In vivo analyses of constitutive and regulated promoters in halophilic archaea

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Dagmar Gregor ◽  
Felicitas Pfeifer
Keyword(s):  
Growth Phase ◽  
Promoter Activity ◽  
Halophilic Archaea ◽  
Stationary Growth Phase ◽  
Halobacterium Salinarum ◽  
Exponential Growth Phase ◽  
Haloferax Mediterranei ◽  
Basal Promoter Activity ◽  
Transcriptional Activator Protein

The two gvpA promoters PcA and PpA of Halobacterium salinarum, and the PmcA promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with β-galactosidase activity, as reporter. For comparison, the Pfdx promoter of the ferredoxin gene of Hbt. salinarum and the PbgaH promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed. Pfdx , driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas PbgaH and the three gvpA promoters yielded the largest activities during the stationary growth phase. Compared to Pfdx , the basal promoter activities of PpA and PmcA were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The PcA promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the PcA -TATA box and the BRE element were the reason for the lack of the basal PcA activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger PpA promoter and investigated in Hfx. volcanii transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the PpA -BRE element was substituted for the PcA -BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring PpA -BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of PmcA -BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of PmcA .

scholarly journals pH-, Lactic Acid-, and Non-Lactic Acid-Dependent Activities of Probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium

2005 ◽  
Vol 71 (10) ◽  
pp. 6008-6013 ◽  
Author(s):  
Domitille Fayol-Messaoudi ◽  
Cédric N. Berger ◽  
Marie-Hélène Coconnier-Polter ◽  
Vanessa Liévin-Le Moal ◽  
Alain L. Servin
Keyword(s):  
Lactic Acid ◽  
Growth Phase ◽  
Salmonella Enterica ◽  
Lactobacillus Rhamnosus ◽  
Stationary Growth Phase ◽  
Exponential Growth Phase ◽  
Killing Activity ◽  
Serovar Typhimurium ◽  
Probiotic Strains ◽  
Probiotic Lactobacilli

ABSTRACT The mechanism(s) underlying the antibacterial activity of probiotic Lactobacillus strains appears to be multifactorial and includes lowering of the pH and the production of lactic acid and of antibacterial compounds, including bacteriocins and nonbacteriocin, non-lactic acid molecules. Addition of Dulbecco's modified Eagle's minimum essential medium to the incubating medium delays the killing activity of lactic acid. We found that the probiotic strains Lactobacillus johnsonii La1, Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029, L. casei DN-114 001, and L. rhamnosus GR1 induced a dramatic decrease in the viability of Salmonella enterica serovar Typhimurium SL1344 mainly attributable to non-lactic acid molecule(s) present in the cell-free culture supernatant (CFCS). These molecules were more active against serovar Typhimurium SL1344 in the exponential growth phase than in the stationary growth phase. We also showed that the production of the non-lactic acid substance(s) responsible for the killing activity was dependent on growth temperature and that both unstable and stable substances with killing activity were present in the CFCSs. We found that the complete inhibition of serovar Typhimurium SL1344 growth results from a pH-lowering effect.

High Hydrostatic Pressure Come-Up Time and Yeast Viability

1998 ◽  
Vol 61 (12) ◽  
pp. 1657-1660 ◽  
Author(s):  
E. PALOU ◽  
A. LÓPEZ-MALO ◽  
G. V. BARBOSA-CÁNOVAS ◽  
J. WELTI-CHANES ◽  
P. M. DAVIDSON ◽  
...  
Keyword(s):  
Growth Phase ◽  
Growth Cycle ◽  
Stationary Growth Phase ◽  
Yeast Cells ◽  
Exponential Growth Phase ◽  
Stationary Growth ◽  
Survival Fraction ◽  
Zygosaccharomyces Bailii ◽  
Yeast Viability ◽  
Increased Sensitivity

