of other anaerobically induced genes including the narGHJI operon which encodes a respiratory nitrate reductase. Genes involved in fermentative growth are controlled by an unidentified FNR-independent regulatory pathway.

Multiple effects induced by unstable mutation in Streptomyces lavendulae.

Arg mutants, isolated from Streptomyces lavendulae at unusually high frequencies, showed several phenotypic characteristics. The characteristics common to all arg mutants include: (1) repression of beta-lactamase production, (2) inhibition of aerial mycelium formation, (3) development of acid pH, (4) low saturation density of growth in liquid culture, (5) a decrease in antibiotic production, (6) an increase in sensitivity to benzylpenicillin and (7) a decrease in production of pigment. These results suggest that the arg mutation concomitantly caused the depression of secondary metabolism in S. lavendulae.

Expression of the kanamycin resistance gene in a kanamycin-producing strain of Streptomyces kanamyceticus.

The previously cloned kanamycin resistance gene (kmr) from Streptomyces kanamyceticus ISP5500 was shown to modify the 30S ribosomal subunit in a subunit exchange experiment. The kmr gene, which was normally repressed in S. kanamyceticus, appeared to be induced under growth conditions which activated kanamycin biosynthesis. S1 mapping analysis revealed that the expression of the kmr gene was regulated at the transcriptional level. Acetylation of kanamycin is another resistance mechanism in the kanamycin producer. However, unlike kmr-mediated resistance, the enzyme which catalyzed acetylation was not regulated coordinately with kanamycin biosynthesis.

Induction of beta-lactamase and penicillin-binding proteins in Escherichia coli by introduction of Streptomyces DNA.

Introduction of hybrid plasmids, which were constructed by ligation of pCR1 or pMN1 vector plasmid and SalI restriction endonuclease cleaved segments of Streptomyces cacaoi chromosome, resulted in the production of new beta-lactamase and penicillin-binding protein in Escherichia coli. The beta-lactamase and penicillin-binding protein were not from S. cacaoi but rather induced by the plasmids. Close relationship was observed between plasmids and penicillin-binding proteins but not with beta-lactamase.

Fusion of protoplasts of Streptomyces lavendulae.

Protoplasts of two different auxotrophic mutants of Streptomyces lavendulae were fused with polyethylene glycol (PEG) 1,000, and allowed to regenerate on selective media. Prototrophic colonies overwhelmed other types of recombinants on any selective media. These prototrophic strains were stable after successive isolation. These results suggest that stable diploid cells were formed by cell fusion, which differed from the case of S. coelicolor.

Pock-forming plasmids isolated from streptomyces roseochromogenus.

A series of high copy number plasmids designated pSRC were isolated from Streptomyces roseochromogenus S264. The pSRC series were found to be self-transmissible by conjugation and to elicit lethal zygosis (Ltz). Using the Ltz phenotype to detect plasmid transformants, the pSRC plasmids were shown to have a wide host range. Among them pSRC1 consisted of two different plasmids with the same molecular weight, pSRC1a and 1b. Information regarding restriction sites suitable for the insertion of DNA was obtained by cloning the thiostrepton resistance gene from pIJ702 into pSRC1b. The single Bgl II site of pSRC1b was nonessential for replication and pock-formation. The pSRC plasmids may be suitable as cloning vectors in Streptomyces.

Cloning of a gene from Streptomyces species complementing argG mutations.

A gene that complements argG mutations was cloned from Streptomyces coelicolor and Streptomyces lividans by using pIJ702 as a vector. The recombinant plasmid pMCP25 complemented argG mutations of S. lividans and S. coelicolor. The inserted DNA of pMCP25 was able to hybridize with argG+ strains of S. lividans, S. coelicolor, S. lavendulae and S. alboniger but not with argG mutants of these strains.

Analysis of the promoter region of the cloned kanamycin resistance gene (kmr) from Streptomyces kanamyceticus.

The appropriate location and orientation of the kanamycin resistance gene (kmr) cloned on multicopy plasmids were determined by subcloning experiments. The transcription start site was identified by high-resolution S1 mapping and the kmr mRNA was shown to have a long leader of about 400 bp. An additional transcript upstream of the kmr gene was also detected, which ran in the opposite direction and overlapped 2 to 3 nucleotides with the kmr transcript. The presumptive promoter region of this physiologically unidentified RNA was similar to the Escherichia coli promoter consensus sequence both in the -10 and -35 regions, whereas the putative promoter region of the kmr gene exhibited sequence similarities in the -10 region to the promoters of the endoglycosidase H (endoH) and the viomycin phosphotransferase (vph) genes from Streptomyces.

