Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow-spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energize their transport into cells and catalyze their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

Culture-Dependent and Metabarcoding Characterization of the Sugar Beet (Beta vulgaris L.) Microbiome for High-Yield Isolation of Bacteria with Plant Growth-Promoting Traits.

The diversity of plant-associated bacteria is vast and can be determined by 16S rRNA gene metabarcoding. Fewer of them have plant-beneficial properties. To harness their benefits for plants, we must isolate them. This study aimed to check whether 16S rRNA gene metabarcoding has predictive power in identifying the majority of known bacteria with plant-beneficial traits that can be isolated from the sugar beet (Beta vulgaris L.) microbiome. Rhizosphere and phyllosphere samples collected during one season at different stages of plant development were analyzed. Bacteria were isolated on rich unselective media and plant-based media enriched with sugar beet leaves or rhizosphere extracts. The isolates were identified by sequencing the 16S rRNA gene and tested in vitro for their plant-beneficial properties (stimulation of germination; exopolysaccharide, siderophore, and HCN production; phosphate solubilization; and activity against sugar beet pathogens). The highest number of co-occurring beneficial traits was eight, found in isolates of five species: Acinetobacter calcoaceticus, Bacillus australimaris, B. pumilus, Enterobacter ludwiigi, and Pantoea ananatis. These species were not detected by metabarcoding and have not previously been described as plant-beneficial inhabitants of sugar beets. Thus, our findings point out the necessity of a culture-dependent microbiome analysis and advocate for low-nutrient plant-based media for high-yield isolation of plant-beneficial taxa with multiple beneficial traits. A culture-dependent and -independent approach is required for community diversity assessment. Still, isolation on plant-based media is the best approach to select isolates for potential use as biofertilizers and biopesticides in sugar beet cultivation.

Bacterial Competition Systems Share a Domain Required for Inner Membrane Transport of the Bacteriocin Pyocin G from Pseudomonas aeruginosa.

Bacteria exploit a variety of attack strategies to gain dominance within ecological niches. Prominent among these are contact-dependent inhibition (CDI), type VI secretion (T6SS), and bacteriocins. The cytotoxic endpoint of these systems is often the delivery of a nuclease to the cytosol. How such nucleases translocate across the cytoplasmic membrane of Gram-negative bacteria is unknown. Here, we identify a small, conserved, 15-kDa domain, which we refer to as the inner membrane translocation (IMT) domain, that is common to T6SS and bacteriocins and linked to nuclease effector domains. Through fluorescence microscopy assays using intact and spheroplasted cells, we demonstrate that the IMT domain of the Pseudomonas aeruginosa-specific bacteriocin pyocin G (PyoG) is required for import of the toxin nuclease domain to the cytoplasm. We also show that translocation of PyoG into the cytosol is dependent on inner membrane proteins FtsH, a AAA+ATPase/protease, and TonB1, the latter more typically associated with transport of bacteriocins across the outer membrane. Our study reveals that the IMT domain directs the cytotoxic nuclease of PyoG to cross the cytoplasmic membrane and, more broadly, has been adapted for the transport of other toxic nucleases delivered into Gram-negative bacteria by both contact-dependent and contact-independent means. IMPORTANCE Nuclease bacteriocins are potential antimicrobials for the treatment of antibiotic-resistant bacterial infections. While the mechanism of outer membrane translocation is beginning to be understood, the mechanism of inner membrane transport is not known. This study uses PyoG as a model nuclease bacteriocin and defines a conserved domain that is essential for inner membrane translocation and is widespread in other bacterial competition systems. Additionally, the presented data link two membrane proteins, FtsH and TonB1, with inner membrane translocation of PyoG. These findings point to the general importance of this domain to the cellular uptake mechanisms of nucleases delivered by otherwise diverse and distinct bacterial competition systems. The work is also of importance for the design of new protein antibiotics.

Targeted Killing of Pseudomonas aeruginosa by Pyocin G Occurs via the Hemin Transporter Hur.

