Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa.

Pseudomonas aeruginosa thrives in the airways of individuals with cystic fibrosis, in part by forming robust biofilms that are resistant to immune clearance or antibiotic treatment. In the cystic fibrosis lung, the thickened mucus layers create an oxygen gradient, often culminating with the formation of anoxic pockets. In this environment, P. aeruginosa can use nitrate instead of oxygen to grow. Current fluorescent reporters for studying P. aeruginosa are limited to the GFP and related analogs. However, these reporters require oxygen for the maturation of their chromophore, making them unsuitable for the study of anaerobically grown P. aeruginosa. To overcome this limitation, we evaluated seven alternative fluorescent proteins, including iLOV, phiLOV2.1, evoglow-Bs2, LucY, UnaG, Fluorescence-Activating and Absorption-Shifting Tag (FAST), and iRFP670, which have been reported to emit light under oxygen-limiting conditions. We generated a series of plasmids encoding these proteins and validated their fluorescence using plate reader assays and confocal microscopy. Six of these proteins successfully labeled P. aeruginosa in anoxia. In particular, phiLOV2.1 and FAST provided superior fluorescence stability and enabled dual-color imaging of both planktonic and biofilm cultures. This study provides a set of fluorescent reporters for monitoring P. aeruginosa under low-oxygen conditions. These reporters will facilitate studies of P. aeruginosa in biofilms or other contexts relevant to its pathogenesis, such as those found in cystic fibrosis airways. Due to the broad host range of our expression vector, the phiLOV2.1 and FAST-based reporters may be applicable to the study of other Gram-negative bacteria that inhabit similar low-oxygen niches.

The T3SS of Shigella: Expression, Structure, Function, and Role in Vacuole Escape.

Shigella spp. are one of the leading causes of infectious diarrheal diseases. They are Escherichia coli pathovars that are characterized by the harboring of a large plasmid that encodes most virulence genes, including a type III secretion system (T3SS). The archetypal element of the T3SS is the injectisome, a syringe-like nanomachine composed of approximately 20 proteins, spanning both bacterial membranes and the cell wall, and topped with a needle. Upon contact of the tip of the needle with the plasma membrane, the injectisome secretes its protein substrates into host cells. Some of these substrates act as translocators or effectors whose functions are key to the invasion of the cytosol and the cell-to-cell spread characterizing the lifestyle of Shigella spp. Here, we review the structure, assembly, function, and methods to measure the activity of the injectisome with a focus on Shigella, but complemented with data from other T3SS if required. We also present the regulatory cascade that controls the expression of T3SS genes in Shigella. Finally, we describe the function of translocators and effectors during cell-to-cell spread, particularly during escape from the vacuole, a key element of Shigella's pathogenesis that has yet to reveal all of its secrets.

Diversity of bacteria associated with corn roots inoculated with Canadian woodland soils, and description of Pseudomonas aylmerense sp. nov.

Bacteria associated with corn roots inoculated with soils collected from the Canadian woodlands were isolated and characterized. Genus-level identification based on 16S rRNA sequence analysis classified the 161 isolates in 19 genera. The majority (64%) of the isolates were affiliated with the genus Pseudomonas. Further analysis of the Pseudomonas isolates based on BLASTn and rpoD-rpoB-gyrB concatenated gene phylogeny revealed three unique clusters that could not be assigned to known species. This study reports the taxonomic description of one of the distinct lineages represented by two strains (S1E40T and S1E44) with P. lurida LMG 21995T, P. costantinii LMG 22119T, P. palleroniana LMG 23076T, P. simiae CCUG 50988T and P. extremorientalis LMG 19695T as the closest taxa. Both strains showed low ANIm (<90%) and genome-based DNA-DNA hybridization (<50%) values, which unequivocally delineated the new strains from the closest relatives. These findings were supported by multilocus sequence analysis (MLSA) and DNA fingerprinting. In addition, growth characteristics and biochemical tests revealed patterns that differed from the related species. Strains S1E40T and S1E44 are Gram-negative, aerobic, rod-shaped and motile by at least one flagellum; and grew optimally at 30 °C. The predominant polar lipid is phosphatidylethanolamine while the major respiratory quinone is ubiquinone-9. Based on phenotypic and genotypic data presented here, strains S1E40T and S1E44 represent a novel species for which the name Pseudomonas aylmerense sp. nov. is proposed. The type strain is S1E40T (= LMG 30784T = DOAB 703T = HAMI 3696T) with a G + C content of 61.6%.

Genome analysis of two novel Pseudomonas strains exhibiting differential hypersensitivity reactions on tobacco seedlings reveals differences in nonflagellar T3SS organization and predicted effector proteins.

Multilocus sequence analysis (MLSA) of two new biological control strains (S1E40 and S3E12) of Pseudomonas was performed to assess their taxonomic position relative to close lineages, and comparative genomics employed to investigate whether these strains differ in key genetic features involved in hypersensitivity responses (HRs). Strain S3E12, at high concentration, incites HRs on tobacco and corn plantlets while S1E40 does not. Phylogenies based on individual genes and 16S rRNA-gyrB-rpoB-rpoD concatenated sequence data show strains S1E40 and S3E12 clustering in distinct groups. Strain S3E12 consistently clustered with Pseudomonas marginalis, a bacterium causing soft rots on plant tissues. MLSA data suggest that strains S1E40 and S3E12 are novel genotypes. This is consistent with the data of genome-based DNA-DNA homology values that are below the proposed cutoff species boundary. Comparative genomics analysis of the two strains revealed major differences in the type III secretion systems (T3SS) as well as the predicted T3SS secreted effector proteins (T3Es). One nonflagellar (NF-T3SS) and two flagellar T3SSs (F-T3SS) clusters were identified in both strains. While F-T3SS clusters in both strains were relatively conserved, the NF-T3SS clusters differed in the number of core components present. The predicted T3Es also differed in the type and number of CDSs with both strains having unique predicted protease-related effectors. In addition, the T1SS organization of the S3E12 genome has protein-coding sequences (CDSs) encoding for key factors such as T1SS secreted agglutinin repeats-toxins (a group of cytolysins and cytotoxins), a membrane fusion protein (LapC), a T1SS ATPase of LssB family (LapB), and T1SS-associated transglutaminase-like cysteine proteinase (LapP). In contrast, strain S1E40 has all CDSs for the seven-gene operon (pelA-pelG) required for Pel biosynthesis but not S3E12, suggesting that biofilm formation in these strains is modulated differently. The data presented here provide an insight of the genome organization of these two phytobacterial strains.