Effects of actin-like proteins encoded by two Bacillus pumilus phages on unstable lysogeny, revealed by genomic analysis.

We characterized two newly isolated myoviruses, Bp8p-C and Bp8p-T, infecting the ginger rhizome rot disease pathogen Bacillus pumilus GR8. The plaque of Bp8p-T exhibited a clear center with a turbid rim, suggesting that Bp8p-T could transform into latent phage. Lysogeny assays showed that both the two phages could form latent states, while Bp8p-T could form latent phage at a higher frequency and stability than Bp8p-C. The genomes of Bp8p-C and Bp8p-T were 151,417 and 151,419 bp, respectively; both encoded 212 putative proteins, and only differed by three nucleotides. Moreover, owing to this difference, Bp8p-C encoded a truncated, putative actin-like plasmid segregation protein Gp27-C. Functional analysis of protein Gp27 showed that Gp27-T encoded by Bp8p-T exhibited higher ATPase activity and assembly ability than Gp27-C. The results indicate that the difference in Gp27 affected the phage lysogenic ability. Structural proteome analysis of Bp8p-C virion resulted in the identification of 14 structural proteins, among which a pectin lyase-like protein, a putative poly-gamma-glutamate hydrolase, and three proteins with unknown function, were firstly identified as components of the phage virion. Both phages exhibited specific lytic ability to the host strain GR8. Bp8p-C showed better control effect on the pathogen in ginger rhizome slices than Bp8p-T, suggesting that Bp8p-C has a potential application in bio-control of ginger rhizome rot disease.

Genomic characteristics and comparative genomics analysis of Penicillium chrysogenum KF-25.

BACKGROUND: Penicillium chrysogenum has been used in producing penicillin and derived ß-lactam antibiotics for many years. Although the genome of the mutant strain P. chrysogenum Wisconsin 54-1255 has already been sequenced, the versatility and genetic diversity of this species still needs to be intensively studied. In this study, the genome of the wild-type P. chrysogenum strain KF-25, which has high activity against Ustilaginoidea virens, was sequenced and characterized. RESULTS: The genome of KF-25 was about 29.9 Mb in size and contained 9,804 putative open reading frames (orfs). Thirteen genes were predicted to encode two-component system proteins, of which six were putatively involved in osmolarity adaption. There were 33 putative secondary metabolism pathways and numerous genes that were essential in metabolite biosynthesis. Several P. chrysogenum virus untranslated region sequences were found in the KF-25 genome, suggesting that there might be a relationship between the virus and P. chrysogenum in evolution. Comparative genome analysis showed that the genomes of KF-25 and Wisconsin 54-1255 were highly similar, except that KF-25 was 2.3 Mb smaller. Three hundred and fifty-five KF-25 specific genes were found and the biological functions of the proteins encoded by these genes were mainly unknown (232, representing 65%), except for some orfs encoding proteins with predicted functions in transport, metabolism, and signal transduction. Numerous KF-25-specific genes were found to be associated with the pathogenicity and virulence of the strains, which were identical to those of wild-type P. chrysogenum NRRL 1951. CONCLUSION: Genome sequencing and comparative analysis are helpful in further understanding the biology, evolution, and environment adaption of P. chrysogenum, and provide a new tool for identifying further functional metabolites.

Genomic analysis of a phage and prophage from a Bacillus thuringiensis strain.

Bacteriophages have been found to be the most abundant and also potentially most diverse biological entities on Earth. In the present study, Bacillus phages were isolated rapidly and shown to have a high degree of diversity. The genomes of a newly isolated phage, phiCM3, and a prophage, proCM3, from the Bacillus thuringiensis strain YM-03 were sequenced and characterized. Comparative genome analysis showed that the phiCM3 genome is highly similar to the genomes of eight other Bacillus phages and seven of these phages were classified as the Wß group of phages. Analysis of the differential evolution of the genes in the Wß-group phages indicated that the genes encoding the antirepressor and tail fibre protein were more highly conserved than those encoding the major capsid protein, DNA replication protein, and RNA polymerase σ factor, which might have diverged to acquire mechanisms suitable for survival in different microbial hosts. Genome analysis of proCM3 revealed that proCM3 might be a defective phage because of mutations in the minor structural protein, and it was not inducible by mitomycin C treatment. The proCM3 genome was similar to those of two lytic Bacillus phages in sequence, but had a different genomic structure, composed of three regions in a different order. These data suggest that the three phages might have had a common ancestor and that genome rearrangement might have occurred during evolution. The findings of this study enrich our current knowledge of Bacillus phage diversity and evolution, especially for the Wß-group and TP21-L-like phages, and may help the development of practical applications of Bacillus phages.

Chromate reduction by a chromate-resistant bacterium, Microbacterium sp.

