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(Received for publication, December 26, 1995; and in revised form, January 23,
1996) From the
Plasmids containing DNA from the green photosynthetic bacterium Chlorobium vibrioforme complement a heme-requiring Escherichia coli hemB mutant that is deficient in
porphobilinogen (PBG) synthase activity. PBG synthase activity was
detected in extract of complemented cells but not in that of cells
transformed with control plasmid. The sequence of the C.
vibrioforme hemB gene predicts a HemB protein that contains 328
amino acids, has a molecular weight of 36,407, and is 53% identical to
the homologous proteins of Synechocystis sp. PCC 6301 and Rhodobacter capsulatus. The response of C. vibrioforme PBG synthase to divalent metals is unlike that of any previously
described PBG synthase; Mg Biosynthesis of porphyrins and related compounds proceeds via a
common set of intermediates from ALA, (
Figure 1:
Early steps of the tetrapyrrole
biosynthetic pathway with the gene products under discussion indicated
for the reactions they catalyze.
Although the early
biosynthetic steps from ALA onward are identical in all species
examined, there are some interesting differences in the properties of
certain enzymes from different sources. For example, PBG synthase (also
known as ALA dehydratase) obtained from different species has different
metal requirements for activity (Jaffe, 1993, 1995). Another difference
is that in some species, the cysG gene product, siroheme
synthase, catalyzes three sequential steps in the conversion of
uroporphyrinogen III to siroheme, whereas in other species, a different
enzyme, S-adenosyl-L-methionine:uroporphyrinogen III
methyltransferase, catalyzes only the first step, forming precorrin-2
(Spencer et al., 1993; Warren et al., 1994).
Interestingly, the amino acid sequence of the methyltransferase is
similar to that of the C-terminal portion of siroheme synthase (Spencer et al., 1993). Genes for the enzymes catalyzing the early
steps of tetrapyrrole biosynthesis have been cloned and sequenced from
several prokaryotic species. Often, two or more of the genes are
arranged in a cluster in the genome, and in some cases the clustered
genes comprise a common transcription unit (Hansson et al.,
1991; Jordan et al., 1988). There is wide variation among
species in the identity and number of clustered hem genes, and
this variation may have both regulatory significance and evolutionary
implications. Chlorobium vibrioforme is a strictly
anaerobic green phototrophic bacterium. Green bacteria have been very
useful for comparative and evolutionary studies of photosynthesis and
related processes because they are only distantly related to other
photosynthetic organisms; their photosynthetic reaction center and
light harvesting apparatus are completely different from those of the
other group of phototrophic anaerobes, the purple bacteria, and their
mode of carbon fixation is totally unlike that of purple bacteria,
cyanobacteria, or plants (Blankenship et al., 1995; Feiler and
Hauska, 1995; Sirevåg, 1995). Previous studies of C.
vibrioforme provided the first information about the structure of
tetrapyrrole biosynthetic enzymes and their genes in any strict
anaerobe. C. vibrioforme has a gene cluster that contains
three hem genes, hemA, hemC, and hemD, which encode glutamyl-tRNA reductase,
hydroxymethylbilane synthase, and uroporphyrinogen III synthase,
respectively (Majumdar et al., 1991; Moberg and Avissar,
1994). These three genes appear to comprise an operon that yields a
transcript of sufficient size to encompass all three open reading
frames (Majumdar et al., 1991). We now communicate the
sequence of the C. vibrioforme hemB gene, describe its
positional relationship to and expressional independence from the hemACD genes, and report on some catalytic properties of its
encoded enzyme, PBG synthase.
DNA sequencing of the 2.8-kilobase insert of pYA4
was done in both directions using an fmol DNA sequencing
system kit (Promega, Madison, Wisc.) according to the
manufacturer's protocol. Sequence information was compiled with
the MacVector DNA sequence analysis program (Eastman Kodak Co.).
