SOS involvement in stress-inducible biofilm formation.

Bacterial biofilm formation can be induced by antimicrobial and DNA damage agents. These agents trigger the SOS response, in which SOS sensor RecA stimulates auto-cleavage of repressor LexA. These observations lead to a hypothesis of a connection between stress-inducible biofilm formation and the RecA-LexA interplay. To test this hypothesis, three biofilm assays were conducted, viz. the standard 96-well assay, confocal laser scanning microscopy, and the newly developed biofilm-on-paper assay. It was found that biofilm stimulation by the DNA replication inhibitor hydroxyurea was dependent on RecA and appeared repressed by the non-cleavable LexA of Pseudomonas aeruginosa. Surprisingly, deletion of lexA led to reduction of both normal and stress-inducible biofilm formation, suggesting that the wild-type LexA contributes to biofilm formation. The decreases was not the result of poor growth of the mutants. These results suggest SOS involvement in hydroxyurea-inducible biofilm formation. In addition, with the paper biofilm assay, it was found that degradation of the biofilm matrix DNA by DNase I appeared to render the biofilms susceptible to the replication inhibitor. The puzzling questions concerning the roles of LexA in DNA release in the biofilm context are discussed.

Role of the mukB gene in chromosome and plasmid partition in Escherichia coli.

The intracellular locations of oriC and oriR1, the replication origins of the chromosome and plasmid R1, respectively, were visualized by fluorescence in situ hybridization (FISH) in exponentially growing populations of Escherichia coli. The locations of oriC and oriR1 (from a Par+ R1 plasmid) were unique and different in the wild-type host. In a mukB mutant, the positions were perturbed for both origins. The position of oriR1 from a plasmid with active partition (Par+) in the mukB host was as randomized as that of oriR1 from the Par- plasmid in a wild-type host. However, this mukB-induced randomization did not result in unstable inheritance of the Par+ plasmid, as measured by the conventional segregation assay. This might result from the preferential association of the Par+ plasmid with the bigger, decondensed nucleoid-containing daughters during cell division of MukB- cells, whereas the Par- plasmids were distributed at random and were lost by frequently ending up in anucleate cells.

Plasmid R1 is present as clusters in the cells of Escherichia coli.

Fluorescence microscopy was used to determine the location(s) of the replication origin of plasmid R1 in exponentially growing cells of Escherichia coli. The number of oriR1 foci per cell was smaller than the number of R1 copies per cell and was found to be the same for a copA mutant of R1 and for the wild-type plasmid. The intensities of individual foci were stronger for the cop mutant than for the wild type. We interpreted these results to imply that the plasmid DNA molecules were localized in small groups/clusters, a result that seems contrary to the earlier observations that plasmid R1 replicates randomly and segregates as a single-copy unit. The implications for the quantitative behavior of plasmid R1 in stability, incompatibility tests, replication, and partition experiments are discussed.

Escherichia coli cell cycle control genes affect chromosome superhelicity.

We have used ethidium bromide titration for direct measurement of the changes in the negative supercoiling of Escherichia coli chromosome caused by mutations inactivating the cell cycle functions mukB and seqA. The amounts of the intercalative agent required to relax the supercoiled chromosome in mukB and seqA mutants were lower and higher, respectively, than for the wild-type parent, confirming that these cell cycle genes modulate the topology of the E. coli chromosome. Plasmid superhelicity measured in these mutant strains showed similar effects albeit of reduced magnitude. As the effects of mukB and seqA mutations were not restricted to the chromosome alone, MukB and SeqA proteins possibly interact with factors involved in the maintenance of intracellular DNA topology. To our knowledge, this is the first direct demonstration of the influence of mukB and seqA genes on the superhelicity of the E. coli chromosome.

Mutual suppression of mukB and seqA phenotypes might arise from their opposing influences on the Escherichia coli nucleoid structure.

A strain of Escherichia coli in which both the seqA and mukB genes were inactivated displayed partial suppressions of their individual phenotypes. Temperature sensitivity, anucleate cell production and poor nucleoid folding seen in the mukB strain were suppressed by the seqA null mutation, whereas filamentation, asymmetric septation and compact folding of the nucleoids observed in the seqA strain were suppressed by inactivation of the mukB gene function. However, the asynchronous initiation of chromosome replication in the seqA strain was not reversed in the mukBseqA double mutant. Membrane-associated nucleoids were isolated from the wild-type, mukB, seqA and mukBseqA strains and their sedimentation rates were compared under identical conditions. Whereas the mukB mutation caused unfolding of the nucleoid, the seqA mutation led to a more compact packaging of the chromosome. The mukBseqA double mutant regained the wild-type nucleoid organization as revealed from its rate of sedimentation. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The mukB mutant was oversensitive to novobiocin and this susceptibility was suppressed in the mukBseqA strain, suggesting possible roles of MukB and SeqA in maintaining chromosome topology. The mutual phenotypic suppression of mukB and seqA alleles thus suggests that these genes have opposing influences on the organization of the bacterial nucleoid.