Characterization of the srfA locus of Bacillus subtilis: only the valine-activating domain of srfA is involved in the establishment of genetic competence

srfA is a locus required for the production of the lipopeptide antibiotic surfactin. This locus is also necessary for efficient sporulation and competence development. Mutations in the 5′ portion of the srfA operon affect all three of these processes, whereas mutations in the 3′ portion of srfA only affect sporulation and surfactin production. Analysis of the proteins encoded by the srfA locus revealed seven large domains which are likely to be responsible for the activation and binding of the seven amino acids of surfactin. Identification of the amino acid that is activated by the srfA domains was determined by amino acid-dependent pyrophosphate exchange reactions on partially purified cell extracts of strains carrying different srfA mutations. These results indicate colin-earity between the order of the domains in the srfA locus and the amino acid sequence of surfactin. The minimal genetic element of srfA required for the establishment of competence was shown to be the 5′ region of the second open reading of srfA, which encodes the valine activation domain. This portion of srfA, when cloned on a plasmid, complemented the competence deficiency of a srfA deletion mutant in trans.     

Genetic evidence for a role of thioesterase domains, integrated in or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in Bacillus subtilis

Next to almost all prokaryotic operons encoding peptide synthetases, which are involved in the nonribosomal synthesis of peptide antibiotics, distinct genes have been detected that encode proteins with strong sequence similarity to type II fatty acid thioesterases of vertebrate origin. Furthermore, sequence analysis of bacterial and fungal peptide synthetases has revealed a region at the C-terminal end of modules that are responsible for adding the last amino acid to the peptide antibiotics; that region also exhibits significant similarities to thioesterases. In order to investigate the function of these putative thioesterases in non-ribosomal peptide synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis, srfA fragments encoding the thioesterase domain of the surfactin synthetase 3 and the thioesterase-like protein SrfA-TE were deleted. This led to a 97 and 84% reduction of the in vivo surfactin production, respectively. In the double mutant, however, no surfaction production was detectable. These findings demonstrate for the first time that the C-terminal thioesterase domains and the SrfA-TE protein are directly involved in nonribosomal peptide biosynthesis. Received: 30 September 1997 / Accepted: 4 December 1997     

Cloning of <Emphasis Type="Italic">srfA</Emphasis> operon from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C9 and its expression in <Emphasis Type="Italic">E. coli</Emphasis>

The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound extracted from the E. coli transformant exhibited a different R _f value of 0.52 from B. subtilis C9 or authentic surfactin (R _f = 0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon.     

Rapid Detection and Characterization of Surfactin-Producing <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and Closely Related Species Based on PCR

The surfactin production genetic locus (sfp) is responsible for the ability of Bacillus subtilis to produce the lipopeptide biosurfactant, surfactin. This report demonstrates the utility of using PCR of the sfp gene as a means of identifying Bacillus species that produce surfactin. We carried out a hemolysis zone assay, quantitative HPLC and NMR in parallel to ensure that the PCR provided correct results. PCR analyses were performed for the sfp gene on 15 standard strains and 20 field-collected Bacillus spp. isolates native to Taiwan. Among the 15 standard strains, surfactin was produced by seven strains of B. subtilis and two closely related species, B. amyloliquefaciens B128 and B. circulans ATCC 4513. Of the 20 field-collected Bacillus spp. isolates; 16 strains yielded surfactin- positive results with PCR and HPLC. A good correlation was observed. Within the 16 field isolates, B. amyloliquefaciens S13 (452.5 mg/L) and B. subtilis S15 (125.6 mg/L) had high productivity of surfactin. The technique is valuable for finding out potential good yields of surfactin-producing strains. The PCR method we used could also be used to find different species or genera containing homologous genes. This is the first report of the detection of surfactin production by B. amyloliquefaciens and B. circulans based on PCR screening.     

Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8

Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1. A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8. When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced. However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides. Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame. The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B. subtilis), lpa-14 (from iturin A- and surfactin-producing B. subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin. The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides. Received: 14 July 1995 / Accepted: 22 December 1995     

Effect of pps disruption and constitutive expression of srfA on surfactin productivity,spreading and antagonistic properties of Bacillus subtilis 168 derivatives

