Research advances in the identification of regulatory mechanisms of surfactin production by Bacillus: a review.

Surfactin is a cyclic hexalipopeptide compound, nonribosomal synthesized by representatives of the Bacillus subtilis species complex which includes B. subtilis group and its closely related species, such as B. subtilis subsp subtilis, B. subtilis subsp spizizenii, B. subtilis subsp inaquosorum, B. atrophaeus, B. amyloliquefaciens, B. velezensis (Steinke mSystems 6: e00057, 2021) It functions as a biosurfactant and signaling molecule and has antibacterial, antiviral, antitumor, and plant disease resistance properties. The Bacillus lipopeptides play an important role in agriculture, oil recovery, cosmetics, food processing and pharmaceuticals, but the natural yield of surfactin synthesized by Bacillus is low. This paper reviews the regulatory pathways and mechanisms that affect surfactin synthesis and release, highlighting the regulatory genes involved in the transcription of the srfAA-AD operon. The several ways to enhance surfactin production, such as governing expression of the genes involved in synthesis and regulation of surfactin synthesis and transport, removal of competitive pathways, optimization of media, and fermentation conditions were commented. This review will provide a theoretical platform for the systematic genetic modification of high-yielding strains of surfactin.

Iron ions regulate antifungal HSAF biosynthesis in Lysobacter enzymogenes by manipulating the DNA-binding affinity of the ferric uptake regulator (Fur).

Heat-stable antifungal factor (HSAF), produced by Lysobacter enzymogenes OH11, is regarded as a potential biological pesticide due to its broad-spectrum antifungal activity and novel mode of action. However, the current production of HSAF is low and cannot meet the requirements for large-scale production. Herein, we discovered that iron ions greatly promoted HSAF production, and the ferric uptake regulator (Fur) was involved in this regulatory process. Fur was also found to participate in the regulation of iron homeostasis in OH11 via the classic inhibition mechanism of Holo-Fur. Furthermore, Fur was collectively observed to directly bind to the promoter of the HSAF biosynthesis gene, and its DNA-binding affinity was attenuated by the addition of iron ions in vitro and in vivo. Its regulatory mechanism followed the uncommon inhibition mechanism of Apo-Fur. In summary, Fur exhibited a bidirectional regulatory mechanism in OH11. This study reveals a novel regulatory mechanism whereby Fur upregulates the biosynthesis of secondary metabolites. These findings contribute to the improvement of HSAF production and may guide its development into biological pesticides. IMPORTANCE HSAF possesses potent and broad antifungal activity with a novel mode of action. The HSAF yield is critical for fermentation production. In this study, iron ions were found to increase HSAF production, and the specific mechanism was elaborated. These results provide theoretical support for genetic transformation to improve HSAF yield, supporting its development into biological pesticides.

Evaluation of the Biocontrol Efficiency of Bacillus subtilis Wettable Powder on Pepper Root Rot Caused by Fusarium solani.

The plant-growth-promoting rhizobacteria (PGPR) B. subtilis PTS-394 has been utilized as a biocontrol agent (in a wettable powder form) due to its excellent ability to suppress tomato soil-borne diseases caused by Fusarium oxysporum and Ralstonia solanacearum. In this study, we evaluated the biocontrol efficiency of Bacillus subtilis PTS-394 wettable powder on pepper root rot in pot experiments and field trials. B. subtilis PTS-394 and its lipopeptide crude extract possessed excellent inhibition activity against Fusarium solani, causing pepper root rot; in an antifungal activity test B. subtilis PTS-394 wettable powder exhibited a good ability to promote pepper seed germination and plant height. The experiments in pots and the field indicated that B. subtilis PTS-394 wettable powder had an excellent control effect at 100-fold dilution, and its biocontrol efficacy reached 69.63% and 74.43%, respectively. In this study, the biocontrol properties of B. subtilis PTS-394 wettable powder on pepper root rot were evaluated and its application method was established. It was concluded that B. subtilis PTS-394 wettable powder is a potential biocontrol agent with an excellent efficiency against pepper root rot.

PafS Containing GGDEF-Domain Regulates Life Activities of Pseudomonas glycinae MS82.

