scholarly journals Inactivation of the pgmA Gene in Streptococcus mutans Significantly Decreases Biofilm-Associated Antimicrobial Tolerance

2019 ◽  
Vol 7 (9) ◽  
pp. 310 ◽  
Author(s):  
Martin Nilsson ◽  
Michael Givskov ◽  
Svante Twetman ◽  
Tim Tolker-Nielsen
Keyword(s):  
Cell Wall ◽  
Streptococcus Mutans ◽  
Congo Red ◽  
Biofilm Formation ◽  
Cell Wall Composition ◽  
Mutant Library ◽  
Wild Type ◽  
Glass Slides ◽  
Increased Sensitivity ◽  
Wall Components

Screening of a Streptococcus mutans mutant library indicated that pgmA mutants displayed a reduced biofilm-associated tolerance toward gentamicin. The biofilms formed by the S. mutans pgmA mutant also displayed decreased tolerance towards linezolid and vancomycin compared to wild-type biofilms. On the contrary, the resistance of planktonic S. mutans pgmA cells to gentamycin, linezolid, and vancomycin was more similar to wild-type levels. Investigations of biofilms grown in microtiter trays and on submerged glass slides showed that pgmA mutants formed roughly the same amount of biofilm as the wild type, indicating that the reduced antimicrobial tolerance of these mutants is not due to diminished biofilm formation. The pgmA gene product is known to be involved in the synthesis of precursors for cell wall components such as teichoic acids and membrane glycolipids. Accordingly, the S. mutans pgmA mutant showed increased sensitivity to Congo Red, indicating that it has impaired cell wall integrity. A changed cell wall composition of the S. mutans pgmA mutant may play a role in the increased sensitivity of S. mutans pgmA biofilms toward antibiotics.

scholarly journals Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Arpan De ◽  
Sumei Liao ◽  
Jacob P. Bitoun ◽  
Randy Roth ◽  
Wandy L. Beatty ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cell Wall ◽  
Cell Division ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Cell Envelope ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Triple Mutant ◽  
Content Type

ABSTRACTStreptococcus mutansis known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen.S. mutansstrains deficient inrgpG, encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. ThergpGdeficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, thergpGmutant existed primarily in chains of swollen, “squarish” dividing cells. Deficiency ofrgpGalso causes significant reduction in biofilm formation (P< 0.01). Double and triple mutants with deficiency inbrpAand/orpsr, genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using thergpGmutant. There were no major differences in growth rates between the wild-type strain and thergpG brpAandrgpG psrdouble mutants, but the growth rate of thergpG brpA psrtriple mutant was reduced drastically (P< 0.001). Under transmission electron microscopy, both double mutants resembled thergpGmutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, thergpGmutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and thebrpAandpsrsingle mutants. These results suggest that RgpG inS. mutansplays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope.IMPORTANCEStreptococcus mutans, a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation byS. mutans, indicative of a significant role of RGP in cell division and biofilm formation inS. mutans. These results are novel not only inS. mutans, but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr inS. mutansare involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation inS. mutans. This study reveals new potential targets to develop anticaries therapeutics.

scholarly journals Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

2009 ◽  
Vol 8 (10) ◽  
pp. 1475-1485 ◽  
Author(s):  
Thanyanuch Kriangkripipat ◽  
Michelle Momany
Keyword(s):  
Cell Wall ◽  
Aspergillus Nidulans ◽  
Double Mutant ◽  
Elevated Temperatures ◽  
Cell Wall Integrity ◽  
Wild Type ◽  
Spatial Cues ◽  
Developmental Patterning ◽  
In The Wild ◽  
Increased Sensitivity

ABSTRACT Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.

scholarly journals Feeding the Walls: How Does Nutrient Availability Regulate Cell Wall Composition?

2018 ◽  
Vol 19 (9) ◽  
pp. 2691 ◽  
Author(s):  
Michael Ogden ◽  
Rainer Hoefgen ◽  
Ute Roessner ◽  
Staffan Persson ◽  
Ghazanfar Khan
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Nutrient Availability ◽  
Cell Walls ◽  
Plant Cell Wall ◽  
Nutrient Status ◽  
Cell Wall Composition ◽  
Nutrient Sensing ◽  
Tissue Type ◽  
Wall Components

Nutrients are critical for plants to grow and develop, and nutrient depletion severely affects crop yield. In order to optimize nutrient acquisition, plants adapt their growth and root architecture. Changes in growth are determined by modifications in the cell walls surrounding every plant cell. The plant cell wall, which is largely composed of complex polysaccharides, is essential for plants to attain their shape and to protect cells against the environment. Within the cell wall, cellulose strands form microfibrils that act as a framework for other wall components, including hemicelluloses, pectins, proteins, and, in some cases, callose, lignin, and suberin. Cell wall composition varies, depending on cell and tissue type. It is governed by synthesis, deposition and remodeling of wall components, and determines the physical and structural properties of the cell wall. How nutrient status affects cell wall synthesis and organization, and thus plant growth and morphology, remains poorly understood. In this review, we aim to summarize and synthesize research on the adaptation of root cell walls in response to nutrient availability and the potential role of cell walls in nutrient sensing.

