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Journal of Bacteriology, December 1998, p. 6415-6418, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Family of Stability Determinants in
Pathogenic Bacteria
Finbarr
Hayes*
Microbiology Unit, Department of
Biochemistry, University of Oxford, Oxford OX1 3QU, England
Received 7 August 1998/Accepted 23 September 1998
 |
ABSTRACT |
A novel segregational stability system was identified on plasmid
R485, which originates from Morganella morganii. The system is composed of two overlapping genes, stbD and
stbE, which potentially encode proteins of 83 and 93 amino
acids, respectively. Homologs of the stbDE genes were
identified on the enterotoxigenic plasmid P307 from Escherichia
coli and on the chromosomes of Vibrio cholerae and
Haemophilus influenzae biogroup aegyptius. The
former two homologs also promote plasmid stability in E. coli. Furthermore, the stbDE genes share homology
with components of the relBEF operon and with the
dnaT gene of E. coli. The organization of the
stbDE cassette is reminiscent of toxin-antitoxin stability cassettes.
| |
TEXT |
Bacterial chromosomes and the
low-copy-number plasmids which bacteria often harbor have developed a
variety of mechanisms which promote their segregational stability
(9, 24, 25). First, active partition systems ensure that
each daughter cell receives a copy of the newly replicated plasmid at
cell division (7, 20, 25). There is increasing evidence that
active partitioning of the chromosome may also occur (7, 13,
18). Second, the resolution by site-specific recombination of
dimers and higher-order multimers which arise by homologous
recombination optimizes the number of chromosomes and plasmids
available for segregation at cell division (23). Third,
toxin-antitoxin systems specified by some plasmids compromise the
survival of those plasmid-free segregants which do arise (6, 10,
11). In this study, a novel two-gene stability system reminiscent
of toxin-antitoxin systems was identified on plasmid R485, which
originates from the nosocomial pathogen Morganella morganii.
This system is also present in a number of other pathogenic bacteria.
Isolation of a stability region from plasmid R485.
Plasmid
R485, which specifies resistance to sulfonamides, originates from
Morganella morganii and can conjugate to and replicate in
Escherichia coli (8). R485 is a member of plasmid
incompatibility group X (3, 8, 12). To isolate segregational
stability determinants from R485, a library of 4- to 6-kb fragments
generated by partial Sau3AI digestion of R485 was
constructed by insertion into the BamHI site of the
stability probe vector pALA136. The latter plasmid harbors both the
moderate-copy-number ColE1 replicon and the unit copy number P1
replicon (16). In a wild-type host, pALA136 replicates by
the ColE1 origin and can be isolated and manipulated with ease.
However, in a polA host, the ColE1 origin is nonfunctional,
and replication switches to a low copy number under the control of the
P1 replicon. As the plasmid does not possess accessory stability genes,
it is unstable in this host in the absence of selective pressure.
Insertion of a stability locus will restabilize the plasmid (16,
21). The library of R485 fragments in pALA136 was transformed
with selection for pALA136-encoded chloramphenicol resistance into the
polA strain, BR825 (15). Colonies from this
transformation were replica plated once on solid medium
containing chloramphenicol and then successively 10 times on medium
without antibiotic selection. At the end of this procedure,
approximately 10% of colonies retained chloramphenicol resistance.
Control experiments with pALA136 resulted in <1% chloramphenicol resistance. The apparent increase in stability of plasmids isolated from chloramphenicol-resistant colonies was retested by retransforming candidate plasmids into the polA strain. Following growth
for approximately 25 generations in the absence of selective pressure as described elsewhere (16), the test plasmids were found to be maintained at levels of 40 to 100% compared to pALA136, which was
maintained at a frequency of <2%. One plasmid with a high level of
segregational stability which was chosen for further study contained a
4.8-kb insert. Subcloning refined the region within this insert
required for stability to a 1,154-bp fragment (Fig.
1).
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FIG. 1.
(A) Genetic organization of the stbDE genes
and flanking regions from plasmid R485. Arrows indicate the length and
orientation of open reading frames. Lines beneath the map indicate
regions cloned in the stability probe vector, pALA136. The levels of
retention conferred by these fragments after approximately 25 generations in the absence of selective pressure in a polA
strain are shown. (B) Nucleotide sequence of the 1,154-bp
ScaI-BglII fragment from plasmid R485. The
inferred amino acid sequences of the four open reading frames are shown
by the single-letter code.
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|
Organization of the R485-derived stability region.
