Project description:Bacteriophage infection of Lactococcus lactis strains used in the manufacture of fermented milk products is a major threat for the dairy industry. A greater understanding of the global molecular response of the bacterial host following phage infection has the potential to identify new targets for the design of phage control measures for biotechnological processes. In this study, we have used whole-genome oligonucleotide microarrays to gain insights into the genomic intelligence driving the instinctive response of L. lactis subsp. lactis IL1403 to the onset of a challenge with the lytic prolate-headed phage c2. Following phage adsorption, the bacterium differentially regulated the expression of 61 genes belonging to 14 functional categories, and mostly to cell envelope (12 genes), regulatory functions (11 genes), and carbohydrate metabolism (7 genes). The nature of the differentially regulated genes suggests the orchestration of a complex response involving induction of cell envelope stress proteins, D-alanylation of cell-wall lipoteichoic acids (LTAs), restoration of the proton motive force (PMF), and energy conservation. Increased D-alanylation of LTAs would act as an adsorption blocking mechanism, which we speculate may allow the survival of a small percent of the cell population when facing more realistic in vivo low titer-phage attacks. The modification of LTAs decoration in response to phage c2 adsorption also suggests these cell wall structures as possible primary receptors for this phage. Restoration of a physiological PMF is achieved by regulating the expression of genes affecting the two main components of the PMF, and serves to reverse a drastic depolarization of the host membrane caused by phage adsorption. Down-regulation of energy-consuming metabolic activities and a switch to anaerobic respiration helps the bacterium to save energy in order to sustain the PMF and the overall response to phage. We finally propose that the overall transcriptional response of L. lactis IL1403 to the phage stimuli is orchestrated by the concerted action of Phage Shock Proteins and of the bivalent transcriptional regulator SpxB following activation by the two-component system CesSR. To our knowledge, this represents the first detailed description in L. lactis, and probably in Gram-positive bacteria, of the molecular mechanisms involved in the host response to the membrane perturbation mediated by phage adsorption. Two-condition experiment: IL1403 vs. Bacteriophage c2-infected IL1403 cells. Biological replicates: 2 controls, 2 infected, independently grown and harvested. Two technical replicates per array.
Project description:Given the gut microbiota involve aging processing, we performed comparative analysis of gut bacteriophage between older and young subjects using next-generation sequencing (NGS). In our previous study, we found that the Ruminococcaceae is higher in aged subjects comparing to young one. To identify the bacteriophage targeting to the Ruminococcaceae, we also access the composition of phage in the Ruminococcaceae (ATCC, TSD-27) after incubated with human stool samples. The Lactobacillus (ATCC, LGG) targeting phage was used as the control. The virome sequencing analysis using NGS indicated that Myoviridae are high enrich in young subjects and predominate in TSD-27 targeting phage.
Project description:Bacteriophage lambda is one of the most extensively studied organisms, and has been a primary model for understanding basic modes of genetic regulation. Here we examine the progress of lambda gene expression during phage development by ribosome profiling, and thereby provide a very high resolution view of lambda gene expression. The known genes are expressed in a predictable fashion, authenticating the analysis. But many previously unappreciated potential open reading frames become apparent in the expression analysis, revealing an unexpected complexity in the pattern of lambda gene function. We chose temperature induction of the classic cI857 repressor mutation in a lysogen of E. coli MG1655 in order to synchronize the lytic process, sampling the lysogen and control non-lysogen both before and 2, 5, 10, and 20 minutes after shifting the temperature from 32M-BM-0 to 42M-BM-0. The last sample time was chosen to be before any significant cell lysis, but during the later stages of lytic gene expression. Total protected nucleotides within open reading frames were summed to determine the density of translation of each reading frame. We take this number to indicate the overall rate of translation, although obviously this assumes that pauses in translation do not excessively affect the overall rate. Since the expression level is not normalized for the copy number of the replicating phage DNA, it thus encompasses both the effect of DNA template availability on mRNA synthesis and the efficiency of utilization of messengers by ribosomes.
Project description:YerA41 is a myoviridae bacteriophage that was originally isolated due its ability to infect Yersinia ruckeri bacteria, the causative agent of enteric redmouth disease of salmonid fish. Several attempts to determine its genomic DNA sequence using traditional and next generation sequencing technologies failed, indicating that the phage genome is modified such way that it is an unsuitable template for PCR amplification and sequencing. To determine the YerA41 genome sequence we isolated RNA from phage-infected Y. ruckeri cells at different time points post-infection, and sequenced it. The host-genome specific reads were substracted and de novo assembly was performed on the unaligned reads.
