Antibacterial action, proteolytic immunity, and in vivo activity of a Vibrio cholerae microcin.

Microcins are small antibacterial proteins that mediate interbacterial competition. Their narrow-spectrum activity provides opportunities to discover microbiome-sparing treatments. However, microcins have been found almost exclusively in Enterobacteriaceae. Their broader existence and potential implications in other pathogens remain unclear. Here, we identify and characterize a microcin active against pathogenic Vibrio cholerae: MvcC. We show that MvcC is reliant on the outer membrane porin OmpT to cross the outer membrane. MvcC then binds the periplasmic protein OppA to reach and disrupt the cytoplasmic membrane. We demonstrate that MvcC's cognate immunity protein is a protease, which precisely cleaves MvcC to neutralize its activity. Importantly, we show that MvcC is active against diverse cholera isolates and in a mouse model of V. cholerae colonization. Our results provide a detailed analysis of a microcin outside of Enterobacteriaceae and its potential to influence V. cholerae infection.

Multiple variants of the type VII secretion system in Gram-positive bacteria.

Type VII secretion systems (T7SS) are found in bacteria across the Bacillota and Actinomycetota phyla and have been well described in Staphylococcus aureus, Bacillus subtilis, and pathogenic mycobacteria. The T7SS from Actinomycetota and Bacillota share two common components, a membrane-bound EccC/EssC ATPase and EsxA, a small helical hairpin protein of the WXG100 family. However, they also have additional phylum-specific components, and as a result they are termed the T7SSa (Actinomycetota) and T7SSb (Bacillota), respectively. Here, we identify additional organizations of the T7SS across these two phyla and describe eight additional T7SS subtypes, which we have named T7SSc-T7SSj. T7SSd is found exclusively in Actinomycetota including the Olselnella and Bifodobacterium genus, whereas the other seven are found only in Bacillota. All of the novel subtypes contain the canonical ATPase (TsxC) and the WXG100-family protein (TsxA). Most of them also contain a small ubiquitin-related protein, TsxB, related to the T7SSb EsaB/YukD component. Protein kinases, phosphatases, and forkhead-associated (FHA) proteins are often encoded in the novel T7SS gene clusters. Candidate substrates of these novel T7SS subtypes include LXG-domain and RHS proteins. Predicted substrates are frequently encoded alongside genes for additional small WXG100-related proteins that we speculate serve as cosecretion partners. Collectively our findings reveal unexpected diversity in the T7SS in Gram-positive bacteria.

Mechanisms of bacterial immunity, protection, and survival during interbacterial warfare.

Most bacteria live in communities, often with closely related strains and species with whom they must compete for space and resources. Consequently, bacteria have acquired or evolved mechanisms to antagonize competitors through the production of antibacterial toxins. Similar to bacterial systems that combat phage infection and mechanisms to thwart antibiotics, bacteria have also acquired and evolved features to protect themselves from antibacterial toxins. Just as there is a large body of research identifying and characterizing antibacterial proteins and toxin delivery systems, studies of bacterial mechanisms to resist and survive assault from competitors' weapons have also expanded tremendously. Emerging data are beginning to reveal protective processes and mechanisms that are as diverse as the toxins themselves. Protection against antibacterial toxins can be acquired by horizontal gene transfer, receptor or target alteration, induction of protective functions, physical barriers, and other diverse processes. Here, we review recent studies in this rapidly expanding field.

Role of Type VI secretion system in pathogenic remodeling of host gut microbiota during Aeromonas veronii infection.

Intestinal microbial disturbance is a direct cause of host disease. The bacterial Type VI secretion system (T6SS) often plays a crucial role in the fitness of pathogenic bacteria by delivering toxic effectors into target cells. However, its impact on the gut microbiota and host pathogenesis is poorly understood. To address this question, we characterized a new T6SS in the pathogenic Aeromonas veronii C4. First, we validated the secretion function of the core machinery of A. veronii C4 T6SS. Second, we found that the pathogenesis and colonization of A. veronii C4 is largely dependent on its T6SS. The effector secretion activity of A. veronii C4 T6SS not only provides an advantage in competition among bacteria in vitro, but also contributes to occupation of an ecological niche in the nutritionally deficient and anaerobic environment of the host intestine. Metagenomic analysis showed that the T6SS directly inhibits or eliminates symbiotic strains from the intestine, resulting in dysregulated gut microbiome homeostasis. In addition, we identified three unknown effectors, Tse1, Tse2, and Tse3, in the T6SS, which contribute to T6SS-mediated bacterial competition and pathogenesis by impairing targeted cell integrity. Our findings highlight that T6SS can remodel the host gut microbiota by intricate interplay between T6SS-mediated bacterial competition and altered host immune responses, which synergistically promote pathogenesis of A. veronii C4. Therefore, this newly characterized T6SS could represent a general interaction mechanism between the host and pathogen, and may offer a potential therapeutic target for controlling bacterial pathogens.

