Cell-to-cell natural transformation in Bacillus subtilis facilitates large scale of genomic exchanges and the transfer of long continuous DNA regions.

Natural transformation is one of the major mechanisms of horizontal gene transfer. Although it is usually studied using purified DNA in the laboratory, recent studies showed that many naturally competent bacteria acquired exogenous DNA from neighboring donor cells. Our previous work indicates that cell-to-cell natural transformation (CTCNT) using two different Bacillus subtilis strains is a highly efficient process; however, the mechanism is unclear. In this study, we further characterized CTCNT and mapped the transferred DNA in the recombinants using whole genome sequencing. We found that a recombinant strain generated by CTCNT received up to 66 transferred DNA segments; the average length of acquired continuous DNA stretches was approximately 27 kb with a maximum length of 347 kb. Moreover, up to 1.54 Mb genomic DNA (37% of the chromosome) was transferred from the donors into one recipient cell. These results suggest that B. subtilis CTCNT facilitates horizontal gene transfer by increasing the transfer of DNA segments and fostering the exchange of large continuous genomic regions. This indicates that the potency of bacterial natural transformation is underestimated using traditional approaches and reveals that DNA donor cells may play an important role in the transformation process in natural environments.

A virus-borne DNA damage signaling pathway controls the lysogeny-induction switch in a group of temperate pleolipoviruses.

Many prokaryotic viruses are temperate and their reactivation is tightly regulated. However, except for a few bacterial model systems, the regulatory circuits underlying the exit from lysogeny are poorly understood, especially in archaea. Here, we report a three-gene module which regulates the switch between lysogeny and replicative cycle in a haloarchaeal virus SNJ2 (family Pleolipoviridae). The SNJ2 orf4 encodes a winged helix-turn-helix DNA binding protein which maintains lysogeny through repressing the expression of the viral integrase gene intSNJ2. To switch to the induced state, two other SNJ2-encoded proteins, Orf7 and Orf8, are required. Orf8 is a homolog of cellular AAA+ ATPase Orc1/Cdc6, which is activated upon mitomycin C-induced DNA damage, possibly through posttranslational modification. Activated Orf8 initiates the expression of Orf7 which, in turn, antagonizes the function of Orf4, leading to the transcription of intSNJ2, thereby switching SNJ2 to the induced state. Comparative genomics analysis revealed that the SNJ2-like Orc1/Cdc6-centered three-gene module is common in haloarchaeal genomes, always present in the context of integrated proviruses. Collectively, our results uncover the first DNA damage signaling pathway encoded by a temperate archaeal virus and reveal an unexpected role of the widely distributed virus-encoded Orc1/Cdc6 homologs.

Tankyrases inhibit innate antiviral response by PARylating VISA/MAVS and priming it for RNF146-mediated ubiquitination and degradation.

During viral infection, sensing of viral RNA by retinoic acid-inducible gene-I-like receptors (RLRs) initiates an antiviral innate immune response, which is mediated by the mitochondrial adaptor protein VISA (virus-induced signal adaptor; also known as mitochondrial antiviral signaling protein [MAVS]). VISA is regulated by various posttranslational modifications (PTMs), such as polyubiquitination, phosphorylation, O-linked ß-d-N-acetylglucosaminylation (O-GlcNAcylation), and monomethylation. However, whether other forms of PTMs regulate VISA-mediated innate immune signaling remains elusive. Here, we report that Poly(ADP-ribosyl)ation (PARylation) is a PTM of VISA, which attenuates innate immune response to RNA viruses. Using a biochemical purification approach, we identified tankyrase 1 (TNKS1) as a VISA-associated protein. Viral infection led to the induction of TNKS1 and its homolog TNKS2, which translocated from cytosol to mitochondria and interacted with VISA. TNKS1 and TNKS2 catalyze the PARylation of VISA at Glu137 residue, thereby priming it for K48-linked polyubiquitination by the E3 ligase Ring figure protein 146 (RNF146) and subsequent degradation. Consistently, TNKS1, TNKS2, or RNF146 deficiency increased the RNA virus-triggered induction of downstream effector genes and impaired the replication of the virus. Moreover, TNKS1- or TNKS2-deficient mice produced higher levels of type I interferons (IFNs) and proinflammatory cytokines after virus infection and markedly reduced virus loads in the brains and lungs. Together, our findings uncover an essential role of PARylation of VISA in virus-triggered innate immune signaling, which represents a mechanism to avoid excessive harmful immune response.

