Oxygen-regulated steps in the Rhodobacter capsulatus tetrapyrrole biosynthetic pathway.

The effect of exogenous aminolevulinate and porphobilinogen on protoporphyrin accumulation in Rhodobacter capsulatus was measured. Oxygen inhibited protoporphyrin accumulation in strain AJB456, a bchH mutant, even in the presence of exogenous aminolevulinate, suggesting that some step in the formation of protoporphyrin from aminolevulinate is regulated by oxygen. In contrast, in the presence of exogenous porphobilinogen, oxygen did not inhibit protoporphyrin accumulation. The results presented in this study indicate that oxygen regulates the formation of porphobilinogen from aminolevulinate.     

Cloning and overexpression of the Rhodobacter capsulatus hemH gene.

In photosynthetically grown Rhodobacter capsulatus, heme is a qualitatively minor end product of the common tetrapyrrole pathway, but it may play a significant regulatory role. Heme is synthesized from protoporphyrin by the product of the hemH gene, ferrochelatase. We have cloned the R. capsulatus hemH gene by complementation of an Escherichia coli hemH mutant. When a plasmid carrying the hemH gene is returned to R. capsulatus, ferrochelatase activity increases, aminolevulinate synthase activity decreases, and bacteriochlorophyll levels are dramatically lowered. This is the first in vivo evidence to suggest that heme feedback inhibits aminolevulinate synthase in R. capsulatus, thereby reducing porphyrin synthesis.     

Cloning of the Rhodobacter capsulatus hemA gene.

Portions of the Rhodobacter capsulatus hemA gene have been cloned from a hemA::Tn5 insertion strain into the lambda bacteriophage derivative EMBL3. A cosmid containing the wild-type R. capsulatus hemA gene was isolated by complementation of the hemA::Tn5 mutant. The cosmid contains a 1.4-kilobase EcoRI fragment that spans the hemA::Tn5 insertion site. The entire hemA gene is contained in this fragment and the adjacent 0.6-kilobase EcoRI fragment.     

Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins.

A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by Tn5 mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between the synthesis of c-type cytochromes and the expression of the tetrapyrrole biosynthetic pathway.     

Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene.

A Tn5-induced mutant of Bradyrhizobium japonicum, strain LORBF1, was isolated on the basis of the formation of fluorescent colonies, and stable derivatives were constructed in backgrounds of strains LO and I110. The stable mutant strains LOek4 and I110ek4 were strictly dependent upon the addition of exogenous hemin for growth in liquid culture and formed fluorescent colonies. The fluorescent compound was identified as protoporphyrin IX, the immediate precursor of protoheme. Cell extracts of strains LOek4 and I110ek4 were deficient in ferrochelatase activity, the enzyme which catalyzes the incorporation of ferrous iron into protoporphyrin IX to produce protoheme. Mutant strain I110ek4 could take up 55Fe from the growth medium, but, unlike the parent strain, no significant incorporation of radiolabel into heme was found. This observation shows that heme was not synthesized in mutant strain I110ek4 and that the heme found in those cells was derived from exogenous hemin in the growth medium. The putative protein encoded by the gene disrupted in strain LORBF1 and its derivatives was homologous to ferrochelatases from eukaryotic organisms. This homology, along with the described mutant phenotype, provides strong evidence that the disrupted gene is hemH, that which encodes ferrochelatase. Mutant strain I110ek4 incited nodules on soybean that did not fix nitrogen, contained few viable bacteria, and did not express leghemoglobin heme or apoprotein. The data show that B. japonicum ferrochelatase is essential for normal nodule development.     

Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis.

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3'-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tumefaciens.     

