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FtsA, a homolog of actin, is essential for cell division of Escherichia coli and is widely conserved among many bacteria. FtsA helps to tether polymers of the bacterial tubulin homolog FtsZ to the cytoplasmic membrane as part of the cytokinetic Z ring. GFP fusions to FtsA have illuminated FtsA's localization in live E. coli, but these fusions have not been fully functional and required the presence of the native FtsA. Here, we characterize "sandwich" fusions of E. coli FtsA to either mCherry or msfGFP that are functional for cell division and exhibit fluorescent rings at midcell that persist throughout constriction until cell separation. FtsA within the Z ring moved circumferentially like FtsZ, and FtsA outside the rings formed highly dynamic patches at the membrane. Notably, both FtsA-mCherry

Summary Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes., Graphical abstract, Highlights • Cryo-EM structures of PNPase in complex with the RNA chaperone Hfq and regulatory RNA • Structural insights into regulatory RNA recognition by Hfq and PNPase • Model for stabilization of regulatory RNAs and facilitation of their functions, The conserved exoribonuclease PNPase contributes to RNA turnover in many organisms, but in bacteria the enzyme can be re-programmed by the RNA chaperone Hfq and regulatory RNA to switch from degradative to chaperoning roles in RNA-mediated gene regulation. Dendooven et al. provide structural insight into the basis for this functional switch and details of the recognition of complex regulatory RNAs by Hfq and PNPase.

Treponema pallidum ssp. pallidum, the causative agent of syphilis, can now be cultured continuously in vitro utilizing a tissue culture system, and the multiplication rates are similar to those obtained in experimental infection of rabbits. In this study, the RNA transcript profiles of the T. pallidum Nichols during in vitro culture and rabbit infection were compared to examine whether gene expression patterns differed in these two environments. To this end, RNA preparations were converted to cDNA and subjected to RNA-seq using high throughput Illumina sequencing; reverse transcriptase quantitative PCR was also performed on selected genes for validation of results. The transcript profiles in the in vivo and in vitro environments were remarkably similar, exhibiting a high degree of concordance overall. However, transcript levels of 94 genes (9%) out of the 1,063 predicted genes in the T. pallidum genome were significantly different during rabbit infection versus in vitro culture, varying by up to 8-fold in the two environments. Genes that exhibited significantly higher transcript levels during rabbit infection included those encoding multiple ribosomal proteins, several prominent membrane proteins, glycolysis-associated enzymes, replication initiator DnaA, rubredoxin, thioredoxin, two putative regulatory proteins, and proteins associated with solute transport. In vitro cultured T. pallidum had higher transcript levels of DNA repair proteins, cofactor synthesis enzymes, and several hypothetical proteins. The overall concordance of the transcript profiles may indicate that these environments are highly similar in terms of their effects on T. pallidum physiology and growth, and may also reflect a relatively low level of transcriptional regulation in this reduced genome organism., Author summary The spiral-shaped bacterium that causes syphilis, Treponema pallidum subsp. pallidum, was first discovered in 1905, but a laboratory system that promotes long-term growth of this tiny organism was not developed until 2017. In this study, we compared the gene expression of T. pallidum grown in this system to organisms recovered from rabbits infected with the bacterium. Gene expression under these two conditions generally was very similar. However, T. pallidum grown in rabbits had more RNA ‘messengers’ for genes encoding important cell membrane proteins and protein making machinery, whereas those grown in vitro (in glass) had higher RNA levels for genes related to fixing DNA breaks and making vitamins. These gene expression patterns may help us understand how T. pallidum can cause infections that last for decades and yet can be so hard to grow in the laboratory.

