Within the highly diverse type four filament (TFF or T4F) superfamily, the machineries of type IVa pili (T4aP) and the type 2 secretion system (T2SS) in diderm bacteria exhibit a substantial sequence similarity despite divergent functions and distinct appearances: T4aP can extend micrometers beyond the outer membrane, whereas the endopili in the T2SS are restricted to the periplasm. The determination of the structure of individual components and entire filaments is crucial to understand how their structure enables them to serve different functions. However, the dynamics of these filaments poses a challenge for their high-resolution structure determination. This review presents different approaches that have been used to study the structure and dynamics of T4aP and T2SS endopili by means of integrative structural biology, cryo-electron microscopy (cryo-EM), and molecular dynamics simulations. Their conserved features and differences are presented. The non-helical stretch in the long-conserved N-terminal helix which is characteristic of all members of the TFF and the impact of calcium on structure, function, and dynamics of these filaments are discussed in detail.

Insects engage in manifold interactions with bacteria that can shift along the parasitism-mutualism continuum. However, only a small number of bacterial taxa managed to successfully colonize a wide diversity of insects, by evolving mechanisms for host-cell entry, immune evasion, germline tropism, reproductive manipulation, and/or by providing benefits to the host that stabilize the symbiotic association. Here we report on the discovery of an Enterobacterales endosymbiont (Symbiodolus, type species S. clandestinus) that is widespread across at least six insect orders and occurs at high prevalence within host populations. Fluorescence in situ hybridization in several Coleopteran and one Dipteran species revealed Symbiodolus\' intracellular presence in all host life stages and across tissues, with a high abundance in female ovaries, indicating transovarial vertical transmission. Symbiont genome sequencing across 16 host taxa revealed a high degree of functional conservation in the eroding and transposon-rich genomes. All sequenced Symbiodolus genomes encode for multiple secretion systems, alongside effectors and toxin-antitoxin systems, which likely facilitate host-cell entry and interactions with the host. However, Symbiodolus-infected insects show no obvious signs of disease, and biosynthetic pathways for several amino acids and cofactors encoded by the bacterial genomes suggest that the symbionts may also be able to provide benefits to the hosts. A lack of host-symbiont cospeciation provides evidence for occasional horizontal transmission, so Symbiodolus\' success is likely based on a mixed transmission mode. Our findings uncover a hitherto undescribed and widespread insect endosymbiont that may present valuable opportunities to unravel the molecular underpinnings of symbiosis establishment and maintenance.

Bacteria in genus Campylobacter are the leading cause of foodborne infections worldwide. Here we describe the roles of extracellular vesicles in the pathogenesis of these bacteria and current knowledge of vesicle biogenesis. We also discuss the advantages of this alternative secretion pathway for bacterial virulence.

Aeromonas caviae is an emerging human enteric pathogen. However, the genomic features and virulence genes of A. caviae strains from human gastroenteritis and other sources have not been fully elucidated. Here, we conducted a genomic analysis of 565 global A. caviae strains isolated from different sources, including 261 strains isolated from faecal samples of gastroenteritis patients, of which 18 genomes were sequenced in this study. The presence of bacterial virulence genes and secretion systems in A. caviae strains from different sources was compared, and the phylogenetic relationship of A. caviae strains was assessed based on the core genome. The complete genome of A. caviae strain A20-9 isolated from a gastroenteritis patient was obtained in this study, from which 300 putative virulence factors and a T4SS-encoding plasmid, pAC, were identified. Genes encoding T4SS were also identified in a novel genomic island, ACI-1, from other T4SS-positive strains. The prevalence of T4SS was significantly lower in A. caviae strains from gastroenteritis patients than in environmental strains (3 %, P<0.0001 vs 14 %, P<0.01). Conversely, the prevalence of T6SS was significantly higher in A. caviae strains isolated from gastroenteritis patients than in environmental strains (25 %, P<0.05 vs 13  %, P<0.01). Four phylogenetic clusters were formed based on the core genome of 565 A. caviae strains, and strains carrying T6SS often showed close phylogenetic relationships. T3SS, aerolysin and thermostable cytotonic enterotoxin were absent in all 565 A. caviae strains. Our findings provide novel information on the genomic features of A. caviae and suggest that T6SS may play a role in A. caviae-induced human gastroenteritis.

