Two promoters and two translation start sites control the expression of the Shigella flexneri outer membrane protease IcsP.

The Shigella flexneri outer membrane protease IcsP proteolytically cleaves the actin-based motility protein IcsA from the bacterial surface. The icsP gene is monocistronic and lies downstream of an unusually large intergenic region on the Shigella virulence plasmid. In silico analysis of this region predicts a second transcription start site 84 bp upstream of the first. Primer extension analyses and beta-galactosidase assays demonstrate that both transcription start sites are used. Both promoters are regulated by the Shigella virulence gene regulator VirB and both respond similarly to conditions known to influence Shigella virulence gene expression (iron concentration, pH, osmotic pressure, and phase of growth). The newly identified promoter lies upstream of a Shine-Dalgarno sequence and second 5'-ATG-3', which is in frame with the annotated icsP gene. The use of either translation start site leads to the production of IcsP capable of proteolytically cleaving IcsA. A bioinformatic scan of the Shigella genome reveals multiple occurrences of in-frame translation start sites associated with putative Shine-Dalgarno sequences, immediately upstream and downstream of annotated open reading frames. Taken together, our observations support the possibility that the use of in-frame translation start sites may generate different protein isoforms, thereby expanding the proteome encoded by bacterial genomes.

Microbially mediated aerobic and anaerobic degradation of acrylamide in a western United States irrigation canal.

Acrylamide (AMD), a neurotoxin and suspected carcinogen, is present at concentrations of up to 0.05% in linear anionic polyacrylamide, which is under evaluation as a temporary sealant in unlined irrigation canal systems across the United States. We examined the microbially mediated degradation of AMD and diversity of AMD-degrading microbial physiotypes in the Rocky Ford Highline Canal, Colorado to better constrain the potential fate ofAMD in a canal environment. Microorganisms able to use AMD (500 mg L(-1)) as a sole nitrogen source were relatively abundant (2.3 x 10(3) to 9.4 x 10(3) cells mL(-1) in water and 4.2 x 10(3) to 2.3 x 10(5) cells g(-1) in sediment). Only sediment samples contained microorganisms able to use AMD as a sole carbon source. Acrylamide (up to 100 mg L(-1)) was efficiently removed from amended canal water and sediment slurries under aerobic conditions, but no AMD degradation was observed in abiotic controls. Anaerobic degradation of AMD by nitrate-, sulfate-, and iron-reducing microorganisms was also tested, with nitrate reducers affecting the highest amounts of AMD removal (70.3-85%) after 60 d. All representatives (n=15) from a collection of 256 AMD-degrading microbial isolates from Rocky Ford Highline Canal were closely related to well characterized environmental bacteria capable of facultative nitrate respiration. Our results demonstrate that natural microbial populations within this canal are capable of AMD degradation under aerobic and anaerobic conditions and that this degradation is performed by naturally abundant bacteria likely to be present in other freshwater irrigation canals or similar lotic habitats.

VirB alleviates H-NS repression of the icsP promoter in Shigella flexneri from sites more than one kilobase upstream of the transcription start site.

The icsP promoter of Shigella spp. is repressed by H-NS and derepressed by VirB. Here, we show that an inverted repeat located between positions -1144 and -1130 relative to the icsP transcription start site is necessary for VirB-dependent derepression. The atypical location of this cis-acting site is discussed.