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All strains were cultured in CASO broth (Sigma-aldrich, Diegem, Belgium) supplemented with 50 µg/mL kanamycin or 30 µg/mL chloramphenicol for the mutant and complementation strain, respectively.

All confocal parameters were set using wild-type biofilm and were used as standard settings for comparison to the biofilms produced by the mutant and complementation strains.

(E ) ChIP-PCR of H3K9me2 marks in heterochromatic loci in kyp mutant and complementation lines inoculated with mock or CaLCuV.

The levels of rRNAs, including 23S, 16S, 5S, and 4.5S rRNAs, in the wild type, cfm4 mutant, and complementation line were determined by Northern blot analysis.

Before studying the effect of XF inactivation on the pathogenesis of S. agalctiae in vivo, it is important to characterize the growth characteristics of the parental, mutant and complementation strains in vitro.

To confirm that the mutant has no effect on the transcription of bilateral genes, qRT-PCR assays was performed to detect the expression of xf gene, xf upstream and downstream ORF genes in the parental, mutant and complementation strains.

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d Quantification of relative expression of the 3′ end of BET11 and BET12 in the WT, double mutants and complementation lines.

As expected, secretion of HrpB2XAC was abolished in the ΔhrpB2 mutants and complementation with pUFR047_hrpB2 restored HrpB2XAC secretion (Fig. 6A, lanes 2 and 3, respectively).

Primers for mutants and complementation lines genotyping.

NIL libraries, mutants and complementation studies are the tools required for this mechanistic understanding.

Future work on the molecular analysis of such genes in the TAM mutants and complementation with the WT gene will confirm the causal link between antenna size and photosynthetic performance, and provide further insight into the basis of the phenotype.

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