Preliminary Characterization of Phage-Like Particles from the Male-Killing Mollicute Spiroplasma poulsonii (an Endosymbiont of Drosophila).

Bacteriophages are vastly abundant, diverse, and influential, but with few exceptions (e.g. the Proteobacteria genera Wolbachia and Hamiltonella), the role of phages in heritable bacteria-arthropod interactions, which are ubiquitous and diverse, remains largely unexplored. Despite prior studies documenting phage-like particles in the mollicute Spiroplasma associated with Drosophila flies, genomic sequences of such phage are lacking, and their effects on the Spiroplasma-Drosophila interaction have not been comprehensively characterized. We used a density step gradient to isolate phage-like particles from the male-killing bacterium Spiroplasma poulsonii (strains NSRO and MSRO-Br) harbored by Drosophila melanogaster. Isolated particles were subjected to DNA sequencing, assembly, and annotation. Several lines of evidence suggest that we recovered phage-like particles of similar features (shape, size, DNA content) to those previously reported in Drosophila-associated Spiroplasma strains. We recovered three ~ 19 kb phage-like contigs (two in NSRO and one in MSRO-Br) containing 21-24 open reading frames, a read-alignment pattern consistent with circular permutation, and terminal redundancy (at least in NSRO). Although our results do not allow us to distinguish whether these phage-like contigs represent infective phage-like particles capable of transmitting their DNA to new hosts, their encoding of several typical phage genes suggests that they are at least remnants of functional phage. We also recovered two smaller non-phage-like contigs encoding a known Spiroplasma toxin (Ribosome Inactivating Protein; RIP), and an insertion element, suggesting that they are packaged into particles. Substantial homology of our particle-derived contigs was found in the genome assemblies of members of the Spiroplasma poulsonii clade.

Bacteriophage P22 SieA-mediated superinfection exclusion.

Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. Salmonella phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the c2 (repressor), gtrABC, sieA, and sieB genes. Here we report that the P22-encoded SieA protein is necessary and sufficient for exclusion by the SieA system and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants that overcome the SieA block were isolated, and they have amino acid changes in the C-terminal regions of the gene 16 and 20 encoded ejection proteins. Three different single-amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in phage target specificity. Our data strongly suggest a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.IMPORTANCEThe ongoing evolutionary battle between bacteria and the viruses that infect them is a critical feature of bacterial ecology on Earth. Viruses can kill bacteria by infecting them. However, when their chromosomes are integrated into a bacterial genome as a prophage, viruses can also protect the host bacterium by expressing genes whose products defend against infection by other viruses. This defense property is called "superinfection exclusion." A significant fraction of bacteria harbor prophages that encode such protective systems, and there are many different molecular strategies by which superinfection exclusion is mediated. This report is the first to describe the mechanism by which bacteriophage P22 SieA superinfection exclusion protein protects its host bacterium from infection by other P22-like phages. The P22 prophage-encoded inner membrane SieA protein prevents infection by blocking transport of superinfecting phage DNA across the inner membrane during injection.

Unraveling the role of the C-terminal helix turn helix of the coat-binding domain of bacteriophage P22 scaffolding protein.

Many viruses encode scaffolding and coat proteins that co-assemble to form procapsids, which are transient precursor structures leading to progeny virions. In bacteriophage P22, the association of scaffolding and coat proteins is mediated mainly by ionic interactions. The coat protein-binding domain of scaffolding protein is a helix turn helix structure near the C terminus with a high number of charged surface residues. Residues Arg-293 and Lys-296 are particularly important for coat protein binding. The two helices contact each other through hydrophobic side chains. In this study, substitution of the residues of the interface between the helices, and the residues in the ß-turn, by aspartic acid was used examine the importance of the conformation of the domain in coat binding. These replacements strongly affected the ability of the scaffolding protein to interact with coat protein. The severity of the defect in the association of scaffolding protein to coat protein was dependent on location, with substitutions at residues in the turn and helix 2 causing the most significant effects. Substituting aspartic acid for hydrophobic interface residues dramatically perturbs the stability of the structure, but similar substitutions in the turn had much less effect on the integrity of this domain, as determined by circular dichroism. We propose that the binding of scaffolding protein to coat protein is dependent on angle of the ß-turn and the orientation of the charged surface on helix 2. Surprisingly, formation of the highly complex procapsid structure depends on a relatively simple interaction.

