- Volume 169, Issue 2, 2023

The mutualistic symbiosis between the Hawaiian bobtail squid Euprymna scolopes and the marine bacterium   Vibrio fischeri   is a powerful experimental system for determining how intercellular interactions impact animal–bacterial associations. In nature, this symbiosis features multiple strains of   V. fischeri   within each adult animal, which indicates that different strains initially colonize each squid. Various studies have demonstrated that certain strains of   V. fischeri   possess a type-VI secretion system (T6SS), which can inhibit other strains from establishing symbiosis within the same host habitat. The T6SS is a bacterial melee weapon that enables a cell to kill adjacent cells by translocating toxic effectors via a lancet-like apparatus. This review describes the progress that has been made in understanding the factors that govern the structure and expression of the T6SS in   V. fischeri   and its effect on the symbiosis.

The incidence of multidrug-resistant bacteria is increasing globally, with efflux pumps being a fundamental platform limiting drug access and synergizing with other mechanisms of resistance. Increased expression of efflux pumps is a key feature of most cells that are resistant to multiple antibiotics. Whilst expression of efflux genes can confer benefits, production of complex efflux systems is energetically costly and the expression of efflux is highly regulated, with cells balancing benefits against costs. This study used TraDIS-Xpress, a genome-wide transposon mutagenesis technology, to identify genes in   Escherichia coli   and   Salmonella   Typhimurium involved in drug efflux and its regulation. We exposed mutant libraries to the canonical efflux substrate acriflavine in the presence and absence of the efflux inhibitor phenylalanine-arginine β-naphthylamide. Comparisons between conditions identified efflux-specific and drug-specific responses. Known efflux-associated genes were easily identified, including acrAB, tolC, marRA, ramRA and soxRS, confirming the specificity of the response. Further genes encoding cell envelope maintenance enzymes and products involved with stringent response activation, DNA housekeeping, respiration and glutathione biosynthesis were also identified as affecting efflux activity in both species. This demonstrates the deep relationship between efflux regulation and other cellular regulatory networks. We identified a conserved set of pathways crucial for efflux activity in these experimental conditions, which expands the list of genes known to impact on efflux efficacy. Responses in both species were similar and we propose that these common results represent a core set of genes likely to be relevant to efflux control across the Enterobacteriaceae.

The twin arginine transport (Tat) pathway exports folded proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of chloroplasts. In Escherichia coli and other Gram-negative bacteria, the Tat machinery comprises TatA, TatB and TatC components. A Tat receptor complex, formed from all three proteins, binds Tat substrates, which triggers receptor organization and recruitment of further TatA molecules to form the active Tat translocon. The polytopic membrane protein TatC forms the core of the Tat receptor and harbours two binding sites for the sequence-related TatA and TatB proteins. A ‘polar’ cluster binding site, formed by TatC transmembrane helices (TMH) 5 and 6 is occupied by TatB in the resting receptor and exchanges for TatA during receptor activation. The second binding site, lying further along TMH6, is occupied by TatA in the resting state, but its functional relevance is unclear. Here we have probed the role of this second binding site through a programme of random and targeted mutagenesis. Characterization of three stably produced TatC variants, P221R, M222R and L225P, each of which is inactive for protein transport, demonstrated that the substitutions did not affect assembly of the Tat receptor. Moreover, the substitutions that we analysed did not abolish TatA or TatB binding to either binding site. Using targeted mutagenesis we introduced bulky substitutions into the TatA binding site. Molecular dynamics simulations and crosslinking analysis indicated that TatA binding at this site was substantially reduced by these amino acid changes, but TatC retained function. While it is not clear whether TatA binding at the TMH6 site is essential for Tat activity, the isolation of inactivating substitutions indicates that this region of the protein has a critical function.

