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).

Bordetella avium is a pathogen of poultry and is phylogenetically distinct from Bordetella bronchiseptica, Bordetella pertussis, and Bordetella parapertussis, which are other species in the Bordetella genus that infect mammals. In order to understand the evolutionary relatedness of Bordetella species and further the understanding of pathogenesis, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors from B. avium was confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present in B. avium but not in B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these.The genus Bordetella comprises eight species: B. pertussis, B. parapertussis, B. bronchiseptica, B. avium, B. hinzii, B. holmesii, B. trematum, and B. petrii. The genus is classified as a member of the beta-proteobacteria. Bordetellae are closely related to the genera Achromobacter and Alcaligenes, which include mostly environmental bacteria with some opportunistic pathogens (reviewed in reference 112). A phylogenetic analysis based on 16S rRNA genes places Alcaligenes as the most ancient, with Achromobacter and Bordetella deriving more recently from a single node and B. petrii and B. avium being most distantly related to all other Bordetella species (112). The very close phylogenetic relationship among B. pertussis, B. parapertussis, and B. bronchiseptica has been well established (110), and it is generally accepted that B. pertussis and B. parapertussis were differentiated from B. bronchiseptica by substantial gene loss (29, 71), diverging as much as 3.5 million years ago (30). B. pertussis, B. parapertussis, and B. bronchiseptica colonize the respiratory tracts of mammals (reviewed in reference 25), but their host ranges and the severity of the diseases they cause differ. B. pertussis is restricted to the human host and is the etiological agent of an acute respiratory disease known as pertussis or whooping cough. B. parapertussis is divided into two phylogenetically distinct subspecies/popula...

Bradyrhizobium japonicum expresses both Fur and Irr, proteins that mediate iron-dependent regulation of gene expression. Control of irr mRNA accumulation by iron was aberrant in a fur mutant strain, and Fur repressed an irr ::lacZ promoter fusion in the presence of iron. Furthermore, metaldependent binding of Fur to an irr gene promoter was demonstrated in a region with no significant similarity to the Fur-binding consensus DNA element. These data suggest that the modest control of irr transcription by iron is mediated by Fur. However, Irr protein levels were regulated normally by iron in the fur strain, indicating that Fur is not required for posttranscriptional control of the irr gene. Accordingly, regulation of hemB, a haem biosynthesis gene regulated by Irr, was controlled normally by iron in a fur strain. In addition, the hemA gene was shown to be controlled by Fur, but not by Irr. It was concluded that Fur cannot be the only protein by which B. japonicum cells sense and respond to iron, and that Irr may be involved in Furindependent signal transduction. Furthermore, iron-dependent regulation of haem biosynthesis involves both Irr and Fur.

Evidence in several microorganisms indicates that Amt proteins are gas channels for NH 3 and CH 3 NH 2 , and this has been confirmed structurally. Chlamydomonas reinhardtii has at least four AMT genes, the most reported for a microorganism. Under nitrogen-limiting conditions all AMT genes are transcribed and Chlamydomonas is sensitive to methylammonium toxicity. All 16 spontaneous methylammonium-resistant mutants that we analyzed had defects in accumulation of [ 14 C]methylammonium. Genetic crosses indicated that 12 had lesions in a single locus, whereas two each had lesions in other loci. Lesions in different loci were correlated with different degrees of defect in [ 14 C]methylammonium uptake. One mutant in the largest class had an insert in the AMT4 gene, and the insert cosegregated with methylammonium resistance in genetic crosses. The other 11 strains in this class also had amt4 lesions, which we characterized at the molecular level. Properties of the amt4 mutants were clearly different from those of rh1 RNAi lines. They indicated that the physiological substrates for Amt and Rh proteins, the only two members of their protein superfamily, are NH 3 and CO 2 , respectively.