As a probe for Northern hybridizations, a 827-bp fragment of
pYA4 containing most of the C. vibrioforme hemB coding region
was prepared by polymerase chain reaction using the oligonucleotides
5`-GCATCGCCCGAGAAG-3` and 5`-TCACCATGGCGTATTCG-3` for the sense and
antisense primers, respectively. The 827-bp fragment was purified by
electrophoresis in low melting point agarose followed by elution and
phenol extraction and labeled with [
The standard
incubation medium contained 100 mM Bis-Tris-Propane-HCl, pH
8.5, 1 mM
To
verify that the complementation was caused by the expression of hemB, complemented RP523 cells were examined for the presence
of PBG synthase activity. This verification was necessary because in
some cases pseudo-complementation can be caused by overexpression of
other hem genes. For example, overexpression of C.
vibrioforme hemA in an E. coli hemL strain leads to
heme-independent growth. ( Extracts of RP523 cells that were complemented with pYA4 had PBG
synthase levels comparable with those of the hemB
In the previous
complementation studies with C. vibrioforme hemA, hemC, and hemD (Avissar and Beale, 1990; Moberg and
Avissar, 1994), there was some uncertainty about whether the
transcription of these genes in E. coli cells was directed by
promoter elements on the C. vibrioforme DNA or from the lac promoter on the pBluescript SK(+) vector. For hemB, the size of the transcript (see below) indicates that
transcription begins well within the inserted DNA, approximately 1750
bp from the beginning of the inserted C. vibrioforme DNA (Fig. 2). Moreover, there is a transcription stop signal between
the end of the hemD gene and the beginning of the hemB gene. Therefore, it is likely that the C. vibrioforme promoter for the hemB gene can function in E. coli sufficiently well to cause complementation and produce measurable
PBG synthase activity in cell extracts.
Figure 2:
Nucleotide sequence of a 2872-bp cloned C. vibrioforme genomic DNA insert in pYA4, the deduced peptide
sequence for the C-terminal 60 amino acids of the hemD gene
product, and the deduced peptide sequence for the complete 328-amino
acid hemB gene product. Stop codons are indicated by asterisks. For hemB, the -10 (Pribnow box)
consensus sequence is indicated by asterisks, and a potential
-35 region consensus sequence is indicated by double
underlining. For HemB, putative metal binding sites B (residues
122-140) and C (residues 228-238) and the active site
lysine (residue 253) are indicated with single underlining.
Putative rho-independent transcription termination sequences downstream
from the ends of hemD and hemB are shown in bold
letters with the palindromic regions indicated by arrowheads. The GenBank accession number is
U38348.
The deduced C. vibrioforme HemB
sequence is significantly similar to all published PBG synthase
sequences. The most similar sequence in the GenBank data base is that
of PBG synthase from the cyanobacterium Synechocystis sp. PCC
6301 (Jones et al., 1994), which is 53.0% identical to C.
vibrioforme HemB (Fig. 3). Even the least similar PBG
synthase, the human enzyme (Wetmur et al., 1986), is 36.9%
identical to C. vibrioforme HemB. In all cases, the similarity
extends throughout the entire polypeptide sequence.
Figure 3:
Comparison of the deduced amino acid
sequence of C. vibrioforme (C. vib.) HemB with the
most similar sequence in the GenBank data base, that of Synechocystis (Syn.) sp. PCC 6301 PBG synthase
(GenBank accession number X70434), and the least similar PBG synthase
sequence in the GenBank data base, that of the human enzyme (GenBank
accession number M13928).
