scholarly journals Identification and Characterization of a Cell Wall Hydrolase for Sporangiospore Maturation in Actinoplanes missouriensis

2019 ◽  
Vol 201 (24) ◽  
Author(s):  
Kyota Mitsuyama ◽  
Takeaki Tezuka ◽  
Yasuo Ohnishi
Keyword(s):  
Cell Wall ◽  
Streptomyces Griseus ◽  
Content Type ◽  
Twin Arginine Translocation ◽  
Cell Wall Hydrolase ◽  
Transmission Electron ◽  
Rare Actinomycetes ◽  
A Cell ◽  
Zymographic Analysis ◽  
Flagellar Biogenesis

ABSTRACT The rare actinomycete Actinoplanes missouriensis grows as substrate mycelium and forms terminal sporangia containing a few hundred spores as dormant cells. Upon contact with water, the sporangia open up and release spores to external environments. Here, we report a cell wall hydrolase, GsmA, that is required for sporangiospore maturation in A. missouriensis. The gsmA gene is conserved among Actinoplanes species and several species of other rare actinomycetes. Transcription of gsmA is activated in the late stage of sporangium formation by the global transcriptional activator TcrA, which is involved in sporangium formation and dehiscence. GsmA is composed of an N-terminal signal peptide for the twin arginine translocation pathway, two tandem bacterial SH3-like domains, and a glucosaminidase domain. Zymographic analysis using a recombinant C-terminal glucosaminidase domain protein showed that GsmA is a hydrolase able to digest cell walls extracted from the vegetative mycelia of A. missouriensis and Streptomyces griseus. A gsmA deletion mutant (ΔgsmA) formed apparently normal sporangia, but they released chains of 2 to 20 spores under sporangium dehiscence-inducing conditions, indicating that spores did not completely mature in the mutant sporangia. From these results, we concluded that GsmA is a cell wall hydrolase for digesting peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in A. missouriensis. Unexpectedly, flagella were observed around the spore chains of the ΔgsmA mutant by transmission electron microscopy. The flagellar formation was strictly restricted to cell-cell interfaces, giving an important insight into the polarity of the flagellar biogenesis in a spherical spore. IMPORTANCE In streptomycetes, an aerial hypha is compartmentalized by multiple septations into prespores, which become spores through a series of maturation processes. However, little is known about these maturation processes. The rare actinomycete Actinoplanes missouriensis produces sporangiospores, which are assumed to be formed also from prespores generated by the compartmentalization of intrasporangium hyphae via septation. The identification of GsmA as a cell wall hydrolase for the separation of adjacent spores sheds light on the almost unknown processes of sporangiospore formation in A. missouriensis. Furthermore, the fact that GsmA orthologues are conserved within the genus Actinoplanes but not in streptomycetes indicates that Actinoplanes has developed an original strategy for the spore maturation in a specific environment, that is, inside a sporangium.

scholarly journals Antimicrobial Activity of Exebacase (Lysin CF-301) against the Most Common Causes of Infective Endocarditis

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Aubrey Watson ◽  
Jun Taek Oh ◽  
Karen Sauve ◽  
Patricia A. Bradford ◽  
Cara Cassino ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Cell Wall ◽  
Infective Endocarditis ◽  
Broth Microdilution ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Microdilution Method ◽  
Cell Wall Hydrolase ◽  
Broth Microdilution Method ◽  
Common Causes

ABSTRACT Exebacase, a recombinantly produced lysin (cell wall hydrolase), and comparator antibiotics were tested by the broth microdilution method against strain sets of Staphylococcus and Streptococcus spp., which are the most common causes of infective endocarditis in humans. Exebacase was active against all Staphylococcus spp. tested, including S. aureus and coagulase-negative staphylococci (MIC50/90, 0.5/1 μg/ml). Activity against Streptococcus spp. was variable, with S. pyogenes, S. agalactiae, and S. dysgalactiae (MIC50/90, 1/2 μg/ml) among the most susceptible.

scholarly journals Effect of Nanoparticles Surface Charge on the Arabidopsis thaliana (L.) Roots Development and Their Movement into the Root Cells and Protoplasts

2019 ◽  
Vol 20 (7) ◽  
pp. 1650 ◽  
Author(s):  
Anna Milewska-Hendel ◽  
Maciej Zubko ◽  
Danuta Stróż ◽  
Ewa Kurczyńska
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Wall ◽  
Surface Charge ◽  
Physical Barrier ◽  
Root Cells ◽  
Plant Body ◽  
Transmission Electron ◽  
A Cell ◽  
Living Organisms ◽  
Positively Charged

Increasing usage of gold nanoparticles (AuNPs) in different industrial areas inevitably leads to their release into the environment. Thus, living organisms, including plants, may be exposed to a direct contact with nanoparticles (NPs). Despite the growing amount of research on this topic, our knowledge about NPs uptake by plants and their influence on different developmental processes is still insufficient. The first physical barrier for NPs penetration to the plant body is a cell wall which protects cytoplasm from external factors and environmental stresses. The absence of a cell wall may facilitate the internalization of various particles including NPs. Our studies have shown that AuNPs, independently of their surface charge, did not cross the cell wall of Arabidopsis thaliana (L.) roots. However, the research carried out with using light and transmission electron microscope revealed that AuNPs with different surface charge caused diverse changes in the root’s histology and ultrastructure. Therefore, we verified whether this is only the wall which protects cells against particles penetration and for this purpose we used protoplasts culture. It has been shown that plasma membrane (PM) is not a barrier for positively charged (+) AuNPs and negatively charged (−) AuNPs, which passage to the cell.

scholarly journals An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
L. Zhang ◽  
K. Ramijan ◽  
V. J. Carrión ◽  
L. T. van der Aart ◽  
J. Willemse ◽  
...  
Keyword(s):  
Cell Wall ◽  
Sequence Similarity ◽  
Streptomyces Coelicolor ◽  
Genomic Analysis ◽  
Environmental Harm ◽  
Cell Wall Synthesis ◽  
Precursor Molecule ◽  
Content Type ◽  
Lipid Ii ◽  
A Cell

ABSTRACT The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces. CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis. IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

scholarly journals Characterization of Zoospore Type IV Pili inActinoplanes missouriensis

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Tomohiro Kimura ◽  
Takeaki Tezuka ◽  
Daisuke Nakane ◽  
Takayuki Nishizaka ◽  
Shin-Ichi Aizawa ◽  
...  
Keyword(s):  
Vegetative Growth ◽  
Physiological State ◽  
Type Iv Pili ◽  
Solid Surfaces ◽  
Cellular Biology ◽  
Content Type ◽  
Type Iv ◽  
Transmission Electron ◽  
Rare Actinomycetes ◽  
Transcriptional Units

ABSTRACTThe rare actinomyceteActinoplanes missouriensisproduces terminal sporangia containing a few hundred flagellated spores. After release from the sporangia, the spores swim rapidly in aquatic environments as zoospores. The zoospores stop swimming and begin to germinate in niches for vegetative growth. Here, we report the characterization and functional analysis of zoospore type IV pili inA. missouriensis. The pilus gene (pil) cluster, consisting of three apparently σFliA-dependent transcriptional units, is activated during sporangium formation similarly to the flagellar gene cluster, indicating that the zoospore has not only flagella but also pili. With a new method in which zoospores were fixed with glutaraldehyde to prevent pilus retraction, zoospore pili were observed relatively easily using transmission electron microscopy, showing 6 ± 3 pili per zoospore (n = 37 piliated zoospores) and a length of 0.62 ± 0.35 μm (n = 206), via observation offliC-deleted, nonflagellated zoospores. No pili were observed in the zoospores of a prepilin-encodingpilAdeletion (ΔpilA) mutant. In addition, the deletion ofpilT, which encodes an ATPase predicted to be involved in pilus retraction, substantially reduced the frequency of pilus retraction. Several adhesion experiments using wild-type and ΔpilAzoospores indicated that the zoospore pili are required for the sufficient adhesion of zoospores to hydrophobic solid surfaces. Many zoospore-forming rare actinomycetes conserve thepilcluster, which indicates that the zoospore pili yield an evolutionary benefit in the adhesion of zoospores to hydrophobic materials as footholds for germination in their mycelial growth.IMPORTANCEBacterial zoospores are interesting cells in that their physiological state changes dynamically: they are dormant in sporangia, show temporary mobility after awakening, and finally stop swimming to germinate in niches for vegetative growth. However, the cellular biology of a zoospore remains largely unknown. This study describes unprecedented zoospore type IV pili in the rare actinomyceteActinoplanes missouriensis. Similar to the case for the usual bacterial type IV pili, zoospore pili appeared to be retractable. Our findings that the zoospore pili have a functional role in the adhesion of zoospores to hydrophobic solid surfaces and that the zoospores use both pili and flagella properly according to their different purposes provide an important insight into the cellular biology of the zoospore.

An enzymatic release of antibiotic-containing peptidoglycan fragments from streptomycin-producing Streptomyces griseus

1980 ◽  
Vol 26 (2) ◽  
pp. 141-145 ◽  
Author(s):  
Gy. Barabás ◽  
I. Szabó ◽  
A. Ottenberger ◽  
V. Zs.-Nagy ◽  
G. Szabó
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Glutamic Acid ◽  
Streptomyces Griseus ◽  
Activity Analysis ◽  
Antibiotic Activity ◽  
Diaminopimelic Acid ◽  
Autolytic Enzyme ◽  
A Cell

A cell wall bound autolytic enzyme of a streptomycin-producing strain of Streptomyces griseus was investigated. The peptidoglycan fragments released by the enzyme showed antibiotic activity. Analysis of these fragments proved that streptomycin is bound to a cell wall peptide. The peptide contained the four amino acids characteristic of Streptomyces cell wall: alanine, glycine, diaminopimelic acid, and glutamine or glutamic acid.

scholarly journals Daptomycin Resistance and Tolerance Due to Loss of Function inStaphylococcus aureusdsp1andasp23

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Elaine M. Barros ◽  
Melissa J. Martin ◽  
Elizabeth M. Selleck ◽  
François Lebreton ◽  
Jorge Luiz M. Sampaio ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antimicrobial Peptides ◽  
Multidrug Resistant ◽  
Loss Of Function ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Transposon Insertion ◽  
Transposon Mutants ◽  
A Cell ◽  
Transposon Insertions

ABSTRACTLipopeptide daptomycin is a last-line cell-membrane-targeting antibiotic to treat multidrug-resistantStaphylococcus aureus. Alarmingly, daptomycin-resistantS. aureusisolates have emerged. The mechanisms underlying daptomycin resistance are diverse and share similarities with resistances to cationic antimicrobial peptides and other lipopeptides, but they remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in sequent type 8 (ST8)S. aureusHG003. Insertions conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss-of-function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to those of wild-type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, theΔdsp1mutant also showed increased resistance to vancomycin, a cell-wall-targeting drug with a different mode of action. Null mutations in bothdsp1andasp23resulted in increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell-wall-targeting endopeptidase. These findings identified two genes core to theS. aureusspecies that make previously uncharacterized contributions to antimicrobial resistance and tolerance inS. aureus.

scholarly journals LysPBC2, a Novel Endolysin Harboring aBacillus cereusSpore Binding Domain

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Minsuk Kong ◽  
Hongjun Na ◽  
Nam-Chul Ha ◽  
Sangryeol Ryu
Keyword(s):  
Cell Wall ◽  
Catalytic Domain ◽  
Binding Activity ◽  
Binding Domain ◽  
Lytic Activity ◽  
Content Type ◽  
Cell Wall Binding ◽  
A Cell ◽  
Cell Wall Binding Domain

ABSTRACTTo control the spore-forming human pathogenBacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolatedB. cereusphage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against allBacillus,Listeria, andClostridiumspecies tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds toB. cereusspores but not to vegetative cells ofB. cereus. Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells ofB. cereuscompared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCEBacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of aBacillus cereusphage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controllingB. cereus.

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