nsd, a locus that affects the Myxococcus xanthus cellular response to nutrient concentration

Abstract

Expression of the previously reported Tn5lac Omega4469 insertion in Myxococcus xanthus cells is regulated by the starvation response. Interested in learning more about the starvation response, we cloned and sequenced the region containing the insertion. Our analysis shows that the gene fusion is located in an open reading frame that we have designated nsd (nutrient sensing/utilizing defective) and that its expression is driven by a sigma70-like promoter. Sequence analysis of the nsd gene product provides no information on the potential structure or function of the encoded protein. In a further effort to learn about the role of nsd in the starvation response, we closely examined the phenotype of cells carrying the nsd::Tn5lac Omega4469 mutation. Our analysis showed that these cells initiate development on medium that contains nutrients sufficient to sustain vegetative growth of wild-type cells. Furthermore, in liquid media these same nutrient concentrations elicit a severe impairment of growth of nsd cells. The data suggest that the nsd cells launch a starvation response when there are enough nutrients to prevent one. In support of this hypothesis, we found that, when grown in these nutrient concentrations, nsd cells accumulate guanosine tetraphosphate, the cellular starvation signal. Therefore, we propose that nsd is used by cells to respond to available nutrient levels.

FIG. 1.

Patterns of Tn5lac Ω4469 expression. The mean β-galactosidase specific activity was determined by at least three independent experiments. Error bars represent the standard deviations of the means. •, β-galactosidase specific activity from DK4469 cells during vegetative growth in CTT broth; ▴, cell density recorded in arbitrary Klett units.

FIG. 2.

Physical map of the nsd locus. Boxes, indicated ORFs (arrows inside indicate the predicted direction of transcription). The site and direction of the Tn5lac (large triangle) insertion were determined by DNA sequencing and Southern blotting, as described in Materials and Methods. Bent arrow, location of the vegA promoter (23); bar labeled pTEA5, cloned region; stippled bar, nsd probe fragment used in RNA slot blot analysis.

FIG. 3.

Sequence analysis of the region upstream from nsd. (A) Sequence of the region upstream of nsd. Bent arrow, predicted start site of the nsd transcript. The putative promoter elements are underlined at the indicated −10 and −35 regions. The predicted start codon for nsd (+30) is in boldface. The sequence complementary to PE1, the primer used to identify the 5′ end of the transcript, is underlined (+40 to +65). (B) Mapping the 5′ end of the nsd transcript by primer extension analysis. A, G, C, and T show the DNA sequencing ladders. Arrows at PE_v and PE_d lanes indicate each product resulting from primer extension with primer PE1 and total RNA prepared from either vegetative (PE_v) or developing (PE_d) DK1622 cells, as described in Materials and Methods. (C) Comparison of the proposed nsd promoter with the E. coli σ⁷⁰consensus sequence and with three known M. xanthus σ⁷⁰ promoters: those for vegA (23), tps (19), and ops (19).

FIG. 4.

Levels of nsd mRNA present in wild-type and Ω4469 cells. The mean RNA levels were determined by at least three independent experiments. Error bars represent standard deviations of the means. Total RNA was isolated from DK1622 (solid bars) and DK4469 (open bars) cells grown under growth, starvation, or developmental conditions. The levels of nsd mRNA were normalized to the amount present in the vegetative DK1622 sample, which was set equal to 1.

FIG. 5.

Development and growth of wild-type and nsd cells. Wild-type DK1622 (A) and nsd::Tn5lac Ω4469 DK4469 (B) cells were compared for development on CF agar. Cells were photographed under a dissecting microscope after 72 h of incubation. Images shown were recorded at 10× magnification; the inset at ×50 shows mature, darkened fruiting bodies. (Bottom) Vegetatively growing cells were photographed at 10× magnification after 24 h of growth on CTT agar.

FIG. 6.

Swarm expansion on nutrient agar plates. Cells were placed in 20-μl aliquots at a density of 5 × 10⁹ cells/ml onto agar plates containing 1% (A), 0.5% (B), or 0.25% (C) Casitone. We measured the diameter of each spot at the indicated time intervals with the aid of a dissecting microscope. Solid bars, sizes of wild-type swarms; open bars, sizes of nsd::Tn5lac Ω4469 swarms. The swarm diameter is given as the average of four independent experiments, and error bars represent the standard deviations of the means.

FIG. 7.

Growth and development on 0.5% CTT agar. Wild-type DK1622 (A) and nsd::Tn5lac Ω4469 DK4469 (B) cells were plated and cultured on 0.5% CTT agar. Growth and development were monitored, and cells were photographed every 24 h at 10× magnification. Insets show mound and fruiting body morphology after 96 h at 50× magnification.

FIG. 8.

Growth and development on 0.25% CTT agar. Wild-type DK1622 (A) and nsd::Tn5lac Ω4469 DK4469 (B) cells were plated and cultured on 0.25% CTT agar. Growth and development were monitored, and cells were photographed as for Fig. 7.

FIG. 9.

Cell growth in liquid nutrient media. Cells were cultured in liquid medium with 1% (A), 0.5% (B), or 0.25% (C) Casitone, as described in Materials and Methods. •, wild-type cell density; ▴, nsd::Tn5lac Ω4469 cell density.

FIG. 10.

Change in cell yield on solid media. Cells were grown on the indicated agar medium for 3 days. The increase in protein concentration was calculated by subtracting the initial protein concentration (cells removed from the plate immediately after spotting) from the final protein concentration (cells removed from the plate after 3 days of growth). Solid bars, wild type; open bars, nsd::Tn5lac Ω4469. Each data point is the average of three independent experiments; error bars show the standard deviations of the means.