Surfing injuries: A US epidemiological study from 2009-2020.

BACKGROUND: The sport of surfing has grown exponentially. Early studies of surfing injuries are outdated as newer and more accessible surf technology has become available. This study's goal was to describe surfing injury patterns, incidence, and disposition of pediatric and adult surfers. STUDY DESIGN: A retrospective review of surfing injuries from 2009 to 2020 of adult (>18 years of age) and pediatric (<18 years of age) patients was performed using the National Electronic Injury Surveillance System (NEISS) database. The consumer product code 1261 (Surfing) was used to identify injury patterns. Chi-squared test was performed on all categorical variables. Logistic regression was used on significant variables from the frequency tables. All analysis was performed with R-statistical programming software. RESULTS: There was an overall decreasing trend of surfing injuries over time. Injuries for both adult and pediatric patients tended to occur most within the summer season (p<0.001). The odds of an adult surfing injury victim being male is 2.89 (95% CI 1.87-4.44). The head/neck/face were the most injured body part in both groups. The pediatric group had a significantly higher rate of concussions at 6.5% compared to the adult group at 3.2%. Overall, the most common injury type was to the skin (p<0.001). Disposition between groups were similar with most patients being discharged home. Mortality was rare with three reported fatalities in the adult group and none in the pediatric group. CONCLUSION: The incidence of surfing injuries is continuing to decline despite more people surfing, revealing the improved safety of the sport over the last decade. Head/neck/face injuries are common injury locations, and pediatric surfers are particularly at increased risk of concussions. Continued education, usage of safety equipment such as protective headgear, and awareness of injury patterns, could further lessen potential injuries.

Small Rotational Changes of the Chest on Portable Chest Films Produce Large Displacements of Test Projectiles in the Cardiac Box.

The cardiac box has been used to guide the management of trauma patients for decades. However, improper imaging can lead to erroneous assumptions about operative management in this patient population. In this study, we used a thoracic model to demonstrate imaging's effect on chest radiography. The data demonstrate that even small changes in rotation can lead to large discrepancies in results.

Educating Residents in Abdominal Wall Closure: An Overview.

Background and Aims: Incisional hernia prevention has become an important concept for surgeons operating on the abdominal wall. Several techniques have been proposed to help decrease incisional hernia formation with suture closure of the abdominal wall being one of the cornerstones. Technical details that have been reported to decrease incisional hernia rates include achieving a 4:1 Suture to Wound length ratio and the use of a small bites technique. Despite evidence to support many of these techniques there appears to be a gap in practice patterns amongst practicing surgeons. Introducing and promoting these principles in surgical residency may help to close this gap. This paper reviews our experience with surgical training for abdominal wall closures at our institution. Materials and Methods: Programs and projects related to abdominal wall closure were reviewed from our institution from 2010-Present. Type of project, intervention, and impact on education was evaluated and summarized. Results: Seven projects were identified relating to surgical training and abdominal wall closure. Three projects dealt with skills training using an abdominal wall simulation model and related to suturing techniques. Two projects were clinical studies focused on suture to wound length ratios and improving outcomes with this variable in a residency training program. Two projects dealt with models relating to abdominal wall closure and education. Conclusion: Implementation of educational programs in surgical residency programs can lead to improvements in technique and knowledge around abdominal wall closure and help in research endeavors.

Bacteriophage P22 SieA mediated superinfection exclusion.

Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. Salmonella phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the c2 (repressor), gtrABC, sieA, and sieB genes. Here we report that the P22-encoded SieA protein is the only phage protein required for exclusion by the SieA system, and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants were isolated that overcome the SieA block, and they have amino acid changes in the C-terminal regions of the gene 16 and 20 encoded ejection proteins. Three different single amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in target specificity. Our data are consistent with a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.

Molecular Architecture of Salmonella Typhimurium Virus P22 Genome Ejection Machinery.