Aims: To analyse the effects of plipastatin operon disruption and constitutive expression of surfactin operon in Bacillus subtilis 168 on surfactin productivity, in vitro invasive growth and antagonism against fungi. Methods and Results: The srfA native promoter was replaced by the constitutive promoter P_repU in B. subtilis 168 after integration of a functional sfp gene. Moreover, the plipastatin synthesis was further disrupted in the B. subtilis 168 derivatives. In liquid media, an earlier and higher expression of P_repU, than that found with P_srfA, led to a specific surfactin production fivefold higher after 6 h of culture. On solid media, not only the invasive growth and the haemolytic activity but also the antifungal activity of the constitutive strains were improved when compared to the parental strain BBG111. As expected, the disruption of the plipastatin operon strongly reduced in vitro antifungal properties but, interestingly, enhanced specific surfactin production (1·47 g g^?1 of biomass), spreading behaviour and haemolytic activity of the strains. Conclusions: This work demonstrates for the first time the interdependency of surfactin and plipastatin regarding their biosynthesis as well as their influence on the biological activities of the producing strain. Significance and Impact of the Study: The constitutive overproduction of surfactin enhances the invasive growth and the in vitro antagonistic activity of the mutant strain. Both properties are known to play an important role in the biocontrol of plant diseases. Plipastatin operon disruption increases the surfactin productivity of mutant strains. These mutants are interesting for use in continuous bioprocesses for surfactin production or in bioremediation.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Recent trends in the biochemistry of surfactin

The name surfactin refers to a bacterial cyclic lipopeptide, primarily renowned for its exceptional surfactant power since it lowers the surface tension of water from 72 mN m⁻¹ to 27 mN m⁻¹ at a concentration as low as 20 μM. Although surfactin was discovered about 30 years ago, there has been a revival of interest in this compound over the past decade, triggered by an increasing demand for effective biosurfactants for difficult contemporary ecological problems. This simple molecule also looks very promising as an antitumoral, antiviral and anti-Mycoplasma agent. Structural characteristics show the presence of a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a β-hydroxy fatty acid with 13–15 C atoms. In solution, the molecule exhibits a characteristic “horse saddle” conformation that accounts for its large spectrum of biological activity, making it very attractive for both industrial applications and academic studies. Surfactin biosynthesis is catalysed non-ribosomally by the action of a large multienzyme complex consisting of four modular building blocks, called the surfactin synthetase. The biosynthetic activity involves the multicarrier thiotemplate mechanism and the enzyme is organized in structural domains that place it in the family of peptide synthetases, a class of enzymes involved in peptidic secondary-metabolite synthesis. The srfA operon, the sfp gene encoding a 4′-phosphopantetheinyltransferase and the comA regulatory gene work together for surfactin biosynthesis, while the gene encoding the acyltransferase remains to be isolated. Concerning surfactin production, there is no indication whether the genetic regulation, involving a quorum-sensing mechanism, overrides other regulation factors promoted by the fermentation conditions. Knowledge of the modular arrangement of the peptide synthetases is of the utmost relevance to combinatorial biosynthetic approaches and has been successfully used at the gene level to modify the surfactin template. Biosynthetic and genetic rationales have been described for building variants. A fine study of the structure/function relationships associated with the three-dimensional structure has led to the recognition of the specific residues required for activity. These studies will assist researchers in the selection of molecules with improved and/or refined properties useful in oil and biomedical industries. Received: 9 October 1998 / Received revision: 29 January 1999 / Accepted: 31 January 1999     

Chromosomal integration of sfp gene in Bacillus subtilis to enhance bioavailability of hydrophobic liquids

Bacillus subtilis C9 effectively degrades aliphatic hydrocarbons up to a chain length of C₁₉ and produces a lipopeptide-type biosurfactant, surfactin, yet it has no genetic competency. Therefore, to obtain a transformable surfactin producer, the sfp gene cloned from B. subtilis C9 was integrated into the chromosome of B. subtilis 168, a non-surfactin producer, by homologous recombination. The transformants reduced the surface tension of the culture broth from 70.0 mN/m to 28.0 mN/m, plus the surface-active compound produced by the transformants exhibited the same R_f value as that from B. subtilis C9 and authentic surfactin in a thin-layer chromatographic analysis. The integration of the sfp gene into the chromosome of B. subtilis 168 was confirmed by Southern hybridization. Like B. subtilis C9, the transformants readily degraded n-hexadecane, although the original strain did not. It was also statistically confirmed that the hydrocarbon degradation of the transformants was highly correlated to their surfactin production by the determination of the correlation coefficient (r²=0.997, P<0.01). Therefore, these results indicate that the surfactin produced from B. subtilis enhances the bioavailability of hydrophobic liquids.     

