We have demonstrated here that CrfA, a novel Caulobacter
sRNA, is required for the cellular stress response to carbon starvation. Expression of CrfA is rapidly induced in response to carbon starvation and rapidly downregulated when the carbon source is replenished. CrfA's induction by carbon starvation is specific in that other forms of nutrient depletion, such as nitrogen and phosphate starvation, do not induce CrfA accumulation. If CrfA is constitutively overproduced when C. crescentus
is grown in rich medium, the culture exhibits a severe growth defect, but CrfA overexpression caused no growth defect in E. coli
. Microarray analysis of a ΔcrfA
strain and a CrfA overpexression strain identified 27 genes that showed a strong response to both CrfA expression and carbon depletion, with CrfA functioning as an activator of its direct or indirect target genes. Most of the genes strongly regulated by CrfA have potential roles in the cell's adaptive response to carbon starvation. One-third of the CrfA activated genes are predicted to encode membrane transport proteins, and the most common among them are the TonB-dependent receptors. These are outer membrane proteins that bind to extracellular substrates and facilitate their transport across the outer membrane and ultimately into the cytoplasm (13
). The upregulation of transport proteins by CrfA during carbon starvation may enable the import of a greater variety of potential carbon sources. One of the CrfA-regulated genes (CC1363) encodes an enzyme with a putative function as a proton pump driven by pyrophosphate hydrolysis. Upregulation of this protein could help the cell maintain its proton electrochemical gradient and continue ATP synthesis during periods of carbon starvation. Other CrfA-activated genes encode enzymes capable of catabolizing alternative carbon sources, such as cyclic-6-aminohexanoate dimers (CC1323), as well as enzymes that modulate intermediary metabolic pathways.
A recent study of the C. crescentus
response to carbon starvation identified 154 genes that are induced (7
). A total of 112 of these genes showed at least partial rpoN
dependence, suggesting a significant role for σ54
's transcriptional response to carbon starvation (7
). Many of the 42 rpoN
-independent and carbon starvation-induced genes identified in that study overlap with the CrfA-regulated genes described here. All seven of the most significantly CrfA-regulated genes (Table , designated +++) overlap with the carbon starvation-induced and rpoN
-independent genes identified in the previous study (7
). This list of seven CrfA-dependent genes (Table ) includes the three most significantly carbon starvation-induced genes identified in the previous study (7
). Thus, σ54
and CrfA appear to be important transcriptional and posttranscriptional regulators, respectively, for Caulobacter
's response to carbon starvation.
While CrfA is strongly induced by carbon starvation, and required for the accumulation of at least 27 mRNA targets during carbon starvation, a ΔcrfA
strain did not exhibit a carbon starvation survival defect. This observation was not surprising, given that many bacterial sRNAs function as redundant components of adaptive stress responses (22
), and there may be additional regulatory factors that substitute for the loss of CrfA function during carbon starvation. Indeed, the C. crescentus
SpoT enzyme has recently been shown to activate the synthesis of the second messenger ppGpp during carbon starvation, and microarray studies have identified a significant number of regulatory factors whose steady-state mRNA levels increase substantially during carbon starvation (7
). Finally, because many of CrfA's mRNA targets encode putative outer membrane transport proteins, CrfA's primary mechanism for promoting survival during carbon starvation may be mediated through the diversification of transport proteins on the cell's surface which thereby enhance the cell's ability to import alternative carbon sources.
The function of CrfA was of particular interest because it was identified in a screen for sRNAs that inhibit growth of C. crescentus during overexpression in rich growth medium. From the list of 27 CrfA-regulated target genes, it is not obvious which target or group of targets is responsible for inhibiting C. crescentus growth. Cells overexpressing CrfA did not exhibit obvious morphological defects and appeared to have simply ceased growth in whatever stage of the cell cycle they occupied at the moment of CrfA overexpression. Since some of the CrfA target genes encode proteins with putative functions in modulating intermediary metabolism, it is possible that the inappropriate upregulation of these proteins during growth in nutrient rich medium, rather than during carbon starvation, disrupts the flux of carbon-containing molecules necessary for maintaining energy production and cellular growth.
