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Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling plant tumor development. Here, we report the structural basis of SghR, a LacI-type transcription factor highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA, and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling cross-talk during Agrobacterium infection, our results pave the way for structure-based inducer analog design, which has potential applications for agricultural industry.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board ‘anti-σ factor’ in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin‐regulating antisense RNA (micF). In bacteria, σ70, together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ70 form a holoenzyme that recognizes ‐35 and ‐10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP‐σ70 at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator‐specific features in activator binding and uncovers distinct roles of the two C‐terminal domains of RNAP α subunit.

Significance Bacterial infection has been extensively investigated; however, little is known about how bacterial pathogens timely shut down infecting machinery after successful infections. Here, a previously unknown sucrose–SghR/SghA–SAG–SA signaling axis was identified which controls the timing to shut off bacterial virulence expression and fine-tune host immune response. Sucrose, salicylic acid (SA), and its storage form SAG are small chemicals produced in plants whereas SghR is a bacterial sensor of sucrose and SghA is a bacterial enzyme that releases SA from SAG. Given that SA is an imperative signaling molecule in defense against a variety of microbial pathogens, these results depict a previously unknown 2-way chemical signaling cross-talk during microbe–host coevolution and shed mechanistic insights into host–bacteria interaction., It is highly intriguing how bacterial pathogens can quickly shut down energy-costly infection machinery once successful infection is established. This study depicts that mutation of repressor SghR increases the expression of hydrolase SghA in Agrobacterium tumefaciens, which releases plant defense signal salicylic acid (SA) from its storage form SA β-glucoside (SAG). Addition of SA substantially reduces gene expression of bacterial virulence. Bacterial vir genes and sghA are differentially transcribed at early and later infection stages, respectively. Plant metabolite sucrose is a signal ligand that inactivates SghR and consequently induces sghA expression. Disruption of sghA leads to increased vir expression in planta and enhances tumor formation whereas mutation of sghR decreases vir expression and tumor formation. These results depict a remarkable mechanism by which A. tumefaciens taps on the reserved pool of plant signal SA to reprogram its virulence upon establishment of infection.

Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA+ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA+ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA+ domain of a bEBP bound to RNAP-σ54-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA+ proteins utilises ATPase activities to allow σ54-dependent transcription initiation.

Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics., eLife digest Bacteria and viruses have long been fighting amongst themselves. Bacteriophages are a type of virus that invade bacteria; their name literally means ‘bacteria eater’. The bacteriophage T7, for example, infects the common bacteria known as Escherichia coli. Once inside, the virus hijacks the bacterium’s cellular machinery, using it to replicate its own genetic material and make more copies of the virus so it can spread. At the same time, the bacteria have found ways to try and defend themselves, which in turn has led some bacteriophages to develop countermeasures to overcome those defences. Many bacteria, for example, have restriction enzymes which recognise certain sections of the bacteriophage DNA and cut it into fragments. However, the T7 bacteriophage has one well-known protein called Ocr which inhibits restriction enzymes. Ocr does this by mimicking DNA, which led Ye et al. to wonder if it could also interrupt other vital processes in a bacterial cell that involve DNA. Transcription is the first step in a coordinated process that turns the genetic information stored in a cell’s DNA into useful proteins. An enzyme called RNA polymerase decodes the DNA sequence into a go-between molecule called messenger RNA, and it was here that Ye et al. thought Ocr might jump in to interfere. To begin, Ye et al. examined the structure of Ocr when it binds to RNA polymerase using an imaging technique called cryo-electron microscopy. Ocr has been well-studied before, its structure previously described, but not when attached to RNA polymerase. The analysis showed that Ocr gets in the way of specific molecules, called sigma factors, that show RNA polymerase where to start transcription. Ocr binds to RNA polymerase in exactly the same pocket as part of sigma factors do, which is also the place where DNA must be to be decoded to make messenger RNA. Ye et al. then performed experiments to show Ocr interfering with binding to RNA polymerase did indeed disrupt transcription. This means Ocr is quite versatile as it interferes with the RNA polymerase of the bacterial host and its restriction enzymes. Ocr’s strategy of mimicking DNA to interrupt transcription could be adopted as an approach to develop new antibiotics to stop bacterial infections. DNA transcription is an essential cellular process – without it, no cell can replicate and survive – and RNA polymerase is already a validated target for drugs. Following Ocr’s lead could provide a new way to stop infections, if the right drug can be designed to fit.

