Aquaporin Z of Escherichia coli : Reassessment of Its Regulation and Physiological Role

Abstract

Transcription of an aqpZ-lac fusion in a single copy on the Escherichia coli chromosome increased as cells entered the stationary growth phase. This was true in a variety of media, and increased transcription in enriched medium required the RpoS sigma factor. Expression of the aqpZ-lac fusion was not affected by up-or downshifts in osmolality. Disruption of aqpZ had no detectable adverse effects.Aquaporins belong to a large family of proteins that increase the rate of diffusion of water and glycerol across cell membranes (18,22,24). They are prominent in multicellular eukaryotic organisms, whose large size and need for rapid water movement make such proteins essential (17,25). Aquaporins (but not glyceroporins) occur only sporadically in bacteria and archaea. For example, aqpZ occurs in all four Escherichia coli strains for which whole genome sequences are available and in Shigella flexneri, but it does not occur in the closely related enteric bacterium Salmonella enterica serovar Typhimurium or in the ␥-proteobacterium Yersinia pestis (E. coli Genome Project, University of Wisconsin-Madison, http://www.genome .wisc.edu/). Moreover, aqpZ appears to be missing from Ͼ80% of the 69 bacterial and archaeal genomes that have been completely sequenced (National Center for Biotechnology Information website, http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez /genom_table_cgi). It is not clear whether small cells that lack internal organelles require aquaporins or whether unmediated diffusion of water across their cytoplasmic membranes is sufficient.The aqpZ gene of E. coli was isolated by homology cloning (3). Calamita and colleagues (4) studied regulation of expression of this gene by using a plasmid carrying an aqpZ-lacZ fusion. They reported that aqpZ expression was induced very sharply in the middle of the exponential growth phase and declined thereafter. Calamita and colleagues reported that an aqpZ-null strain of E. coli (aqpZ::lacZ-kan) formed mostly small colonies on Luria-Bertani (LB) agar containing kanamycin and that it showed decreased viability upon prolonged incubation at osmolalities between 80 and 240 mosmolal.Transcription of aqpZ is increased in stationary phase. To reexamine control of E. coli aqpZ expression, we constructed a strain carrying a single copy of an aqpZ-lac transcriptional fusion stably integrated on the chromosome (7). To create the fusion, aqpZ, which had been amplified by PCR from the genome of strain MG1655, was cleaved with PvuII, resulting in destruction of the sixth codon. The upstream fragment, which carries 400 bp 5Ј of the translational start site for aqpZ, was cloned into pRS551 (19) to yield pJES1320. The fusion was integrated at the trp locus as previously described (7) and then introduced into prototrophic strain NCM1458 (also known as RK4353 [21]) by P1-mediated transduction to yield strain NCM3342 [Kan r -⌽(aqpZЈ-Јlac)]. As reported previously (4

AqpZ-Mediated Water Permeability in Escherichia coli Measured by Stopped-Flow Spectroscopy

“…The increased permeability for the wild type is apparently largely due to the incorporation of AqpZ into the cytoplasmic membrane. The permeability data we report here support the conclusion of Soupene et al that expression of aqpZ increases as cell growth slows and the cells enters stationary phase (9). Our results also indicate that AqpZ mediates water permeability in vivo.…”

“…The observed relationships between the rate of growth and permeability in Fig. 1A, most notably the initial decrease in permeability and the lag that occurs between the observed increase in permeability and the onset of exponential growth, is consistent with the observation that transcription of an aqpZlac fusion on the chromosome of E. coli increases as the cells enter the stationary stage of growth (9).…”

“…Our results also indicate that AqpZ mediates water permeability in vivo. However, these observations do not address the issue of whether AqpZ traffics exclusively in water or whether it might also mediate the permeability of other small molecules, for example, the fermentation end product ethanol, as suggested by Soupene et al (9). We thank Helen I. Zgurskaya and David P. Nagle, Jr., for stimulating discussion and Sydney Kustu for supplying NCM3306.…”

The aquaporin gene aqpX of Brucella abortus is induced in hyperosmotic conditions

“…Surprisingly, the B. abortus gene showed a behaviour different from that of the E. coli aqpZ gene, whose expression was reported to increase significantly in hypo-osmolar conditions, and to decrease in hyperosmolar conditions (Calamita et al, 1998), or found to be unaffected by changes in osmolality (Soupene et al, 2002).…”

“…This result was again similar to that reported for E. coli (Calamita, 2000;Calamita et al, 1998), and indicated that the aqpX gene was subject to growth phase regulation. A recent report on the regulation of expression of the E. coli aqpZ gene using a single-copy chromosomal aqpZ-lacZ fusion indicated that expression of E. coli aqpZ increased in the stationary phase of growth and that this increase was dependent on the RpoS sigma factor (Soupene et al, 2002).…”