Sloths have dense fur on which insects, algae, bacteria and fungi coexist. Previous studies using cultivation-dependent methods and 18S rRNA sequencing revealed that the fungal communities in their furs comprise members of the phyla Ascomycota and Basidiomycota. In this note, we increase the resolution and knowledge of the mycobiome inhabiting the fur of the two- (Choloepus hoffmanni) and three-toed (Bradypus variegatus) sloths. Targeted amplicon metagenomic analysis of ITS2 nrDNA sequences obtained from 10 individuals of each species inhabiting the same site revealed significant differences in the structure of their fungal communities and also in the alpha-diversity estimators. The results suggest a specialization by host species and that the host effect is stronger than that of sex, age and animal weight. Capnodiales were the dominant order in sloths’ fur and Cladosporium and Neodevriesia were the most abundant genera in Bradypus and Choloepus, respectively. The fungal communities suggest that the green algae that inhabit the fur of sloths possibly live lichenized with Ascomycota fungal species. The data shown in this note offer a more detailed view of the fungal content in the fur of these extraordinary animals and could help explain other mutualistic relationships in this complex ecosystem.

One interference mechanism of bacterial competition is the production of antibiotics. Bacteria exposed to antibiotics can resist antibiotic inhibition through intrinsic or acquired mechanisms. Here, we performed a coevolution experiment to understand the long-term consequences of antibiotic production and antibiotic susceptibility for two environmental bacterial strains. We grew five independent lines of the antibiotic-producing environmental strain, Burkholderia thailandensis E264, and the antibiotic-inhibited environmental strain, Flavobacterium johnsoniae UW101, together and separately on agar plates for 7.5 months (1.5 month incubations), transferring each line five times to new agar plates. We observed that the F. johnsoniae ancestor could tolerate the B. thailandensis -produced antibiotic through efflux mechanisms, but that the coevolved lines had reduced susceptibility. We then sequenced genomes from the coevolved and monoculture F. johnsoniae lines, and uncovered mutational ramifications for the long-term antibiotic exposure. The coevolved genomes from F. johnsoniae revealed four potential mutational signatures of reduced antibiotic susceptibility that were not observed in the evolved monoculture lines. Two mutations were found in tolC: one corresponding to a 33 bp deletion and the other corresponding to a nonsynonymous mutation. A third mutation was observed as a 1 bp insertion coding for a RagB/SusD nutrient uptake protein. The last mutation was a G83R nonsynonymous mutation in acetyl-coA carboxylayse carboxyltransferase subunit alpha (AccA). Deleting the 33 bp from tolC in the F. johnsoniae ancestor reduced antibiotic susceptibility, but not to the degree observed in coevolved lines. Furthermore, the accA mutation matched a previously described mutation conferring resistance to B. thailandensis -produced thailandamide. Analysis of B. thailandensis transposon mutants for thailandamide production revealed that thailandamide was bioactive against F. johnsoniae, but also suggested that additional B. thailandensis -produced antibiotics were involved in the inhibition of F. johnsoniae . This study reveals how multi-generational interspecies interactions, mediated through chemical exchange, can result in novel interaction-specific mutations, some of which may contribute to reductions in antibiotic susceptibility.

Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNAfMet to fMet-tRNAfMet is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH2-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using in vivo and in vitro approaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNAfMet. Dihydrofolate (DHF) formed as a by-product in the in vitro assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆fmt strain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment to Escherichia coli showed a decrease in the reduced folate species (THF, 5,10-CH2-THF, 5-CH3-THF, 5,10-CH+-THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNAfMet.