Importance ofeps genes fromBacillus subtilis in biofilm formation and swarming

Unicellular organisms naturally form multicellular communities, differentiate into specialized cells, and synchronize their behaviour under certain conditions. Swarming, defined as a movement of a large mass of bacteria on solid surfaces, is recognized as a preliminary step in the formation of biofilms. The main aim of this work was to study the role of a group of genes involved in exopolysaccharide biosynthesis during pellicle formation and swarming inBacillus subtilis strain 168. To assess the role of particular proteins encoded by the group ofepsI-epsO genes that form theeps operon, we constructed a series of insertional mutants. The results obtained showed that mutations inepsJ-epsN, but not in the last gene of theeps operon (epsO), have a severe effect on pellicle formation under all tested conditions. Moreover, the inactivation of 5 out of the 6 genes analysed caused total inhibition of swarming in strain 168 (that does not produce surfactin) on LB medium. Following restoration of thesfp gene (required for production of surfactin, which is essential for swarming of the wild-type bacteria), thesfp ⁺ strains defective ineps genes (exceptepsO) generated significantly different patterns during swarming on synthetic B medium, as compared to the parental strain 168sfp ⁺.     

Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases

Many peptide antibiotics in prokaryotes and lower eukaryotes are produced non-ribosomally by multi-enzyme complexes. Analysis of gene-derived amino acid sequences of some peptide synthetases of bacterial and fungal origins revealed a high degree of conservation (35-50% identity). The genes encoding those peptide synthetases are clustered into large operons with repetitive domains (about 600 amino acids), in the case of synthetases activating more than one amino acid. We used two 35-mer oligonucleotides derived from two highly conserved regions of known peptide synthetases to identify the surfactin synthetase operon in Bacillus subtilis ATCC 21332, a strain not accessible to genetic manipulation. We show that the derived oligonucleotides can be used not only for the identification of unknown peptide synthetase genes by hybridization experiments but also in sequencing reactions as primers to identify internal domain sequences. Using this method, a 25.8-kb chromosomal DNA fragment bearing a part of the surfactin biosynthesis operon was cloned and partial sequences of two internal domains were obtained.     

Production of lipopeptides among <Emphasis Type="Italic">Bacillus</Emphasis> strains showing growth inhibition of phytopathogenic fungi

The biological activity and the presence of genes sfp and ituD (surfactin and iturin A) among Bacillus strains isolated from the Amazon basin were determined. Bacillus spp. were tested for hemolytic activity and inhibition of fungal growth by agar plate assays in parallel with PCR for identification of sfp and ituD genes. All strains tested produced surface-active compounds, giving evidence by lysis of erythrocytes and emulsifying activity on mineral oil and soybean oil. These strains of Bacillus caused growth inhibition of several phytopathogenic fungi, including Fusarium spp., Aspergillus spp., and Bipolaris sorokiniana. The presence of genes ituD and sfp was confirmed by PCR and sequence analysis. The only exception was Bacillus sp. P34 that lacks sfp gene. Lipopeptides were isolated from culture supernatants and analyzed by mass spectrometry. Characteristic m/z peaks for surfactin and iturin were observed, and some strains also produced fengycin and bacillomycin. The remarkable antifungal activity showed by the strains could be associated with the co-production of three or more lipopeptide antibiotics. Screening for novel bacteria producing useful biosurfactants or biocontrol agents for agriculture is a topic of greatest importance to eliminate chemical pollutants.     

Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution

Background  

Non-ribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a wide range of biologically active natural peptide compounds, of which many are pharmacologically important. Peptide bond formation is catalyzed by the Condensation (C) domain. Various functional subtypes of the C domain exist: An^LC_L domain catalyzes a peptide bond between two L-amino acids, a^DC_L domain links an L-amino acid to a growing peptide ending with a D-amino acid, a Starter C domain (first denominated and classified as a separate subtype here) acylates the first amino acid with a β -hydroxy-carboxylic acid (typically a β -hydroxyl fatty acid), and Heterocyclization (Cyc) domains catalyze both peptide bond formation and subsequent cyclization of cysteine, serine or threonine residues. The homologous Epimerization (E) domain flips the chirality of the last amino acid in the growing peptide; Dual E/C domains catalyze both epimerization and condensation.     

Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping

Analysis of the primary structure of peptide synthetases involved in the non-ribosomal synthesis of peptide antibiotics has revealed a highly conserved and ordered modular arrangement. A module contains at least two domains, involved in ATP-dependent substrate activation and thioester formation. The occurrence and arrangement of these functional building blocks is associated with the number and order of the amino acids incorporated in the peptide product. In this study, we present data on the targeted exchange of the leucine-activating module within the three-module surfactin synthetase 1 (SrfA-A) of Bacillus subtilis. This was achieved by engineering several hybrid srfA-A genes, which were introduced into the surfactin biosynthesis operon by in vivo recombination. We examined the hybrid genes for expression and investigated the enzymatic activities of the resulting recombinant peptide synthetases. For the first time, we demonstrate directly that an individual minimal module, of bacterial or fungal origin, confers its amino acid-specific activity on a multi-modular peptide synthetase. Furthermore, it is shown that directed incorporation of ornithine at the second position of the peptide chain induces a global alteration in the conformation of surfactin and may result in premature cyclization or a branched cyclic structure. Received: 10 July 1997 / Accepted: 11 September 1997     

