Effects on growth and sporulation of inactivation of a Bacillus subtilis gene (ctc) transcribed in vitro by minor vegetative cell RNA polymerases (E-sigma 37, E-sigma 32)

Summary The E-³⁷ gene ctc was inactivated by a site-specific insertion into the Bacillus subtilis chromosome. The resulting mutation inhibited sporulation by 95% at elevated temperatures (48° C). If the ctc ^- mutation is placed in a strain that carries a mutation in the closely linked but distinct spoVC gene, ctc now affects both growth and sporulation at elevated temperatures. Growth of the ctc ^- spoVC285 strain was transiently inhibited when exponentially growing cultures were shifted from 37° C to 48° C. A similar, but less pronounced growth lag, was also seen in a B. subtilis strain carrying only the spoVC-285 mutation. This finding suggests that both the ctc and spoVC products function in vegetatively growing B. subtilis.     

Reactivation of the Bacillus subtilis anti-sigma B antagonist, RsbV, by stress- or starvation-induced phosphatase activities.

sigma B is a secondary sigma factor that controls the general stress regulon in Bacillus subtilis. The regulon is activated when sigma B is released from a complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase. Two separate mechanisms cause sigma B release: an ATP-responsive mechanism that correlates with nutritional stress and an ATP-independent mechanism that responds to environmental insult (e.g., heat shock and ethanol treatment). ATP levels are thought to directly affect RsbW's binding preference. Low levels of ATP cause RsbW to release sigma B and bind to an alternative protein (RsbV), while high levels of ATP favor RsbW-sigma B complex formation and inactivation of RsbV by an RsbW-dependent phosphorylation. During growth, most of the RsbV is phosphorylated (RsbV-P) and inactive. Environmental stress induces the release of sigma B and the formation of the RsbW-RsbV complex, regardless of ATP levels. This pathway requires the products of additional genes encoded within the eight-gene operon (sigB) that includes the genes for sigma B, RsbW, and RsbV. By using isoelectric focusing techniques to distinguish RsbV from RsbV-P and chloramphenicol treatment or pulse-chase labeling to identify preexisting RsbV-P, we have now determined that stress induces the dephosphorylation of RsbV-P to reactivate RsbV. RsbV-P was also found to be dephosphorylated upon a drop in intracellular ATP levels. The stress-dependent and ATP-responsive dephosphorylations of RsbV-P differed in their requirements for the products of the first four genes (rsbR, -S, -T, and -U) of the sigB operon. Both dephosphorylation reactions required at least one of the genes included in a deletion that removed rsbR, -S, and -T; however, only an environmental insult required RsbU to reactivate RsbV.     

Inhibition of bacteriophage M13 and phix174 duplex DNA replication and single-strand synthesis in temperature-sensitive dnaZ mutants of Escherichia coli.

A functional dnaZ product, known to be essential for host DNA polymerization and for the synthesis of M13 and phiX174 parental replicative-form (RF) DNA, is required also for RF replication and single-strand synthesis by both of these phages. All three stages of M13 and phiX174DNA replication (parental RF formation, RF replication, and single-strand synthesis) are inhibited in dnazts mutants at elevated temperatures. In addition, the thermolabile step in M13 parental RF formation appears to occur after RNA priming;i.e., the synthesis of M13 RF DNA proceeded when a dnaZts mutant, infected at a nonpermissive temperature, was transferred to a permissive temperature in the presence of rifampin.     

Synthesis and fractionation properties of SpoIIGA, a protein essential for pro-sigma E processing in Bacillus subtilis.

sigma E, a major sporulation-specific sigma factor of Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma E). The formation of sigma E from pro-sigma E requires the products of several stage II genes, including spoIIGA, a gene that is cotranscribed with the pro-sigma E coding region (spoIIGB, or sigE). SpoIIGA has been hypothesized to be both a membrane-bound protein and the protease which converts pro-sigma E into sigma E. to learn more of its properties, we joined the Escherichia coli lacZ gene to the 3' end of spoIIGA as a translational fusion, creating a gene whose product was found to contain both beta-galactosidase and SpoIIGA activities. Assaying for the beta-galactosidase activity of the chimeric protein as a measure of its abundance, we determined that the spoIIGA::lacZ product accumulated to approximately 10% the level of a spoIIGB::lacZ fusion protein. Using differential centrifugation to fractionate B. subtilis extracts that contained beta-galactosidase fusion proteins, we observed that the beta-galactosidase activity of the spoIIGA::lacZ fusion protein was preferentially associated with a Triton X-100-sensitive, fast-sedimenting portion of the extract, while the beta-galactosidase activity of the spoIIGB::lacZ fusion protein remained primarily in the supernatant fraction. If the properties of the fusion proteins are assumed to be representative of those of the products of the genes to which lacZ is joined, these results support the hypothesis that SpoIIGA is a membrane-bound protein that acts catalytically in the processing of pro-sigma E into sigma E.     