Identification of the potential active site of the septal peptidoglycan polymerase FtsW.

SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.

FtsA acts through FtsW to promote cell wall synthesis during cell division in Escherichia coli.

In Escherichia coli, FtsQLB is required to recruit the essential septal peptidoglycan (sPG) synthase FtsWI to FtsA, which tethers FtsZ filaments to the membrane. The arrival of FtsN switches FtsQLB in the periplasm and FtsA in the cytoplasm from a recruitment role to active forms that synergize to activate FtsWI. Genetic evidence indicates that the active form of FtsQLB has an altered conformation with an exposed domain of FtsL that acts on FtsI to activate FtsW. However, how FtsA contributes to the activation of FtsW is not clear, as it could promote the conformational change in FtsQLB or act directly on FtsW. Here, we show that the overexpression of an activated FtsA (FtsA*) bypasses FtsQ, indicating it can compensate for FtsQ's recruitment function. Consistent with this, FtsA* also rescued FtsL and FtsB mutants deficient in FtsW recruitment. FtsA* also rescued an FtsL mutant unable to deliver the periplasmic signal from FtsN, consistent with FtsA* acting on FtsW. In support of this, an FtsW mutant was isolated that was rescued by an activated FtsQLB but not by FtsA*, indicating it was specifically defective in activation by FtsA. Our results suggest that in response to FtsN, the active form of FtsA acts on FtsW in the cytoplasm and synergizes with the active form of FtsQLB acting on FtsI in the periplasm to activate FtsWI to carry out sPG synthesis.

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes.

SEDS family peptidoglycan (PG) glycosyltransferases, RodA and FtsW, require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try to understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations, as well as others affecting the activity of FtsWI, on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.

ICTV Virus Taxonomy Profile: Sphaerolipoviridae 2023.

Members of the family Sphaerolipoviridae have non-enveloped tailless icosahedral virions with a protein-rich internal lipid membrane. The genome is a linear double-stranded DNA of about 30 kbp with inverted terminal repeats and terminal proteins. The capsid has a pseudo triangulation T=28 dextro symmetry and is built of two major capsid protein types. Spike complexes decorate fivefold vertices. Sphaerolipoviruses have a narrow host range and a lytic life cycle, infecting haloarchaea in the class Halobacteria (phylum Euryarchaeota). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Sphaerolipoviridae, which is available at ictv.global/report/sphaerolipoviridae.

ICTV Virus Taxonomy Profile: Simuloviridae 2023.

The family Simuloviridae includes tailless icosahedral viruses with an internal lipid membrane. The capsid is constructed from two major capsid proteins, both with a single jelly-roll fold. The genome is a circular dsDNA molecule of 16-19 kb. All members infect halophilic archaea in the class Halobacteria (phylum Euryarchaeota) and are temperate viruses, their proviruses residing in host cells as extrachromosomal episomes. Once the lytic life cycle is triggered, production of virions causes cell lysis. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Simuloviridae, which is available at ictv.global/report/simuloviridae.

ORF4 of the Temperate Archaeal Virus SNJ1 Governs the Lysis-Lysogeny Switch and Superinfection Immunity.

