To characterize the nfs locus in more detail, we confirmed the nfs gene expression pattern during glycerol induction, and examined the pattern during a developmental time course.
2.3.1 nfs gene expression and protein accumulation during glycerol-induced sporulation
To confirm the nfs expression pattern observed in the microarray analysis, the cDNA from a glycerol-induced time course was tested by real time PCR. Consistent with the micro array data, nfsA, D and H as well as Ω7536 (exo) were highly up-regulated at 0.5 to two hours and Mxan_3370 (rplC) upstream of nfs appears to be not regulated (Figure 2-9 a).
To determine the pattern of protein accumulation, the glycerol-induced cultures were next examined by immunoblot analysis using anti peptide antibodies (Eurogentec) generated against NfsA to H (Figure 2-9 b). The apparent molecular masses of the Nfs proteins approximately matched the predicted masses (Table 2-13).
(a)
-2 0 2 4 6 8 10 12 14 16
0 2 4 6 8 10 12 14 16
time [h]
log2 induction
nfsD nfsA nfsH Ω7536 (exo) rplC (Mxan_3370)
(b)
α NfsB α NfsC α NfsD α NfsE α NfsG α NfsA
0h 0.5h 1h 2h 4h 8h 12h
wt
∆nfs + -- + +
-- + + -- + +
-- + + -- + +
-- + + - - +
Figure 2-9 nfs gene expression and protein accumulation. (a) Real time PCR analysis on three selected nfs genes. Ω7536 (exo) was used as a highly up-regulated sporulation marker gene. The expression profile of Mxan_3370 (upstream of the nfs locus) was also determined. (b) Anti-Nfs immunoblots on protein samples from glycerol-induced cultures. Equal culture proportions were harvested at the indicated times after induction with glycerol, proteins were released by bead beating, resolved by SDS-PAGE, and probed with anti-sera specific to NfsA - H.
Table 2-13 Number of amino acids, calculated and observed molecular weight of the Nfs proteins.
Protein Amino acids Calculated molecular mass [kDa]
Apparent molecular mass (SDS-PAGE) [kDa]
NfsA 294 32.3 30
NfsB 442 47 53
NfsC 512 57.5 57
NfsD 1219 137 140
NfsE 499 52 55
NfsF 96 10.8 -
NfsG 682 70.4 73
NfsH 200 21.3 -
Antibodies against NfsF and NfsH failed to recognize a specific protein in cell lysates (data not shown). NfsA, B, C, D, E, and G could not be detected in vegetative cells but
started to accumulate 0.5 hours after induction with glycerol. Interestingly, however, while NfsA, B and C were stably accumulated from 0.5 to twelve hours, accumulation of NfsD, E and G began to reduce after two hours and the proteins were absent after eight hours. Since the mRNA transcription profiles of all eight nfs genes are very similar, this may reflect differences in protein stability controlled by proteolysis.
As shown above, the nfs genes are immediately up-regulated during glycerol induction.
To see if every cell activates expression of the nfs locus, a fusion of the putative nfs promoter region with mcherry was generated and the vector introduced into the Mx8 phage attachment site of M. xanthus DK1622. As control strain, mcherry was also fused to the pilA-promoter in order to express the reporter constitutively while the wild type bearing the empty vector served as negative control. The strains were induced with glycerol and imaged by fluorescence microscopy (Figure 2-10).
PH1221 (pilAPr-mcherry) PH1220 (nfsPr-mcherry)
2h
4h
Fluorescence DIC Fluorescence DIC Fluorescence DIC
1h 0.5h 0h
pilAPr-mcherry nfsPr-mcherry empty vector
Figure 2-10 Expression analysis of PH1221 (pilAPr-mcherry, left), PH1220 (nfsPr-mcherry, center), and PH1222 (empty vector control, right) strains grown in CTT and induced with glycerol.
Samples were taken at indicated time points, spotted on Agar slides and imaged with a fluorescence microscope. Bar: 1 µm.
The experiment covers the time in which glycerol-induced vegetative cells convert into spheres as seen in the DIC images. mcherry under control of the pilA-promoter is constitutively expressed as seen for strain PH1221. Both rod shaped cells, shortening rods and spherical spores fluoresce. For strain PH1222 (empty vector control) only weak autofluorescence signals are detectable at any time point. However, in PH1220 where mcherry is fused to the nsf-promoter, fluorescence signals become detectable
after approximately one hour when cells are shortening. The signal is detectable from all cells that round up and increases until four hours. This suggests that nfs expression is below detection limits in vegetative cells but that proteins of nfs promoter controlled genes accumulate one hour after addition of glycerol.
