scholarly journals Mycothiol Import by Mycobacterium smegmatis and Function as a Resource for Metabolic Precursors and Energy Production

2007 ◽  
Vol 189 (19) ◽  
pp. 6796-6805 ◽  
Author(s):  
Krzysztof P. Bzymek ◽  
Gerald L. Newton ◽  
Philong Ta ◽  
Robert C. Fahey
Keyword(s):  
Stationary Phase ◽  
Mycobacterium Smegmatis ◽  
Type Strain ◽  
Energy Production ◽  
Wild Type Strain ◽  
Krebs Cycle ◽  
Wild Type ◽  
Suitable Target ◽  
Cysteine Desulfhydrase ◽  
And Function

ABSTRACT Mycothiol ([MSH] AcCys-GlcN-Ins, where Ac is acetyl) is the major thiol produced by Mycobacterium smegmatis and other actinomycetes. Mutants deficient in MshA (strain 49) or MshC (transposon mutant Tn1) of MSH biosynthesis produce no MSH. However, when stationary phase cultures of these mutants were incubated in medium containing MSH, they actively transported it to generate cellular levels of MSH comparable to or greater than the normal content of the wild-type strain. When these MSH-loaded mutants were transferred to MSH-free preconditioned medium, the cellular MSH was catabolized to generate GlcN-Ins and AcCys. The latter was rapidly converted to Cys by a high deacetylase activity assayed in extracts. The Cys could be converted to pyruvate by a cysteine desulfhydrase or used to regenerate MSH in cells with active MshC. Using MSH labeled with [U-14C]cysteine or with [6-3H]GlcN, it was shown that these residues are catabolized to generate radiolabeled products that are ultimately lost from the cell, indicating extensive catabolism via the glycolytic and Krebs cycle pathways. These findings, coupled with the fact the myo-inositol can serve as a sole carbon source for growth of M. smegmatis, indicate that MSH functions not only as a protective cofactor but also as a reservoir of readily available biosynthetic precursors and energy-generating metabolites potentially important under stress conditions. The half-life of MSH was determined in stationary phase cells to be ∼50 h in strains with active MshC and 16 ± 3 h in the MshC-deficient mutant, suggesting that MSH biosynthesis may be a suitable target for drugs to treat dormant tuberculosis.

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 Characterization of a Legionella pneumophila relA Insertion Mutant and Roles of RelA and RpoS in Virulence Gene Expression

2002 ◽  
Vol 184 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Tal Zusman ◽  
Ohad Gal-Mor ◽  
Gil Segal
Keyword(s):  
Gene Expression ◽  
Stationary Phase ◽  
Gene Product ◽  
Legionella Pneumophila ◽  
Type Strain ◽  
Wild Type Strain ◽  
Insertion Mutant ◽  
Wild Type ◽  
Intracellular Growth ◽  
Virulence Gene Expression

ABSTRACT To investigate the involvement of RelA in the regulation of Legionella pneumophila virulence, a deletion substitution was constructed in the relA gene. The relA knockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when the relA gene product was supplied on a plasmid. The effect of the relA mutation was examined with two systems that are known to be expressed during the stationary phase in L. pneumophila. Pigment production was found to be dependent on the relA gene product, and only one-half as much pigment was produced by the relA mutant as by the wild-type strain. Flagellum gene expression was also found to be dependent on the relA gene product, as determined with a flaA::lacZ fusion. However, the relA gene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii. To determine the involvement of the relA gene product in expression of L. pneumophila genes required for intracellular growth (icm/dot genes), nine icm::lacZ fusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression in relA mutant strains. Expression of only one of the icm::lacZ fusions was moderately reduced in the relA mutant strain. Expression of the nine icm::lacZ fusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained. We concluded that RelA is dispensable for intracellular growth of L. pneumophila in the two hosts examined and that both RelA and RpoS play minor roles in L. pneumophila icm/dot gene expression.

scholarly journals Evidence for Contribution of Neutral Trehalase in Barotolerance of Saccharomyces cerevisiae

2000 ◽  
Vol 66 (12) ◽  
pp. 5182-5185 ◽  
Author(s):  
Hitoshi Iwahashi ◽  
Solomon Nwaka ◽  
Kaoru Obuchi
Keyword(s):  
Hydrostatic Pressure ◽  
Stationary Phase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Glucose Repression ◽  
Logarithmic Phase ◽  
Wild Type ◽  
Neutral Trehalase ◽  
I Gene

ABSTRACT In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression ofNTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.