Recent environmental and metagenomic studies have considerably increased the repertoire of archaeal viruses and suggested that they play important roles in nutrient cycling in the biosphere. However, very little is known about how they regulate their life cycles and interact with their hosts. Here, we report that the life cycle of the temperate haloarchaeal virus SNJ1 is controlled by the product ORF4, a small protein belonging to the antitoxin MazE superfamily. We show that ORF4 controls the lysis-lysogeny switch of SNJ1 and mediates superinfection immunity by repression of genomic DNA replication of the superinfecting viruses. Bioinformatic analysis shows that ORF4 is highly conserved in two SNJ1-like proviruses, suggesting that the mechanisms for lysis-lysogeny switch and superinfection immunity are conserved in this group of viruses. As the lysis-lysogeny switch and superinfection immunity of archaeal viruses have been poorly studied, we suggest that SNJ1 could serve as a model system to study these processes.IMPORTANCE Archaeal viruses are important parts of the virosphere. Understanding how they regulate their life cycles and interact with host cells provide crucial insights into their biological functions and the evolutionary histories of viruses. However, mechanistic studies of the life cycle of archaeal viruses are scarce due to a lack of genetic tools and demanding cultivation conditions. Here, we discover that the temperate haloarchaeal virus SNJ1, which infects Natrinema sp. strain J7, employs a lysis-lysogeny switch and establishes superinfection immunity like bacteriophages. We show that its ORF4 is critical for both processes and acts as a repressor of the replication of SNJ1. These results establish ORF4 as a master regulator of SNJ1 life cycle and provides novel insights on the regulation of life cycles by temperate archaeal viruses and on their interactions with host cells.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

FtsZ filaments have the opposite kinetic polarity of microtubules.

FtsZ is the ancestral homolog of tubulin and assembles into the Z ring that organizes the division machinery to drive cell division in most bacteria. In contrast to tubulin that assembles into 13 stranded microtubules that undergo dynamic instability, FtsZ assembles into single-stranded filaments that treadmill to distribute the peptidoglycan synthetic machinery at the septum. Here, using longitudinal interface mutants of FtsZ, we demonstrate that the kinetic polarity of FtsZ filaments is opposite to that of microtubules. A conformational switch accompanying the assembly of FtsZ generates the kinetic polarity of FtsZ filaments, which explains the toxicity of interface mutants that function as a capper and reveals the mechanism of cooperative assembly. This approach can also be employed to determine the kinetic polarity of other filament-forming proteins.

Disruption of divisome assembly rescued by FtsN-FtsA interaction in Escherichia coli.

Cell division requires the assembly of a protein complex called the divisome. The divisome assembles in a hierarchical manner, with FtsA functioning as a hub to connect the Z-ring with the rest of the divisome and FtsN arriving last to activate the machine to synthesize peptidoglycan. FtsEX arrives as the Z-ring forms and acts on FtsA to initiate recruitment of the other divisome components. In the absence of FtsEX, recruitment is blocked; however, a multitude of conditions allow FtsEX to be bypassed. Here, we find that all such FtsEX bypass conditions, as well as the bypass of FtsK, depend upon the interaction of FtsN with FtsA, which promotes the back-recruitment of the late components of the divisome. Furthermore, our results suggest that these bypass conditions enhance the weak interaction of FtsN with FtsA and its periplasmic partners so that the divisome proteins are brought to the Z-ring when the normal hierarchical pathway is disrupted.

How FtsEX localizes to the Z ring and interacts with FtsA to regulate cell division.

In Escherichia coli, FtsEX, a member of the ABC transporter superfamily, is involved in regulating the assembly and activation of the divisome to couple cell wall synthesis to cell wall hydrolysis at the septum. Genetic studies indicate FtsEX acts on FtsA to begin the recruitment of the downstream division proteins but blocks septal PG synthesis until a signal is received that divisome assembly is complete. However, the details of how FtsEX localizes to the Z ring and how it interacts with FtsA are not clear. Our results show that recruitment of FtsE and FtsX is codependent and suggest that the FtsEX complex is recruited through FtsE interacting with the conserved tail of FtsZ (CCTP), thus adding FtsEX to a growing list of proteins that interacts with the CCTP of FtsZ. Furthermore, we find that the N-terminus of FtsX is not required for FtsEX localization to the Z ring but is required for its functions in cell division indicating that it interacts with FtsA. Taken together, these results suggest that FtsEX first interacts with FtsZ to localize to the Z ring and then interacts with FtsA to promote divisome assembly and regulate septal PG synthesis.

MipZ: one for the pole, two for the DNA.