C. vibrioforme PBG synthase
has a broad pH optimum centered at pH 8.5 (Fig. 4). The C.
vibrioforme enzyme appears to represent a new class of PBG
synthase with respect to the effects of divalent metals. The
gel-filtered enzyme does not require the addition of either
Zn
Figure 4:
pH dependence of C. vibrioforme PBG synthase activity. Gel-filtered C. vibrioforme extract was incubated for 15 min at 32 °C in 0.5 ml of medium
containing 100 mM Bis-Tris-Propane (titrated to the indicated
pH with HCl), 1 mM
In one model that
attempts to relate protein structural features to divalent metal
effects, PBG synthase is proposed to have three metal binding sites
(Jaffe, 1993, 1995). Site A, which is proposed to bind Zn
Figure 5:
Comparison of the amino acid sequences of
putative metal binding regions B and C of PBG synthases from several
sources. The enzymes are grouped on the basis of sequence similarity at
the B site. For the B site sequences, cysteines, histidines, and
residues with carboxyl groups (aspartate and glutamate) are shown in bold letters, and for the C site sequences, residues with
carboxyl groups are shown in bold letters. For conserved
residues shown in the top line, capital letters are
used to indicate absolutely invariant residues, and lowercase
letters are used to indicate residues conserved in all but one or
two sequences. The peptide sequences for the bovine enzyme are taken
from Markham et al.(1993).
The responses of C. vibrioforme PBG
synthase to divalent metals correlate well with the structures of the
putative metal binding sites B and C (Fig. 5). Site B of the C. vibrioforme enzyme most closely resembles that of R.
capsulatus PBG synthase, an enzyme that has no requirements for
divalent metals in the incubation medium. However, site C of the C.
vibrioforme enzyme more closely resembles those of
Zn PBG synthase from some species has been
reported to be allosterically inhibited by heme, a tetrapyrrole end
product (Nandi et al., 1968). The effect of heme on C.
vibrioforme PBG synthase activity was tested in incubations done
at pH 8.5 in the presence of 50 mM K
Figure 6:
Northern blot of C. vibrioforme total RNA hybridized with an 827-bp hemB-specific probe.
RNA size markers were run on a duplicate gel and stained with ethidium
bromide. kb, kilobases.
Figure 7:
A, nucleotide sequence of a region of the C. vibrioforme DNA in pYA4 for the strand complementary to
that which encodes the hemD and hemB genes, and the
deduced peptide sequence of its open reading frame. The stop codon is
indicated by an asterisk. Potential -10 (Pribnow box)
and -35 region consensus sequences are indicated by asterisks and double underlines, respectively. B,
comparison of the deduced peptide sequence in A with the amino
acid sequences of human (GenBank accession number X66922) and E.
coli (GenBank accession number M34828) inositol
monophosphatase.
The clustering of hem genes is common in bacteria (Fig. 8) and may have
regulatory as well as evolutionary implications. For example, the
widespread close clustering and cotranscription of hemC and hemD probably ensures the presence of the hemD product, uroporphyrinogen III synthase, whenever the hemC product, hydroxymethylbilane synthase, is present. The activity of
uroporphyrinogen III synthase is necessary to direct the conversion of
hydroxymethylbilane to the physiologically relevant product
uroporphyrinogen III and prevent its spontaneous conversion to
uroporphyrinogen I, a nonphysiological dead-end product. Extreme
regulatory coordination is found in B. subtilis, where the
genes for all of the enzymes needed for conversion of glutamyl-tRNA to
uroporphyrinogen III (Fig. 1), as well as a gene of uncertain
function, are clustered on the hemAXCDBL operon (Hansson et al., 1991).
Figure 8:
Structures of gene clusters from several
prokaryotic species that contain genes for early tetrapyrrole
biosynthetic steps. The extent of sequenced regions is indicated by horizontal lines, open reading frames of identified and
putative genes are indicated by boxes, and the deduced
directions of transcription are indicated by arrows. For ease
of comparison, the sequences are arbitrarily aligned at the beginning
of the hemC open reading frame, except in the case of the Synechocystis sp. PCC 6301 cluster, which does not contain hemC. In this case, the end of the cobA open reading
frame is aligned with the end of the cysG open reading frames
of C. josui and Mycobacterium leprae to indicate that
the CobA sequence is similar to the C-terminal end of the CysG
sequence. The GenBank accession numbers are: C. vibrioforme (M96364, U38348), B. subtilis (M57676), E. coli (X12614), Pseudomonas aeruginosa (M74844), Synechocystis sp. PCC 6301 (X70434), C. josui (D28503), M. leprae (U00018).