Cyclic dimeric guanosine monophosphate (c-di-GMP) is synthesized by diguanylate cyclase (DGC) with the GGDEF domain. As a ubiquitous bacterial second messenger, it regulates diverse life-activity phenotypes in some bacteria. Although 38 genes encoding GGDEF-domain-containing proteins have been identified in the genome of the Pseudomonas glycinae strain MS82, whether c-di-GMP functions as a facilitator or repressor of life-activity phenotypes is poorly understood. In this study, one of the 38 genes containing a GGDEF domain in MS82, PafS was investigated to explore its regulatory function in bacterial life activities. The PafS-deletion mutant ΔPafS and reversion mutant PafS-comp were constructed by the method of biparental conjugation and homologous recombination. The life activities of the mutants, such as antifungal activity, biofilm formation ability, polysaccharide content, and motor behavior, were explored. The results showed that all life-activity phenotypes were significantly reduced after knocking out PafS, whereas all were significantly restored to a similar level to that of MS82 after the complementation of PafS. These results suggested that PafS plays an important role in the regulation of a range of cellular activities by c-di-GMP in P. glycinae MS82.

Genome-Wide Prediction and Analysis of Oryza Species NRP Genes in Rice Blast Resistance.

Members of the N-rich proteins (NRPs) gene family play important roles in the plant endoplasmic reticulum stress in response, which can be triggered by plant pathogens' infection. Previous studies of the NRP gene family have been limited to only a few plants, such as soybean and Arabidopsis thaliana. Thus, their evolutionary characteristics in the Oryza species and biological functions in rice defense against the pathogenic fungus Magnaporthe oryzae have remained unexplored. In the present study, we demonstrated that the NRP genes family may have originated in the early stages of plant evolution, and that they have been strongly conserved during the evolution of the Oryza species. Domain organization of NRPs was found to be highly conserved within but not between subgroups. OsNRP1, an NRP gene in the Oryza sativa japonica group, was specifically up-regulated during the early stages of rice-M. oryzae interactions-inhibited M. oryzae infection. Predicted protein-protein interaction networks and transcription-factor binding sites revealed a candidate interactor, bZIP50, which may be involved in OsNRP1-mediated rice resistance against M. oryzae infection. Taken together, our results established a basis for future studies of the NRP gene family and provided molecular insights into rice immune responses to M. oryzae.

Identification of Differentially Expressed Genes Reveal Conserved Mechanisms in the Rice-Magnaporthe oryzae Interaction.

Magnaporthe oryzae causes rice blast disease and is responsible for major losses in rice production worldwide. Although numerous studies have focused on the interactions between Oryza sativa and M. oryzae, to date, the conserved mechanisms remain in part unclear. In this study, a comparative analysis of transcriptomes of O. sativa L. ssp. japonica cv. 'Nipponbare' interacting with three M. oryzae strains (248, 235, and 163) were performed to explore the conserved molecular mechanisms. Differentially expressed genes with similar expression patterns in the interactions between cultivar 'Nipponbare' and three M. oryzae strains were defined as Conserved Differentially Expressed Genes (CDEGs). These included 3,647 O. sativa CDEGs and 3,655 M. oryzae CDEGs. Four rice CDEGs (LOC_Os03g19270, LOC_Os07g36600, LOC_Os05g28740, and LOC_Os01g32780) encoding universal stress protein (USP) were induced within 24 h post-inoculation (hpi) by three M. oryzae strains. Meanwhile, overexpression of LOC_Os07g36600 resulted in enhanced rice resistance against M. oryzae. Furthermore, four rice genes coding light-harvesting chlorophyll a/b-binding (LHC) protein (LOC_Os02g52650, LOC_Os09g12540, LOC_Os11g13850, LOC_Os05g22730) were also identified as CDEGs and were induced at 48 hpi, which might contribute to blast resistance through reactive oxygen species (ROS) accumulation. MoCDIP4 is M. oryzae effector inducing rice cell death and were verified that include AA9 CAZy domain (namely GH61 domain). In this study, we found seven MoCDIP4-homologous genes coding proteins with signal peptides and AA9 CAZy domains, which were continuously up-regulated across all infection stages relative to uninoculated control. This study uncovered that genes are required for conserved mechanisms of rice-M. oryzae interaction, which includes rice genes encoding USP proteins and LHC proteins, as well as M. oryzae genes encoding AA9 proteins. This study will help us to understand how O. sativa responds to M. oryzae infections and the molecular mechanisms of M. oryzae pathogenicity.

