Replacement of OmpF's conserved carboxy-terminal phenylalanine with dissimilar amino acids severely impaired its assembly into stable trimers. In some instances, interactions of mutant proteins with the outer membrane were also affected, as judged by their hypersensitivity phenotype. Synthesis of all mutant OmpF proteins elevated the expression of periplasmic protease DegP, and synthesis of most of them made its presence obligatory for cell viability. These results showed a critical role for DegP in the event of aberrant outer membrane protein assembly. The lethal phenotype of mutant OmpF proteins in a degP null background was eliminated when a protease-deficient DegP S210A protein was overproduced. Our data showed that this rescue from lethality and a subsequent increase in mutant protein levels in the envelope did not lead to the proper assembly of the mutant proteins in the outer membrane. Rather, a detergent-soluble and thermolabile OmpF species resembling monomers accumulated in the mutants, and to a lesser extent in the parental strain, when DegP S210A was overproduced. Interestingly, this also led to the localization of a significant amount of mutant polypeptides to the inner membrane, where DegP S210A also fractionated. These results suggested that the DegP S210A -mediated rescue from toxicity involved preferential sequestration of misfolded OmpF monomers from the normal assembly pathway.

A novel mechanism of Escherichia coli porin regulation was discovered from multicopy suppressors that permitted growth of cells expressing a mutant OmpC protein in the absence of DegP. Analyses of two suppressors showed that both substantially lowered OmpC expression. Suppression activities were confined to a short DNA sequence, which we designated ipeX for inhibition of porin expression, and to DNA containing a 3-truncated ompR gene. The major effect of ipeX on ompC expression was exerted posttranscriptionally, whereas the truncated OmpR protein reduced ompC transcription. ipeX was localized within an untranslated region of 247 base pairs between the stop codon of nmpC-a remnant porin gene from the cryptic phage qsr (DLP12) genome-and its predicted Rho-independent transcriptional terminator. Interestingly, another prophage, PA-2, which encodes a porin similar to NmpC, known as Lc, has sequences downstream from lc identical to that of ipeX. PA-2 lysogenization leads to Lc expression and OmpC inhibition. Our data show that the synthesis of the lc transcript, whose 3 end contains the corresponding ipeX sequence, inhibits OmpC expression. Overexpression of ipeX RNA inhibited both OmpC and OmpF expression but not that of OmpA. ompC-phoA chimeric gene constructs revealed a 248-bp untranslated region of ompC required for ipeX-mediated inhibition. However, no sequence complementarity was found between ipeX and this region of ompC, indicating that inhibition may not involve simple base pairing between the two RNA molecules. The effect of ipeX on ompC, but not on ompF, was independent of the RNA chaperone Hfq.

The expression of assembly-defective outer membrane proteins can confer lethality if they are not degraded by envelope proteases. We report here that the expression of a mutant OmpC protein, OmpC 2Cys , which forms disulfide bonds in the periplasm due to the presence of two non-native cysteine residues, is lethal in cells lacking the major periplasmic protease, DegP. This lethality is not observed in dsbA strains that have diminished ability to form periplasmic disulfide bonds. Our data show that this OmpC 2Cys -mediated lethality in a degP::Km r dsbA ؉ background can be reversed by a DegP variant, DegP S210A , that is devoid of its proteolytic activity but retains its reported chaperone activity. However, DegP S210A does not reverse the lethal effect of OmpC 2Cys by correcting its assembly but rather by capturing misfolded mutant OmpC polypeptides and thus removing them from the assembly pathway. Displacement of OmpC 2Cys by DegP S210A also alleviates the negative effect that the mutant OmpC protein has on wild-type OmpF.OmpC and OmpF represent a major class of Escherichia coli outer membrane proteins (OMPs). These proteins form waterfilled channels that permit the diffusion of small solutes into the cell. Because of this property, OmpC and OmpF are called porins or sometimes "classical" porins, which refer to their stable ␤-barrel structure and nonspecific solute diffusion properties (17). Each porin monomer consists of a 16-stranded antiparallel ␤-barrel (22). These porins exist as homotrimers, in which three barrels assemble to produce three separate channels (22).Besides their atomic structures, the assembly and targeting of the outer membrane ␤-barrel proteins have been an intense topic of study (for a recent review, please see reference 6). Based on studies conducted in several laboratories, it has been shown that the assembly of porin proteins begins with the proteolytic cleavage of an amino-terminal signal sequence that yields soluble nascent monomers. Rapid folding of these monomers is followed by their oligomerization into proteaseand heat-sensitive trimers called metastable trimers, which slowly mature into protease-and heat-resistant trimers, the final product of assembly.

Algal cultivation and biomass production for biofuel and other products have many advantages over terrestrial crops. However, sustainable algal cultivation, especially in open ponds, is difficult due to opportunistic zooplankton grazers. There is an urgent need for the development of methods and procedures for early detection and quantification of zooplankton in algal cultures. In this study, the FlowCAM was able to detect and quantify Brachionus calyciflorus, a model organism, in algal cultures with a detection limit of < 1 individual/mL for B. calyciflorus in an algal (Chlorella sp.) cell density of 10 7 cells/mL. The methodology also allowed successful monitoring of rotifer population growth at low density (<1 individual/mL of B. calyciflorus) in dense algal cultures. Furthermore, the methodology was also effective in detecting and quantifying other zooplankton grazers including ciliates, Poterioochromonas sp., and Euplotes sp. in outdoor open raceway cultures.

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