Genetic Organization of the Vibrio harveyi dnaA Gene Region and Analysis of the Function of the V. harveyi DnaA Protein in Escherichia coli

Abstract

The Vibrionaceae family is distantly related to Enterobacteriaceae within the group of bacteria possessing the Dam methylase system. We have cloned, sequenced, and analyzed the dnaA gene region of Vibrio harveyi and found that although the organization of the V. harveyi dnaA region differs from that of Escherichia coli, the expression of both genes is autoregulated and ATP-DnaA binds cooperatively to ATP-DnaA boxes in the dnaA promoter region. The DnaA proteins of V. harveyi and E. coli are interchangeable and function nearly identically in controlling dnaA transcription and the initiation of chromosomal DNA replication despite the evolutionary distance between these bacteria.The DnaA initiator protein and the origin of replication, oriC, with its binding sites for the DnaA protein, the DnaA boxes, are the key elements in controlling the initiation of chromosomal DNA replication (15,23,29). The dnaA operon in Escherichia coli is transcribed from two promoters with a DnaA box located between them (16). dnaA transcription is autoregulated (1,5,19) Several chromosomal origins of replication from Enterobacteriaceae (9) and the origin from Vibrio harveyi-a luminous, motile, and facultatively anaerobic gram-negative rod belonging to the Vibrionaceae-are all functional in E. coli (42), whereas the Pseudomonas putida origin is not (40). In parallel, several dnaA genes from Enterobacteriaceae complement dnaA(Ts) mutations in E. coli (31, 32) but the P. putida dnaA gene does not (17). In the ␥ subdivision of Proteobacteria the families Enterobacteriaceae, Pasteurellaceae, and Vibrionaceae are distinguished from Pseudomonadaceae (10, 26) by having the Dam methylase system in common (4). The Dam methylase methylates the adenine in GATC sequences and plays a role in DNA replication, in mismatch repair, and in gene regulation (27). Overrepresentation of GATC sequences in the oriC and dnaA regions is a conserved feature in E. coli (22,34), in other Enterobacteriaceae (9, 31), and in V. harveyi (Vibrionaceae) (42). It takes a much longer time in E. coli to remethylate GATC sites within the clusters located in the oriC and the dnaA regions than other GATC sites. This delay of remethylation is due to sequestration of the GATC sites, most likely to the cell membrane (7,25). The period with the sequestered origins (the eclipse period) is important for the synchrony of initiation (15), and the SeqA protein (21, 37) is an absolute requisite for the eclipse period (38).We sought an evolutionarily distant dnaA gene that is functional in E. coli. Because neither the dnaA P. putida nor dnaA Haemophilus influenzae gene complements the dnaA46 mutation in E. coli (17; O. Skovgaard, unpublished data), we chose a member of Vibrionaceae, V. harveyi, for this purpose. We investigated whether the basic elements in the control of dnaA transcription regulation and DNA replication in V. harveyi are similar to those of E. coli. After cloning and physical and physiological analysis of the V. harveyi dnaA gene region, we conclude that the basic eleme...

Control of the replication initiator DnaA by an anti-cooperativity factor

“…In addition to these and other proteins that affect replication initiation, the levels of DnaA and its rate of synthesis are important. DnaA is autoregulated and its overproduction leads to over‐replication in B. subtilis (Ogura et al ., 2001) and other organisms (Atlung et al ., 1985; Braun et al ., 1985; Kucherer et al ., 1986; Berenstein et al ., 2002; Salazar et al ., 2003). The combination of DnaA autoregulation and the activities of several regulatory proteins contributes to the complex control of replication initiation (e.g.…”

The bacterial replication initiator DnaA. DnaA andoriC, the bacterial mode to initiate DNA replication

“…The surprising observation was that ATP‐complexed DnaA, but not ADP‐DnaA, has a second sequence specificity for a 6‐mer consensus sequence, the ATP‐DnaA box [13]. This is also true for the dnaA promoter region of Vibrio harveyi [100]. DnaA binding to ‘non‐canonical’ sites was observed before with λ DNA [101].…”

Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant

“…Relevant markers of principal E. coli K-12 strains are AX727 (dnaX2016; Filip et al, 1974), RUC839 (AX727, tna2123 : : Tn10), KM22 [D(recC ptr recB recD) : : Plac-bet exo kan; Murphy, 1998], RUC1227 (KM22, dnaA46 tna2123 : : Tn10), RUC1000 [D(lac)X74; Berenstein et al, 2002], RUC1024 (RUC1000, dnaAp0lacZ; Berenstein et al, 2002), RUC1282 (RUC1000, lsulA0lacZ cI ind att…”

“…The expression of a dnaA gene transcriptionally fused to lacZ is therefore a convenient way of measuring DnaA protein-DnaA-box interaction in vivo (Braun et al, 1985). A dnaA0lacZ gene fusion integrated in the l attachment site attB of the chromosome (Berenstein et al, 2002) was used. This was convenient because the gene dosage of attB changes little with changing growth rates and/or replication times (C-periods).…”