Light-dependent modulation of protein localization and function in living bacteria cells.

Most bacteria lack membrane-enclosed organelles and rely on macromolecular scaffolds at different subcellular locations to recruit proteins for specific functions. Here, we demonstrate that the optogenetic CRY2-CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations with varying efficiencies in live Escherichia coli cells, including the nucleoid, the cell pole, the membrane, and the midcell division plane. Such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E.

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

Ovoid-shaped bacteria, such as (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.

-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.

Alternate routes to mnmsU synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNA contains a variety of xnmsU derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative K12 model.

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

Ovoid-shaped bacteria, such as (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.

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

Unlabelled: The wobble bases of tRNAs that decode split codons are often heavily modified. In Bacteria tRNA contain a variety of xnm s U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative K12 model.

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.

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 D39.

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen (pneumococcus; ) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP( ) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing (MurZ-family homolog of MurA) or that suppress Δ or Δ .

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 , here named MpgA and MpgB, are important in maintaining cell wall integrity.

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 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 (), polynucleotide phosphorylase (), RNase R (), and three proteins with unknown functions.

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.

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 and related 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 [IC], ∼0.

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.

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

The catalase-negative, facultative anaerobe D39 is naturally resistant to hydrogen peroxide (HO) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced HO. We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and that mutants produce significantly lower HO.

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.

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.

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.

Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS-HotDog domain protein (SpxR) in serotype 2 Streptococcus pneumoniae.

spxB-encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signalling, and oxidative stress. We report here the first cis- and trans-acting regulatory elements for spxB transcription.

Regulation of the pspA virulence factor and essential pcsB murein biosynthetic genes by the phosphorylated VicR (YycF) response regulator in Streptococcus pneumoniae.

The VicRK (YycFG) two-component regulatory system (TCS) is required for virulence of the human respiratory pathogen Streptococcus pneumoniae (pneumococcus). The VicR (YycF) response regulator (RR) is essential through its positive regulation of pcsB, which encodes an extracellular protein that mediates murein biosynthesis. To determine other genes that are regulated by VicR, we performed microarray analyses on a unique DeltavicR deletion mutant, which was constructed by uncoupling regulation of pcsB.

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.