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Rapidly identifying and quantifying Gram-positive bacteria are crucial to diagnosing and treating bacterial lower respiratory tract infections (LRTIs). This work presents a field-deployable biosensor for detecting Gram-positive bacteria from exhaled breath condensates (EBCs) based on peptidoglycan recognition using an aptamer. Dielectrophoretic force is employed to enrich the bacteria in 10 s without additional equipment or steps. Concurrently, the measurement of the sensor's interfacial capacitance is coupled to quantify the bacteria during the enrichment process. By incorporation of a semiconductor condenser, the whole detection process, including EBC collection, takes about 3 min. This biosensor has a detection limit of 10 CFU/mL, a linear range of up to 10 5 CFU/mL and a selectivity of 1479:1. It is cost-effective and disposable due to its low cost. The sensor provides a nonstaining, culture-free and PCR-independent solution for noninvasive and real-time diagnosis of Gram-positive bacterial LRTIs.

We isolated and analyzed a novel, Gram-stain-positive, aerobic, rod-shaped, non-motile actinobacterium, designated as strain ZFBP1038 T , from rock sampled on the north slope of Mount Everest. The growth requirements of this strain were 10-37 °C, pH 4-10, and 0-6% (w/v) NaCl. The sole respiratory quinone was MK-9, and the major fatty acids were anteiso-C 15:0 and iso-C 17:0 . Peptidoglycan containing meso-diaminopimelic acid, ribose, and glucose were the major cell wall sugars, while polar lipids included diphosphatidyl glycerol, phosphatidyl glycerol, an unidentified phospholipid, and an unidentified glycolipid. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZFBP1038 T has the highest similarity with Spelaeicoccus albus DSM 26341  T (96.02%). ZFBP1038 T formed a distinct monophyletic clade within the family Brevibacteriaceae and was distantly related to the genus Spelaeicoccus. The G + C content of strain ZFBP1038 T was 63.65 mol% and the genome size was 4.05 Mb. Digital DNA-DNA hybridization, average nucleotide identity, and average amino acid identity values between the genomes of strain ZFBP1038 T and representative reference strains were 19.3-25.2, 68.0-71.0, and 52.8-60.1%, respectively. Phylogenetic, phenotypic, and chemotaxonomic characteristics as well as comparative genome analyses suggested that strain ZFBP1038 T represents a novel species of a new genus, for which the name Saxibacter gen. nov., sp. nov. was assigned with the type strain Saxibacter everestensis ZFBP1038 T (= EE 014  T  = GDMCC 1.3024  T  = JCM 35335  T )., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

The bacterial cell wall is a meshwork of crosslinked peptidoglycan strands, with a thickness of up to 50 nm in Firmicutes. Little is known about how proteins move through the cell wall to find sites of enzymatic activity. Cell wall synthesis for cell elongation involves the integration of new peptidoglycan strands by integral membrane proteins, as well as the degradation of existing strands by so-called autolysins, soluble proteins that are secreted through the cell membrane. Autolysins comprise different classes of proteases and glucanases and mostly contain cell-wall binding domains in addition to their catalytic domain. We have studied dynamics of Bacillus subtilis autolysins LytC, a major endopeptidase required for lateral cell wall growth, and LytF, a peptidase acting at the newly formed division site in order to achieve separation of daughter cells. We show that both proteins, fused to moxVenus are present as three distinct populations of different diffusion constants. The fastest population is compatible with free diffusion in a crowded liquid environment, that is similar to that of cytosolic enzymes, likely reflecting autolysins diffusing through the periplasm. The medium mobile fraction can be explained by constrained motion through a polymeric substance, indicating mobility of autolysins through the wall similar to that of DNA-binding proteins within the nucleoid. The slow-mobile fraction are most likely autolysins bound to their specific substrate sites. We show that LytF is more static during exponential phase, while LytC appears to be more active during the transition to stationary phase. Both autolysins became more static in backgrounds lacking redundant other autolysins, suggesting stochastic competition for binding sites. On the other hand, lack of inhibitor IseA or autolysin CwlS lead to an altered preference for polar localization of LytF within the cell wall, revealing that inhibitors and autolysins also affect each other's pattern of localization, in addition to their activity.

Peptidoglycan is a major and essential component of the bacterial cell envelope that confers cell shape and provides protection against internal osmotic pressure. This complex macromolecule is made of glycan strands cross-linked by short peptides, and its structure is continually modified throughout growth via a process referred to as "remodeling." Peptidoglycan remodeling allows cells to grow, adapt to their environment, and release fragments that can act as signaling molecules during host-pathogen interactions. Preparing peptidoglycan samples for structural analysis first requires purification of the peptidoglycan sacculus, followed by its enzymatic digestion into disaccharide peptides (muropeptides). These muropeptides can then be characterized by liquid chromatography coupled mass spectrometry (LC-MS) and used to infer the structure of intact peptidoglycan sacculi. Due to the presence of unusual crosslinks, noncanonical amino acids, and amino sugars, the analysis of peptidoglycan LC-MS datasets cannot be handled by traditional proteomics software. In this chapter, we describe a protocol to perform the analysis of peptidoglycan LC-MS datasets using the open-source software PGFinder. We provide a step-by-step strategy to deconvolute data from various mass spectrometry instruments, generate muropeptide databases, perform a PGFinder search, and process the data output., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Two aerobic, Gram-staining-positive, rod-shaped, endospore-forming, thermophilic bacterial strains, designated FJAT-47801 T and FJAT-47835, were isolated from the sediment collected from Zhangjiang Estuary Mangrove National Nature Reserve in Fujian Province, China. Growth was observed at 25-55 °C (optimum, 50 °C) and pH 7.0-9.0 (optimum, pH 7.0), with up to 4.0% (w/v) NaCl (optimum, without NaCl). Strains FJAT-47801 T and FJAT-47835 showed the highest 16S rRNA gene sequence similarity to Bacillus oleivorans (98.5%). The 16S rRNA gene sequence similarity between FJAT-47801 T and FJAT-47835 was 99.9% indicating they were the same species. Phylogenetic (based on 16S rRNA gene sequences) and phylogenomic (based on 120 conserved bacterial single-copy genes) trees showed that strains FJAT-47801 T and FJAT-47835 should be affiliated to the genus Bacillus. The of menaquinone of strain FJAT-47801 T was MK-7. The major fatty acids of strain FJAT-47801 T were iso-C 15:0 , anteiso-C 15:0 , iso-C 17:0 , and C 16:0 . The major polar lipids strain FJAT-47801 T were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), and phosphatidylglycerol (PG). The genomic DNA G+C content of strain FJAT-47801 T was 39.3%. The average nucleotide identity (84.3%) and the digital DNA-DNA hybridization value (28.1%) between strain FJAT-47801 T and B. oleivorans CCTCC AB 2013353 T were below the cut-off level for species delineation. Based on the above results, strain FJAT-47801 T represents a novel species of the genus Bacillus, for which the name Bacillus litorisediminis sp. nov., is proposed. The type strain is FJAT-47801 T (=GDMCC 1.2712 T  = JCM 34875 T )., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Peptidoglycan (PG), bacterial spores' major structural component in their cortex layers, was recently found to regulate the spore's water content and deform in response to relative humidity (RH) changes. Here, we report that the cortex PG dominates the Bacillus subtilis spores' water-content-dependent morphological and mechanical properties. When exposed to an environment having RH varied between 10% and 90%, the spores and their cortex PG reversibly expand and contract by 30.7% and 43.2% in volume, which indicates that the cortex PG contributes to 67.3% of a spore's volume change. The spores' and cortex PG's significant volumetric changes also lead to changes in their Young's moduli from 5.7 and 9.0 GPa at 10% RH to 0.62 and 1.2 GPa at 90% RH, respectively. Interestingly, these significant changes in the spores' and cortex PG's morphological and mechanical properties are only caused by a minute amount of the cortex PG's water exchange that occupies 28.0% of the cortex PG's volume. The cortex PG's capability in sensing and responding to environmental RH and effectively changing its structures and properties could provide insight into spores' high desiccation resistance and dormancy mechanisms.

