Streptomyces sp. linear plasmids and linear chromosomes usually contain conserved terminal palindromic sequences bound by the conserved telomeric proteins Tap and Tp, encoded by the tap and tpg genes, respectively, as well as plasmid loci required for DNA replication in circular mode when the telomeres are deleted. These consist of iterons and an adjacent rep gene. By using PCR, we found that 8 of 17 newly detected linear plasmids in Streptomyces strains lack typical telomeric tap and tpg sequences. Instead, two novel telomeres in plasmids pRL1 and pRL2 from the eight strains and one conserved telomere in pFRL1 from the other strains were identified, while multiple short palindromes were also found in the plasmids. The complete nucleotide sequence of pRL2 revealed a gene encoding a protein containing two domains, resembling Tap of Streptomyces and a helicase of Thiobacillus, and an adjacent gene encoding a protein similar to Tpg of Streptomyces and a portion of the telomere terminal protein pTP of adenoviruses. No typical iterons-rep loci were found in the three plasmids. These results indicate an unexpected diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids.Streptomyces species are gram-positive, high-GϩC, myceliumproducing eubacteria. Unlike the case for most eubacteria, linear plasmids and linear chromosomes are common in Streptomyces species (3,8,11,19,20,28). The linear plasmids vary in size between 12 kb (16) and 1,700 kb (19). Their telomeres contain long inverted repeat sequences of 44 bp (7) to 180 kb (21), and the 5Ј telomeric ends are linked covalently to terminal proteins (Tp) (1, 31). The telomeres of the ϳ8-Mb linear Streptomyces chromosomes are 46 bp to 1 Mb long (14,29). With the exceptions of the telomeres of the large linear plasmid SCP1 and the Streptomyces griseus linear chromosome (9, 18), Streptomyces linear plasmids and linear chromosomes usually contain conserved palindromic DNA sequences at their telomeres (13).Unlike the terminal protein-capped linear replicons of adenoviruses and bacteriophage ⌽29 (25), replication of Streptomyces linear plasmids starts at centrally located loci (27) and proceeds bidirectionally toward the telomeres (5). This leaves an ϳ280-nucleotide (nt) single-strand overhang at the 3Ј telomeric end of pSLA2 as a replication intermediate (5). To convert the 3Ј overhang to a double strand, the terminal 144 nt of the telomere contain short palindromes 1 to 5 (22), with palindromes 2/3 being bound by the conserved telomere-associated protein (Tap) to recruit the conserved telomere terminal protein (Tp) (1, 2).Streptomyces linear plasmids can also propagate in circular mode when the telomeres are deleted (5, 10, 24, 27). The centrally located locus for replication of pSLA2 consists of a rep-2 gene (encoding a DNA helicase) and its adjacent iterons within a transcribed rep-1 gene (6). The replication loci of plasmids SCP1 and pSLA2-L also consist of rep genes and different iteron sequences (10, 24). Such iterons-rep loci ...
Unlike most eubacteria, Streptomyces species usually contain linear chromosomes and plasmids (9,16,17). The linear plasmids are 12 to 1,700 kb long (16,27). Their telomeres contain inverted repeat sequences from 44 bp (7) to 180 kb (19), and their 5Ј telomeric ends are linked covalently to terminal proteins (1, 28). Unlike linear replicons of adenoviruses and bacteriophage 29, which also contain terminal proteins linking covalently to 5Ј telomeric DNA ends and undergo replication by a mechanism of strand displacement (23), replication of Streptomyces linear plasmids starts at centrally located loci (5, 25) and continues bidirectionally towards the telomeres-leaving an ϳ280-nucleotide 3Ј single-strand overhang as an intermediate (5). This is converted to a double strand by a postulated "folding back" of multiply short palindromes on the telomere extension (20). The chromosomal telomere-associated protein (TapL, encoded by tapL) binds to the palindromes II/III then to recruit telomere terminal protein (TpgL, encoded by tpgL) (1, 2). Neither tapL nor tpgL homologous genes are carried by the linear plasmids pSLA2 and pSCL1 (1, 2), and the mechanism of recruitment or activation of these chromosomal telomere proteins for plasmid telomere replication is unknown.The centrally located loci of Streptomyces linear plasmids can also maintain propagation in circular mode when the telomeres are deleted (5,8,22,25). The centrally located locus for replication of linear plasmid pSLA2 is composed of the iterons located within the essential genes rep1 (encoding DNA-binding protein) and rep2 (DNA helicase) (6). Experimental evidence shows that the replication origin of linear SCP1 plasmids contains a rep2 pSLA2 -like gene and its adjacent regions contain different iteron sequences (22). Similar loci are also indicated in linear plasmids pSCL1 and SLP2 (11,27). The extent of functional similarity of these individual replicating components, and consequently the extent to which mechanisms of replication are similar among linear plasmids, is not known.The minimal locus required for maintaining the replication of pSLA2 in circular mode cannot allow its propagation in linear mode unless it also contains a new plasmid locus, rlrA pSLA2 (required for linear replication) (21). Plasmids containing rlrA pSLA2 are detrimental for propagation in circular mode, the effect of which can be reversed by an adjacent and divergently transcribed locus, rorA pSLA2 (rlrA override), which resembles korA (kilA override) of Streptomyces circular plasmid pIJ101 (14,26). rlrA pSLA2 and rorA pSLA2 increase inheritance and copy number of pSLA2 circular plasmids, suggesting that they may affect the origin locus (e.g., iterons) (21). Although rorA pSLA2 -homologous genes are found in linear plasmids SCP1, pSLA2-L, and SLP2, no rlrA pSLA2 -homologous loci of the plasmids are found (3,11,18), suggesting that they carry a distinct locus that enables replication in linear mode.SLP2 is a large (50,410-bp) linear plasmid of Streptomyces lividans (7, 10, 11). Here we r...
The structural gene pac in Eschericia coli ATCC11105 encodes penicillin G acylase (PGA). Within the pac gene, there is a regulatory gene pac R, which is transcribed in the opposite direction. Site-directed mutagenesis was performed at base 1045 of pac by replacing a T with a C. This substitution did not alter the amino-acid sequence of PGA, but changed the translation start codon of pac R from AUG to GUG. The expression of the mutant pac R decreased dramatically and the lacZ transcriptional fusion analysis showed that GUG was an extremely poor initiation codon for pac R. The pac R mutation caused PGA expression to be constitutive rather than inductive in two strains (E. coli A56, DH10B). The pac inducer phenylacetic acid (PAA) gave significant induction of PGA production at a concentration of 0.2% in wild type, but PAA at this concentration inhibited both cell growth and PGA production in the pac R mutated strains. The temperature-dependent expression character of pac is preserved in the pacR translationinitiation mutant and the optimum temperature of PGA production was 22 8C in both wild type and mutant. At a higher temperature of 37 8C, the PGA precursor polypeptide could not be matured into subunits and formed inclusion bodies, as revealed by western blot analysis. Our investigations confirmed the hypothesis of pac Rmediated PAA induction for PGA expression and clarified the inhibitory effect of high temperature upon the posttranslational processing of the PGA precursor polypeptide.
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