Katarzyna Potrykus

(p)ppGpp: Still Magical?*

The fundamental details of how nutritional stress leads to elevating (p)ppGpp are questionable. By common usage, the meaning of the stringent response has evolved from the specific response to (p)ppGpp provoked by amino acid starvation to all responses caused by elevating (p)ppGpp by any means. Different responses have similar as well as dissimilar positive and negative effects on gene expression and metabolism. The different ways that different bacteria seem to exploit their capacities to form and respond to (p)ppGpp are already impressive despite an early stage of discovery. Apparently, (p)ppGpp can contribute to regulation of many aspects of microbial cell biology that are sensitive to changing nutrient availability: growth, adaptation, secondary metabolism, survival, persistence, cell division, motility, biofilms, development, competence, and virulence. Many basic questions still exist. This review tries to focus on some issues that linger even for the most widely characterized bacterial strains.

ppGpp is the major source of growth rate control in E. coli.

It is widely accepted that the DNA, RNA and protein content of Enterobacteriaceae is regulated as a function of exponential growth rates; macromolecular content increases with faster growth regardless of specific composition of the growth medium. This phenomenon, called growth rate control, primarily involves regulation of ribosomal RNA and ribosomal protein synthesis. However, it was uncertain whether the global regulator ppGpp is the major determinant for growth rate control. Therefore, here we re-evaluate the effect of ppGpp on macromolecular content for different balanced growth rates in defined media. We find that when ppGpp is absent, RNA/protein and RNA/DNA ratios are equivalent in fast and slow growing cells. Moreover, slow growing ppGpp-deficient cells with increased RNA content, display a normal ribosomal subunit composition although polysome content is reduced when compared with fast growing wild-type cells. From this we conclude that growth rate control does not occur in the absence of ppGpp. Also, artificial elevation of ppGpp or introduction of stringent RNA polymerase mutants in ppGpp-deficient cells restores this control. We believe these findings strongly argue in favour of ppGpp and against redundant regulation of growth rate control by other factors in Escherichia coli and other enteric bacteria.

Antagonistic regulation of Escherichia coli ribosomal RNA rrnB P1 promoter activity by GreA and DksA.

The Escherichia coli proteins DksA, GreA, and GreB are all structural homologs that bind the secondary channel of RNA polymerase (RNAP) but are thought to act at different levels of transcription. DksA, with its co-factor ppGpp, inhibits rrnB P1 transcription initiation, whereas GreA and GreB activate RNAP to cleave back-tracked RNA during elongational pausing. Here, in vivo and in vitro evidence reveals antagonistic regulation of rrnB P1 transcription initiation by Gre factors (particularly GreA) and DksA; GreA activates and DksA inhibits. DksA inhibition is epistatic to GreA activation. Both modes of regulation are ppGpp-independent in vivo but DksA inhibition requires ppGpp in vitro. Kinetic experiments and studies of rrnB P1-RNA polymerase complexes suggest that GreA mediates conformational changes at an initiation step in the absence of NTP substrates, even before DksA acts. GreA effects on rrnB P1 open complex conformation reveal a new feature of GreA distinct from its general function in elongation. Our findings support the idea that a balance of the interactions between the three secondary channel-binding proteins and RNAP can provide a new mode for regulating transcription.

Differential effects of Kid toxin on two modes of replication of lambdoid plasmids suggest that this toxin acts before, but not after, the assembly of the replication complex.

Kid is a small protein that is encoded by plasmid R1. It is a toxin that belongs to a killer system that ensures the stability of the plasmid in host cells. The results of previous studies have suggested that Kid is an inhibitor of DNA replication, possibly acting at the onset of initiation. Here, the authors tested the effects of Kid on orilambda-intitiated and oriJ-initiated replication, which may be driven by both the newly assembled replication complex and the heritable complex. It was found that Kid inhibits only replication that is driven by the newly assembled replication complex. The authors also report that Kid inhibits ColE1-like plasmid replication in vivo, in agreement with the previously reported inhibition of ColE1 during in vitro replication. It is proposed that the Kid toxin acts at the level of replication either by preventing de novo assembly of the replication complex or by impairing the functional interactions of the replication complex at the initiation stage.

