Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes͞operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, Ϸ2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and 70 -dependent promoters.

Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo-and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.

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.

Homologues of the amtB gene of enteric bacteria exist in all three domains of life. Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH 4 ؉ concentrations. We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH 4 ؉ concentrations in batch culture, but only at pH values below 7. In addition, we find that the growth defect of an S. cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium͞methylammonium transport B (AmtB) protein [called methylammonium͞ammonium permeases (MEP)] that was observed at pH 6.1 is relieved at pH 7.1. These results provide direct evidence that AmtB participates in acquisition of NH 4 ؉ ͞NH 3 in bacteria as well as eucarya. Because NH 3 is the species limiting at low pH for a given total concentration of NH 4 ؉ ؉ NH 3 , results with both organisms indicate that AmtB͞MEP proteins function in acquisition of the uncharged form. We confirmed that accumulation of [ 14 C]methylammonium depends on its conversion to ␥-Nmethylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH 4 ؉ ͞NH 3 . Hence, contrary to previous views, we propose that AmtB͞MEP proteins increase the rate of equilibration of the uncharged species, NH 3 , across the cytoplasmic membrane rather than actively transporting-that is, concentrating-the charged species, NH 4 ؉ .

High-pressure liquid chromatography-tandem mass spectrometry was used to obtain a protein profile of Escherichia coli strain MG1655 grown in minimal medium with glycerol as the carbon source. By using cell lysate from only 3 ؋ 10 8 cells, at least four different tryptic peptides were detected for each of 404 proteins in a short 4-h experiment. At least one peptide with a high reliability score was detected for 986 proteins. Because membrane proteins were underrepresented, a second experiment was performed with a preparation enriched in membranes. An additional 161 proteins were detected, of which from half to two-thirds were membrane proteins. Overall, 1,147 different E. coli proteins were identified, almost 4 times as many as had been identified previously by using other tools. The protein list was compared with the transcription profile obtained on Affymetrix GeneChips. Expression of 1,113 (97%) of the genes whose protein products were found was detected at the mRNA level. The arithmetic mean mRNA signal intensity for these genes was 3-fold higher than that for all 4,300 protein-coding genes of E. coli. Thus, GeneChip data confirmed the high reliability of the protein list, which contains about one-fourth of the proteins of E. coli. Detection of even those membrane proteins and proteins of undefined function that are encoded by the same operons (transcriptional units) encoding proteins on the list remained low.

Although Rhesus (Rh) proteins are best known as antigens on human red blood cells, they are not restricted to red cells or to mammals, and hence their primary biochemical functions can be studied in more tractable organisms. We previously established that the Rh1 protein of the green alga Chlamydomonas reinhardtii is highly expressed in cultures bubbled with air containing high CO 2 (3%), conditions under which Chlamydomonas grows rapidly. By RNA interference, we have now obtained Chlamydomonas rh mutants (epigenetic), which are among the first in nonhuman cells. These mutants have essentially no mRNA or protein for RH1 and grow slowly at high CO 2, apparently because they fail to equilibrate this gas rapidly. They grow as well as their parental strain in air and on acetate plus air. However, during growth on acetate, rh1 mutants fail to express three proteins that are known to be down-regulated by high CO 2: periplasmic and mitochondrial carbonic anhydrases and a chloroplast envelope protein. This effect is parsimoniously rationalized if the small amounts of Rh1 protein present in acetate-grown cells of the parental strain facilitate leakage of CO 2 generated internally. Together, these results support our hypothesis that the Rh1 protein is a bidirectional channel for the gas CO 2. Our previous studies in a variety of organisms indicate that the only other members of the Rh superfamily, the ammonium͞methylammonium transport proteins, are bidirectional channels for the gas NH 3. Physiologically, both types of gas channels can apparently function in acquisition of nutrients and͞or waste disposal.T he Rhesus (Rh) blood group substance, one of the most abundant proteins in red cell membranes, was discovered over six decades ago (1). It is composed of the two antigenic Rh30 proteins and the Rh-associated glycoprotein, RhAG (2-9), which is most closely related to the ancestor of all other Rh proteins (ref. 8 and J. Peng and C.-H. Huang, personal communication). The biochemical function of Rh proteins, which are predicted to have 12 transmembrane-spanning segments, remains controversial (10-12). Human RhAG was reported to be an ammonium import͞methylammonium export system when expressed in Saccharomyces cerevisiae (13) and an ammonium-(methylammonium)͞proton exchanger in oocytes injected with RhAG cRNA (14). Moreover, Rh null red blood cells were found to accumulate more of the ammonium analogue [ 14 C]methylammonium than normal red cells and to lose it less rapidly after being preloaded, leading to the proposal that the Rh blood group substance was an ammonium(methylammonium) export system (15). Although inconsistent, all of the studies cited above concluded that Rh proteins, like their only known paralogues, the ammonium͞methylammonium transport (Amt) proteins [also called methylammonium permeases (Mep) in Saccharomyces cerevisiae], were active transport systems for the ion NH 4 ϩ . Disruption of an Rh gene in the slime mold Dictyostelium discoideum yielded no phenotype (16).Contrary to views of others, we have prop...

