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Journal of Bacteriology, December 1998, p. 6412-6414, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification and Characterization of the
Myxococcus xanthus argE Gene
Baruch Z.
Harris and
Mitchell
Singer*
Section of Microbiology, Division of
Biological Sciences, University of California at Davis, Davis,
California 95616
Received 12 June 1998/Accepted 18 September 1998
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ABSTRACT |
The chromosomal acetylornithine deacetylase (argE) gene
of Myxococcus xanthus was identified via homology to
acetylornithine deacetylases from other bacterial species. A mutant
carrying a disruption in argE was unable to grow on minimal
media lacking supplemental arginine and formed fruiting bodies and
spores in response to arginine starvation at high cell density.
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TEXT |
Myxococcus xanthus is a
gram-negative soil bacterium that, upon starvation for essential
nutrients, initiates a complex developmental process culminating in the
formation of multicellular fruiting bodies (for reviews, see references
3 and 15). Within completed fruiting bodies reside several hundred thousand differentiated and
metabolically quiescent myxospores, which are able to withstand environmental stresses and remain dormant for at least 50 years (13). When nutrients are restored, myxospores germinate and form viable colonies. M. xanthus cells are bacteriolytic and
feed on amino acids and peptides from prey bacteria, but they are
unable to use sugars as an energy source due to the absence of key
glycolytic enzymes such as pyruvate kinase (22).
Starvation for amino acids alone results in fruiting body formation and
sporulation. Cells sense amino acid starvation as an indicator of
overall starvation, at least in part, through a stringent response via
the ribosome-associated (p)ppGpp synthetase enzyme RelA. This sensory
mechanism is proposed to be one of the earliest developmental events,
and a block in this mechanism results in an early developmental arrest
and in sporulation deficiency (6, 18). Previous work has
shown that starving cells for any essential amino acids (wild-type
M. xanthus cannot synthesize leucine, isoleucine, or valine)
or depriving an auxotroph of its newly essential amino acid
(9-11) will elicit a developmental response. In this work
we describe the identification and characterization of an M. xanthus open reading frame (ORF) with strong homology to the
Escherichia coli N-acetylornithine deacetylase
gene (argE), which catalyzes the conversion of
acetylornithine to ornithine (5, 12).
Identification of a putative argE homolog.
An ORF
with strong sequence similarity and identity to the E. coli
argE was identified 300 bp upstream of the putative
trcF gene in M. xanthus (4) and is
transcriptionally oriented in the opposite direction with respect to
trcF. A second divergently transcribed ORF was identified
just upstream of this putative argE homolog. A diagram of
this region, including a putative translational start site and
Shine-Dalgarno sequence, is outlined in Fig.
1. Sequence determination and analysis of
this region were performed as previously described (6). DNA
sequence analysis identified two ORFs with >90% third-position GC
codon usage, which is typical for genes in M. xanthus and
other high-G+C-content organisms (1, 16). These ORFs were
designated argE and orf2 (Fig. 1). Analysis of
both ORFs by using BLAST (National Center for Biotechnology Information
[NCBI], National Library of Medicine) revealed that one of these ORFs
bears a high degree of sequence identity to argE in E. coli. An alignment comparison between the E. coli and M. xanthus ArgE homologs showed 33% identity and 46%
similarity (Fig. 2; comparison was
performed with Macaw version 2.0.5, NCBI). No significant identity or
similarity was found between ORF 2 and anything in the NCBI database.
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FIG. 1.
Partial restriction map of the 2.8-kb M. xanthus DK101 argE chromosomal region. Arrows indicate
putative directions of transcription. The region 3' to argE
including the 3' end of M. xanthus trcF is shown (for
reference, see Garza et al. [4]). Restriction enzymes
are abbreviated as follows: A, AccI; B, BamHI; E,
EagI; S, SmaI (not all sites shown); S1,
SacI. Solid lines indicate DNA regions used for insertion
mutagenesis in orf2 (pBAR100) or argE (pBAR113)
and expression of argE on the pBluescript lac
promoter (pBAR114). A possible ribosome binding site (RBS) and
initiator codon (ATG) for argE are shown.
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FIG. 2.
Best-fit alignments of the deduced acetylornithine
deacetylase (argE) amino acid sequences from M. xanthus and E. coli. Black boxes with white lettering
indicate amino acid identities, and gray boxes indicate similarities
within groups by side chain.
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Complementation of an E. coli argE mutant.
To
demonstrate that our putative argE homolog encodes an
acetylornithine deacetylase, we assayed its ability to complement a
known E. coli argE mutant. To test this hypothesis, we
constructed a plasmid containing the entire M. xanthus argE
ORF and putative ribosome binding site under the control of the
Plac promoter from pBluescript KS(+) (Stratagene, La Jolla,
Calif.). A 1,282-bp AccI fragment from pBELF1R
(4) was blunted with DNA polymerase Klenow fragment and
ligated into pBluescript KS(+) linearized with SmaI (14). The orientation of the insert was verified by sequence analysis, and the resulting plasmid was named pBAR114 (Fig. 1). This
plasmid, along with the vector, was transformed into E. coli strain BJW72 (17), which carries a point mutation in
argE. Transformants were then screened for their ability to
grow in the presence and absence of arginine (final concentration, 0.2 mg/ml). As shown in Fig. 3, BJW72 cells
can grow in the absence of arginine only if they carry pBAR114 or are
supplemented with arginine; however, they are unable to grow in the
absence of arginine with the vector pBluescript.