Heavy-metal chromium [Cr(VI)] is a ubiquitous environmental pollutant. Comparing with chemical reduction, microbiological reduction is considered to be a friendly and cheaper way to decrease the damage caused by chromate. A bacterial strain, CR-07, which is resistant to and capable of reducing chromate was isolated from a mud sample of iron ore and identified as a Microbacterium sp. The bacterium had a high degree of tolerance to chromate, and could grow in LB medium containing 4.08 mM of K(2)Cr(2)O(7). It also had a degree of resistance to other heavy metals, e.g. Cd(2+), Pb(2+), Zn(2+), Cu(2+), Co(2+), Hg(2+) and Ag(+). The bacterium could remove 1.02 mM of Cr(VI) from LB medium within 36 h of incubation. Chromate removal was achieved in the supernatant from the bacterial cultures, and corresponded to chromate reduction. The activity of chromate reduction by the bacterium was not related to enzymes or reducing sugars, while fluorometric assay suggested that glutathione, a chromate-reducing substance which was produced by the bacterium, was one of the factors that contributed to the reduction of Cr(VI).

Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis.

A novel δ-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The δ-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 α-helices (domain I); 213 residues forming three antiparallel ß-sheets (domain II); and 134 residues forming a ß-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC50s) were 8.98 µg/ml for the expressed Cry7Ca1, 0.87 µg/ml for the activated toxin 1, and 4.43 µg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.

Isolation and phosphate-solubilizing ability of a fungus, Penicillium sp. from soil of an alum mine.

The use of microorganisms to solubilize elemental phosphorus from insoluble rock phosphate is a promising method to greatly reduce not only environmental pollution but also production costs. Phosphate-solubilizing microorganisms were isolated from soils in China, and a fungus strain (PSM11-5) from a soil sample from an alum mine, with the highest phosphate solubilization potential, was selected and identified as a Penicillium sp. Strain PSM11-5 could grow in buffered medium with pH values between 3.0 and 8.0 and showed phosphate solubilizing activity at pH values from 5.0 to 8.0. It also exhibited a degree of tolerance to the heavy metal ions, Cd(2+), Co(2+), and Cr(6+). PSM11-5 could rapidly solubilize tricalcium phosphate, and a high phosphate-solubilizing efficiency of 98% was achieved in an optimized medium. The strain could solubilize rock phosphate and aluminum phosphate with a solubilizing efficiency of approximately 74.5%, but did not solubilize iron phosphate. Solubilization of phosphate depended on acidification. Analysis of PSM11-5 culture supernatants by capillary electrophoresis showed that tricalcium phosphate was solubilized to PO(4) (3-) and Ca(2+) , and that the organic acid produced by the fungus was mainly gluconic acid at approximately ca. 13 g l(-1). In addition, PSM11-5 produced ca. 830 mg l(-1) of citric acid when it was used to solubilize rock phosphate. These excellent properties of strain PSM11-5 suggest that the fungus has potential for agricultural and industrial utilization.

Comparative transcriptomics characterized the distinct biosynthetic abilities of terpenoid and paeoniflorin biosynthesis in herbaceous peony strains.

The herbaceous peony (Paeonia lactiflora Pall.) is a perennial flowering plant of the Paeoniaceae species that is widely cultivated for medical and ornamental uses. The monoterpene glucoside paeoniflorin and its derivatives are the active compounds of the P. lactiflora roots. However, the gene regulation pathways associated with monoterpene and paeoniflorin biosynthesis in P. lactiflora are still unclear. Here, we selected three genotypes of P. lactiflora with distinct morphologic features and chemical compositions that were a result of long-term reproductive isolation. We performed an RNA-sequencing experiment to profile the transcriptome changes of the shoots and roots. Using de novo assembly analysis, we identified 36,264 unigenes, including 521 genes responsible for encoding transcription factors. We also identified 28,925 unigenes that were differentially expressed in different organs and/or genotypes. Pathway enrichment analysis showed that the P. lactiflora unigenes were significantly overrepresented in several secondary metabolite biosynthesis pathways. We identified and profiled 33 genes responsible for encoding the enzymescontrolling the major catalytic reactions in the terpenoid backbone and in monoterpenoid biosynthesis. Our study identified the candidate genes in the terpenoid biosynthesis pathways, providing useful information for metabolic engineering of P. lactiflora intended for pharmaceutical uses and facilitating the development of strategies to improve marker-assist P. lactiflora in the future.

Proteomic profiling uncovered the cytosolic superoxide dismutase BsSOD1 associated with plant defence in the herbal orchid Bletilla striata.

The herbal orchid Bletilla striata (Thunb.) Rchb.f. has a long cultivation history and has been widely used in medicines and cosmetics. The fungal infection leaf blight (LB) seriously threatens B. striata cultivation. Here, we systemically collected wild B. striata accessions and isolated the accessions with strong resistance against LB. We carried out proteomic profiling analysis of LB-resistant and LB-susceptible accessions, and identified a large number of differentially expressed proteins with significant gene ontology enrichment for 'oxidoreductase activity.' Of the proteins identified in the reactive oxygen species signalling pathway, the protein abundance of the Cu-Zn superoxide dismutase BsSOD1 and its gene expression level were higher in LB-resistant accessions than in LB-susceptible lines. Transient expression of the dismutase fused with yellow fluorescent protein determined that its subcellular localisation is in the cytoplasm. Our study provides new insights into the molecular markers associated with fungal infection in B. striata.

