Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae.

Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNAGlu, Gln, Asp contains a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm5s2U to mnm5s2U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Negative regulation of MurZ and MurA underlies the essentiality of GpsB- and StkP-mediated protein phosphorylation in Streptococcus pneumoniae D39.

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP(Spn) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress ΔgpsB or ΔstkP. These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overproduction of MurZ caused by ΔkhpAB mutations suppressed ΔgpsB or ΔstkP phenotypes to varying extents. ΔgpsB and ΔstkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, ΔgpsB and ΔstkP were not suppressed by ΔclpCP, which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB(Spn), is the only essential requirement for StkP-mediated protein phosphorylation in exponentially growing D39 pneumococcal cells.

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall.

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG's ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

Perceptions of health and coping strategies among temporary migrant workers in East and Southeast Asia: a systematic review.

BACKGROUND: The rate of international migration for the primary purpose of employment has increased exponentially in recent decades. A significant proportion of this global movement takes place across East and Southeast Asia as workers move on a temporary basis from lower-middle-income home countries such as Indonesia, the Philippines, Thailand and Vietnam to high-income host destinations including Hong Kong and Singapore. Relatively little is known about the unique and long-term health needs of this heterogeneous group of people. This systematic review presents an analysis of recent research into the experiences and perceptions of health of temporary migrant workers in the East and Southeast Asian regions. METHODS: Five electronic databases CINAHL Complete (via EbscoHost), EMBASE (including Medline), PsycINFO (via ProQuest), PubMed and Web of Science, were systematically searched for qualitative or mixed methods, peer-reviewed literature published in print or online between January 2010 and December 2020. Quality of the studies was assessed using the Critical Appraisal Checklist for Qualitative Research published by the Joanna Briggs Institute. Findings from the included articles were extracted and synthesised using qualitative thematic analysis. RESULTS: Eight articles were included in the review. Findings from this review indicate that multiple dimensions of workers' health is impacted by the processes of temporary migration. In addition, the research reviewed indicated that migrant workers used various strategies and mechanisms to attempt to address their health-related issues and to take better care of themselves. Such agentic practices could help them manage and maintain their health and wellbeing across physical, psychological and spiritual dimensions within the structural constraints of their employment. CONCLUSIONS: Limited published research has focused on the health perceptions and needs of temporary migrant workers in East and Southeast Asia. The studies included in this review focused on female migrant domestic workers in Hong Kong, Singapore, and the Philippines. These studies provide valuable insights but do not reflect the heterogeneity of migrants moving within these regions. The findings of this systematic review highlight that temporary migrant workers experience high and sustained levels of stress and are exposed to certain health risks which may compromise long-term health outcomes. These workers demonstrate knowledge and skills in managing their own health. This suggests that strength-based approaches to health promotion interventions may be effective in optimising their health over time. These findings are relevant to policy makers and non-government organisations supporting migrant workers.

S1 Domain RNA-Binding Protein CvfD Is a New Posttranscriptional Regulator That Mediates Cold Sensitivity, Phosphate Transport, and Virulence in Streptococcus pneumoniae D39.

