The low-molecular-weight (LMW) penicillin-binding protein, PBP 5, plays a dominant role in determining the uniform cell shape of Escherichia coli. However, the physiological functions of six other LMW PBPs are unknown, even though the existence and enzymatic activities of four of these were established three decades ago. By applying fluorescence-activated cell sorting (FACS) to quantify the cellular dimensions of multiple PBP mutants, we found that the endopeptidases PBP 4 and PBP 7 also influence cell shape in concert with PBP 5. This is the first reported biological function for these two proteins. In addition, the combined loss of three DD-carboxypeptidases, PBPs 5 and 6 and DacD, also impaired cell shape. In contrast to previous reports based on visual inspection alone, FACS analysis revealed aberrant morphology in a mutant lacking only PBP 5, a phenotype not shared by any other strain lacking a single LMW PBP. PBP 5 removes the terminal D-alanine from pentapeptide side chains of muropeptide subunits, and pentapeptides act as donors for cross-linking adjacent side chains. As endopeptidases, PBPs 4 and 7 cleave cross-links in the cell wall. Therefore, overall cell shape may be determined by the existence or location of a specific type of peptide cross-link, with PBP 5 activity influencing how many cross-links are made and PBPs 4 and 7 acting as editing enzymes to remove inappropriate cross-links.
An unexpected observation led us to examine the relationship between L-lactam exposure and synthesis of colonic acid capsular polysaccharide in Escherichia coli. Strains containing a cps-lacZ transcriptional fusion were challenged with antibiotics having various modes of action, and gene expression was detected by a disk-diffusion assay and in broth cultures. The cps genes were induced by a subset of L-lactams but not by agents inhibiting protein synthesis or DNA replication, indicating that cps expression was specific and not due to stresses accompanying cell death or by a general inhibition of peptidoglycan synthesis. A narrow concentration just below the MIC triggered cps expression in liquid culture, suggesting the response may be triggered by near-lethal levels of antibiotic. Because colanic acid is important for maturation of biofilm architecture, antibiotics that increase its synthesis might exacerbate the formation or persistence of biofilms.
Steroidogenic acute regulatory protein (StAR), proposed to be involved in the transport of cholesterol to the inner mitochondrial membrane, has recently been cloned from MA-10 cells. Using reverse transcription-polymerase chain reaction, we generated a complementary DNA encoding 404 base pairs of StAR from ovine luteal tissue to perform studies regarding regulation of the messenger RNA (mRNA) encoding this protein. In Exp 1, ewes were hypophysectomized (HPX) on day 5 of the estrous cycle and administered saline or physiological regimens of LH and/or GH until collection of luteal tissue on day 12 of the estrous cycle (n = 4/group). Luteal concentrations [mean +/- SEM; femtomoles per microgram poly(A)+ RNA] of mRNA encoding StAR were lower (P < 0.05) in the HPX plus saline-treated ewes (26.4 +/- 7.3) than in day 12 pituitary-intact ewes (n = 4; 77.7 +/- 9.3). Replacement of LH (59.1 +/- 13.1), GH (59.1 +/- 12.8), or LH and GH (69.9 +/- 4.5) in HPX ewes increased (P < 0.05) concentrations of mRNA encoding StAR to values not different from those in day 12 controls. In Exp 2, ewes on day 11 or 12 of the estrous cycle were injected with prostaglandin F2 alpha (PGF2 alpha) to induce luteal regression. Corpora lutea were collected 4, 12, or 24 h after injection (n = 4-5/time point) and from untreated control ewes (n = 4) or 24 h after injection of saline (n = 4). Treatment with PGF2 alpha decreased (P < 0.05) concentrations of progesterone in serum 4, 12, and 24 h after injection. Concentrations of StAR mRNA were decreased (P < 0.01) to 47%, 19%, and 8% of control values 4, 12, and 24 h after PGF2 alpha injection, respectively. In Exp 3, ewes received ovarian arterial infusions of saline, PGF2 alpha, or phorbol 12-myristate 13-acetate (PMA), and luteal tissue was collected 0 (no infusion), 4, 12, or 24 h later (n = 3-4/group). Treatment with PGF2 alpha or PMA decreased (P < 0.05) concentrations of progesterone in serum 4, 12, and 24 h postinjection. Steady state concentrations of mRNA encoding StAR (P < 0.05) were 36% and 25% of the control value 12 and 24 h after PGF2 alpha injection. Injection of PMA decreased (P < 0.05) concentrations of StAR mRNA to 75% and 50% of control values at 4 and 12 h, but concentrations of mRNA encoding StAR were not different from control values at 24 h.(ABSTRACT TRUNCATED AT 400 WORDS)