scholarly journals Methionine Sulfoxide Reductase A (MsrA) Deficiency Affects the Survival of Mycobacterium smegmatis within Macrophages

2004 ◽  
Vol 186 (11) ◽  
pp. 3590-3598 ◽  
Author(s):  
T. Douglas ◽  
D. S. Daniel ◽  
B. K. Parida ◽  
C. Jagannath ◽  
S. Dhandayuthapani
Keyword(s):  
Mutant Strain ◽  
Mycobacterium Smegmatis ◽  
Type Strain ◽  
Pathogenic Bacteria ◽  
Wild Type Strain ◽  
Methionine Sulfoxide ◽  
Intracellular Survival ◽  
Methionine Sulfoxide Reductase ◽  
Wild Type ◽  
Methionine Sulfoxide Reductase A

ABSTRACT Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme which reduces oxidized methionine to methionine. Since oxidation of methionine in proteins impairs their function, an absence of MsrA leads to abnormalities in different organisms, including alterations in the adherence patterns and in vivo survival of certain pathogenic bacteria. To understand the role of MsrA in intracellular survival of bacteria, we disrupted the gene encoding MsrA in Mycobacterium smegmatis through homologous recombination. The msrA mutant strain of M. smegmatis exhibited significantly reduced intracellular survival in murine J774A.1 macrophages compared to the survival of its wild-type counterpart. Furthermore, immunofluorescence and immnunoblotting of phagosomes containing M. smegmatis strains revealed that the phagosomes with the msrA mutant strain acquired both p67phox of phagocyte NADPH oxidase and inducible nitric oxide synthase much earlier than the phagosomes with the wild-type strain. In addition, the msrA mutant strain of M. smegmatis was observed to be more sensitive to hydroperoxides than the wild-type strain was in vitro. These results suggest that MsrA plays an important role in both extracellular and intracellular survival of M. smegmatis.

scholarly journals Mycobacterium smegmatis d-Alanine Racemase Mutants Are Not Dependent on d-Alanine for Growth

2002 ◽  
Vol 46 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Ofelia Chacon ◽  
Zhengyu Feng ◽  
N. Beth Harris ◽  
Nancy E. Cáceres ◽  
L. Garry Adams ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mutant Strain ◽  
Mycobacterium Smegmatis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Kanamycin Resistance ◽  
Alanine Racemase ◽  
Wild Type ◽  
Peptidoglycan Biosynthesis ◽  
Insertional Inactivation

ABSTRACT Mycobacterium smegmatis is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. This study focused on the d-alanine racemase gene (alrA), which is involved in the synthesis of d-alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. M. smegmatis alrA null mutants were generated by homologous recombination using a kanamycin resistance marker for insertional inactivation. Mutants were selected on Middlebrook medium supplemented with 50 mM d-alanine and 20 μg of kanamycin per ml. These mutants were also able to grow in standard and minimal media without d-alanine, giving rise to colonies with a drier appearance and more-raised borders than the wild-type strain. The viability of the mutants and independence of d-alanine for growth indicate that inactivation of alrA does not impose an auxotrophic requirement for d-alanine, suggesting the existence of a new pathway of d-alanine biosynthesis in M. smegmatis. Biochemical analysis demonstrated the absence of any detectable d-alanine racemase activity in the mutant strains. In addition, the alrA mutants displayed hypersusceptibility to the antimycobacterial agent d-cycloserine. The MIC of d-cycloserine for the mutant strain was 2.56 μg/ml, 30-fold less than that for the wild-type strain. Furthermore, this hypersusceptibility was confirmed by the bactericidal action of d-cycloserine on broth cultures. The kinetic of killing for the mutant strain followed the same pattern as that for the wild-type strain, but at a 30-fold-lower drug concentration. This effect does not involve a change in the permeability of the cell wall by this drug and is consistent with the identification of d-alanine racemase as a target of d-cycloserine. This outcome is of importance for the design of novel antituberculosis drugs targeting peptidoglycan biosynthesis in mycobacteria.

scholarly journals Secretion and Function of Salmonella SPI-2 Effector SseF Require Its Chaperone, SscB

2004 ◽  
Vol 186 (15) ◽  
pp. 5078-5086 ◽  
Author(s):  
Shipan Dai ◽  
Daoguo Zhou
Keyword(s):  
Type Iii Secretion ◽  
Type Strain ◽  
Wild Type Strain ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Transcriptional Level ◽  
Wild Type ◽  
And Function