Cell growth and division are coordinated, ensuring homeostasis under any given growth condition, with division occurring as cell mass doubles. The signals and controlling circuit(s) between growth and division are not well understood; however, it is known inEscherichia colithat the essential GTPase Era, which is growth rate regulated, coordinates the two functions and may be a checkpoint regulator of both. We have isolated a mutant of Era that separates its effect on growth and division. When overproduced, the mutant protein Era647 is dominant to wild-type Era and blocks division, causing cells to filament. Multicopy suppressors that prevent the filamentation phenotype of Era647 either increase the expression of FtsZ or decrease the expression of the Era647 protein. Excess Era647 induces complete delocalization of Z rings, providing an explanation for why Era647 induces filamentation, but this effect is probably not due to direct interaction between Era647 and FtsZ. The hypermorphicftsZ* allele at the native locus can suppress the effects of Era647 overproduction, indicating that extra FtsZ is not required for the suppression, but another hypermorphic allele that accelerates cell division through periplasmic signaling,ftsL*, cannot. Together, these results suggest that Era647 blocks cell division by destabilizing the Z ring.IMPORTANCEAll cells need to coordinate their growth and division, and small GTPases that are conserved throughout life play a key role in this regulation. One of these, Era, provides an essential function in the assembly of the 30S ribosomal subunit inEscherichia coli, but its role in regulatingE. colicell division is much less well understood. Here, we characterize a novel dominant negative mutant of Era (Era647) that uncouples these two activities when overproduced; it inhibits cell division by disrupting assembly of the Z ring, without significantly affecting ribosome production. The unique properties of this mutant should help to elucidate how Era regulates cell division and coordinates this process with ribosome biogenesis.

Small regulatory RNAs (sRNAs) are an important class of bacterial post-transcriptional regulators that control numerous physiological processes, including stress responses. In Gram-negative bacteria including Escherichia coli, the RNA chaperone Hfq binds many sRNAs and facilitates pairing to target transcripts, resulting in changes in mRNA transcription, translation, or stability. Here, we report that poly(A) polymerase (PAP I), which promotes RNA degradation by exoribonucleases through the addition of poly(A) tails, has a crucial role in the regulation of gene expression by Hfq-dependent sRNAs. Specifically, we show that deletion of pcnB, encoding PAP I, paradoxically resulted in an increased turnover of certain Hfq-dependent sRNAs, including RyhB. RyhB instability in the pcnB deletion strain was suppressed by mutations in hfq or ryhB that disrupt pairing of RyhB with target RNAs, by mutations in the 3′ external transcribed spacer of the glyW-cysT-leuZ transcript (3′ETSLeuZ) involved in pairing with RyhB, or an internal deletion in rne, which encodes the endoribonuclease RNase E. Finally, the reduced stability of RyhB in the pcnB deletion strain resulted in impaired regulation of some of its target mRNAs, specifically sodB and sdhCDAB. Altogether our data support a model where PAP I plays a critical role in ensuring the efficient decay of the 3′ETSLeuZ. In the absence of PAP I, the 3′ETSLeuZ transcripts accumulate, bind Hfq, and pair with RyhB, resulting in its depletion via RNase E-mediated decay. This ultimately leads to a defect in RyhB function in a PAP I deficient strain.

Gene regulation by base pairing between small noncoding RNAs (sRNAs) and their mRNA targets is an important mechanism that allows bacteria to maintain homeostasis and respond to dynamic environments. In Gram-negative bacteria, sRNA pairing and regulation are mediated by several RNA-binding proteins, including the sRNA chaperone Hfq and polynucleotide phosphorylase (PNPase). PNPase and its homolog RNase PH together represent the two 3′ to 5′ phosphorolytic exoribonucleases found in Escherichia coli ; however, the role of RNase PH in sRNA regulation has not yet been explored and reported. Here, we have examined in detail how PNPase and RNase PH interact to support sRNA stability, activity, and base pairing in exponential and stationary growth conditions. Our results indicate that these proteins facilitate the stability and regulatory function of the sRNAs RyhB, CyaR, and MicA during exponential growth. PNPase further appears to contribute to pairing between RyhB and its mRNA targets. During stationary growth, each sRNA responded differently to the absence or presence of PNPase and RNase PH. Finally, our results suggest that PNPase and RNase PH stabilize only Hfq-bound sRNAs. Taken together, these results confirm and extend previous findings that PNPase participates in sRNA regulation and reveal that RNase PH serves a similar, albeit more limited, role as well. These proteins may, therefore, act to protect sRNAs from spurious degradation while also facilitating regulatory pairing with their targets. IMPORTANCE In many bacteria, Hfq-dependent base-pairing sRNAs facilitate rapid changes in gene expression that are critical for maintaining homeostasis and responding to stress and environmental changes. While a role for Hfq in this process was identified more than 2 decades ago, the identity and function of the other proteins required for Hfq-dependent regulation by sRNAs have not been resolved. Here, we demonstrate that PNPase and RNase PH, the two phosphorolytic RNases in E. coli , stabilize sRNAs against premature degradation and, in the case of PNPase, also accelerate regulation by sRNA-mRNA pairings for certain sRNAs. These findings are the first to demonstrate that RNase PH influences and supports sRNA regulation and suggest shared and distinct roles for these phosphorolytic RNases in this process.

Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen and a leading cause of bacterial pneumonia worldwide. Small regulatory RNAs (sRNAs), which often act by posttranscriptionally regulating gene expression, have been shown to be crucial for the virulence of S. pneumoniae and other bacterial pathogens. Over 170 putative sRNAs have been identified in the S. pneumoniae TIGR4 strain (serotype 4) through transcriptomic studies, and a subset of these sRNAs has been further implicated in regulating pneumococcal pathogenesis. However, there is little overlap in the sRNAs identified among these studies, which indicates that the approaches used for sRNA identification were not sufficiently sensitive and robust and that there are likely many more undiscovered sRNAs encoded in the S. pneumoniae genome. Here, we sought to comprehensively identify sRNAs in Avery’s virulent S. pneumoniae strain D39 using two independent RNA sequencing (RNA-seq)-based approaches. We developed an unbiased method for identifying novel sRNAs from bacterial RNA-seq data and have further tested the specificity of our analysis program toward identifying sRNAs encoded by both strains D39 and TIGR4. Interestingly, the genes for 15% of the putative sRNAs identified in strain TIGR4, including ones previously implicated in virulence, are not present in the strain D39 genome, suggesting that the differences in sRNA repertoires between these two serotypes may contribute to their strain-specific virulence properties. Finally, this study has identified 66 new sRNA candidates in strain D39, 30 of which have been further validated, raising the total number of sRNAs that have been identified in strain D39 to 112. IMPORTANCE Recent work has shown that sRNAs play crucial roles in S. pneumoniae pathogenesis, as inactivation of nearly one-third of the putative sRNA genes identified in one study led to reduced fitness or virulence in a murine model. Yet our understanding of sRNA-mediated gene regulation in S. pneumoniae has been hindered by limited knowledge about these regulatory RNAs, including which sRNAs are synthesized by different S. pneumoniae strains. We sought to address this problem by developing a sensitive sRNA detection technique to identify sRNAs in S. pneumoniae D39. A comparison of our data set reported here to those of other RNA-seq studies for S. pneumoniae strain D39 and TIGR4 has provided new insights into the S. pneumoniae sRNA transcriptome.

