Field of Science

Do the rpoD hypercompetence mutations eliminate the normal diauxic shift?

I've been going over the RNA-seq data for our rpoD1 hypercompetence mutant, looking for changes in gene expression that might help us understand why the mutation causes induction of the competence genes in rich medium.

Here's a graph showing the results of DESeq2 analysis of the expression differences between the wildtype strain KW20 and rpoD1 cells at timepoints B1 and B2.  B1 is true log phase growth in rich medium; OD = 0.02.  B2 is OD = 0.6, when the cells are just starting to modify their growth in response to changes they've caused to the medium.  Both axes show how the rpoD1 cells differ from KW20.  The X-axis is differences at B1, and the Y axis is differences at B2.

Phenotypically, RpoD1 cells are not noticeably different from KW20 at timepoint B1. They grow at almost the same rate, and they're not competent.  This similarity is also seen in the lack of horizontal spread of the points in the RNA-seq graph; very few genes are more than twofold different between rpoD1 and KW20.  

But at B2 the  differences are larger, as indicated by the greater spread along the Y axis.  sxy mRNA is up about 3 fold, and the competence genes are increased 3-4-fold (in the green circles).  This is expected, since we know that the rpoD1 cells are competent at this stage.  The gcvB gene (a small regulatory RNA) is also up, but I haven't found any consequences of this (need to look more).  

The only other substantial change (and the largest change) at B2 is a cluster of 7 genes (HI1010-HI1016, in the purple circle) which are down 4-15-fold relative to KW20.  In KW20 these genes are induced briefly at B2 and then shut off again at B3 (OD = 1.0), but in rpoD1 their expression stays low.  The graph below illustrates this for gene HI1010, the first gene in the cluster.  (Look only at the first three time points; the others are cells in the competence-inducing medium MIV.)

What's going on around this time point that could be altered in the rpoD1 mutant?  We know that when cultures of wildtype cells reach this density they have begun to change their gene expression - they're not in true exponential growth any more (not in true 'log phase').  

Bioscreen growth curves of KW20 cultures consistently show a blip around OD = 0.6  (red arrows in the graph above), where growth briefly pauses and then resumes at a slower rate.  This type of growth has been given the name 'diauxy', and the blip represents a 'diauxic shift', a brief slowing or cessation of growth while cells shift from using one one resource to a different resource.

The change in growth rate is more obvious in the version shown below.  It uses a log scale for the Y axis, so periods of exponential growth appears as straight lines.  It's easy to see the initial period of exponential growth (red dashed line), where cell density doubles about every 35 minutes.  After the blip growth resumes, growth is slower but still roughly exponential (blue dashed line), and then gradually stops as conditions become less supportive.

Here's a graph showing all the Bioscreen traces from a different experiment, again showing the diauxic shift.  It appears to occur at a higher OD this time only because the student who made the graph didn't correct for the baseline OD of the culture medium. 

So my hypothesis is that the transient expression of HI1010-HI0116 at the B2 time point is associated with this diauxic shift.  I predict that the lack of the transient expression in the rpoD1 mutant will abolish the diauxic shift - there will be no blip in rpoD1 cultures.

I'm doing a Bioscreen run right now to test this hypothesis, comparing KW20, rpoD1 and rpoD2.  But while I was writing this post I did some digging around and found two relevant results from previous work by undergraduates in the lab.

The first graph compares KW20 to all three types of hypercompetence mutant.  The dark red line is rpoD1; consistent with my hypothesis it's the only strain that doesn't show the diauxic shift.  The second graph compares KW20 to rpoD1 and rpoD2, just like my present run.  This graph uses a log scale  so the shift appears higher in the curve, and appears to occur for all three strains.

And here are my new results (replicates with three slightly different batches of sBHI media):

Conclusion:  I was wrong.  The rpoD mutants are just as likely to show a clear diauzic shift blip as KW20.

Just to further complicate the picture, here's a Bioscreen run using a quite different strain, a clinical strain called 86-028NP, whose DNA sequences differ by 2-3% from the KW20 sequences: there's no sign of a diauxic shift.  Maybe KW20's diauxic shift was selected for by many generations of growth in lab cultures!

Later:  I reran the Bioscreen runs using Medium batch A, this time taking readings every 4 minutes instead of every 10 minutes in order to better resolve the diauxic shift.

The diauxic shift is very evident in the linear-scale (upper) plot, and appears to be identical in all three strains.  The log-scale plot (lower) unexpectedly shows rpoD1 to be growing slower than the others before the shift,.  (Unexpected since this was not seen in the first experiment.)

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