scholarly journals Cell wall composition and biofilm formation of azoles-susceptible and -resistant Candida glabrata strains

2016 ◽  
Vol 29 (3) ◽  
pp. 164-172 ◽  
Author(s):  
Alberto Vitali ◽  
Elisabetta Vavala ◽  
Valeria Marzano ◽  
Claudia Leone ◽  
Massimo Castagnola ◽  
...  
Keyword(s):  
Cell Wall ◽  
Biofilm Formation ◽  
Candida Glabrata ◽  
Cell Wall Composition

scholarly journals LuxS-Mediated Signaling in Streptococcus mutans Is Involved in Regulation of Acid and Oxidative Stress Tolerance and Biofilm Formation

2004 ◽  
Vol 186 (9) ◽  
pp. 2682-2691 ◽  
Author(s):  
Zezhang T. Wen ◽  
Robert A. Burne
Keyword(s):  
Streptococcus Mutans ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Protein A ◽  
Signal Recognition Particle ◽  
Northern Hybridization ◽  
Wild Type ◽  
Increased Sensitivity ◽  
Mutant Phenotypes ◽  
Almost All

ABSTRACT LuxS-mediated quorum sensing has recently been shown to regulate important physiologic functions and virulence in a variety of bacteria. In this study, the role of luxS of Streptococcus mutans in the regulation of traits crucial to pathogenesis was investigated. Reporter gene fusions showed that inactivation of luxS resulted in a down-regulation of fructanase, a demonstrated virulence determinant, by more than 50%. The LuxS-deficient strain (TW26) showed increased sensitivity to acid killing but could still undergo acid adaptation. Northern hybridization revealed that the expression of RecA, SmnA (AP endonuclease), and Nth (endonuclease) were down-regulated in TW26, especially in early-exponential-phase cells. Other down-regulated genes included ffh (a signal recognition particle subunit) and brpA (biofilm regulatory protein A). Interestingly, the luxS mutant showed an increase in survival rate in the presence of hydrogen peroxide (58.8 mM). The luxS mutant formed less biofilm on hydroxylapatite disks, especially when grown in biofilm medium with sucrose, and the mutant biofilms appeared loose and hive-like, whereas the biofilms of the wild type were smooth and confluent. The mutant phenotypes were complemented by exposure to supernatants from wild-type cultures. Two loci, smu486 and smu487, were identified and predicted to encode a histidine kinase and a response regulator. The phenotypes of the smu486 smu487 mutant were, in almost all cases, similar to those of the luxS mutant, although our results suggest that this is not due to AI-2 signal transduction via Smu486 and Smu487. This study demonstrates that luxS-dependent signaling plays critical roles in modulating key virulence properties of S. mutans.

scholarly journals Poly-N-Acetylglucosamine Expression by Wild-Type Yersinia pestis Is Maximal at Mammalian, Not Flea, Temperatures

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
Author(s):  
Pauline Yoong ◽  
Colette Cywes-Bentley ◽  
Gerald B. Pier
Keyword(s):  
Yersinia Pestis ◽  
Congo Red ◽  
Biofilm Formation ◽  
Mammalian Host ◽  
Virulence Plasmid ◽  
Insect Vector ◽  
Wild Type ◽  
Content Type ◽  
Higher Temperature ◽  
Mammalian Infection