Posttranscriptional gene regulation often involves RNA-binding proteins that modulate mRNA translation and/or stability either directly through protein-RNA interactions or indirectly by facilitating the annealing of small regulatory RNAs (sRNAs). The human pathogen Streptococcus pneumoniae D39 (pneumococcus) does not encode homologs to RNA-binding proteins known to be involved in promoting sRNA stability and function, such as Hfq or ProQ, even though it contains genes for at least 112 sRNAs. However, the pneumococcal genome contains genes for other RNA-binding proteins, including at least six S1 domain proteins: ribosomal protein S1 (rpsA), polynucleotide phosphorylase (pnpA), RNase R (rnr), and three proteins with unknown functions. Here, we characterize the function of one of these conserved, yet uncharacterized, S1 domain proteins, SPD_1366, which we have renamed CvfD (conserved virulence factor D), since loss of the protein results in attenuation of virulence in a murine pneumonia model. We report that deletion of cvfD impacts the expression of 144 transcripts, including the pst1 operon, encoding phosphate transport system 1 in S. pneumoniae We further show that CvfD posttranscriptionally regulates the PhoU2 master regulator of the pneumococcal dual-phosphate transport system by binding phoU2 mRNA and impacting PhoU2 translation. CvfD not only controls expression of phosphate transporter genes but also functions as a pleiotropic regulator that impacts cold sensitivity and the expression of sRNAs and genes involved in diverse cellular functions, including manganese uptake and zinc efflux. Together, our data show that CvfD exerts a broad impact on pneumococcal physiology and virulence, partly by posttranscriptional gene regulation.IMPORTANCE Recent advances have led to the identification of numerous sRNAs in the major human respiratory pathogen S. pneumoniae However, little is known about the functions of most sRNAs or RNA-binding proteins involved in RNA biology in pneumococcus. In this paper, we characterize the phenotypes and one target of the S1 domain RNA-binding protein CvfD, a homolog of general stress protein 13 identified, but not extensively characterized, in other Firmicutes species. Pneumococcal CvfD is a broadly pleiotropic regulator, whose absence results in misregulation of divalent cation homeostasis, reduced translation of the PhoU2 master regulator of phosphate uptake, altered metabolism and sRNA amounts, cold sensitivity, and attenuation of virulence. These findings underscore the critical roles of RNA biology in pneumococcal physiology and virulence.

The Opp (AmiACDEF) Oligopeptide Transporter Mediates Resistance of Serotype 2 Streptococcus pneumoniae D39 to Killing by Chemokine CXCL10 and Other Antimicrobial Peptides.

Antimicrobial peptides (AMPs), including chemokines, are produced during infections to kill pathogenic bacteria. To fill in gaps in knowledge about the sensitivities of Streptococcus pneumoniae and related Streptococcus species to chemokines and AMPs, we performed a systematic, quantitative study of inhibition by chemokine CXCL10 and the AMPs LL-37 and nisin. In a standard Tris-glucose buffer (TGS), all strains assayed lacked metabolic activity, as determined by resazurin (alamarBlue) reduction, and were extremely sensitive to CXCL10 and AMPs (50% inhibitory concentration [IC50], ∼0.04 µM). In TGS, changes in sensitivities caused by mutations were undetectable. In contrast, strains that retained reductive metabolic activity in a different assay buffer (NPB [10 mM sodium phosphate {pH 7.4}, 1% {vol/vol} brain heart infusion {BHI} broth]) were less sensitive to CXCL10 and AMPs than in TGS. In NPB, mutants known to respond to AMPs, such as Δdlt mutants lacking d-alanylation of teichoic acids, exhibited the expected increased sensitivity. S. pneumoniae serotype 2 strain D39 was much (∼10-fold) less sensitive to CXCL10 killing in NPB than serotype 4 strain TIGR4, and the sensitivity of TIGR4 was unaffected by the absence of capsule. Candidate screening of strain D39 revealed that mutants lacking Opp (ΔamiACDEF) oligopeptide permease were significantly more resistant to CXCL10 than the wild-type strain. This increased resistance could indicate that Opp is a target for CXCL10 binding or that it transports CXCL10 into cells. Finally, ΔftsX or ΔftsE mutants of Bacillus subtilis or amino acid changes that interfere with FtsX function in S. pneumoniae did not impart resistance to CXCL10, in contrast to previous results for Bacillus anthracis, indicating that FtsX is not a general target for CXCL10 binding.IMPORTANCES. pneumoniae (pneumococcus) is a human commensal bacterium and major opportunistic respiratory pathogen that causes serious invasive diseases, killing millions of people worldwide annually. Because of its increasing antibiotic resistance, S. pneumoniae is now listed as a "superbug" for which new antibiotics are urgently needed. This report fills in knowledge gaps and resolves inconsistencies in the scientific literature about the sensitivity of S. pneumoniae and related Streptococcus pathogens to chemokines and AMPs. It also reveals a new mechanism by which S. pneumoniae can acquire resistance to chemokine CXCL10. This mechanism involves the Opp (AmiACDEF) oligopeptide transporter, which plays additional pleiotropic roles in pneumococcal physiology, quorum sensing, and virulence. Taking the results together, this work provides new information about the way chemokines kill pneumococcal cells.

