No abstract

In gram-positive bacteria, HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), is phosphorylated by an ATP-dependent, metabolite-activated protein kinase on seryl residue 46. In a Bacillus subtilis mutant strain in which Ser-46 of HPr was replaced with a nonphosphorylatable alanyl residue (ptsHl mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-l-P dehydrogenase and the mannitol-specific PTS permease was completely relieved from repression by glucose, fructose, or mannitol, whereas synthesis of inositol dehydrogenase was partially relieved from catabolite repression and synthesis of oa-glucosidase and glycerol kinase was still subject to catabolite repression. When the S46A mutation in HPr was reverted to give S46 wild-type HPr, expression of gluconate kinase and glucitol dehydrogenase regained full sensitivity to repression by PTS sugars. These results suggest that phosphorylation of HPr at Ser-46 is directly or indirectly involved in catabolite repression. A strain deleted for the ptsGHI genes was transformed with plasmids expressing either the wild-type ptsH gene or various S46 mutant ptsH genes (S46A or S46D). Expression of the gene encoding S46D HPr, having a structure similar to that of P-ser-HPr according to nuclear magnetic resonance data, caused significant reduction of gluconate kinase activity, whereas expression of the genes encoding wild-type or S46A HPr had no effect on this enzyme activity. When the promoterless lacZ gene was put under the control of the gnt promoter and was subsequently incorporated into the amyE gene on the B. subtilis chromosome, expression of 1-galactosidase was inducible by gluconate and repressed by glucose. However, we observed no repression of I-galactosidase activity in a strain carrying the ptsHl mutation. Additionally, we investigated a ccpA mutant strain and observed that all of the enzymes which we found to be relieved from carbon catabolite repression in the ptsHl mutant strain were also insensitive to catabolite repression in the ccpA mutant. Enzymes that were repressed in the ptsHl mutant were also repressed in the ccpA mutant.Catabolite-repressible genes in the gram-positive bacterium Bacillus subtilis are controlled by multiple mechanisms rather than by a single global regulatory system. These mechanisms are known to differ from those operating in enteric bacteria such as Escherichia coli, but the molecular details are poorly understood (15,40,46

SummaryThe dynamic instability of microtubules plays a key role in controlling their organization and function, but the cellular mechanisms regulating this process are poorly understood. Here, we show that cytoplasmic linker-associated proteins (CLASPs) suppress transitions from microtubule growth to shortening, termed catastrophes, including those induced by microtubule-destabilizing agents and physical barriers. Mammalian CLASPs encompass three TOG-like domains, TOG1, TOG2, and TOG3, none of which bind to free tubulin. TOG2 is essential for catastrophe suppression, whereas TOG3 mildly enhances rescues but cannot suppress catastrophes. These functions are inhibited by the C-terminal domain of CLASP2, while the TOG1 domain can release this auto-inhibition. TOG2 fused to a positively charged microtubule-binding peptide autonomously accumulates at growing but not shrinking ends, suppresses catastrophes, and stimulates rescues. CLASPs suppress catastrophes by stabilizing growing microtubule ends, including incomplete ones, preventing their depolymerization and promoting their recovery into complete tubes. TOG2 domain is the key determinant of these activities.

Laulimalide and peloruside A are microtubule-stabilizing agents (MSAs), the mechanism of action on microtubules of which is poorly defined. Here, using X-ray crystallography it is shown that laulimalide and peloruside A bind to a unique non-taxane site on β-tubulin and use their respective macrolide core structures to interact with a second tubulin dimer across protofilaments. At the same time, they allosterically stabilize the taxane-site M-loop that establishes lateral tubulin contacts in microtubules. Structures of ternary complexes of tubulin with laulimalide/peloruside A and epothilone A are also solved, and a crosstalk between the laulimalide/peloruside and taxane sites via the M-loop of β-tubulin is found. Together, the data define the mechanism of action of laulimalide and peloruside A on tubulin and microtubules. The data further provide a structural framework for understanding the synergy observed between two classes of MSAs in tubulin assembly and the inhibition of cancer cell growth.

Steinmetz and colleagues solved the structure of tubulin in complex with the cis isomer of combretastatin A4, a potent anti-tumor microtubule-destabilizing agent, at 2.4 Å resolution by X-ray crystallography. They subsequently computed the cisto-trans switch of the bound molecule by using atomistic simulations, revealing the structural basis for the large difference in activity between the two combretastatin isomers. The results could aid the development of combretastatin derivatives with superior chemical and biological properties.

