Download Nitrate respiration in the actinomycete Streptomyces coelicolor

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Biochemical Society Transactions (2005) Volume 33, part 1
Nitrate respiration in the actinomycete
Streptomyces coelicolor
G. van Keulen, J. Alderson, J. White and R.G. Sawers1
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, U.K.
Abstract
Streptomyces coelicolor is an obligate aerobic, filamentous soil-dwelling bacterium. Remarkably, the
genome of S. coelicolor has three copies of the narGHJI operon that encodes respiratory nitrate reductase.
This review summarizes our current views on the requirements for multiple nitrate reductases in S. coelicolor.
Introduction
Streptomycetes are ubiquitous actinomycetes that have had
a major impact on the biotechnological and pharmaceutical
industries. They undergo a complex developmental life-cycle
and exhibit a remarkable metabolic diversity. As well as
having a wealth of genes encoding products involved in secondary metabolic pathways, analysis of the 8.6 Mb genome
of Streptomyces coelicolor has revealed the presence of some
rather unexpected operons, in particular three operons encoding respiratory NARs (respiratory nitrate reductases) [1].
Despite being classified as an obligate aerobe, the presence of
an enzyme in anaerobic respiration suggests that S. coelicolor
has at least the capacity to grow, or generate energy, using
nitrate as an alternative electron acceptor and under oxygenlimiting conditions in standing liquid culture [2]. Indeed,
nitrate reduction has been demonstrated for a number of
other Streptomyces spp. ([3], and references therein).
Members of the NAR class of nitrate reductase are predicted to form a membrane-associated enzyme complex,
which has the site of nitrate reduction on the cytoplasmic
face of the membrane [4]. The energy-conserving enzyme
complex comprises three different protein subunits and the
structure of the enzyme from Escherichia coli has been
recently determined [5,6]. The NarG subunit harbours the
bis-MGD (bis-molybdenum guanine dinucleotide) containing catalytic site, while the NarH subunit is an electron
transfer component which binds 4 iron–sulphur clusters. The
whole complex is anchored to the membrane via the di-bhaem integral membrane NarI quinol dehydrogenase subunit.
The fourth protein encoded in the operon, NarJ, is proposed
to have a chaperone function, necessary for maturation of the
enzyme.
Nitrate respiratory capacity in S. coelicolor
Although originally characterized extensively in Gram negative bacteria, the characteristic narGHJI operon structure
Key words: anaerobic respiration, energy conservation, nitrate reductase, phylogeny, soil
bacteria, Streptomycetes.
Abbreviations used: bis-MGD, bis-molybdenum guanine dinucleotide; NAR, respiratory nitrate
reductase.
1
To whom correspondence should be addressed (email [email protected]).
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is also highly conserved in the genomes of a number of
Gram positive bacteria (Figure 1). Perhaps unsurprisingly,
the degree of amino acid sequence conservation between the
three NarG polypeptides of S. coelicolor and that of the actinomycete Mycobacterium tuberculosis and that of Bacillus
subtilis is, respectively, 65 and 50%. However, the S. coelicolor
NarG proteins also share 45–48% amino acid similarity with
the E. coli NarG polypeptide. Remarkably, when compared
against each other, the NarG polypeptides of S. coelicolor
only share approx. 65% amino acid identity.
As well as having three narGHJI operons, S. coelicolor
also has two NarK orthologues. NarK belongs to the major
facilitator superfamily of transmembrane transporters.
Phylogenetic analyses suggest that there are two classes of
NarK: the type I class is proposed to catalyse nitrate/proton
symport and nitrate/nitrite antiport, while the type II class
probably functions in nitrite efflux [7]. The narK gene is
often, but not always, found upstream of the narGHJI
operon. The narK2 gene in S. coelicolor is found near the
nar2 operon (Figure 1) and NarK2 falls within the type I
subgroup along with NarK2 from M. tuberculosis and NarK
from B. subtilis [7]. The narK1 gene product from S. coelicolor
falls within the type II class along with NarK1, NarK3 and
NarU from M. tuberculosis and NarK and NarU from E. coli.