Developing molecular models to capture the complex physicochemical architecture of the bacterial cell wall and to study the interaction with antibacterial molecules is an important aspect of assessing and developing novel antimicrobial molecules. We carried out molecular dynamics simulations using an atomistic model of peptidoglycan to represent the architecture for Gram-positive S. aureus. The model is developed to capture various structural features of the Staphylococcal cell wall, such as the peptide orientation, area per disaccharide, glycan length distribution, cross-linking, and pore size. A comparison of the cell wall density and electrostatic potentials is made with a previously developed cell wall model of Gram-negative bacteria, E. coli, and properties for both single and multilayered structures of the Staphylococcal cell wall are studied. We investigated the interactions of the antimicrobial peptide melittin with peptidoglycan structures. The depth of melittin binding to peptidoglycan is more pronounced in E. coli than in S. aureus, and consequently, melittin has greater contacts with glycan units of E. coli. Contacts of melittin with the amino acids of peptidoglycan are comparable across both the strains, and the D-Ala residues, which are sites for transpeptidation, show enhanced interactions with melittin. A low energetic barrier is observed for translocation of a naturally occurring antimicrobial thymol with the four-layered peptidoglycan model. The molecular model developed for Gram-positive peptidoglycan allows us to compare and contrast the cell wall penetrating properties with Gram-negative strains and assess for the first time binding and translocation of antimicrobial molecules for Gram-positive cell walls.

A novel actinomycete strain, designated H8589 T , was isolated from a lake sediment sample, and a polyphasic approach was employed to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene indicated that strain H8589 T formed a monophyletic clade within the genus Sphaerisporangium and was most closely related to Sphaerisporangium siamense DSM 45784  T (97.9% similarity) and Sphaerisporangium rufum DSM 46862  T (97.7% similarity). The draft genome had a length of 10,134,050 bp with a G + C content of 71.2%. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain H8589 T and its closely related Sphaerisporangium species were 80.6 ~ 83.2%, 73.9 ~ 78.4% and 24.5 ~ 29.0%, respectively, which were significantly lower than the widely accepted species-defined threshold. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Whole-cell sugars were glucose, ribose and madurose. The menaquinones were MK-9(H 4 ), MK-9(H 2 ), MK-9(H 6 ) and MK-9. The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, unidentified phospholipids and unidentified aminophospholipids. The major fatty acids were identified as iso-C 16:0 , 10-methyl-C 17:0 and C 17:0. The results of phenotypic properties, genotypic distinctiveness and chemotaxonomic features indicated that strain H8589 T should represent a novel species within the genus Sphaerisporangium, Sphaerisporangium fuscum sp.nov. The type strain is H8589 T (= JCM 34848  T  = CICC 25115  T )., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence., (© 2022. The Author(s).)

Fluorescence microscopy is a method of choice for studying peptidoglycan assembly, but it presents two major challenges: the peptidoglycan must be labeled with a probe that will not perturb the physiological process, and the spatial resolution must reach the nanometer scale to reveal fine details of the synthesis process. This protocol meets both challenges by combining biorthogonal metabolic labeling of peptidoglycan in Streptococcus pneumoniae with super-resolution fluorescence microscopy (dSTORM), also providing cues to adapt it to other bacteria. For complete details on the use and execution of this protocol, please refer to Trouve et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The imaging of peptidoglycan (PGN) dynamics in living bacteria facilitates the understanding of PGN biosynthesis and wall-targeting antibiotics. The main tools for imaging bacterial PGN are fluorescent probes, such as the well-known PGN metabolic labeling probes. However, fluorescent small-molecule probes for labeling key PGN-synthesizing enzymes, especially for transglycosylases (TGases), remain to be explored. In this work, the first imaging probe for labeling TGase in bacterial cell wall studies is reported. We synthesized various fluorescent MoeA-based molecules by derivatizing the natural antibiotic moenomycin A (MoeA), and used them to label TGases in living bacteria, monitor bacterial growth and division cycles by time-lapse imaging, and study cell wall growth in the mecA-carrying methicillin-resistant Staphylococcus aureus (MRSA) strains when the β-lactam-based probes were unsuitable., (© 2021 Wiley-VCH GmbH.)

We studied the effect of bacterial wall peptidoglycan of 7 bacterial species on the competitive properties of human-associated microorganisms. Addition of peptidoglycan to the culture medium did not change the growth characteristics of the test cultures; however, an increase in the antagonism and hydrophobicity of Bifidobacterium sp. and Enterococcus sp. was observed, while the effect on enterobacteria was predominantly indifferent or inhibitory. The effect did not depend much on the source of peptidoglycan and was equally manifested on both indigenous and probiotic strains. The observed new property of peptidoglycan indicates its participation in the formation and functioning of microbiota. The obtained data on the regulation of the properties of microorganisms provide new possibilities for the correction and maintenance of host homeostasis through host-associated microbiota., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

The antigens for acellular pertussis vaccines are made up of protein components that are purified directly from Bordetella pertussis (B. pertussis) bacterial fermentation. As such, there are additional B. pertussis toxins that must be monitored as residuals during process optimization. This paper describes a liquid chromatography mass spectrometry (LC-MS) method for simultaneous analysis of residual protein toxins adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), as well as a small molecule glycopeptide, tracheal cytotoxin (TCT) in a Pertussis toxin vaccine antigen. A targeted LC-MS technique called multiple reaction monitoring (MRM) is used for quantitation of ACT and TCT, which have established limits in drug product formulations. However, DNT is currently monitored in an animal test, which does not have an established quantitative threshold. New approaches for DNT testing are discussed, including a novel standard based on concatenated quantitation sequences for ACT and DNT. Collectively, the method represents a "3-in-1" analytical simplification for monitoring process-related residuals during development of B. pertussis vaccines.

Aims: The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance., Methods and Results: Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units. Morphological examination of bacterial cells was conducted using phase-contrast and scanning electron microscopy. The enzymes EndoT5, endolysin of bacteriophage T5, EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49 turned out to be much less bacteriospecific than the corresponding Escherichia coli phages; they lysed bacteria of the genera Bacillus, Cellulomonas and Sporosarcina, whose PGs had different structures (A1γ, A4α and A4β) and chemical modifications (amidation). The specific lytic activity of phage enzymes was independent of the antibiotic resistance of bacterial cells and was higher when the cells were in the exponential, rather than stationary, growth phase. The analysis of morphological changes showed that the intermediate stage of the endolysin-induced lysis of bacterial cells was the formation of spheroplasts and protoplasts., Conclusions: Endolysins of colibacteriophages RB49, RB43 and T5 have a wide spectrum of antibacterial action, which includes a number of diverse micro-organisms with different PG structures., Significance and Impact of the Study: This is a study of the bacterial selectivity of enzymes degrading bacterial cell wall in relation to the chemical structure of PG. It is shown that endolysins of bacteriophages RB49 and RB43 efficiently lyse cell wall of Gram-positive bacteria of the genus Bacillus and Gram-negative bacteria of the genus Pseudomonas (including an antibiotic-resistant strain). The number of bacterial cells is reduced by 3-6 orders of magnitude, which indicates good prospects for using these enzymes in biotechnology., (© 2020 The Society for Applied Microbiology.)