Bacteriophage P22 is a prototypical member of the Podoviridae superfamily. Since its discovery in 1952, P22 has become a paradigm for phage transduction and a model for icosahedral viral capsid assembly. Here, we describe the complete architecture of the P22 tail apparatus (gp1, gp4, gp10, gp9, and gp26) and the potential location and organization of P22 ejection proteins (gp7, gp20, and gp16), determined using cryo-EM localized reconstruction, genetic knockouts, and biochemical analysis. We found that the tail apparatus exists in two equivalent conformations, rotated by ∼6° relative to the capsid. Portal protomers make unique contacts with coat subunits in both conformations, explaining the 12:5 symmetry mismatch. The tail assembles around the hexameric tail hub (gp10), which folds into an interrupted ß-propeller characterized by an apical insertion domain. The tail hub connects proximally to the dodecameric portal protein and head-to-tail adapter (gp4), distally to the trimeric tail needle (gp26), and laterally to six trimeric tailspikes (gp9) that attach asymmetrically to gp10 insertion domain. Cryo-EM analysis of P22 mutants lacking the ejection proteins gp7 or gp20 and biochemical analysis of purified recombinant proteins suggest that gp7 and gp20 form a molecular complex associated with the tail apparatus via the portal protein barrel. We identified a putative signal transduction pathway from the tailspike to the tail needle, mediated by three flexible loops in the tail hub, that explains how lipopolysaccharide (LPS) is sufficient to trigger the ejection of the P22 DNA in vitro.

Intravirion DNA Can Access the Space Occupied by the Bacteriophage P22 Ejection Proteins.

Tailed double-stranded DNA bacteriophages inject some proteins with their dsDNA during infection. Phage P22 injects about 12, 12, and 30 molecules of the proteins encoded by genes 7, 16 and 20, respectively. After their ejection from the virion, they assemble into a trans-periplasmic conduit through which the DNA passes to enter the cytoplasm. The location of these proteins in the virion before injection is not well understood, although we recently showed they reside near the portal protein barrel in DNA-filled heads. In this report we show that when these proteins are missing from the virion, a longer than normal DNA molecule is encapsidated by the P22 headful DNA packaging machinery. Thus, the ejection proteins occupy positions within the virion that can be occupied by packaged DNA when they are absent.

Use of regional analgesia and risk of delirium in older adults with multiple rib fractures: An Eastern Association for the Surgery of Trauma multicenter study.

BACKGROUND: Single-center data demonstrates that regional analgesia (RA) techniques are associated with reduced risk of delirium in older patients with multiple rib fractures. We hypothesized that a similar effect between RA and delirium would be identified in a larger cohort of patients from multiple level I trauma centers. METHODS: Retrospective data from seven level I trauma centers were collected for intensive care unit (ICU) patients 65 years or older with ≥3 rib fractures from January 2012 to December 2016. Those with a head and/or spine injury Abbreviated Injury Scale (AIS) score of ≥ 3 or a history of dementia were excluded. Delirium was defined as one positive Confusion Assessment Method for the Intensive Care Unit score in the first 7 days of ICU care. Poisson regression with robust standard errors was used to determine the association of RA (thoracic epidural or paravertebral catheter) with delirium incidence. RESULTS: Data of 574 patients with a median age of 75 years (interquartile range [IQR], 69-83), Injury Severity Score of 14 (IQR, 11-18), and ICU length of stay of 3 days (IQR, 2-6 days) were analyzed. Among the patients, 38.9% were women, 15.3% were non-White, and 31.4% required a chest tube. Regional analgesia was used in 19.3% patients. Patient characteristics did not differ by RA use; however, patients with RA had more severe chest injury (chest AIS, flail segment, hemopneumothorax, thoracostomy tube). In univariate analysis, there was no difference in the likelihood of delirium between the RA and no RA groups (18.9% vs. 23.8% p = 0.28). After adjusting for age, sex, Injury Severity Score, maximum chest AIS, thoracostomy tube, ICU length of stay, and trauma center, RA was associated with reduced risk of delirium (incident rate ratio [IRR], 0.65; 95% confidence interval [CI], 0.44-0.94) but not with in-hospital mortality (IRR, 0.42; 95% CI, 0.14-1.26) or respiratory complications (IRR, 0.70; 95% CI, 0.42-1.16). CONCLUSION: In this multicenter cohort of injured older adults with multiple rib fractures, RA use was associated with a 35% lower risk of delirium. Further studies are needed to standardize protocols for optimal pain management and prevention of delirium in older adults with severe thoracic injury. LEVEL OF EVIDENCE: Therapeutic, level IV; Epidemiologic, level III.

Genome Sequence of Salmonella enterica Serovar Typhimurium Bacteriophage MG40.

We report the complete genome sequence of P22-like Salmonella enterica serovar Typhimurium phage MG40, whose prophage repressor specificity is different from that of other known temperate phages.