A type VI secretion system effector TseG of Pantoea ananatis is involved in virulence and antibacterial activity.

The type VI secretion system (T6SS) of many gram-negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram-negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ-12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS-1, T6SS-2, and T6SS-3. Interestingly, only T6SS-1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS-1 system in detail and identified an unknown T6SS-1-secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS-1-secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins.

Xanthomonas immunity proteins protect against the cis-toxic effects of their cognate T4SS effectors.

Many bacteria kill rival species by translocating toxic effectors into target cells. Effectors are often encoded along with cognate immunity proteins that could (i) protect against "friendly-fire" (trans-intoxication) from neighboring sister cells and/or (ii) protect against internal cis-intoxication (suicide). Here, we distinguish between these two mechanisms in the case of the bactericidal Xanthomonas citri Type IV Secretion System (X-T4SS). We use a set of X. citri mutants lacking multiple effector/immunity protein (X-Tfe/X-Tfi) pairs to show that X-Tfis are not absolutely required to protect against trans-intoxication by wild-type cells. Our investigation then focused on the in vivo function of the lysozyme-like effector X-TfeXAC2609 and its cognate immunity protein X-TfiXAC2610. In the absence of X-TfiXAC2610, we observe X-TfeXAC2609-dependent and X-T4SS-independent accumulation of damage in the X. citri cell envelope, cell death, and inhibition of biofilm formation. While immunity proteins in other systems have been shown to protect against attacks by sister cells (trans-intoxication), this is an example of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis-intoxication.

Identification and distribution of new candidate T6SS effectors encoded in Salmonella Pathogenicity Island 6.

The type VI secretion system (T6SS) is a contact-dependent contractile multiprotein apparatus widely distributed in Gram-negative bacteria. These systems can deliver different effector proteins into target bacterial and/or eukaryotic cells, contributing to the environmental fitness and virulence of many bacterial pathogens. Salmonella harbors five different T6SSs encoded in different genomic islands. The T6SS encoded in Salmonella Pathogenicity Island 6 (SPI-6) contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, information regarding the total number of effector proteins encoded within SPI-6 and its distribution among different Salmonella enterica serotypes is limited. In this work, we performed bioinformatic and comparative genomics analyses of the SPI-6 T6SS gene cluster to expand our knowledge regarding the T6SS effector repertoire and the global distribution of these effectors in Salmonella. The analysis of a curated dataset of 60 Salmonella enterica genomes from the Secret6 database revealed the presence of 23 new putative T6SS effector/immunity protein (E/I) modules. These effectors were concentrated in the variable regions 1 to 3 (VR1-3) of the SPI-6 T6SS gene cluster. VR1-2 were enriched in candidate effectors with predicted peptidoglycan hydrolase activity, while VR3 was enriched in candidate effectors of the Rhs family with C-terminal extensions with predicted DNase, RNase, deaminase, or ADP-ribosyltransferase activity. A global analysis of known and candidate effector proteins in Salmonella enterica genomes from the NCBI database revealed that T6SS effector proteins are differentially distributed among Salmonella serotypes. While some effectors are present in over 200 serotypes, others are found in less than a dozen. A hierarchical clustering analysis identified Salmonella serotypes with distinct profiles of T6SS effectors and candidate effectors, highlighting the diversity of T6SS effector repertoires in Salmonella enterica. The existence of different repertoires of effector proteins suggests that different effector protein combinations may have a differential impact on the environmental fitness and pathogenic potential of these strains.

A Novel Deoxyribonuclease Low-Molecular-Weight Bacteriocin, Carocin S4, from Pectobacterium carotovorum subsp. carotovorum.

