Nanobodies as potential tools for microbiological testing of live biotherapeutic products.

Nanobodies are highly specific binding domains derived from naturally occurring single chain camelid antibodies. Live biotherapeutic products (LBPs) are biological products containing preparations of live organisms, such as Lactobacillus, that are intended for use as drugs, i.e. to address a specific disease or condition. Demonstrating potency of multi-strain LBPs can be challenging. The approach investigated here is to use strain-specific nanobody reagents in LBP potency assays. Llamas were immunized with radiation-killed Lactobacillus jensenii or L. crispatus whole cell preparations. A nanobody phage-display library was constructed and panned against bacterial preparations to identify nanobodies specific for each species. Nanobody-encoding DNA sequences were subcloned and the nanobodies were expressed, purified, and characterized. Colony immunoblots and flow cytometry showed that binding by Lj75 and Lj94 nanobodies were limited to a subset of L. jensenii strains while binding by Lc38 and Lc58 nanobodies were limited to L. crispatus strains. Mass spectrometry was used to demonstrate that Lj75 specifically bound a peptidase of L. jensenii, and that Lc58 bound an S-layer protein of L. crispatus. The utility of fluorescent nanobodies in evaluating multi-strain LBP potency assays was assessed by evaluating a L. crispatus and L. jensenii mixture by fluorescence microscopy, flow cytometry, and colony immunoblots. Our results showed that the fluorescent nanobody labelling enabled differentiation and quantitation of the strains in mixture by these methods. Development of these nanobody reagents represents a potential advance in LBP testing, informing the advancement of future LBP potency assays and, thereby, facilitation of clinical investigation of LBPs.

Characterization of Lactobacilli Phage Endolysins and Their Functional Domains-Potential Live Biotherapeutic Testing Reagents.

Phage endolysin-specific binding characteristics and killing activity support their potential use in biotechnological applications, including potency and purity testing of live biotherapeutic products (LBPs). LBPs contain live organisms, such as lactic acid bacteria (LAB), and are intended for use as drugs. Our approach uses the endolysin cell wall binding domains (CBD) for LBP potency assays and the endolysin killing activity for purity assays. CBDs of the following five lactobacilli phage lysins were characterized: CL1, Jlb1, Lj965, LL-H, and ΦJB. They exhibited different bindings to 27 LAB strains and were found to bind peptidoglycan or surface polymers. Flow cytometry based on CBD binding was used to enumerate viable counts of two strains in the mixture. CL1-lys, jlb1-lys, and ΦJB-lys and their enzymatic domains (EADs) exhibited cell wall digestive activity and lytic activity against LAB. Jlb1-EAD and ΦJB-EAD were more sensitive than their respective hololysins to buffer pH and NaCl changes. The ΦJB-EAD exhibited stronger lytic activity than ΦJB-lys, possibly due to ΦJB-CBD-mediated sequestration of ΦJB-lys by cell debris. CBD multiplex assays indicate that these proteins may be useful LBP potency reagents, and the lytic activity suggests that CL1-lys, jlb1-lys, and ΦJB-lys and their EADs are good candidates for LBP purity reagent development.

Pseudomonas chlororaphis Produces Multiple R-Tailocin Particles That Broaden the Killing Spectrum and Contribute to Persistence in Rhizosphere Communities.

R-tailocins are high-molecular-weight bacteriocins resembling bacteriophage tails. Pseudomonas chlororaphis 30-84 is a plant growth-promoting rhizobacterial (PGPR) strain that produces two distinct R-tailocin particles with different killing spectra. The two R-tailocins have different evolutionary histories but are released by the same lysis cassette. A previous study showed that both tailocins are important for pairwise competition with susceptible rhizosphere-colonizing strains; however, the broader role of tailocins in competition with the native rhizosphere microbiome was not tested. Genomic analysis of the P. chlororaphis 30-84 R-tailocin gene cluster uncovered the presence of three tail fiber genes in the tailocin 2 genetic module that could potentially result in tailocin 2 particles having different tail fibers and thus a wider killing spectrum. In this study, the tail fibers were found to incorporate onto different tailocin 2 particles, each with a distinct killing spectrum. A loss of production of one or both tailocins resulted in decreased P. chlororaphis 30-84 persistence within the wheat rhizosphere when in competition with the native microflora but not bulk soil. The capacity to produce three different versions of a single tailocin, each having one of three different types of tail fibers, is a previously unreported mechanism that leads to a broader R-tailocin killing spectrum. This study also provides evidence for the function of R-tailocins in competition with rhizosphere microbiome communities but not in bulk soil.IMPORTANCE Although R-tailocin gene clusters typically encode one tail fiber protein, three tail fiber-resembling genes were identified in association with one of the two sets of R-tailocin genes within the tailocin cluster of P. chlororaphis 30-84 and other sequenced P. chlororaphis strain genomes. This study confirmed that P. chlororaphis 30-84 not only produces two distinct tailocins, but that one of them is produced with three different types of tail fibers. This is a previously unreported strategy to increase the breadth of strains targeted by an R-tailocin. Our finding that R-tailocins produced by a PGPR Pseudomonas strain enhanced its persistence within the wheat rhizosphere microbiome confirms that R-tailocin production contributes to the population dynamics of rhizobacterial communities.