Absence of the KhpA and KhpB (JAG/EloR) RNA-binding proteins suppresses the requirement for PBP2b by overproduction of FtsA in Streptococcus pneumoniae D39.

Suppressor mutations were isolated that obviate the requirement for essential PBP2b in peripheral elongation of peptidoglycan from the midcells of dividing Streptococcus pneumoniae D39 background cells. One suppressor was in a gene encoding a single KH-domain protein (KhpA). ΔkhpA suppresses deletions in most, but not all (mltG), genes involved in peripheral PG synthesis and in the gpsB regulatory gene. ΔkhpA mutations reduce growth rate, decrease cell size, minimally affect shape and induce expression of the WalRK cell-wall stress regulon. Reciprocal co-immunoprecipitations show that KhpA forms a complex in cells with another KH-domain protein (KhpB/JAG/EloR). ΔkhpA and ΔkhpB mutants phenocopy each other exactly, consistent with a direct interaction. RNA-immunoprecipitation showed that KhpA/KhpB bind an overlapping set of RNAs in cells. Phosphorylation of KhpB reported previously does not affect KhpB function in the D39 progenitor background. A chromosome duplication implicated FtsA overproduction in Δpbp2b suppression. We show that cellular FtsA concentration is negatively regulated by KhpA/B at the post-transcriptional level and that FtsA overproduction is necessary and sufficient for suppression of Δpbp2b. However, increased FtsA only partially accounts for the phenotypes of ΔkhpA mutants. Together, these results suggest that multimeric KhpA/B may function as a pleiotropic RNA chaperone controlling pneumococcal cell division.

Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39.

In ellipsoid-shaped ovococcus bacteria, such as the pathogen Streptococcus pneumoniae (pneumococcus), side-wall (peripheral) peptidoglycan (PG) synthesis emanates from midcells and is catalyzed by the essential class B penicillin-binding protein PBP2b transpeptidase (TP). We report that mutations that inactivate the pneumococcal YceG-domain protein, Spd_1346 (renamed MltG), remove the requirement for PBP2b. ΔmltG mutants in unencapsulated strains accumulate inactivation mutations of class A PBP1a, which possesses TP and transglycosylase (TG) activities. The 'synthetic viable' genetic relationship between Δpbp1a and ΔmltG mutations extends to essential ΔmreCD and ΔrodZ mutations that misregulate peripheral PG synthesis. Remarkably, the single MltG(Y488D) change suppresses the requirement for PBP2b, MreCD, RodZ and RodA. Structural modeling and comparisons, catalytic-site changes and an interspecies chimera indicate that pneumococcal MltG is the functional homologue of the recently reported MltG endo-lytic transglycosylase of Escherichia coli. Depletion of pneumococcal MltG or mltG(Y488D) increases sphericity of cells, and MltG localizes with peripheral PG synthesis proteins during division. Finally, growth of Δpbp1a ΔmltG or mltG(Y488D) mutants depends on induction of expression of the WalRK TCS regulon of PG hydrolases. These results fit a model in which MltG releases anchored PG glycan strands synthesized by PBP1a for crosslinking by a PBP2b:RodA complex in peripheral PG synthesis.

A new structural paradigm in copper resistance in Streptococcus pneumoniae.

Copper resistance has emerged as an important virulence determinant of microbial pathogens. In Streptococcus pneumoniae, copper resistance is mediated by the copper-responsive repressor CopY, CupA and the copper-effluxing P(1B)-type ATPase CopA. We show here that CupA is a previously uncharacterized cell membrane-anchored Cu(I) chaperone and that a Cu(I) binding-competent, membrane-localized CupA is obligatory for copper resistance. The crystal structures of the soluble domain of CupA and the N-terminal metal-binding domain (MBD) of CopA (CopA(MBD)) reveal isostructural cupredoxin-like folds that each harbor a binuclear Cu(I) cluster unprecedented in bacterial copper trafficking. NMR studies reveal unidirectional Cu(I) transfer from the low-affinity site on the soluble domain of CupA to the high-affinity site of CopA(MBD). However, copper binding by CopA(MBD) is not essential for cellular copper resistance, consistent with a primary role of CupA in cytoplasmic Cu(I) sequestration and/or direct delivery to the transmembrane site of CopA for cellular efflux.

