Isolation of the Staphylococcus aureus hemCDBL gene cluster coding for early steps in heme biosynthesis.

We have recently reported [Kafala, B., Sasarman, A., 1994. Can. J. Microbiol. 40, 651 657] the cloning and sequencing of the Staphylococcus aureus hemB gene. This gene purportedly encodes the delta-aminolevulinic acid dehydratase of the heme pathway. In this present communication, we report the sequences and identities of three putative hem genes. Two of these genes are located immediately upstream from hemB. Complementation analysis of Escherichia coli and Salmonella typhimurium hemC and hemD mutants and the comparison of the Sa nucleotide sequences with those of Bacillus subtilis and Ec showed that these two open reading frames, ORF1 and ORF2, are likely to be the hemC gene coding for porphobilinogen deaminase and the hemD gene coding for uroporphyrinogen III synthase, respectively. The third hem gene, hemL, is located immediately downstream of hemB, and encodes glutamate 1-semialdehyde 2,1-aminotransferase. Sequencing of the region which extends past hemL indicates that no further hem genes are located downstream of hemL. In Sa, hemC, hemD, hemB and hemL are proposed to constitute a hem cluster encoding enzymes required for the synthesis of uroporphyrinogen III from glutamate 1-semialdehyde (GSA).

Non-iron porphyrins cause tumbling to blue light by an Escherichia coli mutant defective in hemG.

Previously we showed that an Escherichia coli hemH mutant, defective in the ultimate step of heme synthesis, ferrochelatase, is somewhat better than 100-fold more sensitive than its wild-type parent in tumbling to blue light. Here we explore the effect of a hemG mutant, defective in the penultimate step, protoporphyrinogen oxidase. We found that a hemG mutant also is somewhat better than 100-fold more sensitive in tumbling to blue light compared to its wild-type parent. The amount of non-iron porphyrins accumulated in hemG or hemH mutants was more than 100-fold greater than in wild type. The nature of these accumulated porphyrins is described. When heme was present, as in the wild type, the non-iron (non-heme) porphyrins were maintained at a relatively low concentration and tumbling to blue light at an intensity effective for hemG or hemH did not occur. The function of tumbling to light is most likely to allow escape from the lethality of intense light.

Cloning and sequence analysis of the hemB gene of Staphylococcus aureus.

The hemB gene is a member of the family of genes encoding enzymes of the porphyrin biosynthetic pathway and codes for the enzyme porphobilinogen synthase, which is responsible for the conversion of delta-aminolevulinic acid to porphobilinogen. To clone the hemB gene of Staphylococcus aureus we used Tn917-mediated transposon mutagenesis. Tn917 confers resistance to erythromycin and is carried by plasmid pTV1ts, which has thermosensitive replication. Hem mutants were selected by growth in the presence of kanamycin and erythromycin at 43 degrees C. Preliminary identification of the hem mutants was based on their dwarf colony growth, which could be restored to normal by hemin. DNA extracted from one of the hem mutants was digested with several restriction endonucleases and hybridized to a probe representing the XbaI-AvaI end of Tn917. A BglII-EcoRI fragment of 4.5 kb gave a positive signal and was cloned into pUC18. Transformants were identified by colony hybridization with the Tn917 probe. The positive clones were sequenced, starting from the transposon end. The results allowed us to identify an open reading frame whose nucleotide sequence presented a homology of 63% to the sequence of the hemB gene of Bacillus subtilis and of 55% to the sequence of the hemB gene of Escherichia coli K12. No other nucleotide sequences, except those belonging to known hemB genes, presented significant homologies to our sequence. The cloning of the hemB gene of S. aureus was confirmed by the ability of the gene to complement a hemB mutant of E. coli K12. To our knowledge, this is the first report of the cloning of a hem gene in S. aureus.

Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of Escherichia coli K12.

The hemG gene of Escherichia coli K12 is involved in the activity of protoporphyrinogen oxidase, the enzyme responsible for the conversion of protoporphyrinogen IX into protoporphyrin IX during heme and chlorophyll biosynthesis. The gene is located at min 87 on the genetic map of E. coli K12. The hemG gene was isolated by a mini-Mu in vivo cloning procedure. As expected, the hemG gene is able to restore normal growth to the hemG mutant, and the transformed cells display strong protoporphyrinogen oxidase activity. Sequencing of the hemG gene allowed us to identify an open reading frame of 546 nucleotides (181 amino acids), within the minimal fragment able to complement the mutant. The presumed molecular mass of the HemG protein is 21,202 Da, in agreement with values found by SDS-PAGE, in a DNA-directed coupled transcription-translation system. The identity of the first 18 amino acids at the amino-terminal end of the protein was confirmed by microsequencing. To our knowledge, this is the first cloning of a gene involved in the protoporphyrinogen oxidase activity of E. coli.