Composition of the lambda plasmid heritable replication complex.

Previous studies indicated during replication of plasmids derived from bacteriophage lambda (the so-called lambda plasmids), that, once assembled, replication complex can be inherited by one of the two daughter plasmid copies after each replication round, and may function in subsequent replication rounds. It seems that similar processes occur during replication of other DNA molecules, including chromosomes of the yeast Saccharomyces cerevisiae. However, apart from some suggestions based on genetic experiments, composition of the lambda heritable replication complex remains unknown. In amino acid-starved Escherichia coli relA mutants, replication of lambda plasmid DNA is carried out exclusively by the heritable replication complex as assembly of new complexes is impaired due to inhibition of protein synthesis. Here, using a procedure based on in vivo cross-linking, cell lysis, immunoprecipitation with specific sera, de-cross-linking and PCR analysis, we demonstrate that the lambda heritable replication complex consists of O, P, DnaB and, perhaps surprisingly, DnaK proteins.

λpo, a promoter for oop RNA synthesis, has a role in replication of plasmids derived from bacteriophage λ

Abstract Transcription initiated at the bacteriophage λpo promoter gives a short RNA, called oop RNA. Early studies led to a proposal that this transcript plays a role in the initiation of λ DNA replication. In fact, the po promoter is located in the λ replication region and it was suggested that oop RNA may be a primer for replication proceeding leftward from oriλ. However, since in vitro experiments demonstrated that primers for λ DNA replication are produced by the dnaG gene product (DnaG primase) and subsequent in vivo studies indicated that oop RNA is an antisense RNA for the λ cII gene expression, the above-mentioned hypothesis has fallen into oblivion. Nevertheless, here we demonstrate that the po promoter plays a role in λ DNA replication, indeed. We found that λ plasmids bearing a mutation that inactivates po occur in Escherichia coli cells in a copy number significantly lower than wild-type λ plasmids. Amplification of λpo− plasmids during the relaxed response was less efficient relative to λpo+ plasmids suggesting less frequent initiation of replication from oriλ in the absence of transcription from po. This suggestion was confirmed by measurement of incorporation of [3H]thymidine into λ plasmid DNA during pulse-labeling experiments. Therefore, we propose that transcription from the po promoter stimulates replication initiation at oriλ as suggested a long time ago, however, contrary to that suggestion, we assume that the process of po-initiated transcription per se but not the transcription product (oop RNA) might play a role at early steps of λ DNA replication.

Growth at best and worst of times

It has been assumed that bacteria adapt to nutrient limitation by adjusting the number of ribosomes, no matter what they are being starved for. Instead, two recent studies show that Escherichia coli uses different approaches depending on whether its growth is limited by the availability of carbon, nitrogen or phosphate.

A search for the in trans role of GraL, an Escherichia coli small RNA

Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraLs targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed. Only one targets activity (nudE) decreased under those conditions and was thus selected for further analysis. In the absence of GraL and greA, the nudE::lacZ fusions β-galactosidase activity was increased. However, a similar effect was also visible in the strain deleted only for greA. Furthermore, overproduction of GreA alone increased the nudE::lacZ fusions activity as well. This suggests existence of complex regulatory loop-like interactions between GreA, GraL and nudE mRNA. To further dissect this relationship, we performed in vitro EMSA experiments employing GraL and nudE mRNA. However, stable GraL-nudE complexes were not detected, even though the detectable amount of unbound GraL decreased as increasing amounts of nudE mRNA were added. Interestingly, GraL is being bound by Hfq, but nudE easily displaces it. We also conducted a search for genes that are synthetic lethal when deleted along with GraL. This revealed 40 genes that are rendered essential by GraL deletion, however, they are involved in many different cellular processes and no clear correlation was found. The obtained data suggest that GraLs mechanism of action is non-canonical, unique and requires further research.