scholarly journals Isolation and Characterization of Bacillus subtilis sigB Operon Mutations That Suppress the Loss of the Negative Regulator RsbX

1998 ◽  
Vol 180 (14) ◽  
pp. 3671-3680 ◽  
Author(s):  
Natalya Smirnova ◽  
Janelle Scott ◽  
Uwe Voelker ◽  
W. G. Haldenwang
Keyword(s):  
Bacillus Subtilis ◽  
Negative Regulator ◽  
General Stress Response ◽  
Balanced Growth ◽  
Isolation And Characterization ◽  
Suppressor Mutations ◽  
Positive Regulators ◽  
High Level ◽  
Stress Dependent

ABSTRACT ςB, a transcription factor that controls theBacillus subtilis general stress response regulon, is activated by either a drop in intracellular ATP or exposure to environmental stress. RsbX, one of seven ςB regulators (Rsb proteins) whose genes are cotranscribed with ςB, is a negative regulator in the stress-dependent activation pathway. To better define the interactions that take place among the Rsb proteins, we analyzed sigB operon mutations which suppress the high-level ςB activity that normally accompanies the loss of RsbX. Each of these mutations was in one of three genes (rsbT, -U, and -V) which encode positive regulators of ςB, and they all defined amino acid changes which either compromised the activities of the mutant Rsbs or affected their ability to accumulate. ςB activity remained inducible by ethanol in several of the RsbX−suppressor strains. This finding supports the notion that RsbX is not needed as the target for ςB activation by at least some stresses. ςB activity in several RsbX−strains with suppressor mutations in rsbT or -Uwas high during growth and underwent a continued, rather than a transient, increase following stress. Thus, RsbX is likely responsible for maintaining low ςB activity during balanced growth and for reestablishing ςB activity at prestress levels following induction. Although RsbX likely participates in limiting the ςB induction response, a second mechanism for curtailing unrestricted ςB activation was suggested by the ςB induction profile in two suppressor strains with mutations in rsbV. ςB activity in these mutants was stress inducible but transient, even in the absence of RsbX.

scholarly journals Isolation and Characterization of Dominant Mutations in the Bacillus subtilis Stressosome Components RsbR and RsbS

2006 ◽  
Vol 189 (5) ◽  
pp. 1531-1541 ◽  
Author(s):  
Adam Reeves ◽  
W. G. Haldenwang
Keyword(s):  
Bacillus Subtilis ◽  
Inhibitor Protein ◽  
Wild Type ◽  
Homologous Proteins ◽  
Stress Induction ◽  
General Stress Response ◽  
Isolation And Characterization ◽  
Activity State ◽  
Downstream Processes

ABSTRACT The general stress response of Bacillus subtilis is controlled by the activity state of the σB transcription factor. Physical stress is communicated to σB via a large-molecular-mass (>106-Da) structure (the stressosome) formed by one or more members of a family of homologous proteins (RsbR, YkoB, YojH, YqhA). The positive regulator (RsbT) of the σB stress induction pathway is incorporated into the complex bound to an inhibitor protein (RsbS). Exposure to stress empowers an RsbT-dependent phosphorylation of RsbR and RsbS, with the subsequent release of RsbT to activate downstream processes. The mechanism by which stress initiates these reactions is unknown. In an attempt to identify changes in stressosome components that could lead to σB activation, a DNA segment encoding these proteins was mutagenized and placed into B. subtilis to create a merodiploid strain for these genes. Eight mutations that allowed heightened σB activity in the presence of their wild-type counterparts were isolated. Two of the mutations are missense changes in rsbR, and six are amino acid changes in rsbS. Additional experiments suggested that both of the rsbR mutations and three of the rsbS mutations likely enhance σB activity by elevating the level of RsbS phosphorylation. All of the mutations were found to be dominant over wild-type alleles only when they are cotranscribed within an rsbR rsbS rsbT operon. The data suggest that changes in RsbR can initiate the downstream events that lead to σB activation and that RsbR, RsbS, and RsbT likely interact with each other concomitantly with their synthesis.

