Kiyoshi Mizobuchi

Systematic sequencing of the Escherichia coli genome: analysis of the 0 – 2.4 min region

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.

Complete genome sequence of the incompatibility group I1 plasmid R64.

A streptomycin and tetracycline resistance plasmid R64 isolated from Salmonella enterica serovar Typhimurium belongs to the incompatibility group I1 (IncI1). The DNA sequence of the R64 conjugative transfer region was described previously (Komano et al., 2000). Here, we report the complete genome sequence of R64. In the circular double-stranded R64 genome with 120,826bp, 126 complete ORFs are predicted. In addition, 2 and 6 different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of five major regions: replication, drug resistance, stability, transfer leading, and conjugative transfer regions in clockwise order. The nucleotide sequence essential for autonomous replication of R64 is completely identical to that of IncI1 colicinogenic plasmid ColIb-P9, an indication that these two plasmids share the same mechanisms for replication and copy number control. Tetracycline and streptomycin resistance genes are encoded in transposons Tn10 and Tn6082, respectively. These transposons and two insertion elements, IS2 and IS1133, were inserted stepwise into the arsenic-resistant gene, arsA1, present in the drug resistance region. The stability and transfer leading regions contain various important genes such as parAB, resD, ardA, psiAB, or ssb for plasmid maintenance, recombination and transfer reactions. When the genome of R64 was compared with those of other plasmids, varying levels of similarity were observed. It is suggested that genetic recombinations including the site-specific rfsF-ResD system have played an important role in diversity of genomes related to R64. It was found that R64 exhibits highly organized genome structure.

Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site.

Replication of a low‐copy‐number IncIα plasmid ColIb‐P9 depends on expression of the repZ gene encoding the replication initiator protein. repZ expression is negatively controlled by the small antisense Inc RNA, and requires formation of a pseudoknot in the RepZ mRNA consisting of stem–loop I, the Inc RNA target, and a downstream sequence complementary to the loop I. The loop I sequence comprises 5′‐rUUGGCG‐3′, conserved in many prokaryotic antisense systems, and was proposed to be the important site of copy number control. Here we show that the level of repZ expression is rate‐limiting for replication and thus copy number, by comparing the levels of repZ expression and copy number from different mutant ColIb‐P9 derivatives defective in Inc RNA and pseudoknot formation. Kinetic analyses using in vitro transcribed RNAs indicate that Inc RNA binding and the pseudoknot formation are competitive at the level of initial base paring to loop I. This initial interaction is stimulated by the presence of the loop U residue in the 5′‐rUUGGCG‐3′ motif. These results indicate that the competition between the two RNA–RNA interactions at the specific site is a novel regulatory mechanism for establishing the constant level of repZ expression and thus copy number.

Structural Analysis of Late Intermediate Complex Formed between Plasmid ColIb-P9 Inc RNA and Its Target RNA HOW DOES A SINGLE ANTISENSE RNA REPRESS TRANSLATION OF TWO GENES AT DIFFERENT RATES?

The antisense Inc RNA encoded by the IncIα ColIb-P9 plasmid replicon controls the translation of repZencoding the replication initiator and its leader peptiderepY at different rates with different mechanisms. The initial loop-loop base pairing between Inc RNA and the target in therepZ mRNA leader inhibits formation of a pseudoknot required for repZ translation. A subsequent base pairing at the 5′ leader of Inc RNA blocks repY translation. To delineate the molecular basis for the differential control, we analyzed the intermediate complexes formed between RepZ mRNA and Inc RNA54, a 5′-truncated Inc RNA derivative. We found that the initial base pairing at the loops transforms into a more stable intermediate complex by its propagation in both directions. The resulting extensive base pairing indicates that the inhibition of the pseudoknot formation is established at this stage. Furthermore, the region of extensive base pairing includes bases different in related plasmids showing different incompatibility. Thus, the observed extensive base pairing is important for determining the incompatibility of the low-copy-number plasmids. We discuss the evolution of replication control systems found in IncIα, IncB, and IncFII group plasmids.

The plasmid F OmpP protease, a homologue of OmpT, as a potential obstacle to E. coli‐based protein production

OmpT, an outer membrane‐localized protease of Escherichia coli, cleaves a number of exogenous and endogenous proteins during their purification. SecY, an endogenous membrane protein, is a target of this artificial proteolysis in vitro. Here we report that SecY cleavage occurs even in cell extracts from ompT‐disrupted cells, if they carry an F plasmid derivative. A gene, ompP, on the F plasmid was shown to be responsible for this proteolysis. These results indicate that the absence of an F‐like plasmid should be checked when choosing a host strain for E. coli‐based protein production.

The Plasmid ColIb-P9 Antisense Inc RNA Controls Expression of the RepZ Replication Protein and Its Positive Regulator repYwith Different Mechanisms

The autonomous replication region of plasmid ColIb-P9 contains repZ encoding the RepZ replication protein, and inc and repY as the negative and positive regulators of repZ translation, respectively. inc encodes the antisense Inc RNA, andrepY is a short open reading frame upstream ofrepZ. Translation of repY enablesrepZ translation by inducing formation of a pseudoknot containing stem-loop I, which base pairs with the sequence preceding the repZ start codon. Inc RNA inhibits bothrepY translation and formation of the pseudoknot by binding to the loop I. To investigate control of repY expression by Inc RNA, we isolated a number of mutations that expressrepY in the presence of Inc RNA. One class of mutations delete a part of another stem-loop (II), which derepressesrepY expression by initiating translation at codon 10 (GUG), located within this structure. Point mutations in stem-loop II can also derepress repY translation, and the introduction of compensatory base-changes restores control of repYtranslation. These results not only indicate that suppressing a cryptic start codon by secondary structure is important for maintaining the translational control of repZ but also demonstrate that the position of start site for repY translation is critical for its control by Inc RNA. Thus, Inc RNA controls repYtranslation by binding in the vicinity of the start codon, in contrast to the control of repZ expression at the level of loop-loop interaction.