Tomato microbiome under long-term organic and conventional farming.

The compartment niche is the main reason behind the shifts in endophytic bacterial communities. Long-term organic greenhouse exerted limited influence on the variations of endophytic bacterial communities. Organic greenhouse and root had more complex co-occurrence networks than conventional greenhouse and stem, respectively. Cultivable method results found that Protecbacteria, Bacteriodes, and Actinobacteria are the dominant phyla in the endophytes.

Tomato Endophytic Bacteria Composition and Mechanism of Suppressiveness of Wilt Disease (Fusarium oxysporum).

Tomato wilt disease, caused by the Fusarium oxysporum is an ever-increasing threat for agricultural production, and unreasonable fertilization and pesticide abuse caused environmental challenge. Increasing evidence suggested that microbiomes or those associated with crops, played key roles on plant health. Plant disease dynamics were affected by multiple biotic and abiotic factors including phytopathogen population density, the genetic type of the pathogen and the host, in particular, the composition and assembly of the host-associated microbiome. However, it was unclear how pathogen invasion interaction and correlate with endophytic bacterial communities in natural field conditions. To study this, we sampled temporally the tomato plants that were exposed to F. oxysporum invasions over one crop season. High-throughput sequencing were performed to explore the correlation between agricultural practice, pathogen invasion, and endophytic microbiota communities. Results showed that pathogen invasion had clear effect on the endophytic and a strong link between increased pathogen densities and reduced abundance of Bacillus sp., which are crucial taxonomy for suppressiveness to F. oxysporum in vitro and in greenhouse condition. In summary, monitoring the dynamics of endophytic bacteria communities and densities of pathogen could thus open new avenue for more accurate disease diagnostics and high-efficiency screening antagonisms methods in the future, and our results will broaden the agricultural view of beneficial microbiota as biological control agents against plant pathogen.

Phenazine-1-carboxylic Acid Produced by Pseudomonas chlororaphis YL-1 Is Effective against Acidovorax citrulli.

The bacterial pathogen Acidovorax citrulli causes the destructive fruit blotch (BFB) on cucurbit plants. Pseudomonas chlororaphis YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors some antimicrobial-related gene clusters, such as phenazine, pyrrolnitrin, and pyoverdine. Here, we evaluated the antimicrobial activity of strain YL-1 as compared with its deficient mutants of antimicrobial-related genes, which were obtained using a sacB-based site-specific mutagenesis strategy. We found that only phenazine-deficient mutants ΔphzE and ΔphzF almost lost the inhibitory effects against A. citrulli in LB plates compared with the wild-type strain YL-1, and that the main antibacterial compound produced by strain YL-1 in LB medium was phenazine-1-carboxylic acid (PCA) based on the liquid chromatography-mass spectrometry (LC-MS) analysis. Gene expression analyses revealed that PCA enhanced the accumulation of reactive oxygen species (ROS) and increased the activity of catalase (CAT) in A. citrulli. The inhibition effect of PCA against A. citrulli was lowered by adding exogenous CAT. PCA significantly upregulated the transcript level of katB from 6 to 10 h, which encodes CAT that helps to protect the bacteria against oxidative stress. Collectively, the findings of this research suggest PCA is one of the key antimicrobial metabolites of bacterial strain YL-1, a promising biocontrol agent for disease management of BFB of cucurbit plants.

Pyricularia sp. jiangsuensis, a new cryptic rice panicle blast pathogen from rice fields in Jiangsu Province, China.

Pyricularia oryzae is a multi-host pathogen causing cereal disease, including the devastating rice blast. Panicle blast is a serious stage, leading to severe yield loss. Thirty-one isolates (average 4.1%) were collected from the rice panicle lesions at nine locations covering Jiangsu province from 2010 to 2017. These isolates were characterized as Pyricularia sp. jiangsuensis distinct from known Pyricularia species. The representative strain 18-2 can infect rice panicle, root and five kinds of grasses. Intriguingly, strain 18-2 can co-infect rice leaf with P. oryzae Guy11. The whole genome of P. sp. jiangsuensis 18-2 was sequenced. Nine effectors were distributed in translocation or inversion region, which may link to the rapid evolution of effectors. Twenty-one homologues of known blast-effectors were identified in strain 18-2, seven effectors including the homologues of SLP1, BAS2, BAS113, CDIP2/3, MoHEG16 and Avr-Pi54, were upregulated in the sample of inoculated panicle with strain 18-2 at 24 hpi compared with inoculation at 8 hpi. Our results provide evidences that P. sp. jiangsuensis represents an addition to the mycobiota of blast disease. This study advances our understanding of the pathogenicity of P. sp. jiangsuensis to hosts, which sheds new light on the adaptability in the co-evolution of pathogen and host.

