Inactivation of Cronobacter malonaticus cells and inhibition of its biofilm formation exposed to hydrogen peroxide stress.

Presence of Cronobacter malonaticus in powdered infant formula (PIF) poses a high risk to infant and public health. Cronobacter malonaticus has been widely distributed in food and food processing environments, and the true origin of C. malonaticus in PIF is poorly understood. Control and prevention of C. malonaticus is necessary for achieving microbial safety of PIF. However, little information about decontamination of C. malonaticus is available. In this study, effects of hydrogen peroxide on inactivation and morphological changes of C. malonaticus cells were determined. Furthermore, inhibitory effects of H2O2 on biofilm formation in C. malonaticus were also performed. Results indicated that H2O2 could completely inactivate C. malonaticus in sterile water with 0.06% H2O2 for 25 min, 0.08% H2O2 for 15 min, and 0.10% for 10 min, respectively, whereas the survival rates of C. malonaticus in tryptic soy broth medium significantly increased with the same treatment time and concentration of H2O2. In addition, morphological changes of C. malonaticus cells, including cell shrinkage, disruption of cells, cell intercession, and leakage of intercellular material in sterile water after H2O2 treatment, were more predominant than those in tryptic soy broth. Finally, significant reduction in biofilm formation by H2O2 was found using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy detection compared with control samples. This is the first report to determine the effects of H2O2 on C. malonaticus cells and biofilm formation. The findings provided valuable information for practical application of H2O2 for decontamination of C. malonaticus in dairy processing.

Transformation of rice with large maize genomic DNA fragments containing high content repetitive sequences.

KEY MESSAGE: Large and complex maize BIBAC inserts, even with a length of about 164 kb and repeat sequences of 88.1%, were transferred into rice. The BIBAC vector has been established to clone large DNA fragments and directly transfer them into plants. Previously, we have constructed a maize B73 BIBAC library and demonstrated that the BIBAC clones were stable in Agrobacterium. In this study, we demonstrated that the maize BIBAC clones could be used for rice genetic transformation through Agrobacterium-mediated method, although the average transformation efficiency for the BIBAC clones (0.86%) is much lower than that for generally used binary vectors containing small DNA fragments (15.24%). The 164-kb B73 genomic DNA insert of the BIBAC clone B2-6 containing five maize gene models and 88.1% of repetitive sequences was transferred into rice. In 18.75% (3/16) of the T1, 13.79% (4/29) of the T2, and 5.26% (1/19) of the T3 generation transgenic rice plants positive for the GUS and HYG marker genes, all the five maize genes can be detected. To our knowledge, this is the largest and highest content of repeat sequence-containing DNA fragment that was successfully transferred into plants. Gene expression analysis (RT-PCR) showed that the expression of three out of five genes could be detected in the leaves of the transgenic rice plants. Our study showed a potential to massively use maize genome resource for rice breeding by mass transformation of rice with large maize genomic DNA fragment BIBAC clones.

A pair of new BAC and BIBAC vectors that facilitate BAC/BIBAC library construction and intact large genomic DNA insert exchange.

BACKGROUND: Large-insert BAC and BIBAC libraries are important tools for structural and functional genomics studies of eukaryotic genomes. To facilitate the construction of BAC and BIBAC libraries and the transfer of complete large BAC inserts into BIBAC vectors, which is desired in positional cloning, we developed a pair of new BAC and BIBAC vectors. RESULTS: The new BAC vector pIndigoBAC536-S and the new BIBAC vector BIBAC-S have the following features: 1) both contain two 18-bp non-palindromic I-SceI sites in an inverted orientation at positions that flank an identical DNA fragment containing the lacZ selection marker and the cloning site. Large DNA inserts can be excised from the vectors as single fragments by cutting with I-SceI, allowing the inserts to be easily sized. More importantly, because the two vectors contain different antibiotic resistance genes for transformant selection and produce the same non-complementary 3' protruding ATAA ends by I-SceI that suppress self- and inter-ligations, the exchange of intact large genomic DNA inserts between the BAC and BIBAC vectors is straightforward; 2) both were constructed as high-copy composite vectors. Reliable linearized and dephosphorylated original low-copy pIndigoBAC536-S and BIBAC-S vectors that are ready for library construction can be prepared from the high-copy composite vectors pHZAUBAC1 and pHZAUBIBAC1, respectively, without the need for additional preparation steps or special reagents, thus simplifying the construction of BAC and BIBAC libraries. BIBAC clones constructed with the new BIBAC-S vector are stable in both E. coli and Agrobacterium. The vectors can be accessed through our website http://GResource.hzau.edu.cn. CONCLUSIONS: The two new vectors and their respective high-copy composite vectors can largely facilitate the construction and characterization of BAC and BIBAC libraries. The transfer of complete large genomic DNA inserts from one vector to the other is made straightforward.