Plant viruses are excellent tools for studying microbial-plant interactions as well as the complexities of host activities. Our study focuses on the role of C2 encoded by Beet severe curly top virus (BSCTV) in the virus-plant interaction. Using BSCTV C2 as bait in a yeast two-hybrid screen, a C2-interacting protein, S-adenosyl-methionine decarboxylase 1 (SAMDC1), was identified from an Arabidopsis thaliana cDNA library. The interaction was confirmed by an in vitro pull-down assay and a firefly luciferase complemention imaging assay in planta. Biochemical analysis further showed that the degradation of the SAMDC1 protein was inhibited by MG132, a 26S proteasome inhibitor, and that C2 could attenuate the degradation of the SAMDC1 protein. Genetic analysis showed that loss of function of SAMDC1 resulted in reduced susceptibility to BSCTV infection and reduced viral DNA accumulation, similar to the effect of BSCTV C2 deficiency. Bisulfite sequencing analysis further showed that C2 deficiency caused enhanced DNA methylation of the viral genome in infected plants. We also showed that C2 can suppress de novo methylation in the FWA transgenic assay in the C2 transgene background. Overexpression of SAMDC1 can mimic the suppressive activity of C2 against green fluorescent protein-directed silencing. These results suggest that C2 interferes with the host defense mechanism of DNA methylation-mediated gene silencing by attenuating the 26S proteasome-mediated degradation of SAMDC1.

SummaryThe C4 protein from Curtovirus is known as a major symptom determinant, but the mode of action of the C4 protein remains unclear. To understand the mechanism of involvement of C4 protein in virus-plant interactions, we introduced the C4 gene from Beet severe curly top virus (BSCTV) into Arabidopsis under a conditional expression promoter; the resulting overexpression of BSCTV C4 led to abnormal host cell division. RKP, a RING finger protein, which is a homolog of the human cell cycle regulator KPC1, was discovered to be induced by BSCTV C4 protein. Mutation of RKP reduced the susceptibility to BSCTV in Arabidopsis and impaired BSCTV replication in plant cells. Callus formation is impaired in rkp mutants, indicating a role of RKP in the plant cell cycle. RKP was demonstrated to be a functional ubiquitin E3 ligase and is able to interact with cell-cycle inhibitor ICK/KRP proteins in vitro. Accumulation of the protein ICK2/KRP2 was found increased in the rkp mutant. The above results strengthen the possibility that RKP might regulate the degradation of ICK/ KRP proteins. In addition, the protein level of ICK2/KRP2 was decreased upon BSCTV infection. Overexpression of ICK1/KRP1 in Arabidopsis could reduce the susceptibility to BSCTV. In conclusion, we found that RKP is induced by BSCTV C4 and may affect BSCTV infection by regulating the host cell cycle.

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are widely produced by Gram-positive bacteria, including many species of the Bacillus group. In the present study, one novel gene cluster coding lantibiotic cerecidins was unveiled in Bacillus cereus strain As 1.1846 through genomic mining and PCR screening. The designated cer locus is different from that of conventional class II lantibiotics in that it included seven tandem precursor cerA genes, one modification gene (cerM), two processing genes (cerT and cerP), one orphan regulator gene (cerR), and two immunity genes (cerF and cerE). In addition, one unprecedented quorum sensing component, comQXPA, was inserted between cerM and cerR. The expression of cerecidins was not detected in this strain of B. cereus, which might be due to repressed transcription of cerM. We constitutively coexpressed cerA genes and cerM in Escherichia coli, and purified precerecidins were proteolytically processed with the endoproteinase GluC and a truncated version of putative serine protease CerP. Thus, two natural variants of cerecidins A1 and A7 were obtained which contained two terminal nonoverlapping thioether rings rarely found in lantibiotics. Both cerecidins A1 and A7 were active against a broad spectrum of Gram-positive bacteria. Cerecidin A7, especially its mutant Dhb13A, showed remarkable efficacy against multidrug-resistant Staphylococcus aureus (MDRSA), vancomycin-resistant Enterococcus faecalis (VRE), and even Streptomyces.

Lactobacillus plantarum is a widespread bacterial species and is commonly used as a probiotic. L. plantarum PFM105 was isolated from the rectum of a healthy sow. Here we found that L. plantarum PFM105 showed probiotic effect on weaning piglets in which intestinal inflammation and unbalanced gut microbiota happened frequently. L. plantarum PFM105 was identified to improve the growth of weaning piglet and promote the development of small intestinal villi. Antibiotics are often used in weaning piglet to prevent intestinal infection and promote the growth of animal. We found that weaning piglets feeding with L. plantarum PFM105 showed similar growth promotion but decreased diarrhea incidence compared with those feeding with antibiotics. High-throughput sequencing was used to analyze the gut microbiota in weaning piglets treated with L. plantarum PFM105 or antibiotics. The relative abundance of beneficial microbes Prevotellaceae and Bifidobacteriaceae were increased in colon of weaning piglet feeding L. plantarum PFM105, while antibiotics increased the relative abundance of bacteria associated with pathogenicity, such as Spirochaeta and Campylobacteraceae. L. plantarum PFM 105 increased indicators of intestinal health including serum levels of IgM, IL-10, and TGF-β, and colonic levels of SCFAs. We found strong correlations between the alterations in gut microbiota composition caused by feeding antibiotics and probiotics and the measured growth and health parameters in weaning piglets. The addition of L. plantarum PFM105 could significantly increase the relative abundance of metabolic genes which may important to intestinal microbiota maturation. Altogether, we demonstrated here that L. plantarum PFM 105 could promote intestinal development through modulation of gut microbiota in weaning piglets.