From an evolutionary perspective, it is of
interest that wherever hem genes are clustered, the sequential
arrangement of the genes within the cluster is generally conserved in
the order ACDBL (Fig. 8). This arrangement does not follow the
order of their products as enzymes catalyzing early steps of the
biosynthetic pathway, which is ALBCD (Fig. 1). It is especially
interesting that the sequential arrangement of these hem genes
has been maintained even when they are not within the same
transcription unit and despite the presence of intervening genes
between hem genes, such as in C. vibrioforme where a
putative inositol monophosphatase-encoding gene is between hemD and hemB and is oriented in the opposite direction. The
order of the clustered hem genes is conserved even in the
extreme case of Clostridium josui, where there has been
apparent fusion of different parts of a split cysG homolog to
the hemA and hemD genes (Fujino et al.,
1995). The high degree of conservation of the order of clustered hem genes in these phylogenetically diverse organisms strongly
suggests that there are functional constraints on their relative
genomic position. Critical assessment of this suggestion will require
information about the structure of hem gene clusters in
additional species. The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U38348[GenBank].
Volume 271,
Number 14,
Issue of April 5, 1996 pp. 8176-8182
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
stimulates but is not
required for activity, and Zn neither stimulates nor
is required. This response correlates with predicted sequences of two
putative variable metal binding regions of C. vibrioforme HemB. The C. vibrioforme hemB open reading frame begins
1585 bases downstream from the end of the hemD open reading
frame and is transcribed in the same direction as hemA, hemC, and hemD. However, hemB is not part of
the same transcription unit as these genes, and the hemB transcript is approximately the same size as the hemB gene alone. Between hemD and hemB there is an
intervening open reading frame that is oriented in the opposite
direction and encodes a protein with a predicted amino acid sequence
significantly similar to that of inositol monophosphatase, an enzyme
that is not involved in tetrapyrrole biosynthesis. The gene order
within hem gene clusters is highly conserved in
phylogenetically diverse prokaryotic organisms. This conservation
suggests that there are functional constraints on the relative order of
the hem genes.
)the first universal
precursor, through the first cyclic tetrapyrrole, uroporphyrinogen III,
at which point the pathway splits into two branches, one leading to
reduced products such as siroheme and vitamin B
and the
other leading to oxidized end products, including hemes, bilins,
chlorophylls, and bacteriochlorophylls (Fig. 1). There are two
routes to ALA, one that involves the condensation of glycine and
succinyl-coenzyme A and occurs exclusively in 
-proteobacteria and
nonphototrophic eukaryotes and the other more common pathway that
begins with glutamyl-tRNA (Beale, 1995).
C. vibrioforme Cell Culture
C. vibrioforme f. thiosulfatophilum NCIB 8327 was obtained from J. G.
Ormerod (University of Oslo) and grown at 30 °C in the medium
described by Rieble et al.(1989) in completely filled 60- or
2400-ml bottles. Light was supplied by incandescent lamps at an
intensity of 50-100 microeinsteins m
s
. Cell growth was monitored by measuring the A
.Cloning and Sequence Analysis of C. vibrioforme PBG
Synthase-encoding DNA
Escherichia coli RP523, a hemB strain (Li et al., 1988), and C600, the
hemin-permeable parental strain, were obtained from B. J. Bachmann (E. coli Genetic Stock Center, Yale University, New Haven,
CT). Recombinant plasmid pYA1 containing 5.8 kilobases of Sau3AI-digested C. vibrioforme genomic DNA inserted
into the BamHI site of pBluescript SK(+) vector
(Stratagene, La Jolla, CA) was constructed as described previously
(Avissar and Beale, 1990). Plasmid pYA4 containing 2.8 kilobases of C. vibrioforme genomic DNA was derived from pYA1 by digestion
with SalI and ligation with SalI-digested pBluescript
SK(+) vector.Analysis of the hemB Transcript
C. vibrioforme cells were harvested in the mid-exponential growth phase (A = 0.5-0.6) by centrifugation at
5000 
g for 15 min at 4 °C. Total cellular RNA was
isolated using the TRI reagent (Molecular Research Center, Inc.,
Cincinnati, Ohio) according to the manufacturer's protocol.