Characteristics and optimised fermentation of a novel magnetotactic bacterium, Magnetospirillum sp. ME-1.

Magnetotactic bacteria (MTB) can biosynthesise magnetosomes, which have great potential for commercial applications. A new MTB strain, Magnetospirillum sp. ME-1, was isolated and cultivated from freshwater sediments of East Lake (Wuhan, China) using the limiting dilution method. ME-1 had a chain of 17 ± 4 magnetosomes in the form of cubooctahedral crystals with a shape factor of 0.89. ME-1 was closest to Magnetospirillum sp. XM-1 according to 16S rRNA gene sequence similarity. Compared with XM-1, ME-1 possessed an additional copy of mamPA and a larger mamO in magnetosome-specific genes. ME-1 had an intact citric acid cycle, and complete pathway models of ammonium assimilation and dissimilatory nitrate reduction. Potential carbon and nitrogen sources in these pathways were confirmed to be used in ME-1. Adipate was determined to be used in the fermentation medium as a new kind of dicarboxylic acid. The optimised fermentation medium was determined by orthogonal tests. The large-scale production of magnetosomes was achieved and the magnetosome yield (wet weight) reached 120 mg L-1 by fed-batch cultivation of ME-1 at 49 h in a 10-L fermenter with the optimised fermentation medium. This study may provide insights into the isolation and cultivation of other new MTB strains and the production of magnetosomes.

Complete Genome Sequence of Magnetospirillum sp. ME-1, a Novel Magnetotactic Bacterium Isolated from East Lake, Wuhan, China.

A novel spiral magnetotactic bacterium, Magnetospirillum sp. ME-1, was isolated from East Lake in China. Here we report the complete genome of ME-1, which contains a 4,551,873-bp circular chromosome and a 5,222-bp circular plasmid. The magnetosome biogenesis-specific genes are located in a 97,664-bp magnetosome genomic island.

Cellular responses in Bacillus thuringiensis CS33 during bacteriophage BtCS33 infection.

Bacillus thuringiensis (Bt) has been widely used for 50years as a biopesticide for controlling insect pests. However, bacteriophage infection can cause failures in 50%-80% of the batches during Bt fermentation, resulting in severe losses. In the present work, the physiological and biochemical impacts of Bt strain CS33 have been studied during bacteriophage infection. This study adopted a gel-based proteomics approach to probe the sequential changed proteins in phage-infected Bt cells. To phage, it depressed the host energy metabolism by suppressing the respiration chain, the TCA cycle, and the utilization of PHB on one hand; on the other hand, it hijacked the host translational machine for its own macromolecular synthesis. To host, superinfection exclusion might be triggered by the changes of S-layer protein and flagella related proteins, which were located on the cell surface and might play as the candidates for the phage recognition. More importantly, the growth rate, cell mass, and ICPs yield were significantly decreased. The low yield of ICPs was mainly due to the suppressed utilization of PHB granules. Further functional study on these altered proteins may lead to a better understanding of the pathogenic mechanisms and the identification of new targets for phage control. BIOLOGICAL SIGNIFICANCE: B. thuringiensis (Bt) has been widely used for 50years as a safe biopesticide for controlling agricultural and sanitary insect pests. However, bacteriophage infection can cause severe losses during B. thuringiensis fermentation. The processes and consequences of interactions between bacteriophage and Bt were still poorly understood, and the molecular mechanisms involved were more unknown. This study adopted a gel-based proteomics approach to probe the physiological and biochemical impacts of Bt strain CS33 after phage-infection. The interactions between phage BtCS33 and its host Bt strain CS33 occurred mainly on four aspects. First, phage synthesized its nucleic acids through metabolic regulation by increasing the amount of NDK. Second, it is reasonable to infer that a phage resistance or superinfection exclusion was triggered by several increased or decreased proteins (SLP, FliD, FlaB), which were located on the cell surface and might play as candidates for the phage recognition. Third, combining the decreased flavoproteins (SdhA and EtfB) and the down regulated Fe-S cluster biosynthesis pathway together, it can be suggested that the respiration chain was weakened after phage infection. Additionally, three key enzymes (AcnB, FumC and AdhA) involved in the TCA cycle were all decreased, indicating the TCA cycle was seriously inhibited after infection. Fourth, the growth rate, cell mass and ICPs yield of the host were significantly decreased. To the best of our knowledge, this work represents the first systematic study on the interactions of an insecticidal bacterium with its phage, and has contributed novel information to understand the molecular events in the important biological pesticide producer, B. thuringiensis, in response to phage challenge.

Genome characteristics of a novel phage from Bacillus thuringiensis showing high similarity with phage from Bacillus cereus.

Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the "late" region, the "lysogeny-lysis" region and the "early" region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wß and some variants of Wß, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wß and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wß may have a common distant ancestor.