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae.

Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed non-processive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

O-glycosylation of intrinsically disordered regions regulates homeostasis of membrane proteins in streptococci.

Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane proteins in streptococci. We demonstrate that these IDRs are O-glycosylated with glucose by glycosyltransferases GtrB and PgtC2 in Streptococcus pyogenes and Streptococcus pneumoniae, and with N-acetylgalactosamine by a Pgf-dependent mechanism in Streptococcus mutans. Absence of glycosylation leads to a defect in biofilm formation under ethanol-stressed conditions in S. mutans. We link this phenotype to the C-terminal IDR of a post-translocation secretion chaperone PrsA. O-glycosylation of the IDR protects this region from proteolytic degradation. The IDR length attenuates the efficiency of glycosylation and, consequently, the expression level of PrsA. Taken together, our data reveal that O-glycosylation of IDRs functions as a dynamic switch of protein homeostasis in streptococci.

Inter-observer and intra-observer reliability between manual segmentation and semi-automated segmentation for carotid vessel wall volume measurements on three-dimensional ultrasonography.

PURPOSE: Carotid vessel wall volume (VWV) measurement on three-dimensional ultrasonography (3DUS) outperforms conventional two-dimensional ultrasonography for carotid atherosclerosis evaluation. Although time-saving semi-automated algorithms have been introduced, their clinical availability remains limited due to a lack of validation, particularly an extensive reliability analysis. This study compared inter-observer and intra-observer reliability between manual segmentation and semi-automated segmentation for carotid VWV measurements on 3DUS. METHODS: Thirty-one 3DUS volume datasets were prospectively acquired from 20 healthy subjects, aged >18 years, without previous stroke, transient ischemic attack, or cardiovascular disease. Five observers segmented all volume datasets both manually and semi-automatically. The process was repeated five times. Reliability was expressed by the intraclass correlation coefficient, supplemented by the coefficient of variation. RESULTS: Carotid VWV measurements using the common carotid artery (CCA) were more reliable than those using the internal carotid artery (ICA) or external carotid artery (ECA) for both manual and semiautomated segmentation (manual segmentation, CCA: inter-observer, 0.935; intra-observer, 0.934 to 0.966; ICA: inter-observer, 0.784; intra-observer, 0.756 to 0.878; ECA: inter-observer, 0.732; intraobserver, 0.919 to 0.962; semi-automated segmentation, CCA: inter-observer, 0.986; intra-observer, 0.954 to 0.993; ICA: inter-observer, 0.977; intra-observer, 0.958 to 0.978; ECA: inter-observer, 0.966; intra-observer, 0.884 to 0.937). Total carotid VWV measurements by manual (inter-observer, 0.922; intra-observer, 0.927 to 0.961) and semi-automated segmentation (inter-observer, 0.987; intra-observer, 0.968 to 0.989) were highly reliable. Semi-automated segmentation showed higher reliability than manual segmentation for both individual and total carotid VWV measurements. CONCLUSION: 3DUS carotid VWV measurements of the CCA are more reliable than measurements of the ICA and ECA. Total carotid VWV measurements are highly reliable. Semi-automated segmentation has higher reliability than manual segmentation.

Chromosomal Duplications of MurZ (MurA2) or MurA (MurA1), Amino Acid Substitutions in MurZ (MurA2), and Absence of KhpAB Obviate the Requirement for Protein Phosphorylation in Streptococcus pneumoniae D39.

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn ) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP( Spn ) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress Δ gpsB or Δ stkP . These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overexpression of MurZ caused by Δ khpAB mutations suppressed Δ gpsB or Δ stkP phenotypes to varying extents. Δ gpsB and Δ stkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, Δ gpsB and Δ stkP were not suppressed by Δ clpCP , which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB( Spn ), is the only essential requirement for protein phosphorylation in exponentially growing D39 pneumococcal cells.