Effects of antibiotics on synthesis and persistence of sigma E in sporulating Bacillus subtilis.

A potential regulatory link between the activation of a sporulation-specific sigma factor (sigma E) and forespore septum formation was investigated by treating Bacillus subtilis with inhibitors of protein or peptidoglycan synthesis and monitoring the consequences of these treatments on sigma E activation and septation. Western blot (immunoblot) and electron microscopic analyses revealed that both the formation of sigma E and septation were inhibited to a similar degree when either rifampin or chloramphenicol was added at different times before the second hour into sporulation but that penicillin preferentially blocked septation. We interpret these results as indicating that the syntheses of the gene products for both septation and sigma E activation occur at approximately the same time in development but that synthesis of an intact septum is unlikely to be a prerequisite for the formation of sigma E. We observed that penicillin could not only block septation but, depending on the time of its addition, could also inhibit both the activation of sigma E and the synthesis of its precursor. The basis of this effect is unknown, but it is not due to an overall disruption of protein synthesis. The incorporation of [35S] methionine by the sporulating cultures was unaffected by penicillin treatment. A time course study of the effects of rifampin and chloramphenicol treatments on sigma E levels revealed that both the synthesis of sigma E and its disappearance from sporulating cultures is inhibited by these antibiotics. This suggests that ongoing macromolecular synthesis is required for the turnover of sigma E.     

Role of ultrasonography in diagnosing early rheumatoid arthritis and remission of rheumatoid arthritis - a systematic review of the literature

Introduction

Ultrasonography (US) might have an added value to clinical examination in diagnosing early rheumatoid arthritis (RA) and assessing remission of RA. We aimed to clarify the added value of US in RA in these situations performing a systematic review.

Methods

A systematic literature search was performed for RA, US, diagnosis and remission. Methodological quality was assessed; the wide variability in the design of studies prohibited pooling of results.

Results

Six papers on the added value of US diagnosing early RA were found, in which at least bilateral metacarpophalangeal (MCP), wrists and metatarsophalangeal (MTP) joints were scanned. Compared to clinical examination, US was superior with regard to detecting synovitis and predicting progression to persistent arthritis or RA. Eleven papers on assessing remission were identified, in which at least the wrist and the MCP joints of the dominant hand were scanned. Often US detected inflammation in patients clinically in remission, irrespective of the remission criteria used. Power Doppler signs of synovitis predicted X-ray progression and future flare in patients clinically in remission.

Conclusions

US appears to have added value to clinical examination for diagnosing of RA when scanning at least MCP, wrist and MTP joints, and, when evaluating remission of RA, scanning at least wrist and MCP joints of the dominant hand. For both purposes primarily power Doppler US might be used since its results are less equivocal than those of greyscale US.     

HrcA is a negative regulator of the dnaK and groESL operons of Streptococcus pyogenes

The genome of Streptococcus pyogenes, an important human pathogen, encodes homologs of the principal bacterial heat shock proteins DnaK and GroES, -EL, as well as HrcA, a negative regulator of dnaK and groESL expression in other Gram-positive bacteria. Using nuclease protection assays to measure dnaK/groESL mRNA abundance and a "non-polar" insertion to disrupt hrcA, we demonstrate that heat shock triggers a 4- to 8-fold increase in dnaK and groESL-specific mRNAs within 5 min of the temperature shift and that HrcA is a negative regulator of S. pyogenes dnaK/groESL mRNA abundance in unstressed S. pyogenes. Although the loss of HrcA elevated dnaK and groESL mRNA levels under non-heat shock conditions, the relative abundance of these RNAs increased further in heat shocked S. pyogenes, suggesting an additional element contributing to their synthesis or stability.