The N-terminus of an Ustilaginoidea virens Ser-Thr-rich glycosylphosphatidylinositol-anchored protein elicits plant immunity as a MAMP.

Many pathogens infect hosts through specific organs, such as Ustilaginoidea virens, which infects rice panicles. Here, we show that a microbe-associated molecular pattern (MAMP), Ser-Thr-rich Glycosyl-phosphatidyl-inositol-anchored protein (SGP1) from U. virens, induces immune responses in rice leaves but not panicles. SGP1 is widely distributed among fungi and acts as a proteinaceous, thermostable elicitor of BAK1-dependent defense responses in N. benthamiana. Plants specifically recognize a 22 amino acid peptide (SGP1 N terminus peptide 22, SNP22) in its N-terminus that induces cell death, oxidative burst, and defense-related gene expression. Exposure to SNP22 enhances rice immunity signaling and resistance to infection by multiple fungal and bacterial pathogens. Interestingly, while SGP1 can activate immune responses in leaves, SGP1 is required for U. virens infection of rice panicles in vivo, showing it contributes to the virulence of a panicle adapted pathogen.

Pyoverdines Are Essential for the Antibacterial Activity of Pseudomonas chlororaphis YL-1 under Low-Iron Conditions.

Pseudomonas chlororaphis YL-1 has extensive antimicrobial activities against phytopathogens, and its genome harbors a pyoverdine (PVD) biosynthesis gene cluster. The alternative sigma factor PvdS in Pseudomonas aeruginosa PAO1 acts as a critical regulator in response to iron starvation. The assembly of the PVD backbone starts with peptide synthetase enzyme PvdL. PvdF catalyzes formylation of l-OH-Orn to produce l-N5-hydroxyornithine. Here, we describe the characterization of PVD production in YL-1 and its antimicrobial activity in comparison with that of its PVD-deficient ΔpvdS, ΔpvdF, and ΔpvdL mutants, which were obtained using a sacB-based site-specific mutagenesis strategy. Using in vitro methods, we examined the effect of exogenous iron under low-iron conditions and an iron-chelating agent under iron-sufficient conditions on PVD production, antibacterial activity, and the relative expression of the PVD transcription factor gene pvdS in YL-1. We found that strain YL-1, the ΔpvdF mutant, and the ΔpvdS(pUCP26-pvdS) complemented strain produced visible PVDs and demonstrated a wide range of inhibitory effects against Gram-negative and Gram-positive bacteria in vitro under low-iron conditions and that with the increase of iron, its PVD production and antibacterial activity were reduced. The antibacterial compounds produced by strain YL-1 under low-iron conditions were PVDs based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Moreover, the antibacterial activity observed in vitro was correlated with in vivo control efficacies of strain YL-1 against rice bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae. Collectively, PVDs are responsible for the antibacterial activities of strain YL-1 under both natural and induced low-iron conditions.IMPORTANCE The results demonstrated that PVDs are essential for the broad-spectrum antibacterial activities of strain YL-1 against both Gram-positive and Gram-negative bacteria under low-iron conditions. Our findings also highlight the effect of exogenous iron on the production of PVD and the importance of this bacterial product in bacterial interactions. As a biocontrol agent, PVDs can directly inhibit the proliferation of the tested bacteria in addition to participating in iron competition.

Addition of plant-growth-promoting Bacillus subtilis PTS-394 on tomato rhizosphere has no durable impact on composition of root microbiome.

BACKGROUND: Representatives of the genus Bacillus are increasingly used in agriculture to promote plant growth and to protect against plant pathogens. Unfortunately, hitherto the impact of Bacillus inoculants on the indigenous plant microbiota has been investigated exclusively for the species Bacillus amyloliquefaciens and was limited to prokaryotes, whilst eukaryotic member of this community, e.g. fungi, were not considered. RESULTS: The root-colonizing Bacillus subtilis PTS-394 supported growth of tomato plants and suppressed soil-borne diseases. Roche 454 pyrosequencing revealed that PTS-394 has only a transient impact on the microbiota community of the tomato rhizosphere. The impact on eukaryota could last up to 14 days, while that on bacterial communities lasted for 3 days only. CONCLUSIONS: Ecological adaptation and microbial community-preserving capacity are important criteria when assessing suitability of bio-inoculants for commercial development. As shown here, B. subtilis PTS-394 is acting as an environmentally compatible plant protective agent without permanent effects on rhizosphere microbial community.