Structures of domains I and IV from YbbR are representative of a widely distributed protein family

YbbR domains are widespread throughout Eubacteria and are expressed as monomeric units, linked in tandem repeats or cotranslated with other domains. Although the precise role of these domains remains undefined, the location of the multiple YbbR domain‐encoding ybbR gene in the Bacillus subtilis glmM operon and its previous identification as a substrate for a surfactin‐type phosphopantetheinyl transferase suggests a role in cell growth, division, and virulence. To further characterize the YbbR domains, structures of two of the four domains (I and IV) from the YbbR‐like protein of Desulfitobacterium hafniense Y51 were solved by solution nuclear magnetic resonance and X‐ray crystallography. The structures show the domains to have nearly identical topologies despite a low amino acid identity (23%). The topology is dominated by β‐strands, roughly following a “figure 8” pattern with some strands coiling around the domain perimeter and others crossing the center. A similar topology is found in the C‐terminal domain of two stress‐responsive bacterial ribosomal proteins, TL5 and L25. Based on these models, a structurally guided amino acid alignment identifies features of the YbbR domains that are not evident from naïve amino acid sequence alignments. A structurally conserved cis‐proline (cis‐Pro) residue was identified in both domains, though the local structure in the immediate vicinities surrounding this residue differed between the two models. The conservation and location of this cis‐Pro, plus anchoring Val residues, suggest this motif may be significant to protein function.     

Cloning and Sequence Analysis of cDNA for Diuretic Hormone Receptor from the Bombyx mori

The insect diuretic hormone (DH) binds to their receptor in malpighian tubules, and stimulates water secretion and cAMP synthesis. Complementary DNA encoding a diuretic hormone receptor was cloned from the malpighian tubules of Bombyx mori. The cloned cDNA encodes a protein consisting of 391 amino acid residues with the seven transmembrane domains. The receptor protein is homologous with that of other insects, and is structurally related to G-protein coupled receptors such as corticotropin relating factor (CRF), secretin, and vasoactive intestinal peptide.     

Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants

Summary 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the target of the herbicide glyphosate, catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis. We have cloned an EPSP synthase gene from Arabidopsis thaliana by hybridization with a petunia cDNA probe. The Arabidopsis gene is highly homologous to the petunia gene within the mature enzyme but is only 23% homologous in the chloroplast transit peptide portion. The Arabidopsis gene contains seven introns in exactly the same positions as those in the petunia gene. The introns are, however, significantly smaller in the Arabidopsis gene. This reduction accounts for the significantly smaller size of the gene as compared to the petunia gene. We have fused the gene to the cauliflower mosaic virus 35 S promoter and reintroduced the chimeric gene into Arabidopsis. The resultant overproduction of EPSPs leads to glyphosate tolerance in transformed callus and plants.     

A small gene, designated comS, located within the coding region of the fourth amino acid-activation domain of srfA, is required for competence development in Bacillus subtilis

The valine-activation domain-encoding portion of the srfA locus (srfA-d4) is not only involved in the non-ribosomal synthesis of surfactin, but is also required for the regulation of competence development. In this study we show that impairment of the adenylation activity of the valine-activating domain did not affect competence development. Deletion analysis and complementation studies delineated the competence-required portion of srfA-d4 to a 168bp fragment, which contains a small open reading frame (ORF), designated comS, encoding a polypeptide of 46 amino acids, embedded within, but translated in, a frame different from that of srfA-d4. Introduction of an amber mutation in the comS-coding frame prevented competence development, demonstrating the involvement of comS in this prokaryotic specialization process.     

Nucleotide sequence of psbC,the gene encoding the CP-43 chlorophyll a-binding protein of Photosystem II,in the cyanobacterium Synechocystis 6803

The nucleotide sequence for the Photosystem II gene psbC has been determined for the cyanobacterium Synechocystis 6803. The gene overlaps the last 50 bases of the psbD gene, and both genes are transcribed in the same direction, but read in different frames. This arrangement is identical to that found in all chloroplast genomes for which psbC has been sequenced. The Synechocystis nucleotide sequence is 70% homologous to the tobacco gene and the predicted amino acid sequence shows 85% homology. A possible alternative translation start site for psbC has been conserved between seven plant sequences and the cyanobacterial sequence. The hydropathy plot for the cyanobacterial protein is very similar to plots determined for six plant species. Pairs of histidines that may play a role in binding chlorophyll are conserved between the cyanobacterial and plant amino acid sequences.