Sourdough is widely used for the preparation of traditional sourdough breads in Europe and as an important inoculum for steamed buns prepared in Asian countries, especially China. This study aims to characterize Chinese traditional sourdoughs in terms of their taxonomic compositions, physicochemical characteristics, bacterial diversity and functional features. Thirty geographically diverse sourdoughs were collected from three regions across China. High-throughput sequencing and bioinformatics were used to characterize the bacterial diversity of the sourdoughs from the different sampling sites. The results showed that the West group was significantly different from the North and South groups in the dominant genera (mainly Lactobacillus, Pediococcus and Leuconostoc) and metabolic pathways (metabolism of terpenoids and polyketides, MTP, and xenobiotics biodegradation and metabolism, XBM), whereas samples from the North and South groups were not significantly different from each other. This is the first report of the genus Clostridium in sourdough and the first report of metabolic pathways related to MTP and XBM in sourdough. This research provides a thorough description and analysis of the relationship between bacterial diversity and geographic region in sourdough.

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-β4 loop, pore loop 1, and the presensor 1-β hairpin (PS1-βH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO₂ fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures. Ministry of Education (MOE) Nanyang Technological University This work was funded by a Nanyang Technological University startup grant (to O.M.-C.) and by Ministry of Education (MOE) of Singapore Tier 2 grants to O.M.-C. (MOE2016-T2-2-088), Y.-G.G. (MOE2015-T2-1-078), and S.B. (MOE2017-T2-2-089).

Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (m3C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of trm141 as the second gene responsible for m3C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m3C formation in mammals as well. Here, we report the discovery and characterization of three distinct m3C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m3C in specific tRNAs and that METTL8 only contributes m3C to mRNA. MS analysis revealed that there is an ∼30–40% and ∼10–15% reduction, respectively, in METTL2 and -6 null-mutant cells, of m3C in total tRNA, and primer extension analysis located METTL2-modified m3C at position 32 of tRNAThr isoacceptors and tRNAArg(CCU). We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. METTL8, however, modified only mRNA, as determined by biochemical and genetic analyses in Mettl8 null-mutant mice and two human METTL8 mutant cell lines. Our findings provide the first evidence of the existence of m3C modification in mRNA, and the discovery of METTL8 as an mRNA m3C writer enzyme opens the door to future studies of other m3C epitranscriptomic reader and eraser functions. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version

Summary Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo., Graphical Abstract, Highlights • RNA polymerase closed complex (RPc) structure reveals DNA distortions by σ • Intermediate complex (RPi) structure reveals the roles of AAA activator • DNA distortion and opening are initiated in RPc and RPi before entering the RNAP • RNAP conformation in RPi is significantly different from closed or open complex, Glyde et al. report structures of σ54-dependent RNA polymerase (RNAP) closed (RPc) and intermediate (RPi) complexes. Promoter DNA distortions occur in RPc and RPi. Transitions from RPc to RPi are accompanied by significant conformational changes in RNAP and σ54. Direct interactions between AAA activators and DNA contribute to DNA distortions.

Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances.

Iridoviruses are large DNA viruses that infect invertebrates and poikilothermic vertebrates, and result in significant economic losses in aquaculture production, and drastic declines in amphibian populations. Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in farm-raised soft-shelled turtles (Trionyx sinensis). In the present study, the mechanisms of STIV-induced cell death and the roles of the mitogen-activated protein kinase (MAPK) signaling pathway were investigated. STIV infection evoked typical apoptosis in fish cells, as demonstrated by the formation of apoptotic bodies, positive terminal deoxynucleotidyl transferase-mediated nicked-end labeling, and caspase-3 activation. The translocation of cytochrome c from mitochondria to cytoplasm, and caspase-9 activation suggested that a mitochondria-mediated pathway was involved in STIV-induced apoptosis. Moreover, MAPK pathways, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK signaling were activated during STIV infection. Using specific inhibitors, we found that MAPK signaling molecules, including ERK, JNK and p38 MAPK, were important for virus release, whereas, only ERK and p38 MAPK were involved in STIV-induced apoptosis by modulating caspase-3 activity. Taken together, our findings shed light on the roles of the MAPK signaling pathway in iridovirus-induced apoptosis and virus replication, which provides new insights into understanding iridovirus-host interaction.

The crystallization of the novel virulence factors SghA and SghR is reported., Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction.