The effects of the come-up time at selected pressures (50 to 689 MPa) on Saccharomyces cerevisiae and Zygosaccharomyces bailii viability were evaluated at 21°C. For Z. bailii the effects of the water activity (aw) of the suspension media and the stage of the growth cycle were also investigated. Pressure come-up times exerted an important effect on the yeast survival fraction, decreasing counts as pressure increased. An increased sensitivity to pressure treatments was observed with yeast cells from the exponential growth phase. Lethality increased as aw of the suspension media increased. For an aw of 0.98 and cells from the stationary growth phase, pressure treatments at less than 200 MPa had no effect on Z. bailii viability; however, no survivors (<10 CFU/ml) were observed in treatments applied only for the time needed to reach pressures greater than 517 MPa. Yeast survivor curves showed an excellent fit (r > 0.996) when described by a phenomenological model based on the Fermi equation, S(P) = 1/|1 + exp[(P − Pc)/k]|, where S(P) is the survival fraction, P is the pressure, Pc is a critical pressure corresponding to 50% survival, and k is a constant representing the steepness of the curve.

scholarly journals An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6365-6371 ◽  
Author(s):  
Kwang-Hwan Jung ◽  
Elena N. Spudich ◽  
Vishwa D. Trivedi ◽  
John L. Spudich
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Current Model ◽  
Halophilic Archaea ◽  
Halobacterium Salinarum ◽  
Cytoplasmic Domains ◽  
Transmembrane Segments ◽  
C Terminus ◽  
Reaction Cycles

ABSTRACT Halophilic archaea, such as Halobacterium salinarumand Natronobacterium pharaonis, alter their swimming behavior by phototaxis responses to changes in light intensity and color using visual pigment-like sensory rhodopsins (SRs). In N. pharaonis, SRII (NpSRII) mediates photorepellent responses through its transducer protein, NpHtrII. Here we report the expression of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial cytoplasmic domains and analyze their function in vivo in haloarchaea and in eubacteria. A fusion in which the C terminus of NpSRII is connected by a short flexible linker to NpHtrII is active in phototaxis signaling for H. salinarum, showing that the fusion does not inhibit functional receptor-transducer interactions. We replaced the cytoplasmic portions of this fusion protein with the cytoplasmic domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria. Purification of the fusion protein from H. salinarum and Tar fusion chimera from Escherichia coli membranes shows that the proteins are not cleaved and exhibit absorption spectra characteristic of wild-type membranes. Their photochemical reaction cycles in H. salinarum and E. coli membranes, respectively, are similar to those of native NpSRII in N. pharaonis. These fusion chimeras mediate retinal-dependent phototaxis responses by Escherichia coli, establishing that the nine-helix membrane portion of the receptor-transducer complex is a modular functional unit able to signal in heterologous membranes. This result confirms a current model for SR-Htr signal transduction in which the Htr transducers are proposed to interact physically and functionally with their cognate sensory rhodopsins via helix-helix contacts between their transmembrane segments.

scholarly journals Adherence of Staphylococcus aureus to Endothelial Cells: Influence of Capsular Polysaccharide, Global Regulatoragr, and Bacterial Growth Phase

2000 ◽  
Vol 68 (9) ◽  
pp. 4865-4871 ◽  
Author(s):  
Petra Pöhlmann-Dietze ◽  
Martina Ulrich ◽  
Kevin B. Kiser ◽  
Gerd Döring ◽  
Jean C. Lee ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Endothelial Cells ◽  
Bacterial Growth ◽  
Growth Phase ◽  
Parental Strain ◽  
Double Mutant ◽  
Capsular Polysaccharide ◽  
Stationary Growth Phase ◽  
Exponential Growth Phase ◽  
Logarithmic Phase

ABSTRACT The adherence of Staphylococcus aureus to human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulator agr, and the bacterial growth phase onS. aureus adherence to EC. Whereas S. aureusNewman showed maximal adherence to EC in the logarithmic phase of growth, an isogenic agr mutant showed maximal adherence in the stationary growth phase. S. aureus adherence to EC and CP5 expression were negatively correlated: a mutation in theagr locus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC.S. aureus Newman cells that adhered to EC did not express CP5. A Newman cap5O mutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P < 0.005). Complementation of the cap5Omutation in trans restored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by the cap5O mutant was similar in magnitude to that of the agr mutant or the cap5O agr double mutant. Cells of the cap5O mutant andcap5O agr double mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential and agr-independent expression by S. aureus of at least one putative EC adhesin, whose binding domain may be masked by CP5.