Bovicin HJ50, a lantibiotic produced by Streptococcus bovis HJ50, is featured by the presence of a disulfide bridge. This study described a simplified in vitro synthetic strategy for producing bovicin HJ50 totally based on Escherichia coli expression system. In this strategy termed as Semi-in vitro biosynthesis (SIVB), prepeptide BovA and modification enzyme BovM were co-expressed to generate posttranslationally modified BovA. Then a specific protease BovT150 was employed to remove the leader peptide in vitro and produce biologically active bovicin HJ50. Via SIVB, a series of ring-broken bovicin mutants C13A, C21A, C29A and T10A/C32A were prepared by introducing site-directed mutations into bovA gene. Further, we analyzed the bridging patterns of these mutants through chemical modification and successfully clarified the bridging pattern of bovicin HJ50. The results showed that two thioether bridges exist between Thr8 and Cys13, and Thr10 and Cys32, respectively, and that the disulfide bond bridging Cys21 and 29 is very relevant for the antimicrobial activity of bovicin HJ50. This is the first study that reports the bridging pattern of bovicin HJ50.

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SUMMARYGeminiviruses include a large number of single-stranded DNA viruses that are emerging as useful tools to dissect many fundamental processes in plant hosts. However, there have been no reports yet regarding the genetic dissection of the geminivirus-plant interaction. Here, a high-throughput approach was developed to screen Arabidopsis activation-tagged mutants which are resistant to geminivirus Beet severe curly top virus (BSCTV) infection. A mutant, lsb1 (less susceptible to BSCTV 1), was identified, in which BSCTV replication was impaired and BSCTV infectivity was reduced. We found that the three genes closest to the T-DNA were up-regulated in lsb1, and the phenotypes of lsb1 could only be recapitulated by the overexpression of GDU3 (GLUTAMINE DUMPER 3), a gene implicated in amino acid transport. We further demonstrated that activation of LSB1/GDU3 increased the expression of components in the salicylic acid (SA) pathway, which is known to counter geminivirus infection, including the upstream regulator ACD6. These data indicate that up-regulation of LSB1/GDU3 affects BSCTV infection by activating the SA pathway. This study thus provides a new approach to study of the geminivirus-host interaction.

Lactic acid bacteria that exist in the urinogenital system play an important role in maintaining the health of the host. Here, we report the finished and annotated genome of a Lactococcus strain that was isolated from the vaginas of healthy women and shows probiotic properties, including nisin A production and adhesion to vaginal epithelial cells.The vast diversity of lactic acid bacteria (LAB) allows them to occupy different kinds of ecological niches, such as dairy products, meats, the gastrointestinal tract, and the vagina (5). As the dominant bacteria in the vaginal microflora of healthy women, Lactobacillus spp. play an important role in maintaining the natural balance of microflora (2). So far, only a few probiotic Lactococcus strains, like L. lactis subsp. lactis HV219, have been found in the vaginal microflora (6). In this study, we identified a Lactococcus strain named L. lactis subsp. lactis CV56 from vaginal secretions of healthy women. This strain not only exhibits strong antimicrobial activity by producing bacteriocin nisin A but also shows a greater ability to adhere to vaginal epithelial cells than other Lactococcus strains, such as L. lactis MG1363. The whole-genome sequencing of L. lactis CV56 will help us identify the genomic diversity, metabolic diversity, and probiotic and adaptation properties of Lactococcus species in different environments.The whole genome was sequenced by using the Roche GS FLX system. A total of 233,256 reads, counting up to 87,594,199 bases, were obtained, providing 36-fold coverage. Assembly was performed by use of Newbler software (454 Sequencing) and produced 77 contigs. The contig order was first determined through a BLASTN search against the reference strain L. lactis IL1403 and then confirmed by PCR. The relationships of contigs that cannot be ordered by reference were determined by multiplex PCR. Gaps were closed by sequencing PCR fragments from the genomic DNA template by using ABI 3730. The Phred/Phrap/Consed software package (2a, 2b) was used for final assembly and edition, and lowquality regions of the genome were resequenced.Putative protein-coding sequences were identified by Glimmer3 (1), and those shorter than 90 bp were eliminated. All predicted proteins were searched against KEGG databases, the NCBI nonredundant protein database (nr), and the Conserved Domain Database (CDD), using BLASTP or RPS-BLAST. The tRNA genes were predicted by tRNA-scan SE1.21 (4), and the rRNA genes were detected using the RNAmmer 1.2 server (3). Functional classification of proteins was conducted using NCBI clusters of orthologous groups (COG).The complete genome of L. lactis CV56 consists of a single, circular chromosome (2,399,458 bp, 35.24% GC content) and five plasmids, namely, pCV56A (44,098 bp, 32.08% GC content), pCV56B (35,934 bp, 34.54% GC content), pCV56C (31,442 bp, 32.49% GC content), pCV56D (5,543 bp, 32.24% GC content), and pCV56E (2,262 bp, 33.82% GC content). The chromosome of L. lactis CV56 contains 2,352 predicted protein-coding genes, 6 rRNA operons, and 62 t...