scholarly journals Conversion of Methionine to Cysteine in Bacillus subtilis and Its Regulation

2006 ◽  
Vol 189 (1) ◽  
pp. 187-197 ◽  
Author(s):  
Marie-Françoise Hullo ◽  
Sandrine Auger ◽  
Olga Soutourina ◽  
Octavian Barzu ◽  
Mireille Yvon ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Promoter Region ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sulfur Source ◽  
Wild Type ◽  
Lyase Activity ◽  
Recycling Pathway ◽  
Dna Complex

ABSTRACT Bacillus subtilis can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in Escherichia coli were tested. Both CysK and YrhA have an O-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine β-synthase activity using O-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine γ-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for cysK or yrhB grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an ΔyrhA mutant or a luxS mutant, inactivated for the S-ribosyl-homocysteinase step of the S-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a ΔyrhA ΔcysK or cysE mutant did not grow at all under the same conditions. The yrhB and yrhA genes form an operon together with yrrT, mtnN, and yrhC. The expression of the yrrT operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the yrrT promoter region in gel-shift experiments. The addition of O-acetyl-serine prevented the formation of this protein-DNA complex.

scholarly journals Bacillus subtilis Phosphorylated PhoP: Direct Activation of the EσA- and Repression of the EσE-Responsive phoB-PS+V Promoters during Pho Response

2005 ◽  
Vol 187 (15) ◽  
pp. 5166-5178 ◽  
Author(s):  
Wael R. Abdel-Fattah ◽  
Yinghua Chen ◽  
Amr Eldakak ◽  
F. Marion Hulett
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pho Regulon ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphate Deprivation

ABSTRACT The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-PS, was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-PV. EσE was necessary and sufficient for PS expression in vitro. PS expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of σE is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP∼P) repressed PS in vitro via direct binding to the promoter, the first example of an EσE-responsive promoter that is repressed by PhoP∼P. Whereas either PhoP or PhoP∼P in the presence of EσA was sufficient to stimulate transcription from the phoB-PV promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP∼P were required for PV promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during Pi -replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

scholarly journals Construction of an In Vivo Nonsense Readthrough Assay System and Functional Analysis of Ribosomal Proteins S12, S4, and S5 in Bacillus subtilis

2001 ◽  
Vol 183 (17) ◽  
pp. 4958-4963 ◽  
Author(s):  
Takashi Inaoka ◽  
Koji Kasai ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Ribosomal Proteins ◽  
Type Strain ◽  
Wild Type Strain ◽  
Assay System ◽  
Wild Type ◽  
Translational Accuracy ◽  
Free Translation

ABSTRACT To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant. These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in aram phenotype in B. subtilis. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.

scholarly journals Construction and Characterization of aMycobacterium tuberculosis Mutant Lacking the Alternate Sigma Factor Gene, sigF

2000 ◽  
Vol 68 (10) ◽  
pp. 5575-5580 ◽  
Author(s):  
Ping Chen ◽  
Rafael E. Ruiz ◽  
Qing Li ◽  
Richard F. Silver ◽  
William R. Bishai
Keyword(s):  
Stationary Phase ◽  
Sigma Factor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Axenic Culture ◽  
Wild Type ◽  
Intracellular Growth ◽  
Factor Gene ◽  
Time To Death

ABSTRACT The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain ofMycobacterium tuberculosis. The growth rate of the ΔsigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the ΔsigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the ΔsigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the ΔsigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

scholarly journals Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi

2006 ◽  
Vol 50 (2) ◽  
pp. 445-452 ◽  
Author(s):  
Daniel Criswell ◽  
Virginia L. Tobiason ◽  
J. Stephen Lodmell ◽  
D. Scott Samuels
Keyword(s):  
16S Rrna ◽  
Borrelia Burgdorferi ◽  
Type Strain ◽  
Wild Type Strain ◽  
Small Subunit ◽  
Wild Type ◽  
Spectinomycin Resistance ◽  
E Coli ◽  
Resistant Mutants

ABSTRACT We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

NEW LOCI IN DICTYOSTELIUM DISCOIDEUM DETERMINING PIGMENT FORMATION AND GROWTH ON BACILLUS SUBTILIS

Genetics ◽  
1980 ◽  
Vol 96 (1) ◽  
pp. 115-123
Author(s):  
James H Morrissey ◽  
Steven Wheeler ◽  
William F Loomis
Keyword(s):  
Bacillus Subtilis ◽  
Dictyostelium Discoideum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Linkage Groups ◽  
New Mutation ◽  
Pigment Formation ◽  
Complementation Groups ◽  
First Time

ABSTRACT Seventeen independently isolated pigmentless (white) mutations in Dictyostelium discoideum are all recessive and fall into three complementation groups identifying two new whi loci in addition to the previously characterized whiA locus. whiB and whiC map to linkage groups III and IV, respectively. In addition, it was discovered that our laboratory stock of NC4, the wild-type strain from which these mutants were derived, has spontaneously lost the ability to grow on Bacillus subtilis. This new mutation, bsgB500, maps to linkage group VII and is not allelic to bsgA. bsgB500 is the first spontaneously derived mutation in D. discoideum that can be used to select heterozygous diploids, and for the first time allows genetic analysis to be routinely performed on strains derived from an unmutagenized background.