Pseudomonas aeruginosa is a priority pathogen for the development of new antibiotics, particularly because multi-drug-resistant strains of this bacterium cause serious nosocomial infections and are the leading cause of death in cystic fibrosis patients. Pyocins, bacteriocins of P. aeruginosa, are potent and diverse protein antibiotics that are deployed during bacterial competition. Pyocins are produced by more than 90% of P. aeruginosa strains and may have utility as last resort antibiotics against this bacterium. In this study, we explore the antimicrobial activity of a newly discovered pyocin called pyocin G (PyoG). We demonstrate that PyoG has broad killing activity against a collection of clinical P. aeruginosa isolates and is active in a Galleria mellonella infection model. We go on to identify cell envelope proteins that are necessary for the import of PyoG and its killing activity. PyoG recognizes bacterial cells by binding to Hur, an outer-membrane TonB-dependent transporter. Both pyocin and Hur interact with TonB1, which in complex with ExbB-ExbD links the proton motive force generated across the inner membrane with energy-dependent pyocin translocation across the outer membrane. Inner-membrane translocation of PyoG is dependent on the conserved inner-membrane AAA+ ATPase/protease, FtsH. We also report a functional exploration of the PyoG receptor. We demonstrate that Hur can bind to hemin in vitro and that this interaction is blocked by PyoG, confirming the role of Hur in hemin acquisition.

Tools and Approaches for Dissecting Protein Bacteriocin Import in Gram-Negative Bacteria.

Bacteriocins of Gram-negative bacteria are typically multi-domain proteins that target and kill bacteria of the same or closely related species. There is increasing interest in protein bacteriocin import; from a fundamental perspective to understand how folded proteins are imported into bacteria and from an applications perspective as species-specific antibiotics to combat multidrug resistant bacteria. In order to translocate across the cell envelope and cause cell death, protein bacteriocins hijack nutrient uptake pathways. Their import is energized by parasitizing intermembrane protein complexes coupled to the proton motive force, which delivers a toxic domain into the cell. A plethora of genetic, structural, biochemical, and biophysical methods have been applied to find cell envelope components involved in bacteriocin import since their discovery almost a century ago. Here, we review the various approaches that now exist for investigating how protein bacteriocins translocate into Gram-negative bacteria and highlight areas of research that will need methodological innovations to fully understand this process. We also highlight recent studies demonstrating how bacteriocins can be used to probe organization and architecture of the Gram-negative cell envelope itself.

Silencing of antibiotic resistance in E. coli with engineered phage bearing small regulatory RNAs.

In response to emergent antibiotic resistance, new strategies are needed to enhance the effectiveness of existing antibiotics. Here, we describe a phagemid-delivered, RNA-mediated system capable of directly knocking down antibiotic resistance phenotypes. Small regulatory RNAs (sRNAs) were designed to specifically inhibit translation of chloramphenicol acetyltransferase and kanamycin phosphotransferase. Nonlytic phagemids coding for sRNA expression were able to infect and restore chloramphenicol and kanamycin sensitivity to populations of otherwise resistant E. coli. This modular system could easily be extended to other bacteria with resistance profiles that depend on specific transcripts.

In situ characterization of mycobacterial growth inhibition by lytic enzymes expressed in vectorized E. coli.

The emergence of extremely drug resistant Mycobacterium tuberculosis necessitates new strategies to combat the pathogen. Engineered bacteria may serve as vectors to deliver proteins to human cells, including mycobacteria-infected macrophages. In this work, we target Mycobacterium smegmatis, a nonpathogenic tuberculosis model, with E. coli modified to express trehalose dimycolate hydrolase (TDMH), a membrane-lysing serine esterase. We show that TDMH-expressing E. coli are capable of lysing mycobacteria in vitro and at low pH. Vectorized E. coli producing TDMH were found suppress the proliferation of mycobacteria in infected macrophages.