A gene cluster encoding five enzymes of the mevalonate pathway had been cloned from Streptomyces sp. strain CL190. This gene cluster contained an additional ORF, orfD, encoding an unknown protein ...that was detected in some archaebacteria and some Gram-positive bacteria including Staphylococcus aureus. The recombinant product of orfD was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 37 kDa by SDS-polyacrylamide gel electrophoresis and 155 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a tetramer. The purified enzyme contained flavin mononucleotide (FMN) with the amount per tetramer being 1.4 to 1.6 mol/mol. The enzyme catalyzed the isomerization of isopentenyl diphosphate (IPP) to produce dimethylallyl diphosphate (DMAPP) in the presence of both FMN and NADPH. The Escherichia coli plasmid expressing orfD could complement the disrupted IPP isomerase gene in E. coli. These results indicate that orfD encodes an unusual IPP isomerase showing no sequence similarity to those of IPP isomerases identified to date. Based on the difference in enzymatic properties, we classify the IPP isomerases into two types: Type 2 for FMN- and NAD(P)H-dependent enzymes, and type 1 for the others. In view of the critical role of this isomerase in S. aureus and of the different enzymatic properties of mammalian (type 1) and S. aureus (type 2) isomerases, this unusual enzyme is considered to be a suitable molecular target for the screening of antibacterial drugs specific to S. aureus.
2-
C-Methyl-
d-erythritol 4-phosphate is transformed to 4-(cytidine 5′-diphospho)-2-
C-methyl-
d-erythritol in the presence of cytidine 5′-triphosphate by a novel
Escherichia coli enzyme, 2-
...C-methyl-
d-erythritol 4-phosphate cytidylyltransferase, involved in the nonmevalonate pathway.
A nonmevalonate pathway intermediate, 4-(cytidine 5′-diphospho)-2-
C-methyl-
d-erythritol, is transformed to its 2-phospho-derivative in the presence of ATP by a novel
Escherichia coli enzyme, ...4-(cytidine 5′-diphospho)-2-
C-methyl-
d-erythritol kinase.
2-Phospho-4-(cytidine 5′-diphospho)-2-
C-methyl-
d-erythritol was transformed to 2-
C-methyl-
d-erythritol 2,4-cyclodiphosphate by a novel
Escherichia coli enzyme involved in the nonmevalonate ...pathway.
We previously identified the fni gene of Streptomyces sp. strain CL190 as type 2 isopentenyl diphosphate (IPP) isomerase, which needs both FMN and NADPH for enzyme activity. An fni gene homolog, ...ypgA, was detected in the database of the Bacillus subtilis genome. However, the ypgA product was about 140 amino acids shorter in the N-terminal than the Streptomyces fni gene product. A database search found three new putative start codons in 129, 225, and 411 bases upstream of the original start codon of the ypgA gene. The longest gene product, which was named ypgA3, showed the most significant homology to the Streptomyces fni gene product. The ypgA3 gene was expressed with an N-terminal His-tag in Escherichia coli and the purified soluble protein was characterized in detail. The ypgA3 protein converted IPP to its isomer dimethylallyl diphosphate in the presence of both FMN and NADPH. The enzyme also catalyzed the reverse reaction in the presence of both the cofactors. Disruption of the ypgA3 gene was not lethal to B. subtilis. These results indicate that Bacillus ypgA3 gene is fni, a nonessential gene encoding type 2 IPP isomerase.
In 2003, a Leptospira survey was performed on Yoroshima Island of the Amami Islands located in the southwestern part of Japan. Seven Leptospira strains were isolated from the field rat Rattus rattus, ...which were identified as L. borgpetersenii by flaB sequencing, 16S rDNA sequencing and gyrB sequencing, and serovar Javanica was determined by a microscopic agglutination test. NotI‐long restriction fragment analysis indicated that these isolates were genetically indistinguishable from an isolate from the Okinawa Islands. The present results suggest that L. borgpetersenii is migrating into the Amami Islands in Japan.