A TonB-dependent receptor regulates antifungal HSAF biosynthesis in Lysobacter.

Lysobacter species are Gram-negative bacteria that are emerging as new sources of antibiotics, including HSAF (Heat Stable Antifungal Factor), which was identified from L. enzymogenes with a new mode of action. LesR, a LuxR solo, was recently shown to regulate the HSAF biosynthesis via an unidentified mechanism in L. enzymogenes OH11. Here, we used a comparative proteomic approach to identify the LesR targets and found that LesR influenced the expression of 33 proteins belonging to 10 functional groups, with 9 proteins belonging to the TBDR (TonB-Dependent Receptor) family. The fundamental role of bacterial TBDR in nutrient uptake motivates us to explore their potential regulation on HSAF biosynthesis which is also modulated by nutrient condition. Six out of 9 TBDR coding genes were individually in-frame deleted. Phenotypic and gene-expression assays showed that TBDR7, whose level was lower in a strain overexpressing lesR, was involved in regulating HSAF yield. TBDR7 was not involved in the growth, but played a vital role in transcribing the key HSAF biosynthetic gene. Taken together, the current lesR-based proteomic study provides the first report that TBDR7 plays a key role in regulating antibiotic (HSAF) biosynthesis, a function which has never been found for TBDRs in bacteria.

SecG is required for antibiotic activities of Pseudomonas sp. YL23 against Erwinia amylovora and Dickeya chrysanthemi.

Strain YL23 was isolated from soybean root tips and identified to be Pseudomonas sp. This strain showed broad-spectrum antibacterial activity against bacterial pathogens that are economically important in agriculture. To characterize the genes dedicated to antibacterial activities against microbial phytopathogens, a Tn5-mutation library of YL23 was constructed. Plate bioassays revealed that the mutant YL23-93 lost its antibacterial activities against Erwinia amylovora and Dickeya chrysanthemi as compared with its wild type strain. Genetic and sequencing analyses localized the transposon in a homolog of the secG gene in the mutant YL23-93. Constitutive expression plasmid pUCP26-secG was constructed and electroporated into the mutant YL23-93. Introduction of the plasmid pUCP26-secG restored antibacterial activities of the mutant YL23-93 to E. amylovora and D. chrysanthemi. As expected, empty plasmid pUCP26 could not complement the phenotype of the antibacterial activity in the mutant. Thus the secG gene, belonging to the Sec protein translocation system, is required for antibacterial activity of strain YL23 against E. amylovora and D. chrysanthemi.

Draft Genome Sequence of Pseudomonas chlororaphis YL-1, a Biocontrol Strain Suppressing Plant Microbial Pathogens.

Pseudomonas chlororaphis YL-1 was isolated from soybean root tips and showed a broad range of antagonistic activities to microbial plant pathogens. Here, we report the high-quality draft genome sequence of YL-1, which consists of a chromosome with an estimated size of 6.8 Mb with a G+C value of 63.09%.

Draft Genome Sequence of Root-Colonizing Bacterium Bacillus sp. Strain PTS-394.

Here, we report the high-quality draft genome sequence of Bacillus sp. strain PTS-394, isolated from the rhizosphere of tomatoes grown on Putuo Mountain (Xiamen, Fujian province, China), which exhibited excellent colonization ability on plant roots. The 4.0-Mb genome uncovered the mechanism for its potential root colonization ability and may provide novel insights into plant-bacterium interactions.

Three non-aspartate amino acid mutations in the ComA Response regulator receiver motif severely decrease surfactin production, competence development and spore formation in Bacillus subtilis.