An aerobic, Gram-staining-positive, rod-shaped, endospore-forming and motile bacterial strain, designated SJY2 T , was isolated from the rhizosphere soil of tea plants (Camellia sinensis var. assamica) collected in the organic tea garden of the Jingmai Pu-erh tea district in Pu'er city, Yunnan, southwest China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Paenibacillus. The closest phylogenetic relative was Paenibacillus filicis DSM 23916 T (98.1% similarity). The major fatty acids (> 10% of the total fatty acids) were anteiso-C 15:0 and isoC 16:0 . The major respiratory quinone was MK-7 and the major polar lipid was diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The peptidoglycan contained glutamic acid, serine, alanine and meso-diaminopimelic acid. Genome sequencing revealed a genome size of 6.71 Mbp and a G + C content of 53.1%. Pairwise determined whole genome average nucleotide identity (gANI) values and digital DNA-DNA hybridization (dDDH) values suggested that strain SJY2 T represents a new species, for which we propose the name Paenibacillus puerhi sp. nov. with the type strain SJY2 T (= CGMCC 1.17156 T  = KCTC 43242 T ).

A Gram-staining positive aerobic bacterium, designated TLY-12 T , was isolated from the Pu-erh tea pile-fermentation process in Pu'er city, Yunnan, China. Strain TLY-12 T grew at 15-37 °C (optimum, 30 °C), pH 6.0-11.0 (optimum, pH 9.0) and 0-9.0% (w/v) NaCl (optimum, 3.0%). The major cellular fatty acids were anteiso-C 15:0 , C 16:0 and iso-C 16:0 . The respiratory quinone were menaquinones MK-9 (H 2 ) and MK-9 (H 4 ). The polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), phosphoglycolipid (PGL), glycolipid (GL) and an unidentified phospholipid (PL). The peptidoglycan contained glutamic acid, aspartic acid, alanine and lysine, with the last named being the diagnostic diamino acid. Whole-cell sugars of the isolate were ribose, galactose and glucose. Phylogenetic analyses of 16S rRNA gene showed that this strain belonged to the family Promicromonosporaceae, and was most closely related to Isoptericola cucumis DSM 101603  T , which gave sequence similarity of 97.9%. Genome sequencing revealed a genome size of 3.91 Mbp and a G + C content of 75.0%. Average nucleotide identity and digital DNA-DNA hybridization values were all below the species threshold of described Promicromonosporaceae species. Genome phylogenetic analysis showed that strain TLY-12 T formed a separate evolutionary branch, and was parallel to other related genera of Promicromonosporaceae. Based on the phylogenetic, phenotypic, chemotaxonomic and genome pairwise data, strain TLY-12 T is considered to represent a novel species in a new genus in the family Promicromonosporaceae, for which the name Puerhibacterium puerhi gen. nov, sp. nov. is proposed. The type strain is TLY-12 T (= CGMCC 1.17157 T  = KCTC 49467 T ).

A gram-stain positive, aerobic, motile, rod-shaped bacterium, designated strain LAM7117 T , was isolated from a sulfonylurea herbicides degrading consortium enriched with birch forest soil. The optimal temperature and pH for the growth of strain LAM7117 T were 35 °C and 7.5, respectively. Strain LAM7117 T could grow in the presence of NaCl with concentration up to 9% (w/v). Strain LAM7117 T formed a distinct phylogenetic subclade within the genus Arthrobacter in the phylogenetic trees built with 16S rRNA gene sequences and shared the highest similarity with A. crystallopoietes JCM 2522 T (97.7%). The values of digital DNA-DNA relatedness and Avery Nucleotide Identity based on the genome sequences between LAM7117 T and A. crystallopoietes JCM 2522 T were 21.4 and 77.4%, respectively. The genomic DNA G + C content was 65.9 mol%. The major cellular fatty acids were anteiso-C 15:0 , iso-C 16:0 and anteiso-C 17:0 . The cell wall peptidoglycan contained the amino acids as glycine, lysine, alanine and glutamic acid. The major polar lipids present in strain LAM7117 T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidyl inositol, two unidentified glycolipids and one unidentified lipid. The predominant menaquinones of strain LAM7117 T were MK-8 and MK-9. Based on the phenotypic characteristics, chemotaxonomic data and genotypic analyses, strain LAM7117 T should be classified as a novel species of genus Arthrobacter, for which the name Arthrobacter sulfonylureivorans sp. nov. is proposed. The type strain is LAM7117 T (= JCM 32824 T  = CGMCC 1.16681 T ).

Clostridium difficile is an anaerobic and spore-forming bacterium responsible for 15-25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The C. difficile peptidoglycan is largely N -deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as N -deacetylases, and this N -deacetylation modulates host-pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. C. difficile genome analysis showed that 12 genes potentially encode N -deacetylases; however, which of these N -deacetylases are involved in peptidoglycan N- deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan N -deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the N- deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan N -deacetylation in C. difficile and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine N -deacetylase. However, its impact on N -deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan N -deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of N -deacetylation in C. difficile is not linked to a decrease in virulence., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Coullon et al.)

The interaction between host immunity and bacterial cells plays a pivotal role in a variety of human diseases. The bacterial cell wall component peptidoglycan (PG) is known to stimulate an immune response, which makes PG a distinctive recognition element for unveiling these complicated molecular interactions. Pattern recognition receptor (PRR) proteins are among the critical components of this system that initially recognize molecular patterns associated with microorganisms such as bacteria and fungi. These molecular patterns are mostly embedded in the bacterial or fungal cell wall structure and can be released and presented to the immune system in various situations. Nonetheless, detailed knowledge of this recognition is limited due to the diversity among the PG polymer and its fragments; the subsequent responses by multiple hosts add more complexity. Here, we discuss how our understanding of the role and molecular mechanisms of the well-studied PRR, the NOD-like receptors (NLRs), in the human immune system has evolved in recent years. We highlight the instances of other classes of proteins with similar behavior in the recognition of PG that have been identified in other microorganisms such as yeasts. These proteins are particularly interesting because a network of cellular interactions exists between human host cells, bacteria and yeast as a part of the normal human flora. To support our understanding of these interactions, we provide insight into the chemist's toolbox of peptidoglycan probes that aid in the investigations of the behaviors of these proteins and other biological contexts relevant to the sensing and recognition of peptidoglycan. The importance of these interactions in human health for the development of biomarkers and biotherapy is highlighted.

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph-mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