scholarly journals Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Jan Kampf ◽  
Jan Gerwig ◽  
Kerstin Kruse ◽  
Robert Cleverley ◽  
Miriam Dormeyer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Selective Pressure ◽  
Wild Type ◽  
Master Regulator ◽  
Form Complex ◽  
Content Type

ABSTRACT Biofilm formation by Bacillus subtilis requires the expression of genes encoding enzymes for extracellular polysaccharide synthesis and for an amyloid-like protein. The master regulator SinR represses all the corresponding genes, and repression of these key biofilm genes is lifted when SinR interacts with its cognate antagonist proteins. The YmdB phosphodiesterase is a recently discovered factor that is involved in the control of SinR activity: cells lacking YmdB exhibit hyperactive SinR and are unable to relieve the repression of the biofilm genes. In this study, we have examined the dynamics of gene expression patterns in wild-type and ymdB mutant cells by microfluidic analysis coupled to time-lapse microscopy. Our results confirm the bistable expression pattern for motility and biofilm genes in the wild-type strain and the loss of biofilm gene expression in the mutant. Moreover, we demonstrated dynamic behavior in subpopulations of the wild-type strain that is characterized by switches in sets of the expressed genes. In order to gain further insights into the role of YmdB, we isolated a set of spontaneous suppressor mutants derived from ymdB mutants that had regained the ability to form complex colonies and biofilms. Interestingly, all of the mutations affected SinR. In some mutants, large genomic regions encompassing sinR were deleted, whereas others had alleles encoding SinR variants. Functional and biochemical studies with these SinR variants revealed how these proteins allowed biofilm gene expression in the ymdB mutant strains. IMPORTANCE Many bacteria are able to choose between two mutually exclusive lifestyles: biofilm formation and motility. In the model bacterium Bacillus subtilis, this choice is made by each individual cell rather than at the population level. The transcriptional repressor SinR is the master regulator in this decision-making process. The regulation of SinR activity involves complex control of its own expression and of its interaction with antagonist proteins. We show that the YmdB phosphodiesterase is required to allow the expression of SinR-repressed genes in a subpopulation of cells and that such subpopulations can switch between different SinR activity states. Suppressor analyses revealed that ymdB mutants readily acquire mutations affecting SinR, thus restoring biofilm formation. These findings suggest that B. subtilis cells experience selective pressure to form the extracellular matrix that is characteristic of biofilms and that YmdB is required for the homeostasis of SinR and/or its antagonists.

scholarly journals In VivoEfficacy of Human Simulated Regimens of Carbapenems and Comparator Agents against NDM-1-Producing Enterobacteriaceae

2013 ◽  
Vol 58 (3) ◽  
pp. 1671-1677 ◽  
Author(s):  
Dora E. Wiskirchen ◽  
Patrice Nordmann ◽  
Jared L. Crandon ◽  
David P. Nicolau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Similar Efficacy ◽  
Infection Model ◽  
High Dose ◽  
Wild Type ◽  
Prolonged Infusion ◽  
Content Type

ABSTRACTDoripenem and ertapenem have demonstrated efficacy against several NDM-1-producing isolatesin vivo, despite having high MICs. In this study, we sought to further characterize the efficacy profiles of humanized regimens of standard (500 mg given every 8 h) and high-dose, prolonged infusion of doripenem (2 g given every 8 h, 4-h infusion) and 1 g of ertapenem given intravenously every 24 h and the comparator regimens of ceftazidime at 2 g given every 8 h (2-h infusion), levofloxacin at 500 mg every 24 h, and aztreonam at 2 g every 6 h (1-h infusion) against a wider range of isolates in a murine thigh infection model. An isogenic wild-type strain and NDM-1-producingKlebsiella pneumoniaeand eight clinical NDM-1-producing members of the familyEnterobacteriaceaewere tested in immunocompetent- and neutropenic-mouse models. The wild-type strain was susceptible to all of the agents, while the isogenic NDM-1-producing strain was resistant to ceftazidime, doripenem, and ertapenem. Clinical NDM-1-producing strains were resistant to nearly all five of the agents (two were susceptible to levofloxacin). In immunocompetent mice, all of the agents produced ≥1-log10CFU reductions of the isogenic wild-type and NDM-1-producing strains after 24 h. Minimal efficacy of ceftazidime, aztreonam, and levofloxacin against the clinical NDM-1-producing strains was observed. However, despitein vitroresistance, ≥1-log10CFU reductions of six of eight clinical strains were achieved with high-dose, prolonged infusion of doripenem and ertapenem. Slight enhancements of doripenem activity over the standard doses were obtained with high-dose, prolonged infusion for three of the four isolates tested. Similar efficacy observations were noted in neutropenic mice. These data suggest that carbapenems are a viable treatment option for infections caused by NDM-1-producingEnterobacteriaceae.