Pseudomonas chlororaphis Produces Two Distinct R-Tailocins That Contribute to Bacterial Competition in Biofilms and on Roots.

R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many Pseudomonas species. In this study, analysis of the P. chlororaphis 30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of P. chlororaphis 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to Pseudomonas species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few Pseudomonas species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of P. chlororaphis 30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that Pseudomonas species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated Pseudomonas in biofilms and the rhizosphere.IMPORTANCE Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated Pseudomonas but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that Pseudomonas strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated Pseudomonas species in bacterial population dynamics within surface-attached biofilms and on roots.

The Phenazine 2-Hydroxy-Phenazine-1-Carboxylic Acid Promotes Extracellular DNA Release and Has Broad Transcriptomic Consequences in Pseudomonas chlororaphis 30-84.

Enhanced production of 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the biological control strain Pseudomonas chlororaphis 30-84 derivative 30-84O* was shown previously to promote cell adhesion and alter the three-dimensional structure of surface-attached biofilms compared to the wild type. The current study demonstrates that production of 2-OH-PCA promotes the release of extracellular DNA, which is correlated with the production of structured biofilm matrix. Moreover, the essential role of the extracellular DNA in maintaining the mass and structure of the 30-84 biofilm matrix is demonstrated. To better understand the role of different phenazines in biofilm matrix production and gene expression, transcriptomic analyses were conducted comparing gene expression patterns of populations of wild type, 30-84O* and a derivative of 30-84 producing only PCA (30-84PCA) to a phenazine defective mutant (30-84ZN) when grown in static cultures. RNA-Seq analyses identified a group of 802 genes that were differentially expressed by the phenazine producing derivatives compared to 30-84ZN, including 240 genes shared by the two 2-OH-PCA producing derivatives, the wild type and 30-84O*. A gene cluster encoding a bacteriophage-derived pyocin and its lysis cassette was upregulated in 2-OH-PCA producing derivatives. A holin encoded in this gene cluster was found to contribute to the release of eDNA in 30-84 biofilm matrices, demonstrating that the influence of 2-OH-PCA on eDNA production is due in part to cell autolysis as a result of pyocin production and release. The results expand the current understanding of the functions different phenazines play in the survival of bacteria in biofilm-forming communities.

Adaptation genomics of a small-colony variant in a Pseudomonas chlororaphis 30-84 biofilm.

The rhizosphere-colonizing bacterium Pseudomonas chlororaphis 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we characterize a small-colony variant (SCV) isolated from a P. chlororaphis 30-84 biofilm. The SCV exhibited pleiotropic phenotypes, including small cell size, slow growth and motility, low levels of phenazine production, and increased biofilm formation and resistance to antimicrobials. To better understand the genetic alterations underlying these phenotypes, RNA and whole-genome sequencing analyses were conducted comparing an SCV to the wild-type strain. Of the genome's 5,971 genes, transcriptomic profiling indicated that 1,098 (18.4%) have undergone substantial reprograming of gene expression in the SCV. Whole-genome sequence analysis revealed multiple alterations in the SCV, including mutations in yfiR (cyclic-di-GMP production), fusA (elongation factor), and cyoE (heme synthesis) and a 70-kb deletion. Genetic analysis revealed that the yfiR locus plays a major role in controlling SCV phenotypes, including colony size, growth, motility, and biofilm formation. Moreover, a point mutation in the fusA gene contributed to kanamycin resistance. Interestingly, the SCV can partially switch back to wild-type morphologies under specific conditions. Our data also support the idea that phenotypic switching in P. chlororaphis is not due to simple genetic reversions but may involve multiple secondary mutations. The emergence of these highly adherent and antibiotic-resistant SCVs within the biofilm might play key roles in P. chlororaphis natural persistence.