A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a ...flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence. ---End of Page 8---
The relationships between
Borrelia species and the vector ticks,
Ixodes persulcatus and
Ixodes ricinus were examined in a molecular epidemiological study. We conducted a survey in the Moscow region ...which is a sympatric region for both species of tick. We examined 630 unfed
I. ricinus and
I. persulcatus, ticks collected from four different regions around Moscow within an area of 250
km
2. Eighty-four ticks were culture positive (13.3%) and the prevalence rate varied in each region from 5.7% to 42.3%. No difference was found between the total prevalence rate for both species. Eight
Borrelia afzelii-like variant isolates from
I. ricinus and
Clethrionomys glareolus were identified as
B. afzelii by flagellin gene and 16S rDNA sequence analyses. Most isolates from
I. ricinus were identified as
Borrelia garinii type 20047 and
B. afzelii. Two isolates were identified as
Borrelia burgdorferi sensu stricto (s.s.) and
Borrelia valaisiana, respectively, but no
B. garinii type NT29 was found. In contrast, isolates from
I. persulcatus were identified as both types 20047 and NT29 of
B. garinii, and
B. afzelii. No
B. burgdorferi s.s. isolate was found among isolates from
I. persulcatus.
A gene cluster encoding five enzymes of the mevalonate pathway had
been cloned from
Streptomyces
sp. strain CL190. This
gene cluster contained an additional ORF,
orfD
, encoding
an unknown protein ...that was detected in some archaebacteria and some
Gram-positive bacteria including
Staphylococcus aureus
.
The recombinant product of
orfD
was purified as a
soluble protein and characterized. The molecular mass of the enzyme was
estimated to be 37 kDa by SDS-polyacrylamide gel electrophoresis and
155 kDa by gel filtration chromatography, suggesting that the enzyme is
most likely to be a tetramer. The purified enzyme contained flavin
mononucleotide (FMN) with the amount per tetramer being 1.4 to 1.6
mol/mol. The enzyme catalyzed the isomerization of isopentenyl
diphosphate (IPP) to produce dimethylallyl diphosphate (DMAPP) in the
presence of both FMN and NADPH. The
Escherichia coli
plasmid expressing
orfD
could complement the disrupted
IPP isomerase gene in
E. coli
. These results indicate
that
orfD
encodes an unusual IPP isomerase showing no
sequence similarity to those of IPP isomerases identified to date.
Based on the difference in enzymatic properties, we classify the IPP
isomerases into two types: Type 2 for FMN- and NAD(P)H-dependent
enzymes, and type 1 for the others. In view of the critical role of
this isomerase in
S. aureus
and of the different
enzymatic properties of mammalian (type 1) and
S. aureus
(type 2) isomerases, this unusual enzyme is considered to be a suitable
molecular target for the screening of antibacterial drugs specific to
S. aureus
.
Previously, a novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium, which infests tortoises (Testudo graeca), by using Barbour-Stoenner-Kelly (BSK) II medium; the tick ...samples were taken from the Istanbul area in northwestern Turkey Güner et al. (2003). Microbiology 149, 2539-2544. Here is presented a detailed characterization of the spirochaete. Electron microscopy revealed that strain IST7T is morphologically similar to other spirochaetes of the genus Borrelia and possesses 15 to 16 flagellae that emerge from both polar regions. PFGE analysis revealed the genome to comprise a linear chromosome of approximately 1 Mb; two large linear plasmids of approximately 145 and 140 kb, and several small plasmids ranging from 50 to 20 kb in size were also found. The 16S rRNA gene sequence of this Borrelia isolate exhibited 99.4 to 99.8 % identity with other strains isolated from H. aegyptium and less than 99 % similarity with those of other Borrelia species. A phylogenetic tree, generated from 16S rRNA gene sequences, demonstrated that the spirochaete isolates from H. aegyptium clustered together and branched off from both Lyme-disease-related and relapsing-fever-associated Borrelia species. A single copy of the rrs gene was detected in the genome of strain IST7T by Southern hybridization. DNA-DNA hybridization results showed that strain IST7T was distinct from Lyme-disease-related Borrelia, Borrelia burgdorferi and the relapsing-fever-associated species Borrelia hermsii. The G+C content of strain IST7T is 30.0 mol%. From these genetic features, a novel Borrelia species, Borrelia turcica sp. nov., is proposed; the type strain is IST7T (= JCM 11958T = DSM 16138T).
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