Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation.

We present genetic and structural analyses of a fix operon conserved among rhizobia, fixGHI from Rhizobium meliloti. The nucleotide sequence of the operon suggests it may contain a fourth gene, fixS. Adjacent open reading frames of this operon showed an overlap between TGA stop codons and ATG start codons in the form of an ATGA motif suggestive of translational coupling. All four predicted gene products contained probable transmembrane sequences. FixG contained two cysteine clusters typical of iron-sulfur centers and is predicted to be involved in a redox process. FixI was found to be homologous with P-type ATPases, particularly with K+ pumps from Escherichia coli and Streptococcus faecalis but also with eucaryotic Ca2+, Na+/K+, H+/K+, and H+ pumps, which implies that FixI is a pump of a specific cation involved in symbiotic nitrogen fixation. Since prototrophic growth of fixI mutants appeared to be unimpaired, the predicted FixI cation pump probably has a specifically symbiotic function. We suggest that the four proteins FixG, FixH, FixI, and FixS may participate in a membrane-bound complex coupling the FixI cation pump with a redox process catalyzed by FixG.     

CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus.

The czcR gene, one of the two control genes responsible for induction of resistance to Co2+, Zn2+, and Cd2+ (czc system) in the Alcaligenes eutrophus plasmid pMOL30, was cloned and characterized. The 1,376-bp sequence upstream of the czcCBAD structural genes encodes a 41.4-kDa protein, the czcR gene product, transcribed in the opposite direction of that of the czcCBAD genes. The putative CzcR polypeptide (355 amino acid residues) contains 11 cysteine and 14 histidine residues which might form metal cation-binding sites. A czcC::lacZ reporter gene translational fusion was constructed, inserted into plasmid pMOL30 in A. eutrophus, and expressed under the control of CzcR. Zn2+, Co2+, and Cd2+, as well as Ni2+, Cu2+, Hg2+, and Mn2+ and even Al3+, served as inducers of beta-galactosidase activity. Besides the CzcR protein, the membrane-bound CzcD protein was essential for induction of czc. The CzcR and CzcD proteins display no sequence similarity to two-component regulatory systems of a sensor and a response activator type; however, CzcD has 34% identity with the ZRC-1 protein, which mediates zinc resistance in Saccharomyces cerevisiae (A. Kamizomo, M. Nishizawa, Y. Teranishi, K. Murata, and A. Kimura, Mol. Gen. Genet. 219:161-167, 1989).     

Supraoperonic clustering of pca genes for catabolism of the phenolic compound protocatechuate in Agrobacterium tumefaciens.

The protocatechuate branch of the beta-ketoadipate pathway comprises the last six enzymatic steps in the catabolism of diverse phenolic compounds to citric acid cycle intermediates. In this paper, the regulation and tight supraoperonic clustering of the protocatechuate (pca) genes from Agrobacterium tumefaciens A348 are elucidated. A previous study found that the pcaD gene is controlled by an adjacent regulatory gene, pcaQ, which encodes an activator. The activator responded to beta-carboxy-cis,cis-muconate and was shown to control the synthesis of at least three genes (pcaD and pcaHG). In this work, eight genes required for the catabolism of protocatechuate were localized within a 13.5-kb SalI region of DNA. Isolation and characterization of transposon Tn5 mutant strains facilitated the localization of pca genes. Five structural genes were found to respond to the tricarboxylic acid and to be contiguous in an operon transcribed in the order pcaDCHGB. These genes encode enzymes beta-ketoadipate enol-lactone hydrolase, gamma-carboxymuconolactone decarboxylase, protocatechuate 3,4-dioxygenase (pcaHG), and beta-carboxy-cis,cis-muconate lactonizing enzyme, respectively. Approximately 4 kb from the pcaD gene are the pcaIJ genes, which encode beta-ketoadipate succinyl-coenzyme A transferase for the next-to-last step of the pathway. The pcaIJ genes are transcribed divergently from the pcaDCHGB operon and are expressed in response to beta-ketoadipate. The pattern of induction of pca genes by beta-carboxy-cis,cis-muconate and beta-ketoadipate in A. tumefaciens is similar to that observed in Rhizobium leguminosarum bv. trifolii and is distinct from induction patterns for the genes from other microbial groups.     

