Structure of Full-Length SMC and Rearrangements Required for Chromosome Organization.

Multi-subunit SMC complexes control chromosome superstructure and promote chromosome disjunction, conceivably by actively translocating along DNA double helices. SMC subunits comprise an ABC ATPase "head" and a "hinge" dimerization domain connected by a 49 nm coiled-coil "arm." The heads undergo ATP-dependent engagement and disengagement to drive SMC action on the chromosome. Here, we elucidate the architecture of prokaryotic Smc dimers by high-throughput cysteine cross-linking and crystallography. Co-alignment of the Smc arms tightly closes the interarm space and misaligns the Smc head domains at the end of the rod by close apposition of their ABC signature motifs. Sandwiching of ATP molecules between Smc heads requires them to substantially tilt and translate relative to each other, thereby opening up the Smc arms. We show that this mechanochemical gating reaction regulates chromosome targeting and propose a mechanism for DNA translocation based on the merging of DNA loops upon closure of Smc arms.

The 1.7 Å crystal structure of the C5a peptidase from Streptococcus agalactiae (ScpB) reveals an active site competent for catalysis.

A 1.7 Å structure is presented for an active form of the virulence factor ScpB, the C5a peptidase from Streptococcus agalactiae. The previously reported structure of the ScpB active site mutant exhibited a large separation (~20 Å) between the catalytic His and Ser residues. Significant differences are observed in the catalytic domain between the current and mutant ScpB structures resulting with a high RMSDCα (4.6 Å). The fold of the active form of ScpB is nearly identical to ScpA (RMSDCα 0.2 Å), the C5a-peptidase from Streptococcus pyogenes. Both ScpA and ScpB have comparable activity against human C5a, indicating neither enzyme require host proteins for C5a-ase activity. These studies are a first step in resolving reported differences in the specificities of these enzymes.

Digital gene expression analysis in the liver of ScpB-vaccinated and Streptococcus agalactiae-challenged Nile tilapia.

In recent years, streptococcal diseases have severely threatened the development of tilapia aquaculture, but effective prevention and control methods have not yet been established. To understand the immune responses of vaccinated Nile tilapia (Oreochromis niloticus), digital gene expression (DGE) technology was applied in this study to detect the gene expression profile of the Nile tilapia (O. niloticus) liver in response to ScpB (Streptococcal C5a peptidase from group B Streptococcus, ScpB) vaccination and a Streptococcus agalactiae-challenge. The control and the ScpB-vaccinated Nile tilapia yielded a total of 25,788,734 and 27,088,598 clean reads, respectively. A total of 1234 significant differentially expressed unigenes were detected (P < 0.05), of which 236 were significantly up-regulated, and 269 were significantly down-regulated (P < 0.05, |fold|>2, FDR<0.05). Of the differentially expressed gene, the identified genes which were enriched using databases of GO and KEGG could be categorized into a total of 67 functional groups and were mapped to 153 signaling pathways including 15 immune-related pathways. The differentially expressed genes (TLR1, TLR2, TLR3, TLR5, TLR9, MyD88, C3, IL-1ß, IL-10) were detected in the expression profiles, and this was subsequently verified via quantitative real-time PCR (qPCR). The results of this study can serve as a basis for future research not only on the molecular mechanism of S. agalactiae invasion, but also on the anti-S. agalactiae mechanism in targeted tissues of Nile tilapia.

Evaluating the Analgesic Efficacy of Superficial Cervical Plexus Block for Head and Neck Surgeries: A Comparative Randomized Control Study.

