Motility in Periweissella Species: Genomic and Phenotypic Characterization and Update on Motility in Lactobacillaceae.

The genus Weissella and the recently described genus Periweissella, to which some previously named Weissella species have been reclassified as a result of a taxogenomic assessment, includes lactic acid bacteria species with high biotechnological and probiotic potential. Only one species, namely, Periweissella (P.) beninensis, whose type strain has been shown to possess probiotic features, has so far been described to be motile. However, the availability of numerous genome sequences of Weissella and Periweissella species prompted the possibility to screen for the presence of the genetic determinants encoding motility in Weissella and Periweissellas spp. other than P. beninensis. Herein, we performed a comprehensive genomic analysis to identify motility-related proteins in all Weissella and Periweissella species described so far, and extended the analysis to the recently sequenced Lactobacillaceae spp. Furthermore, we performed motility assays and transmission electron microscopy (TEM) on Periweissella type strains to confirm the genomic prediction. The homology-based analysis revealed genes coding for motility proteins only in the type strains of P. beninensis, P. fabalis, P. fabaria and P. ghanensis genomes. However, only the P. beninensis type strain was positive in the motility assay and displayed run-and-tumble behavior. Many peritrichous and long flagella on bacterial cells were visualized via TEM, as well. As for the Lactobacillaceae, in addition to the species previously described to harbor motility proteins, the genetic determinants of motility were also found in the genomes of the type strains of Lactobacillus rogosae and Ligilactobacillus salitolerans. This study, which is one of the first to analyze the genomes of Weissella, Periweissella and the recently sequenced Lactobacillaceae spp. for the presence of genes coding for motility proteins and which assesses the associated motility phenotypes, provides novel results that expand knowledge on these genera and are useful in the further characterization of lactic acid bacteria.

Characterisation of the Novel Filamentous Phage PMBT54 Infecting the Milk Spoilage Bacteria Pseudomonas carnis and Pseudomonas lactis.

Filamentous bacteriophages are lysogenic and pseudo-lysogenic viruses that do not lyse their host but are often continuously secreted from the infected cell. They belong to the order Tubulavirales, which encompasses three families, with the Inoviridae being the largest. While the number of identified inoviral sequences has greatly increased in recent years due to metagenomic studies, morphological and physiological characterisation is still restricted to only a few members of the filamentous phages. Here, we describe the novel filamentous phage PMBT54, which infects the spoilage-relevant Pseudomonas species P. carnis and P. lactis. Its genome is 7320 bp in size, has a mol% GC content of 48.37, and codes for 13 open-reading frames, two of which are located on the (-) strand. The virion exhibits a typical filamentous morphology and is secreted from the host cell at various lengths. The phage was shown to promote biofilm formation in both host strains and, therefore, has potential implications for milk spoilage, as biofilms are a major concern in the dairy industry.

Pheno- and Genotyping of Three Novel Bacteriophage Genera That Target a Wheat Phyllosphere Sphingomonas Genus.

Bacteriophages are viral agents that infect and replicate within bacterial cells. Despite the increasing importance of phage ecology, environmental phages-particularly those targeting phyllosphere-associated bacteria-remain underexplored, and current genomic databases lack high-quality phage genome sequences linked to specific environmentally important bacteria, such as the ubiquitous sphingomonads. Here, we isolated three novel phages from a Danish wastewater treatment facility. Notably, these phages are among the first discovered to target and regulate a Sphingomonas genus within the wheat phyllosphere microbiome. Two of the phages displayed a non-prolate Siphovirus morphotype and demonstrated a narrow host range when tested against additional Sphingomonas strains. Intergenomic studies revealed limited nucleotide sequence similarity within the isolated phage genomes and to publicly available metagenome data of their closest relatives. Particularly intriguing was the limited homology observed between the DNA polymerase encoding genes of the isolated phages and their closest relatives. Based on these findings, we propose three newly identified genera of viruses: Longusvirus carli, Vexovirus birtae, and Molestusvirus kimi, following the latest ICTV binomial nomenclature for virus species. These results contribute to our current understanding of phage genetic diversity in natural environments and hold promising implications for phage applications in phyllosphere microbiome manipulation strategies.

Cas1 and Fen1 Display Equivalent Functions During Archaeal DNA Repair.

CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways, either by regulating their expression or via direct protein-protein contact and enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but an additional role in cellular DNA repair pathways has been proposed previously. Here, we analysed the capacity of an archaeal Cas1 from Haloferax volcanii to act upon DNA damage induced by oxidative stress and found that a deletion of the cas1 gene led to reduced survival rates following stress induction. In addition, our results indicate that Cas1 is directly involved in DNA repair as the enzymatically active site of the protein is crucial for growth under oxidative conditions. Based on biochemical assays, we propose a mechanism by which Cas1 plays a similar function to DNA repair protein Fen1 by cleaving branched intermediate structures. The present study broadens our understanding of the functional link between CRISPR-Cas immunity and DNA repair by demonstrating that Cas1 and Fen1 display equivalent roles during archaeal DNA damage repair.

The Biology of CRISPR-Cas: Backward and Forward.

In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system against phages and other foreign genetic elements. Here, we review the biology of the diverse CRISPR-Cas systems and the major progress achieved in recent years in understanding the underlying mechanisms of the three stages of CRISPR-Cas immunity: adaptation, crRNA biogenesis, and interference. The ecology and regulation of CRISPR-Cas in the context of phage infection, the roles of these systems beyond immunity, and the open questions that propel the field forward are also discussed.

CRISPR-Cas: biology, mechanisms and relevance.

Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes-termed spacers-into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent.This article is part of the themed issue 'The new bacteriology'.