Biomedical subjects
N Connell
Publications and source records attributed to N Connell.
Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity.
Antimicrobial peptides are proposed to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against pathogenic microbes. Pleurocidin, a new 25-residue linear antimicrobial peptide, was recently isolated from the skin secretions of winter flounder (Pleuronectes americanus). The present study identifies the cDNA and gene encoding pleurocidin. The pleurocidin gene comprises four exons. Its upstream region demonstrates consensus binding sequences for transcription factors found in host defense genes in mammals, including sequences identical to the NF-IL6 and alpha and gamma interferon response elements. Pleurocidin is predicted to exist as a 68-residue prepropeptide that undergoes proteolytic cleavage of its amino-terminal signal and carboxy-terminal anionic propiece to form the active, mature peptide. Transmission electron microscopy localized pleurocidin to the mucin granules of skin and intestinal goblet cells. Significant synergy was shown to occur between pleurocidin and D-cycloserine targeting Mycobacterium smegmatis. Pleurocidin was functionally active at physiologic concentrations of magnesium and calcium; however, high concentrations of these divalent cations ablated pleurocidin's activity against a standard test strain, Escherichia coli D31. Pleurocidin was tested against bacterial and fungal clinical isolates and showed broad-spectrum antimicrobial activity. Together, these data support the hypothesis that pleurocidin participates in innate mucosal immunity, and it may prove to be a beneficial therapeutic agent.
Old microbes with new faces: molecular biology and the design of new vaccines.
Rational approaches to the design of live attenuated bacterial and viral recombinant vaccine strains are leading to the manipulation of old vaccines and the generation of new ones. The two basic problems to be solved are attenuation of pathogenic strains, and the stable expression of foreign antigens.
Stationary-phase-inducible "gearbox" promoters: differential effects of katF mutations and role of sigma 70.
Many of the changes in gene expression observed when Escherichia coli cells enter stationary phase are regulated at the level of transcription initiation. A group of stationary-phase-inducible promoters, known as "gearbox" promoter, display a characteristic sequence in the -10 region which differs greatly from the consensus sequence for sigma 70-dependent promoters. Here we describe our studies on the gearbox promoters bolAp1 and mcbAp, responsible for the temporally regulated transcription of bolA and the genes involved in the synthesis of the peptide antibiotic microcin B17, respectively. Deletion analysis of mcbAp demonstrated that the stationary-phase-inducible properties of this promoter are found in a DNA fragment extending from -54 to +11 bp, surrounding the transcriptional start site, and are separable from DNA sequences responsible for the OmpR-dependent stimulation of transcription of mcbAp. In vitro transcription studies indicate that the RNA polymerase holoenzyme involved in the transcription of mcbAp contains sigma 70. In this and an accompanying paper (R. Lange and R. Hengge-Aronis, J. Bacteriol. 173: 4474-4481, 1991), experiments are described which show that the product of katF, a global regulator of stationary-phase gene expression and a putative sigma factor, is required for the expression of bolAp1 fused to the reporter gene lacZ. In contrast, mcbAp appears to be negatively regulated by katF. We discuss the implications of these results for postexponential gene expression and the role of gearbox sequences in the regulation of promoter activity.
An E. coli promoter induced by the cessation of growth.
The production of the bacterial DNA replication inhibitor Microcin B17 is induced as cultures enter stationary phase. Using S1 nuclease protection assays we have shown that this induction is the result of increased levels of transcription initiation from a promoter located upstream from mcbA, the structural gene for Microcin B17. Upstream from the start site of transcription there is a rather typical -35 region. However, there is no good homology to the consensus -10 region. While most of the cell's transcription is shut off as a result of the cessation of growth, transcription from the mcbA promoter continues for several hours in stationary phase. A single-copy gene fusion between mcbA and lacZ was used to monitor the response of the promoter to different nutritional conditions and in different host backgrounds altered in metabolic regulatory loci. Starvation for nitrogen, phosphate or carbon sources all induced transcription from the promoter. Levels of transcription were reduced in ompR backgrounds. In contrast, mutations in other global regulatory loci, fnr, relA and cya had little or no effect.