INTRODUCTION: In the present study, the advantages of superficial cervical plexus block (SCPB) were evaluated using a landmark-guided method. Our primary aim was to evaluate the analgesic efficacy of SCPB in various head and neck surgeries by observing intra- and postoperative requirements of the total dose of the systemic analgesic, visual analog scale (VAS) score, and the total duration of analgesia; vital parameters and associated perioperative complications were also observed. MATERIALS AND METHODS: Sample size was calculated using reference by taking the parameter pain score at 12 hours using MedCalc software v. 19.5.1 (MedCalc Software, Ostend, Belgium) with a mean difference of score 3±3.5 between two groups, 80% power, and 95% confidence interval (CI); the sample size for each group was 21. There were 30 patients in each group of ASA I, II, and III who were posted for mandibular, tympanomastoid and clavicular surgeries. Group A received general anaesthesia with systemic analgesia and Group B received general anaesthesia followed by SCPB with an injection of bupivacaine 0.25% 10ml on each side according to the site of surgery. VAS score, intra and postoperative analgesic requirement in 24 hours, time of first demand bolus, and peri-operative complications were noted. RESULTS: Intraoperative fentanyl requirement for group A was 97.5±13.75 µg as compared to group B (70.16±13.09 µg), postoperative injection paracetamol requirement was also significantly higher in group A (2566.66±504 mg) as compared to group B (833.33±874.28 mg). The total duration of analgesia was significantly higher in Group B (1191.33±375.36 min) as compared to Group A (122.0±50.88 min) with a p-value <0.0001. No significant complications were noted in any patient. CONCLUSION: SCPB provides better perioperative analgesia by decreasing intraoperative as well as postoperative systemic analgesic requirements and their associated side effects, with no significant perioperative complications in various head and neck region surgeries.

Alterations in Genes rib, scpB and Pilus Island Decrease the Prevalence of Predominant Serotype V, Not III and VI, of Streptococcus agalactiae from 2008 to 2012.

Streptococcus agalactiae (GBS) can infect newborns, pregnant women and immunocompromised or elderly people. This study aimed to investigate differences in three pilus genes and virulence genes pavA, cfb, rib and scpB and changes in predominant serotypes III, V and VI from 2008 to 2012. The susceptibilities to penicillin, ceftriaxone, azithromycin, erythromycin, clindamycin, levofloxacin and moxifloxacin of 145 GBS strains of serotype III, V and VI strains from 2008 and 2012 were determined using disc diffusion method. PCR identification of ST-17, the pilus genes and virulence genes; multilocus sequence typing (MLST); and conserved domain and phylogenetic analysis of scpB-1 and scpB-2 proteins were performed. A dramatic number reduction was observed in serotype V, not III and V, from 2008 to 2012. The rate of resistance to azithromycin, clindamycin and erythromycin was the highest in serotype V. ST-17 was only found in serotype III with pilus genes PI-1+PI-2b. The major pilus genotype was PI-1+PI-2a. Serotype V without the rib gene was reduced in number between two studied years. Compared to scpB-1,&nbsp;scpB-2 had a 128-bp deletion in a PA C5a-like peptidase domain and putative integrin-binding motif RGD. In conclusion, reduction in serotype V may be due to presence of scpB-2 or lack of genes scpB and rib.

A joint-ParB interface promotes Smc DNA recruitment.

Chromosomes readily unlink and segregate to daughter cells during cell division, highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes promote DNA organization by loop extrusion. In most bacteria, chromosome folding initiates at dedicated start sites marked by the ParB/parS partition complexes. Whether SMC complexes recognize a specific DNA structure in the partition complex or a protein component is unclear. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate foreign chromosomes. Using chimeric proteins and chemical cross-linking, we find that ParB directly binds the Smc subunit. We map an interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex, presumably to initiate DNA loop extrusion.

Gradual opening of Smc arms in prokaryotic condensin.

