The Biosynthesis of Differentiation-Inducing Factor, a Chlorinated Signal Molecule Regulating DictyosteliumDevelopment

Abstract

Differentiation-inducing factor (DIF)-1 is a chlorinated alkyl phenone released by developing Dictyostelium amoebae, which induces them to differentiate into stalk cells. A biosynthetic pathway for DIF-1 is proposed from labeling, inhibitor, and enzymological experiments. Cells incorporate 36 Cl؊ into DIF-1 during development, showing that the chlorine atoms originate from chloride ions; peak incorporation is at the first finger stage. DIF-1 synthesis can be blocked by cerulenin, a polyketide synthase inhibitor, suggesting that it is made from a polyketide. This is most likely the C 12 polyketide (2,4,6-trihydroxyphenyl)-1-hexan-1-one (THPH). Feeding experiments confirm that living cells can convert THPH to DIF-1. Conversion requires both chlorination and methylation of THPH, and enzymatic activities able to do this exist in cell lysates. The chlorinating activity, assayed using 36 Cl ؊ , is stimulated by H 2 O 2 and requires both soluble and particulate components. It is specific for THPH and does not use this compound after O-methylation. The methyltransferase is soluble, uses S-adenosyl-L-methionine as a co-substrate, has a K m for dichloro-THPH of about 1.1 M, and strongly prefers this substrate to close analogues. Both chlorinating and methyltransferase activities increase in development in parallel with DIF-1 production, and both are greatly reduced in a mutant strain that makes little DIF-1. It is proposed that DIF-1 is made by the initial assembly of a C 12 polyketide skeleton, which is then chlorinated and methylated.

A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1

“…Transcription of chlA is up-regulated during early development in synchrony with stlB and dmtA. Low levels of chlA transcription were also observed at 0 and 4 h consistent with a previous observation that lysates of undeveloped cells exhibit very weak in vitro chlorinating activity (15).…”

“…Not only is chlorination of THPH a prerequisite for methylation by DmtA (15), but DIF-1 is 25 times more potent than its monochloro analogue (the known metabolite DIF-3) in inducing stalk cell differentiation (17). The chlorination of THPH has only been observed as an enzymatic activity in crude lysates of Dictyostelium (15). Chlorinating activity is low in vegetative cells but rises substantially at the end of aggregation, mimicking the rise in enzymatic activity seen for the methyltransferase.…”

“…Early studies in Dictyostelium helped to define a three-step biosynthetic pathway for production of DIF-1 (15). Formation of the core polyketide (2,4,6-trihydroxyphenyl)-1-hexan-1-one (THPH) is followed in order by dichlorination of the aromatic ring and O-methylation.…”

Evolution of cooperation and control of cheating in a social microbe

“…One of the delights in working with a microbial system is the accessibility of mechanisms. Whether a cell becomes spore or stalk is mediated by DIF-1, a small, secreted, chlorinated alkyl phenone (79). Stronger cells that are immune to its effects at biological levels produce DIF-1.…”

Steely Enzymes Are Involved in Prestalk and Prespore Pattern Formation

“…The Dictyostelium diguanylate cyclase DgcA, which produces c-di-GMP, is expressed in the slug tip region, which consists of pstA cells. Therefore pstA cell inducers, which should be polyketides, 24) have an important role in Dictyostelium development. We tried to identify the PKS that regulates pstA differentiation.…”