The genome of Halobacterium strain 63-R2 was recently reported and provides the opportunity to resolve long-standing issues regarding the source of two widely used model strains of Hbt. salinarum, NRC-1 and R1. Strain 63-R2 was isolated in 1934 from a salted buffalo hide (epithet ‘cutirubra’), along with another strain from a salted cowhide (91-R6T, epithet ‘salinaria’, the type strain of Halobacterium salinarum). Both strains belong to the same species according to genome-based taxonomy analysis (TYGS), with chromosome sequences showing 99.64% identity over 1.85 Mb. The chromosome of strain 63-R2 is 99.99% identical to the two laboratory strains NRC-1 and R1, with only 5 indels, excluding the mobilome. The two reported plasmids of strain 63-R2 share their architecture with plasmids of strain R1 (pHcu43/pHS4, 99.89% identity; pHcu235/pHS3, 100.0% identity). We detected and assembled additional plasmids, using PacBio reads deposited at the SRA database, further corroborating that strain differences are minimal. One plasmid, pHcu190 (190,816 bp) corresponds to pHS1 (strain R1) but is even more similar in architecture to pNRC100 (strain NRC-1). Another plasmid, pHcu229, assembled partially and completed in silico (229,124 bp), shares most of its architecture with pHS2 (strain R1). In deviating regions, it corresponds to pNRC200 (strain NRC-1). Further architectural differences between the laboratory strain plasmids are not unique but are present in strain 63-R2, which contains characteristics from both of them. Based on these observations, it is proposed that the early 20th-century isolate 63-R2 is the immediate ancestor of the twin laboratory strains NRC-1 and R1.

Human exposures to the Gram-negative, halophilic environmental pathogen, Vibrio vulnificus, are on the rise, yet pathogenicity and virulence mechanisms remain poorly understood. There are hundreds of cases of Vibrio vulnificus-related infections yearly, and these infections result in hospitalizations 92% of the time, with a mortality rate of 35%. This infection is severe, with patients typically contracting the bacteria via consumption of contaminated food, or through an open wound while swimming in contaminated water, which can result in necrotizing fasciitis and amputation of infected tissue. Several genes have been implicated in contributing to the pathogenicity of this organism (rtxA1, vvpE, vvhA), but no defined mechanism for pathogenicity has been discovered. Here, we focus on environmentally isolated Vibrio vulnificus strains and use a Zebrafish model (Danio rerio) to address the virulence capabilities of these strains. We found that virulence varied greatly between individual strains, and the commonly used marker gene of disease-causing strains of vcgC, did not accurately predict the more virulent strains. To this point, the least virulent strain from the study, V. vulnificus Sept WR1-BW6, which was positive for vcgC, vvhA, and rtxA1, did not produce severe disease in the fish and was the only strain that did not produce a single mortality throughout the study. Our study shows that virulence between different environmental strains varies greatly, and cannot accurately be predicted based on genotype alone.

The regulation of the central metabolism and fermentation pathways and its effect on antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is not completely understood. In this study, we show that B. fragilis encodes for two iron-dependent redox-sensitive regulatory pirin protein genes, pir1 and pir2, whose mRNA expression is upregulated following oxygen exposure and growth in iron-limiting conditions. Pir1 and Pir2 modulate short-chain fatty acids production and alter susceptibility to metronidazole (MTZ), and to amixicile (AMIX), a novel inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobes. Consistent with this, we showed that Pir1 forms direct protein-protein interactions with PFOR as determined by two-hybrid system assays. In addition, AlphaFold2-based structural analysis predicts that Pir1 and Pir2 form stable interactions with several enzymes of the central metabolism including the 2-ketoglutarate:ferredoxin oxidoreductases Kor1AB and Kor2CDAEBG. A series of metabolic mutants and electron transport chain inhibitors were used to show a wide-ranging effect of bacterial metabolism on MTZ and AMIX susceptibility. Furthermore, we show that AMIX is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. This investigation led to the discovery that the kor2AEBG genes are essential for growth, and we present evidence that kor2AEBG genes have dual functions including the reductive synthesis of 2-ketoglutarate via reverse TCA cycle. However, the metabolic activity that bypasses KorAEBG function remains to be defined. Collectively our investigation reveals new information on B. fragilis central metabolism and its modulatory control by pirin proteins which may be leveraged for the future development of new narrow-spectrum antimicrobials

The persistence and efficacy of biocontrol agents (BCAs) in agricultural fields are crucial for sustainable crop production. In this study, using a novel qPCR probe protocol, we investigated the persistence of the introduced bacterial strain Pseudomonas sivasensis CF10PS3 in the wheat phyllosphere. The CF10PS3 strain, known for its biocontrol properties against wheat pathogens, was applied through foliar spray, and its persistence was monitored over seven weeks. Our qPCR assays, designed to specifically detect CF10PS3, distinguished it from naturally occurring P. sivasensis strains, providing precise insights into its dynamics in the field. The experimental results indicated that CF10PS3 was already present on the wheat leaves before application, suggesting its natural adaptation to the foliar environment. Following initial application, a significant increase in CF10PS3 was observed, though subsequent environmental factors such as rain and wind caused notable fluctuations in its population. Despite these variations, the introduced strain showed considerable persistence, with population levels significantly higher than those in untreated plots by the end of the study period. This research underscores the importance of understanding BCA dynamics in the field, highlighting the influence of environmental conditions on their efficacy. Specific qPCR probes proved effective in monitoring introduced strains, offering valuable insights for optimizing BCA application strategies. Our findings contribute to developing robust biocontrol methods, promoting sustainable agricultural practices and enhancing crop protection.