Global Annotation, Expression Analysis, and Stability of Candidate sRNAs in Group B Streptococcus.

Small, noncoding RNAs (sRNAs) are being increasingly identified as important regulatory molecules in prokaryotes. Due to the prevalence of next-generation sequencing-based techniques, such as RNA sequencing (RNA-seq), there is potential for increased discovery of sRNAs within bacterial genomes; however, these elements are rarely included in annotation files. Consequently, expression values for sRNAs are omitted from most transcriptomic analyses, and mechanistic studies have lagged behind those of protein regulators in numerous bacteria. Two previous studies have identified sRNAs in the human pathogen group B Streptococcus (GBS). Here, we utilize the data from these studies to create updated genome annotation files for the model GBS strains NEM316 and COH1. Using the updated COH1 annotation file, we reanalyze publicly available GBS RNA-seq whole-transcriptome data from GenBank to monitor GBS sRNA expression under a variety of conditions and genetic backgrounds. This analysis generated expression values for 232 putative sRNAs that were overlooked in previous transcriptomic analyses in 21 unique comparisons. To demonstrate the utility of these data, we identify an sRNA that is upregulated during vaginal colonization and demonstrate that overexpression of this sRNA leads to increased bacterial invasion into host epithelial cells. Finally, to monitor RNA degradation, we perform a transcript stability assay to identify highly stable sRNAs and compare stability profiles of sRNA- and protein-coding genes. Collectively, these data provide a wealth of transcriptomic data for putative sRNAs in GBS and a platform for future mechanistic studies. IMPORTANCE In recent years, sRNAs have emerged as potent regulatory molecules in bacteria, including numerous streptococcal species, and contribute to diverse processes, including stress response, metabolism, housekeeping, and virulence regulation. Improvements in sequencing technologies and in silico analyses have facilitated identification of these regulatory molecules as well as improved attempts to determine the location of sRNA genes on the genome. However, despite these advancements, sRNAs are rarely included in genome annotation files. Consequently, these molecules are often omitted from transcriptomic data analyses and are commonly repeat identified across multiple studies. Updating current genomes to include sRNA genes is therefore critical for better understanding bacterial regulation.

Streptococcus pneumoniae, S. pyogenes and S. agalactiae membrane phospholipid remodelling in response to human serum.

Streptococcus pneumoniae, S. pyogenes (Group A Streptococcus; GAS) and S. agalactiae (Group B Streptococcus; GBS) are major aetiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization MS, we characterized the phospholipids and glycolipids of S. pneumoniae, GAS and GBS in routine undefined laboratory medium, streptococcal defined medium and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodelling by the major pathogenic streptococci in response to metabolites present in human serum.

Characterization of the nucleotide-binding domain NsrF from the BceAB-type ABC-transporter NsrFP from the human pathogen Streptococcus agalactiae.

Treatment of bacterial infections is a great challenge of our era due to the various resistance mechanisms against antibiotics. Antimicrobial peptides are considered to be potential novel compound as antibiotic treatment. However, some bacteria, especially many human pathogens, are inherently resistant to these compounds, due to the expression of BceAB-type ABC transporters. This rather new transporter family is not very well studied. Here, we report the first full characterization of the nucleotide binding domain of a BceAB type transporter from Streptococcus agalactiae, namely SaNsrF of the transporter SaNsrFP, which confers resistance against nisin and gallidermin. We determined the NTP hydrolysis kinetics and used molecular modeling and simulations in combination with small angle X-ray scattering to obtain structural models of the SaNsrF monomer and dimer. The fact that the SaNsrFH202A variant displayed no ATPase activity was rationalized in terms of changes of the structural dynamics of the dimeric interface. Kinetic data show a clear preference for ATP as a substrate, and the prediction of binding modes allowed us to explain this selectivity over other NTPs.

Structure-Guided Design of a Group†B Streptococcus Type†III Synthetic Glycan-Conjugate Vaccine.