Induction of the Bacillus subtilis sacB gene and sacPA operon and Escherichia coli bgl operon is mediated by structurally homologous antiterminators encoded by the sacY, sacT, and bhgG genes, respectively. When activated, these proteins prevent early transcription termination at terminators located in the leader regions of the three operons. BglG was previously shown to bind in vitro to an imperfectly palindromic 29-nucleotide RNA sequence located upstream of the terminator and partially overlapping with it [Houman, F., DiazTorres, M. R. & Wright, A. (1990) Cell 62, 1153-11631. Similar motifs, here termed ribonucleic antiterminators (RATs), strongly conserved in sequence and in position, are found in the leader of both sacB and sacPA. Mutations were created in sacB RAT and tested in B. subtilis; this showed that sacB RAT is the target for SacY-mediated induction of sacB and that a stem-oop structure in the mRNA is required for regulatory function. Mutations increasing the similarity of the sacB RAT with those of sacPA or bgl rendered sacB inducible by SacT or BglG, respectively; most of these changes did not strongly affect induction by SacY, suggesting that the nucleotides at these variable positions act as negative specificity determinants.Induction of the Bacillus subtilis levansucrase (sacB) gene by sucrose and the Escherichia coli f3-glucoside (bgt) operon by (3-glucosides is controlled by similar transcriptional antitermination mechanisms (1-3). In both systems, transcription initiates constitutively from the promoter; in the absence of inducer, most transcripts terminate at a terminator located in the leader region, between the promoter and the first gene; in the presence of inducer, a trans-acting protein (antiterminator) is activated and prevents early transcription termination (1, 2). Sucrose induction of the B. subtilis sucrase (sacPA) operon appears to follow a similar mechanism (4, 5). The antiterminator BglG is encoded by the first gene of the bgl operon. Between bglG and the second gene (bglF) lies a second BglG-controlled terminator (6). In B. subtilis, the sac Y and sacT gene products are required for induction of sacB and sacPA, respectively (3-5). BglG, SacY, and SacT are very similar proteins, with 48% identity between SacY and SacT and 30%1o between SacY and BglG (5). It was shown in vitro that BglG is an RNA-binding protein that recognizes a specific sequence located upstream of each of the two bgl conditional terminators (7). These 29-nucleotide sequences partially overlap with the terminators; thus, binding of BglG to these sequences, hereafter referred to as ribonucleic antiterminator (RAT) sequences, might prevent the formation ofthe termination structures. Similar RAT sequences are present in the sacB and sacPA leader regions and overlap with the terminator sequences in exactly the same way (5). This suggests that they are binding sites for SacY and SacT in the leader of sacB and sacPA mRNAs, respectively. The same model can therefore be proposed for the three systems: when ac...

The target of the induction by sucrose of the levansucrase gene is a transcription terminator (sacRt) located upstream from the coding sequence, sacB. The two-gene locus sacX-sacY (formerly sacS) and the ptsI gene were previously shown to be involved in this induction. ptsI encodes enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. SacX is strongly homologous to sucrose-specific phosphotransferase system-dependent permeases. SacY is a positive regulator of sacB. Here we show that SacY is probably an antiterminator interacting directly with sacRt, since in Escherichia coli the presence of the sacY gene stimulates the expression of a reporter gene fused downstream from sacRt. Missense mutations affecting sacY were sequenced, and the sacB regulation was studied in isogenic strains carrying these mutations or in vitro-generated mutations affecting sacX, sacY, or ptsI. The phenotype of double mutants suggests a model in which SacX might be a sucrose sensor that would be phosphorylated by the phosphotransferase system and, in this state, could inhibit the SacY antiterminator. Exogenous sucrose, or a mutation inactivating the phosphotransferase system, would dephosphorylate SacX and allow antitermination at sacRt.

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of BaciUus subtilis for use in the positive selection and isolation of insertion sequence (IS)Y'elements in gram-negative bacteria. Cells containing pUCD80 do not grow on medium containing 5% sucrose tinless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NTlRE by selection for growth on sucrose medium. This putative IS element'appears to be unique to Agrobacterium strains.