Pectobacterium carotovorum subsp. carotovorum (Pcc) is known to produce different types of bacteriocins, active protein substances that inhibit or kill related strains and are known to be induced by several factors. In this paper, we report the discovery, isolation, characterization, and functional analysis of Carocin S4, a novel low-molecular-weight bacteriocin (LMWB) from Pcc. A 2750 bp gene fragment was isolated from the chromosomal DNA of Pcc mutant strain rif-TO6, a rifampicin-resistant strain of TO6. The gene contains caroS4K and caroS4I within two open reading frames, which encode CaroS4K and CaroS4I, with molecular weights of about 90 kD and 10 kD, respectively. The unique characteristics of Carocin S4 were revealed after homology analysis with the previously discovered bacteriocins from Pcc. CaroS4K, which shares 23% and 85% homology with CaroS1K and CaroS3K, respectively, is also a deoxyribonuclease. However, unlike the two which can only hydrolyze genomic DNA, CaroS4K hydrolyzes both genomic and plasmid DNA. On the other hand, CaroS4K was found to be 90% homologous with CaroS2K but works differently in killing the target cell, as the latter is a ribonuclease. The optimal reaction temperature for CaroS4K to hydrolyze dsDNA is approximately 50 °C and requires the divalent metal ions Mg2+, Ca2+, and Zn2+ to catalyze its DNase activity. This study reveals another nuclease type of bacteriocin in Pcc, with CaroS4K and CaroS4I functioning as killer and immunity proteins, respectively.

A novel variant of the Listeria monocytogenes type VII secretion system EssC component is associated with an Rhs toxin.

The type VIIb protein secretion system (T7SSb) is found in Bacillota (firmicute) bacteria and has been shown to mediate interbacterial competition. EssC is a membrane-bound ATPase that is a critical component of the T7SSb and plays a key role in substrate recognition. Prior analysis of available genome sequences of the foodborne bacterial pathogen Listeria monocytogenes has shown that although the T7SSb was encoded as part of the core genome, EssC could be found as one of seven different sequence variants. While each sequence variant was associated with a specific suite of candidate substrate proteins encoded immediately downstream of essC, many LXG-domain proteins were encoded across multiple essC sequence variants. Here, we have extended this analysis using a diverse collection of 37 930 L. monocytogenes genomes. We have identified a rare eighth variant of EssC present in ten L. monocytogenes lineage III genomes. These genomes also encode a large toxin of the rearrangement hotspot (Rhs) repeat family adjacent to essC8, along with a probable immunity protein and three small accessory proteins. We have further identified nine novel LXG-domain proteins, and four additional chromosomal hotspots across L. monocytogenes genomes where LXG proteins can be encoded. The eight L. monocytogenes EssC variants were also found in other Listeria species, with additional novel EssC types also identified. Across the genus, species frequently encoded multiple EssC types, indicating that T7SSb diversity is a primary feature of the genus Listeria.

A Clickable Photosystem I, Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration.

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b6 f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI-Fd and PSI-Fd-FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI-PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7-PSI was coupled with the E7-Fd or E7-Fd-FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.

Identification of atypical T4SS effector proteins mediating bacterial defense.

To remain competitive, proteobacteria use various contact-dependent weapon systems to defend against microbial competitors. The bacterial-killing type IV secretion system (T4SS) is one such powerful weapon. It commonly controls the killing/competition between species by secreting the lethal T4SS effector (T4E) proteins carrying conserved XVIPCD domains into competing cells. In this study, we sought knowledge to understand whether the bacterial-killing T4SS-producing bacteria encode T4E-like proteins and further explore their biological functions. To achieve this, we designed a T4E-guided approach to discover T4E-like proteins that are designated as atypical T4Es. Initially, this approach required scientists to perform simple BlastP search to identify T4E homologs that lack the XVIPCD domain in the genomes of T4SS-producing bacteria. These homologous genes were then screened in Escherichia coli to identify antibacterial candidates (atypical T4Es) and their neighboring detoxification proteins, followed by testing their gene cotranscription and validating their physical interactions. Using this approach, we did discover two atypical T4E proteins from the plant-beneficial Lysobacter enzymogenes and the phytopathogen Xanthomonas citri. We also provided substantial evidence to show that the atypical T4E protein Le1637-mediated bacterial defense in interspecies interactions between L. enzymogenes and its competitors. Therefore, the newly designed T4E-guided approach holds promise for detecting functional atypical T4E proteins in bacterial cells.

