Characterization of vB_SalS_PSa2, a T5-like Demerecviridae bacteriophage, and its potential use in food matrices.

This study characterizes a newly isolated Demerecviridae phage, named vB_SalS_PSa2, belonging to the phage T5 group. The main variations between vB_SalS_PSa2 and T5 concern structural proteins related to morphology and host recognition. vB_SalS_PSa2 is infective to 19 out of the 25 tested Salmonella enterica (including the rare "Sendai" and "Equine" serotypes) and Escherichia coli isolates, most of them being multidrug resistant. vB_SalS_PSa2 displayed good thermal stability (4-60 °C) and broad pH stability (4.0-12.0). It also exhibited antibacterial activity against S. enterica sv. Paratyphi A Enb50 at 4 °C in milk during the whole tested period (5 d), and for 3-6 h at both 25 and 37 °C. Furthermore, vB_SalS_PSa2 was able to inhibit biofilm formation and to show degradation activity on mature biofilms of E. coli K12 and S. enterica sv. Paratyphi Enb50 in both LB and milk. Altogether, these results indicate that phage vB_SalS_PSa2 is a valuable candidate for controlling foodborne S. enterica and E. coli pathogens.

Interspecies Horizontal Transfer and Specific Integration of the Mosquitocidal Toxin-Encoding Plasmid pTAND672-2 from Bacillus thuringiensis subsp. israelensis to Lysinibacillus sphaericus.

pTAND672-2, a 144-kb resident plasmid of Bacillus thuringiensis serovar israelensis strain TAND672, was sequenced and characterized. This extrachromosomal element carries mosquitocidal toxin-, conjugation-, and recombinase-encoding genes, together with a putative arbitrium system, a genetic module recently discovered in temperate phages controlling lysogeny-lysis transition and in mobile genetic elements (MGEs) where its function remains clarified. Using conjugation experiments, pTAND672-2 is shown to be a novel integrative and conjugative element (ICE), which can horizontally transfer from B. thuringiensis serovar israelensis to Lysinibacillus sphaericus, another mosquitocidal bacterium, where it integrates into the chromosome. Its integration and circularization are reversible and involve a single-cross recombination between 33-bp specific sites, attB in the chromosome of L. sphaericus and attP in pTAND672-2. CDS143, coding for the putative tyrosine integrase Int143 distantly related to site-specific tyrosine Xer recombinases and phage integrases, can mediate the integration of pTAND672-2 to attB. The B. thuringiensis mosquito-killing genes carried by pTAND672-2 are efficiently transcribed and expressed in L. sphaericus, displaying a slight increased toxicity in this bacterium against Aedes albopictus larvae. The occurrence of pTAND672-2-like plasmids within the Bacillus cereus group was also explored and indicated that they all share a similar genetic backbone with diverse plasmid sizes, ranging from 58 to 225 kb. Interestingly, among them, the pEFR-4-4 plasmid of Bacillus paranthracis EFR-4 and p5 of B. thuringiensis BT-59 also display conjugative capability; moreover, like pTAND672-2 displays a chimeric structure between the pCH_133-e- and pBtoxis-like plasmids, pBTHD789-3 also appears to be mosaic of two plasmids. IMPORTANCE Horizontal transfer of mobile genetic elements carrying mosquitocidal toxin genes may play a driving role in the diversity of mosquitocidal bacteria. Here, the 144-kb mosquitocidal toxin-encoding plasmid pTAND672-2 is the first verified integrative and conjugative element (ICE) identified in Bacillus thuringiensis serovar israelensis. The key tyrosine integrase Int143, involved in the specific integration, is distantly related to other tyrosine recombinases. The study also reports the occurrence and potential interspecies transmission of pTAND672-2-like plasmids with varied sizes in B. thuringiensis, Bacillus paranthracis, and Bacillus wiedmannii isolates belonging to the Bacillus cereus group. This study is important for further understanding the evolution and ecology of mosquitocidal bacteria, as well as for providing new direction for the genetic engineering of biopesticides in the control of disease-transmitting mosquitoes.

Influence of the Phagemid PfNC7401 on Cereulide-Producing Bacillus cereus NC7401.

