N6-Adenine DNA methyltransferases associated with some Type I and Type III restriction-modification (R-M) systems are able to undergo phase variation, randomly switching expression ON or OFF by varying the length of locus-encoded simple sequence repeats (SSRs). This variation of methyltransferase expression results in genome-wide methylation differences and global changes in gene expression. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria. A distinct switching system has also been described in Type I R-M systems, based on recombination-driven changes in hsdS genes, which dictate the DNA target site. In order to determine the prevalence of recombination-driven phasevarions, we generated a program called RecombinationRepeatSearch to interrogate REBASE and identify the presence and number of inverted repeats of hsdS downstream of Type I R-M loci. We report that 3.9% of Type I R-M systems have duplicated variable hsdS genes containing inverted repeats capable of phase variation. We report the presence of these systems in the major pathogens Enterococcus faecalis and Listeria monocytogenes, which could have important implications for pathogenesis and vaccine development. These data suggest that in addition to SSR-driven phasevarions, many bacteria have independently evolved phase-variable Type I R-M systems via recombination between multiple, variable hsdS genes. IMPORTANCE Many bacterial species contain DNA methyltransferases that have random on/off switching of expression. These systems, called phasevarions (phase-variable regulons), control the expression of multiple genes by global methylation changes. In every previously characterized phasevarion, genes involved in pathobiology, antibiotic resistance, and potential vaccine candidates are randomly varied in their expression, commensurate with methyltransferase switching. Our systematic study to determine the extent of phasevarions controlled by invertible Type I R-M systems will provide valuable information for understanding how bacteria regulate genes and is key to the study of physiology, virulence, and vaccine development; therefore, it is critical to identify and characterize phase-variable methyltransferases controlling phasevarions.

Over recent years several examples of randomly switching methyltransferases, associated with Type III restriction-modification (R-M) systems, have been described in pathogenic bacteria. In every case examined, changes in simple DNA sequence repeats result in variable methyltransferase expression and result in global changes in gene expression, and differentiation of the bacterial cell into distinct phenotypes.These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria, with 17.4% of Type III R-M system containing simple DNA sequence repeats. A distinct, recombination-driven random switching system has also been described in Streptococci in Type I R-M systems that also regulate gene expression. Here, we interrogate the most extensive and well-curated database of R-M systems, REBASE, by searching for all possible simple DNA sequence repeats in the hsdRMS genes that encode Type I R-M systems. We report that 7.9% of hsdS, 2% of hsdM, and of 4.3% of hsdR genes contain simple sequence repeats that are capable of mediating phase variation. Phase variation of both hsdM and hsdS genes will lead to differential methyltransferase expression or specificity, and thereby the potential to control phasevarions. These data suggest that in addition to well characterized phasevarions controlled by Type III mod genes, and the previously described Streptococcal Type I R-M systems that switch via recombination, approximately 10% of all Type I R-M systems surveyed herein have independently evolved the ability to randomly switch expression via simple DNA sequence repeats. K E Y W O R D Sbacterial pathogenesis, epigenetics, phase variation, phasevarion, R-M systems | 1039 ATACK eT Al.

To investigate responses of milk protein synthesis and mammary AA metabolism to a graded decrease of postruminal Lys supply, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h and then received a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period; a mammary biopsy was performed in the last hour. Treatments were graded decrease of lysine content in the infusate to 100 (complete), 60, 30, or 0% as in casein. Lysine-removed infusions linearly decreased milk yield, tended to decrease lactose yield, and tended to increase milk fat to protein ratio. Milk protein content and yield were linearly decreased by graded Lys deficiency. Mammary Lys uptake was concomitantly decreased, but linear regression analysis found no significant relationship between mammary Lys uptake and milk protein yield. Treatments had no effects on phosphorylation levels of the downstream proteins measured in the mammalian target or rapamycin pathway except for a tended quadratic effect on that of eukaryotic initiation factor 2, which was increased and then decreased by graded Lys deficiency. Removal of Lys from the infusate linearly increased circulating glucagon and glucose. Removal of Lys from the infusate linearly decreased arterial and venous concentrations of Lys. Treatments also had a significant quadratic effect on venous Lys, suggesting mechanisms to stabilize circulating Lys at a certain range. The 2 infusions partially removing Lys resulted in a similar 20% decrease, whereas the 0% Lys infusion resulted in an abrupt 70% decrease in mammary Lys uptake compared with that of the full-AA mixture infusion. Consistent with the abrupt decrease, mammary Lys uptake-to-output ratio decreased from 2.2 to 0.92, suggesting catabolism of Lys in the mammary gland could be completely prevented when the animal faced severe Lys deficiency. Mammary blood flow was linearly increased, consistent with the linearly increased circulating nitric oxide by graded Lys deficiency, indicating mechanisms to ensure the priority of the mammary gland in acquiring AA for milk protein synthesis. Infusions with Lys removed increased mammary clearance rate of Lys numerically by 2 to 3 fold. In conclusion, the decreased milk protein yield by graded Lys deficiency was mainly a result of the varied physiological status, as indicated by the elevated circulating glucagon and glucose, rather than a result of the decreased mammary Lys uptake or depressed signals in the mTOR pathway. Mechanisms of Lys deficiency to promote glucagon secretion and mammary blood flow and glucagon to depress milk protein synthesis need to be clarified by future studies.

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that contain duplicated, variable hsdS specificity genes that randomly switch 2 methyltransferase specificity by recombination. 3 4 5 Abstract 29 N 6 -adenine DNA methyltransferases associated with some Type I and Type III restriction-30 modification (R-M) systems are able to randomly switch expression by variation in the length of 31 locus-encoded simple sequence repeats (SSRs). SSR tract-length variation causes ON/OFF 32 switching of methyltransferase expression, resulting in genome-wide methylation differences, and 33 global changes in gene expression. These epigenetic regulatory systems are called phasevarions, 34 phase-variable regulons, and are widespread in bacteria. A distinct switching system has also been 35 described in Type I R-M systems, based on recombination-driven changes in hsdS genes, which 36 dictate the DNA target site. In order to determine the prevalence of recombination-driven 37 phasevarions, we generated a program called RecombinationRepeatSearch to interrogate REBASE 38 and identify the presence and number of inverted repeats of hsdS downstream of Type I R-M loci. 39We report that 5.9% of Type I R-M systems have duplicated variable hsdS genes containing 40 inverted repeats capable of phase-variation. We report the presence of these systems in the major 41 pathogens Enterococcus faecalis and Listeria monocytogenes, which will have important 42 implications for pathogenesis and vaccine development. These data suggest that in addition to SSR-43 driven phasevarions, many bacteria have independently evolved phase-variable Type I R-M 44 systems via recombination between multiple, variable hsdS genes. 45 Importance 46Many bacterial species contain DNA methyltransferases that have random on/off switching of 47 expression. These systems called phasevarions (phase-variable regulons) control the expression of 48 multiple genes by global methylation changes. In every previously characterised phasevarion, genes 49 involved in pathobiology, antibiotic resistance, and potential vaccine candidates are randomly 50 varied in their expression, commensurate with methyltransferase switching. A systematic study to 51 determine the extent of phasevarions controlled by invertible Type I R-M systems has never before 52 been performed. Understanding how bacteria regulate genes is key to the study of physiology, 53 virulence, and vaccine development; therefore it is critical to identify and characterize phase-54 variable methyltransferases controlling phasevarions. 55 56