Cloning and nucleotide sequence of the hemA gene of Agrobacterium radiobacter.

The hemA gene of Agrobacterium radiobacter ATCC4718 was identified by hybridization with a hemA probe from Rhizobium meliloti and cloned by complementation of a hemA mutant of Escherichia coli K12. E. coli hemA transformants carrying the hemA gene of Agrobacterium showed delta-aminolevulinic acid synthetase (delta-ALAS) activity in vitro. The hemA gene was carried on a 4.4 kb EcoRI fragment which could be reduced to a 2.6 kb EcoRI-SstI fragment without affecting its complementing or delta-ALAS activity. The sequence of the hemA gene showed an open reading frame of 1215 nucleotides, which could code for a protein of 44,361 Da. This is very close to the molecular weight of the HemA protein obtained using an in vitro coupled transcription-translation system (45,000 Da). Comparison of amino acid sequences of the delta-ALAS of A. radiobacter and Bradyrhizobium japonicum showed strong homology between the two enzymes; less, but still significant, homology was observed when A. radiobacter and human delta-ALAS were compared. Primer extension experiments enabled us to identify two promoters for the hemA gene of A. radiobacter. One of these promoters shows some similarity to the first promoter of the hemA gene of R. meliloti.

Isolation and nucleotide sequence of the hemA gene of Escherichia coli K12.

The hemA gene of Escherichia coli K12 was cloned by complementation of a hemA mutant of this organism. Subcloning of the initial 6.0 kb HindIII fragment allowed the isolation of a 1.5 kb NheI-AvaI fragment which retained the ability to complement the hemA mutant. DNA sequencing by the dideoxy chain terminator method of Sanger showed the presence of an open reading frame (ORF) of 1254 nucleotides, which ends 6 nucleotides beyond the AvaI site. Primer extension experiments showed the existence of a putative transcription initiation site for the hemA gene, located at position 130. A possible promoter sequence was identified upstream from this transcription initiation site, and its functional activity was confirmed by the use of the pK01 promoter-probe vector. Protein synthesis in an in vitro coupled transcription-translation system showed a 46 kDa protein, which corresponds to the mol. wt. of the hemA protein, as deduced from the nucleotide sequence of the gene. No homology was found between the amino acid sequence of the hemA protein of E. coli K12 and known sequences of other delta-aminolevulinic acid synthases (delta-ALAS), suggesting that this protein is different from other delta-ALAS enzymes.

Nucleotide sequence of the hemB gene of Escherichia coli K12.

The hemB gene of Escherichia coli K12, coding for porphobilinogen synthase (PBG-S; syn., 5-aminolevulinic acid dehydratase, ALA-D), was cloned following insertion of an EcoRI fragment of plasmid F'13 into the mobilizable vector pCR1. The hybrid plasmid carrying the hemB gene was able to complement a hemB mutant of E. coli K12: not only was the PBG-S activity of the mutant restored after the acquisition of the hemB gene, but it was about ten times higher than that of the wild type. Subcloning of the original EcoRI fragment (14.6 kb) enabled us to locate the hemB gene on an NruI-HpaI fragment of about 1.1 kb. The hemB promoter was located toward the NruI end of the fragment, as shown by the use of the pKO promoter-probe series of vectors. Sequencing of the hemB gene indicated the presence of an open reading frame (ORF) of 1051 nucleotides, which should correspond to the HemB protein. Primer extension experiments enabled us to identify the 5' end of the hemB mRNA, and to deduce the -10 and -35 regions of the hemB promoter. Protein synthesis performed by an in vitro coupled transcription-translation system, showed the presence of a protein of about 35 kDa. This is in agreement with the molecular weight of the HemB protein (35.6 kDa), as deduced from the nucleotide sequence of the gene. Comparison of the amino acid sequences of E. coli and human PBG-S allowed the detection of several regions of strong homology between the two proteins. Two of these regions correspond, as expected, to the putative zinc-binding and catalytic sites of the human PBG-S.

Molecular cloning and sequencing of the hemD gene of Escherichia coli K-12 and preliminary data on the Uro operon.