Glyoxal-denatured total cellular RNA (8 µg) was separated on 0.9%
(w/v) agarose gels in duplicate. One set, including an RNA marker (Life
Technologies, Inc.) lane was stained with ethidium bromide to confirm
the RNA integrity and to determine the size of the transcript. The
other set was transferred to a Nytran
nylon membrane
(Schleicher & Schuell) in 20 SSPE. The blot was UV
cross-linked and probed in 50% (v/v) formamide/5 SSPE/2
Denhardt's reagent/5% (w/v) SDS/50 µg/ml salmon sperm DNA at
45 °C and washed in 2 SSPE/0.1% (w/v) SDS at 50 °C
followed by a final wash in 0.2 SSPE/0.1% (w/v) SDS at 55
°C.
P]dATP by
the nick translation method using a kit obtained from Life
Technologies, Inc.PBG Synthase Enzyme Assay
E. coli and C. vibrioforme cells were harvested in the mid-exponential
growth phase by centrifugation for 15 min at 5000 g and resuspended in extraction buffer consisting of 100 mM Bis-Tris-Propane-HCl, pH 6.8, 10 mM 
-mercaptoethanol, and 0.004% (w/v) phenylmethylsulfonyl
fluoride at a concentration of 1 g of packed cells/2 ml of buffer. 1 g
of 5-µm diameter glass beads was added per g of packed cells, and
the cells were broken by sonication (ten 15-s bursts separated by 45-s
cooling periods). Cell debris and glass powder were removed by
centrifugation for 10 min at 10,000 g. The pellet was
washed once with an equal volume of extraction buffer and centrifuged,
and the supernatants were combined. The C. vibrioforme extract
was purified further by centrifugation for 60 min at 285,000 g, and the soluble proteins in the supernatant were separated
from small molecules by chromatography through a Sephadex G-25 column
that had been pre-equilibrated with extraction buffer. Extracts were
stored as 1-ml aliquots at -80 °C until use.-mercaptoethanol, 10 mM ALA, 50 mM KCl, 10 mM MgCl
, and cell extract (additions
and variations are described under ``Results and
Discussion''). Incubation was for 15 or 30 min at 32 °C in a
total volume of 0.5 ml. Incubations were stopped by the addition of 0.5
ml of modified Ehrlich-Hg reagent (Urata and Granick, 1963) and mixing.
The mixture was clarified by centrifugation for 2 min in the
microcentrifuge, and the absorbance was recorded at 555 nm between 5
and 15 min after the addition of the modified Ehrlich-Hg reagent. The
PBG concentration was calculated using a molar absorption value of
68,000 (Urata and Granick, 1963).Chemicals
All chemicals whose source was not
specifically identified above were from Fisher, Sigma, or Research
Organics (Cleveland, Ohio).
Cloning of C. vibrioforme hemB
It was previously
shown that pYA1 contains 5.8 kilobase pairs of C. vibrioforme genomic DNA that is capable of complementing heme-dependent E.
coli strains that are deficient in glutamyl-tRNA reductase (hemA) and uroporphyrinogen III synthase (hemD), and
a Salmonella typhimurium strain that is deficient in
hydroxymethylbilane synthase (hemC) (Majumdar et al.,
1991; Moberg and Avissar, 1994). pYA1 was subcloned into approximately
equal size halves, pYA2 and pYA4 (Avissar and Beale, 1990). pYA2 was
able to complement an E. coli hemA strain, and partial
sequencing showed that it contains an open reading frame encoding the
entire C. vibrioforme hemA gene (Majumdar et al.,
1991). Further sequencing revealed that hemA, hemC,
and hemD form a compact cluster in the C. vibrioforme genome (Majumdar et al., 1991). ()We now
report that both pYA1 and its subclone pYA4 complement a heme-requiring E. coli hemB strain RP523 (Li et al., 1988) (Table 1). In contrast, neither the pBluescript SK(+) vector
nor pYA3, a subclone of pYA1 that contains C. vibrioforme hemA and part of hemC, was able to complement RP523.