A phagemid-cured strain, NC7401-∆Pf, was constructed to survey the biological function of the plasmidal prophage PfNC7401 in cereulide-producing Bacillus cereus NC7401. The transcriptome analysis between the mutant and the wild strains revealed a series of differentially expressed genes mainly involved in different function classifications, including the two-component signal transduction system, bacterial structure, transporters, related antibiotic response, purine biosynthesis, non-ribosomal peptide synthetases (NRPS) and related secondary metabolites, and aromatic or other amino acid synthesis. BIOLOG and phenotypic experiment analyses confirmed that PfNC7401 may affect phage immunity and the metabolism of several amino acids, including L-Alanine, which was suggested to be related to one precursor (D-Alanine) of cereulide synthesis. However, neither the transcription levels of the cereulide production-related genes (e.g., ilvB, cesA, cesB, and cesH) nor the cereulide production nor cell cytotoxicity were affected by the presence or absence of PfNC7401, corresponding with the transcriptome data, in which only four genes unrelated to cereulide synthesis on the plasmid-carrying ces gene cluster were affected by the curing of PfNC7401.

Characterization and heterologous expression of a novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from Lysinibacillus sphaericus.

This study aims to explore the potential aminopeptidases of Lysinibacillus sphaericus based on the unique metabolic characteristics of this species which cannot metabolize carbohydrates and may have a strong ability to metabolize amino acids. Fifteen peptidase-encoding genes predicted in L. sphaericus C3-41 have been heterologously expressed in Escherichia coli BL21, and of these genes, only Amp0279 shows a high ability to hydrolyze L-leucine-4-nitroanilide (Leu-pNA). Phylogenetic analysis, 3D-structure modeling, and enzyme assays indicated that Amp0279 should be a novel Co2+-dependent aminopeptidase belonging to the M29 family. The optimal conditions of Amp0279 were determined to be 50 °C and pH 8.0 with the addition of 100 µM Co2+, and under this condition, the specific activity of Amp0279 matched that of Flavourzyme® (3.54 × 104 vs. 3.37 × 104 U/mg for the protein ingredient of Flavourzyme®). Amp0279 is mainly expressed in the middle sporulation phase in wild-type L. sphaericus or in Bacillus subtilis under the control of the sporulation-dependent strong promoter pcry8E, which is carried by the recombinant vector pHT315-8E21b. Furthermore, the secretory expression systems based on B. subtilis and Corynebacterium glutamicum were used to enhance the soluble expression of Amp0279. Obvious expression and enzymatic activity were detected from the crude supernatant media of both host bacteria without further concentration and purification. Moreover, expression can occur in the vegetative phase in B. subtilis under the control of the Pgrac promoter. KEY POINTS: • A novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from L. sphaericus was characterized. • The activity of Amp0279 as a leucyl aminopeptidase matches that of Flavourzyme® under optimal conditions. • B. subtilis- and C. glutamicum-based expression systems are built to promote secretory (soluble) Amp0279 expression.

[Advances of phage receptor binding proteins].

Bacteriophages bind to the bacteria receptor through the receptor binding proteins (RBPs), a process that requires the involvement of complex atomic structures and conformational changes. In response to bacteriophage infection, bacteria have developed a variety of resistance mechanisms, while bacteriophages have also evolved multiple antagonistic mechanisms to escape host resistance. The exploration of the "adsorption-anti adsorption-escape process" between bacteriophages and bacteria helps us better understand the co-evolution process of bacteriophages and bacteria, which is important for the development of phage therapeutic technologies and phage-based biotechnologies. This review summarizes the bacteriophage adsorption related proteins, how bacteriophages escape host resistance based on the RBP alternations, and the recent progress of RBP-related biotechnologies.

Characterization of two newly isolated bacteriophages PW2 and PW4 and derived endolysins with lysis activity against Bacillus cereus group strains.

This study characterized two novel Siphoviridae phages, PW2 and PW4, that can infect 52% and 44% of the tested Bacillus cereus group isolates and display relatively high activity against four cereulide-producing isolates belonging to B. weihenstephanensis and B. paranthracis. The genome sequences of PW2 and PW4 are similar to six known phages infecting B. cereus group isolates, which can be classified into two conserved groups, with the PW2 genome harboring conserved coding sequences (CDSs) from both groups. Two phage-derived endolysins, LysPW2 and LysPW4, which are predicted to encode N-acetylmuramoyl-L-alanine amidase, and their enzymatically active domains (EADs), LysPW2-EAD and LysPW4-EAD, were heterologously expressed. Both LysPW2 and LysPW4, especially the former, show a much wider host range than the phages, albeit still limited to the B. cereus group for the tested bacteria. The optimal temperature and pH for LysPW2 ability is 37 °C and pH 8.0 and for LysPW4 is 50 °C and pH 9.0. Neither LysPW2-EAD nor LysPW4-EAD show any lytic activity against vegetative cells of the tested B. cereus group isolates but can inhibit germination in 66.3% and 65.7% of spores, respectively. In addition, both LysPW2-EAD and LysPW4-EAD exhibit spore-binding capabilities.