DNA of plasmid pSAS1002TH (F' ilv+ hemD+ hemC+ cya+) was used to clone the hemD gene of Escherichia coli K-12. Due to poor transformability of the heme-deficient mutants, the restriction fragments of the F' plasmid were first cloned into a mobilizable derivative of pBR322, pSAS1211LP, which was then mobilized into a hemD recA mutant (E. coli SASX419AN). One recombinant plasmid, carrying a HindIII fragment of about 5 kilobases (kb), was shown to complement the hemD mutant and also a cya mutant of E. coli K-12, as well as a hemC mutant of Salmonella typhimurium LT2. Further subcloning of the insert enabled us to locate the hemD gene to a BamHI-PstI fragment (approximately 2.3 kb) which also carried the hemC gene. The hemD gene occupies a region close to the PstI end, since the deletion of a 0.6-kb fragment from this end resulted in loss of the ability to complement the hemD mutation. The use of the promoter-probe vector pK01 and the results of complementation showed that the hemD gene was transcribed under physiological conditions from the same promoter as the hemC gene, the direction of transcription being hemC-hemD. This allows us to define a new polycistronic operon of E. coli K-12, for which we propose the designation Uro operon. Sequencing of the hemD gene showed the presence of an open reading frame (ORF) of 738 nucleotides which could code for a protein with a molecular weight of 27,766, which should correspond to the hemD protein; the ORF starts with the last nucleotide of the hemC gene, the two genes having different reading frames. An ORF of at least 480 base pairs follows the hemD gene after a few nucleotides. The corresponding gene X, the function of which is unknown, might represent a third member of the Uro operon.

Construction of a shuttle vector and expression in Streptomyces lividans of the sulfonamide-resistance gene derived from Escherichia coli plasmid pSAS1206.

We have constructed a hybrid plasmid using Streptomyces lividans plasmid p1J101 and Escherichia coli plasmid pSAS1206. This plasmid, designated pFSH102, is able to replicate in both hosts and the sulfonamide-resistance gene encoded by pSAS1206 is phenotypically expressed in S. lividans.

A detailed physical and genetic map of two R.ColBM IncFIII plasmids.

The cleavage and genetic maps of two closely related R.ColBM IncFIII plasmids, designated pSAS1201 and pSAS1203, are presented. Restriction analysis of both plasmids with SstI, EcoRI, Bg/II, XhoI, HindIII, and Sa/I indicated that the maps of these two plasmids are superimposable with the exception of a 1.70-MDa DNA segment absent in pSAS1203. Our results provide the first physical and genetic maps of plasmids belonging to the IncFIII incompatibility group.

Suspected epidemiological relationships among strains of Escherichia coli O55:B5 confirmed by restriction pattern analysis of the carried plasmid (RColBM IncFIII).

Seven strains of Escherichia coli O55:B5g, isolated from independent cases of infantile diarrhoea, contained similar RColBM plasmids beloning to incompatibility group IncFIII. Suspecting an epidemiological link among all these cases we compared the restriction pattern of the corresponding RColBM plasmids after digestion with endonucleases EcoRI and BglII. These patterns were similar, confirming the suspected relationships among the seven cases of infantile diarrhoea. The patterns obtained with the RColBM plasmids from children were different from those obtained with two RColBM plasmids isolated from adults. The probable source of the seven cases of infantile diarrhoea was identified as a nursery.

Naturally occurring R.ColBM plasmids belonging to the IncFIII incompatibility group.

Two Escherichia coli strains isolated from urinary tract infections were resistant to streptomycin, kanamycin, neomycin, tetracycline and sulphonamides. The strains also produced colicins B and M. The resistance to streptomycin, kanamycin and neomycin and the ability to produce colicins B and M could be transferred to an E. coli K12 recipient. Resistance and colicinogeny markers were transferred together by conjugation, and did not segregate even after interrupted mating or phage P1-mediated transduction. Hence, the drug-resistance and colicinogeny markers were carried by the same plasmid, designated as R.ColBM plasmid. The two R.ColBM plasmids were Fi+ and produced an F-like pilus. They belonged to the IncFIII incompatibility group, being thus the first R plasmids identified in this group. The two plasmids were isolated and their molecular sizes were determined by electrophoresis in agarose gels and by contour length measurements. Both methods showed that both plasmids were about 52 megadaltons.

Mapping of a new hem gene in Escherichia coli K12.

A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection. The mutant, designated SASX38, accumulated uroporphyrin, coproporphyrin and protoporphyrin. Since it possessed normal ferrochelatase activity, it was assumed to be deficient in protoporphyrinogen oxidase activity. The gene affected in the mutant was designated hemG. Mapping of the hemG gene by phage P1-mediated transduction showed that it was located very close to the chlB gene (frequency of cotransduction 78.7%), between the metE and rha markers. This location is distinct from the other known hem loci in E. coli K12.