Multi-subunit SMC ATPases control chromosome superstructure apparently by catalyzing a DNA-loop-extrusion reaction. SMC proteins harbor an ABC-type ATPase "head" and a "hinge" dimerization domain connected by a coiled coil "arm." Two arms in a SMC dimer can co-align, thereby forming a rod-shaped particle. Upon ATP binding, SMC heads engage, and arms are thought to separate. Here, we study the shape of Bacillus subtilis Smc-ScpAB by electron-spin resonance spectroscopy. Arm separation is readily detected proximal to the heads in the absence of ligands, and separation near the hinge largely depends on ATP and DNA. Artificial blockage of arm opening eliminates DNA stimulation of ATP hydrolysis but does not prevent basal ATPase activity. We report an arm contact as being important for controlling the transformations. Point mutations at this arm interface eliminated Smc function. We propose that partially open, intermediary conformations provide directionality to SMC DNA translocation by (un)binding suitable DNA substrates.

Evidence for binary Smc complexes lacking kite subunits in archaea.

SMC complexes play a central role in chromosome organization in all domains of life. The bacterial Smc-ScpAB complex is a three-subunit complex composed of Smc, ScpA and ScpB. ScpA bridges the two ATPase domains of the Smc homodimer, while ScpB, which belongs to the kite family of proteins, interacts with ScpA. The three subunits are known to be equally important for the function of Smc-ScpAB in bacteria. From crystallographic and biochemical studies, evidence is provided that six archaeal ScpA proteins are unable to interact with the only putative ScpB found in these species. Structure-based sequence alignment reveals that these archaeal ScpAs lack the ScpB-binding segment that is commonly present in the middle of bacterial ScpA sequences, which is thus responsible for their inability to interact with ScpB. ScpA proteins lacking the ScpB-binding segment are found to prevail in archaea. Moreover, two archaeal ScpA proteins with a longer middle region also failed to bind their putative ScpB partner. Furthermore, all or most species belonging to five out of 14 euryarchaeotal orders contain Smc and ScpA but not a detectable ScpB homologue. These data support the notion that archaeal Smc-based complexes generally function as a two-subunit complex composed of only Smc and ScpA.

Functional and Proteomic Analyses Reveal That ScpBXv Is Involved in Bacterial Growth, Virulence, and Biofilm Formation in Xanthomonas campestris pv. vesicatoria.

Segregation and condensation protein B (ScpB) is essential for replication and segregation in living organisms. Here, we reported the functions of ScpBXv (ScpB-like protein in Xanthomonas campestris pv. vesicatoria) using phenotypic and proteomic analyses. Growth of XcvΔscpBXv (ScpBXv knockout mutant) was reduced under both slow and fast growth conditions in rich medium, but comparable to this of the wild-type in plant-mimic conditions. Interestingly, the mutant was significantly less virulent than the wild-type in tomato, indicating that ScpBXv is involved in virulence. To investigate ScpBXv-associated mechanisms, comparative proteomic analyses were carried out and the abundance of 187 proteins was altered. Among them, diverse transcriptional regulators involved in biofilm formation and virulence were abundant in the wild-type. We further showed that biofilm formation of XcvΔscpBXv was reduced. This study provides new insights into the functions of ScpBXv in bacterial replication and biofilm formation, which may contribute to the virulence of Xcv.

Overall Shapes of the SMC-ScpAB Complex Are Determined by Balance between Constraint and Relaxation of Its Structural Parts.

The SMC-ScpAB complex plays a crucial role in chromosome organization and segregation in many bacteria. It is composed of a V-shaped SMC dimer and an ScpAB subcomplex that bridges the two Structural Maintenance of Chromosomes (SMC) head domains. Despite its functional significance, the mechanistic details of SMC-ScpAB remain obscure. Here we provide evidence that ATP-dependent head-head engagement induces a lever movement of the SMC neck region, which might help to separate juxtaposed coiled-coil arms. Binding of the ScpA N-terminal domain (NTD) to the SMC neck region is negatively regulated by the ScpB C-terminal domain. Mutations in the ScpA NTD compromise this regulation and profoundly affect the overall shape of the complex. The SMC hinge domain is structurally relaxed when free from coiled-coil juxtaposition. Taken together, we propose that the structural parts of SMC-ScpAB are subjected to the balance between constraint and relaxation, cooperating to modulate dynamic conformational changes of the whole complex.