Mu-induced polarity in the Escherichia coli K-12 ent gene cluster: evidence for a gene (entG) involved in the biosynthesis of enterochelin.

A strain of Escherichia coli K-12 has been isolated that carries a Mu bacteriophage-induced mutation in the ent gene cluster. Nutritional tests together with examination of the compounds accumulated by the mutant strain indicated that the mutant was blocked both in the synthesis of 2,3-dihydroxy-benzoate and its subsequent conversion into enterochelin. Enzymic complementation assays of the mutant with several mutants each affected in one of the ent genes showed that the Mu-induced mutant was entA-, entB-, entC+, entD+, entE+, and entF+. Since the mutant produced the entD, entE, and entF gene products but was unable to produce enterochelin from 2,3-dihydroxybenzoate, it must therefore be affected in an additional protein concerned with this conversion. It is therefore postulated that the Mu-induced mutation affects a previously unrecognized gene, entG. Genetic experiments indicate that the mutation in strain AN462 which affects the three ent genes is the result of a single insertion of Mu in the ent gene cluster. This polarity mutant therefore provides evidence that three of the ent genes are part of an operon.     

The Propanediol Utilization (pdu) Operon of Salmonella enterica Serovar Typhimurium LT2 Includes Genes Necessary for Formation of Polyhedral Organelles Involved in Coenzyme B12-Dependent 1,2-Propanediol Degradation

The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 contains genes needed for the coenzyme B(12)-dependent catabolism of 1,2-propanediol. Here the completed DNA sequence of the pdu operon is presented. Analyses of previously unpublished pdu DNA sequence substantiated previous studies indicating that the pdu operon was acquired by horizontal gene transfer and allowed the identification of 16 hypothetical genes. This brings the total number of genes in the pdu operon to 21 and the total number of genes at the pdu locus to 23. Of these, six encode proteins of unknown function and are not closely related to sequences of known function found in GenBank. Two encode proteins involved in transport and regulation. Six probably encode enzymes needed for the pathway of 1,2-propanediol degradation. Two encode proteins related to those used for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase. Five encode proteins related to those involved in the formation of polyhedral organelles known as carboxysomes, and two encode proteins that appear distantly related to those involved in carboxysome formation. In addition, it is shown that S. enterica forms polyhedral bodies that are involved in the degradation of 1,2-propanediol. Polyhedra are formed during either aerobic or anaerobic growth on propanediol, but not during growth on other carbon sources. Genetic tests demonstrate that genes of the pdu operon are required for polyhedral body formation, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with these polyhedra. This is the first evidence for a B(12)-dependent enzyme associated with a polyhedral body. It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual polyhedral bodies was not determined, but some possibilities are discussed.     

Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12.

Previous studies have shown that narL+ is required for nitrate induction of nitrate reductase synthesis and for nitrate inhibition of fumarate reductase synthesis in Escherichia coli. We cloned narL on a 5.1-kilobase HindIII fragment. Our clone also contained a previously unidentified gene, which we propose to designate as narX, as well as a portion of narK. Maxicell experiments indicated that narL and narX encode proteins with approximate MrS of 28,000 and 66,000, respectively. narX insertion mutations reduced nitrate reductase structural gene expression by less than twofold. Expression of phi (narL-lacZ) operon fusions was weakly induced by nitrate but was indifferent to aerobiosis and independent of fnr. Expression of phi (narX-lacZ) operon fusions was induced by nitrate and was decreased by narL and fnr mutations. A phi (narK-lacZ) operon fusion was induced by nitrate, and its expression was fully dependent on narL+ and fnr+. Analysis of these operon fusions indicated that narL and narX are transcribed counterclockwise with respect to the E. coli genetic map and that narK is transcribed clockwise.     

