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

We have recently reported [Kafala, B., Sasarman, A., 1994.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

Role of menaquinone in nitrate respiration in Staphylococcus aureus.

Two menaquinone-deficient and one aromatic-deficient mutants of Staphylococcus aureus were unable to reduce nitrate to nitrite. Reinitiation of menaquinone synthesis in the aromatic-deficient mutant by growing it with shikimic acid restored its nitrate respiratory activity. The results clearly demonstrate a role for menaquinone in nitrate respiration in Staphylococcus aureus.