ABSTRACTNumerous bacteria, includingYersinia pestis, express the poly-N-acetylglucosamine (PNAG) surface carbohydrate, a major component of biofilms often associated with a specific appearance of colonies on Congo red agar. Biofilm formation and PNAG synthesis byY. pestishave been reported to be maximal at 21 to 28°C or “flea temperatures,” facilitating the regurgitation ofY. pestisinto a mammalian host during feeding, but production is diminished at 37°C and thus presumed to be decreased during mammalian infection. Most studies of PNAG expression and biofilm formation byY. pestishave used a low-virulence derivative of strain KIM, designated KIM6+, that lacks the pCD1 virulence plasmid, and an isogenic mutant without the pigmentation locus, which contains the hemin storage genes that encode PNAG biosynthetic proteins. Using confocal microscopy, fluorescence-activated cell sorter analysis and growth on Congo red agar, we confirmed prior findings regarding PNAG production with the KIM6+ strain. However, we found that fully virulent wild-type (WT) strains KIM and CO92 had maximal PNAG expression at 37°C, with lower PNAG production at 28°C both in broth medium and on Congo red agar plates. Notably, the typical dark colony morphology appearing on Congo red agar was maintained at 28°C, indicating that this phenotype is not associated with PNAG expression in WTY. pestis. Extracts of WT sylvaticY. pestisstrains from the Russian Federation confirmed the maximal expression of PNAG at 37°C. PNAG production by WTY. pestisis maximal at mammalian and not insect vector temperatures, suggesting that this factor may have a role during mammalian infection.IMPORTANCEYersinia pestistransitions from low-temperature residence and replication in insect vectors to higher-temperature replication in mammalian hosts. Prior findings based primarily on an avirulent derivative of WT (wild-type) KIM, named KIM6+, showed that biofilm formation associated with synthesis of poly-N-acetylglucosamine (PNAG) is maximal at 21 to 28°C and decreased at 37°C. Biofilm formation was purported to facilitate the transmission ofY. pestisfrom fleas to mammals while having little importance in mammalian infection. Here we found that for WT strains KIM and CO92, maximal PNAG production occurs at 37°C, indicating that temperature regulation of PNAG production in WTY. pestisis not mimicked by strain KIM6+. Additionally, we found that Congo red binding does not always correlate with PNAG production, despite its widespread use as an indicator of biofilm production. Taken together, the findings show that a role for PNAG in WTY. pestisinfection should not be disregarded and warrants further study.

scholarly journals Mutation of luxS Affects Biofilm Formation in Streptococcus mutans

2003 ◽  
Vol 71 (4) ◽  
pp. 1972-1979 ◽  
Author(s):  
Justin Merritt ◽  
Fengxia Qi ◽  
Steven D. Goodman ◽  
Maxwell H. Anderson ◽  
Wenyuan Shi
Keyword(s):  
Quorum Sensing ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Microscopic Analysis ◽  
Homoserine Lactone ◽  
Good Evidence ◽  
Genome Project ◽  
Bacterial Biofilm ◽  
Wild Type

ABSTRACT Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www.genome.ou.edu/smutans.html ). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5α. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.

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 Disruption of L-Rhamnose Biosynthesis Results in Severe Growth Defects in Streptococcus mutans

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
Andrew P. Bischer ◽  
Christopher J. Kovacs ◽  
Roberta C. Faustoferri ◽  
Robert G. Quivey
Keyword(s):  
Cell Wall ◽  
Dental Caries ◽  
Oral Cavity ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Etiologic Agent ◽  
Growth Defect ◽  
Cell Wall Polysaccharide ◽  
Content Type ◽  
Severe Growth

ABSTRACT The rhamnose-glucose cell wall polysaccharide (RGP) of Streptococcus mutans plays a significant role in cell division, virulence, and stress protection. Prior studies examined function of the RGP using strains carrying deletions in the machinery involved in RGP assembly. In this study, we explored loss of the substrate for RGP, l-rhamnose, via deletion of rmlD (encoding the protein responsible for the terminal step in l-rhamnose biosynthesis). We demonstrate that loss of rhamnose biosynthesis causes a phenotype similar to strains with disrupted RGP assembly (ΔrgpG and ΔrgpF strains). Deletion of rmlD not only caused a severe growth defect under nonstress growth conditions but also elevated susceptibility of the strain to acid and oxidative stress, common conditions found in the oral cavity. A genetic complement of the ΔrmlD strain completely restored wild-type levels of growth, whereas addition of exogenous rhamnose did not. The loss of rhamnose production also significantly disrupted biofilm formation, an important aspect of S. mutans growth in the oral cavity. Further, we demonstrate that loss of either rmlD or rgpG results in ablation of rhamnose content in the S. mutans cell wall. Taken together, these results highlight the importance of rhamnose production in both the fitness and the ability of S. mutans to overcome environmental stresses. IMPORTANCE Streptococcus mutans is a pathogenic bacterium that is the primary etiologic agent of dental caries, a disease that affects billions yearly. Rhamnose biosynthesis is conserved not only in streptococcal species but in other Gram-positive, as well as Gram-negative, organisms. This study highlights the importance of rhamnose biosynthesis in RGP production for protection of the organism against acid and oxidative stresses, the two major stressors that the organism encounters in the oral cavity. Loss of RGP also severely impacts biofilm formation, the first step in the onset of dental caries. The high conservation of the rhamnose synthesis enzymes, as well as their importance in S. mutans and other organisms, makes them favorable antibiotic targets for the treatment of disease.

Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae

1983 ◽  
Vol 3 (5) ◽  
pp. 922-930
Author(s):  
R L Roberts ◽  
B Bowers ◽  
M L Slater ◽  
E Cabib
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Wall ◽  
Permissive Temperature ◽  
Wild Type ◽  
Chitin Synthesis ◽  
Specific Staining ◽  
Septal Region ◽  
Wall Components ◽  
Generalized Distribution

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.

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