[Presence of larva migrans in sand boxes of public elementary schools, Araçatuba, Brazil]. / Ocorrência de larva migrans na areia de áreas de lazer das escolas municipais de ensino infantil, Araçatuba, SP, Brasil.

There are sandboxes in public elementary school playground areas in Brazil, which can be harmful to children. They are at risk of cutaneous and visceral larva migrans infection caused by Ancylostoma spp. and Toxocara spp., respectively. The study was designed to investigate contamination by Toxocara spp. and/or their eggs and Ancylostoma spp. larvae in sand samples collected from the schools' sandboxes. Five hundred and thirty-five sand samples from 28 public elementary schools were collected during summer and winter and analyzed by both Baerman's method and centrifugal flotation technique. Ancylostoma spp. larvae were found in 35.7% (10/28) schools in summer time and in 46.4% (13/28) schools in the winter time. Eggs of Toxocara spp. could not be recovered from the samples analyzed and eggs from Ancylostoma spp. were seen in 0.56% (3/535) of the samples.

Mutational analysis of the regulatory region of the srfA operon in Bacillus subtilis.

Transcription of the Bacillus subtilis srfA operon is dependent on the transcriptional activator ComA. Mutational analysis of the srfA regulatory region suggests that two regions of dyad symmetry upstream of the srfA promoter may function in transcriptional activation by facilitating a cooperative interaction between ComA dimers.

The primary role of comA in establishment of the competent state in Bacillus subtilis is to activate expression of srfA.

The establishment of genetic competence in Bacillus subtilis requires the genes of the competence regulon which function in the binding, processing, and transport of DNA. Their expression is governed by multiple regulatory pathways that are composed of the comA, comP, sin, abrB, spo0H, spo0K, spo0A, degU, and srfA gene products. Among these, srfA is thought to occupy an intermediate position in one of the pathways that controls late competence gene expression. The full expression of srfA requires the gene products of comP, comA, and spo0K. To determine the role of these genes in the regulation of competence development, the expression of the srfA operon was placed under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter Pspac and the expression of the Pspac-srfA construct was examined in mutants blocked in early competence. By monitoring the IPTG-induced expression of Pspac-srfA with a srfA-lacZ operon fusion, it was observed that srfA expression was no longer dependent on the products of comP, comA, and spo0K. Production of the lipopeptide antibiotic surfactin in Pspac-srfA-bearing cells was induced in the presence of IPTG and was independent of ComP and ComA. Competence development was induced by IPTG and was independent of comP, comA, and spo0K in cells carrying Pspac-srfA. These results suggest that the ComP-ComA signal transduction pathway as well as Spo0K is required for the expression of srfA in the regulatory cascade of competence development. Studies of Pspac-srfA also examined the involvement of srfA in the growth stage-specific and nutritional regulation of a late competence gene.

Cloning and characterization of srfB, a regulatory gene involved in surfactin production and competence in Bacillus subtilis.

A Tn917 insertion mutation srfB impairs the production of the lipopeptide antibiotic surfactin in Bacillus subtilis. srfB is located between aroG and ald in the B. subtilis genome, as determined by phage PBS1 transduction mapping, and is not linked to the previously described surfactin loci sfp or srfA. A srfB mutant was found to be also deficient in the establishment of competence. SP beta phage-mediated complementation analysis showed that both competence and surfactin production were restored in the srfB mutant by a single DNA fragment of 1.5 kilobase pairs. The sequence of the complementing DNA revealed that the srfB gene is comA, an early competence gene which codes for a product similar to that of the activator class of bacterial two-component regulatory systems. The srfB mutation impaired the expression of a srfA-lacZ fusion, suggesting that surfactin production is positively regulated at the transcriptional level by the srfB (comA) gene product.

Anaerobic growth of a "strict aerobe" (Bacillus subtilis).

There was a long-held belief that the gram-positive soil bacterium Bacillus subtilis is a strict aerobe. But recent studies have shown that B. subtilis will grow anaerobically, either by using nitrate or nitrite as a terminal electron acceptor, or by fermentation. How B. subtilis alters its metabolic activity according to the availability of oxygen and alternative electron acceptors is but one focus of study. A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, occupies an early stage in the regulatory pathway governing anaerobic respiration. One of the essential roles of ResD and ResE in anaerobic gene regulation is induction of fnr transcription upon oxygen limitation. FNR is a transcriptional activator for anaerobically induced genes, including those for respiratory nitrate reductase, narGHJI.B. subtilis has two distinct nitrate reductases, one for the assimilation of nitrate nitrogen and the other for nitrate respiration. In contrast, one nitrite reductase functions both in nitrite nitrogen assimilation and nitrite respiration. Unlike many anaerobes, which use pyruvate formate lyase, B. subtilis can carry out fermentation in the absence of external electron acceptors wherein pyruvate dehydrogenase is utilized to metabolize pyruvate.

