Improved lux reporters for use in Staphylococcus aureus

The use of luxABCDE (lux) offers certain advantages over other reporters, such as: lacZ and xylE. It is real time and its signal generation is produced without the requirement for any additional substrates. In some bacteria such as Staphylococcus spp, light production by luciferase is restricted because of a limited availability of endogenous substrates such as fatty acid aldehyde. We describe the construction of promoterless-lux cloning vectors, pGYlux and pAmilux. S. aureus carrying B. subtilis xyl/tetO promoter fused to the lux genes of pGYlux gave up to a 2.5-fold enhancement of luminescence over S. aureus carrying the xyl/tetO promoter fused to lux genes of the previously published parent vector pAL2. Furthermore, pAmilux showed a 6-fold enhancement of lux expression when compared to pGYlux in S. aureus. This was achieved by cloning the constitutive ami promoter upstream of the luxCDE genes to increase endogenous fatty acid aldehyde production while maintaining its reporter functionality by fusing promoters to the luxAB genes.     

Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters

Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 α promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish.     

Cloning of type 8 capsule genes and analysis of gene clusters for the production of different capsular polysaccharides in Staphylococcus aureus.

Eleven serotypes of capsular polysaccharide from Staphylococcus aureus have been reported. We have previously cloned a cluster of type 1 capsule (cap1) genes responsible for type 1 capsular polysaccharide biosynthesis in S. aureus M. To clone the type 8 capsule (cap8) genes, a plasmid library of type 8 strain Becker was screened with a labelled DNA fragment containing the cap1 genes under low-stringency conditions. One recombinant plasmid containing a 14-kb insert was chosen for further study and found to complement 14 of the 18 type 8 capsule-negative (Cap8-) mutants used in the study. Additional library screening, subcloning, and complementation experiments showed that all of the 18 Cap8- mutants were complemented by DNA fragments derived from a 20.5-kb contiguous region of the Becker chromosome. The mutants were mapped into six complementation groups, indicating that the cap8 genes are clustered. By Southern hybridization analyses under high-stringency conditions, we found that DNA fragments containing the cap8 gene cluster show extensive homology with all 17 strains tested, including type 1 strains. By further Southern analyses and cloning of the cap8-related homolog from strain M, we show that strain M carries an additional capsule gene cluster different from the cap1 gene cluster. In addition, by using DNA fragments containing different regions of the cap8 gene cluster as probes to hybridize DNA from different strains, we found that the central region of the cap8 gene cluster hybridizes only to DNAs from certain strains tested whereas the flanking regions hybridize to DNAs of all strains tested. Thus, the cap8 gene clusters and its closely related homologs are likely to have organizations similar to those of the encapsulation genes of other bacterial systems.     

Nucleotide sequence of the Vibrio alginolyticus glnA region

The nucleotide sequence of a 4 kb fragment containing the Vibrio alginolyticus glnA, ntrB and ntrC genes was determined. The upstream region of the glnA gene contained tandem promoters. The upstream promoter resembled the consensus sequence for Escherichia coli ⁷⁰ promoters whereas the presumptive downstream promoter showed homology with nitrogen regulated promoters. Four putative NR_I binding sites were located between the tandem promoters. The ntrB gene was preceded by a single presumptive NR_I binding site. The ntrC gene was located 45 base pairs downstream from the ntrB gene. The V. alginolyticus ntrB and ntrC genes were able to complement ntrB, ntrC deletions in E. coli.Abbreviations bp base pair(s) - CAP catabolite-activating protein - GS glutamine synthetase - kb kilobase(s) - ORF open reading frame - SD Shine-Dalgarno