Reporter gene technology has facilitated greatly the analysis of gene expression and the study of individual promoters and their regulation. Although various reporter gene systems are available, none of them are universally applicable and consequently, studies aimed at screening of new reporters are continuing. Toward this end, an acid phosphatase, designated SapS, was identified and characterized from the culture supernatant of a Staphylococcus aureus strain isolated from vegetables. Biochemical characterization of the 30-kDa monomeric enzyme indicated that it displayed optimum activity at 40°C and pH 5, using p-nitrophenyl phosphate (pNPP) as substrate. The enzymatic activity was enhanced by Mg2+, but was inhibited by EDTA and molybdate. Based on its properties and amino acid sequence analyses, SapS was classified as a new member of the bacterial class C family of non-specific acid phosphatases. The S. aureus SapS enzyme was subsequently evaluated as a reporter for host strain evaluation and cell surface display. Bacillus halodurans of which the major cell wall protease gene (wprA) was inactivated was used as expression host, and the cell wall-binding domain of the cwlC gene from B. halodurans was used as an anchoring motif for cell surface display. The results from in vitro enzyme activity assays indicated that extracellular production of the SapS reporter enzyme was improved 3.5-fold in the mutant compared to wild-type B. halodurans strain. Zymographic detection of SapS activity showed that the SapS-CwlC fusion protein was localized in the B. halodurans cell wall fraction, thus demonstrating the potential of SapS as a reporter for cell surface display of heterologous proteins. The versatility of the SapS enzyme as a reporter for gene expression and protein secretion in both Gram-positive and Gram-negative bacteria was also investigated. Transcriptional and translational fusions of the sapS gene with selected heterologous promoters and signal sequences were constructed, and expressed in Escherichia coli, B. subtilis and B. halodurans. The strongest promoter for heterologous protein production in each of the host strains was identified, i.e. the E. coli lacZ promoter in E. coli, the B. halodurans alkaline protease promoter in B. subtilis, and the B. halodurans σD promoter in B. halodurans. / Thesis (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

In eukaryotic cells, some mRNAs localize to distinct areas of the cell where RNA is translated and the encoded protein is specifically localized. Recent studies have suggested that even though prokaryotic cells lack internal compartmentalization, different RNAs can localize to distinct regions of the bacterial cell. Our lab is developing methods for labeling and detecting RNA with the goal of determining localization of endogenous RNAs within single cells. We currently employ an eIF4a protein-specific aptamer for RNA labeling using one of two methods. (1) Target RNA is tagged with the aptamer sequence at the 3' end and the aptamer triggers protein complementation of two fusion proteins, each containing split EGFP and split eIF4A proteins. (2) Two RNA probes, each containing a half of a split eIF4a-specific aptamer and an antisense sequence complementary to the target RNA, bind the unmodified transcript through complementary interactions. This binding brings the two fragments of the split aptamer in close proximity and allows proper folding of a split aptamer. A fluorescent signal is generated by the aptamer-driven reassociation of the fusion proteins. In this work, we investigate the sensitivity of the first method for detecting transcripts expressed from their natural chromosomal loci, and describe attempts to increase the sensitivity of the method by using multiple aptamer tagging. We also present results suggesting that the second method, combining protein complementation and split aptamer approach, provides high sensitivity enabling detection of endogenous bacterial RNAs expressed at low level.

Aeolian dust exerts a notable influence on atmospheric and oceanic conditions and human health, particularly in arid and semi-arid regions like Saudi Arabia. Dust is often characterized by its mineral and chemical composition, but there is a microbiological component of natural aerosols which has received comparatively little attention. Moreover, the amount of materials suspended in the atmosphere is highly variable from day to day. Thus, knowing the loads of dust and suspended microbes and its variability over the year is essential to understand the possible effects of dust on the Red Sea ecosystem. Here, we present the first estimates of dust and microbial loads at a coastal side on the Red Sea over a two-year period supplemented with information from dust samples collected along the Red Sea in offshore water and their variability. Weekly average dust loads ranged from 4.63 to 646.11 μg m-3, while the abundance of airborne prokaryotic cells and viral particles ranged from 31,457 to 608,333 cells m-3 and from 69,615.5 to 3,104,758 particles m-3, respectively. These are the first estimates of airborne microbial abundance that we are aware of in this region. The large number of dust particles and suspended microbes found in the air indicates that airborne microbes may have a large impact on our health and that of the Red Sea ecosystem.

Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains v, 61 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 50-56).