2.3.2 Activation during starvation-induced sporulation
To determine nfs promoter activation during starvation-induced development, quanti-tative fluorescence measurements of developing submerged cultures were performed on the above strains.
Strains containing no mcherry display an approximately 2.9-fold gain in signal intensity over time which correlates with increasing autofluorescence of developing cultures (data not shown). Signal intensity of Mcherry derived from the pilA-promoter starts approximately twofold higher than in the control strain. The signal intensity increases and peaks at 36 hours 2.7-fold higher than at 0 hours. nfs promoter driven mcherry-expression starts at same levels as the negative control but increases 6.6-fold at 36 hours (Figure 2-11 a) correlating with darkening of the fruiting bodies (Figure 2-11 b).
(a)
0 4,000 8,000 12,000 16,000 20,000
0 6 12 18 24 30 36 42 48 time [h]
fluorescence intensity
vector nfsPr-mcherry pilAPr-mcherry
(b)
nfsPr-mcherry vector
6h 18h 24h 36h 48h
pilAPr-mcherry
Figure 2-11 Quantitative fluorescence measurements of developing cultures. 2.5 ⋅ 108 cells were developed in black 24-well glass bottom dishes in submerged culture. (a) Mcherry specific fluorescence was determined every six hours. Experiments were carried out in triplicate. (b) Stereo microscope images of the developing cultures. The strains were imaged to confirm proper development (selected time points are shown). Bar: 1 mm.
These results suggest that nfs promoter driven proteins start to accumulate after 18 hours of development and reach highest levels at 36 hours reflecting continued expression. Otherwise, the curve of the signal intensity would adopt the slope of the control strain or decrease.
To distinguish which subpopulations of developing cells express nfs promoted mcherry, cultures that have developed for 36 hours were harvested and fruiting bodies were separated from peripheral rods by centrifugation (O'Connor & Zusman, 1991). The separated populations were then imaged with a fluorescence microscope.
While both rods and spores of the empty vector control strain displayed only weak autofluorescence signals, rods and spores from PH1221 (pilAPr-mcherry) produced strong fluorescence signals. In PH1220 (nfsPr-mcherry), only the background signal is detected from rods whereas spores emitted strong fluorescence suggesting that nfs is only expressed in the spore forming subpopulation of developing cells. This observation was confirmed by confocal laser microscopy of fruiting bodies. Fluorescing rods were only visible in strain PH1221 (pilAPr-mcherry) but not in PH1220 (nfsPr-mcherry) and the empty vector control (Figure 2-12 a).
We next sought to determine the patterns of Nfs protein accumulation during a starvation-induced time course. Only NfsA and B could be detected by immunoblot analysis (Figure 2-12 b). The proteins could be observed after 24 hours of development and they are still detected after one week. NfsC - H were not detectable by immunoblot.
Therefore, it cannot be ruled out that these proteins display different expression and stability patterns during starvation-induced development.
PH1221 (pilAPr-mcherry)
fluorescence DIC
PH1220 (nfsPr-mcherry)
DIC fluorescence
PH1222 (vector control)
DIC fluorescence
Fruiting bodies (CLSM)
(a) (b)
Peripheral rods Spores
Figure 2-12 Fluorescence micrographs of cells from submerged culture. (a) Separated spores (left) and peripheral rods (right) after 36 hours development. Bar: 1 µm. (b) Confocal laser scanning microscope images of fruiting bodies after 48 hours (microscopy was performed by Dr. Kai Thormann).
α NfsA α NfsB
0h 18h 24h 30h 36h 42h
wt ∆nsf wt ∆nsf wt ∆nsf wt ∆nsf wt ∆nsf wt ∆nsf wt ∆nsf wt ∆nsf wt ∆nsf 48h 1wk glycerol
Figure 2-13 Anti-Nfs immunoblots on samples from developing cultures. Equal culture volumes were harvested at the indicated times of development, proteins were released by bead beating, resolved by poly-acrylamide gel electrophoresis, and probed with anti-sera specific to NfsA - H.