Tuberculosis killed 1.5 million people in 2018. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the most deadly infectious bacteria in the world. A strength of mycobacterial pathogens - their formidable cell wall - could also be one of their greatest molecular vulnerabilities. As in other bacteria, peptidoglycan (PG) maintenance and integrity is essential to mycobacterial survival. But Mtb PG is unique, and a better understanding of its biosynthetic machinery could lead to new drugs or more effective treatment regimens. Such investigations are being accelerated by the application of fluorescent probes, including those based on vancomycin, β-lactams, PG stem mimics, d-amino acids, and reactive glycans. This review will describe how fluorescent probes are being used to uncover new information on the regulation and drug susceptibility of two classes of enzymes that fortify the Mtb PG: the penicillin-binding proteins and the L,D-transpeptidases., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A Gram-staining-positive and aerobic coccus with the ability to degrade petroleum bacterium, designated Y42 T , was isolated from the Lenghu oil field located in the northern margin of the Qaidam Basin. Phylogenetic and signature nucleotides analyses revealed that strain Y42 T belongs to the genus Planococcus. The multiple sequence alignments of 16S rRNA and housekeeping genes showed that strain Y42 T formed a distinct lineage with the other Planococcus clade. The average nucleotide identity (ANI) and DNA-DNA hybridization values (DDH) between strain Y42 T and the reference strains were 69.5-70.1 and 19.4-21.7%, respectively, which values were below the threshold for species delineation. The major fatty acids of strain Y42 T were anteiso-C 15:0 . The respiratory quinone was MK-7 (71.8%) as the predominant menaquinone followed the MK-6 (28.2%) and the cell-wall hydrolysates contained LL-diaminopimelic acid. The polar lipid was composed of diphosphatidyl glycerol, phosphatidyl glycerol, phosphoglycolipid, aminophospholipid and four unidentified lipids. The peptidoglycan type was A4α (L-Lys-D-Glu). The strain Y42 T possessed larger genome (approximately 4 MB) and revealed obvious differences for the abundance of the COG categories compared with the other Planococcus bacteria. Also, the strain Y42 T also possessed more unique orthologous proteins. The structural characteristics of the strain Y42 T genome provided a competitive advantage for better survival in petroleum-polluted environments. Combined with the 16S rRNA gene and genome sequence, phenotypic as well as chemotaxonomic characterisations, strain Y42 T is considered to represent a novel species of the genus Planococcus, for which the name Planococcus lenghuensis sp. nov. be proposed. The type strain is Y42 T (= CGMCC 1.15921 T  = JCM 32719 T ).

A novel aerobic marine actinobacterium (strain S5-52 T ) belonging to the genus Glutamicibacter was isolated from the coral Favia veroni sampled from the Andaman Sea, India. Cells are Gram stain positive and rod shaped. The DNA G+C content was 58.7 mol%. The major quinones were MK-8 and MK-9. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, glycolipid, trimannosyldiacylglycerol, phospholipid and dimannosylglyceride. The peptidoglycan type was A4α. Strain S5-52 T showed a maximum 16S rRNA similarity of 99.36% with Glutamicibacter halophytocola DSM 101718 T . The genome of strain S5-52 T was 3.57 Mb that contains 3274 protein coding sequences (CDS). DNA-DNA similarity and ANI values between S5-52 T and the reference strains were below 70% and 95-96%, respectively. Analysis of genomic reduction events in the evolutionary path from the LUCA (last universal common ancestor) to G. mishrai LMG 29155 T and G. halophytocola DSM 101718 T exhibit a number of genes involved in amino acid metabolism, cell wall biogenesis and replication, recombination and repair mechanism that reduced in both the species. Based on phenotypic, chemotaxonomic properties and comparative genomic studies, the strain S5-52 T is considered a novel species of the genus Glutamicibacter, for which the name Glutamicibacter mishrai sp. nov. is proposed. The type strain is S5-52 T (= KCTC 39846 T  = LMG 29155 T ).

A strictly anaerobic bacterium, designated as strain KGMB-03357 T , was isolated from the faeces of a healthy Korean selected by Bundang Seoul National University based on health status. Cells of strain KGMB03357 T are Gram-stain-positive, non-motile, non-spore-forming, and observed as straight or curved rods. The isolate grew at 10-45°C (optimum temperature of 40°C) and a pH range of 5.1-10.5 (optimum pH of 6.8). Analysis of phylogenetic trees based on the 16S rRNA gene sequences revealed that strain KGMB03357 T forms a lineage within the genus Anaerotignum, and is most closely related to Anaerotignum lactatifermentans G17 T (= KCTC 15066 T , 96.1%), Anaerotignum propionicum DSM 1682T (= KCTC 5582 T , 94.9%), Anaerotignum neopropionicum DSM 03847 T (= KCTC 15564T, 94.9%), and Anaerotignum aminivorans SH021 T (= KCTC 15705T, 94.8%). The ANI values between strain KGMB 03357T and members of the genus Anaerotignum were 73.3-71.0%, which are below the ANI criterion for interspecies identity. The DNA G + C content based on the whole-genome sequence is 47.3 mol%. The major cellular fatty acids of strain KGMB03357 T are C 16:0 , C 18:0 , C 18∶1 cis 9, and anteiso-C 15∶0 . Strain KGMB03357T contains meso-diaminopimelic acid as the diagnostic amino acid in the cell wall peptidoglycan. Based on the phenotypic, phylogenetic, and genomic properties, strain KGMB 03357 T represents a novel species of the genus Anaerotignum, for which the name Anaerotignum faecicola sp. nov. is proposed. The type strain is KGMB03357 T (= KCTC 15736T = DSM 107953 T ).

A Gram-stain positive, short rod-shaped, aerobic, motile by means of gliding, yellow-pigmented actinobacterium, designated strain DD4a T , was isolated from dry yellow foxtail. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DD4a T is closely related to Amnibacterium soli MB78 T (98.4% similarity), Amnibacterium kyonggiense KSL51201-037 T (98.2%) and Amnibacterium endophyticum 1T4Z-3 T (97.43%). Strain DD4a T forms yellow colonies on R2A agar medium. The peptidoglycan was found to contains diaminopimelic acid (which is a diagnostic cell wall diamino acid), alanine, glutamic acid and lysine. The polar lipids diphosphatidylglycerol, phosphatidylglycerol, six unidentified glycolipids and an unidentified polar lipid were found to be present in strain DD4a T . The major cellular fatty acids anteiso-C 15:0 (42.9%) and iso-C 16:0 (34.6%) were found in strain DD4a T . The predominant respiratory quinones were found to be MK-11 and MK-12. The DNA G+C content of strain DD4a T is 73.9 mol%. DNA-DNA relatedness of strain DD4a T with A. soli MB78 T , A. kyonggiense KSL51201-037 T , and A. endophyticum 1T4Z-3 T were 53.3% (± 1.1%), 47.0% (± 0.5%), and 47.9% (± 0.9%), respectively. The digital DNA-DNA hybridisation and average nucleotide identity values between strain DD4a T and A. kyonggiense KSL51201-037 T were determined to be 26.1% and 82.7%. On the basis of phenotypic, genotypic, chemotaxonomic and phylogenetic analysis, DD4a T represents a novel member of the genus Amnibacterium, for which the name Amnibacterium setariae sp. nov., is proposed. The type strain of Amnibacterium setariae is DD4a T (= KACC 19817 T  = JCM 32878 T ).

