26Bacteria initially respond to conditions that attenuate their growth by eliciting large-scale 27 transcriptional changes. The accompanying changes in gene expression and metabolism allow 28 the bacterial cells to effectively adapt to the growth attenuated state. How the transcriptome 29 subsequently changes as growth attenuation ensues is not well understood. We used nitrogen 30 (N) starvation as a model nutrient starvation condition to study the transcriptome of growth 31 attenuated Escherichia coli. The results reveal that the transcriptome of nitrogen starvation-32 induced growth attenuated E. coli remains dynamic and perturbations to it compromise the 33 viability of growth attenuated bacteria and their ability to effectively recover growth when N 34 starvation conditions become alleviated. We further reveal that, over time, N starvation-35 induced growth attenuated bacteria rely on the degradation of allantoin for optimal growth 36 recovery when N becomes replenished. This study provides insights into the temporally 37 coordinated adaptive responses that occur in E. coli experiencing sustained N starvation. 38 39 IMPORTANCE 40Bacteria in their natural environments seldom encounter conditions that support continuous 41 growth. Hence, many bacteria spend the majority of their time in states of little or no growth 42 due to starvation of essential nutrients. To cope with prolonged periods of nutrient starvation, 43 bacteria have evolved several strategies, primarily manifesting themselves through changes in 44 how the information in their genes is accessed. How these coping strategies change over time 45 under nutrient starvation is not well understood and this knowledge is not only important to 46 broaden our understanding of bacterial cell function, but also to potentially find ways to 47 manage harmful bacteria. This study provides insights into how nitrogen starved Escherichia 48 coli bacteria rely on different genes during long term nitrogen starvation. 49 50 3 KEYWORDS 51
T7 development in Escherichia coli requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. While the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growing E. coli as a consequence of buildup of guanosine pentaphosphate ((p)ppGpp) during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.Significance statementVirus that infect bacteria (phages) represent the most abundant living entities on the planet and many aspects of our fundamental knowledge of phage-bacteria relationships have been derived in the context of exponentially growing bacteria. In the case of the prototypical Escherichia coli phage T7, specific inhibition of the housekeeping form of the RNA polymerase (Eσ70) by a T7 protein, called Gp2, is essential for the development of viral progeny. We now reveal that T7 uses a second specific inhibitor that selectively inhibits the stationary phase RNAP (EσS), which enables T7 to develop well in exponentially growing and stationary phase bacteria. The results have broad implications for our understanding of phage-bacteria relationships and therapeutic application of phages.
SPO1 phage infection of Bacillus subtilis results in a comprehensive remodelling of processes leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins for the specific shut down of various host processes including transcription, DNA synthesis and cell division. However, the properties and bacterial targets of many genes of the SPO1 host takeover module remain elusive. Through a systematic analysis of gene products encoded by the SPO1 host takeover module we identified eight gene products which attenuated B. subtilis growth. Out of the eight gene products that attenuated bacterial growth, a 25 kDa protein, called Gp53, was shown to interact with the AAA+ chaperone protein ClpC of the ClpCP protease of B. subtilis. Results reveal that Gp53 functions like a phage encoded adaptor protein and thereby appears to alter the substrate specificity of the ClpCP protease to modulate the proteome of the infected cell to benefit efficient SPO1 phage progeny development. It seems that Gp53 represents a novel strategy used by phages to acquire their bacterial prey.Significance statementViruses of bacteria (phages) represent the most abundant living entities on the planet, and many aspects of our fundamental knowledge of phage–bacteria relationships remain elusive. Many phages encode specialised small proteins, which modulate essential physiological processes in bacteria in order to convert the bacterial cell into a ‘factory’ for phage progeny production – ultimately leading to the demise of the bacterial cell. We describe the identification of several antibacterial proteins produced by a prototypical phage that infects Bacillus subtilis and describe how one such protein subverts the protein control system of its host to benefit phage progeny development. The results have broad implications for our understanding of phage–bacteria relationships and the therapeutic application of phages and their gene products.
While transcriptional reprogramming is perhaps the most well understood form of controlling gene expression in response to nitrogen starvation in bacteria, how post-transcriptional regulation (PTR) of gene expression contributes to this adaptive response remains elusive. Small regulatory RNAs (sRNAs) are the major post-transcriptional regulators of gene expression in bacteria. They regulate gene expression by base pairing to target mRNAs, leading to enhanced translation or inhibition of translation and/or alteration of mRNA stability. To form productive interactions with target mRNAs, most sRNAs require an RNA chaperone. In many bacteria of diverse lineages, the RNA chaperone Hfq plays a central and integral role in the PTR of gene expression by stabilising sRNAs and promoting their interactions with cognate mRNAs. Comparative analysis of the transcriptomes of Escherichia coli at different stages of nitrogen starvation reveal that levels of sRNA vary throughout starvation. We used Hfq as a surrogate to study sRNA-mediated PTR of gene expression during sustained nitrogen starvation. Our results indicate that sRNAs-mediated PTR of gene expression plays a major role in the adaptive response to sustained nitrogen starvation. Intriguingly, using single-molecule PALM, we reveal that Hfq is involved in the formation of intracellular structures which functionally might resemble processing (P) bodies found in eukaryotic cells involved in mRNA turnover.
The canonical function of a bacterial sigma factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the sigma 54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the sigma 54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. The genes that rely upon sigma 54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by sigma 54, is not yet exhaustive. By comparing the non-planktonic growth properties of prototypical enteropathogenic, uropathogenic and non-pathogenic Escherichia coli strains devoid of sigma 54, we uncovered sigma 54 as a determinant of homogenous non-planktonic growth specifically in the uropathogenic strain. Notably, bacteria devoid of individual activator ATPases of the sigma 54-RNAP do not phenocopy the sigma 54 mutant strain. It seems that sigma 54's role as a determinant of homogenous non-planktonic growth represents a putative non-canonical function of sigma 54 in regulating genetic information flow.
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