The function of the Rhesus (Rh) complex in the human red cell membrane has been unknown for six decades. Based on the organismal, organ, and tissue distribution of Rh proteins, and on our evidence that their only known paralogues, the ammonium and methylammonium transport proteins (also called methylammonium permeases), are gas channels for NH 3, we recently speculated that Rh proteins are biological gas channels for CO 2. Like NH 3, CO2 differs from other gases in being readily hydrated. We have now tested our speculation by studying expression of the RH1 gene in the photosynthetic microbe Chlamydomonas reinhardtii. Expression of RH1 was high for cells grown in air supplemented with 3% CO 2 or shifted from air to high CO2 (3%) for 3 h. Conversely, RH1 expression was low for cells grown in air (0.035% CO 2) or shifted from high CO2 to air for 3 h. These results make viable the hypothesis that Rh1 and Rh proteins generally are gas channels for CO 2.T he Rhesus (Rh) blood group substance is the second most abundant protein in human red cell membranes (Ϸ10 5 copies per cell) (1). The related RhAG and Rh30 proteins, which constitute this complex (2, 3), have only one known paralogue, the ammonium and methylammonium transport (Amt) proteins [also called methylammonium permeases (MEP)] (4). Marini and colleagues (5) reported that both Amt͞MEP proteins and the human RhAG and RhCG proteins are active transporters for NH 4 ϩ . Their conclusion regarding Rh proteins was based on the properties of Saccharomyces cerevisiae strains lacking function of its three MEP proteins and carrying cloned human Rh genes. Contrary to the views of Marini et al., we have provided several lines of evidence that Amt and MEP proteins are gas channels for NH 3 and have speculated that Rh proteins are gas channels for CO 2 (6-9). To test the viability of our speculation regarding Rh, we have studied expression of the RH1 gene in the green alga Chlamydomonas reinhardtii, one of the few microbes to have RH genes. Materials and MethodsMedia and Growth Conditions. C. reinhardtii strains CC125 (137c; nit1 nit2 mtϩ) (10), 4Aϩ (nit1 nit2 mtϩ), and CC124 (nit1 nit2 mtϪ) were maintained at 24°C in TAP medium (11), under continuous illumination (40 mol photons m Ϫ2 s Ϫ1 ). Strain 4Aϩ was kindly provided by J.-D. Rochaix (Univ. of Geneva, Switzerland). For growth in high CO 2 , cells were cultured in 1 l bottles containing 700 ml of TP(-N) medium (TAP medium without acetate and nitrogen; ref. 11) under constant illumination (170 mol photons m Ϫ2 s Ϫ1 ) and were bubbled with air enriched with 3% (vol/vol) CO 2 . The nitrogen source was NH 4 Cl (10 mM), arginine (2.5 mM), or hypoxanthine (2.5 mM), as indicated. For growth in low CO 2 , cultures were bubbled with ordinary air [0.035% (vol/vol) CO 2 ]. Chlorophyll aϩb content was estimated after extracting cells with 96% (vol/vol) ethanol (12).

In indeterminate alfalfa nodules, the establishment of the senescent zone IV, in which both symbionts undergo simultaneous degeneration, has been considered, until now, as the end point of the symbiotic interaction. However, we now describe an additional zone, zone V, proximal to the senescent zone IV and present in alfalfa nodules more than 6 weeks old. In zone V, a new round of bacterial release occurs from remaining infection threads, leading to the reinvasion of plant cells that have completely senesced. These intracellular rhizobia are rod shaped and do not display the ultrastructural differentiation features of bac-teroids observed in the more distal zones of the nodule. Interestingly, we have found that oxygen is available in zone V at a concentration compatible with both bacterial development and nitrogen fixation gene expression in newly released rhizobia. However, this expression is not correlated with acetylene reduction. Moreover, the pattern of nifH expression in this zone, as well as new data relating to expression in zone II, strongly suggest that nifH transcription in the nodule is under the control of a negative regulator in addition to oxygen. Our results support the conclusion that zone V is an ecological niche where intracellular rhizobia take advantage of the interaction for their exclusive benefit and live as parallel saprophytic partners. The demonstration of such an advantage for rhizobia in nodules was the missing evidence that Rhizobium-legume interactions are indeed symbiotic and, in particular, suggests that benefits to the two partners are associated with different developmental stages within the nodule.