scholarly journals A Bile Salt Hydrolase of Brucella abortus Contributes to the Establishment of a Successful Infection through the Oral Route in Mice

2006 ◽  
Vol 75 (1) ◽  
pp. 299-305 ◽  
Author(s):  
M. Victoria Delpino ◽  
María I. Marchesini ◽  
Silvia M. Estein ◽  
Diego J. Comerci ◽  
Juliana Cassataro ◽  
...  
Keyword(s):  
Bile Salt ◽  
Brucella Abortus ◽  
Type Strain ◽  
Wild Type Strain ◽  
Interleukin 2 ◽  
Oral Route ◽  
Bile Salt Hydrolase ◽  
Wild Type ◽  
Successful Infection

ABSTRACT Choloylglycine hydrolase (CGH), a bile salt hydrolase, has been annotated in all the available genomes of Brucella species. We obtained the Brucella CGH in recombinant form and demonstrated in vitro its capacity to cleave glycocholate into glycine and cholate. Brucella abortus 2308 (wild type) and its isogenic Δcgh deletion mutant exhibited similar growth rates in tryptic soy broth in the absence of bile. In contrast, the growth of the Δcgh mutant was notably impaired by both 5% and 10% bile. The bile resistance of the complemented mutant was similar to that of the wild-type strain. In mice infected through the intragastric or the intraperitoneal route, splenic infection was significantly lower at 10 and 20 days postinfection in animals infected with the Δcgh mutant than in those infected with the wild-type strain. For both routes, no differences in spleen CFU were found between animals infected with the wild-type strain and those infected with the complemented mutant. Mice immunized intragastrically with recombinant CGH mixed with cholera toxin (CGH+CT) developed a specific mucosal humoral (immunoglobulin G [IgG] and IgA) and cellular (interleukin-2) immune responses. Fifteen days after challenge by the same route with live B. abortus 2308 cells, splenic CFU counts were 10-fold lower in mice immunized with CGH+CT than in mice immunized with CT or phosphate-buffered saline. This study shows that CGH confers on Brucella the ability to resist the antimicrobial action of bile salts. The results also suggest that CGH may contribute to the ability of Brucella to infect the host through the oral route.

scholarly journals Lipooligosaccharide Structure Contributes to Multiple Steps in the Virulence of Neisseria meningitidis

2006 ◽  
Vol 74 (2) ◽  
pp. 1360-1367 ◽  
Author(s):  
Laura Plant ◽  
Johanna Sundqvist ◽  
Susu Zughaier ◽  
Lena Lövkvist ◽  
David S. Stephens ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Neisseria Meningitidis ◽  
Type Strain ◽  
Lipid A ◽  
Wild Type Strain ◽  
Inner Core ◽  
Wild Type ◽  
Proinflammatory Response

ABSTRACT Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galβ-GlcNAc-Galβ-Glcβ-Hep2 (GlcNAc, Glcα) 3-deoxy-d-manno-2-octulosonic acid (KDO2)-lipid A; 1,4′ bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.

scholarly journals Identification of Concomitant Infection with Chlamydia trachomatis IncA-Negative Mutant and Wild-Type Strains by Genomic, Transcriptional, and Biological Characterizations

2008 ◽  
Vol 76 (12) ◽  
pp. 5438-5446 ◽  
Author(s):  
Robert J. Suchland ◽  
Brendan M. Jeffrey ◽  
Minsheng Xia ◽  
Ajay Bhatia ◽  
Hencelyn G. Chu ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Genome Sequence ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Sequence ◽  
Genomic Structure ◽  
Transcriptional Analysis ◽  
Clinical Samples ◽  
Wild Type

ABSTRACT Clinical isolates of Chlamydia trachomatis that lack IncA on their inclusion membrane form nonfusogenic inclusions and have been associated with milder, subclinical infections in patients. The molecular events associated with the generation of IncA-negative strains and their roles in chlamydial sexually transmitted infections are not clear. We explored the biology of the IncA-negative strains by analyzing their genomic structure, transcription, and growth characteristics in vitro and in vivo in comparison with IncA-positive C. trachomatis strains. Three clinical samples were identified that contained a mixture of IncA-positive and -negative same-serovar C. trachomatis populations, and two more such pairs were found in serial isolates from persistently infected individuals. Genomic sequence analysis of individual strains from each of two serovar-matched pairs showed that these pairs were very similar genetically. In contrast, the genome sequence of an unmatched IncA-negative strain contained over 5,000 nucleotide polymorphisms relative to the genome sequence of a serovar-matched but otherwise unlinked strain. Transcriptional analysis, in vitro culture kinetics, and animal modeling demonstrated that IncA-negative strains isolated in the presence of a serovar-matched wild-type strain are phenotypically more similar to the wild-type strain than are IncA-negative strains isolated in the absence of a serovar-matched wild-type strain. These studies support a model suggesting that a change from an IncA-positive strain to the previously described IncA-negative phenotype may involve multiple steps, the first of which involves a translational inactivation of incA, associated with subsequent unidentified steps that lead to the observed decrease in transcript level, differences in growth rate, and differences in mouse infectivity.

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