In this issue of Molecular Cell,Kiekebusch et al. (2012) show that a novel regulatory mechanism of the ATPase cycle of MipZ, together with nonspecific DNA binding, generates the MipZ gradient that spatially regulates Z ring formation in Caulobacter crescentus.

MinC and FtsZ mutant analysis provides insight into MinC/MinD-mediated Z ring disassembly.

The Min system negatively regulates the position of the Z ring, which serves as a scaffold for the divisome that mediates bacterial cytokinesis. In Escherichia coli, this system consists of MinC, which antagonizes assembly of the tubulin homologue FtsZ. MinC is recruited to the membrane by MinD and induced by MinE to oscillate between the cell poles. MinC is a dimer with each monomer consisting of functionally distinct MinCN and MinCC domains, both of which contact FtsZ. According to one model, MinCC/MinD binding to the FtsZ tail positions MinCN at the junction of two GDP-containing subunits in the filament, leading to filament breakage. Others posit that MinC sequesters FtsZ-GDP monomers or that MinCN caps the minus end of FtsZ polymers and that MinCC interferes with lateral interactions between FtsZ filaments. Here, we isolated minC mutations that impair MinCN function and analyzed FtsZ mutants resistant to MinC/MinD. Surprisingly, we found mutations in both minC and ftsZ that differentiate inhibition by MinC from inhibition by MinC/MinD. Analysis of these mutations suggests that inhibition of the Z ring by MinC alone is due to sequestration, whereas inhibition by MinC/MinD is not. In conclusion, our genetic and biochemical data support the model that MinC/MinD fragments FtsZ filaments.

Prophage LambdaSo uses replication interference to suppress reproduction of coexisting temperate phage MuSo2 in Shewanella oneidensis MR-1.

Many bacterial genomes carry multiple prophages that compete with each other, potentially affecting the physiology, fitness, and pathogenicity of their hosts. However, molecular mechanisms of such prophage-prophage conflicts remain poorly understood. The genome of Shewanella oneidensis MR-1, a Gammaproteobacterium residing in aquatic environments and notable for its ability to reduce metal ions, harbours four prophages, two of which (LambdaSo and MuSo2) form infectious virions during biofilm formation. Here, we constructed indicator strains of LambdaSo and MuSo2 by deleting the corresponding prophages from the MR-1 chromosome and investigated their reproduction. Interestingly, the fitness of MuSo2 increased in the absence of LambdaSo, suggesting that prophage LambdaSo repressed MuSo2 reproduction. Partial deletion of LambdaSo from the MR-1 chromosome revealed that gene cluster R of LambdaSo, which was responsible for the switch to the lytic cycle and LambdaSo genome replication initiation, was necessary and sufficient to repress MuSo2. Furthermore, activation of cluster R genes facilitated replication of cluster R-encoding DNA and inhibited host and MuSo2 DNA replication. These findings suggest that LambdaSo represses MuSo2 propagation by inhibiting DNA replication during simultaneous induction. We predict that such a mechanism of inter-prophage interference is more widespread in bacteria than currently appreciated.