Complete Genome Sequence of Serratia Phage Muldoon.

Serratia marcescens is a ubiquitous Gram-negative bacterium that is linked with emerging opportunistic infections. In this report, we describe the isolation and annotation of an S. marcescens myophage called Muldoon. Related to T4-like phages, such as Serratia phage PS2, Muldoon contains 257 predicted protein-coding genes and 4 tRNA genes.

Complete Genome Sequence of Serratia marcescens Siphophage Slocum.

Serratia marcescens is a ubiquitous Gram-negative opportunistic pathogen. This announcement describes the isolation and genome annotation of S. marcescens T5-like siphophage Slocum. Terminal repeats, 170 protein-coding genes, and 23 tRNAs were predicted in the 112,436-bp Slocum genome.

Complete Genome Sequence of Serratia marcescens Podophage Pila.

Multidrug-resistant Serratia marcescens strains cause serious nosocomial infections in humans. Here, we present the annotated genome sequence of S. marcescens podophage Pila. Similar to its closest relative, Enterobacteria phage T7, Pila has a 38,678-bp genome, predicted to encode 51 protein-coding genes, and contains 148-bp direct terminal repeats.

Complete Genome Sequence of Stenotrophomonas Phage Mendera.

Stenotrophomonas maltophilia is an emerging opportunistic human pathogen. In this report, we describe the isolation and genomic annotation of the S. maltophilia-infecting bacteriophage Mendera. A myophage of 159,961 base pairs, Mendera is T4-like and related most closely to Stenotrophomonas phage IME-SM1.

Complete Genome Sequence of Stenotrophomonas maltophilia Myophage Moby.

Stenotrophomonas maltophilia is a prevalent nosocomial pathogen with multidrug resistance. Here, we describe the complete genome of S. maltophilia myophage Moby, which shares characteristics with Enterobacteria phage T4 and is closely related to Stenotrophomonas phage IME-SM1. Moby has a 159,365-bp genome with 271 predicted protein-coding genes and 24 predicted tRNAs.

Genome Sequence of Escherichia coli Tailed Phage Utah.

Escherichia coli bacteriophage Utah is a member of the chi-like tailed phage cluster in the Siphoviridae family. We report here the complete 59,024-bp sequence of the genome of phage Utah.

Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging.

UNLABELLED: The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three "ejection proteins" (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins' locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection. IMPORTANCE: While it has long been appreciated that capsids serve as delivery vehicles for viral genomes, there is now growing awareness that viruses also deliver proteins into their host cells. P22 has three such proteins (ejection proteins [E-proteins]), whose initial locations in the virion have remained unknown despite their copious amounts (total of 2.5 MDa). This study succeeded in localizing them by the novel technique of bubblegram imaging. The P22 E-proteins are seen to be distributed around the orifice of the portal barrel. Interestingly, this barrel, 15 nm long in a crystal structure, is only about half as long in situ: the remaining, disordered, portion appears to present binding sites for E-proteins. These observations document a spectacular example of a regulatory order-disorder transition in a supramolecular system and demonstrate the potential of bubblegram imaging to map the components of other viruses as well as cellular complexes.

Genome Sequence of Salmonella Phage 9NA.

The virulent double-stranded DNA (dsDNA) bacteriophage 9NA infects Salmonella enterica serovar Typhimurium and has a long noncontractile tail. We report its complete 52,869-bp genome sequence. Phage 9NA and two closely related S. enterica serovar Newport phages represent a tailed phage type whose molecular lifestyle has not yet been studied in detail.

Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor.

Bacteriophage Sf6 DNA packaging series initiate at many locations across a 2kbp region. Our in vivo studies show that Sf6 small terminase subunit (TerS) protein recognizes a specific packaging (pac) site near the center of this region, that this site lies within the portion of the Sf6 gene that encodes the DNA-binding domain of TerS protein, that this domain of the TerS protein is responsible for the imprecision in Sf6 packaging initiation, and that the DNA-binding domain of TerS must be covalently attached to the domain that interacts with the rest of the packaging motor. The TerS DNA-binding domain is self-contained in that it apparently does not interact closely with the rest of the motor and it binds to a recognition site that lies within the DNA that encodes the domain. This arrangement has allowed the horizontal exchange of terS genes among phages to be very successful.