Identification of glycan functional epitopes is of paramount importance for rational design of glycoconjugate vaccines. We recently mapped the structural epitope of the capsular polysaccharide from type III Group B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment (composed of two pentasaccharide repeating units) obtained by depolymerization complexed with a protective mAb. Although reported data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a proper glycosylation degree elicited functional antibodies comparably to the full-length conjugated polysaccharide. Here, starting from the X-ray crystallographic structure of the polysaccharide fragment-mAb complex, we synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive surface plasmon resonance and saturation transfer difference NMR spectroscopy as well as in-silico modeling, we demonstrated that this synthetic glycan was recognized by the mAb similarly to the dimer. The hexasaccharide conjugated to CRM197 , a mutant of diphtheria toxin, elicited a robust functional immune response that was not inferior to the polysaccharide conjugate, indicating that it may suffice as a vaccine antigen. This is the first evidence of an X-ray crystallography-guided design of a synthetic carbohydrate-based conjugate vaccine.

Small-molecule inhibitors of nisin resistance protein NSR from the human pathogen Streptococcus agalactiae.

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria and active in the nanomolar range. Nisin is the most intensely studied and used lantibiotic, with applications as food preservative and recognized potential for clinical usage. However, different bacteria that are pathogenic for humans and do not produce nisin, including Streptococcus agalactiae, show an innate resistance that has been related to the nisin resistance protein (NSR), a membrane-associated protease. Here, we report the first-in-class small-molecule inhibitors of SaNSR identified by virtual screening based on a previously derived structural model of the nisin/NSR complex. The inhibitors belong to three different chemotypes, of which the halogenated phenyl-urea derivative NPG9 is the most potent one. Co-administration of NPG9 with nisin yields increased potency compared to nisin alone in SaNSR-expressing bacteria. The binding mode of NPG9, predicted with molecular docking and validated by extensive molecular dynamics simulations, confirms a structure-activity relationship derived from the in vivo data. Saturation transfer difference-NMR experiments demonstrate direct binding of NPG9 to SaNSR and agree with the predicted binding mode. Our results demonstrate the potential to overcome SaNSR-related lantibiotic resistance by small molecules.

Chemical synthesis of the dimeric repeating unit of type Ia group B Streptococcus capsular polysaccharide.

The first chemical synthesis of the dimeric repeating unit of serotype Ia group B Streptococcus capsular polysaccharide was achieved. The on-site elongation and dual glycosylation strategies were utilized to construct two sialotrisaccharide branches based on a hexasaccharide containing adjacent 3,4-di-branched Gal units, which were synthesized via a preactivation-based one-pot glycosylation method.

Identification of a proteomic biomarker associated with invasive ST1, serotype VI Group B Streptococcus by MALDI-TOF MS.

BACKGROUND: Group B Streptococcus (GBS) is an important invasive pathogen in neonates, pregnant women and the elderly. Serotype VI GBS, which has been rarely reported globally, has emerged as a significant pathogen in Asia. However, traditional serologic latex agglutination (LA) methods may fail to type isolates that lack of or low expression of CPS. METHODS: A total of 104 GBS strains were analyzed by MALDI-TOF MS. Multiplex PCR and multilocus sequence typing (MLST) were also performed to confirm their strains. The protein markers were purified with gel electrophoresis and LC-column, followed by identification with nanoLC-MS/MS analysis. RESULTS: Protein peak of 6251-Da was appeared in most (20/24, 92%) serotypes VI (94% ST-1 or single locus variant of ST-1), and protein peak of 6891-Da was appeared in most serotypes III (15/18, 83%) and Ib (19/23, 83%) strains. The protein peak of 6251-Da and 6891-Da were identified as CsbD family protein and UPF0337 protein gbs0600, respectively. CONCLUSIONS: The protein peak of 6251 Da may play a role of emergence of ST-1 clone, serotype VI GBS in central Taiwan and could be useful in rapid identifying invasive serotype VI from III isolates, which is hardly achieved by LA.

Molecular dynamics simulation of metal free structure of Lmb, a laminin-binding adhesin of Streptococcus agalactiae: metal removal and its structural implications.