4-Hydroxyphenylpyruvate Dioxygenase Thermolability Is Responsible for Temperature-Dependent Melanogenesis in Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a major pathogen affecting fisheries worldwide and is a well-known pigmented member of the Aeromonas genus. This subspecies produces melanin at ≤22°C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30°C to 35°C, while bacterial growth is unaffected. The mechanism and biological significance of this temperature-dependent melanogenesis remain unclear. Heterologous expression of an A. salmonicida subsp. salmonicida 4-hydroxyphenylpyruvate dioxygenase (HppD), the most critical enzyme in the homogentisic acid (HGA)-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli, suggesting that HppD plays a key role in this process. In this study, we demonstrated that the thermolability of HppD is responsible for the temperature-dependent melanization of A. salmonicida subsp. salmonicida Substitutions of three residues, S18T, P103Q, and L119P, in A. salmonicida subsp. salmonicida HppD increased the thermostability of this enzyme and resulted in temperature-independent melanogenesis. Moreover, the replacement of the corresponding residues in HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A. salmonicida subsp. salmonicida HppD resulted in thermosensitive melanogenesis. A structural analysis suggested that mutations at these sites, especially at position P103, strengthen the secondary structure of HppD and greatly improve its thermal stability. Additionally, we found that the HppD sequences of all A. salmonicida subsp. salmonicida isolates were identical and that two of the three residues were clearly distinct from those of other Aeromonas strains.IMPORTANCEAeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a bacterial septicemia of cold-water fish of the Salmonidae family. Although other Aeromonas species can produce melanin, A. salmonicida subsp. salmonicida is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here, we demonstrated that thermosensitive melanogenesis in A. salmonicida subsp. salmonicida strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). Additionally, we confirmed that this thermolabile HppD exhibited higher activity at low temperatures than its mesophilic homologues, suggesting this as an adaptive strategy of this enzyme to the psychrophilic lifestyle of A. salmonicida subsp. salmonicida The strictly conserved hppD sequences among A. salmonicida subsp. salmonicida isolates and the specific possession of P103 and L119 residues could be used as a reference for the identification of A. salmonicida subsp. salmonicida isolates.

FtsEX acts on FtsA to regulate divisome assembly and activity.

Bacterial cell division is driven by the divisome, a ring-shaped protein complex organized by the bacterial tubulin homolog FtsZ. Although most of the division proteins in Escherichia coli have been identified, how they assemble into the divisome and synthesize the septum remains poorly understood. Recent studies suggest that the bacterial actin homolog FtsA plays a critical role in divisome assembly and acts synergistically with the FtsQLB complex to regulate the activity of the divisome. FtsEX, an ATP-binding cassette transporter-like complex, is also necessary for divisome assembly and inhibits division when its ATPase activity is inactivated. However, its role in division is not clear. Here, we find that FtsEX acts on FtsA to regulate both divisome assembly and activity. FtsX interacts with FtsA and this interaction is required for divisome assembly and inhibition of divisome function by ATPase mutants of FtsEX. Our results suggest that FtsEX antagonizes FtsA polymerization to promote divisome assembly and the ATPase mutants of FtsEX block divisome activity by locking FtsA in the inactive form or preventing FtsA from communicating with other divisome proteins. Because FtsEX is known to govern cell wall hydrolysis at the septum, our findings indicate that FtsEX acts on FtsA to promote divisome assembly and to coordinate cell wall synthesis and hydrolysis at the septum. Furthermore, our study provides evidence that FtsA mutants impaired for self-interaction are favored for division, and FtsW plays a critical role in divisome activation in addition to the FtsQLB complex.

The N-succinyl-l,l-diaminopimelic acid desuccinylase DapE acts through ZapB to promote septum formation in Escherichia coli.

Spatial regulation of cell division in Escherichia coli occurs at the stage of Z ring formation. It consists of negative (the Min and NO systems) and positive (Ter signal mediated by MatP/ZapA/ZapB) regulators. Here, we find that N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) facilitates functional Z ring formation by strengthening the Ter signal via ZapB. DapE depends on ZapB to localize to the Z ring and its overproduction suppresses the division defect caused by loss of both the Min and NO systems. DapE shows a strong interaction with ZapB and requires the presence of ZapB to exert its function in division. Consistent with the idea that DapE strengthens the Ter signal, overproduction of DapE supports cell division with reduced FtsZ levels and provides some resistance to the FtsZ inhibitors MinCD and SulA, while deletion of dapE, like deletion of zapB, exacerbates the phenotypes of cells impaired in Z ring formation such as ftsZ84 or a min mutant. Taken together, our results report DapE as a new component of the divisome that promotes the integrity of the Z ring by acting through ZapB and raises the possibility of the existence of additional divisome proteins that also function in other cellular processes.

Assembly and activation of the Escherichia coli divisome.

Cell division in Escherichia coli is mediated by a large protein complex called the divisome. Most of the divisome proteins have been identified, but how they assemble onto the Z ring scaffold to form the divisome and work together to synthesize the septum is not well understood. In this review, we summarize the latest findings on divisome assembly and activation as well as provide our perspective on how these two processes might be regulated.