Project description:Antibiotic use can lead to expansion of multi-drug resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, we describe a Klebsiella PhageBank that enables the rapid design of antimicrobial bacteriophage cocktails to treat multi-drug resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K. pneumoniae, we identified host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank and experimental evolution strategies, we formulated combinations of phages that minimize the occurrence of phage resistance in vitro. Optimized bacteriophage cocktails selectively suppressed the burden of multi-drug resistant K. pneumoniae in the mouse gut microbiome and drove bacterial populations to lose key virulence factors that act as phage receptors. Further, phage-mediated diversification of bacterial populations in the gut enabled co-evolution of phage variants with higher virulence and a broader host range. Altogether, the Klebsiella PhageBank represents a roadmap for both phage researchers and clinicians to enable phage therapy against a critical multidrug-resistant human pathogen.
Project description:The present work describes LC-ESI-MS/MS analyses of tryptic digestion peptides from phages that infect Staphylococcus aureus-causing mastitis, and isolated from dairy products. A total of 1935 non-redundant peptides belonging to 1282 proteins were identified and analyzed. Among them, 80 staphylococcal peptides from phages were confirmed. These peptides belong to proteins such as phage repressors, structural phage proteins, uncharacterized phage proteins and complement inhibitors. Moreover, of the phage origin peptides found, eighteen of them were specific to S. aureus strains. These diagnostic peptides could be useful for the identification and characterization of S. aureus strains that cause mastitis. Furthermore, a study of bacteriophage phylogeny and the relationship among the identified phage peptides and the bacteria they infect was also performed. The results show the specific peptides which are present in closely related phages, and the existing links between bacteriophage phylogeny and the respective Staphylococcus spp. infected.
Project description:Bacteriophage infection of Lactococcus lactis strains used in the manufacture of fermented milk products is a major threat for the dairy industry. A greater understanding of the global molecular response of the bacterial host following phage infection has the potential to identify new targets for the design of phage control measures for biotechnological processes. In this study, we have used whole-genome oligonucleotide microarrays to gain insights into the genomic intelligence driving the instinctive response of L. lactis subsp. lactis IL1403 to the onset of a challenge with the lytic prolate-headed phage c2. Following phage adsorption, the bacterium differentially regulated the expression of 61 genes belonging to 14 functional categories, and mostly to cell envelope (12 genes), regulatory functions (11 genes), and carbohydrate metabolism (7 genes). The nature of the differentially regulated genes suggests the orchestration of a complex response involving induction of cell envelope stress proteins, D-alanylation of cell-wall lipoteichoic acids (LTAs), restoration of the proton motive force (PMF), and energy conservation. Increased D-alanylation of LTAs would act as an adsorption blocking mechanism, which we speculate may allow the survival of a small percent of the cell population when facing more realistic in vivo low titer-phage attacks. The modification of LTAs decoration in response to phage c2 adsorption also suggests these cell wall structures as possible primary receptors for this phage. Restoration of a physiological PMF is achieved by regulating the expression of genes affecting the two main components of the PMF, and serves to reverse a drastic depolarization of the host membrane caused by phage adsorption. Down-regulation of energy-consuming metabolic activities and a switch to anaerobic respiration helps the bacterium to save energy in order to sustain the PMF and the overall response to phage. We finally propose that the overall transcriptional response of L. lactis IL1403 to the phage stimuli is orchestrated by the concerted action of Phage Shock Proteins and of the bivalent transcriptional regulator SpxB following activation by the two-component system CesSR. To our knowledge, this represents the first detailed description in L. lactis, and probably in Gram-positive bacteria, of the molecular mechanisms involved in the host response to the membrane perturbation mediated by phage adsorption.
Project description:This microarray serie represents the complete gene expression study of bacteriophage BFK 20 during the infection of its host Brevibacterium flavum CCM 251. Gene expression was measured at fourteen time intervals (0, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50, 60, 80 and 120 min) during phage infection according to proposed loop. Each sample was directly compared to the previous and to the next sample (point of total RNA isolation).