Isolation and characterization of a halotolerant Bacillus subtilis BBK-1 which produces three kinds of lipopeptides: bacillomycin L, plipastatin, and surfactin

Extremophiles ◽  
2002 ◽  
Vol 6 (6) ◽  
pp. 499-506 ◽  
Author(s):  
Niran Roongsawang ◽  
Jiraporn Thaniyavarn ◽  
Suthep Thaniyavarn ◽  
Takayuki Kameyama ◽  
Mitsuru Haruki ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Isolation And Characterization ◽  
Bacillomycin L

scholarly journals Isolation and characterization of the SPT2 gene, a negative regulator of Ty-controlled yeast gene expression.

1985 ◽  
Vol 5 (7) ◽  
pp. 1543-1553 ◽  
Author(s):  
G S Roeder ◽  
C Beard ◽  
M Smith ◽  
S Keranen
Keyword(s):  
Gene Product ◽  
Regulatory Region ◽  
Negative Regulator ◽  
Transcriptional Level ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Insertion Mutations ◽  
His4 Gene

The his4-917 mutation of Saccharomyces cerevisiae results from the insertion of the Ty element Ty917 into the regulatory region of the HIS4 gene and renders the cell His-. The hist4-912 delta mutant, which carries a solo delta in the 5'-noncoding region of HIS4, is His+ at 37 degrees C but His- at 23 degrees C. Both these mutations interfere with HIS4 expression at the transcriptional level. The His- phenotype of both insertion mutations is suppressed by mutations at the SPT2 locus. The product of the wild-type SPT2 gene apparently represses HIS4 transcription in these mutant strains; this repression is relieved when the SPT2 gene is destroyed by mutation. The repression of transcription by SPT2 presumably results from an interaction between the SPT2+ gene product and Ty or delta sequences. In this paper, we report the cloning and DNA sequence analysis of the wild-type SPT2 gene and show that the gene is capable of encoding a protein of 333 amino acids in length. In addition, we show that a dominant mutation of the SPT2 gene results from the generation of an ochre codon which is presumed to lead to a shortened SPT2 gene product.

Cloning and expression of DiT33 from Dirofilaria immitis: a specific and early marker of heartworm infection

Parasitology ◽  
1996 ◽  
Vol 112 (3) ◽  
pp. 331-338 ◽  
Author(s):  
X. Q. Hong ◽  
J. Santiago Mejia ◽  
S. Kumar ◽  
F. B. Perler ◽  
C. K. S. Carlow
Keyword(s):  
Dirofilaria Immitis ◽  
Cloning And Expression ◽  
E Coli ◽  
Spliced Leader ◽  
Isolation And Characterization ◽  
The Family ◽  
Heartworm Infection ◽  
High Level ◽  
Control And Management

SUMMARYDirofilaria immitis is an important filarial parasite of dogs and cats, and a useful model for human filariasis. Current diagnostic tests for heartworm infection in animals rely on the presence of fecund female worms (usually found 6·5 months post-infection or later) and therefore fail to detect pre-patent infections. Putative pepsin inhibitors from 2 filarial parasites of humans namely Onchocerca volvulus (Ov33, Oc3.6, OvDSB) and Brugia malayi (Bm33), have been shown to be useful in diagnosis of onchocerciasis and lymphatic filariasis, respectively. Previous studies have suggested that a homologue exists in D. immitis (DiT33), which may have potential in diagnosis of heartworm infection. In this study, the isolation and characterization of a cDNA clone encoding DiT33 is described.‡ This cDNA contains 12 bases of the nematode-specific 22 nucleotide spliced leader sequence and encodes a 26·4 kDa-protein with a high level of similarity (87–89%) to other filarial members of the family. DJT33 was over-expressed in E. coli as a fusion with the maltose-binding protein and serological analysis was performed using a panel of clinically defined dog sera. The findings of this study indicate that DiT33 is a promising antigen for the early detection of D. immitis and may be a valuable tool in the control and management of heartworm infection.

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