Alternate routes to mnm 5 s 2 U synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In Bacteria tRNA Glu, Gln, Asp contain a variety of xnm 5 s 2 U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm 5 s 2 U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the installation of this modification. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the Radical Sam superfamily was found to be involved in the synthesis of mnm 5 s 2 U in both Bacillus subtilis and Streptococcus mutans . This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm 5 s 2 U into mnm 5 s 2 U in B. subtilis . Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathways intermediates in regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. The occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in nature. Importance: The xnm 5 s 2 U modifications found in several tRNAs at the wobble base position are widespread in Bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile Radical SAM superfamily and is involved in the synthesis of mnm 5 s 2 U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Identification and characterization of noncoding small RNAs in Streptococcus pneumoniae serotype 2 strain D39.

We report a search for small RNAs (sRNAs) in the low-GC, gram-positive human pathogen Streptococcus pneumoniae. Based on bioinformatic analyses by Livny et al. (J. Livny, A. Brencic, S. Lory, and M. K. Waldor, Nucleic Acids Res. 34:3484-3493, 2006), we tested 40 candidates by Northern blotting and confirmed the expression of nine new and one previously reported (CcnA) sRNAs in strain D39. CcnA is one of five redundant sRNAs reported by Halfmann et al. (A. Halfmann, M. Kovacs, R. Hakenbeck, and R. Bruckner, Mol. Microbiol. 66:110-126, 2007) that are positively controlled by the CiaR response regulator. We characterized 3 of these 14 sRNAs: Spd-sr17 (144 nucleotides [nt]; decreased in stationary phase), Spd-sr37 (80 nt; strongly expressed in all growth phases), and CcnA (93 nt; induced by competence stimulatory peptide). Spd-sr17 and CcnA likely fold into structures containing single-stranded regions between hairpin structures, whereas Spd-sr37 forms a base-paired structure. Primer extension mapping and ectopic expression in deletion/insertion mutants confirmed the independent expression of the three sRNAs. Microarray analyses indicated that insertion/deletion mutants in spd-sr37 and ccnA exerted strong cis-acting effects on the transcription of adjacent genes, indicating that these sRNA regions are also cotranscribed in operons. Deletion or overexpression of the three sRNAs did not cause changes in growth, certain stress responses, global transcription, or virulence. Constitutive ectopic expression of CcnA reversed some phenotypes of D39 Delta ciaR mutants, but attempts to link CcnA to -E to comC as a target were inconclusive in ciaR(+) strains. These results show that S. pneumoniae, which lacks known RNA chaperones, expresses numerous sRNAs, but three of these sRNAs do not strongly affect common phenotypes or transcription patterns.

The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes.

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here, we describe critical structural features for the interaction of GpsB with peptidoglycan synthases from three bacterial species (Bacillus subtilis, Listeria monocytogenes and Streptococcus pneumoniae) and suggest their importance for cell wall growth and viability in L. monocytogenes and S. pneumoniae. We use these structural motifs to identify novel partners of GpsB in B. subtilis and extend the members of the GpsB interactome in all three bacterial species. Our results support that GpsB functions as an adaptor protein that mediates the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner.

Biological and Chemical Adaptation to Endogenous Hydrogen Peroxide Production in Streptococcus pneumoniae D39.

The catalase-negative, facultative anaerobe Streptococcus pneumoniae D39 is naturally resistant to hydrogen peroxide (H2O2) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced H2O2. We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and that ΔlctO mutants produce significantly lower H2O2. In addition, both the SpxB pathway and a candidate pyruvate dehydrogenase complex (PDHC) pathway contribute to acetyl coenzyme A (acetyl-CoA) production during aerobic growth, and the pyruvate format lyase (PFL) pathway is the major acetyl-CoA pathway during anaerobic growth. Microarray analysis of the D39 strain cultured under aerobic versus strict anaerobic conditions shows upregulation of spxB, a gene encoding a rhodanese-like protein (locus tag spd0091), tpxD, sodA, piuB, piuD, and an Fe-S protein biogenesis operon under H2O2-producing conditions. Proteome profiling of H2O2-induced sulfenylation reveals that sulfenylation levels correlate with cellular H2O2 production, with endogenous sulfenylation of ≈50 proteins. Deletion of tpxD increases cellular sulfenylation 5-fold and has an inhibitory effect on ATP generation. Two major targets of protein sulfenylation are glyceraldehyde-3-phosphate dehydrogenase (GapA) and SpxB itself, but targets also include pyruvate kinase, LctO, AdhE, and acetate kinase (AckA). Sulfenylation of GapA is inhibitory, while the effect on SpxB activity is negligible. Strikingly, four enzymes of capsular polysaccharide biosynthesis are sulfenylated, as are enzymes associated with nucleotide biosynthesis via ribulose-5-phosphate. We propose that LctO/SpxB-generated H2O2 functions as a signaling molecule to downregulate capsule production and drive altered flux through sugar utilization pathways. IMPORTANCE Adaptation to endogenous oxidative stress is an integral aspect of Streptococcus pneumoniae colonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous H2O2 and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in S. pneumoniae.

