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Mutational Analysis of the Myxococcus xanthus  4400
Promoter Region Provides Insight into Developmental Gene Regulation by C
Signaling.Deborah R. Yoder, 2004.Myxococcus xanthus utilizes extracellular signals during development to coordinate cell movement, differentiation, and changes in gene expression . One of these signals, the C signal, regulates the expression of many genes, including 4400,
a gene identified by an insertion of Tn5 lac into the
chromosome . Expression of Tn5 lac
4400
is reduced in csgA mutant cells, which fail to perform C
signaling, and the promoter region has several sequences similar to
sequences found in the regulatory regions of other C-signal-dependent
genes . One such gene,
4403,
depends absolutely on the C signal for expression, and its promoter
region has been characterized previously by mutational analysis . To
determine if the similar sequences within the
4400
and
4403
regulatory regions function in the same way, deletion analysis and
site-directed mutagenesis of the
4400
promoter region were performed . A 7-bp sequence centered at -49 bp,
termed a C box, is identical in the
4400
and
4403
promoter regions, yet mutations in the individual base pairs affected
expression from the two promoters very differently . Also, a
single-base-pair change within a similar 5-bp element, which is
centered at -61 bp in both promoter regions, had very different
effects on the activities of the two promoters . Further mutational
analysis showed that two regions are important for
4400
expression; one region, from -63 to -31 bp, is required for
4400
expression, and the other, from -86 to -81 bp, exerts a two- to
fourfold effect on expression and is at least partially responsible
for the C signal dependence of the
4400
promoter . Mutations in sigD and sigE, which are genes
that encode
factors, abolished and reduced
4400
expression, respectively . Expression of
4400
in actB or actC mutants correlated well with the altered
levels of C signal produced in these mutants . Our results provide
the first detailed analysis of an M . xanthus regulatory region
that depends partially on C signaling for expression and indicate
that similar DNA sequences in the
4400
and
4403
promoter regions function differently .
Helix 4 Mutants of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa Display Altered Pore-Forming Abilities. Vincent Vachon, 2004.The role played by  -helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis . The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa . Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix . Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation . For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-D-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation . These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores . They provide further evidence that
-helix 4 plays a crucial role in the mechanism of pore formation .
High Genetic Variability of the agr Locus in Staphylococcus Species. Philippe Dufour, 2002.The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced . Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S . epidermidis and S . lugdunensis . In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity . Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested . Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment . This variability involved all three open reading frames involved in quorum sensing and signal transduction . However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S . intermedius of pigeon origin, which contained a serine in place of cysteine at this position . We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences .
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