Pseudomonas aeruginosa relies on cell motility and ability to form biofilms to establish infections; however, the mechanism of regulation remains obscure. Here we report that BswR, a xenobiotic response element-type transcriptional regulator, plays a critical role in regulation of bacterial motility and biofilm formation in P. aeruginosa. Transcriptomic and biochemical analyses showed that BswR counteracts the repressor activity of MvaT, controls the transcription of small RNA rsmZ and regulates the biogenesis of bacterial flagella. The crystal structure of BswR was determined at 2.3 Å resolution; the monomer comprises a DNA-binding domain with a helix-turn-helix motif in the N terminus and two helices (α6 and α7) with a V-shaped arrangement in the C-terminus. In addition to the contacts between the parallel helices α5 of two monomers, the two helical extensions (α6 and α7) intertwine together to form a homodimer, which is the biological function unit. Based on the result of DNase I protection assay together with structural analysis of BswR homodimer, we proposed a BswR–DNA model, which suggests a molecular mechanism with which BswR could interact with DNA. Taken together, our results unveiled a novel regulatory mechanism, in which BswR controls the motility and biofilm formation of P. aeruginosa by modulating the transcription of small RNA rsmZ. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. During the virus-host co-evolution, host Rab7 was also exploited by virus to complete their life cycle. To date, however, the roles of fish Rab7 in virus infection remained largely unknown. Here, we cloned and characterized a Rab7 gene from grouper, Epinephelus coioides (Ec-Rab7). The full-length Ec-Rab7 cDNA was composed of 1182 bp and encoded a polypeptide of 207 amino acids which shared 99% identity with that from Anoplopoma fimbria or Oreochromis niloticus. Ec-Rab7 contained five conserved domains of Rab GTPase family including GTP-binding or GTPase regions as well as an effector site. RT-PCR analysis revealed that Ec-Rab7 ubiquitously expressed in all detected tissues and its transcript in spleen was up-regulated after challenge with Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-Rab7 was distributed in the cytoplasm as spots and mostly colocalized with lysosomes. Notably, the ectopic expressed Ec-Rab7 partly aggregated into the viral factories in cells infected by SGIV. Furthermore, overexpression of Ec-Rab7 accelerated the occurrence of cytopathic effect (CPE) induced by SGIV infection and promoted viral gene transcription. In addition, far western blotting assay revealed that Ec-Rab7 might interact with viral proteins, including SGIV VP69 and VP101. Taken together, our data suggested that Ec-Rab7 might be potentially involved in SGIV replication. (C) 2013 Elsevier Ltd. All rights reserved.

p38 mitogen-activated protein kinases (MAPKs) are broadly expressed signaling molecules that involves in the regulation of cellular responsible for various extracellular stimuli. In this study, three p38 MAPK genes (Ec-p38a, p38b and p38β) were cloned from grouper, Epinephelus coioides and their characteristics were investigated in vitro. Although Ec-p38a, p38b and p38β showed high homologies to other fish p38a MPAK, p38b MAPK and p38β MAPK, respectively, they all contained the conserved structures of Thr-Gly-Tyr (TGY) motif and substrate binding site Ala-Thr-Arg-Trp (ATRW). Phylogenetic analysis indicated that Ec-p38a, p38b and p38β are more closely related to those from fish than mammals. The tissue distribution patterns of Ec-p38a, p38b and p38β were different, and Ec-p38β was up-regulated most obviously in head kidney after Singapore grouper iridovirus (SGIV) infection. Overexpression of Ec-p38β in FHM cells delayed the occurrence of CPE induced by SGIV infection. Further analysis indicated that overexpression of Ec-p38β inhibited viral gene transcription and protein synthesis, as well as SGIV induced typical apoptosis in fish cells. Taken together, our data indicated that Ec-p38β played a crucial role in regulating apoptosis and virus replication during iridovirus infection.

Pseudomonas aeruginosa relies on cell motility and ability to form biofilms to establish infections, however, the mechanism of regulation remains obscure. Here we report that BswR, a XRE (Xenobiotic Response Element) type transcriptional regulator, plays a critical role in regulation of bacterial motility and biofilm formation in P. aeruginosa. Transcriptomic and biochemical analyses showed that BswR counteracts the repressor activity of MvaT, controls the transcription of small RNA rsmZ and regulates the biogenesis of bacterial flagella. The crystal structure of BswR was determined at 2.3 Å resolution; the monomer comprises a DNA-binding domain with a helix-turn-helix (HTH) motif in the N terminus and two helices (6 and 7) with a V-shaped arrangement in the C-terminus. In addition to the contacts between the parallel helices α5 of two monomers, the two helical extensions (6 and 7) intertwine together to form a homodimer, which is the biological function unit. Based on the result of DNase I protection assay together with structural analysis BswR homodimer, we proposed a BswR-DNA model, which suggests a molecular mechanism with which BswR could interact with DNA. Taken together, our results unveiled a novel regulatory mechanism, in which BswR controls the motility and biofilm formation of P. aeruginosa by modulating the transcription of small RNA rsmZ.