scholarly journals Involvement of the Detoxifying Enzyme Lactoylglutathione Lyase in Streptococcus mutans Aciduricity

2007 ◽  
Vol 189 (21) ◽  
pp. 7586-7592 ◽  
Author(s):  
Bryan Korithoski ◽  
Céline M. Lévesque ◽  
Dennis G. Cvitkovitch
Keyword(s):  
Streptococcus Mutans ◽  
Growth Phase ◽  
Proteome Analysis ◽  
Stationary Growth Phase ◽  
Low Ph ◽  
Transcriptional Analysis ◽  
Exponential Growth Phase ◽  
Expression Studies ◽  
Insertional Inactivation ◽  
Gene Expression Studies

ABSTRACT Streptococcus mutans, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Its main virulence factors are acidogenicity and aciduricity, the abilities to produce acid and to survive and grow at low pH, respectively. Metabolic processes are finely regulated following acid exposure in S. mutans. Proteome analysis of S. mutans demonstrated that lactoylglutathione lyase (LGL) was up-regulated during acid challenge. The LGL enzyme catalyzes the conversion of toxic methylglyoxal, derived from glycolysis, to S-d-lactoylglutathione. Methylglyoxal inhibits the growth of cells in all types of organisms. The current study aimed to investigate the relationship between LGL and aciduricity and acidogenicity in S. mutans. An S. mutans isogenic mutant defective in lgl (LGLKO) was created, and its growth kinetics were characterized. Insertional inactivation of lgl resulted in an acid-sensitive phenotype. However, the glycolytic rate at pH 5.0 was greater for LGLKO than for S. mutans UA159 wild-type cells. LGL was involved in the detoxification of methylglyoxal, illustrated by the absence of enzyme activity in LGLKO and the hypersensitivity of LGLKO to methylglyoxal, compared with UA159 (MIC of 3.9 and 15.6 mM, respectively). Transcriptional analysis of lgl conducted by quantitative real-time PCR revealed that lgl was up-regulated (approximately sevenfold) during the exponential growth phase compared with that in the stationary growth phase. Gene expression studies conducted at low pH demonstrated that lgl was induced during acidic growth (∼3.5-fold) and following acid adaptation (∼2-fold).This study demonstrates that in S. mutans, LGL functions in the detoxification of methylglyoxal, resulting in increased aciduricity.

scholarly journals Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Wenchao Feng ◽  
Dylan Minor ◽  
Mengyao Liu ◽  
Jinquan Li ◽  
Suzanne L. Ishaq ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Phase ◽  
Virulence Genes ◽  
Expression Patterns ◽  
Mouse Skin ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Subcutaneous Infection ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [SseA+] and normal SpeB activity [SpeBA+]). SseA+ SpeBA+ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an SseA+ SpeBA+ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other SseA+ SpeBA+ isolates also had nonsense mutations or small indels in rocA. RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.

scholarly journals Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

2010 ◽  
Vol 192 (22) ◽  
pp. 6039-6044 ◽  
Author(s):  
B. Singaravelan ◽  
B. R. Roshini ◽  
M. Hussain Munavar
Keyword(s):  
Escherichia Coli ◽  
Growth Phase ◽  
Stationary Growth Phase ◽  
Luciferase Assay ◽  
Logarithmic Phase ◽  
Nonsense Mutations ◽  
Translational Readthrough ◽  
Amber Suppressor ◽  
Nonsense Codons

ABSTRACT Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.

Triated Water Uptake and Desorption Kinetics in a Primary Producer: Chlorella Pyrenoidosa

1984 ◽  
Vol 19 (1) ◽  
pp. 101-110
Author(s):  
Thomas G. Dunstall
Keyword(s):  
Growth Phase ◽  
Tritiated Water ◽  
Chlorella Pyrenoidosa ◽  
Stationary Growth Phase ◽  
Exponential Growth Phase ◽  
Desorption Kinetics ◽  
Initial Growth ◽  
Organically Bound Tritium ◽  
Primary Producer ◽  
Aquatic Food