)This pseudo-complementation
presumably results from nonenzymatic conversion of
glutamate-1-semialdehyde to ALA, which can occur at high
glutamate-1-semialdehyde concentrations (Hoober et al., 1988). parental strain C600 (Table 2). In contrast, extracts of
RP523 cells that were complemented with the pBluescript SK(+)
vector were devoid of PBG synthase activity. Because it was necessary
to add heme to the medium of uncomplemented RP523 cells to obtain
growth, it was possible that the absence of PBG synthase in RP523 cell
extract was due to repression of its formation or inhibition of its
activity caused by the added heme. Therefore, PBG synthase activity was
determined in extracts of RP523 cells that were transformed with pYA4
but grown in the presence of added heme. These cells had levels of PBG
synthase activity equal to that of complemented cells grown without
added heme. We therefore conclude that the complete absence of PBG
synthase activity in uncomplemented RP523 cells is not caused by the
heme added to the medium and that the activity in RP523 cells
complemented by pYA4 is attributable to the expression of C.
vibrioforme hemB in the transformed cells.
Sequence of C. vibrioforme hemB
The sequence of
the C. vibrioforme DNA in pYA4 indicates that its size is 2872
bp and that it contains the 3` portion of the hemD gene
encoding the C-terminal 60 amino acids of uroporphyrinogen III synthase
and a downstream open reading frame encoding the complete hemB gene product, PBG synthase (Fig. 2). The partial hemD sequence is identical to that of the hemD sequence
reported previously. The beginning of the hemB open reading frame is separated from the end of the hemD open reading frame by 1585 bp. Potential -10 (Pribnow box)
and -35 region consensus sequences are present upstream of the
beginning of the hemB open reading frame. However, a
recognizable translation initiation (Shine-Dalgarno) sequence was not
found. It should be noted that a Shine-Dalgarno sequence also could not
be identified for the C. vibrioforme hemA gene (Majumdar et al., 1991).
Characterization of C. vibrioforme PBG
Synthase
Despite the overall sequence similarity of all PBG
synthases, there are significant differences among the enzymes from
different species with regard to pH optimum for activity and the
requirement for or enhancement of activity by divalent metals (Jaffe,
1993). The pH optimum for PBG synthase from different species ranges
from approximately 6.5 to 8.5. It is hypothesized that all PBG
synthases contain tightly bound Zn atoms that are not
released under normal conditions of protein purification and exposure
to chelators such as EDTA (Jaffe, 1993, 1995). In addition, PBG
synthase from all mammals and birds examined, yeast, and some bacteria
has a requirement for Zn in the micromolar
concentration range, which is presumably bound at a second site. In
contrast, PBG synthase from plants and some bacteria has no
demonstrable requirement for Zn in the incubation
medium but instead requires micromolar concentrations of Mg for activity. For the Rhodobacter sphaeroides enzyme,
the Mg requirement can be met by K (Burnham and Lascelles, 1963; Nandi et al., 1968). Both
classes of PBG synthase that require divalent metal ions in the
incubation medium are inhibited by EDTA, which presumably removes the
comparatively loosely bound metal atoms at the second site. Among the
PBG synthases that require Zn in the incubation
medium, some (e.g. that from E. coli) but not all are
stimulated approximately 2-fold by Mg, which is
proposed to bind at a third site on the protein. Finally, a fourth
class of PBG synthase, represented by the enzyme from Rhodobacter
capsulatus, neither requires nor is stimulated by micromolar
concentrations of either Zn or Mg (Nandi and Shemin, 1973). or Mg for activity, but it is
stimulated approximately 2-3-fold (depending on the incubation
pH) by Mg (Table 3). Zn is
inhibitory. K is somewhat stimulatory in the absence
of Mg, especially at the higher incubation pH, but
K does not stimulate in incubations containing
Mg. The absence of inhibition by EDTA reinforces the
conclusion that activity does not require Zn or
Mg in the incubation medium.