Metal-binding receptors are one of the extracellular components of ATP-binding cassette transporters that are essential for regulation of metal homeostasis in bacteria. Laminin-binding adhesin (Lmb) of Streptococcus agalactiae falls under this class of solute binding proteins. It binds to zinc with a high affinity. Crystal structure of Lmb solved previously by our group reveals that the zinc is tetrahedrally coordinated by three histidines and a glutamate at the interdomain cleft. Lmb contains a long disordered loop close to the metal-binding site whose precise function is unknown. Several experimental attempts to produce apo-Lmb failed and this prompted us to carry out in silico studies to analyse the structural importance of the metal in Lmb. Here, we present the results of the molecular dynamics (MD) simulation studies of native, apo-(metal removed) and the long loop truncated Lmb models along with a homologous protein, TroA from Treponema pallidum that was taken up for validating the MD results of Lmb. Absence of a metal results in significant structural changes in Lmb, particularly at the metal-binding pocket and with the long loop, although the overall fold is retained. This study thus revealed that the Lmb can exist in different conformational states with subtle differences in the overall fold based on the presence or absence of the metal. This could be functionally important for a putative metal uptake and release and also for the adhesive function of Lmb in recognizing laminin, which contains a high number of zinc finger motifs.

Capsular polysaccharide of Streptococcus agalactiae is an essential virulence factor for infection in Nile tilapia (Oreochromis niloticus Linn.).

Streptococcus agalactiae (Group B Streptococcus, GBS) is associated with diverse diseases in aquatic animals. The capsule polysaccharide (CPS) encoded by the cps gene cluster is the major virulence factor of S. agalactiae; however, limited information is available regarding the pathogenic role of the CPS of serotype Ia piscine GBS strains in fish. Here, a non-encapsulated mutant (Δcps) was constructed by insertional mutagenesis of the cps gene cluster. Mutant pathogenicity was evaluated in vitro based on the killing of whole blood from tilapia, in vivo infections, measuring mutant survival in tilapia spleen tissues and pathological analysis. Compared to wild-type (WT) GBS strain, the Δcps mutant had lower resistance to fresh tilapia whole blood in vitro (p < 0.01), and more easily cleared in tilapia spleen tissue, and was highly attenuated in tilapia and zebrafish. Additionally, compared to the Δcps mutant, numerous GBS strains and severe tissue necrosis were observed in the tilapia spleen tissue infected with WT strains. These results indicated that the CPS is essential for GBS pathogenicity and may serve as a target for attenuation in vaccine development. Gaining a better understanding of the role, the GBS pathogenicity in fish will provide insight into related pathogenesis and host-pathogen interactions.

Stable Expression of Modified Green Fluorescent Protein in Group B Streptococci To Enable Visualization in Experimental Systems.

Group B streptococcus (GBS) is a Gram-positive bacterium associated with various diseases in humans and animals. Many studies have examined GBS physiology, virulence, and microbe-host interactions using diverse imaging approaches, including fluorescence microscopy. Strategies to label and visualize GBS using fluorescence biomarkers have been limited to antibody-based methods or nonspecific stains that bind DNA or protein; an effective plasmid-based system to label GBS with a fluorescence biomarker would represent a useful visualization tool. In this study, we developed and validated a green fluorescent protein (GFP)-variant-expressing plasmid, pGU2664, which can be applied as a marker to visualize GBS in experimental studies. The synthetic constitutively active CP25 promoter drives strong and stable expression of the GFPmut3 biomarker in GBS strains carrying pGU2664. GBS maintains GFPmut3 activity at different phases of growth. The application of fluorescence polarization enables easy discrimination of GBS GFPmut3 activity from the autofluorescence of culture media commonly used to grow GBS. Differential interference contrast microscopy, in combination with epifluorescence microscopy to detect GFPmut3 in GBS, enabled visualization of bacterial attachment to live human epithelial cells in real time. Plasmid pGU2664 was also used to visualize phenotypic differences in the adherence of wild-type GBS and an isogenic gene-deficient mutant strain lacking CovR (the control of virulence regulator) in adhesion assays. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications. We discuss the advantages and consider the limitations of this fluorescent biomarker system developed for GBS.IMPORTANCE Group B streptococcus (GBS) is a bacterium associated with various diseases in humans and animals. This study describes the development of a strategy to label and visualize GBS using a fluorescence biomarker, termed GFPmut3. We show that this biomarker can be successfully applied to track the growth of bacteria in liquid medium, and it enables the detailed visualization of GBS in the context of live human cells in real-time microscopic analysis. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications.

Group B Streptococcus Biofilm Regulatory Protein A Contributes to Bacterial Physiology and Innate Immune Resistance.