Almost 60 years ago, Severo Ochoa was awarded the Nobel Prize in Physiology or Medicine for his discovery of the enzymatic synthesis of RNA by polynucleotide phosphorylase (PNPase). Although this discovery provided an important tool for deciphering the genetic code, subsequent work revealed that the predominant function of PNPase in bacteria and eukaryotes is catalyzing the reverse reaction, i.e., the release of ribonucleotides from RNA. PNPase has a crucial role in RNA metabolism in bacteria and eukaryotes mainly through its roles in processing and degrading RNAs, but additional functions in RNA metabolism have recently been reported for this enzyme. Here, we discuss these established and noncanonical functions for PNPase and the possibility that the major impact of PNPase on cell physiology is through its unorthodox roles., Author summary Widely distributed among bacteria and eukaryotes, including humans, polynucleotide phosphorylase (PNPase) is a critical enzyme in RNA metabolism that functions in most organisms as a 3ʹ to 5ʹ exoribonuclease. In bacteria, inactivation of the gene encoding PNPase results in a wide range of consequences, including impaired growth, diminished stress responses, and loss of virulence. In mammals, PNPase has an essential role in mitochondrial function. Mutations in the gene encoding the human PNPase (hPNPase) that reduce its activity can lead to hereditary hearing loss, encephalomyopathy, severe axonal neuropathy, delayed myelination, and Leigh syndrome. In this review, we highlight both the canonical and unorthodox activities that have been reported for PNPase. Specifically, we examine its role in bacterial mRNA and rRNA decay, RNA processing, and small regulatory RNA (sRNA) degradation and stabilization. Furthermore, we explore the recently reported findings on the function of hPNPase in mitochondrial RNA import and degradation and cytoplasmic mRNA and noncoding RNA decay. Despite being discovered more than six decades ago, we are still only beginning to grasp the breadth of mechanisms by which the enzymatic activities of PNPase contribute to cellular and organismal physiology.

The genetic transformation of plant cells by Agrobacterium tumefaciens results from the transfer of DNA and proteins via a specific virulence ( vir ) -induced type IV secretion system (T4SS). To better understand T4SS function, we analyzed the localization of its structural components and substrates by deconvolution fluorescence microscopy. GFP fusions to T4SS proteins with cytoplasmic tails, VirB8 and VirD4, or cytoplasmic T4SS substrate proteins, VirD2, VirE2, and VirF, localize in a helical pattern of fluorescent foci around the perimeter of the bacterial cell. All fusion proteins were expressed at native levels of vir induction. Importantly, most fusion proteins are functional and do not exhibit dominant-negative effects on DNA transfer to plant cells. Further, GFP-VirB8 complements a virB8 deletion strain. We also detect native VirB8 localization as a helical array of foci by immunofluorescence microscopy. T4SS foci likely use an existing helical scaffold during their assembly. Indeed, the bacterial cytoskeletal component MinD colocalizes with GFP-VirB8. Helical arrays of foci are found at all times investigated between 12 and 48 h post vir induction at 19 °C. These data lead to a model with multiple T4SSs around the bacterial cell that likely facilitate host cell attachment and DNA transfer. In support, we find multiple T pili around vir -induced bacterial cells.

With common bacterial pathogens becoming increasingly resistant to the current therapeutic arsenal, there is a growing need to utilize alternative strategies when developing new antibacterial drugs. In this issue of Chemistry & Biology , Smith et al. explore the idea of antivirulence drugs by developing inhibitors of the type IV secretion system in Brucella .

The accessory Sec system of Streptococcus gordonii is comprised of SecY2, SecA2, and five proteins (Asp1 through -5) that are required for the export of a serine-rich glycoprotein, GspB. We have previously shown that a number of the Asps interact with GspB, SecA2, or each other. To further define the roles of these Asps in export, we examined their subcellular localization in S. gordonii and in Escherichia coli expressing the streptococcal accessory Sec system. In particular, we assessed how the locations of these accessory Sec proteins were altered by the presence of other components. Using fluorescence microscopy, we found in E. coli that SecA2 localized within multiple foci at the cell membrane, regardless of whether other accessory Sec proteins were expressed. Asp2 alone localized to the cell poles but formed a similar punctate pattern at the membrane when SecA2 was present. Asp1 and Asp3 localized diffusely in the cytosol when expressed alone or with SecA2. However, these proteins redistributed to the membrane in a punctate arrangement when all of the accessory Sec components were present. Cell fractionation studies with S. gordonii further corroborated these microscopy results. Collectively, these findings indicate that Asp1 to -3 are not integral membrane proteins that form structural parts of the translocation channel. Instead, SecA2 serves as a docking site for Asp2, which in turn attracts a complex of Asp1 and Asp3 to the membrane. These protein interactions may be important for the trafficking of GspB to the cell membrane and its subsequent translocation.