scholarly journals Septal Localization of Penicillin-Binding Protein 1 in Bacillus subtilis

1999 ◽  
Vol 181 (10) ◽  
pp. 3201-3211 ◽  
Author(s):  
Lotte B. Pedersen ◽  
Esther R. Angert ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Immunofluorescence Microscopy ◽  
Binding Protein ◽  
Divalent Cations ◽  
Significant Proportion ◽  
Growth Media ◽  
Weight Class ◽  
Penicillin Binding Protein ◽  
Septal Localization ◽  
Mutant Cells

ABSTRACT Previous studies have shown that Bacillus subtiliscells lacking penicillin-binding protein 1 (PBP1), encoded byponA, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells. It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg2+, suggesting a possible involvement of PBP1 in septum formation under these conditions. Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of B. subtilis. In addition, we have used fluorescence and electron microscopy to show that growingponA mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most ponA mutant cells, a significant proportion of ponA mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly. These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in B. subtilis. This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.

scholarly journals Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a

1998 ◽  
Vol 180 (24) ◽  
pp. 6493-6502 ◽  
Author(s):  
Thomas Murray ◽  
David L. Popham ◽  
Christine B. Pearson ◽  
Arthur R. Hand ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Cell Elongation ◽  
Binding Protein ◽  
Divalent Cations ◽  
Cross Linking ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Spore Outgrowth ◽  
Partial Redundancy

ABSTRACT The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect inpbpA spore outgrowth have shown that (i) outgrowingpbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpAspores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpAspores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.

scholarly journals Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

1998 ◽  
Vol 180 (17) ◽  
pp. 4555-4563 ◽  
Author(s):  
Thomas Murray ◽  
David L. Popham ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Cell Growth ◽  
Divalent Cation ◽  
Vegetative Growth ◽  
Binding Protein ◽  
Divalent Cations ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Deficient Medium ◽  
Spore Outgrowth

ABSTRACT Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg2+ or Ca2+ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg2+ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg2+ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restoreponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg2+ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.

Characterization of ywhE, which encodes a putative high-molecular-weight class A penicillin-binding protein in Bacillus subtilis

Gene ◽  
2000 ◽  
Vol 246 (1-2) ◽  
pp. 187-196 ◽  
Author(s):  
L.B. Pedersen ◽  
K. Ragkousi ◽  
T.J. Cammett ◽  
E. Melly ◽  
A. Sekowska ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
High Molecular Weight ◽  
Binding Protein ◽  
Weight Class ◽  
Penicillin Binding Protein ◽  
Class A

scholarly journals The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a dd-carboxypeptidase in vitro

1985 ◽  
Vol 230 (3) ◽  
pp. 825-828 ◽  
Author(s):  
John A. Todd ◽  
Eileen J. Bone ◽  
David J. Ellar
Keyword(s):  
Bacillus Subtilis ◽  
Binding Protein ◽  
Penicillin Binding Protein

The sporulation-specific penicillin-binding protein 5a was purified from Bacillus subtilis and shown to possess dd-carboxypeptidase activity in vitro.

scholarly journals FtsI and FtsW Are Localized to the Septum inEscherichia coli

1998 ◽  
Vol 180 (11) ◽  
pp. 2810-2816 ◽  
Author(s):  
Lilin Wang ◽  
Medhat K. Khattar ◽  
W. D. Donachie ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Immunofluorescence Microscopy ◽  
Binding Protein ◽  
Specific Inhibitor ◽  
Inescherichia Coli ◽  
Penicillin Binding Protein ◽  
Cell Division Protein

ABSTRACT The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in Escherichia coli, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed inftsZ or ftsA mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.

scholarly journals On-Chip Isoniazid Exposure of Mycobacterium smegmatis Penicillin-Binding Protein (PBP) Mutant Using Time-Lapse Fluorescent Microscopy

Micromachines ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 561
Author(s):  
Meltem Elitas
Keyword(s):  
Antibiotic Resistance ◽  
Single Cell ◽  
Mycobacterium Smegmatis ◽  
Single Cell Analysis ◽  
Binding Protein ◽  
Cell Analysis ◽  
Penicillin Binding Protein ◽  
Intact Cells ◽  
The Impact ◽  
Mutant Cells

Antibiotic resistance has been one of the biggest threats to global health. Despite the available prevention and control strategies and efforts in developing new antibiotics, the need remains for effective approaches against antibiotic resistance. Efficient strategies to cope with antimicrobial resistance require a quantitative and deeper understanding of microbial behavior, which can be obtained using different techniques to provide the missing pieces of the current antibiotic-resistance puzzle. Microfluidic-microscopy techniques are among the most promising methods that contribute modernization of traditional assays in microbiology. They provide monitoring and manipulation of cells at micro-scale volumes. Here, we combined population-level, culture-based assays with single-cell resolution, microfluidic-microscopy systems to investigate isoniazid response of Mycobacterium smegmatis penicillin-binding protein (PBP) mutant. This mutant exhibited normal growth in plain medium and sensitivity to stress responses when treated with thermal stress (45 °C), detergent stress (0.1% sodium dodecyl sulfate), acid stress (pH 4.5), and nutrient starvation (1XPBS). The impact of msm0031 transposon insertion on drug-mediated killing was determined for isoniazid (INH, 50 µg/mL), rifampicin (RIF, 200 µg/mL), ethionamide (ETH, 200 µg/mL), and ethambutol (EMB, 5 µg/mL). The PBP mutant demonstrated remarkable isoniazid-killing phenotype in batch culture. Therefore, we hypothesized that single-cell analysis will show increased lysis kinetics and fewer intact cells after drug treatment. However, the single-cell analysis data showed that upon isoniazid exposure, the percentage of the intact PBP mutant cells was 24%, while the percentage of the intact wild-type cells was 4.6%. The PBP mutant cells exhibited decreased cell-lysis profile. Therefore, the traditional culture-based assays were not sufficient to provide insights about the subpopulation of viable but non-culture cells. Consequently, we need more adequate tools to be able to comprehend and fight the antibiotic resistance of bacteria.

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