Pseudomonas aeruginosa is a versatile bacterium capable of adapting to a wide range of stress factors, including solar UVA radiation (400–315 nm). High UVA doses produce lethal effects due to the action of reactive oxygen species. Sublethal UVA doses also induces oxidative damage, but, in addition, it triggers a variety of adaptive responses, including the overexpression of pelA and pslA genes in P. aeruginosa . These genes encode the synthesis of Pel and Psl, which are essential polysaccharides in biofilm formation. The present study analysed the role of Pel and Psl in the adaptive responses generated by exposure to low UVA doses, and their importance in the response to lethal doses of UVA, hydrogen peroxide (H2O2), and sodium hypochlorite, in both planktonic cells and submerged and air–liquid interface (ALI) biofilms. It also studied the roles of Pel and Psl in P. aeruginosa–Staphylococcus aureus interaction. The results demonstrate that the capacity of sublethal UVA exposure to increase cell hydrophobicity and cell attachment and generate cross-protection phenomena in P. aeruginosa depends on the presence of Pel and Psl. The study also shows that Pel and Psl have a key role in the tolerance to lethal doses of UVA radiation, sodium hypochlorite and H2O2, in both biofilms and planktonic cells. Finally, co-culture assays showed total inhibition of S. aureus growth in presence of P. aeruginosa . This phenomenon depends, at least in part, on the simultaneous presence of Pel and Psl in planktonic cells and biofilms, suggesting a relevant role of these polysaccharides in the interaction between these species.

The in vivo temperature can vary according to the host tissue and the response to infection. Streptococcus pneumoniae has evolved mechanisms to survive these temperature differences, but neither the consequences of different temperatures for pneumococcal phenotype nor the genetic basis of thermal adaptation are known in detail. In our previous study [ 16 ], we found that CiaR, which is a part of two-component regulatory system CiaRH, as well as 17 genes known to be controlled by CiaRH, were identified to be differentially expressed with temperature. One of the CiaRH-regulated genes shown to be differentially regulated by temperature is for the high-temperature requirement protein (HtrA), coded by SPD_2068 (htrA). In this study, we hypothesized that the CiaRH system plays an important role in pneumococcal thermal adaptation through its control over htrA. This hypothesis was evaluated by testing strains mutated or overexpressing ciaR and/or htrA, in in vitro and in vivo assays. The results showed that in the absence of ciaR, the growth, haemolytic activity, amount of capsule and biofilm formation were considerably diminished at 40 °C only, while the cell size and virulence were affected at both 34 and 40 °C. The overexpression of htrA in the ∆ciaR background reconstituted the growth at all temperatures, and the haemolytic activity, biofilm formation and virulence of ∆ciaR partially at 40 °C. We also showed that overexpression of htrA in the wild-type promoted pneumococcal virulence at 40 °C, while the increase of capsule was observed at 34 °C, suggesting that the role of htrA changes at different temperatures. Our data suggest that CiaR and HtrA play an important role in pneumococcal thermal adaptation.

Many saprophytic bacteria have LuxR-I-type acyl-homoserine lactone (AHL) quorum-sensing systems that may be important for competing with other bacteria in complex soil communities. LuxR AHL receptors specifically interact with cognate AHLs to cause changes in expression of target genes. Some LuxR-type AHL receptors have relaxed specificity and are responsive to non-cognate AHLs. These promiscuous receptors might be used to sense and respond to AHLs produced by other bacteria by eavesdropping. We are interested in understanding the role of eavesdropping during interspecies competition. The soil saprophyte Chromobacterium subtsugae has a single AHL circuit, CviR-I, which produces and responds to N-hexanoyl-HSL (C6-HSL). The AHL receptor CviR can respond to a variety of AHLs in addition to C6-HSL. In prior studies we have utilized a coculture model with C. subtsugae and another soil saprophyte, Burkholderia thailandensis. Using this model, we previously showed that promiscuous activation of CviR by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Here, we show that B. thailandensis AHLs activate transcription of dozens of genes in C. subtsugae, including the hcnABC genes coding for production of hydrogen cyanide. We show that hydrogen cyanide production is population density-dependent and demonstrate that the cross-induction of hydrogen cyanide by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Our results provide new information on C. subtsugae quorum sensing and are the basis for future studies aimed at understanding the role of eavesdropping in interspecies competition.