Two novel actinobacterial strains, MS1-9 T and NGC1-4, were isolated from roots of Musa (ABB) cv. 'Kluai Namwa', collected from Chachoengsao province, and Musa (ABB) cv. 'Kluai Chang', from Suphan Buri province, Thailand, respectively. Comparative analysis of 16S rRNA gene (98.0 to 98.9% similarity), gyrase subunit B (gyrB) gene and whole-genome sequences emphasised that the strains MS1-9 T and NGC1-4 showed closely related with Micromonospora peucetia DSM 43363 T , M. krabiensis JCM 12869 T and M. avicenniae DSM 45758 T , respectively. Strains MS1-9 T and NGC1-4 contained meso-diaminopimelic acid in cell-wall peptidoglycan. Whole-cell sugars were glucose, xylose, mannose, and ribose. The acyl type of peptidoglycan was glycolyl. MK-10(H 6 ), MK-9(H 6 ), and MK-10(H 8 ) were presented as the major menaquinones. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol were detected as predominant phospholipid profiles. The major cellular fatty acids consisted of iso-C 15:0 , anteiso-C 15:0 , anteiso-C 17:0 , iso-C 17:0 and C 17:0 . The DNA G+C content of strains MS1-9 T and NGC1-4 were 72.2 and 72.3mol%, respectively. Draft genome sequences indicated by ANI values and digital DNA-DNA hybridisation analysis asserted that the strains MS1-9 T and NGC1-4 should be represented as a novel species within the genus Micromonospora for which the name Micromonospora musae sp. nov. is proposed. The type strain is MS1-9 T (=JCM 32149 T =TISTR 2659 T )., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Fifteen bifidobacterial strains were obtained from faeces of Rousettus aegyptiacus; after grouping them by RAPD PCR only eight were selected and characterized. Analysis of 16S rRNA and of five housekeeping (hsp60, rpoB, clpC, dnaJ, dna G) genes revealed that these eight strains were classified into five clusters: Cluster I (RST 8 and RST 16 T ), Cluster II (RST 9 T and RST 27), Cluster III (RST 7 and RST 11), Cluster IV (RST 19), Cluster V (RST 17) were closest to Bifidobacterium avesanii DSM 100685 T (96.3%), Bifidobacterium callitrichos DSM 23973 T (99.2% and 99.7%), Bifidobacterium tissieri DSM 100201 T (99.7 and 99.2%), Bifidobacterium reuteri DSM 23975 T (98.9%) and Bifidobacterium myosotis DSM 100196 T (99.3%), respectively. Strains in Cluster I and strain RST 9 in Cluster II could not be placed within any recognized species while the other ones were identified as known species. The average nucleotide identity values between two novel strains, RST 16 T and RST 9 T and their closest relatives were lower than 79% and 89%, respectively. In silico DNA-DNA hybridization values for those closest relatives were 32.5 and 42.1%, respectively. Phenotypic and genotypic tests demonstrated that strains in Cluster I and RST 9 T in Cluster II represent two novel species for which the names Bifidobacterium vespertilionis sp. nov. (RST 16 T =BCRC 81138 T =NBRC 113380 T =DSM 106025 T ; RST 8=BCRC 81135=NBRC 113377) and Bifidobacterium rousetti sp. nov. (RST 9 T =BCRC 81136 T =NBRC 113378 T =DSM 106027 T ) are proposed., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Microbiota analysis of blown pack spoiled salami revealed five distinguishable Lactobacillus isolates we could not assign to a known species. Two of the isolates (TMW 1.2172 T and TMW 1.1920) are rod-shaped, whilst three isolates (TMW 1.2098 T , TMW 1.2118 and TMW 1.2188) appear coccus shaped or as short rods. All isolates are Gram-stain positive, facultative anaerobic, catalase and oxidase negative, non-motile and non-sporulating. Phylogenetic analysis of the 16S rRNA, dnaK, pheS and rpoA gene sequences revealed two distinct lineages within the genus Lactobacillus (L.). The isolates are members of the Lactobacillus alimentarius group with Lactobacillus ginsenosidimutans DSM 24154 T (99.4% 16S similarity), Lactobacillus versmoldensis DSM 14857 T (97.9%) and Lactobacillus furfuricola DSM 27174 T (97.7%) as phylogenetic closest related species and L. alimentarius DSM 20249 T (97.7%) and Lactobacillus paralimentarius DSM 13961 T (97.5%) as closest relatives, respectively. Average Nucleotide Identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the isolates and their close related type strains are lower than 80% and 25%, respectively. For both designated type strains, the peptidoglycan type is A4α l-Lys-d-Asp and the major fatty acids are C 16:0 , C 18:1ω9c and summed feature 7. Based on phylogenetic, phenotypic and chemotaxonomic analysis we demonstrated that the investigated isolates belong to two novel Lactobacillus species for which we propose the names Lactobacillus salsicarnum with the type strain TMW 1.2098 T =DSM 109451 T =LMG 31401 T and Lactobacillus halodurans with the type strain TMW 1.2172 T =DSM 109452 T =LMG 31402 T ., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non-soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N-acetyl-muramidase, cleaving the β 1→4 bound between N-acetylglucosamine and N-acetylmuramic acid. Here, we report the use of CZE-MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID-MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A Gram-negative, aerobic, short rodshaped, asporogenous bacterium, designated CBS5Q-3 T , was isolated from a surface-sterilised root of Ficus microcarpa Linn. f. collected from Guangxi, China and investigated by a polyphasic approach to determine its taxonomic position. Strain CBS5Q-3 T was found to grow optimally with 2% (w/v) NaCl at 30 °C, pH 7.0-8.0. Substrate mycelia and aerial mycelia were not formed, and no diffusible pigments were observed on the media tested. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CBS5Q-3 T is closely related to species of genus Jiella and shares high 16S rRNA gene sequence similarity of 98.1% with Jiella aquimaris JCM 30119 T . The average nucleotide identity and in silico DNA-DNA hybridization values between strain CBS5Q-3 T and J. aquimaris JCM 30119 T were 82.8% and 26.0%, respectively. The DNA G + C content of strain CBS5Q-3 T was determined to be 66.5 mol %. The cell wall peptidoglycan was found to contain meso-diaminopimelic acid and ubiquinone Q-10 identified as the respiratory lipoquinone. The polar lipids were found to be comprised of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidylethanolamine and three unidentified aminolipids, while the major fatty acids were identified as C 18:1 ω7c and cyclo-C 19:0 ω8c. On the basis of phylogenetic, chemotaxonomic and phenotypic data, strain CBS5Q-3 T can be concluded to represent a novel species of the genus Jiella, for which the name Jiella endophytica sp. nov. is proposed. The type strain is CBS5Q-3 T (= JCM 33167 T  = CGMCC 1.13863 T ).

A strain of a Gram-positive, strictly aerobic, motile, rod-shaped, endospore forming bacterium was originally isolated from rhizospheric soil of a pepper plant when screening and bioprospecting for plant beneficial microorganisms. Phylogenetic analysis of the 16S rRNA gene sequences indicated that this strain, PB300 T , is closely related to Lysinibacillus macroides DMS 54 T (99.6%) and Lysinibacillus xylanilyticus DSM 23493 T (99.4%). In phenotypic characterisation, the novel strain was found to grow between 15 and 40 °C and tolerate up to 10% (w/v) NaCl. Furthermore, the strain was found to grow in media with pH 5 to 10 (optimal growth at pH 7.0). The predominant cellular fatty acids were observed to be iso-C 15 : 0 (56.6 %), anteiso-C 15 : 0 (14.6%), C 16 :1 ω7C alcohol (9.3%) and C 16 : 0 (7.1%). The cell wall peptidoglycan contains lysine-aspartic acid, as in its close relatives. A draft genome was completed and the DNA G + C content was determined to be 37.5% (mol content). A phylogenomic analysis of the core genome of the new strain and 5 closely related type strains of the genus Lysinibacillus revealed that this strain formed a distinct monophyletic clade with the nearest neighbour being Lysinibacillus boronitolerans. DNA-DNA relatedness studies using in silico DNA-DNA hybridizations (DDH) showed relationships for the new strain were below the species threshold of 70%. Based upon the consensus of phylogenetic and phenotypic analyses, we conclude that this strain represents a novel species within the genus Lysinibacillus, for which the name Lysinibacillus capsici sp. nov. is proposed, with type strain PB300 T (= NRRL B-65515 T , = CCUG 72241 T ).