Background: Streptococcus agalactiae (group B Streptococcus [GBS]) asymptomatically colonizes approximately 20% of adults; however, GBS causes severe disease in susceptible populations, including newborns, pregnant women, and elderly individuals. In shifting between commensal and pathogenic states, GBS reveals multiple mechanisms of virulence factor control. Here we describe a GBS protein that we named "biofilm regulatory protein A" (BrpA) on the basis of its homology with BrpA from Streptococcus mutans. Methods: We coupled phenotypic assays, RNA sequencing, human neutrophil and whole-blood killing assays, and murine infection models to investigate the contribution of BrpA to GBS physiology and virulence. Results: Sequence analysis identified BrpA as a LytR-CpsA-Psr enzyme. Targeted mutagenesis yielded a GBS mutant (ΔbrpA) with normal ultrastructural morphology but a 6-fold increase in chain length, a biofilm defect, and decreased acid tolerance. GBS ΔbrpA stimulated increased neutrophil reactive oxygen species and proved more susceptible to human and murine blood and neutrophil killing. Notably, the wild-type parent outcompeted ΔbrpA GBS in murine sepsis and vaginal colonization models. RNA sequencing of ΔbrpA uncovered multiple differences from the wild-type parent, including pathways of cell wall synthesis and cellular metabolism. Conclusions: We propose that BrpA is an important virulence regulator and potential target for design of novel antibacterial therapeutics against GBS.

Crystal structure of the Streptococcus agalactiae CAMP factor provides insights into its membrane-permeabilizing activity.

Streptococcus agalactiae is an important human opportunistic pathogen that can cause serious health problems, particularly among newborns and older individuals. S. agalactiae contains the CAMP factor, a pore-forming toxin first identified in this bacterium. The CAMP reaction is based on the co-hemolytic activity of the CAMP factor and is commonly used to identify S. agalactiae in the clinic. Closely related proteins are present also in other Gram-positive pathogens. Although the CAMP toxin was discovered more than a half century ago, no structure from this toxin family has been reported, and the mechanism of action of this toxin remains unclear. Here, we report the first structure of this toxin family, revealing a structural fold composed of 5 + 3-helix bundles. Further analysis by protein truncation and site-directed mutagenesis indicated that the N-terminal 5-helix bundle is responsible for membrane permeabilization, whereas the C-terminal 3-helix bundle is likely responsible for host receptor binding. Interestingly, the C-terminal domain inhibited the activity of both full-length toxin and its N-terminal domain. Moreover, we observed that the linker region is highly conserved and has a conserved DLXXXDXAT sequence motif. Structurally, this linker region extensively interacted with both terminal CAMP factor domains, and mutagenesis disclosed that the conserved sequence motif is required for CAMP factor's co-hemolytic activity. In conclusion, our results reveal a unique structure of this bacterial toxin and help clarify the molecular mechanism of its co-hemolytic activity.

Antibody Kinetics and Response to Routine Vaccinations in Infants Born to Women Who Received an Investigational Trivalent Group B Streptococcus Polysaccharide CRM197-Conjugate Vaccine During Pregnancy.

BACKGROUND: Maternal vaccination against group B Streptococcus (GBS) might provide protection against invasive GBS disease in infants. We investigated the kinetics of transplacentally transferred GBS serotype-specific capsular antibodies in the infants and their immune response to diphtheria toxoid and pneumococcal vaccination. METHODS: This phase 1b/2, observer-blind, single-center study (NCT01193920) enrolled infants born to women previously randomized (1:1:1:1) to receive either GBS vaccine at dosages of 0.5, 2.5, or 5.0 µg of each of 3 CRM197-glycoconjugates (serotypes Ia, Ib, and III), or placebo. Infants received routine immunization: combination diphtheria vaccine (diphtheria-tetanus-acellular pertussis-inactivated poliovirus/Haemophilus influenzae type b vaccine; age 6/10/ 14 weeks) and 13-valent pneumococcal CRM197-conjugate vaccine (PCV13; age 6/14 weeks and 9 months). Antibody levels were assessed at birth, day (D) 43, and D91 for GBS serotypes; 1 month postdose 3 (D127) for diphtheria; and 1 month postprimary (D127) and postbooster (D301) doses for pneumococcal serotypes. RESULTS: Of 317 infants enrolled, 295 completed the study. In infants of GBS vaccine recipients, GBS serotype-specific antibody geometric mean concentrations were significantly higher than in the placebo group at all timepoints and predictably decreased to 41%-61% and 26%-76% of birth levels by D43 and D91, respectively. Across all groups, ≥95% of infants were seroprotected against diphtheria at D127 and ≥91% of infants had seroprotective antibody levels against each PCV13 pneumococcal serotype at D301. CONCLUSIONS: Maternal vaccination with an investigational CRM197-glycoconjugate GBS vaccine elicited higher GBS serotype-specific antibody levels in infants until 90 days of age, compared with a placebo group, and did not affect infant immune responses to diphtheria toxoid and pneumococcal vaccination. CLINICAL TRIALS REGISTRATION: NCT01193920.

Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide.

Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.

Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

Discovery, Production and Modification of Five Novel Lantibiotics Using the Promiscuous Nisin Modification Machinery.

To find the right conditions to isolate natively expressed antimicrobial peptides from a wide range of different microorganisms can be a challenge. Here, we exploited a heterologous expression system to produce and characterize several novel lantibiotics. We identified 54 novel putative class I and class II lantibiotics after inspecting all publicly available prokaryotic genomes using the in-house developed mining tool BAGEL3. The genes encoding these new lantibiotics fused to the nisin leader peptide gene sequence were synthesized, and the constructs were plugged into the nisin expression and modification system. Using this approach 30 peptides could be expressed, 27 of which were dehydrated by NisBC on at least 1 predicted position. Good antimicrobial activity against several pathogenic bacteria could be demonstrated for 5 novel heterologously modified lantibiotics. Lantibiotics from Corynebacterium lipophiloflavum DSM 44291 and Streptococcus agalactiae ATCC 13813, named flavucin and agalacticin, respectively, were fully modified and displayed high antimicrobial activity. The efficiency of functional expression was significantly enhanced when we made use of the native nisin leader cleavage site, instead of an artificial factor Xa site. Thus, we describe an efficient way for heterologous production of active lantibiotics, facilitating a rapid identification of promising molecules.

Structural and Functional Analysis of Cell Wall-anchored Polypeptide Adhesin BspA in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus, GBS) is the predominant cause of early-onset infectious disease in neonates and is responsible for life-threatening infections in elderly and immunocompromised individuals. Clinical manifestations of GBS infection include sepsis, pneumonia, and meningitis. Here, we describe BspA, a deviant antigen I/II family polypeptide that confers adhesive properties linked to pathogenesis in GBS. Heterologous expression of BspA on the surface of the non-adherent bacterium Lactococcus lactis confers adherence to scavenger receptor gp340, human vaginal epithelium, and to the fungus Candida albicans Complementary crystallographic and biophysical characterization of BspA reveal a novel ß-sandwich adhesion domain and unique asparagine-dependent super-helical stalk. Collectively, these findings establish a new bacterial adhesin structure that has in effect been hijacked by a pathogenic Streptococcus species to provide competitive advantage in human mucosal infections.

Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance.

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria. Interestingly, several clinically relevant and human pathogenic strains are inherently resistant towards lantibiotics. The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators. Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding.

Overexpression, purification, crystallization and preliminary X-ray diffraction of the nisin resistance protein from Streptococcus agalactiae.

Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively.

Multiparametric AFM reveals turgor-responsive net-like peptidoglycan architecture in live streptococci.

Cell-wall peptidoglycan (PG) of Gram-positive bacteria is a strong and elastic multi-layer designed to resist turgor pressure and determine the cell shape and growth. Despite its crucial role, its architecture remains largely unknown. Here using high-resolution multiparametric atomic force microscopy (AFM), we studied how the structure and elasticity of PG change when subjected to increasing turgor pressure in live Group B Streptococcus. We show a new net-like arrangement of PG, which stretches and stiffens following osmotic challenge. The same structure also exists in isogenic mutants lacking surface appendages. Cell aging does not alter the elasticity of the cell wall, yet destroys the net architecture and exposes single segmented strands with the same circumferential orientation as predicted for intact glycans. Together, we show a new functional PG architecture in live Gram-positive bacteria.