Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus.

The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.     

Three functions of bacteriophage P1 involved in cell lysis.

Amber and deletion mutants were used to assign functions in cell lysis to three late genes of bacteriophage P1. Two of these genes, lydA and lydB of the dar operon, are 330 and 444 bp in length, respectively, with the stop codon of lydA overlapping the start codon of lydB. The third, gene 17, is 558 bp in length and is located in an otherwise uncharacterized operon. A search with the predicted amino acid sequence of LydA for secondary motifs revealed a holin protein-like structure. Comparison of the deduced amino acid sequence of gene 17 with sequences of proteins in the SwissProt database revealed homologies with the proteins of the T4 lysozyme family. The sequence of lydB is novel and exhibited no known extended homology. To study the effect of gp17, LydA, and LydB in vivo, their genes were cloned in a single operon under the control of the inducible T7 promoter, resulting in plasmid pAW1440. A second plasmid, pAW1442, is identical to pAW1440 but has lydB deleted. Induction of the T7 promoter resulted in a rapid lysis of cells harboring pAW1442. In contrast, cells harboring pAW1440 revealed only a small decrease in optical density at 600 nm compared with cells harboring vector alone. The rapid lysis phenotype in the absence of active LydB suggests that this novel protein might be an antagonist of the holin LydA.     

Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4.

The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.     

Regulation of the Escherichia coli allantoin regulon: coordinated function of the repressor AllR and the activator AllS

The allantoin regulon of Escherichia coli, formed by three operons expressed from promoters allA(P), gcl(P) and allD(P), is involved in the anaerobic utilization of allantoin as nitrogen source. The expression of these operons is under the control of the repressor AllR. The hyperinduction of one of these promoters (allD(P)) by allantoin in an AllR defective mutant suggested the action of another regulator, presumably of activator type. In this work we have identified ybbS (proposed gene name allS), divergently transcribed from allA, as the gene encoding this activator. Analysis of the expression of the three structural operons in DeltaallS mutant showed that the expression from allD(P) was abolished, suggesting that AllS is essential for the expression of the corresponding operon. In a wild-type strain expression of allS takes place mainly anaerobically and is hyperinduced when the nitrogen source limits growth. However, expression of allS is independent of regulators of the Ntr response, NtrC or Nac. Band shift experiments showed that AllR binds to DNA containing the allS-allA intergenic region and the gcl(P) promoter and its binding is abolished by glyoxylate. Both DNA fragments contain a highly conserved inverted repeat, which after site-directed mutagenesis, has been proven to be the AllR-binding site. This site displays similarity with the IclR family recognized consensus. Interaction of AllR with the single operator present in the allS-allA intergenic region prevented binding of RNA polymerase to either of the two divergent promoters. The regulator AllS interacts only with allD(P) even in the absence of allantoin. Analysis of this promoter allowed us to identify an inverted repeat as a motif for AllS binding. We propose a model for the coordinate control of the allantoin regulon by AllR and AllS.