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PG Bb ) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PG Bb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PG Bb , which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PG Bb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PG Bb In addition, systemic administration of PG Bb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PG Bb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PG Bb fragments during growth suggests a potential role for PG Bb in the immunopathogenesis of other Lyme disease manifestations., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

A strictly anaerobic, Gram-stain-positive, catalase-negative, non-motile, rod-shaped bacterium, designated SG0102 T , was isolated from the small intestine of a swine. Optimal growth occurred at 37°C and pH 7.0. Furthermore, growth was observed in the presence of up to 3% (w/v) NaCl but not at salinity levels higher than 4%. The comparative analysis of 16S rRNA gene sequences showed that strain SG0102 T was most closely related to Kandleria vitulina DSM 20405 T (93.3%), followed by Catenibacterium mitsuokai KCTC 5053 T (91.1%), Sharpea azabuensis KCTC 15217 T (91.0%), and Eggerthia catenaformis DSM 5348 T (89.6%). The average nucleotide identity values between strain SG0102 T and related species, K. vitulina DSM 20405 T , C. mitsuokai KCTC 5053 T , S. azabuensis KCTC 15217 T , and E. catenaformis DSM 5348 T , were 71.0, 69.3, 70.0, and 69.2%, respectively. The phylogenetic analysis based on 16S rRNA gene sequence revealed that strain SG0102 T belonged to the family Erysipelotrichaceae in the class Erysipelotrichia. The DNA G + C content of the strain SG0102 T was 39.5 mol%. The major cellular fatty acids (> 10%) of strain SG0102 T were C 16:0 , C 16:0 dimethyl acetal, and C 18:2 ω 9/12c. The cell wall peptidoglycan of strain SG0102 T contained the meso-diaminopimelic acid. The strain SG0102 T produced lactic acid as a major end product of fermentation. These distinct phenotypic and phylogenetic properties suggest that strain SG0102 T represents a novel species in a novel genus of the family Erysipelotrichaceae, for which the name Intestinibaculum porci gen. nov. sp. nov. is proposed. The type strain is SG0102 T (= KCTC 15725 T = NBRC 113396 T ).

The bacterial cell wall is composed of the peptidoglycan (PG), a large polymer that maintains the integrity of the bacterial cell. Due to its multi-gigadalton size, heterogeneity, and dynamics, atomic-resolution studies are inherently complex. Solid-state NMR is an important technique to gain insight into its structure, dynamics and interactions. Here, we explore the possibilities to study the PG with ultra-fast (100 kHz) magic-angle spinning NMR. We demonstrate that highly resolved spectra can be obtained, and show strategies to obtain site-specific resonance assignments and distance information. We also explore the use of proton-proton correlation experiments, thus opening the way for NMR studies of intact cell walls without the need for isotope labeling., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A bacterial strain, S13-1-2-1 T , was isolated from a soil sample collected in Gyeongsangnam-do province, South Korea. Cells were observed to be Gram-stain negative, short rod-shaped and colonies to be pale pink in colour. Analysis of 16S rRNA gene sequences identified this strain as a member of the genus Deinococcus in the family Deinococcaceae, with high levels of sequence similarity with Deinococcus ficus CC-FR2-10 T (97.9%) and Deinococcus enclensis NIO-1023 T (95.4%). Growth of strain S13-1-2-1 T was observed at 10-42 °C, pH 6-8, and in the presence of 0-1.0% NaCl. The isolate was found to exhibit resistance to gamma radiation (D 10 10.1 KGy) and UV-light (D 10 612 J/m 2 ). The major peptidoglycan amino acids were identified as D-glutamic acid, glycine, alanine and L-ornithine. The predominant respiratory quinone of the strain was identified as menaquinone-8, the major fatty acids were found to be C 16:1 ω7c (31.4%), C 16:0 (18.4%), and C 17:1 ω8c (17.4%) and the major polar lipids were observed to be an unidentified phosphoglycolipid and an unidentified glycolipid. The genomic DNA G + C content of the strain was determined to be 69.2 mol%. DNA-DNA hybridization with D. ficus showed a relatedness value of 31.5 ± 4.2%. The DNA-DNA hybridization result and the differentiating phenotypic properties clearly indicate that strain S13-1-2-1 T represents a novel species in the genus Deinococcus, for which the name Deinococcus terrigena sp. nov. is proposed. The type strain is S13-1-2-1 T (= KCTC 33939 T  = JCM 32248 T ).

Strain CQ16Z1 T was isolated from jamiecosley, a traditional Chinese pickle. The isolate was Gram-positive, non-motile, non-spore-forming, facultatively anaerobic, catalase-negative, and long rod-shaped. The optimal temperature for growth was 37 °C and the DNA G + C content was 39.1 mol%. The results of 16S rRNA and rpoA gene sequencing, DNA-DNA hybridization, and peptidoglycan type analyses indicated that strain CQ16Z1 T belongs to the recognized species Lactobacillus futsaii. However, the analysis results of pheS gene sequencing, amplified fragment length polymorphism, phenotypic profiles, cellular fatty acid, cell-wall monosaccharide determination, and cell morphology revealed that the novel strain was obviously different from the type strain L. futsaii JCM17355 T , and had genetic relationship with Weissella cibaria to a certain degree, suggesting that the novel strain represents a novel subspecies of L. futsaii, for which the following names are proposed: L. futsaii subsp. futsaii subsp. nov. (type strain YM0097 T  = JCM 17355 T  = BCRC 80278 T ) and L. futsaii subsp. chongqingii subsp.nov., with the type strain CQ16Z1 T (= CCTCC AB2017187 T  = KCTC 21089 T ).

A moderately halophilic and alkalitolerant bacterial strain NKC1-1 T was isolated from commercial kimchi in Korea. Strain NKC1-1 T was Gram-stain-positive, aerobic, rod-shaped, non-motile, and contained diaminopimelic acid-type murein. Cell growth was observed in a medium containing 0-25% (w/v) NaCl (optimal at 10% [w/v]), at 20-40°C (optimal at 37°C) and pH 6.5-10.0 (optimal at pH 9.0). The major isoprenoid quinone of the isolate was menaquinone-7, and the major polar lipids were phosphatidylglycerol and unidentified phospholipids. Cell membrane of the strain contained iso-C 17:0 and anteiso-C 15:0 as the major fatty acids. Its DNA G + C content was 45.2 mol%. Phylogenetic analysis indicated the strain to be most closely related to Geomicrobium halophilum with 92.7-92.9% 16S rRNA gene sequence similarity. Based on polyphasic taxonomic evaluation with phenotypic, phylogenetic, and chemotaxonomic analyses, the strain represents a novel species in a new genus, for which the name Salicibibacter kimchii gen. nov., sp. nov. is proposed (= CECT 9537 T ; KCCM 43276 T ).

Background: Multidrug-resistant (MDR) Streptococcus pneumonia constitute a major worldwide public health concern., Materials and Methods: In our preliminary study, PEGylated nano-self-assemblies of bacitracin A (PEGylated Nano-BA 12K ) showed strong antibacterial potency against reference S. pneumonia strain (ATCC 49619). In this study, the possibility of applying PEGylated Nano-BA 12K against penicillin-resistant S. pneumonia was further investigated. In addition, the underlying antibacterial mechanism of PEGylated Nano-BA 12K against both sensitive and resistant S. pneumonia was also clarified systematically, since S. pneumonia was naturally resistant to its unassembled counterpart bacitracin A (BA)., Results: PEGylated Nano-BA 12K showed strong antibacterial potency against 13 clinical isolates of S. pneumonia , including five penicillin-resistant strains. Structural changes, partial collapse, and even lysis of both penicillin-sensitive and penicillin-resistant bacteria were observed after incubation with PEGylated Nano-BA 12K via transmission electron microscopy and atomic force microscopy. Thus, the cell wall or/and cell membrane might be the main target of PEGylated Nano-BA 12K against S. pneumonia . PEGylated Nano-BA 12K exhibited limited effect on the permeabilization and peptidoglycan content of cell wall. Surface pressure measurement suggested that PEGylated Nano-BA 12K was much more tensioactive than BA, which was usually translated into a good membranolytic effect, and is helpful to permeabilize the cell membrane and damage membrane integrity, as evidenced by depolarization of the membrane potential, permeabilization of membrane and leakage of calcein from liposomes., Conclusion: Collectively, great cell membrane permeability and formidable membrane disruption may work together for the strong antibacterial activity of PEGylated Nano-BA 12K against S. pneumonia . Taken together, PEGylated Nano-BA 12K has excellent potential against both penicillin-sensitive and penicillin-resistant S. pneumonia and might be suitable for the treatment of S. pneumonia infectious diseases., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Peptidoglycan (PG) is the main structural component of bacterial envelopes. It protects bacterial cells against variations in osmotic pressure and cell lysis. The newly discovered Escherichia coli factor ElyC has been shown to be important for peptidoglycan biosynthesis at low temperatures. PG production in Δ elyC mutant cells is totally blocked after a few hours of growth at 21°C, triggering cell lysis. In this study, we took a candidate approach to identify genetic suppressors of the Δ elyC mutant cell lysis phenotype. We identified the periplasmic proteins DsbG and Spy as multicopy suppressors and showed that their overproduction restores PG biosynthesis in the Δ elyC mutant. Interestingly, we found that DsbG acts by a novel mechanism, which is independent of its known reductase activity and substrates. DsbG, like Spy, acts as a chaperone to reduce the amounts of protein aggregates in the envelopes of Δ elyC cells. In fact, we found that the amount of protein aggregates was greater in the Δ elyC mutant than in the wild type. Taken together, our results show a protein-folding defect in the envelope compartments of Δ elyC cells that blocks PG production, and they reveal a new physiological activity of DsbG. IMPORTANCE Peptidoglycan biosynthesis is a dynamic and well-controlled pathway. The molecular assembly of PG and the regulatory pathways ensuring its maintenance are still not well understood. Here we studied the newly discovered Escherichia coli factor ElyC, which is important for PG biosynthesis at low temperatures. We revealed an important protein-folding defect in the Δ elyC mutant and showed that overproduction of the periplasmic chaperone DsbG or Spy was sufficient to correct the protein-folding defect and restore PG biosynthesis. These results show that the PG defect in the absence of ElyC is caused, at least in part, by a protein-folding problem in the cell envelope. Furthermore, we showed, for the first time, that the periplasmic protein DsbG has chaperone activity in vivo ., (Copyright © 2018 American Society for Microbiology.)

A novel, Gram-stain-positive, microaerophilic to aerobic, non-endospore-forming, no-motile and rod-shaped bacterium designated Q14 T was isolated from mangrove soil samples collected on chengmai, Hainan province, China. Strain Q14 T was able to grow at 10-40 °C (optimum 30 °C), pH 5.5-10.0 (optimum 6.5-8.0) and with 0.5-6% (w/v) NaCl (optimum 1%). The genomic DNA G+C content was 70.1%. The chemotaxonomic analysis showed that the predominant isoprenoid quinone was MK-12 and the major fatty acids were anteiso-C 15:0 , iso-C 17:0 and anteiso-C 17:0 . The major polar lipids of strain Q14 T were diphosphatidylglycerol, phosphatidylglycerol and one glycolipid. The strain Q14 T contained 2,4-diaminobutylic acid (A 2 bu), alanine acid, glutamic acid and glycine in the peptidoglycans. The phylogenetic analysis and DNA-DNA hybridization, along with the phenotypic and chemotaxonomic characteristics, indicate that strain Q14 T as a novel species of the genus Agromyces, for which the name Agromyces mangrovi sp. nov. is proposed. The type strain is Q14 T (= MCCC 1K03191 T  = KCTC 39814 T ).

SYP-B2174 T is a yellow-pigmented, Gram-positive, non-motile, and rod-shaped actinobacterium isolated from the rhizospheric soil of Aquilegia viridiflora Pall. collected from the Xinjiang uygur autonomous region of China. The strain's growth temperature ranges from 1 to 35°C, with an optimal growth being observed at 28°C. Growth occurs from 0 to 5% NaCl and at pH 6-8, with optimal growth being observed in 1% NaCl at pH 7. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Leifsonia kafniensis JCM 17021 T and Leifsonia psychrotolerans DSM 22824 T with similarities of 97.8 and 97.6%, respectively. The DNA-DNA hybridization values of the strain SYP-B2174 T to its closest phylogenetic neighbors were significantly lower than 35.7%. The strain was identified as a novel species of the genus Leifsonia judging by the coryneform morphology, peptidoglycans based upon 2,4-diaminobutyric acid, principal phospholipids phosphatidylglycerol and diphosphatidylglycerol, major menaquinone MK-11, predominant fatty acids of anteiso-C 15:0 , anteiso-C 17:0 , and iso-C 16:0 , and a DNA G + C base composition of 68.7 mol%, for which the name Leifsonia flava sp. nov. is proposed. The type strain is SYP-B2174 T (= CGMCC 1.15856 T = DSM 105144 T = KCTC 39963T).

In the present work, Principal coordinate analysis (PCoA) is introduced to develop a robust model to classify the chromatographic data sets of peptidoglycan sample. PcoA captures the heterogeneity present in the data sets by using the dissimilarity matrix as input. Thus, in principle, it can even capture the subtle differences in the bacterial peptidoglycan composition and can provide a more robust and fast approach for classifying the bacterial collection and identifying the novel cell wall targets for further biological and clinical studies. The utility of the proposed approach is successfully demonstrated by analysing the two different kind of bacterial collections. The first set comprised of peptidoglycan sample belonging to different subclasses of Alphaproteobacteria. Whereas, the second set that is relatively more intricate for the chemometric analysis consist of different wild type Vibrio Cholerae and its mutants having subtle differences in their peptidoglycan composition. The present work clearly proposes a useful approach that can classify the chromatographic data sets of chromatographic peptidoglycan samples having subtle differences. Furthermore, present work clearly suggest that PCoA can be a method of choice in any data analysis workflow., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: PBP4 is typically considered unimportant for conferring high-level β-lactam resistance in Staphylococcus aureus. Mutations in PBP4 have been associated with β-lactam non-susceptibility among natural strains of S. aureus. We have previously shown that PBP4 can mediate high-level β-lactam resistance in laboratory-generated strains passaged in β-lactam antibiotics. Mutations in the pbp4 promoter that up-regulate its expression and missense mutations that surround PBP4's active site were detected in high frequencies among passaged strains, suggesting PBP4 plays a key role in resistance. How these mutations participate in PBP4's ability to provide high-level β-lactam resistance is unknown., Objectives: To determine whether enzymatic activity of PBP4 is required for high-level β-lactam resistance and to investigate how the pbp4-associated mutations provide β-lactam resistance., Methods: The catalytic activity of PBP4 was disabled through introduction of a serine to alanine point mutation in its active site (Ser-75→Ala) in a representative and well-studied passaged strain, CRB. pbp4 promoter and missense mutations detected in CRB were reconstituted in a WT strain individually and in combination. β-Lactam resistance of the resultant strains was evaluated by population analysis. Bacterial peptidoglycan composition of the pbp4 mutants was evaluated with and without antibiotic treatment using LC., Results: PBP4 inactivation imparted complete β-lactam susceptibility of CRB. Reconstitution of PBP4 missense mutations alone did not impart β-lactam resistance, but did so in synergism with pbp4 promoter mutation. A similar synergistic interaction of pbp4 mutations was observed in enhanced peptidoglycan cross-linking upon antibiotic treatment., Conclusions: PBP4's activity and overexpression both contribute to high-level β-lactam resistance.

