Complementation of sporulation and motility defects in a prokaryote by a eukaryotic GTPase

Patricia L. Hartzell

doi:10.1073/pnas.94.18.9881

New Research In

Communicated by A. Dale Kaiser, Stanford University School of Medicine, Stanford, CA (received for review December 3, 1996)

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Figure 1
MglA shares 24% identity with Sar1p. The complete amino acid sequence for MglA and Sar1p is shown. The primary sequence of the 22-kDa MglA contains 195 amino acids, and the 21-kDa Sar1p contains 190 amino acids. Residues that comprise the conserved GTP consensus ae underlined in bold. | indicates identity; ∗ indicates similarity. # indicates the site of the mutation in mglA8. The method of Altschul et al. (44) was used to align the sequences.
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Figure 2
The 22-kDa yeast protein Sar1p is produced in M. xanthus. Immunoblot of extracts from S. cerevisiae and M. xanthus strains was probed with a 1:2,000 dilution of rabbit anti-Sar1p antibody. Lane 1, Bio-Rad Kaleidoscope prestained standards; carbonic anhydrase (42,600); soybean trypsin inhibitor (29,900) and lysozyme (17,100). Lane 2, M. xanthus DK1622 (wild type). Lane 3, M. xanthus DK6204 (ΔmglA). Lane 4, M. xanthus MxH1048 (ΔmglA SAR1). Lane 5, S. cerevisiae. Lane 6, M. xanthus MxH1056 (ΔmglA SAR1 rpm). Lane 7, M. xanthus MxH1116 (ΔmglA SAR1 lys36thr).
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Figure 3
A second-site mutation restores motility to ΔmglA SAR1. Morphology of M. xanthus colonies growing on CTPM 1.5% agar plates. (A) M. xanthus DK1622. (B) ΔmglA (DK6204). (C) ΔmglA SAR1 (MxH1048). (D) ΔmglA SAR1 rpm (MxH1056). Photographs were taken with a Nikon SMZ-U stereomicroscope. (Bar = 3 mm.)
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Figure 4
The motile ΔmglA SAR1 rpm strain forms fruiting aggregates. Morphogenesis of fruiting bodies on TPM starvation medium. (A) M. xanthus DK1622. (B) ΔmglA (DK6204). (C) ΔmglA SAR1 (MxH1048). (D) ΔmglA SAR1 rpm (MxH1056). The parent, ΔmglA SAR1, fails to aggregate (C), although heat-resistant spores are produced. Cells with the ΔmglA mutation (B) fail to aggregate and fail to differentiate into spores. Photographs were taken 48 hr after the onset of development with a Nikon SMZ-U stereomicroscope. (Bar = 50 mm.)

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Table 1

SAR1 rescues the sporulation defect of a ΔmglA strain

Strain	Genotype	Phenotype^*		Viable spores, ml	Viable spores^†, wild type
Strain	Genotype	Motility	Fruiting	Viable spores, ml	Viable spores^†, wild type
DK1622	Wild type	+	+	5 × 10⁸	100.00%
DK6204	ΔmglA	−	−	5 × 10⁴	0.01%
MxH1048	ΔmglA SAR1	−	−	4.6 × 10⁸	92.00%
MxH1116	Δmgl SAR1 K36T	−	−	9 × 10⁴	0.02%
MxH1091	ΔmglA Ha-RAS	−	−	2 × 10⁵	0.04%

↵* Assays for motility and morphogenesis are described in Materials and Methods.
↵† Approximately 5-10% of wild-type cells differentiate into heat-resistant spores by day 5.

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Table 2

Disruption of SAR1 in the ΔmglA SAR1 rpm strain abolishes gliding motility

Tet^R donor	Kan^R recipient	Tet^R transductants^*
		Total Tet^R	Kan^R		Kan^S
		Total Tet^R	Motile	Nonmotile	Motile	Nonmotile
Ω1901 mglA⁺ (motile)	ΔmglA SAR1	113	0	10	103	0
	ΔmglA SAR1 rpm	63	63^†	0	0	0
Ω1901 ΔmglA (nonmotile)	ΔmglA SAR1	78	0	11	0	67
	ΔmglA SAR1 rpm	49	49^†	0	0	0
Ω1901 mglA8 (nonmotile)	ΔmglA SAR1	89	0	9	0	80
	ΔmglA SAR1 rpm	44	12^†	0	0	32^‡

↵* Transductants initially were selected on plates with tetracycline and subsequently were scored for resistance to kanamycin as described in Materials and Methods.
↵† Motile transductants were identical to the ΔmglA SAR1 rpm recipient and did not carry the donor mglA allele.
↵‡ DNA from eight transductants was assayed for the presence of the mglA8 and SAR1 genes. In all eight, the mglA8 allele had replaced the integrated copy of SAR1.