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.

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 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 Characterization of dacC, Which Encodes a New Low-Molecular-Weight Penicillin-Binding Protein in Bacillus subtilis

1998 ◽  
Vol 180 (18) ◽  
pp. 4967-4973 ◽  
Author(s):  
Lotte B. Pedersen ◽  
Thomas Murray ◽  
David L. Popham ◽  
Peter Setlow
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Binding Protein ◽  
Low Molecular Weight ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Genome Sequencing Project ◽  
Penicillin Binding Proteins ◽  
Sequencing Project

ABSTRACT The pbp gene (renamed dacC), identified by the Bacillus subtilis genome sequencing project, encodes a putative 491-residue protein with sequence homology to low-molecular-weight penicillin-binding proteins. Use of a transcriptional dacC-lacZ fusion revealed thatdacC expression (i) is initiated at the end of stationary phase; (ii) depends strongly on transcription factor ςH; and (iii) appears to be initiated from a promoter located immediately upstream of yoxA, a gene of unknown function located upstream of dacC on the B. subtilis chromosome. A B. subtilis dacCinsertional mutant grew and sporulated identically to wild-type cells, and dacC and wild-type spores had the same heat resistance, cortex structure, and germination and outgrowth kinetics. Expression ofdacC in Escherichia coli showed that this gene encodes an ∼59-kDa membrane-associated penicillin-binding protein which is highly toxic when overexpressed.

scholarly journals A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth.

1992 ◽  
Vol 12 (3) ◽  
pp. 1162-1178 ◽  
Author(s):  
C Costigan ◽  
S Gehrung ◽  
M Snyder
Keyword(s):  
Cell Growth ◽  
Vegetative Growth ◽  
Sequence Similarity ◽  
Growth Control ◽  
Cell Morphogenesis ◽  
Wild Type ◽  
Extra Copy ◽  
Synthetic Lethal ◽  
Cell Biol ◽  
Regulatory Link

The Saccharomyces cerevisiae SPA2 protein localizes at sites involved in polarized cell growth in budding cells and mating cells. spa2 mutants have defects in projection formation during mating but are healthy during vegetative growth. A synthetic lethal screen was devised to identify mutants that require the SPA2 gene for vegetative growth. One mutant, called slk-1 (for synthetic lethal kinase), has been characterized extensively. The SLK1 gene has been cloned, and sequence analysis predicts that the SLK1 protein is 1,478 amino acid residues in length. Approximately 300 amino acids at the carboxy terminus exhibit sequence similarity with the catalytic domains of protein kinases. Disruption mutations have been constructed in the SLK1 gene. slk1 null mutants cannot grow at 37 degrees C, but many cells can grow at 30, 24, and 17 degrees C. Dead slk1 mutant cells usually have aberrant cell morphologies, and many cells are very small, approximately one-half the diameter of wild-type cells. Surviving slk1 cells also exhibit morphogenic defects; these cells are impaired in their ability to form projections upon exposure to mating pheromones. During vegetative growth, a higher fraction of slk1 cells are unbudded compared with wild-type cells, and under nutrient limiting conditions, slk1 cells exhibit defects in cell cycle arrest. The different slk1 mutant defects are partially rescued by an extra copy of the SSD1/SRK1 gene. SSD1/SRK1 has been independently isolated as a suppressor of mutations in genes involved in growth control, sit4, pde2, bcy1, and ins1 (A. Sutton, D. Immanuel, and K.T. Arnat, Mol. Cell. Biol. 11:2133-2148, 1991; R.B. Wilson, A.A. Brenner, T.B. White, M.J. Engler, J.P. Gaughran, and K. Tatchell, Mol. Cell. Biol. 11:3369-3373, 1991). These data suggest that SLK1 plays a role in both cell morphogenesis and the control of cell growth. We speculate that SLK1 may be a regulatory link for these two cellular processes.

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

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.

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