The
1,154-bp fragment contained four open reading frames which would be
transcribed in the same direction (Fig. 1). One of these open reading
frames extends beyond the region which was sequenced. Subcloning
further delineated the stability locus to a 597-bp region which harbors
two overlapping open reading frames (stbD and
stbE) which potentially encode proteins of 83 and 93 amino
acids, respectively. The stbD gene alone, as present on a
ScaI-MluI fragment, was insufficient to confer
stability (Fig. 1). Attempts to clone the stbE gene alone in
pALA136 were unsuccessful. Plasmid constructs which were stabilized as
a result of the presence of stbDE contained less than 50 bp
of R485-derived sequences upstream of stbD (Fig. 1B). These
sequences contained no close match to the E. coli
70 consensus promoter sequence. It is likely that, in
these constructs, the stbDE genes are expressed from a
vector-derived promoter.
Identification of stbDE homologs.
Database
searching identified stbDE homologs of hitherto unknown
function on the enterotoxigenic plasmid P307 (22) (GenBank accession no. M26308) and on the chromosomes of Vibrio
cholerae (1) (GenBank accession no. X64097), the
causative agent of cholera, and Haemophilus influenzae
biogroup aegyptius (GenBank accession no. AF018635), the
causative agent of Brazilian purpuric fever, which is a highly fatal
pediatric septicemia (Fig. 2A). The
stbE nucleotide sequence of the latter requires a +1
frameshift to maintain its integrity. In each case the stbD
and stbE genes overlap, albeit by different numbers of
nucleotides. Pairs of StbD proteins are collinear and share 37 to 54%
identity; pairs of StbE proteins are collinear and share 55 to 60%
identity (Fig. 3). Furthermore, StbE is
collinear with and ~60% identical to the RelE protein encoded by the
E. coli chromosome whereas the 26 C-terminal amino acids of
StbD and RelB are 46% identical (Fig. 2 and 3). The relB
gene is positioned upstream of and overlaps with relE (Fig.
2A). Curiously, the third gene, in this operon is homologous to the
hok gene, which encodes the toxin component of the Hok-Sok
toxin-antitoxin stability system of plasmid R1 (5). The
relBEF operon is involved in the stringent response to amino
acid starvation (2). Components of this operon may have been
recruited to form parts of chromosome stabilization mechanisms.
Alternatively, as the relBEF operon appears not to be highly
conserved, at least among those bacteria whose genomes have been
sequenced, the chromosomal rel genes in E. coli
may have been captured from plasmid-borne sequences.
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FIG. 2.
(A) Comparative organizations of the stbDE
genes from plasmid R485 and homologous genes from plasmid P307 and from
the chromosomes of V. cholerae, E. coli, and
H. influenzae biogroup aegyptius. Homologous
regions are denoted by similar shadings. The asterisk indicates the
position of the  1 frameshift in the stbE gene of H. influenzae biogroup aegyptius. (B) Homology between the
RelB, DnaT, and putative StbD proteins. The DnaT and StbD proteins are
aligned from their N termini, whereas the RelB and StbD proteins are
aligned from their C termini. No gaps were introduced into the
alignments. Shaded lines represent amino acids which are identical
between proteins.
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FIG. 3.
Homology in the StbD (A) and StbE (B) families of
proteins. Residues which are identical in a majority of family members
are shaded. Dashes indicate gaps introduced to optimize the alignments.
Protein sequences were aligned with the PILEUP program (4)
with subsequent manual modification. Note that for the StbE' protein
from H. influenzae biogroup aegyptius, a
hypothetical +1 frameshift, indicated by an asterisk in the protein
sequence, was introduced in the nucleotide sequence to maintain the
integrity of the gene.
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|
Whereas the C-terminal one-third of StbD and RelB share significant
identity, the 58 N-terminal amino acids of StbD are 36% identical to
the 58 N-terminal amino acids of DnaT, also encoded by the E. coli chromosome (Fig. 2). The homology between orf3.1 of V. cholerae and dnaT has previously been noted
(1). StbD appears to be a natural hybrid between DnaT and RelB.
The stbDE homologs are functional stability
cassettes.
The stbDE homologs from plasmid P307 and
V. cholerae were subcloned as a 1,101-bp
BglII-XmnI fragment in
BamHI-EcoRV-cleaved pALA136 and as a 1,133-bp
NsiI-BamHI fragment in
PstI-BamHI-cleaved pFH450, respectively. Plasmid
pFH450 is a derivative of pALA136 with a multiple cloning site. The
P307- and V. cholerae-derived fragments conferred
approximately 90 and 70% retention, respectively, following growth
under nonselective conditions for approximately 25 generations in the
polA strain, BR825. In contrast, the stbDE homolog from H. influenzae biogroup aegyptius,
which was cloned as an end-filled 981-bp
PmlI-BstXI fragment in HpaI-cleaved
pFH450, conferred no detectable stability, which probably reflects the 1 frameshift in the N-terminal segment of the stbE gene.