-mercaptoethanol, 10 mM ALA,
50 mM KCl, 10 mM MgCl, and cell extract
(43 µg of protein).
very tightly in all PBG synthases, has not been identified. Site
B (Fig. 5) in the Zn-requiring enzymes has
several cysteine and histidine residues, which are replaced by
carboxyl-containing residues in the Mg-requiring
enzymes. Site C has several carboxyl-containing residues in the
Zn-requiring enzymes that are stimulated by
Mg, and these residues are absent from the enzymes
that are not stimulated by Mg. For the
Mg-requiring enzymes, it is difficult to determine
experimentally whether Mg additionally stimulates by
binding at site C.
-requiring PBG synthases that are stimulated by
Mg. In summary, these results for C. vibrioforme PBG synthase provide strong support for the model identifying
metal binding sites B and C. and 10
mM Mg. In this experiment, hemin was added
to the incubation mixture from a concentrated stock solution in
dimethyl sulfoxide, and all incubations contained the same dimethyl
sulfoxide concentration (2%, v/v). PBG synthase activity was not
inhibited even by 100 µM heme, the highest concentration
tested (data not shown).Expression of C. vibrioforme hemB
A Northern blot
of C. vibrioforme RNA probed with a hemB-specific
probe reveals a single 1.0-kilobase band (Fig. 6). Because this hemB transcript corresponds in size to the hemB gene,
this result indicates that hemB mRNA is not a part of a
larger, multigene transcript. This result is consistent with the
absence of significant open reading frames between the end of the hemD gene and the beginning of the hemB gene, a
distance of 1585 bp, and the presence of rho-independent transcription
terminators closely downstream from both hemD and hemB (Fig. 2). In contrast, transcription terminators are not
present between hemA and hemC or between hemC and hemD, and these three genes appear to be
co-transcribed (Majumdar et al., 1991). In summary, although C. vibrioforme hemB is located near other hem genes,
it appears to be independently transcribed, in contrast to the
situation in some other organisms with clustered hem genes,
such as Bacillus subtilis, in which all of the genes for
enzymes that convert glutamyl-tRNA to uroporphyrinogen III are parts of
a single operon (Hansson et al., 1991).
Location of C. vibrioforme hemB Relative to Other hem
Genes
The sequence of pYA4 indicates that hemB is
located 1585 bp away from the end of hemD and is transcribed
in the same direction as the genes in the hemACD cluster (Fig. 2). However, the distance between hemD and hemB suggests that although the genes are clustered on the C. vibrioforme chromosome, they are unlikely to be part of the
same operon. Moreover, between hemD and hemB there is
a 786-bp open reading frame oriented in the opposite direction. This
open reading frame encodes a protein that has a high degree of
similarity to inositol monophosphatase (Matsuhisa et al.,
1995; McAllister et al., 1992), an enzyme that is unrelated to
tetrapyrrole biosynthesis (Fig. 7). The putative C.
vibrioforme inositol monophosphatase is 28.9 and 27.8% identical
to the human and E. coli enzymes, respectively. The
significance of the location of this gene between two hem genes in C. vibrioforme is unknown.
)
-aminolevulinic acid; PBG, porphobilinogen; bp, base pair(s).))
We thank B. J. Bachmann and J. G. Ormerod for
supplying strains and D. L. Bollivar, E. K. Jaffe, G. L. Matters, M.
O'Connor, and J. Park for helpful advice and discussions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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