Lei Sun’s research while affiliated with University of Kent and other places

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE, and hence MicA, in the absence of RfaH. Additional work confirms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression.

The enzyme product of the dddD gene, found in several different marine bacteria, acts on dimethylsulfoniopropionate (DMSP), liberating dimethyl sulfide (DMS) and generating 3-OH-propionate as the initially detected C3 product. In many bacteria, dddD is near genes whose sequence suggests that they encode a DMSP transporter. These are of two very different types, in the BCCT (betaine-carnitine-choline transporter) family or resembling members of the ABC super-family that import betaines. Even within these two families, the amino acid sequences of these putative transporters are not particularly similar to each other. Genes for the predicted DMSP transporters of Halomonas and Marinomonas (both BCCT type) and of Burkholderia ambifaria AMMD (ABC-type) were each cloned and introduced into an Escherichia coli mutant (MKH13) that is defective in betaine uptake, and so fails to catabolise DMSP even when a cloned dddD gene was present, due to the failure of the substrate to be imported. DMSP-dependent DMS production (Ddd+ phenotype) was restored by introducing any of these cloned transporters into MKH13 containing dddD. Other marine bacteria use a range of enzymes, called DddL, DddP, DddQ, DddW and DddY, to cleave DMSP, but the various ddd genes that encode them are usually unlinked to any that are predicted to encode betaine transporters. We identified one gene in Sulfitobacter sp. EE-36 and two in Roseovarius nubinhibens ISM, which, when cloned and introduced into E. coli MKH13, overcame its osmotic sensitivity when it was grown with DMSP or other exogenous betaines. These genes all encoded BCCT transporters, but were unlinked to any known genes involved in DMSP catabolism in these two strains of α-proteobacteria. KeywordsABC transporter–BCCT transporter– ddd Genes–DMSP– Halomonas – Marinomonas – Roseovarius – Sulfitobacter

We have demonstrated that SlyA activates fimB expression and hence type 1 fimbriation, a virulence factor in Escherichia coli. SlyA is shown to bind to two operator sites (OSA1 and OSA2), situated between 194 and 167 base pairs upstream of the fimB transcriptional start site. fimB expression is derepressed in an hns mutant and diminished by a slyA mutation in the presence of H-NS only. H-NS binds to multiple sites in the promoter region, including two sites (H-NS2 and H-NS3) that overlap OSA1 and OSA2, respectively. Mutations that disrupt either OSA1 or OSA2 eliminate or reduce the activating effect of SlyA but have different effects on the level of expression. We interpret these results as reflecting the relative competition between SlyA and H-NS binding. Moreover we show that SlyA is capable of displacing H-NS from its binding sites in vitro. We suggest SlyA binding prevents H-NS binding to H-NS2 and H-NS3 and the subsequent oligomerization of H-NS necessary for full inhibition of fimB expression. In addition, we show that SlyA activates fimB expression independently of two other known regulators of fimB expression, NanR and NagC. It is demonstrated that the rarely used UUG initiation codon limits slyA expression and that low SlyA levels limit fimB expression. Furthermore, Western blot analysis shows that cells grown in rich-defined medium contain ∼1000 SlyA dimers per cell whereas those grown in minimal medium contain >20% more SlyA. This study extends our understanding of the role that SlyA plays in the host-bacterial relationship.

SummaryA bacterium in the genus Halomonas that grew on dimethylsulfoniopropionate (DMSP) or acrylate as sole carbon sources and that liberated the climate-changing gas dimethyl sulfide in media containing DMSP was obtained from the phylloplane of the macroalga Ulva. We identified a cluster that contains genes specifically involved in DMSP catabolism (dddD, dddT) or in degrading acrylate (acuN, acuK) or that are required to break down both substrates (dddC, dddA). Using NMR and HPLC analyses to trace 13C- or 14C-labelled acrylate and DMSP in strains of Escherichia coli with various combinations of cloned ddd and/or acu genes, we deduced that DMSP is imported by the BCCT-type transporter DddT, then converted by DddD to 3-OH-propionate (3HP), liberating dimethyl sulfide in the process. As DddD is a predicted acyl CoA transferase, there may be an earlier, unidentified catabolite of DMSP. Acrylate is also converted to 3HP, via a CoA transferase (AcuN) and a hydratase (AcuK). The 3HP is predicted to be catabolized by an alcohol dehydrogenase, DddA, to malonate semialdehyde, thence by an aldehyde dehydrogenase, DddC, to acyl CoA plus CO2. The regulation of the ddd and acu genes is unusual, as a catabolite, 3HP, was a co-inducer of their transcription. This first description of genes involved in acrylate catabolism in any organism shows that the relationship between the catabolic pathways of acrylate and DMSP differs from that which had been suggested in other bacteria.

This paper describes the ddd genes that are involved in the production of the gas dimethyl sulphide from the substrate dimethylsulphoniopropionate (DMSP), an abundant molecule that is a stress protectant in many marine algae and a few genera of angiosperms. What is known of the arrangement of the ddd genes in different bacteria that can undertake this reaction is reviewed here, stressing the fact that these genes are probably subject to horizontal gene transfer and that the same functions (e.g. DMSP transport) may be accomplished by very different mechanisms. A surprising number of DMS-emitting bacteria are associated with the roots of higher plants, these including strains of Rhizobium and some rhizosphere bacteria in the genus Burkholderia. One newly identified strain that is predicted to make DMS is B. phymatum which is a highly unusual β-proteobacterium that forms N2-fixing nodules on some tropical legumes, in this case, the tree Machaerium lunatum, which inhabits mangroves. The importance of DMSP catabolism and DMS production is discussed, not only in terms of nutritional acquisition by the bacteria but also in a speculative scheme (the ‘messy eater’ model) in which the bacteria may make DMS as an info-chemical to attract other organisms, including invertebrates and other plankton.

Dimethyl sulfide (DMS) is a key compound in global sulfur and carbon cycles. DMS oxidation products cause cloud nucleation and may affect weather and climate. DMS is generated largely by bacterial catabolism of dimethylsulfoniopropionate (DMSP), a secondary metabolite made by marine algae. We demonstrate that the bacterial gene dddD is required for this process and that its transcription is induced by the DMSP substrate. Cloned dddD from the marine bacterium Marinomonas and from two bacterial strains that associate with higher plants, the N2-fixing symbiont Rhizobium NGR234 and the root-colonizing Burkholderia cepacia AMMD, conferred to Escherichia coli the ability to make DMS from DMSP. The inferred enzymatic mechanism for DMS liberation involves an initial step in which DMSP is modified by addition of acyl coenzyme A, rather than the immediate release of DMS by a DMSP lyase, the previously suggested mechanism.