Horizontal transfer of large plasmid with type IV secretion system and mosquitocidal genomic island with excision and integration capabilities in Lysinibacillus sphaericus.

We identified a ~30-kb genomic island (named GI8) carrying the binary toxin gene operon binA/binB on both the chromosome and large pBsph plasmid in the mosquitocidal Lysinibacillus sphaericus C3-41 strain. We found that GI8 is related to the occurrence of binA/binB within L. sphaericus and displays excision and integration capability by recognizing the attB region, which consists of a 2-nt target site (AT) flanked by an 11-nt imperfect inverted repeat. pBsph and two pBsph-like plasmids (p2362 and p1593) were found to carry a type IV secretion system (T4SS) and displayed transmissibility within a narrow host range specific to L. sphaericus. GI8 can be co-transferred with pBsph as a composite element by integration into its attB site, then excised from pBsph and re-integrated into the chromosomal attB site in the new host. The potential hosts of GI8, regardless of whether they are toxic or non-toxic to mosquito larvae, share good collinearity at the chromosomal level. Data indicated that the appearance of the mosquitocidal L. sphaericus lineage was driven by horizontal transfer of the T4SS-type conjugative plasmid and GI8 with excision and specific integration capability.

Application of Bacillus thuringiensis strains with conjugal and mobilizing capability drives gene transmissibility within Bacillus cereus group populations in confined habitats.

BACKGROUND: Bacillus thuringiensis bacteria share similar genetic, physiological, and biochemical characteristics with other members of the Bacillus cereus group. Their diversity and entomopathogenic origin are related to their mobile genetic elements. However, the effects of wide-spread application of B. thuringiensis-based pesticides on genetically related B. cereus group populations present in the environment remain poorly understood. RESULTS: We first identified pBMB76 from B. thuringiensis tenebrionis as a new conjugative plasmid. Mixed mating experiments suggested that pBMB76 may compete with pHT73, another known conjugative plasmid. Applications of single (tenebrionis 4AA1 and kurstaki HD73 carrying pBMB76 and pHT73, respectively) and mixed (4AA1 + HD73) B. thuringiensis strains were performed in confined plot habitats (soil and leaf) over two planting seasons. In total, 684 B. cereus group isolates were randomly selected from different treatment sets, and the transmissibility and occurrence rate of potential conjugative plasmids were surveyed. Results showed that the percentage of isolates with plasmid mobility was markedly enhanced in the B. thuringiensis-sprayed groups. Furthermore, we performed multi-locus sequence typing (MLST) for a subset of 291 isolates, which indicated that the dominant sequence types in the treated habitats were identical or related to the corresponding sprayed formulations. CONCLUSIONS: The application of B. thuringiensis strains with conjugal and mobilizing capability drove gene transmissibility within the B. cereus group populations in confined habitats and potentially modified the population structure.

CesH Represses Cereulide Synthesis as an Alpha/Beta Fold Hydrolase in Bacillus cereus.

Cereulide is notorious as a heat-stable emetic toxin produced by Bacillus cereus and glucose is supposed to be an ingredient supporting its formation. This study showed that glucose addition benefited on cell growth and the early transcription of genes involved in substrate accumulation and toxin synthesis, but it played a negative role in the final production of cereulide. Meanwhile, a lasting enhancement of cesH transcription was observed with the addition of glucose. Moreover, the cereulide production in ΔcesH was obviously higher than that in the wild type. This indicates that CesH has a repression effect on cereulide production. Bioinformatics analysis revealed that CesH was an alpha/beta hydrolase that probably associated with the cell membrane, which was verified by subcellular localization. The esterase activity against para-nitrophenyl acetate (PNPC2) of the recombinant CesH was confirmed. Although no sign of ester bond cleavage in cereulide or valinomycin was demonstrated in in vitro assays, CesH could reverse the cereulide analogue sensitivity of Bacillus subtilis in vivo, by which toxin degradation was facilitated. Moreover, site directed mutations identified that the conserved catalytic triad of CesH might consist of Serine 86, Glutamate 199, and Histidine 227. These results help us to understand the regulation of cereulide production and provide clues for developing control measurements.

vB_LspM-01: a novel myovirus displaying pseudolysogeny in Lysinibacillus sphaericus C3-41.