Antimicrobial Weapons of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a robust and versatile organism capable of surviving and prospering in a diverse array of environments and is an opportunistic pathogen of humans. One reason for the success of this pathogen is the large arsenal of antimicrobial weapons that it possesses. Here we focus our attention on these antimicrobial weapons and how they give P. aeruginosa a survival edge in polymicrobial environments. We define antimicrobial weapons as components produced by P. aeruginosa that are used to kill, inhibit growth and/or subvert key cellular functions in other microbes. P. aeruginosa has a large and complex genome and encodes an armament of antimicrobial weapons that fall into two subclasses; those that are delivered directly to competing microbes using a contact-dependent method, and those that are secreted in a contact-independent manner into the environment to then be available to target neighbouring cells. This chapter provides an overview of the major antimicrobial weapons possessed by P. aeruginosa, captures recent advances in the field and discusses how these could be targeted as a therapeutic intervention, or potentially harnessed to combat infection.

Lamprey immunity protein enables early detection and recurrence monitoring for bladder cancer through recognizing Neu5Gc-modified uromodulin glycoprotein in urine.

The clinical management of bladder cancer (BCa) is hindered by the lack of reliable biomarkers. We aimed to investigate the potential of lamprey immunity protein (LIP), a lectin that specifically binds to multi-antennary sialylated N-glycolylneuraminic acid (Neu5Gc) structures on UMOD glycoproteins in the urine of BCa patients. Primary BCa patients had higher levels of LIP-bound Neu5Gc in urine than healthy participants and patients receiving postoperative treatment did. In addition, lectin chip assay and mass spectrometry were used to analyze the glycan chain structure, which can recognize the UMOD glycoprotein decorated with multi-antennary sialylated Neu5Gc structures. Furthermore, compared with urine samples from healthy patients (N = 2821, T/C = 0.12 ± 0.09) or benign patients (N = 360, T/C = 0.11 ± 0.08), the range of the urine T/C ratio detected using LIP test paper was 1.97 ± 0.32 in patients with bladder cancer (N = 518) with significant difference (P < 0.0001). Our results indicate that LIP may be a tool for early BCa identification, diagnosis, and monitoring. Neu5Gc-modified UMOD glycoproteins in urine and Neu5Gc-modified N-glycochains and sialyltransferases may function as potential markers in clinical trials.

Unraveling the Uncharacterized Domain of Carocin S2: A Ribonuclease Pectobacterium carotovorum subsp. carotovorum Bacteriocin.

Carocin S2 is a bacteriocin with a low molecular weight generated by Pectobacterium carotovorum subsp. carotovorum 3F3 strain. The caroS2K gene, which is found in the genomic DNA alongside the caroS2I gene, which codes for an immunity protein, encodes this bacteriocin. We explored the residues responsible for Carocin S2's cytotoxic or RNA-se activity using a structure-based mutagenesis approach. The minimal antibiotic functional region starts at Lys691 and ends at Arg783, according to mutational research. Two residues in the identified region, Phe760 and Ser762, however, are unable to demonstrate this activity, suggesting that these sites may interact with another domain. Small modifications in the secondary structure of mutant caroS2K were revealed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence (ITF), showing ribosomal RNA cleavage in the active site. A co-immunoprecipitation test indicated that the immunity protein CaroS2I binds to CaroS2K's C-terminus, while a region under the uncharacterized Domain III inhibits association of N-terminally truncated CaroS2K from interacting with CaroS2I. Carocin S2, a ribosomal ribonuclease bacteriocin, is the first to be identified with a domain III that encodes the cytotoxic residues as well as the binding sites between its immunity and killer proteins.

Identification of Type VI Secretion Systems Effector Proteins That Contribute to Interbacterial Competition in Salmonella Dublin.