ABSTRACT Salmonella strains utilize a type III secretion system for their successful survival and replications inside host cells. SseF is one of the several effector proteins that are required for conferring this survival ability by altering the trafficking of the Salmonella-containing vacuoles. These effector proteins often require appropriate chaperones to maintain their stabilities inside the bacteria. These chaperones are also known to assist the subsequent secretion and translocation of their substrates. We report here that SscB acts as the chaperone for SseF, an effector for the Salmonella pathogenicity island 2 (SPI-2). We found that the sscB gene is required for the formation of Salmonella sp.-induced continuous filaments in epithelial cells. Efficient Salmonella replication in macrophages requires SscB function. Intracellular and secretion levels of SseF are greatly reduced in an sscB mutant strain compared to the wild-type strain. A protein stability assay demonstrated that the half-life of SseF is significantly shortened in the absence of SscB. Transcriptional analysis of the sseF gene showed that the effect of SscB on the SseF level is not at the transcriptional level. A coprecipitation experiment indicated that SscB interacts with SseF. In summary, our results indicate that SscB is a chaperone for SPI-2 effector SseF to facilitate its secretion and function inside the host cells.

scholarly journals Role of EspB in Experimental Human EnteropathogenicEscherichia coli Infection

2000 ◽  
Vol 68 (6) ◽  
pp. 3689-3695 ◽  
Author(s):  
Carol O. Tacket ◽  
Marcelo B. Sztein ◽  
Genevieve Losonsky ◽  
Akio Abe ◽  
B. Brett Finlay ◽  
...  
Keyword(s):  
Host Cell ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Structure ◽  
Wild Type ◽  
Double Blind ◽  
Host Cytoplasm ◽  
And Function

ABSTRACT Enteropathogenic Escherichia coli (EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic ΔespB mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the ΔespB mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the ΔespB mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.

scholarly journals Nucleotide Sequence and Characterization ofcdrA, a Cell Division-Related Gene ofHelicobacter pylori

1998 ◽  
Vol 180 (19) ◽  
pp. 5263-5268 ◽  
Author(s):  
Hiroaki Takeuchi ◽  
Mutsunori Shirai ◽  
Junko K. Akada ◽  
Masataka Tsuda ◽  
Teruko Nakazawa
Keyword(s):  
Cell Division ◽  
Stationary Phase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Related Gene ◽  
Wild Type ◽  
Putative Gene ◽  
Allelic Exchange ◽  
H Pylori ◽  
High Level

ABSTRACT We identified cell division-related gene cdrA inHelicobacter pylori HPK5. The putative gene product, CdrA, is a 367-amino-acid polypeptide that exhibited a high level of homology to conserved hypothetical ATP-binding protein HP0066 of H. pylori 26695, except in the N-terminal region, and showed some similarity to the FtsK/SpoIIIE family proteins. We isolated acdrA-disrupted mutant by allelic exchange mutagenesis. Because of the low transformation frequency, the possibility that a suppressing mutation would be found in the obtained cdrAmutant was discussed. A repressive role for CdrA on cell division was suggested by the observations that the wild-type strain formed filamentous cells in a high-salt level medium at early stationary phase, while a cdrA-disrupted mutant did not show such an abnormality. In addition, the wild-type strain adopted coccoid forms in the stationary phase, whereas the cdrA-disrupted mutant remained mostly as short rods. Furthermore, thecdrA-disrupted mutant regained the filamentation phenotype when the intact cdrA gene was introduced by allelic exchange. Taken together, these observations show that thecdrA gene plays an important role in the cell growth ofH. pylori.

scholarly journals Roles of Mycobacterium smegmatisd-Alanine:d-Alanine Ligase and d-Alanine Racemase in the Mechanisms of Action of and Resistance to the Peptidoglycan Inhibitor d-Cycloserine

2003 ◽  
Vol 47 (1) ◽  
pp. 283-291 ◽  
Author(s):  
Zhengyu Feng ◽  
Raúl G. Barletta
Keyword(s):  
Mycobacterium Smegmatis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Mechanisms Of Action ◽  
Alanine Racemase ◽  
Wild Type ◽  
Peptidoglycan Biosynthesis ◽  
Novel Drugs ◽  
Twofold Excess ◽  
Fold Excess

ABSTRACT d-Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes d-alanine racemase (Alr) and d-alanine:d-alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Alr in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis. We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of d-alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts of l- and d-alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess of l- over d-alanine. In all strains, DCS treatment led to a significant accumulation of l-alanine and a concomitant decease of d-alanine, with approximately a 20-fold excess of l-alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Alr. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the d-alanine branch of mycobacterial peptidoglycan biosynthesis.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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