A new anti-CRISPR gene promotes the spread of drug-resistance plasmids in Klebsiella pneumoniae.

The Klebsiella pneumoniae (K. pneumoniae, Kp) populations carrying both resistance-encoding and virulence-encoding mobile genetic elements (MGEs) significantly threaten global health. In this study, we identified a new anti-CRISPR gene (acrIE10) on a conjugative plasmid with self-target sequence in K. pneumoniae with type I-E* CRISPR-Cas system. AcrIE10 interacts with the Cas7* subunit of K. pneumoniae I-E* CRISPR-Cas system. The crystal structure of the AcrIE10-KpCas7* complex suggests that AcrIE10 suppresses the I-E* CRISPR-Cas by binding directly to Cas7 to prevent its hexamerization, thereby preventing the surveillance complex assembly and crRNA loading. Bioinformatic and functional analyses revealed that AcrIE10 is functionally widespread across diverse species. Our study reports a novel anti-CRISPR and highlights its potential role in spreading resistance and virulence among pathogens.

Isolation and characterization of novel Fusobacterium nucleatum bacteriophages.

Fusobacterium nucleatum is a strictly anaerobic, Gram-negative bacterial species that is a member of the commensal flora in the oral cavity and gut. Recent studies suggested that the increase of abundance is associated with the development of various diseases, among which colorectal cancer is of the biggest concerns. Phage therapy is regarded as a potential approach to control the number of F. nucleatum, which may contribute to the prevention and treatment of related diseases. In this study, we isolated five isolates of bacteriophage targeting F. nucleatum. The morphological, biological, genomic and functional characteristics of five bacteriophages were investigated. Transmission electron microscopy revealed that JD-Fnp1 ~ JD-Fnp5 are all myoviruses. The size of the JD-Fnp1 ~ JD-Fnp5 genomes was 180,066 bp (JD-Fnp1), 41,329 bp (JD-Fnp2), 38,962 bp (JD-Fnp3), 180,231 bp (JD-Fnp4), and 41,353 bp (JD-Fnp5) respectively. The biological features including pH and heat stability, host range, growth characteristics of JD-Fnp1 ~ JD-Fnp5 displayed different patterns. Among them, JD-Fnp4 is considered to have the greatest clinical application value. The identification and characterization of JD-Fnp1 ~ JD-Fnp5 provides a basis for subsequent therapeutic strategy exploration of F. nucleatum-related diseases.

A Plasmid With Conserved Phage Genes Helps Klebsiella pneumoniae Defend Against the Invasion of Transferable DNA Elements at the Cost of Reduced Virulence.

Klebsiella pneumoniae exhibits extensive phenotypic and genetic diversity. Higher plasmid loads in the cell were supposed to play an key role in its genome diversity. Although some plasmids are widely distributed in Kp populations, they are poorly recognized. A plasmid named p2 in strain Kp1604 was predicted to be an intact prophage like Salmonella phage SSU5. However, our study showed that p2 was specifically packaged into membrane vesicles (MVs) rather than phage particles triggered by mitomycin C and subinhibitory concentrations of antibiotics. p2-minus mutant Kp1604Δp2 did not affect MV production. Compared with Kp1604, the capacity of plasmid uptake and the amount of phage burst of Kp1604Δp2 were improved. Moreover, virulence of Kp1604Δp2 also increased. Our results indicated that p2 could contribute to the host defense against the invasion of transferable DNA elements at the cost of reduced virulence. Further study on the mechanism will help us understand how it provides adaptive phenotypes to host evolution.

Biodegradation of kraft lignin by newly isolated Klebsiella pneumoniae, Pseudomonas putida, and Ochrobactrum tritici strains.

Bacterial systems have drawn an increasing amount of attention on lignin valorization due to their rapid growth and powerful environmental adaptability. In this study, Klebsiella pneumoniae NX-1, Pseudomonas putida NX-1, and Ochrobactrum tritici NX-1 with ligninolytic potential were isolated from leaf mold samples. Their ligninolytic capabilities were determined by measuring (1) the cell growth on kraft lignin as the sole carbon source, (2) the decolorization of kraft lignin and lignin-mimicking dyes, (3) the micro-morphology changes and transformations of chemical groups in kraft lignin, and (4) the ligninolytic enzyme activities of these three isolates. To the best of our knowledge, this is the first report that Ochrobactrum tritici species can depolymerize and metabolize lignin. Moreover, laccase, lignin peroxidase, and Mn-peroxidase showed high activities in P. putida NX-1. Due to their excellent ligninolytic capabilities, these three bacteria are important supplements to ligninolytic bacteria library and could be valuable in lignin valorization.

Integrated bioethanol production from mixtures of corn and corn stover.

Conversion of lignocellulosic biomass, such as corn stover (CS), to ethanol has encountered issues of inhibition from degradation products, low ethanol titer and low ethanol productivity. This work integrated CS into corn ethanol process for effective conversion. CS was pretreated using either dilute alkali or dilute acid pretreatment. The pretreated CS was enzymatically hydrolyzed and then mixed with liquefied corn for ethanol fermentation. Fermentation strains, substrate mixing ratios and fed-batch strategy were investigated. The mixture of alkali pretreated CS and corn at solids loadings of 10% and 20%, respectively, resulted in 92.30 g/L ethanol. Ethanol titer was further improved to 96.43 g/L with a fed-batch strategy. The mixture of dilute acid pretreated CS and corn achieved a better performance, leading to 104.9 g/L ethanol with 80.47% ethanol yield and a productivity as high as 2.19 g/L/h. This work demonstrated effective conversion of CS and corn together to ethanol.