Involvement of ResE phosphatase activity in down-regulation of ResD-controlled genes in Bacillus subtilis during aerobic growth.

The ResD-ResE signal transduction system is required for aerobic and anaerobic respiration in Bacillus subtilis. The histidine sensor kinase ResE, by functioning as a kinase and a phosphatase for the cognate response regulator ResD, controls the level of phosphorylated ResD. A high level of phosphorylated ResD is postulated to cause a dramatic increase in transcription of ResDE-controlled genes under anaerobic conditions. A mutant ResE, which retains autophosphorylation and ResD phosphorylation activities but is defective in ResD dephosphorylation, allowed partially derepressed aerobic expression of the ResDE-controlled genes. The result indicates that phosphatase activity of ResE is regulated by oxygen availability and anaerobic induction of the ResDE regulon is partly due to a reduction of the ResE phosphatase activity during anaerobiosis. That elimination of phosphatase activity does not result in complete aerobic derepression suggests that the ResE kinase activity is also subject to control in response to oxygen limitation.

Molecular biology of antibiotic production in Bacillus.

Several species of the genus Bacillus produce peptide antibiotics which are synthesized either through a ribosomal or non-ribosomal mechanism. The antibiotics gramicidin, tyrocidine, and bacitracin are synthesized nonribosomally by the multienzyme thiotemplate mechanism. Surfactin and mycobacillin are also synthesized nonribosomally but by a mechanism that, apparently, is distinct from that of the multienzyme thiotemplate. Other antibiotics such as subtilin are gene encoded and are ribosomally synthesized. Molecular genetic and DNA sequence analysis have shown that biosynthesis genes for some antibiotics are clustered into polycistronic transcription units and are under the control of global regulatory systems that govern the expression of genes that are induced when Bacillus cells enter stationary phase of growth. Future experiments involving the molecular dissection of peptide antibiotic biosynthesis genes in Bacillus will be attempted in hopes of further examining the mechanism and regulation of antibiotic production.

Nitrogen regulation of nasA and the nasB operon, which encode genes required for nitrate assimilation in Bacillus subtilis.

The divergently transcribed nasA gene and nasB operon are required for nitrate and nitrite assimilation in Bacillus subtilis. The beta-galactosidase activity of transcriptional lacZ fusions from the nasA and nasB promoters was high when cells were grown in minimal glucose medium containing poor nitrogen sources such as nitrate, proline, or glutamate. The expression was very low when ammonium or glutamine was used as the sole nitrogen source. The repression of the genes during growth on good sources of nitrogen required wild-type glutamine synthetase (GlnA), but not GlnR, the repressor of the glnRA operon. Primer extension analysis showed that the -10 region of each promoter resembles those of sigma A-recognized promoters. Between the divergently oriented nasA and nasB promoters is a region of dyad symmetry. Mutational analysis led to the conclusion that this sequence is required in cis for the activation of both nasA and nasB. The derepression of these genes in a glnA mutant also required this sequence. These results suggest that an unidentified transcriptional activator and glutamine synthetase function in the regulation of nasA and the nasB operon.

Characterization of the incompatibility region of Streptomyces plasmid pSL1.

The incompatibility region of the Streptomyces plasmid pSL1 was identified as a 240-bp segment, though some other function from the vector plasmid was also necessary. A 540-bp fragment including the 240-bp region was enough for full expression of incompatibility. Inserted mutation analysis led to a more detailed location of the region essential for replication.

Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis.

The sfp gene is required for cells of Bacillus subtilis to become producers of the lipopeptide antibiotic surfactin. sfp was isolated and its nucleotide sequence was determined. sfp was expressed in Escherichia coli and its putative product was purified for use in antibody production and in amino acid sequence analysis. The gene was plasmid-amplified in B. subtilis, where it conferred a Srf+ phenotype on sfp0 (surfactin non-producing) cells. Overproduction of Sfp in B. subtilis did not cause production of an increased amount of surfactin and resulted in the repression of a lacZ transcriptional fusion of the srfA operon, which encodes enzymes that catalyze surfactin synthesis. We propose that sfp represents an essential component of peptide synthesis systems and also plays a role, either directly or indirectly, in the regulation of surfactin biosynthesis gene expression.