Molybdenum cofactor biosynthesis
A major requirement for the synthesis of catalytically active
nitrate reductase is the machinery for the biosynthesis of the
bis-MGD cofactor. The S. coelicolor genome encodes all of
the proteins required for the uptake of molybdenum and the
biosynthesis of bis-MGD.
Differential expression of the nar operons
We have determined through transcript analysis that all three
nar operons in S. coelicolor are expressed. Although we
have not identified conditions in the laboratory that allow
S. coelicolor to grow anaerobically, our transcript analysis
suggests that at least one of the operons (nar2) is regulated in
response to fluctuations in oxygen levels. Surprisingly, however, we have not yet identified conditions under which
expression of any of the nar operons responds to nitrate.
10th Nitrogen Cycle Meeting 2004
Figure 1 Schematic representation of the nar gene operons in
S. coelicolor
Shown is the organization of the nar operons in S. coelicolor, M.
tuberculosis and B. subtilis. Genes whose products exhibit a high degree
of amino acid identity (>60%) within and between species share the
same colour. Genes shown in white or light blue and without associated
names are not related to the nar genes. The transcriptional orientation of
the genes in the respective chromosomes is depicted by the orientation
of the arrows. The location of the S. coelicolor nar operons on the 8.6 Mb
genome is shown in brackets. The narK orthologues (narK1 in S. coelicolor, and all four in M. tuberculosis) shown in isolation are located in
distinct and unrelated regions of the respective genomes.
Phylogenetic relationships
An alignment of full-length NarG sequences obtained from
the sequenced and annotated genomes in the databases was
used to construct a phylogenetic tree using distance matrix
methods (Figure 2). A number of major clades were supported
by the analysis and reflect reasonably well the findings
reported recently for partial NarG sequences [8,9]. The three
NarG sequences from Streptomyces coelicolor cluster together
and form a clade with other Actinobacteria, in particular
NarG from the Mycobacteria. Genome sequence projects for
other actinomycetes, for example S. scabies and Nocardia
farcinica, indicate the presence of at least partial narG genes,
suggesting that nitrate reduction is widespread in this group
of bacteria.
Interestingly, NarG sequences from Rhodococcus sp.
RHA1 do not cluster with those from other Actinomycetes,
but form a deeply branched cluster, which indicates that the
NARs from these bacteria diverged from a common ancestor
in the distant evolutionary past.
Perspectives
Future challenges will be to determine the roles of the
respective nitrate reductases in S. coelicolor physiology and
to identify the source of the reducing power that drives
nitrate reduction. Preliminary data indicate that the three
narGHJI operons are differentially regulated. A detailed
analysis of expression levels throughout the growth cycle may
Figure 2 Phylogenetic relationship of translated, full-length narG gene products
The phylogenetic tree was constructed using the Treecon v 1.3b program [10] inferred from alignments created by the
ClustalX program [11]. Phylogenetic distances were determined by neighbour-joining analysis; numbers on the branching
points are bootstrap values with 100 replicates.
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Biochemical Society Transactions (2005) Volume 33, part 1
reveal the conditions under which each NAR is required.
Clearly, the construction and analysis of specific narGHJI
operon knock-out mutants will aid functional assignment of
the NAR enzyme complexes. It remains to be established
whether there is functional significance in the fact that the
NarG polypeptides in S. coelicolor share only 65% amino acid
sequence similarity. Indeed, the NarH and NarI polypeptides
share even less similarity.
The complex nature of the soil implies that oxygen supply
is often limited for the growth of ‘aerobes’. Consequently,
Streptomyces spp. will have had to evolve a broad range of
metabolic pathways that deliver sufficient energy to maintain
the membrane potential, or to sustain growth, when oxygen
gets depleted [2]. The capacity to respire with nitrate may
give Streptomycetes a selective advantage in this complex
environment.
The work in our laboratory is supported by the BBSRC (EGH16080)
and through a Marie Curie Postdoctoral Fellowship to G.v.K. (MEIFCT-2004-506056).
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