Bacillus subtilis strains produce a broad spectrum of bioactive peptides. The lipopeptide surfactin belongs to one wellknown class, which includes amphiphilic membrane-active biosurfactants and peptide antibiotics. Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Bs-M49, obtained by means of low-energy ion implantation in wild-type Bs-916, produced significantly lower levels of surfactin, and had no obvious effects against R. solani. Occasionally, we found strain Bs- M49 decreased spore formation and the development of competence. Blast comparison of the sequences from Bs- 916 and M49 indicate that there is no difference in the srfA operon promoter PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild-type Bs-916 and mutant M49 strains were significantly different. When we integrated the comA ORF into the chromosome of M49 at the amyE locus, M49 restored hemolytic activity and antifungal activity. Then, HPLC analyses results also showed the comA-complemented strain had a similar ability to produce surfactin with wild-type strain Bs-916. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis. ComA protein 3D structure prediction and motif search prediction indicated that ComA has two obvious motifs common to response regulator proteins, which are the Nterminal response regulator receiver motif and the Cterminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural prediction results implicate that three mutated residues in the ComA protein may play an important role in the formation of a saltbridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.

[Construction and function analysis of Bac operon mutants of bio-control strain Bacillus subtilis Bs-916].

Bacillus subtilis Bs-916 is an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. We identified and analyzed the operon Bac in Bs-916 responsible for synthesis of iturin-like lipopeptides. The research plays an important part in genetic engineered Bs-916 for further improving its bio-control activity. Taking advantage of homologous recombination method, the one mutant obtained by replacement of the Bac original promoter by a constitutive promoter P (repU) and designated BGG104; the other mutant was obtained by disruption of the Bac promoter by insertion and designated BGG105. The biological activities results showed the mutant BGG104 enhanced antagonistic activities against several pathogenic fungi and also clearly increased in hemolytic activities. However, the mutant BGG105 decreased clearly in both antagonistic activities and hemolytic activities. Crude lipopeptides were extracted with methanol from precipitates, which were obtained by adding 6 mol/L HCl to the cell-free culture broth. The results of reversed-phase high-performance liquid chromatography (HPLC) analysis of crude lipopeptides showed lipopeptides produced by Bs-916 have a different retention time compared to iturinA. The mutant BGG104 produced up to 3-fold more lipopeptides than Bs-916. However, the mutant BGG105 has not been detected countpart lipopeptides production. The molecular weights of the lipopeptides synthesized by Bac determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) were 1007.7 Da, 1021.7 Da and 1035.7 Da. They were presumed to belong to homologues differed by a structure of --CH2 and their molecular weights were not the same as the other iturin-like liopeptides', such as iturinA, mycosubtilin and bacillomycin, molecular weights. In conclusion, this paper showed the lipopeptides synthesized by Bac play crucial part in Bs-916 antifungal activities and the lipopeptides overproduction were able to enhance Bs-916 bio-control activities.

[Sporulation, competence development and biopesticide activity of a Bacillus subtilis mutant].

UNLABELLED: Bacillus subtilis Bs-916 has obvious effects against Rhizoctonia solani. We demonstrated that the mutant strain, M49 obtained by means of low energy ion implantation in strain Bs-916, which produces significantly lower levels of surfactin, had no obvious effects against R. solani. OBJECTIVE: In order to identify the influencing factors of surfactin-decrease mutant strain M49, its phenotype and related gene expression levels were studied. METHODS: Strains to be tested for sporulation were grown for 24 h in sporulation medium. Plasmid pDG1728 (1 microg/mL) was used for DNA transformation to test competence development of M49 and Bs-916 strains. RT-PCR (Semi-quantitative reverse transcriptase-polymerase chain-reaction) was used to determine the expression levels of selected genes involved in competence and sporulation in both the wildtype Bs-916 and mutant M49 strains, such as comS, rapA, rapC gene. RESULTS: Our data showed that mutant strain M49 confers a leaky competence phenotype, typified by a ten-fold reduction. This indicated a fact that DNA fragments are more easily transformed to wildtype strain than M49 mutant. The M49 strain also appeared to exhibit a sporulation-deficient phenotype, compared with the wild-type Bs-916, its spore's number declined by about 75%. RT-PCR results showed that both the comS and rapC genes were expressed in the Bs-916 strain but not in the M49 strain. CONCLUSIONS: Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Blast comparison of the sequences from Bs-916 and M49 indicate that there is no difference in the srfA operon promoter, PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild type Bs-916 and mutant M49 strains is significantly different. When we integrated comA orf into the chromosome of M49 at amyE locus, M49 restored antifungal activity. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis.