scholarly journals Characterization of Conserved and Novel Septal Factors inMycobacterium smegmatis

2018 ◽  
Vol 200 (6) ◽  
Author(s):  
Katherine J. Wu ◽  
Jenna Zhang ◽  
Catherine Baranowski ◽  
Vivian Leung ◽  
E. Hesper Rego ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Model Organisms ◽  
Wall Structure ◽  
Cross Wall ◽  
Content Type ◽  
A Cell ◽  
Mediate Cell ◽  
Mycobacterial Cell Wall ◽  
Regulatory Functions

ABSTRACTSeptation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, usingMycobacterium smegmatis, we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs inEscherichia coli. We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31.IMPORTANCEThe enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such asEscherichia coli, but other proteins have critical regulatory functions that may be unique to the actinomycetes.

scholarly journals Critical Roles for Lipomannan and Lipoarabinomannan in Cell Wall Integrity of Mycobacteria and Pathogenesis of Tuberculosis

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Takeshi Fukuda ◽  
Takayuki Matsumura ◽  
Manabu Ato ◽  
Maho Hamasaki ◽  
Yukiko Nishiuchi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Critical Role ◽  
Protective Role ◽  
Cell Wall Integrity ◽  
Structural Defects ◽  
Wall Structure ◽  
Content Type ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

ABSTRACTLipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. InMycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalentMycobacterium tuberculosismutants became more sensitive to β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis.IMPORTANCETuberculosis (TB) is a global burden, affecting millions of people worldwide.Mycobacterium tuberculosisis a causative agent of TB, and understanding the biology ofM. tuberculosisis essential for tackling this devastating disease. The cell wall ofM. tuberculosisis highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in allMycobacteriumspecies, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target.

scholarly journals YtfB, an OapA Domain-Containing Protein, Is a New Cell Division Protein in Escherichia coli

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Matthew A. Jorgenson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Division ◽  
Wall Structure ◽  
Bacterial Cell Division ◽  
Negative Bacterium ◽  
Gram Negative ◽  
Content Type ◽  
Cell Division Protein ◽  
Inner Membrane Protein

ABSTRACT While screening the Pfam database for novel peptidoglycan (PG) binding modules, we identified the OapA domain, which is annotated as a LysM-like domain. LysM domains bind PG and mediate localization to the septal ring. In the Gram-negative bacterium Escherichia coli , an OapA domain is present in YtfB, an inner membrane protein of unknown function but whose overproduction causes cells to filament. Together, these observations suggested that YtfB directly affects cell division, most likely through its OapA domain. Here, we show that YtfB accumulates at the septal ring and that its action requires the division-initiating protein FtsZ and, to a lesser extent, ZipA, an early recruit to the septalsome. While the loss of YtfB had no discernible impact, a mutant lacking both YtfB and DedD (a known cell division protein) grew as filamentous cells. The YtfB OapA domain by itself also localized to sites of division, and this localization was enhanced by the presence of denuded PGs. Finally, the OapA domain bound PG, though binding did not depend on the formation of denuded glycans. Collectively, our findings demonstrate that YtfB is a cell division protein whose function is related to cell wall hydrolases. IMPORTANCE All living cells must divide in order to thrive. In bacteria, this involves the coordinated activities of a large number of proteins that work in concert to constrict the cell. Knowing which proteins contribute to this process and how they function is fundamental. Here, we identify a new member of the cell division apparatus in the Gram-negative bacterium Escherichia coli whose function is related to the generation of a transient cell wall structure. These findings deepen our understanding of bacterial cell division.

scholarly journals Attachment of Enterohemorrhagic Escherichia coli to Host Cells Reduces O Antigen Chain Length at the Infection Site That Promotes Infection

mBio ◽  
2021 ◽  
Author(s):  
Bin Liu ◽  
Chengqian Qian ◽  
Pan Wu ◽  
Xiaodan Li ◽  
Yutao Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Infection Process ◽  
Enterohemorrhagic Escherichia Coli ◽  
Cell Wall Structure ◽  
Host Cells ◽  
Intestinal Lumen ◽  
Wall Structure ◽  
Content Type ◽  
Enteropathogenic Bacteria

Little is known about the regulation of cell wall structure of enteropathogenic bacteria within the host. Here, we report that enterohemorrhagic Escherichia coli regulates its cell wall structure during the infection process, which balances its survival in the intestinal lumen and infection of intestinal epithelial cells.

scholarly journals High-Resolution Analysis of the Peptidoglycan Composition inStreptomyces coelicolor

2018 ◽  
Vol 200 (20) ◽  
Author(s):  
Lizah T. van der Aart ◽  
Gerwin K. Spijksma ◽  
Amy Harms ◽  
Waldemar Vollmer ◽  
Thomas Hankemeier ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycelial Growth ◽  
Antibiotic Production ◽  
Hydrolytic Enzymes ◽  
Morphological Differentiation ◽  
Model Organisms ◽  
Major Constituent ◽  
Single Spore ◽  
Content Type ◽  
Liquid Chromatography Tandem Mass

ABSTRACTThe bacterial cell wall maintains cell shape and protects against bursting by turgor. A major constituent of the cell wall is peptidoglycan (PG), which is continuously modified to enable cell growth and differentiation through the concerted activity of biosynthetic and hydrolytic enzymes. Streptomycetes are Gram-positive bacteria with a complex multicellular life style alternating between mycelial growth and the formation of reproductive spores. This involves cell wall remodeling at apical sites of the hyphae during cell elongation and autolytic degradation of the vegetative mycelium during the onset of development and antibiotic production. Here, we show that there are distinct differences in the cross-linking and maturation of the PGs between exponentially growing vegetative hyphae and the aerial hyphae that undergo sporulation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified over 80 different muropeptides, revealing that major PG hydrolysis takes place over the course of mycelial growth. Half of the dimers lacked one of the disaccharide units in transition-phase cells, most likely due to autolytic activity. The deacetylation of MurNAc to MurN was particularly pronounced in spores and strongly reduced in sporulation mutants with a deletion ofbldDorwhiG, suggesting that MurN is developmentally regulated. Altogether, our work highlights the dynamic and growth phase-dependent changes in the composition of the PG inStreptomyces.IMPORTANCEStreptomycetes are bacteria with a complex lifestyle and are model organisms for bacterial multicellularity. From a single spore, a large multigenomic multicellular mycelium is formed, which differentiates to form spores. Programmed cell death is an important event during the onset of morphological differentiation. In this work, we provide new insights into the changes in the peptidoglycan composition and over time, highlighting changes over the course of development and between growing mycelia and spores. This revealed dynamic changes in the peptidoglycan when the mycelia aged, with extensive peptidoglycan hydrolysis and, in particular, an increase in the proportion of 3-3 cross-links. Additionally, we identified a muropeptide that accumulates predominantly in the spores and may provide clues toward spore development.

scholarly journals Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Heather J. Beck ◽  
Gary R. Janssen
Keyword(s):  
Escherichia Coli ◽  
Translation Initiation ◽  
Translational Regulation ◽  
Current Knowledge ◽  
Upstream Open Reading Frame ◽  
Model Organisms ◽  
Regulation Mechanism ◽  
Rna Regulation ◽  
Content Type ◽  
E Coli

ABSTRACT Alternative translation initiation mechanisms, distinct from the Shine-Dalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coli ptrB instead requires an AUG triplet at the 5′ terminus of its mRNA. The 5′-terminal AUG (5′-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5′-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5′-uAUG. Additional sequences within the ptrB 5′ untranslated region (5′-UTR) work cumulatively with the 5′-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5′-UTRs from other mRNAs with the ptrB 5′-UTR sequence showed a similar dependence on the 5′-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5′-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5′-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5′ termini of E. coli mRNAs and the ability of ptrB 5′-UTR regulation to function independently of gene context, the regulatory effects of 5′-uAUGs on downstream CDSs may be widespread throughout the E. coli genome. IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.

scholarly journals Enumeration of Gut-Homing β7-Positive, Pathogen-Specific Antibody-Secreting Cells in Whole Blood from Enterotoxigenic Escherichia coli- and Vibrio cholerae-Infected Patients, Determined Using an Enzyme-Linked Immunosorbent Spot Assay Technique

2015 ◽  
Vol 23 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Taufiqur Rahman Bhuiyan ◽  
Mohammad Rubel Hoq ◽  
Naoshin Sharmin Nishat ◽  
Deena Al Mahbuba ◽  
Rasheduzzaman Rashu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Vibrio Cholerae ◽  
Elispot Assay ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Content Type ◽  
A Cell ◽  
Mucosal Immune Responses ◽  
Antibody Secreting Cells

ABSTRACTVibrio choleraeand enterotoxigenicEscherichia coli(ETEC) are noninvasive mucosal pathogens that cause acute watery diarrhea in people in developing countries. Direct assessment of the mucosal immune responses to these pathogens is problematic. Surrogate markers of local mucosal responses in blood are increasingly being studied to determine the mucosal immune responses after infection. However, the volume of blood available in children and infants has limited this approach. We assessed whether an approach that first isolates β7-positive cells from a small volume of blood would allow measurement of the antigen-specific immune responses in patients with cholera and ETEC infection. β7 is a cell surface marker associated with mucosal homing. We isolated β7-expressing cells from blood on days 2, 7, and 30 and used an enzyme-linked immunosorbent spot (ELISPOT) assay to assess the gut-homing antibody-secreting cells (ASCs) specific to pathogen antigens. Patients with ETEC diarrhea showed a significant increase in toxin-specific gut-homing ASCs at day 7 compared to the levels at days 2 and 30 after onset of illness and to the levels in healthy controls. Similar elevations of responses to the ETEC colonization factors (CFs) CS6 and CFA/I were observed in patients infected with CS6- and CFA/I-positive ETEC strains. Antigen-specific gut-homing ASCs to the B subunit of cholera toxin and cholera-specific lipopolysaccharides (LPS) were also observed on day 7 after the onset of cholera using this approach. This study demonstrates that a simple ELISPOT assay can be used to study the mucosal immunity to specific antigens using a cell-sorting protocol to isolate mucosal homing cells, facilitating measurement of mucosal responses in children following infection or vaccination.

scholarly journals Affinity, life cycle, and intracellular complexity of organic-walled microfossils from the Mesoproterozoic of Shanxi, China

2015 ◽  
Vol 89 (1) ◽  
pp. 28-50 ◽  
Author(s):  
Heda Agić ◽  
Małgorzata Moczydłowska ◽  
Lei-Ming Yin
Keyword(s):  
Cell Wall ◽  
Life Cycle ◽  
Structural Complexity ◽  
Protective Role ◽  
Life Cycles ◽  
Wall Structure ◽  
Phenotypic Characters ◽  
Biological Affinity ◽  
New Material ◽  
A Cell

AbstractLight microscope and scanning electron microscope observations on new material of unicellular microfossilsDictyosphaera macroreticulataandShuiyousphaeridium macroreticulatum,from the Mesoproterozoic Ruyang Group in China, provide insights into the microorganisms’ biological affinity, life cycle and cellular complexity.Gigantosphaeridium fibratumn. gen. et sp., is described and is one of the largest Mesoproterozoic microfossils recorded. Phenotypic characters of vesicle ornamentation and excystment structures, properties of resistance and cell wall structure inDictyosphaeraandShuiyousphaeridiumare all diagnostic of microalgal cysts. The wide size ranges of the various morphotypes indicate growth phases compatible with the development of reproductive cysts. Conspecific biologically, each morphotype represents an asexual (resting cyst) or sexual (zygotic cyst) stage in the life cycle, respectively. We reconstruct this hypothetical life cycle and infer that the organism demonstrates a reproductive strategy of alternation of heteromorphic generations. Similarly inGigantosphaeridium,a metabolically expensive vesicle with processes suggests its protective role as a zygotic cyst. In combination with all these characters and from the resemblance to extant green algae, we propose the placement of these ancient microorganisms in the stem group of Chloroplastida (Viridiplantae). A cell wall composed of primary and secondary layers inDictyosphaeraandShuiyouisphaeridiumrequired a high cellular complexity for their synthesis and the presence of an endomembrane system and the Golgi apparatus. The plastid was also present, accepting the organism was photosynthetic. The biota reveals a high degree of morphological and cell structural complexity, and provides an insight into ongoing eukaryotic evolution and the development of complex life cycles with sexual reproduction by 1200 Ma.

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 The aceE involves in mycolic acid synthesis and biofilm formation in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Suting Chen ◽  
Tianlu Teng ◽  
Shuan Wen ◽  
Tingting Zhang ◽  
Hairong Huang
Keyword(s):  
Cell Wall ◽  
Biofilm Formation ◽  
Morphological Changes ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Colony Morphology ◽  
Wall Structure ◽  
Wild Type ◽  
Mycobacterial Cell Wall

Abstract Background: The integrity of cell wall structure is highly significant for the in vivo survival for mycobacteria. However, the mechanisms underlying the biosynthesis of mycobacterial cell wall remain poorly understood. aceE encodes the E1 component of pyruvate dehydrogenase (PDH)complex. This study aimed to know the functional role of aceE gene in cell wall biosynthesis in M. smegmatis.Results: We observed that the colony morphology of aceE-deficient mutants(aceE-mut)was quite different from the wild-type(WT) strain during the transposon library screening of M.smegmatis, smaller and smoother on the solid culture medium. Notably, the aceE-mut lost its ability of growing aggregately and biofilm forming, which are two very important features of mycobacteria.The morphological changes of the aceE-mut strain were further confirmed by electron microscopy that presented shorter, smoother and thinner images in contrast withWT strain.Additionally, the analysis of mycolic acid(MA)using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceEgene restored the composition of MA. Conclusions: Overall, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology and biofilm formation of M.smegmatis.

scholarly journals Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Matthew B. McNeil ◽  
Theresa O’Malley ◽  
Devon Dennison ◽  
Catherine D. Shelton ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
New Drugs ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Primary Mechanism ◽  
Resistant Mutants ◽  
Mycobacterial Cell Wall

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

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