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ABSTRACT The effort to discover novel phages infecting Staphylococcus epidermidis contributes to both the development of phage therapy and the expansion of genome-based phage phylogeny. Here, we report the genome of an S. epidermidis -infecting phage, Lacachita, and compare its genome with those of five other phages with high sequence identity. These phages represent a novel siphovirus genus, which was recently reported in the literature. The published member of this group was favorably evaluated as a phage therapeutic agent, but Lacachita is capable of transducing antibiotic resistance and conferring phage resistance to transduced cells. Members of this genus may be maintained within their host as extrachromosomal plasmid prophages, through stable lysogeny or pseudolysogeny. Therefore, we conclude that Lacachita may be temperate and members of this novel genus are not suitable for phage therapy. IMPORTANCE This project describes the discovery of a culturable bacteriophage infecting Staphylococcus epidermidis that is a member of a rapidly growing novel siphovirus genus. A member of this genus was recently characterized and proposed for phage therapy, as there are few phages currently available to treat S. epidermidis infections. Our data contradict this, as we show Lacachita is capable of moving DNA from one bacterium to another, and it may be capable of maintaining itself in a plasmid-like state in infected cells. These phages’ putative plasmid-like extrachromosomal state appears to be due to a simplified maintenance mechanism found in true plasmids of Staphylococcus and related hosts. We suggest Lacachita and other identified members of this novel genus are not suitable for phage therapy. 

ABSTRACT The annotated whole-genome sequences of five cultured phietaviruses infecting Staphylococcus aureus are presented. They are closely related to prophages that were previously sequenced as part of S. aureus genomes. 

ABSTRACT Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins. Genes coding for Fe-S proteins can be found in nearly every sequenced genome. Fe-S proteins have a wide variety of functions, and therefore, defective assembly of Fe-S proteins results in cell death or global metabolic defects. Compared to alternative essential cellular processes, there is less known about Fe-S cluster synthesis and Fe-S protein maturation. Moreover, new factors involved in Fe-S protein assembly continue to be discovered. These facts highlight the growing need to develop a deeper biological understanding of Fe-S cluster synthesis, holo-protein maturation, and Fe-S cluster repair. Here, we outline bacterial strategies used to assemble Fe-S proteins and the genetic regulation of these processes. We focus on recent and relevant findings and discuss future directions, including the proposal of using Fe-S protein assembly as an antipathogen target. 

ABSTRACT Mercury (Hg) is a widely distributed, toxic heavy metal with no known cellular role. Mercury toxicity has been linked to the production of reactive oxygen species (ROS), but Hg does not directly perform redox chemistry with oxygen. How exposure to the ionic form, Hg(II), generates ROS is unknown. Exposure of Thermus thermophilus to Hg(II) triggered ROS accumulation and increased transcription and activity of superoxide dismutase (Sod) and pseudocatalase (Pcat); however, Hg(II) inactivated Sod and Pcat. Strains lacking Sod or Pcat had increased oxidized bacillithiol (BSH) levels and were more sensitive to Hg(II) than the wild type. The Δ bshA Δ sod and Δ bshA Δ pcat double mutant strains were as sensitive to Hg(II) as the Δ bshA strain that lacks bacillithiol, suggesting that the increased sensitivity to Hg(II) in the Δ sod and Δ pcat mutant strains is due to a decrease of reduced BSH. Treatment of T. thermophilus with Hg(II) decreased aconitase activity and increased the intracellular concentration of free Fe, and these phenotypes were exacerbated in Δ sod and Δ pcat mutant strains. Treatment with Hg(II) also increased DNA damage. We conclude that sequestration of the redox buffering thiol BSH by Hg(II), in conjunction with direct inactivation of ROS-scavenging enzymes, impairs the ability of T. thermophilus to effectively metabolize ROS generated as a normal consequence of growth in aerobic environments. IMPORTANCE Thermus thermophilus is a deep-branching thermophilic aerobe. It is a member of the Deinococcus - Thermus phylum that, together with the Aquificae , constitute the earliest branching aerobic bacterial lineages; therefore, this organism serves as a model for early diverged bacteria (R. K. Hartmann, J. Wolters, B. Kröger, S. Schultze, et al., Syst Appl Microbiol 11:243–249, 1989, https://doi.org/10.1016/S0723-2020(89)80020-7 ) whose natural heated habitat may contain mercury of geological origins (G. G. Geesey, T. Barkay, and S. King, Sci Total Environ 569-570:321–331, 2016, https://doi.org/10.1016/j.scitotenv.2016.06.080 ). T. thermophilus likely arose shortly after the oxidation of the biosphere 2.4 billion years ago. Studying T. thermophilus physiology provides clues about the origin and evolution of mechanisms for mercury and oxidative stress responses, the latter being critical for the survival and function of all extant aerobes. 