The Type VI Secretion System (T6SS) is a multiprotein device that has emerged as an important fitness and virulence factor for many Gram-negative bacteria through the injection of effector proteins into prokaryotic or eukaryotic cells via a contractile mechanism. While some effector proteins specifically target bacterial or eukaryotic cells, others can target both types of cells (trans-kingdom effectors). In Salmonella, five T6SS gene clusters have been identified within pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22, which are differentially distributed among serotypes. Salmonella enterica serotype Dublin (S. Dublin) is a cattle-adapted pathogen that harbors both T6SSSPI-6 and T6SSSPI-19. Interestingly, while both systems have been linked to virulence and host colonization in S. Dublin, an antibacterial activity has not been detected for T6SSSPI-6 in this serotype. In addition, there is limited information regarding the repertoire of effector proteins encoded within T6SSSPI-6 and T6SSSPI-19 gene clusters in S. Dublin. In the present study, we demonstrate that T6SSSPI-6 and T6SSSPI-19 of S. Dublin CT_02021853 contribute to interbacterial competition. Bioinformatic and comparative genomic analyses allowed us to identify genes encoding three candidate antibacterial effectors located within SPI-6 and two candidate effectors located within SPI-19. Each antibacterial effector gene is located upstream of a gene encoding a hypothetic immunity protein, thus conforming an effector/immunity (E/I) module. Of note, the genes encoding these effectors and immunity proteins are widely distributed in Salmonella genomes, suggesting a relevant role in interbacterial competition and virulence. Finally, we demonstrate that E/I modules SED_RS01930/SED_RS01935 (encoded in SPI-6), SED_RS06235/SED_RS06230, and SED_RS06335/SED_RS06340 (both encoded in SPI-19) contribute to interbacterial competition in S. Dublin CT_02021853.

Formylglycine-Generating Enzyme-Like Proteins Constitute a Novel Family of Widespread Type VI Secretion System Immunity Proteins.

Competition is a critical aspect of bacterial life, as it enables niche establishment and facilitates the acquisition of essential nutrients. Warfare between Gram-negative bacteria is largely mediated by the type VI secretion system (T6SS), a dynamic nanoweapon that delivers toxic effector proteins from an attacking cell to adjacent bacteria in a contact-dependent manner. Effector-encoding bacteria prevent self-intoxication and kin cell killing by the expression of immunity proteins, which neutralize effector toxicity by specifically binding their cognate effector and either occluding its active site or preventing the structural rearrangements necessary for effector activation. In this study, we investigate Tsi3, a previously uncharacterized T6SS immunity protein present in multiple strains of the human pathogen Acinetobacter baumannii. We show that Tsi3 is the cognate immunity protein of an antibacterial effector of unknown function, Tse3. Our bioinformatic analyses indicate that Tsi3 homologs are widespread among Gram-negative bacteria, often encoded within T6SS effector-immunity modules. Surprisingly, we found that Tsi3 homologs are predicted to possess a characteristic formylglycine-generating enzyme (FGE) domain, which is present in various enzymatic proteins. Our data show that Tsi3-mediated immunity is dependent on Tse3-Tsi3 protein-protein interactions and that Tsi3 homologs from various bacteria do not provide immunity against nonkin Tse3. Thus, we conclude that Tsi3 homologs are unlikely to be functional enzymes. Collectively, our work identifies FGE domain-containing proteins as important mediators of immunity against T6SS attacks and indicates that the FGE domain can be coopted as a scaffold in multiple proteins to carry out diverse functions. IMPORTANCE Despite the wealth of knowledge on the diversity of biochemical activities carried out by T6SS effectors, comparably little is known about the various strategies that bacteria employ to prevent susceptibility to T6SS-dependent bacterial killing. Our work establishes a novel family of T6SS immunity proteins with a characteristic FGE domain. This domain is present in enzymatic proteins with various catalytic activities. Our characterization of Tsi3 expands the known functions carried out by FGE-like proteins to include defense during T6SS-mediated bacterial warfare. Moreover, it highlights the evolution of FGE domain-containing proteins to carry out diverse biological functions.

Extreme genetic diversity in the type VII secretion system of Listeria monocytogenes suggests a role in bacterial antagonism.

