Insertions that increase capsule biosynthesis

Since only a small fraction (six of 127: ~5 %) of transposon mutants resulted in an increase in capsule expression (Table 4.1), each insertion was of considerable interest. Although all six strains produced opaque-like colonies, none of the strains produced entirely capsulated populations; each one continued to produce a significant proportion of non-capsulated cells. Insertions increasing the proportion of capsulated cells were obtained in four genomic loci: Pflu3841 (JG30), purU/Pflu4939 (JG76, JG113 and JG148), ndk (JG176) and Pflu5438 (JG178). Calcofluor binding assays revealed that –

with the exception of the insertion in Pflu5438 – these insertions greatly reduced ACP production (Figure 4.14). Presumably, this results from a lower proportion of ACP- synthesizing non-capsulated cells (see section 3.4.1).

Figure 4.14: Calcofluor binding by 1w4 transposon mutants with insertions in genes that increase

capsule expression. Images were taken after 16 hours growth on KB+calcofluor agar, at x40 or x100

magnification under a fluorescence microscope (left to right): 1w4, JG30, JG76, JG113 (top row); JG148, JG148-ΔCre, JG176 JG178 (bottom row).

As none of the four loci above presented an obvious connection to capsule biosynthesis, investigative BLASTP and Pfam domain searches were employed (Table 4.5). The product of Pflu3841 is a hypothetical integral membrane protein unique to P. fluorescens SBW25 and contains one domain of unknown function (DUF). Pflu5438 encodes an aromatic acid decarboxylase that is conserved across many bacterial species. The nature of the involvement of both Pflu3841 and Pflu5438 in the capsule phenotype remains unknown. The purU-Pflu4939 locus encodes both PurU, an enzyme involved in methionine and purine nucleotide biosynthesis, and a predicted transcriptional regulatory protein. Finally, the product of ndk is nucleoside diphosphate kinase, a highly conserved protein involved in the biosynthesis of nucleotides. The purU-Pflu4939 and

ndk insertions are discussed in more detail in the following sections.

1w4 Pflu3841 purU promoter purU promoter

Protein Pflua Ins.b Sizec Pfam domainsd E-valued Homologse Pflu3841 3841 1 128 DUF2282 1.0 x 10-14 SBW25 purU 4938 282 ACT Formyl_trans_N 1.2 x 10-7 1.8 x 10-47 Conserved Pflu4939 4939 3

124 None n/a Pseudomonas

ndk 5061 1 141 NDK 1.3 x 10-75 Conserved

Pflu5438 5438 1 209 Flavoprotein 6.1 x 10-43 Conserved

Table 4.5: Predicted function, domain characteristics and homologs of proteins identified as

increasing capsules. aPflu refers to the numeric name of the gene encoding the protein in the SBW25

genome. bIns. refers to the number of independent transposon insertions obtained in the gene encoding each protein. cNumber of amino acids. dPfam domain searches (including associated E-values) were performed through the Sanger Pfam website. eConservation level of the protein using a BLASTP homolog search (Conserved=conserved in bacterial genomes outside the Pseudomonas genus).

4.3.1.5.1 Insertions in the purU-Pflu4939 locus

Three independently obtained transposon mutants contain insertions in the purU- Pflu4939 genomic locus. The locus consists of two divergently transcribed genes, purU

(Pflu4938) and Pflu4939. JG76 and JG113 were found to contain insertions at precisely the same nucleotide of the intergenic region, while JG148 contains an insertion at the 3ʹ′

end of Pflu4939 (Figure 4.15). The transcriptional regulator encoded by Pflu4939 belongs to the Pseudomonas-specific MvaT family, members of which have been shown to regulate a wide range of genes. These include genes involved in mevalonate metabolism in Pseudomonas mevalonii (Rosenthal & Rodwell, 1998), the fimbrial cup

genes of P. aeruginosa (Vallet et al., 2004), and genes encoding the antimicrobial exoproteins of P. fluorescens CHA0 (Baehler et al., 2006). Therefore, it is conceivable that Pflu4939 regulates the expression of genes required for capsule biosynthesis.

Figure 4.15: Distribution of transposon insertions in the purU-Pflu4939 genomic locus. Transposons

are indicated as triangles. Numbers refer to JG genotype numbers.

purU Pflu4939 sbcB

Pflu4937

76,113 148

A non-polar mutation was generated in Pflu4939, giving strain JG148-ΔCre, and a capsule counting assay revealed that JG148-ΔCre generated almost exclusively non- capsulated cells (median 0, Appendix A2.2). This phenotype was unexpected, given that JG148 was isolated as a result of increased capsule production. The flipping of the phenotype on Cre-deletion is consistent with the hypothesis that the original capsulated phenotype was the result of increased purU expression from the nptII promoter of the transposon, and the non-capsulated phenotype was the result of correspondingly low

purU expression levels. The purU gene encodes formyltetrahydrofolate deformylase, an enzyme required in methionine biosynthesis and nucleotide pools (see section 4.3.1.4.3). Interestingly, there are multiple purU genes in the SBW25 genome: purU1

(Pflu2321), purU2 (Pflu2332), purU (Pflu4938) and purU (Pflu5647). It is unclear at this stage why the purU gene influences capsule expression.

4.3.1.5.2 Insertion in ndk

A single insertion was obtained at the 5ʹ′ end of ndk (Pflu5061). Ndk is a highly conserved protein that synthesizes all ribo- and deoxyribonucleoside triphosphates. A non-polar mutant was constructed (JG176-ΔCre), and a counting assay estimated the median proportion of capsulated cells in JG176-ΔCre to be 0.544 (Appendix A2.2). This was a significantly greater proportion than that obtained for 1w4 (M-W-W test P=3.97 x 10-3). Precisely how this phenotype emerges is unknown at present. However, targeted deletion of E. coli ndk has been shown to cause nucleotide pool aberrations, which in turn affect RNA, DNA, polysaccharide and signalling molecule biosynthesis (Lu et al., 1995; Bernard et al., 2000). Accordingly, a range of phenotypes has been reported for

ndk mutants, including a mutator phenotype in E. coli (Miller et al., 2002) and alteration of biosynthesis of the polysaccharide alginate in P. aeruginosa (Sundin et al., 1996a; Sundin et al., 1996b). Further insight into the role of the ndk gene in the 1w4 switching phenotype is provided in sections 6.4.3.1, 6.4.3.2.4 and 6.4.4.2.1.

4.4 Discussion