Gram-stain-positive, catalase and oxidase-negative and short rod-shaped bacterium C10 with occasional branching was isolated under strictly anaerobic conditions from the rumen fluid of a red deer (Cervus elaphus) in the course of study attempting to uncover new xylanolytic and cellulolytic rumen bacteria inhabiting the digestive tract of wild ruminants in the Czech Republic. The anaerobic M10 medium containing bovine rumen fluid and carboxymethylcellulose as a defined source of organic carbon was used in the process of bacterial isolation. The 16S rRNA gene similarity revealed recently characterized new species Actinomyces succiniciruminis Am4 T (GenBank accession number of the gene retrieved from the complete genome: LK995506) and Actinomyces glycerinitolerans G10 T (GenBank accession number from the complete genome: NZFQTT01000017) as the closest relatives (99.7 and 99.6% gene pairwise identity, respectively), followed by the Actinomyces ruminicola DSM 27982 T (97.2%, in all compared fragment of 41468 pb). Due to the taxonomic affinity of the examined strain to both species A. succiniciruminis and A. glycerinitolerans, its taxonomic status towards these species was evaluated using variable regions of rpsA (length of 519 bp) and rplB (597 bp) gene sequences amplified based on specific primers designed so as to be applicable in differentiation, classification, and phylogeny of Actinomyces species/strains. Comparative analyses using rpsA and rplB showed 98.5 and 97.9% similarities of C10 to A. succiniciruminis, respectively, and 97.5 and 97.6% similarities to A. glycerinitolerans, respectively. Thus, gene identities revealed that the evaluated isolate C10 (=DSM 100236 = LMG 28777) is a little more related to the species A. succiniciruminis isolated from the rumen of a Holstein-Friesian cow than A. glycerinitolerans. Phylogenetic analyses confirmed affinity of strain C10 to both recently characterized species. Unfortunately, they did not allow the bacterial strain to be classified into a particular species. Phenotypic characterization suggested similar conclusions. This brief contribution is aimed at classification and detailed phenotypic characterization of bacterial strain C10 isolated from the rumen of a wild red deer exhibiting, from the point of view of Actinomyces species, noteworthy cellulolytic and xylanolytic activities.

As part of an antibiotic screening program, an actinobacterium, strain HMC13 T , was isolated from soil collected from the banks of the Gamka River, Western Cape Province, South Africa. The isolate was found to produce branched mycelia that differentiated into spiral spore chains with spiny spores. 16S rRNA gene sequence analysis showed the strain to be closely related to Streptomyces caelestis NRRL 2418 T (99.72%) and Streptomyces azureus NBRC 12744 T (99.51%). Chemotaxonomic analyses confirmed the classification of the strain as a member of the genus Streptomyces: LL-DAP in the peptidoglycan, no diagnostic sugars in the whole cell sugar pattern, dominant menaquinones including MK9(H 8 ), MK9(H 6 ), and the polar lipids detected included phosphatidylethanolamine. The fatty acid profile revealed the presence of mostly branched, saturated fatty acids: iso-C 15:0 (14.4%), anteiso-C 15:0 (21.1%), iso-C 16:0 (16.8%), C 16:1 ω7c/2-OH iso-C 15:0 (5.8%), C 16:0 (6.2%), iso-C 17:1 ω9c (5.8%), iso-C 17:0 (5.9%), and anteiso-C 17:0 (9.6%). Strain HMC13 T is a tyrosinase producer and exhibits very strong antibiosis against Mycobacterium aurum A+ and Staphylococcus aureus subsp. aureus ATCC 33591 (methicillin resistant), while only weak activity was observed against Bacillus cereus ATCC 10876, Enterococcus faecium VanA (vancomycin resistant), Enterococcus faecalis ATCC 51299 (vancomycin resistant) and Candida tropicalis ATCC 750 T . Strain HMC13 T (= LMG 28849 T  = NRRL B-65294 T ) is proposed as the type strain of a new species, to be named Streptomyces swartbergensis sp. nov.

A Gram-positive, non-motile, asporogenous and aerobic bacterium, designated YIM 98012 T , was isolated from a salt lake in China. Strain YIM 98012 T was found to be catalase and oxidase positive. Optimal growth of strain YIM 98012 T was observed at 37 °C and pH 7.0 and it was found to grow in the presence of 5-20% (w/v) NaCl (optimum 10% NaCl). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the novel strain is affiliated with the family Bacillaceae of the phylum Firmicutes and that it shares high (94.7%) sequence similarity with Alteribacillus persepolensis DSM 21632 T and does not show sequence similarities of more than 94.0% to known members of other related genera. The major fatty acids (> 10%) were identified as anteiso-C 15:0 , anteiso-C 17:0 , iso-C 16:0 and C 16:0 . The genomic DNA G+C content was determined to be 41.0 mol% and the dominated respiratory quinone was identified as MK-7. The cell wall peptidoglycan of strain YIM 98012 T was found to contain meso-diaminopimelic acid, while the polar lipids profile was found to include diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine. Based on physiological and chemotaxonomic characteristics, strain YIM 98012 T is concluded to be the type strain of the type species of a novel genus in the family Bacillaceae for which the name Aidingibacillus halophilus gen. nov., sp. nov. is proposed. The type strain is YIM 98012 T (= KCTC 33868 T  = DSM 104332 T ).

Peptidoglycan (PG) is a major component of the cell wall in Enterococcus faecalis. Accurate analysis of PG composition provides crucial insights into the bacterium's cellular functions and responses to external stimuli, but this analysis remains challenging because of various chemical modifications to PG-repeat subunits. We characterized changes to the PG composition of E. faecalis grown as planktonic bacteria and biofilm by developing "stable isotope labeling by amino acids in bacterial culture" (SILAB), optimized for bacterial cultures with incomplete amino acid labeling. This comparative analysis by mass spectrometry was performed by labeling E. faecalis in biofilm with heavy Lys (l-[ 13 C 6 , 2 D 9 , 15 N 2 ]Lys) and planktonic bacteria with natural abundance l-Lys, then mixing equal amounts of bacteria from each condition, and performing cell wall isolation and mutanolysin digestion necessary for liquid chromatography and mass spectrometry. An analytical method was developed to determine muropeptide abundances using correction factors to compensate for incomplete heavy Lys isotopic enrichment (98.33 ± 0.05%) and incorporation (83.23 ± 1.16%). Forty-seven pairs of PG fragment ions from isolated cell walls of planktonic and biofilm samples were selected for SILAB analysis. We found that the PG in biofilm showed an increased level of PG cross-linking, an increased level of N-deacetylation of GlcNAc, a decreased level of O-acetylation of MurNAc, and an increased number of stem modifications by d,d- and l,d-carboxypeptidases.

The filamentous anoxygenic phototrophic bacterium Oscillochloris trichoides DG-6 has been studied, and it has been shown that there are no lipopolysaccharides on the cell surface. Fatty acids hydroxylated at the C3 position, amino sugars and phosphate-containing compounds characteristic of lipid A have also not been found. The genes encoding for proteins responsible for the synthesis of lipopolysaccharides and the genes for the transport system, usually localized in the outer membrane of Gram-negative bacteria, have not been detected in the genome. The rigid layer of the cell wall contains a peptidoglycan consisting of alanine, glutamine, ornithine and glycine, in the respective ratio 1.8 : 1.5 : 1.0 : 0.6. Thus, the investigated bacterium, Osc. trichoides, is a monoderm. The cell wall also contains a branched α-1,4-d-glucan with a repeating unit consisting of glucose residues linked by α-1→4 bonds (α-1→6 at the branching sites). Such polymers have not previously been reported in phototrophic bacteria.