Nucleotide sequence of the thymidylate synthase B and dihydrofolate reductase genes contained in one Bacillus subtilis operon

The nucleotide sequence of the thymidylate synthase B (thyB) and dihydrofolate reductase (dfrA) gene regions from wild-type and trimethoprim-resistant (TpR) mutant strains of Bacillus subtilis 168 was determined. The sequenced region contains two open reading frames, ORF1 and ORF2, which correspond to thyB and dfrA, respectively, and overlap by one nucleotide. The thyB-dfrA genes encode 267 and 168 amino acid polypeptides, respectively, and are present in the order of thyB - dfrA in 5'----3' orientation. This gene order differs from those which have been found in other organisms so far. S1 mapping analysis indicated that both genes were transcribed from a single promoter located upstream from the thyB gene. Thus, the genes belong to an operon. A nucleotide substitution from 'A' in the wild type to 'C' in the TpR mutant was located in the dfrA gene region, with predicted conversion of isoleucine-95 (wild type) to leucine-95 (mutant) in dihydrofolate reductase (DHFR). It is suggested that the affinity between DHFR and Tp is reduced by this alteration.     

Identification of a locus involved in the utilization of iron by Actinobacillus pleuropneumoniae

Abstract The cloned afu locus of Actinobacillus pleuropneumoniae restored the ability of an Escherichia coli K-12 mutant ( aroB ) to grow on iron-limited media. DNA sequence analysis of the fragment showed that there are three genes designated afuA, afuB and afuC (Actinobacillus ferric uptake) that encode products similar to the SfuABC proteins of Serratia marcescens , the HitABC proteins of Haemophilia influenzae , the FbpABC proteins of Neisseria gonorrhoeae and the YfuABC proteins of Yersinia enterocolitica . The three genes encode a periplasmic iron-binding protein (AfuA), a highly hydrophobic integral cytoplasmic membrane protein with two consensus permease motifs (AfuB) and one hydrophilic peripheral cytoplasmic membrane protein with Walker ATP-binding motifs (AfuC), respectively. This system has been shown to constitute a periplasmic binding protein-dependent iron transport system in these organisms. The afuABC operon is locating approximately 200 bp upstream of apxIC gene, but transcribed in opposite direction to the ApxI-toxin genes.     

Molecular characterization, nucleotide sequence, and expression of the fliO, fliP, fliQ, and fliR genes of Escherichia coli.

The fliL operon of Escherichia coli contains seven genes that are involved in the biosynthesis and functioning of the flagellar organelle. DNA sequences for the first three genes of this operon have been reported previously. A 2.2-kb PstI restriction fragment was shown to complement known mutant alleles of the fliO, fliP, fliQ, and fliR genes, the four remaining genes of the fliL operon. Four open reading frames were identified by DNA sequence analysis and correlated to their corresponding genes by complementation analysis. These genes were found to encode very hydrophobic polypeptides with molecular masses of 11.1, 26.9, 9.6, and 28.5 kDa for FliO, FliP, FliQ, and FliR, respectively. Analysis of recombinant plasmids in a T7 promoter-polymerase expression system enabled us to identify three of the four gene products. On the basis of DNA sequence analysis and in vivo protein expression, it appears that the fliP gene product is synthesized as a precursor protein with an N-terminal signal peptide of 21 amino acids. The FliP protein was homologous to proteins encoded by a DNA sequence upstream of the flaA gene of Rhizobium meliloti, to a gene involved in pathogenicity in Xanthomonas campestris pv. glycines, and to the spa24 gene of the Shigella flexneri. The latter two genes encode proteins that appear to be involved in protein translocation, suggesting that the FliP protein may have a similar function.