PMID:19432803

Citation	Rotman, E, Bratcher, P and Kuzminov, A (2009) Reduced lipopolysaccharide phosphorylation in Escherichia coli lowers the elevated ori/ter ratio in seqA mutants. Mol. Microbiol. 72:1273-92
Abstract	The seqA defect in Escherichia coli increases the ori/ter ratio and causes chromosomal fragmentation, making seqA mutants dependent on recombinational repair (the seqA recA colethality). To understand the nature of this chromosomal fragmentation, we characterized DeltaseqA mutants and isolated suppressors of the DeltaseqA recA lethality. We demonstrate that our DeltaseqA alleles have normal function of the downstream pgm gene and normal ratios of the major phospholipids in the membranes, but increased surface lipopolysaccharide (LPS) phosphorylation. The predominant class of DeltaseqA recA suppressors disrupts the rfaQGP genes, reducing phosphorylation of the inner core region of LPS. The rfaQGP suppressors also reduce the elevated ori/ter ratio of the DeltaseqA mutants but, unexpectedly, the suppressed mutants still exhibit the high levels of chromosomal fragmentation and SOS induction, characteristic of the DeltaseqA mutants. We also found that colethality of rfaP with defects in the production of acidic phospholipids is suppressed by alternative initiation of chromosomal replication, suggesting that LPS phosphorylation stimulates replication initiation. The rfaQGP suppression of the seqA recA lethality provides genetic support for the surprising physical evidence that the oriC DNA forms complexes with the outer membrane.
Links	PubMed PMC2691451 Online version:10.1111/j.1365-2958.2009.06725.x
Keywords	Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Chromosomes, Bacterial/genetics; DNA Fragmentation; DNA Replication; DNA, Bacterial/metabolism; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Expression Regulation, Bacterial; Genes, Bacterial; Lipopolysaccharides/metabolism; Mutagenesis, Insertional; Mutation; Origin Recognition Complex/metabolism; Phosphorylation; SOS Response (Genetics)
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Significance

This results of this article suggest that the mechanisms behind chromosomal fragmentation are somehow linked to a novel phenomena associated with DNA replication stress and replication fork failure.

Methods & Results

(1) Characterization of the ΔseqA mutants for possible pgm defect (Fig 2)

• Lu and Kleckner’s (1994) ΔseqA::tet has a polar effect on pgm, the downstream gene of seqA, so precise replacement and deletions of seqA were created.

• pRL27, a plasmid containing a hyperactive Tn5 transposase gene was used for random insertion mutagenesis.

• SDS testing: Putative outer membrane defects, as in the case of pgm mutant, make the cells sensitive to anionic detergents like SDS. So cells were plated on LB+SDS to test for pgm defect.

• Iodine staining: Colonies grown till size reaches 1 mm in diameter, overlaid with M9 Iodine agar. pgm mutants turn blue because of the accumulation of maltodextrin.

Results & Conclusion:

The two seqA mutants used in this study didn’t affect the downstream gene pgm.

(2) Testing the ΔseqA dependence on recombinational repair i.e. characterizing the co-lethality of ΔseqA recA mutants. (Fig 3)

• ΔrecA304: a deletion generated by Tn10 in the slr-recA region; highly UV sensitive and recombination deficient.

• recA629{Cs): D32G,E38K, 1298V: deficient at low temperatures (28°C)but only moderately defective at higher temperatures (42°C.

Results & Conclusion:

The double mutant ΔseqA21 recA629(Cs) can grow at 42°C but not 28-30°C. This mutant used as background strain for insertional mutagenesis.

(3) Isolating the rfa suppressors of ΔseqA recA lethality (Fig 4)

• Selection was made for colonies that grew at 30°C.

• Suppressors were verified by Pl transductions back into original double mutant.

• Test for loss of suppression by complementation of specific genes.

Results & Conclusion:

18 of the suppressors were identified as insertions in rfaQ, rfaG and rfaP; the first three genes of the rfaQGPSBIJYZK operon, responsible for lipopolysaccharide biosynthesis.

(4) Characterization of the rfa suppressors (Fig 5)

• ΔseqA21 recA629(Cs) and respective rfaQ, rfaG, and rfaP suppressors were grown in the MOPS minimal medium and labeled the cells with (10µCi) 32P-orthophosphoric acid for 5 minutes.

• Phospholipids and RNA removed using the total DNA isolation protocol. DNA, LPS and polyphosphates.

• Separated on a 1.1% alkaline agarose gel run at 20V for about 10 hrs.

• The suppressor sensitivity for SDS was tested on LB plates with 1%SDS.

Results & Conclusion:

One of the proximal changes in the seqA mutants that lead to their synthetic lethality with recA could be this increased LPS phosphorylation, compensated by the rfaQGP defect.

(5) Composition of phospholipids in the ΔseqA mutants. (Fig 6)

• Total phospholipids extracted by the method of Bligh and Dyer (1959.

• ³²P-labeled cells were pelleted, mixed with methanol and chloroform and centrifuged to separate the phases. After repeated extractions with KCl and water, the remaining organic phase was dried under a stream of nitrogen and dissolved in (2:1) chloroform:methanol. The extract was used for determination of radioisotope incorporation into phospholipids and for analysis by TLC.

Results & Conclusion:

No significant differences in phospholipid composition were found between the wild-type strains and Δseq. So the rfa suppressors are not compensating for any changes in the phospholipid composition.

(6) The effect of rfa mutants on ori/ter ratio, SOS induction and chromosomal fragmentation (Fig 7)

• Total DNA extracted by phenol chloroform method, denatured in NaOH and spotted onto Nylon membrane. DNA was cross-linked to the membrane with UV, separated into two halves and each half probed for hybridization with either an origin-proximal probe or a terminus­ proximal probe.

• The rfa suppressors were introduced into the seqA recBC(Ts) double and recBC(Ts) single mutants.

• Chromosomal fragmentation was quantified using pulse field gel electrophoresis.

• All mutants to be tested were P1 transduced into a PsfiA-lacZ reporter background. When the cells are under SOS-induced stress, both by the mutation or external DNA damage, the promoter is expressed, and thus the level of β-galactosidase can be quantitative measure of SOS response. As a positive control, wild type cells were treated with 100 ng/ml Mitomycin C.

Results & Conclusion:

The rfa suppressors are likely to reduce the ori/ter ratio in the chromosomal DNA of seqA mutants. This effect does not necessarily translate into reduced chromosomal fragmentation, but instead allows the seqA recA mutants to tolerate the levels of chromosomal fragmentation. The rfa suppressors do not decrease SOS in the seqA mutants.

(7) How does LPS phosphorylation affect replication initiation? (Fig 8C&D)

• The rfaP mutant was introduced into the double pgsA lpxB mutant. This mutant synthesizes reduced amounts of PG and CL at low temperatures and totally stops their synthesis at 42°C, thus has replication initiation defect at 42°C.

• To confirm the growth problems were due to replication initiation defects, IPTG-induced plasmid replication origin was inserted near oriC and tested for growth in presence and absence of IPTG.

Results & Conclusion:

It is suggested that rfaQGP inactivation suppresses the seqA recA colethality by lowering the ori/ter ratio to manageable levels via decreasing replication initiation, working outside of the two known regulation cycles and without direct interactions with DnaA itself.

Annotations

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References

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