Characterization of the Global Transcriptional Responses to Different Types of DNA Damage and Disruption of Replication in Bacillus subtilisGoranov, Alexi I.; Kuester-Schoeck, Elke; Wang, Jue D.; Grossman, Alan D.
doi: 10.1128/JB.00342-06pmid: 16855250
DNA damage and perturbations in DNA replication can induce global transcriptional responses that can help organisms repair the damage and survive. RecA is known to mediate transcriptional responses to DNA damage in several bacterial species by inactivating the repressor LexA and phage repressors. To gain insight into how Bacillus subtilis responds to various types of DNA damage, we measured the effects of DNA damage and perturbations in replication on mRNA levels by using DNA microarrays. We perturbed replication either directly with p -hydroxyphenylazo-uracil (HPUra), an inhibitor of DNA polymerase, or indirectly with the DNA-damaging reagents mitomycin C (MMC) and UV irradiation. Our results indicate that the transcriptional responses to HPUra, MMC, and UV are only partially overlapping. recA is the major transcriptional regulator under all of the tested conditions, and LexA appears to directly repress the expression of 63 genes in 26 operons, including the 18 operons previously identified as LexA targets. MMC and HPUra treatments caused induction of an integrative and conjugative element (ICE Bs1 ) and resident prophages (PBSX and SPß), which affected the expression of many host genes. Consistent with previous results, the induction of these mobile elements required recA . Induction of the phage appeared to require inactivation of LexA. Unrepaired UV damage and treatment with MMC also affected the expression of some of the genes that are controlled by DnaA. Furthermore, MMC treatment caused an increase in origin-proximal gene dosage. Our results indicate that different types of DNA damage have different effects on replication and on the global transcriptional profile.
Daughter Cell Separation by Penicillin-Binding Proteins and Peptidoglycan Amidases in Escherichia coliPriyadarshini, Richa; Popham, David L.; Young, Kevin D.
doi: 10.1128/JB.00476-06pmid: 16855223
As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli , this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the DD -carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.
A New D,L-Endopeptidase Gene Product, YojL (Renamed CwlS), Plays a Role in Cell Separation with LytE and LytF in Bacillus subtilisFukushima, Tatsuya; Afkham, Anahita; Kurosawa, Shin-ichirou; Tanabe, Taichi; Yamamoto, Hiroki; Sekiguchi, Junichi
doi: 10.1128/JB.00188-06pmid: 16855244
A new peptidoglycan hydrolase, Bacillus subtilis YojL ( c ell w all- l ytic enzyme associated with cell s eparation, renamed CwlS), exhibits high amino acid sequence similarity to LytE (CwlF) and LytF (CwlE), which are associated with cell separation. The N-terminal region of CwlS has four tandem repeat regions (LysM repeats) predicted to be a peptidoglycan-binding module. The C-terminal region exhibits high similarity to the cell wall hydrolase domains of LytE and LytF at their C-terminal ends. The C-terminal region of CwlS produced in Escherichia coli could hydrolyze the linkage of D - -glutamyl- meso -diaminopimelic acid in the cell wall of B. subtilis , suggesting that CwlS is a D , L -endopeptidase. ß-Galactosidase fusion experiments and Northern hybridization analysis suggested that the cwlS gene is transcribed during the late vegetative and early stationary phases. A cwlS mutant exhibited a cell shape similar to that of the wild type; however, a lytE lytF cwlS triple mutant exhibited aggregated microfiber formation. Moreover, immunofluorescence microscopy showed that FLAG-tagged CwlS was localized at cell separation sites and cell poles during the late vegetative phase. The localization sites are similar to those of LytF and LytE, indicating that CwlS is involved in cell separation with LytF and LytE. These specific localizations may be dependent on the LysM repeats in their N-terminal domains. The roles of CwlS, LytF, and LytE in cell separation are discussed.
Uropathogenic Escherichia coli Strains Generally Lack Functional Trg and Tap Chemoreceptors Found in the Majority of E. coli Strains Strictly Residing in the GutLane, M. Chelsea; Lloyd, Amanda L.; Markyvech, Tiffany A.; Hagan, Erin C.; Mobley, Harry L. T.
doi: 10.1128/JB.00449-06pmid: 16855252
The prevalence and function of four chemoreceptors, Tsr, Tar, Trg, and Tap, were determined for a collection of uropathogenic, fecal-commensal, and diarrheagenic Escherichia coli strains. tar and tsr were present or functional in nearly all isolates. However, trg and tap were significantly less prevalent or functional among the uropathogenic E. coli strains (both in 6% of strains) than among fecal-commensal strains (both in 50% of strains) or diarrheal strains (both in 75% of strains) ( P < 0.02).
The MgtC Virulence Factor of Salmonella enterica Serovar Typhimurium Activates Na+,K+-ATPaseGunzel, Dorothee; Kucharski, Lisa M.; Kehres, David G.; Romero, Michael F.; Maguire, Michael E.
doi: 10.1128/JB.00296-06pmid: 16855249
The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg 2+ -transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg 2+ transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H + , Mg 2+ , or Ca 2+ , thus ruling out a previously postulated function as a Mg 2+ /H + antiporter. However, addition of extracellular K + markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na + ,K + -ATPase completely prevented hyperpolarization and restored the normal K + -induced depolarization response. These results suggested that the Na + ,K + -ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na + ,K + -ATPase. Consistent with the involvement of Na + ,K + -ATPase, oocytes expressing MgtC exhibited an increased rate of 86 Rb + uptake and had increased intracellular free K + and decreased free Na + and ATP. The free concentrations of Mg 2+ and Ca 2+ and cytosolic pH were unchanged, although the total intracellular Ca 2+ content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg 2+ or another ion.
UvrD Helicase Suppresses Recombination and DNA Damage-Induced DeletionsKang, Josephine; Blaser, Martin J.
doi: 10.1128/JB.00275-06pmid: 16855234
UvrD, a highly conserved helicase involved in mismatch repair, nucleotide excision repair (NER), and recombinational repair, plays a critical role in maintaining genomic stability and facilitating DNA lesion repair in many prokaryotic species. In this report, we focus on the UvrD homolog in Helicobacter pylori , a genetically diverse organism that lacks many known DNA repair proteins, including those involved in mismatch repair and recombinational repair, and that is noted for high levels of inter- and intragenomic recombination and mutation. H. pylori contains numerous DNA repeats in its compact genome and inhabits an environment rich in DNA-damaging agents that can lead to increased rearrangements between such repeats. We find that H. pylori UvrD functions to repair DNA damage and limit homologous recombination and DNA damage-induced genomic rearrangements between DNA repeats. Our results suggest that UvrD and other NER pathway proteins play a prominent role in maintaining genome integrity, especially after DNA damage; thus, NER may be especially critical in organisms such as H. pylori that face high-level genotoxic stress in vivo.