Specificity of the heme requirement for growth of Bacteroides ruminicola

Caldwell, D. R. (U.S. Department of Agriculture, Beltsville, Md.), D. C. White, M. P. Bryant, and R. N. Doetsch. Specificity of the heme requirement for growth of Bacteroides ruminicola. J. Bacteriol. 90:1645-1654. 1965.-Previous studies suggested that most strains of Bacteroides ruminicola subsp. ruminicola require heme for growth. Present studies with heme-requiring strain 23 showed that protoheme was replaced by various porphyrins, uroporphyrinogen, coproporphyrinogen, certain iron-free metalloporphyrins, hemes, and certain heme-proteins containing readily removable hemes. Strain 23 utilized a wider range of tetrapyrroles than hemin-requiring bacteria previously studied. Inactive compounds included porphyrin biosynthesis intermediates preceding the tetrapyrrole stage and related compounds; uroporphyrin, chlorophyll, pheophytin, phycoerythrin, bilirubin, pyrrole, FeSO(4) with or without chelating agents; and representative ferrichrome compounds. Strain 23, two other strains representing predominant biotypes of B. ruminicola subsp. ruminicola, and one closely related strain grew in media containing heme-free protoporphyrin, mesoporphyrin, hematoporphyrin, or deuteroporphyrin, apparently inserting iron into several nonvinyl porphyrins. Porphobilinogen and porphyrin synthesis, apparently via the commonly known heme synthesis pathway, occurred during growth of heme-independent B. ruminicola subsp. brevis strain GA33 in a tetrapyrrole-free medium containing delta-aminolevulinic acid, but delta-aminolevulinic acid metabolism to porphobilinogen or porphyrins could not be detected in cells of heme-requiring strain 23 grown in the same medium with hemin added. Growth of strain 23 with uroporphyrinogen, coproporphyrinogen, or protoporphyrin IX replacing hemin suggests that part of the commonly known heme-biosynthesis pathway is present in this strain, but nutritional and metabolic evidence indicates that some or all of the enzymes synthesizing the tetrapyrrole nucleus from linear molecules are lacking or inactive.     

Isolation and Genetic Analysis of an Agrobacterium tumefaciens Avirulent Mutant with a Chromosomal Mutation Produced by Transposon Mutagenesis

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harbouring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent- mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3′-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tu?efaciens.     

Mutant Strains of Rhodopseudomonas spheroides Lacking δ-Aminolevulinate Synthase: Growth, Heme, and Bacteriochlorophyll Synthesis

Two mutant strains of Rhodopseudomonas spheroides were described which lacked delta-aminolevulinate synthase activity. They required delta-aminolevulinate for growth; they did not respond to protoporphyrin or magnesium photoporphyrin, and only poorly to hemin. Synthesis of cytochromes and heme by mutant H-4 was dependent upon delta-aminolevulinate; this strain did not form bacteriochlorophyll either with or without delta-aminolevulinate and, consequently, grew only under aerobic conditions. Mutant H-5 formed bacteriochlorophyll in response to delta-aminolevulinate and grew both anaerobically in the light and aerobically in the dark; the amount of delta-aminolevulinate needed for optimal anaerobic growth was higher than that required aerobically. Synthesis of bacteriochlorophyll and heme by suspensions of mutant H-5 incubated anaerobically in the light was dependent upon delta-aminolevulinate; bacteriochlorophyll production was completely inhibited by high aeration and by puromycin. The mutants differed in their ability to take up radioactive delta-aminolevulinate from the external environment; mutant H-5 was less active than mutant H-4 or the wild type. It was suggested that R. spheroides made only one form of delta-aminolevulinate synthase, which provided delta-aminolevulinate for bacteriochlorophyll and heme synthesis.     

Isolation of a Rhodobacter capsulatus bioB mutant and cloning of the bioB gene.

We isolated a Tn5 insertion mutant of Rhodobacter capsulatus which requires biotin for growth. Crossfeeding studies with Escherichia coli bio mutants indicated that the insertion is in the bioB gene. A cosmid that complements the bioB insertion was isolated, and the bioB gene was localized to a 2.85-kilobase EcoRI-PstI fragment.     