We have explored the effect of CrfA on one of its target genes, CC3461. In the absence of carbon starvation, both CrfA and CC3461 mRNA accumulation levels were maintained at a low level. Once CrfA was allowed to accumulate in the absence of glucose, CC3461 mRNA was stabilized. Deletion of a region of secondary structure in the 5′-UTR of CC3461 mRNA, which is distal to the Shine-Dalgarno sequence, resulted in loss of CC3461 mRNA stabilization. This region of the 5′-UTR is complementary to a stretch of 31 bases in CrfA, shown in Fig. and . A site-specific mutation in CrfA's complementary sequence, G52A, and a 3-base deletion, Δ52-54, abrogate CrfA function, possibly by destabilizing CrfA's ability to hybridize to the CC3461 5′-UTR. These observations suggest that CrfA binds directly to the 5′-UTR of CC3461 mRNA, thereby disrupting the secondary structure and inhibiting the degradation of the transcript. In fact, we observed that in the presence of CrfA, there was a 12-fold increase in the CC3461 mRNA half-life and an accompanying increase in the steady-state level of CC3461 mRNA.
It is not yet known how many of the other strongly responsive targets of CrfA regulation identified by the microarray studies are subject to direct regulation. Aligning CrfA and the CC3461 mRNA 5′-UTR using CLUSTALW identified a 31-nucleotide region of complementarity in which 21 bases out of 31 were paired. We used CLUSTALW to identify potential interactions between the CrfA region of complementarity and the other six CrfA-regulated genes identified by all three microarray experiments. For all six genes (CC1348, CC3161, CC2804, CC3336, CC1323, and CC1363), complementarity was detected between CrfA and the putative UTRs. For example, an alignment of the CC1363 mRNA 5′-UTR with CrfA identified complementarity for 19 out of 31 bases.
The most common form of posttranscriptional regulation by bacterial sRNAs involves the negative regulation of the target mRNA (34
). Negative regulation occurs when the sRNA hybridizes to its target and obscures the Shine-Dalgarno sequence. Binding in this manner inhibits translation by preventing mRNA interaction with the ribosome. In addition, it creates a target for RNases, such as RNase E (21
) or RNase III (4
), leading to activated degradation of the mRNA target. A less common form of posttranscriptional regulation by sRNAs involves the positive regulation of the target mRNA (9
). In these rare cases, the 5′-UTR of the mRNA forms an inhibitory stem-loop structure in the region corresponding to the Shine-Dalgarno sequence. This stem-loop prevents ribosome binding and reduces the translation efficiency of the transcript. The activating sRNA hybridizes to a region of the 5′-UTR that overlaps one arm of the stem-loop and results in the loss of secondary structure and the release of the Shine-Dalgarno sequence from the inhibitory stem-loop configuration. The exposed Shine-Dalgarno sequence is then free to bind ribosomes, and the translation efficiency and stability of the target mRNA increase. The direct stabilization and consequent activation of CC3461 mRNA by CrfA present a potentially novel mechanism for posttranscriptional activation by sRNAs. The Shine-Dalgarno sequence of the CC3461 mRNA is not obscured by an inhibitory stem-loop structure (Fig. ). The only significant secondary structure within the CC3461 mRNA 5′-UTR is located at the extreme 5′ end of the transcript. Virtually all of the bases within the 5′-UTR predicted to hybridize with CrfA are located within this stem-loop, and virtually no hybridization is predicted at or around the Shine-Dalgarno sequence. The predicted CrfA hybridization site in the 5′-UTR was verified by deleting a portion of the UTR predicted to form the stem-loop. A CC3461 Δ 5′-UTR strain behaved identically to a ΔcrfA
strain and was largely unable to regulate CC3461 mRNA levels in response to carbon starvation. Since the hybridization of CrfA to the extreme 5′ end of the 5′-UTR and away from the Shine-Dalgarno sequence is not predicted to alter ribosome binding, then activation of CC3461 mRNA by CrfA occurs by a unique mechanism by which CrfA binding at the 5′-UTR stabilizes the transcript against RNase degradation, ultimately leading to mRNA accumulation.