The multi-drug resistant pathogen Acinetobacter baumannii has gained global attention as an important clinical challenge. Owing to its ability to survive on surfaces, its capacity for horizontal gene transfer, and its resistance to front-line antibiotics, A. baumannii has established itself as a successful pathogen. Bacterial conjugation is a central mechanism for pathogen evolution. The epidemic multidrug-resistant A. baumannii ACICU harbours a plasmid encoding a Type IV Secretion System (T4SS) with homology to the E. coli F-plasmid, and plasmids with homologous gene clusters have been identified in several A. baumannii sequence types. However the genetic and host strain diversity, global distribution, and functional ability of this group of plasmids is not fully understood. Using systematic analysis, we show that pACICU2 belongs to a group of almost 120 T4SS-encoding plasmids within four different species of Acinetobacter and one strain of Klebsiella pneumoniae from human and environmental origin, and globally distributed across 20 countries spanning 4 continents. Genetic diversity was observed both outside and within the T4SS-encoding cluster, and 47% of plasmids harboured resistance determinants, with two plasmids harbouring eleven. Conjugation studies with an extensively drug-resistant (XDR) strain showed that the XDR plasmid could be successfully transferred to a more divergent A. baumanii, and transconjugants exhibited the resistance phenotype of the plasmid. Collectively, this demonstrates that these T4SS-encoding plasmids are globally distributed and more widespread among Acinetobacter than previously thought, and that they represent an important potential reservoir for future clinical concern.

The symbioses between leguminous plants and nitrogen-fixing bacteria known as rhizobia are well known for promoting plant growth and sustainably increasing soil nitrogen. Recent evidence indicates that hopanoids, a family of steroid-like lipids, promote Bradyrhizobium symbioses with tropical legumes. To characterize hopanoids in Bradyrhizobium symbiosis with soybean, we validated a recently published cumate-inducible hopanoid mutant of Bradyrhizobium diazoefficiens USDA110, Pcu-shc::∆shc. GC-MS analysis showed that this strain does not produce hopanoids without cumate induction, and under this condition, is impaired in growth in rich medium and under osmotic, temperature, and pH stress. In planta, Pcu-shc::∆shc is an inefficient soybean symbiont with significantly lower rates of nitrogen fixation and low survival within the host tissue. RNA-seq revealed that hopanoid loss reduces the expression of flagellar motility and chemotaxis-related genes, further confirmed by swim plate assays, and enhances the expression of genes related to nitrogen metabolism and protein secretion. These results suggest that hopanoids provide a significant fitness advantage to B. diazoefficiens in legume hosts and provide a foundation for future mechanistic studies of hopanoid function in protein secretion and motility.
A major problem for global sustainability is feeding our exponentially growing human population while available arable land decreases. Harnessing the power of plant-beneficial microbes is a potential solution, including increasing our reliance on the symbioses of leguminous plants and nitrogen-fixing rhizobia. This study examines the role of hopanoid lipids in the symbiosis between Bradyrhizobium diazoefficiens USDA110, an important commercial inoculant strain, and its economically significant host soybean. Our research extends our knowledge of the functions of bacterial lipids in symbiosis to an agricultural context, which may one day help improve the practical applications of plant-beneficial microbes in agriculture.

Super-resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single-molecule resolution on a millisecond time scale. Here we review recent applications of super-resolution fluorescence microscopy with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of molecular machines in action.

Bacterial effector proteins are secreted by a variety of protein secretion systems and play an important role in the interaction between the host and pathogenic bacteria. Therefore, it is important to find a fast and inexpensive method to discover bacterial effectors. In this study, we propose a multi-type secretion effector adaptive random forest (TSE-ARF) to adaptively identify secretion effectors across T1SE-T4SE and T6SE based only on protein sequences. First, we proposed two new feature descriptors by considering some characteristic protein information and fused them with some universal features to form a 290-dimensional feature vector with good versatility. Then, the TSE-ARF model was used to make classification predictions by parameter adaptation of different secretion effectors integrating Shuffled Frog Leaping Algorithm and random forest. The perfect performance in TSE-ARF under different data sets and settings shows its considerable generalization ability, with which more candidate effectors were screened in the whole genome. Source code is available at https://github.com/AIMOVE/TSE-ARF.

OBJECTIVE: T6SS has received attention due to its significance in mediating interorganismal competition through contact-dependent release of effector molecules into prokaryotic and eukaryotic cells. Reverse-genetic studies have indicated the role of T6SS in virulence in a variety of plant pathogenic bacteria, including the one studied here, Xanthomonas. However, it is not clear whether such effect on virulence is merely due to a shift in the microbiome-mediated protection or if T6SS is involved in a complex virulence regulatory network. In this study, we conducted in vitro transcriptome profiling in minimal medium to decipher the signaling pathways regulated by tssM-i3* in X. perforans AL65. We show that TssM-i3* regulates the expression of a suite of genes associated with virulence and metabolism either directly or indirectly by altering the transcription of several regulators. These findings further expand our knowledge on the intricate molecular circuits regulated by T6SS in phytopathogenic bacteria.

Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Due to its size and mechanism of action, the T6SS was intuitively thought to be energetically costly. Here, using a combination of mutants and growth measurements in liquid medium, on plates, and in competition experiments, we show that the T6SS does not entail a growth cost to enteroaggregative Escherichia coli.