DNA repair mutants of Rhodobacter sphaeroides

Abstract

The genome of the photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1 comprises two chromosomes and five endogenous plasmids and has a 65% G؉C base composition. Because of these characteristics of genome architecture, as well as the physiological advantages that allow this organism to live in sunlight when in an anaerobic environment, the sensitivity of R. sphaeroides to UV radiation was compared with that of the more extensively studied bacterium Escherichia coli. R. sphaeroides was found to be more resistant, being killed at about 60% of the rate of E. coli. To begin to analyze the basis for this increased resistance, a derivative of R. sphaeroides, strain 2.4.1⌬S, which lacks the 42-kb plasmid, was mutagenized with a derivative of Tn5, and the transposon insertion mutants were screened for increased UV sensitivity (UV s ). Eight UV s strains were isolated, and the insertion sites were determined by contour-clamped homogeneous electric field pulsed-field gel electrophoresis. These mapped to at least five different locations in chromosome I. Preliminary analysis suggested that these mutants were deficient in the repair of DNA damage. This was confirmed for three loci by DNA sequence analysis, which showed the insertions to be within genes homologous to uvrA, uvrB, and uvrC, the subunits of the nuclease responsible for excising UV damage.The photosynthetic bacterium Rhodobacter sphaeroides was the first prokaryote shown to have multiple chromosomes (48). Wild-type strain 2.4.1 comprises a genome of two circular chromosomes, as well as five additional plasmids with unknown functions. This genome architecture raises significant questions pertaining to DNA metabolism, an area about which virtually nothing is known in this multichromosomal prokaryote. Furthermore, the nature of DNA replication, repair, and recombination in R. sphaeroides is of interest because of other aspects of the lifestyle of this organism. Being photosynthetic, it is subjected to potentially lethal UV irradiation, making its mode of DNA repair of special interest. The high GϩC content (65%) of the R. sphaeroides genome makes this of further interest because of the expected higher content of Z DNA, the greater need for uracil repair following cytosine deamination, and the role of the latter process in UV mutagenesis when it occurs in pyrimidine dimers (49). In addition, R. sphaeroides can use a range of compounds as terminal electron acceptors, including toxic metal oxides and organic compounds with a potential for free radical formation (34). Thus, it is not only likely but probable that there are specific mechanisms that protect the genome against damage from these compounds.The current paradigm for prokaryotic DNA replication, repair, and recombination is based on DNA metabolism in Escherichia coli. Since this is an enteric organism with exposure to a limited environment and has a single chromosome with a base composition of about 50% GϩC, there is reason to expect that the E. coli model is incomplete or not entirely appropriate for free...

Isolation and expression of the Rhodobacter sphaeroides gene (pgsA) encoding phosphatidylglycerophosphate synthase

“…The DNA insert in cosmid pLAB14 maps to AseI fragment B on chromosome I of R. sphaeroides (41). DNA sequence analysis revealed the presence of one complete ORF with a high degree of homology between PgsA Rs and the products of the pgsA genes of not only E. coli (24,61) but also a number of other bacteria as shown in Fig.…”

“…The complete DNA sequence (derived from both strands) of the PstI fragment can be obtained from GenBank (accession number U29587); several errors reported in the initial sequence for part of this region (41) have been corrected. …”

“…PCRs were performed by following the manufacturer's instructions and using Taq polymerase (Promega). A primer (5Ј-TTCAGGAC GCTACTTGTGTA-3Ј) complementary to the end of the IS50 segment of plasmid pBS492 (41) and the T7 primer (Promega) were used in a reaction with plasmid pBS492 in order to isolate a DNA fragment carrying the potential pgsA Rs gene. A Perkin-Elmer Cetus thermal cycler was used, and conditions were the following: 7 min of incubation at 95ЊC, followed by 30 cycles of 95ЊC for 1 min, 50ЊC for 2 min, and 75ЊC for 1 min.…”

Fate of Heterotrophic Microbes in Pelagic Habitats: Focus on Populations

“…Some alpine lakes exposed to intense levels of UV-B radiation (148) feature conspicuously high abundances of actinobacteria, a phylogenetic lineage of mainly gram-positive bacteria with a high genomic GϩC content (91,263,310). Gram-positive bacterial isolates are often less affected by UV radiation than gram-negative bacteria (8), and a high genomic GϩC content has been suggested to mediate higher resistance to radiation damage (166). Viral lysis.…”

TheVibrio harveyiGTPase CgtAVIs Essential and Is Associated with the 50S Ribosomal Subunit

“…The plasmid pAC1, encoding at least part of the cgtA V gene, was obtained by digestion of BB7X DNA with EcoRI, ligation to pUC19, and selection for both trimethoprim and ampicillin resistance (5). The expectation was that trimethoprim-resistant transformants would harbor both the Tn5 transposon (encoding dhfrII, the dihydrofolate reductase gene [21]) and flanking chromosomal DNA. Using a primer complementary to the IS50 element of Tn5Tp r MCS (Tn, 5Ј-TTCAGGACGCTACTT GTGTA-3Ј), the sequence of the N-terminal 99 amino acids of cgtA V was obtained, and it was concluded that BB7X contained a cgtA V ::Tn5Tp r MCS insertion mutation (5).…”