Colonias de hormigas

Analysis of open reading frames (ORFs) in the pIJ12404 nucleotide sequence identified a putative biosynthetic gene cluster of nine genes (Figure III.12.A.).

Upstream of cypA and divergently transcribed from it is orf1, which encodes a putative transcriptional regulator. Seven genes with predicted biosynthetic and transport functions lie directly downstream of cypA, the first six of which are likely to be co-transcribed since their coding sequences overlap by several nucleotides (i.e. they appear to be translationally coupled). The first gene downstream of cypA is cypH, which encodes a product with no significant homology to functionally characterized proteins. CypH is likely a two-domain protein with partial homology to a conserved horizontally transferred transmembrane helix domain at its N-terminus (Schultz 2004) and a possible α/β hydrolase fold located towards its C-terminus.

Following cypH is cypL, again with no functionally identified homologs. The next gene, cypD, encodes a decarboxylase of the HFCD family (Kupke et al. 2000), and could be responsible for the introduction of AviCys, analogous to LanD enzymes in lantibiotic biosynthesis. cypM is the fourth gene downstream of cypA and encodes an S-adenosyl methionine (SAM)-dependent methyltransferase presumably required for methylation of the N-terminal alanine of cypemycin. These biosynthetic genes are followed by an adenosine triphosphate (ATP)-binding subunit of an ATP-binding cassette (ABC) transporter, designated cypT. The last gene in which the start codon overlaps with the upstream ORF is cypP, encoding a 516 amino acid protein with 12 predicted transmembrane helices and with no functionally annotated homologues in the public databases (Figure III.12.B.). Because of its location downstream of cypT, it was suggested that this protein forms a pore in the cytoplasmic membrane to allow cypemycin export from cell. No leader peptidase domains were found in CypT or CypP. cypI is the last gene that is transcribed in the same direction, but its start codon does not overlap with the coding sequence of cypP. CypI is a member of the large DUF255 family of conserved proteins with a thioredoxin domain. Analysis of the remaining ORFs in the pIJ12404 cosmid sequence did not identify any lantibiotic dehydratase homologues indicating, contrary to previous classification, that cypemycin is not a member of the lantibiotic family of modified peptides (Chatterjee et al. 2005).

A

B

Figure III.12. A) Schematic representation of the cypemycin biosynthetic gene cluster.

Flanking genes not expected to be involved in cypemycin biosynthesis are in grey. Vertical arrows delineate the putative cyp gene cluster used to generate a minimal gene set. B) CypP is a putative pore protein. TMHMM analysis identified 12 putative transmembrane domains (red). Predicted cytoplasmic domains are indicated in blue and exterior domains in pink.

regulation? modification transport modification

orf1 cypA cypH cypL cypH cypP cypI

cypD cypT

regulation? modification transport modification

orf1 cypA cypH cypL cypH cypP cypI

cypD cypT

regulation? modification transport modification

orf1 cypA cypH cypL cypH cypP cypI

cypD cypT

regulation? modification transport modification

orf1 cypA cypH cypL cypH cypP cypI

cypD cypT

III.7. Discussion

Various attempts were made to identify the gene cluster responsible for the biosynthesis of the unusual post-translationally modified peptide antibiotic cypemycin. This section is devoted to a discussion of these approaches. Since the next chapter describes the genetic characterisation of the cypemycin gene cluster, the reader is referred to Chapter IV.6. for a detailed discussion of the functions of individual genes.

Several experiments described in this chapter relied on the use of degenerate primers or probes to obtain sequence information for cypA. The main factors that likely contributed to the failure of these experiments were the short length and degree of degeneracy of the primers/probes, and the reduced sensitivity of DIG-labelling compared to radioactive DIG-labelling. The predicted CypA propeptide sequence turned out to be correct apart from the occurrence of a Cys at position 19 where Ser was expected (compare Figure III.3. and III.6.B.). Although this probably had only a minor effect on Southern hybridisations with Cyp probe 2, the PCR reaction using Cyp deg R would have been markedly compromised.

Alternative approaches that could have been used to identify the cypemycin gene cluster could have included a genetic screen for likely biosynthetic enzymes and an activity based screen. However, when employed for cypemycin, both would have been problematic. Starting from the assumption that cypemycin was a lantibiotic, a genetic screen could have been directed at the identification of lantibiotic dehydratases, for example by using primers complementary to conserved regions found in the corresponding genes (Dodd et al. 2006). Since it later emerged that cypemycin belongs to a different peptide family, this would have been a futile exercise. An activity based screen could have been performed by introducing the S. sp. OH-4156 cosmid library in a heterologous host. Lantibiotic and other post-translationally modified peptide gene clusters are relatively small and there is a reasonable chance that at least a couple cosmids from a genome library will contain all of the necessary genes. However, it is difficult to predict whether the cluster will be expressed, and if so, at what level. In the case of cypemycin, heterologous production was observed in S. venezuelae, S. lividans and S.

coelicolor, but production levels varied markedly between these strains and the expression levels were generally too low to allow for a screen with single colonies against M. luteus.

In summary, with the benefit of hindsight, it can be concluded that the genome scanning approach was particularly useful for the identification of the cypemycin gene cluster. The unusual precursor peptide and the novelty of the biosynthetic enzymes would have made the cluster difficult to find using other means.