Expression of metabotropic glutamate receptors in rat meningeal and brain microvasculature and choroid plexus.

This study investigated the distribution of metabotropic glutamate receptors (mGluRs) in meningeal and parenchymal microvasculature and in choroid plexus by means of Western blot analysis and immunohistochemistry. Western blot analysis demonstrated mGluR expression in both rat and human leptomeningeal tissues. In the rat, mGluR expression was developmentally regulated, with only mGluR2/3 showing expression at the embryonic day 19 developmental stage. In contrast, mGluR1 alpha, mGluR2/3, mGluR4a, and mGluR7 were expressed in leptomeninges from adult rats. Immunohistochemical analyses showed intense mGluR1 alpha immunoreactivity in the pia mater and blood vessels in the subarachnoid space and in the arachnoid layer of the meninges. mGluR2/3, mGluR4a, mGluR5, and mGluR7 were also expressed in meningeal microvasculature. In addition, the parenchymal microvasculature and choroid plexus were strongly immunoreactive for mGluR1 alpha, mGluR2/3, mGluR4a, mGluR5, and mGluR7. We used antibodies specific for phenotypic markers of microvascular and glial cells to characterize the cell type(s) immunopositive for mGluRs. Comparison of staining with anti-von Willebrand factor antibody and anti-mGluR antibodies revealed that mGluR immunoreactivity was present in cells that surrounded the luminal surface labeled by the endothelial cell marker. In these cells, smooth muscle actin and mGluR immunoreactivity overlapped, suggesting that, in addition to endothelial cells, pericytes within the microvasculature also express mGluRs. Furthermore, expression of mGluR1 alpha was also observed in pure pericyte cultures isolated from bovine retina. These data suggest that glutamate by means of activation of mGluRs may have a broad sphere of physiological influence in the brain which in addition to modulating synaptic transmission may also have a role in determining microvascular function and dysfunction.

Assessing local patients' knowledge and awareness of radiation dose and risks associated with medical imaging: a questionnaire study.

INTRODUCTION: To assess the awareness of radiation dose and associated risks caused by radiological procedures among local patients. METHODS: All subjects were recruited by randomly sampling the patients receiving radiological examinations. These subjects were stratified on age, sex and education. The questionnaire was in Chinese and consisted of 28 questions mostly in multiple choice/true-or-false format, divided into three sections examining demographic data, radiation knowledge/awareness and expectations. RESULTS: A total of 173 questionnaires were returned (83 females and 84 females; mean age of 53). Of these, 32.6% had attended college, 32.6% had completed matriculation and 24.4% secondary school. Most subjects underwent CT (75), MRI (70) and PET-CT (18). Education significantly affected the radiation knowledge (P = 0.013). 60.7% and 32.7% were not aware of the radiation-free nature of MRI and USG, respectively. Respectively, 45.4% and 43.5% were of the misconception that Barium enema and Barium swallow studies do not involve radiation. Moreover, 77.6% and 87.9% were aware of the radiation-laden nature of CT and plain X-rays, respectively. Furthermore, 34% and 50%, respectively, think that they are not exposed to radiation at home and on a plane. Regarding the fatal cancer risk from CT, 17.8% chose the correct answer and 62% underestimated the risk. 32.2% correctly estimated the equivalent dose of CT in terms of number of conventional X-rays and 43.2% underestimated the dose. Most (98.2%) were told of the indication, and 42.7% were told the associated radiation dose. CONCLUSION: Patient radiation awareness is unsatisfactory. There is need to increase patient radiation awareness, and to provide them with the necessary information.