Escherichia coli strain MG1655 was chosen for sequencing because the few mutations it carries (ilvG rfb-50 rph-1) were considered innocuous. However, it has a number of growth defects. Internal pyrimidine starvation due to polarity of the rph-1 allele on pyrE was problematic in continuous culture. Moreover, the isolate of MG1655 obtained from the E. coli Genetic Stock Center also carries a large deletion around the fnr (fumaratenitrate respiration) regulatory gene. Although studies on DNA microarrays revealed apparent cross-regulation of gene expression between galactose and lactose metabolism in the Stock Center isolate of MG1655, this was due to the occurrence of mutations that increased lacY expression and suppressed slow growth on galactose. The explanation for apparent cross-regulation between galactose and N-acetylglucosamine metabolism was similar. By contrast, cross-regulation between lactose and maltose metabolism appeared to be due to generation of internal maltosaccharides in lactose-grown cells and may be physiologically significant. Lactose is of restricted distribution: it is normally found together with maltosaccharides, which are starch degradation products, in the mammalian intestine. Strains designated MG1655 and obtained from other sources differed from the Stock Center isolate and each other in several respects. We confirmed that use of other E. coli strains with MG1655-based DNA microarrays works well, and hence these arrays can be used to study any strain of interest. The responses to nitrogen limitation of two urinary tract isolates and an intestinal commensal strain isolated recently from humans were remarkably similar to those of MG1655.

The ammonium͞methylammonium transport (Amt) proteins of enteric bacteria and their homologues, the methylammonium͞ ammonium permeases of Saccharomyces cerevisiae, are required for fast growth at very low concentrations of the uncharged species NH3. For example, they are essential at low ammonium (NH4 ؉ ؉ NH3) concentrations under acidic conditions. Based on growth studies in batch culture, the Amt protein of Salmonella typhimurium (AmtB) cannot concentrate either NH3 or NH4 ؉ and this organism appears to have no means of doing so. We now show that S. typhimurium releases ammonium into the medium when grown on the alternative nitrogen source arginine and that outward diffusion of ammonium is enhanced by the activity of AmtB. The latter result indicates that AmtB acts bidirectionally. We also confirm a prediction that the AmtB protein would be required at pH 7.0 in ammonium-limited continuous culture, i.e., when the concentration of NH3 is <50 nM. Together with our previous studies, current results are in accord with the view that Amt and methylammonium͞ammonium permease proteins increase the rate of diffusion of the uncharged species NH3 across the cytoplasmic membrane. These proteins are examples of protein facilitators for a gas. Members of the ammonium͞methylammonium transport (Amt) protein family are found in all three domains of life (1, 2). These cytoplasmic membrane proteins are involved in acquisition of ammonium, often the best nitrogen source for microbes. As a function of pH, ammonium (NH 3 ϩ NH 4 ϩ ) exists as a mixture of uncharged (gaseous) and protonated forms. [The pKa of ammonium is 9.25 (3).] Because the uncharged species, NH 3 , diffuses freely across phospholipid bilayers, there has been controversy about the need for a transport system(s) for ammonium and about the species being transported. Although many reports propose that Amt and methylammonium͞ammonium permease (MEP) proteins actively transport the charged species NH 4 ϩ (4, 5), there are several lines of evidence against this proposal (6, 7). First, apparent concentration of the ammonium analog, [ 14 C]methylammonium ( 14 CH 3 NH 2 ϩ 14 CH 3 NH 3 ϩ ), in an Amt͞MEP-dependent manner, was accounted for by metabolic trapping in enteric bacteria and by diffusion trapping into vacuoles and other acidic compartments in fungi. In each case accumulation depended on a subsequent energy-requiring reaction, one catalyzed by glutamine synthetase in bacteria or the V-type H ϩ -ATPase in fungi. Hence neither example provides evidence for active transport of methylammonium. Second, growth studies in Salmonella typhimurium indicated that the Amt protein of this organism (AmtB) did not concentrate ammonium and that the organism had no means of doing so. Third, in batch culture, amt mutants of enteric bacteria and mep mutants of Saccharomyces cerevisiae had a profound growth defect at low ammonium concentrations only at acid pH, conditions under which the concentration of the uncharged species NH 3 is very low. This finding was in accord with the view th...

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

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