Conclusions.
Plasmid R485 from the nosocomial pathogen
M. morganii contains a pair of overlapping genes
(stbDE) which confers a high level of segregational
stability on a low-copy-number heterologous replicon in E. coli. Closely related genes, two of which were shown to be
functional plasmid stability determinants, are present in a number of
other gram-negative pathogens. The organization of the stbDE
cassette is highly reminiscent of toxin-antitoxin stability cassettes
which have been identified on a number of plasmids, viz., two
overlapping genes encoding approximately 10-kDa proteins (6, 10,
11). The inability to clone stbE in the absence of
stbD further suggests that the StbE protein may be toxic to its host. In toxin-antitoxin stability systems, the toxic activity of
one protein is normally repressed by the partner antitoxin, which may
be either a protein or an antisense RNA. When a plasmid-free variant
arises, the antitoxin decays more rapidly than the toxin. This releases
the latter to act on its intracellular target, which results in cell
death or stasis. A number of different intracellular targets have been
identified for different toxin-antitoxin systems (10). If
the stbDE system is indeed proven to be a toxin-antitoxin system, the lack of homology between its components and components of
other toxin-antitoxin systems suggests that the intracellular target of
StbD-StbE differs from heretofore characterized targets. The homology
between StbD and the essential host protein DnaT (17)
fortuitously may provide a clue as to the target in this case: StbD may
interact with a protein with which DnaT normally interacts, thereby
poisoning the primosome of which DnaT is a component throughout
replication (14, 19). Alternatively, the homology between
StbD and DnaT may indicate that StbE can interact with both of these
proteins through homologous residues or regions on these proteins. StbD
and StbE may normally be physically associated, but in the absence of
StbD, for example, following plasmid loss and StbD decay, StbE may
substitute for a component of the primosome with which DnaT interacts,
thereby disrupting primosome assembly or maturation. These hypotheses
remain to be tested.
StbD appears to be a hybrid between DnaT and RelB, whereas StbE is
homologous to RelE (Fig. 2). The potential significance of the homology
between StbD and DnaT has been alluded to in the preceding section. The
questions of the importance of the homology which the stbDE
genes share with the relBEF operon, which itself does not
function as a plasmid stability cassette (5), and why
components of the relBEF operon have been hijacked as parts of both the stbDE and R1 hok-sok (5)
stability systems remain to be elucidated. However, the homology which
StbE and RelE share suggests a mechanism different from that proposed
above by which the stbDE cassette might promote stability:
StbE may interact with a factor with which RelE usually interacts,
thereby disrupting a critical cellular function.
The stbDE homologs identified to date have been found only
in pathogenic bacteria. This suggests that these genes may function in
virulence or its control. In the case of the plasmid-located genes, the
stbDE genes simply may contribute to virulence plasmid segregational stability. This hypothesis could be tested by
inactivating the stbDE cassette on plasmids R485 and P307
and determining the effect on plasmid stability either in laboratory
strains or in their natural hosts. In the instance of chromosomal
stbDE genes, it is less clear how a toxin-antitoxin system
can promote chromosome stability, although putative toxin-antitoxin
systems also have been identified on the E. coli chromosome
(10). The construction of chromosomal stbDE
mutants in V. cholerae and the characteristics and virulence
of the resulting mutants will assist in determining the biological
significance of the chromosomal stbDE genes.
Nucleotide sequence accession number.
The nucleotide sequence
of the 1,154-bp ScaI-BglII fragment from plasmid
R485 has been deposited in the GenBank database under accession no.
AF072126.
| |
ACKNOWLEDGMENTS |
This work was funded by Wellcome Trust Research Career Development
Fellowship 040822/Z/94/Z.
I thank Stuart Austin, Martine Couturier, Paul Manning, Leonard Mayer,
Lyndsay Radnedge, and Andrew Spiers for generous gifts of strains and plasmids.
| |
FOOTNOTES |
*
Present address: Department of Biomolecular Sciences,
University of Manchester Institute of Science and Technology (UMIST), P.O. Box 88, Manchester M60 1QD, England. Phone: 44-(0)161-2004200. Fax: 44-(0)161-2360409. E-mail: Finbarr.hayes{at}mail.bi.umist.ac.uk.
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Journal of Bacteriology, December 1998, p. 6415-6418, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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