To persist within the host and cause disease, Staphylococcus aureus relies on its ability to precisely fine-tune virulence factor expression in response to rapidly-changing environments. During an unbiased transposon mutant screen, we observed that disruption of the two-gene operon, yjbIH , resulted in decreased pigmentation and aureolysin activity relative to the wild-type strain. Further analyses revealed that YjbH, a predicted thioredoxin-like oxidoreductase, is mostly responsible for the observed yjbIH mutant phenotypes, though a minor role exists for the putative truncated hemoglobin YjbI. These differences were due to significantly decreased expression of crtOPQMN and aur . Previous studies found that YjbH targets the disulfide- and oxidative-stress responsive regulator Spx for degradation by ClpXP. The absence of yjbH or yjbI resulted in altered sensitivities to nitrosative and oxidative stress and iron deprivation. Additionally, aconitase activity was altered in the yjbH and yjbI mutant strains. Decreased pigmentation and Aur activity in the yjbH mutant was found to be Spx-dependent. Lastly, we used a murine sepsis model to determine the effect of the yjbIH deletion on pathogenesis and found that the mutant was better able to colonize the kidneys and spleens during an acute infection than the wild-type strain. These studies identify changes in pigmentation and protease activity in response to YjbIH and are the first to show a role for these proteins during infection. 

S. aureusUSA300 isolates utilize thecopBLandcopAZgene products to prevent Cu intoxication. We created and examined a ΔcopAZΔcopBLmutant strain (cop‐). Thecop‐ strain was sensitive to Cu and accumulated intracellular Cu. We screened a transposon (Tn) mutant library in thecop‐ background and isolated strains with Tn insertions in themntABCoperon that permitted growth in the presence of Cu. The mutations were inmntAand they were recessive. Under the growth conditions utilized, MntABC functioned in manganese (Mn) import. When cultured with Cu, strains containing amntA::Tnaccumulated less Cu than the parent strain. Mn(II) supplementation improved growth whencop‐ was cultured with Cu and this phenotype was dependent upon the presence of MntR, which is a repressor ofmntABCtranscription. A ΔmntRstrain had an increased Cu load and decreased growth in the presence of Cu, which was abrogated by the introduction ofmntA::Tn. Over‐expression ofmntABCincreased cellular Cu load and sensitivity to Cu. The presence of amntA::Tnmutation protected iron‐sulfur (FeS) enzymes from inactivation by Cu. The data presented are consistent with a model wherein defective MntABC results in decreased cellular Cu accumulation and protection to FeS enzymes from Cu poisoning.

 

Mercury (Hg) is a highly toxic and widely distributed heavy metal, which some Bacteria and Archaea detoxify by the reduction of ionic Hg (Hg[II]) to the elemental volatile form, Hg(0). This activity is specified by themeroperon. Themeroperon of the deeply branching thermophileThermus thermophilusHB27 encodes for, anO‐acetyl‐l‐homoacetylserine sulfhydrylase (Oah2), a transcriptional regulator (MerR), a hypothetical protein (hp) and a mercuric reductase (MerA). Here, we show that this operon has two convergently expressed and differentially regulated promoters. An upstream promoter, P_oah, controls the constitutive transcription of the entire operon and a second promoter (P_mer), located withinmerR, is responsive to Hg(II). In the absence of Hg(II), the transcription ofmerAis basal and when Hg(II) is present,merAtranscription is induced. This response to Hg(II) is controlled by MerR and genetic evidence suggests that MerR acts as a repressor and activator of P_mer. When the wholemerR, including P_mer, is removed,merAis transcribed from P_oahindependently of Hg(II). These results suggest that the transcriptional regulation ofmerinT.thermophilusis both similar to, and different from, the well‐documented regulation of proteobacterialmersystems, possibly representing an early step in the evolution ofmer‐operon regulation.