Previously, we constructed a set of mutants from which eight penicillin binding protein (PBP) genes were deleted in 192 combinations from Escherichia coli (S. A. Denome, P. K. Elf, T. A. Henderson, D. E. Nelson, and K. D. Young, J. Bacteriol. 181:3981-3993, 1999). Although these mutants were constructed correctly as determined by restriction mapping and the absence of relevant protein products, we recently discovered by PCR mapping that strains from which mrcA (PBP 1a) was deleted were also missing two neighboring genes of unknown function (yrfE and yrfF). We created a new deletion mutation in mrcA and reconstructed 63 strains lacking PBP 1a and other PBP mutant combinations. The new mrcA mutants do not exhibit mucoidy, phage resistance, temperature sensitivity, growth rate defects, or antibiotic resistance, suggesting that these phenotypes require the loss of either yrfE or yrfF alone or in combination with the absence of multiple PBPs.Four high-molecular-weight penicillin binding proteins (PBPs) of Escherichia coli (PBPs 1a, 1b, 2, and 3) are responsible for synthesizing and assembling the peptidoglycan sacculus that forms the rigid bacterial cell wall (5, 6). However, E. coli also possesses at least seven low-molecular-weight PBPs (PBPs 4, 5, 6, and 7 and DacD, AmpC, and AmpH), the biological functions of which are either poorly characterized or completely unknown (2, 5, 6).To address this question of physiological function, we constructed a set of multiply mutated strains in which one to seven PBPs were deleted in every viable combination (2). At the time of construction, each strain was tested by restriction mapping and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm that the correct genes and protein products had been deleted. Recently, we found we were unable to PCR amplify the mutated mrcA gene (encoding PBP 1a) from chromosomal preparations when using oligonucleotide primers hybridizing to sequences just upstream and downstream of the putative deletion endpoints. Primers further away from the mutated site did give an amplification product (data not shown), suggesting that a larger fragment had been deleted than was reported previously. DNA sequencing confirmed that one open reading frame (ORF) (yrfE) and the promoter and 5Ј end of a second open reading frame (yrfF) were deleted in addition to most of the mrcA gene (data not shown). The extent of the deletion is pictured schematically in Fig. 1B. Thus, every strain designated as ⌬mrcA in our previous publication (2) is actually a ⌬(mrcA-yrfE-yrfF) deletion. All other PBP gene deletions were correct as reported (data not shown).The data in Fig. 1 illustrates why the deletion was not correctly characterized earlier. When creating the original mrcA mutation we observed a single XhoI-BspDI DNA fragment, leading us to believe that one BspDI site existed in the cloned segment. However, after the mutants were constructed, the complete genomic sequence of E. coli (1) revealed there were three BspDI sites-two so close together t...
To investigate the regulation of ovine luteal receptors for prostaglandin F2 alpha (PGF2 alpha), reverse transcription-polymerase chain reaction was used to produce a 284-bp partial cDNA that was 98% identical to that reported for the bovine PGF2 alpha receptor (PGF2 alpha-R). In situ hybridization localized mRNA for PGF2 alpha-R specifically to large luteal cells. In experiment 1, pools of luteal tissue (n = 4/day) collected from ewes on Days 3, 6, 9, 12, and 15 of the estrous cycle were analyzed for mRNA encoding PGF2 alpha-R. There was no difference in mean steady-state concentrations of mRNA encoding PGF2 alpha-R among any of the days studied (range = 2.3 +/- 0.3 to 3.5 +/- 0.7 fmol PGF2 alpha-R mRNA/ microgram poly[A]+ RNA as assessed by slot-blot hybridization). In experiment 2, ewes on Day 11 or Day 12 of the estrous cycle were administered PGF2 alpha, and corpora lutea were collected 4, 12, or 24 h later (n = 4-5 per time point). Nontreated (n = 4) or saline-treated (n = 4) ewes served as controls. Luteal concentrations of mRNA encoding PGF2 alpha-R were decreased (p < 0.05) at 4, 12, and 24 h after injection of PGF2 alpha. In experiment 3, ewes (midluteal phase) were administered saline, PGF2 alpha, phorbol 12-myristate 13-acetate (PMA), or LH via ovarian arterial injection, and luteal tissue was collected 0, 4, 12, or 24 h later (n = 3-4 per treatment per time). Steady-state concentrations of mRNA encoding PGF2 alpha-R were decreased (p < 0.05) by PGF2 alpha and PMA treatment (4 and 12 h) but were increased (p < 0.05) at 24 h after LH treatment. In summary, 1) mRNA encoding PGF2 alpha-R was localized to large luteal cells; 2) concentrations of mRNA encoding PGF2 alpha-R did not vary during the estrous cycle; 3) treatment with PGF2 alpha or PMA to activate protein kinase C decreased concentrations of PGF2 alpha-R mRNA within 4 h of treatment; and 4) administration LH increased concentrations of mRNA encoding PGF2 alpha-R 24 h following injection.
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