Uroporphyrin- and coproporphyrin I-accumulating mutant of Escherichia coli K12.

A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection. The mutant was deficient in uroporphyrinogen III cosynthase activity as indicated by the accumulation of uroporphyrin I and coproporphyrin. The mapping of the corresponding hemD gene by P1-mediated transduction showed that the new gene was located between ilv and cya, at min 83 on the chromosomal map of Escherichia coli K12.

Mapping of the hemE locus in Salmonella typhimurium.

A new type of heme-deficient mutant was isolated in Salmonella typhimurium by neomycin selection. The mutant was deficient in uroporphyrinogen decarboxylase activity, coded by the hemE gene. The hemE gene was located between the genes rif and thi at 128 min on the chromosomal map of S. typhimurium.

Uroporphyrinogen III cosynthase-deficient mutant of Salmonella typhimurium LT2.

A new type of heme-deficient mutant of Salmonella typhimurium LT2 was isolated using neomycin. The mutant, designated as strain SASY74, accumulated uroporphyrin I and coproporphyrin I. Extracts of the mutant converted 5-aminolevulinic acid to uroporphyrin I. Extracts of the mutant SASY74 and of the uroporphyrinogen synthase-deficient mutant SASY32 complemented each other and converted, when incubated together, 5-aminolevulinic acid to protoporphyrin. This finding excludes the possibility that uroporphyrinogen I synthase in strain SASY74 is deficient in its cosynthase-binding ability. Hence, the most probable explanation for the accumulation of uroporphyrin I and coproporphyrin I by the mutant is the lack of the uroporphyrinogen III cosynthase activity. This mutant is the first isolated in bacteria with such deficiency, and the mutation is analogous, as far as porphyrin synthesis is concerned, to human congenital porphyria. Mapping of the corresponding gene (hemD) by conjugation and P22-mediated transduction suggests the following gene order on the chromosome: ilv....hemC, hemD, cya....metE. The hemC and hemD genes are probably adjacent; this is the first case in which two hem genes of Enterobacteriaceae are contiguous on the chromosomal map.

Porphobilinogen-accumulating mutants of Salmonella typhimurium LT2.

Four independent porphobilinogen-accumulating mutants of Salmonella typhimurium LT2 were isolated by selecting for dwarf colony formation on neomycin agar media. Cell-free extracts of the parent strain, but not of the mutants, were able to convert 5-aminolaevulinic acid or porphobilinogen to porphyrins. The results indicated that the mutants were deficient in uroporphyrinogen I synthase (EC. 4.3.I. 8) activity: these are the first mutants of this type reported in S. typhimurium LT2. Mapping of the hemC locus (for uroporphyrinogen I synthase) by F-mediated conjugation and by P22-mediated transduction showed the gene sequence ilvEDAC-hemC-cya-metE.

Uroporphyrin-accumulating mutant of Escherichia coli K-12.

An uroporphyrin III-accumulating mutant of Escherichia coli K-12 was isolated by neomycin. The mutant, designated SASQ85, was catalase deficient and formed dwarf colonies on usual media. Comparative extraction by cyclohexanone and ethyl acetate showed the superiority of the former for the extraction of the uroporphyrin accumulated by the mutant. Cell-free extracts of SASQ85 were able to convert 5-aminolevulinic acid and porphobilinogen to uroporphyrinogen, but not to copro- or protoporphyrinogen. Under the same conditions cell-free extracts of the parent strain converted 5-aminolevulinic to uroporphyringen, coproporphyrinogen, and protoporphyrinogen. The conversion of porphobilinogen to uroporphyrinogen by cell-free extracts of the mutant was inhibited 98 and 95%, respectively, by p-chloromercuribenzoate and p-chloromercuriphenyl-sulfonate, indicating the presence of uroporphyrinogen synthetase activity in the extracts. Spontaneous transformation of porphobilinogen to uroporphyrin was not detectable under the experimental conditions used [4 h at 37 C in tris(hydroxymethyl)aminomethane-potassium phosphate buffer, pH 8.2]. The results indicate a deficient uroporphyrinogen decarboxylase activity of SASQ85 which is thus the first uroporphyrinogen decarboxylase-deficient mutant isolated in E. coli K-12. Mapping of the corresponding locus by P1-mediated transduction revealed the frequent joint transduction of hemE and thiA markers (frequency of co-transduction, 41 to 44%). The results of the genetic analysis suggest the gene order rif, hemE, thiA, metA; however, they do not totally exclude the gene order rif, thiA, hemE, metA.