Role of PUG1 in inducible porphyrin and heme transport in Saccharomyces cerevisiae

Unlike pathogenic fungi, the budding yeast Saccharomyces cerevisiae is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of PUG1 (protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing PUG1 exhibited decreased accumulation of [(55)Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of PUG1 overexpression on intracellular heme pools, we used a CYC1-lacZ reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.     

Construction of a single-transposon-insertion mutant in Rhizobium sp. strain TAL1145 from a double-insertion mutant

A method for developing a single-transposon-insertion mutant from a double-insertion mutant in Rhizobium is described. An exopolysaccharide (EPS)-defective mutant containing two Tn 5-lacZ insertions was complemented with cloned wild-type DNA for EPS synthesis. One of the Tn 5-lacZ insertions from the mutant was transferred to the complementing plasmid by homologous recombination. The plasmid containing the Tn 5-lacZ insertion in the gene involved in EPS synthesis was transferred into the wild-type strain and the Tn 5-lacZ was homogenized to obtain an EPS-defective mutant with a single Tn 5-lacZ insertion.     

先天性红细胞生成性卟啉症患者酶活性的研究

先天性红细胞生成性卟啉症（ｃｏｎｇｅｎｉｔａｌｅｒｙ－ｔｈｒｏｐｏｉｅｔｉｃｐｏｒｐｈｙｒｉａ,ＣＥＰ）是Ｇｕｎｔｈｅｒ于１９１１年首先提出并加以描述,有时亦称Ｇｕｎｔｈｅｒ病．该病是因遗传性缺陷所致卟啉代谢中有关酶的异常造成的卟啉代谢紊乱而发生的一...     

An Azorhizobium caulinodans ORS571 locus involved in lipopolysaccharide production and nodule formation on Sesbania rostrata stems and roots.

Azorhizobium caulinodans ORS571 is able to nodulate roots and stems of the tropical legume Sesbania rostrata. An ORS571 Tn5 insertion mutant, strain ORS571-X15, had a rough colony morphology, was nonmotile, and showed clumping behavior on various media. When this pleiotropic mutant was inoculated on roots or stems of the host, no nodules developed (Nod-). Compared with the wild type, strain ORS571-X15 produced lipopolysaccharides (LPS) with an altered ladder pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggestive of a different O-antigen structure with a lower degree of polymerization. A cosmid clone, pRG20, that fully complemented all phenotypes of ORS571-X15 was isolated. With a 6-kb EcoRI subfragment of pRG20, clumping was relieved and nodulation was almost completely restored, but the strain was still nonmotile. LPS preparations from these complemented strains resembled the wild-type LPS, although minor quantitative and qualitative differences were evident. The sequence of the locus hit by the Tn5 in ORS571-X15 (the oac locus) revealed a striking homology with the rfb locus of Salmonella typhimurium, which is involved in O-antigen biosynthesis. The Tn5 insertion position was mapped to the oac3 gene, homologous to rfbA, encoding dTDP-D-glucose synthase. Biochemical assaying showed that ORS571-X15 is indeed defective in dTDP-D-glucose synthase activity, essential for the production of particular deoxyhexoses. Therefore, it was proposed that the O antigen of the mutant strain is devoid of such sugars.     

Sensitivity of a Salmonella typhimurium aspC mutant to sulfometuron methyl, a potent inhibitor of acetolactate synthase II.

Sulfometuron methyl is a potent and specific inhibitor of acetolactate synthase II in Salmonella typhimurium. Mutant strains sensitive to sulfometuron methyl on minimal medium were isolated following mutagenesis with Tn10. A conditionally auxotrophic insertion mutant, strain SMS409, which required aspartate at high temperatures or in the presence of tyrosine, was found among the 15 mutants isolated. The Tn10 insertion in strain SMS409 was mapped by conjugation and transduction to the region between aroA and pncB at 20 min on the chromosome of S. typhimurium; this location is similar to the genetic location of aspC in Escherichia coli. The specific activity of the aspC product, aspartate aminotransferase, was severely reduced in strain SMS409. This indicated that the Tn10 insertion in strain SMS409 inactivated aspC. An aspC mutant of E. coli was also inhibited by either sulfometuron methyl or tyrosine. We present a hypothesis which relates the observed alpha-ketobutyrate accumulation in sulfometuron methyl-inhibited cultures of strain SMS409 to aspartate starvation.     

Trehalase of Escherichia coli. Mapping and cloning of its structural gene and identification of the enzyme as a periplasmic protein induced under high osmolarity growth conditions

Escherichia coli can use the nonreducing disaccharide trehalose as a sole source of carbon and energy. Trehalose transport into the cell is mediated via the phosphotransferase system, and a mutant depleted in the nonspecific proteins enzyme I, HPr, and enzyme IIIGlc of this system was not only unable to grow on glucose or mannitol but also was strongly reduced in its ability to grow on trehalose. A pseudorevertant (PPA69) of such a deletion mutant was isolated that could again grow on glucose but not on mannitol. This revertant could now also use trehalose as a carbon source due to a constitutive galactose permease. PPA69 was subjected to Tn10 insertional mutagenesis, and a mutant (UE5) was isolated that no longer could use trehalose as a carbon source but could still grow on glucose. UE5 lacked a periplasmic trehalase that was present in PPA69. P1-mediated transduction of this Tn10 insertion (treA::Tn10) into a pts+ wild-type strain (MC4100) had no effect on the ability of MC4100 to grow on trehalose but resulted in loss of the periplasmic trehalase activity. The Tn10 insertion was mapped at 26 min on the E. coli linkage map and was 3% cotransducible with trp, in the order treA::Tn10, trp, cys. Trehalase activity in MC4100 was not induced by growth in the presence of trehalose but increased by about 10-fold when 0.6 M sucrose was added to minimal growth medium. Using the in vivo mini-Mu cloning system and growth on trehalose as selection, we cloned the treA gene. A 9-kilobase EcoRI fragment containing treA was subcloned into pBR322. Strains carrying this plasmid (pTRE5) contained about 100-fold higher periplasmic trehalase activity than PPA69 or MC4100. Using polyacrylamide gel electrophoresis, we found a protein of molecular weight 58,000 among the periplasmic proteins of the pTRE5-carrying strain that was absent in UE5. This protein was purified by ammonium sulfate precipitation and DEAE-Sepharose ion-exchange chromatography and contained all the trehalase activity. Minicells containing the treA+ plasmid produced, in addition to three other proteins, the 58,000-dalton protein. Thus, the plasmid carries the structural gene for the periplasmic trehalase and not just a gene involved in the regulation of the enzyme.     

Agrobacterium rhizogenes mutants that fail to bind to plant cells.

Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria to bind to carrot cells. The parent strain bound to carrot cells in greatest numbers in low-ionic-strength media such as 4% sucrose but still showed significant binding in Murashige-Skoog tissue culture medium. All of the mutants showed reduced binding in 4% sucrose after 2 h of incubation with carrot cells. One mutant was delayed in binding in 4% sucrose. This mutant and one other mutant also showed reduced binding to carrot cells in Murashige-Skoog medium. To determine whether the Tn5 insertion was responsible for the mutant phenotype, DNA containing the Tn5 insertion was cloned from the mutant bacteria and used to introduce Tn5 into the parent strain in the same location as in the original mutant by marker exchange. The resulting transconjugants had the same avirulent, nonattaching phenotype as the original mutants, suggesting that the mutant phenotype was due to the Tn5 insertion. The cloned DNA containing the Tn5 insertion was also tested for homology to DNA of known genes that affect attachment of Agrobacterium tumefaciens to plant cells by DNA hybridization. No homology to chv, att, or pscA clones was observed. In addition, cloned chv, att, and pscA genes from A. tumefaciens were unable to complement the attachment-minus A. rhizogenes mutants. Thus, the A. rhizogenes nonattaching mutants appear to be different from the previously described A. tumefaciens mutants.