Lysinibacillus sphaericus has great application potential not only in the biocontrol of mosquitoes but also in the bioremediation of toxic metals. Phages contribute to the genetic diversity and niche adaptation of bacteria, playing essential roles in their life cycle, but may also cause economic damage for industrially important bacteria through phage contamination during fermentation. In this study, the L. sphaericus phage vB_LspM-01, which belongs to the Myoviridae family, was isolated and characterized. Results showed that vB_LspM-01 could specifically infect most tested L. sphaericus isolates but was not active against isolates belonging to other species. Furthermore, phage-born endolysin exhibited a broader antimicrobial spectrum than the host range of the phage. The vB_LspM-01 genome had no obvious similarity with that of its host, and ca. 22.6% of putative ORFs could not get a match with the public databases. Phylogenic analysis based on the putative terminase large subunit showed high similarity with the phages identified with pac-type headful packaging. The vB_LspM-01 encoding genes were only detected in a tiny percentage of L. sphaericus C3-41 individual cells in the wild population, whereas they showed much higher frequency in the resistant population grown within the plaques; however, the phage genes could not be stably inherited during host cell division. Additionally, the vB_LspM-01 encoding genes were only detected in the host population during the logarithmic growth phase. The mitomycin C induction helped the propagation and lysogeny-lysis switch of vB_LspM-01. The study demonstrated that vB_LspM-01 can be present in a pseudolysogenic state in L. sphaericus C3-41 populations.

Identification and genomic comparison of temperate bacteriophages derived from emetic Bacillus cereus.

Cereulide-producing Bacillus cereus isolates can cause serious emetic (vomiting) syndrome and even acute lethality. As mobile genetic elements, the exploration of prophages derived from emetic B. cereus isolates will help in our understanding of the genetic diversity and evolution of these pathogens. In this study, five temperate phages derived from cereulide-producing B. cereus strains were induced, with four of them undergoing genomic sequencing. Sequencing revealed that they all belong to the Siphoviridae family, but presented in different forms in their hosts. PfNC7401 and PfIS075 have typical icosahedral heads, probably existing alone as phagemids in the host with self-replicating capability in the lysogenic state. PfEFR-4, PfEFR-5, and PfATCC7953 have elongated heads, with the genomes of the former two identified as linear dsDNA, which could be integrated into the host genome during the lysogenic state. Genomic comparison of the four phages with others also derived from emetic B. cereus isolates showed similar genome structures and core genes, thus displaying host spectrum specificity. In addition, phylogenic analysis based on the complete genome and conserved tail fiber proteins of 36 Bacillus species-derived phages confirmed that the phages derived from emetic B. cereus strains were highly similar. Furthermore, one endolysin LysPfEFR-4 was cloned and showed lytic activity against all tested emetic B. cereus strains and cross-lytic activity against some other pathogenic bacteria, implying a potential to control bacterial contamination in the food supply.

Characterization of three autolysins with activity against cereulide-producing Bacillus isolates in food matrices.

Bacillus cereus is a pathogen related with diarrhoeal or emetic food poisoning cases, of which the latter caused by the cereulide-producing isolates are more severe with several reported lethal cases. It is therefore necessary to develop an effective strategy to prevent the propagation of B. cereus in the food supply. In this study, three autolysins from the cereulide-producing B. cereus group isolates, LysIS075, LysF8819.1 and LysCER057, were identified and characterized. The results showed that the three autolysins were highly lytic and bactericidal to the tested cereulide-producing B. cereus group strains and cross-lytic against other tested B. cereus group strains, and they could inhibit the spore germination and propagation of their tested derived emetic strains. Physical and chemical characterization showed that all the three autolysins were alkalophilic with the optimal activity at pH9.0 or 9.5 with one exception of LysF8819.1 also having significant lytic activity at pH5.0, and they all had relative strong lytic activity at 37-50°C during the 30minute assay. However, LysCER057 showed relative susceptibility to thermo-condition. Remarkably, the separate or cock-tail addition of the three autolysins in food matrices (milk and rice porridge) showed effective bactericidal activity within the tested 2h. All the results revealed that the three autolysins might be potential candidates to control emetic B. cereus strains in different applications.