The type VII protein secretion system (T7SS) has been characterized in members of the phyla Actinobacteria and Firmicutes. In mycobacteria the T7SS is intimately linked with pathogenesis and intracellular survival, while in Firmicutes there is mounting evidence that the system plays a key role in interbacterial competition. A conserved membrane-bound ATPase protein, termed EssC in Staphylococcus aureus, is a critical component of the T7SS and is the primary receptor for substrate proteins. Genetic diversity in the essC gene of S. aureus has previously been reported, resulting in four protein variants that are linked to specific subsets of substrates. Here we have analysed the genetic diversity of the T7SS-encoding genes and substrate proteins across Listeria monocytogenes genome sequences. We find that there are seven EssC variants across the species that differ in their C-terminal region; each variant is correlated with a distinct subset of genes for likely substrate and accessory proteins. EssC1 is most common and is exclusively linked with polymorphic toxins harbouring a YeeF domain, whereas EssC5, EssC6 and EssC7 variants all code for an LXG domain protein adjacent to essC. Some essC1 variant strains encode an additional, truncated essC at their T7 gene cluster. The truncated EssC, comprising only the C-terminal half of the protein, matches the sequence of either EssC2, EssC3 or EssC4. In each case the truncated gene directly precedes a cluster of substrate/accessory protein genes acquired from the corresponding strain. Across L. monocytogenes strains we identified 40 LXG domain proteins, most of which are encoded at conserved genomic loci. These loci also harbour genes encoding immunity proteins and sometimes additional toxin fragments. Collectively our findings strongly suggest that the T7SS plays an important role in bacterial antagonism in this species.

Two PAAR Proteins with Different C-Terminal Extended Domains Have Distinct Ecological Functions in Myxococcus xanthus.

Bacterial proline-alanine-alanine-arginine (PAAR) proteins are located at the top of the type VI secretion system (T6SS) nanomachine and carry and deliver effectors into neighboring cells. Many PAAR proteins are fused with a variable C-terminal extended domain (CTD). Here, we report that two paar-ctd genes (MXAN_RS08765 and MXAN_RS36995) located in two homologous operons are involved in different ecological functions of Myxococcus xanthusMXAN_RS08765 inhibited the growth of plant-pathogenic fungi, while MXAN_RS36995 was associated with the colony-merger incompatibility of M. xanthus cells. These two PAAR-CTD proteins were both toxic to Escherichia coli cells, while MXAN_RS08765, but not MXAN_RS36995, was also toxic to Saccharomyces cerevisiae cells. Their downstream adjacent genes, i.e., MXAN_RS08760 and MXAN_RS24590, protected against the toxicities. The MXAN_RS36995 protein was demonstrated to have nuclease activity, and the activity was inhibited by the presence of MXAN_RS24590. Our results highlight that the PAAR proteins diversify the CTDs to play divergent roles in M. xanthusIMPORTANCE The type VI secretion system (T6SS) is a bacterial cell contact-dependent weapon capable of delivering protein effectors into neighboring cells. The PAAR protein is located at the top of the nanomachine and carries an effector for delivery. Many PAAR proteins are extended with a diverse C-terminal sequence with an unknown structure and function. Here, we report two paar-ctd genes located in two homologous operons involved in different ecological functions of Myxococcus xanthus; one has antifungal activity, and the other is associated with the kin discrimination phenotype. The PAAR-CTD proteins and the proteins encoded by their downstream genes form two toxin-immunity protein pairs. We demonstrated that the C-terminal diversification of the PAAR-CTD proteins enriches the ecological functions of bacterial cells.

The membrane topology of immunity proteins for the two-peptide bacteriocins carnobacteriocin XY, lactococcin G, and lactococcin MN shows structural diversity.

The two-peptide bacteriocins produced by Gram-positive bacteria require two different peptides, present in equimolar amounts, to elicit optimal antimicrobial activity. Producer organisms are protected from their bacteriocin by a dedicated immunity protein. The immunity proteins for two-peptide bacteriocins contain putative transmembrane domains (TMDs) and might therefore be associated with the membrane. The immunity protein CbnZ for the two-peptide bacteriocin carnobacteriocin XY (CbnXY) was identified by heterologously expressing the cbnZ gene in sensitive host strains. Using protein topology prediction methods and the dual pho-lac reporter system, we mapped out the membrane topology of CbnZ, along with those of the immunity proteins LagC and LciM for the two-peptide bacteriocins lactococcin G and lactococcin MN, respectively. Our results reveal wide structural variety between these immunity proteins that can contain as little as one TMD or as many as four TMDs.

Bacteria-Killing Type IV Secretion Systems.

Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I-IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitors.