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Cronobacter species are linked with life-treating diseases in neonates and show strong tolerances to environmental stress. However, the information about factors involved in oxidative tolerance in Cronobacter remains elusive. Here, factors involved in oxidative tolerance in C. malonaticus were identified using a transposon mutagenesis. Eight mutants were successfully screened based on a comparison of the growth of strains from mutant library (n = 215) and wild type (WT) strain under 1.0 mM H2O2. Mutating sites including thioredoxin 2, glutaredoxin 3, pantothenate kinase, serine/threonine protein kinase, pyruvate kinase, phospholipase A, ferrous iron transport protein A, and alanine racemase 2 were successfully identified by arbitrary PCR and sequencing alignment. Furthermore, the comparison about quantity and structure of biofilms formation among eight mutants and WT was determined using crystal violet staining (CVS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Results showed that the biofilms of eight mutants significantly decreased within 48 h compared to that of WT, suggesting that mutating genes play important roles in biofilm formation under oxidative stress. The findings provide valuable information for deeply understanding molecular mechanism about oxidative tolerance of C. malonaticus.

Cronobacter species are linked with life-treating diseases in neonates and show strong tolerances to environmental stress. However, the information about factors involved in oxidative tolerance in Cronobacter remains elusive. Here, factors involved in oxidative tolerance in C. malonaticus were identified using a transposon mutagenesis. Eight mutants were successfully screened based on a comparison of the growth of strains from mutant library (n = 215) and wild type (WT) strain under 1.0 mM H2O2. Mutating sites including thioredoxin 2, glutaredoxin 3, pantothenate kinase, serine/threonine protein kinase, pyruvate kinase, phospholipase A, ferrous iron transport protein A, and alanine racemase 2 were successfully identified by arbitrary PCR and sequencing alignment. Furthermore, the comparison about quantity and structure of biofilms formation among eight mutants and WT was determined using crystal violet staining (CVS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Results showed that the biofilms of eight mutants significantly decreased within 48 h compared to that of WT, suggesting that mutating genes play important roles in biofilm formation under oxidative stress. The findings provide valuable information for deeply understanding molecular mechanism about oxidative tolerance of C. malonaticus. [ABSTRACT FROM AUTHOR]

Carrot ( L.) chromosomes are small and poorly differentiated in size and morphology. Here we demonstrate that fluorescent in situ hybridization (FISH) signals derived from arbitrary PCR probes can be used for chromosome identification in carrot. To prepare probes, we searched for nonpolymorphic products abundantly amplified with arbitrary decamer primers in a group of accessions representing carrot genetic diversity. As a result, 13 fragments ranging in size from 517 to 1758 bp were selected, sequenced, and used as probes for fluorescent in situ hybridization. Four of these probes produced clear and reproducible hybridization signals. The sequences showed similarity to a number of carrot BAC-end sequences, indicating their repetitive character. Three of them were similar to internal portions of gypsy and copia LTR retrotransposons previously identified in plants. Hybridization signals for the four probes were observed as dotted tracks on chromosomes, differing in distribution and intensity. Generally, they were present in pericentromeric and (or) interstitial localizations on chromosome arms. The use of the four probes allowed discrimination of chromosome pairs and construction of more detailed karyotypes and idiograms of carrot. [ABSTRACT FROM AUTHOR]

Experimental studies of transcriptional regulation in bacteria require the ability to precisely measure changes in gene expression, often accomplished through the use of reporter genes. However, the boundaries of promoter sequences required for transcription are often unknown, thus complicating the construction of reporters and genetic analysis of transcriptional regulation. Here, we analyze reporter libraries to define the promoter boundaries of the luxCDABE bioluminescence operon and the betIBA-proXWV osmotic stress operon in Vibrio harveyi. We describe a new method called rapid arbitrary PCR insertion libraries (RAIL) that combines the power of arbitrary PCR and isothermal DNA assembly to rapidly clone promoter fragments of various lengths upstream of reporter genes to generate large libraries. To demonstrate the versatility and efficiency of RAIL, we analyzed the promoters driving expression of the luxCDABE and betIBA-proXWV operons and created libraries of DNA fragments from these loci fused to fluorescent reporters. Using flow cytometry sorting and deep sequencing, we identified the DNA regions necessary and sufficient for maximum gene expression for each promoter. These analyses uncovered previously unknown regulatory sequences and validated known transcription factor binding sites. We applied this high-throughput method to gfp, mCherry, and lacZ reporters and multiple promoters in V. harveyi. We anticipate that the RAIL method will be easily applicable to other model systems for genetic, molecular, and cell biological applications. [ABSTRACT FROM AUTHOR]