Complementation of a mutant of Rhodobacter sphaeroides defective in photosynthetic CO 2 reduction led to the identification of a gene which encodes a protein that is related to a class of sensor kinases involved in bacterial signal transduction. The nucleotide sequence and deduced amino acid sequence led to the finding that the gene which complemented the mutant is the regB (prrB) gene, previously isolated from both R. sphaeroides and Rhodobacter capsulatus and shown to regulate the anaerobic expression of structural genes required for the synthesis of the reaction center and light-harvesting systems of these organisms. The current investigation indicates that in addition to its role in the regulation of photosystem biosynthesis, regB (prrB) of R. sphaeroides is intimately involved in the positive regulation of the cbb I and cbb II Calvin cycle CO 2 fixation operons. In addition to regulating the expression of structural genes encoding enzymes of the primary pathway for CO 2 fixation in R. sphaeroides, regB was also found to be required for the expression of a gene(s) important for the putative alternative CO 2 fixation pathway(s) of this organism. A mutation in regB also blocked expression of structural genes of the cbb regulon in a strain of R. sphaeroides capable of aerobic CO 2 -dependent growth in the dark. It is thus apparent that regB is part of a two-component system and encodes a sensor kinase involved in the global regulation of both anoxygenic light-dependent-and oxygenic light-independent CO 2 fixation as well as anoxygenic photosystem biosynthesis.Rhodobacter sphaeroides is a nonsulfur purple bacterium capable of both anoxygenic photosynthetic growth and vigorous aerobic growth in the dark. Under photolithoautotrophic growth conditions, this organism may be cultured with H 2 being used as an electron donor and CO 2 being used as the electron acceptor and sole carbon source. R. sphaeroides also employs various organic compounds as electron donors and major carbon sources (photoheterotrophic growth), and several organic acids, such as malate, succinate, and butyrate, are especially well suited for this growth mode. Although the role of CO 2 fixation is diminished under photoheterotrophic conditions, CO 2 is still assimilated primarily through the Calvin cycle (33). Thus, ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), the key enzyme of the primary CO 2 fixation pathway (14), is essential for photosynthetic growth when CO 2 is the major carbon source or prime electron acceptor. Interestingly, there are two distinct forms of Rubisco in R. sphaeroides, each of which, by itself, is capable of supporting photolithoautotrophic and photoheterotrophic growth (7). When the form I and form II Rubisco genes are both deleted, as in the case of R. sphaeroides 16 (a Rubisco double deletion mutant, cbbL cbbS cbbM), the organism is no longer capable of photolithoautotrophic growth in an H 2 -CO 2 atmosphere; under photoheterotrophic growth conditions in the presence of malate, growth is also impossible unl...

The diphtheria toxin repressor (DtxR) uses Fe 2؉ as a corepressor and inhibits transcription from ironregulated promoters (IRPs) in Corynebacterium diphtheriae. A new IRP, designated IRP6, was cloned from C. diphtheriae by a SELEX-like procedure. DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in an Escherichia coli system was iron dependent. The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria. IRP1 and IRP6 were repressed under high-iron conditions in wild-type C. diphtheriae C7(␤), but they were expressed constitutively in C7(␤) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake. A clone of the wild-type irp6 operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-type irp1 operon failed to complement any of these strains.Complementation by subclones of pCM6ABC demonstrated that mutant alleles of irp6A, irp6C, and irp6B were responsible for the phenotypes of HC1, HC4, and HC5, respectively. The irp6A allele in HC1 and the irp6B allele in HC5 encoded single amino acid substitutions in their predicted protein products, and the irp6C allele in HC4 caused premature chain termination of its predicted protein product. Strain HC3 was found to have a chain-terminating mutation in dtxR in addition to a missense mutation in its irp6B allele. These findings demonstrated that the irp6 operon in C. diphtheriae encodes a putative ABC transporter, that specific mutant alleles of irp6A, irp6B, and irp6C are associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by the irp6 operon.

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Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na ؉ /melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.