Abstract Chlorella pyrenoidosa, grown in batch culture under chronic exposure to tritiated water, was used to model behavior of tritium at the primary producer level of an aquatic food chain. The ratio of organically-bound tritium to tritium in the medium increased during initial growth, eventually reaching an asymptotic value of 0.59 after seven cell doublings. Loss of previously formed organically-bound tritium from cells transferred to tritium-free media averaged less than 5% for exponential growth phase cultures after three cell doublings. Over a comparable time period, organically-bound tritium lost from senescent cells averaged 13% which was attributed to increased degradative metabolism in stationary growth phase cultures. The concentration of tritium in organically-bound form may exceed environmental concentrations of tritium in water under dynamic conditions in which a pulse of tritiated water to the environment is dissipated over time.

scholarly journals Characterization of key enzymes involved in triacylglycerol biosynthesis in mycobacteria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Agostina Crotta Asis ◽  
Franco Savoretti ◽  
Matías Cabruja ◽  
Hugo Gramajo ◽  
Gabriela Gago
Keyword(s):  
Phosphatidic Acid ◽  
Growth Phase ◽  
De Novo ◽  
Cell Envelope ◽  
Exponential Growth Phase ◽  
Genes Encoding ◽  
Concomitant Reduction ◽  
Tag Biosynthesis ◽  
Odd Chain Fatty Acids

AbstractPhosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. PAP activity has a key role in the regulation of PA flux towards TAG or glycerophospholipid synthesis. In this work we have characterized two Mycobacterium smegmatis genes encoding for functional PAP proteins. Disruption of both genes provoked a sharp reduction in de novo TAG biosynthesis in early growth phase cultures under stress conditions. In vivo labeling experiments demonstrated that TAG biosynthesis was restored in the ∆PAP mutant when bacteria reached exponential growth phase, with a concomitant reduction of phospholipid synthesis. In addition, comparative lipidomic analysis showed that the ∆PAP strain had increased levels of odd chain fatty acids esterified into TAGs, suggesting that the absence of PAP activity triggered other rearrangements of lipid metabolism, like phospholipid recycling, in order to maintain the wild type levels of TAG. Finally, the lipid changes observed in the ∆PAP mutant led to defective biofilm formation. Understanding the interaction between TAG synthesis and the lipid composition of mycobacterial cell envelope is a key step to better understand how lipid homeostasis is regulated during Mycobacterium tuberculosis infection.

scholarly journals CcpA-Independent Regulation of Expression of the Mg2+-Citrate Transporter Gene citM by Arginine Metabolism in Bacillus subtilis

2003 ◽  
Vol 185 (3) ◽  
pp. 854-859 ◽  
Author(s):  
Jessica B. Warner ◽  
Christian Magni ◽  
Juke S. Lolkema
Keyword(s):  
Bacillus Subtilis ◽  
Growth Phase ◽  
Exponential Growth ◽  
Stationary Growth Phase ◽  
Luria Bertani ◽  
Transition Phase ◽  
Exponential Growth Phase ◽  
Uptake System ◽  
Regulation Of Expression ◽  
Citrate Transporter

ABSTRACT Transcriptional regulation of the Mg2+-citrate transporter, CitM, the main citrate uptake system of Bacillus subtilis, was studied during growth in rich medium. Citrate in the growth medium was required for induction under all growth conditions. In Luria-Bertani medium containing citrate, citM expression was completely repressed during the exponential growth phase, marginally expressed in the transition phase, and highly expressed in the stationary growth phase. The repression was relieved when the cells were grown in spent Luria-Bertani medium. The addition of a mixture of 18 amino acids restored repression. l-Arginine in the mixture appeared to be solely responsible for the repression, and ornithine appeared to be an equally potent repressor of citM expression. Studies of mutant strains deficient in RocR and SigL, proteins required for the expression of the enzymes of the arginase pathway, confirmed that uptake into the cell and, most likely, conversion of arginine to ornithine were required for repression. Arginine-mediated repression was independent of a functional CcpA, the global regulator protein in carbon catabolite repression (CCR). Nevertheless, CCR-mediated repression was the major mechanism controlling the expression during exponential growth, while the newly described, CcpA-independent arginine-mediated repression was specifically apparent during the transition phase of growth.

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