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FIG. 3.
Complementation of an E. coli argE mutation
by the M. xanthus argE gene. E. coli cells
carrying a point mutation in argE were transformed with
either pBluescript KS(+) (pBS KS+) or pBAR114 and assayed for growth on
M9 minimal agar plates (14) with or without supplemental
arginine (Arg). (Each quadrant of a single plate contains a separate
isolate, and corresponding quadrants of the two plates contain the same
isolate.) Both strains show growth on plates with supplemental
arginine, but only the strain with pBAR114 grows in the absence of
arginine.
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M. xanthus argE is essential for arginine
biosynthesis.
To test whether argE is essential for
arginine biosynthesis in M. xanthus, we constructed an
argE null mutant and tested whether it required arginine for
growth on A1 minimal agarose plates (2). The argE
null mutation was constructed by plasmid-insertion mutagenesis procedures, as previously described (6). This procedure
leaves a tandem duplication of two truncated, nonfunctional genes. For the argE insertion, an EagI fragment internal to
the argE ORF was cloned into pBGS18 (19), to give rise to
the deletion plasmid pBAR113. This plasmid was then transformed into
M. xanthus DK101 via electroporation (7). Because
M. xanthus does not maintain pBGS18 as an extrachromosomal
plasmid, kanamycin-resistant colonies result from plasmid insertions
into the M. xanthus chromosome (20). The presence
of a single copy of pBAR113 at the chromosomal locus was confirmed by
Southern blot (data not shown). The argE null strain was
designated MS2014. A similar approach was taken to construct an
orf2 null mutant by using an internal BamHI to SacI fragment within orf2. The resulting null
mutant was designated pBAR100.
Growth of the argE null mutant was tested on A1 agarose
plates with or without 0.1 mg of arginine supplement/ml. Strain MS2014 was able to grow on A1 agarose only when supplemented with arginine, demonstrating that this strain is an arginine auxotroph. When the
strain was grown in rich medium, such as CTT medium (6), no
detectable growth phenotype was observed. In addition, the orf2 null strain MS2008 was examined for its ability to grow
in the presence and absence of arginine. No detectable growth phenotype was observed, demonstrating that this gene is not involved in arginine biosynthesis.
Developmental phenotypes of the argE and
orf2 null mutants.
To determine whether either the
argE null mutant or orf2 null mutant was
defective in development under standard laboratory conditions
(8), both strains, along with the parent strain, DK101, were
assayed for their ability to form fruiting bodies on TPM agar plates
(6, 21). No defect in fruiting body formation was observed for either
mutant strain as compared to the parental strain. Viable spore assays
were also performed after 3 and 5 days, and the numbers of heat- and
sonication-resistant CFU were determined (21). The
argE null mutant formed viable spores at a level two- to
threefold greater than that by the wild type; the mutant showed 229%
(± 56%) wild-type developing cells after 3 days and 297% (± 57%)
wild-type developing cells after 5 days, while the orf2 null
mutant showed wild-type sporulation at 87% (± 22%) after 3 days and
91% (± 25%) after 5 days.
Previous work with M. xanthus auxotrophs showed that
development could be initiated by simply removing the required amino acid (9-11). This work led to the hypothesis that
starvation for any amino acid could lead to initiation of development.
To further test this hypothesis, we examined whether the
argE null mutant strain MS2014 could be induced to form
fruiting bodies if placed at high cell density on A1 agarose plates in
the absence of supplemental arginine. Under such conditions, MS2014
forms multicellular fruiting bodies while DK101 remains as a normal
vegetative colony (Fig. 4). Inspection of
the MS2014 cells after heating and sonication shows that these
developing cells also form viable spores, approximately 4 × 106 spores/ml after 5 days. This is approximately 20% of
the number of spores observed under complete starvation condition on
TPM agar (21) as described above. Though the number of
viable spores produced by MS2014 is lower under arginine-starvation
conditions than that produced by complete starvation, it is
significantly higher than the number of spores produced by wild-type
cells under identical conditions, which is 100 spores/ml. These studies
support the hypothesis that starvation of M. xanthus for any
required amino acid at high cell density can initiate the developmental process.
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FIG. 4.
Fruiting body formation in the absence of supplemental
arginine (Arg). Cells were spotted at high density (5 × 109 cells/ml) on 0.8% agarose A1 plates with or without
0.1 mg of supplemental arginine/ml. M. xanthus MS2014
(sglA1 argE::pBAR113) forms fruiting
bodies on A1 minimal media in response to arginine starvation, while
strain DK101 (sglA1) does not. Magnification, ×8. Both
strains show normal growth in the presence of supplemental arginine.
Development of strain DK101 on TPM starvation agar is shown for
reference. Magnification, ×6.
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Nucleotide sequence accession number.
The DNA sequence of the
argE region has been submitted to GenBank under accession
no. AF055904.
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ACKNOWLEDGMENTS |
We thank Margaret Baer, Anthony Garza, and Jeff Pollack for helpful
discussions and readings of this work. In addition, we thank Stacia
Hoover and Dean Lavell for technical assistance with sequencing and
loaning of computer software.
This research was supported in part by National Institutes of Health
grant GM54592 to M.S.
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FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Microbiology, Division of Biological Sciences, University of California at Davis, 1 Shields Ave., Davis, CA 95616. Phone: (530) 752-9005. Fax:
(530) 752-9014. E-mail: mhsinger{at}ucdavis.edu.
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Journal of Bacteriology, December 1998, p. 6412-6414, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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