Integrated regulation involving quorum sensing, a two-component system, a GGDEF/EAL domain protein and a post-transcriptional regulator controls swarming and RhlA-dependent surfactant biosynthesis in Serratia

Serratia sp. ATCC 39006 ( Serratia 39006) is a Gram-negative bacterium which produces the secondary metabolite antibiotics, prodigiosin and 1-carbapen-2-em-3-carboxylic acid and secretes plant cell wall degrading enzymes. In this study we have identified mutations in the genes, pigX , rap and rsmA , which caused increased production of a previously unidentified surfactant and flagella-dependent swarming phenotype in Serratia 39006. Analysis of both the biosynthesis and regulation of surfactant production and swarming, revealed FlhC, quorum sensing, a GGDEF/EAL domain protein (PigX), a GacAS two-component system, an Rsm system and Rap as key regulators. In addition, surfactant biosynthesis required a protein similar to RhlA, involved in rhamnolipid synthesis in Pseudomonas aeruginosa . Homologues of RhlA have not previously been identified in members of the Enterobacteriaceae . Furthermore, we provide evidence that the surfactant may be responsible for dispersal of the antimicrobial pigment, prodigiosin. This study demonstrates the complex regulatory inputs into the coordinated multicellular swarming phenotype in Serratia .     

Pseudomonas aeruginosa cupA-encoded fimbriae expression is regulated by a GGDEF and EAL domain-dependent modulation of the intracellular level of cyclic diguanylate

Cyclic-diguanylate (c-di-GMP) is a widespread bacterial signal molecule that plays a major role in the modulation of cellular surface components, such as exopolysaccharides and fimbriae, and in the establishment of a sessile life style. Here, we report that intracellular c-di-GMP levels influence cupA-encoded fimbriae expression in Pseudomonas aeruginosa. In an autoaggregative P. aeruginosa small colony variant (SCV) CupA fimbriae and the intracellular c-di-GMP concentration were found to be enhanced as compared with the clonal wild-type. The SCV morphology and the expression of CupA fimbriae were dependent on a functional PA1120 and morA gene both encoding a GGDEF domain. Overexpression of the GGDEF domain protein PA1120 complemented the PA1120 and the morA mutant with respect to CupA fimbriae expression. In agreement with these findings, overexpression of the EAL domain containing phenotypic variance regulator (PvrR) in the SCV resulted in a decreased intracellular level of c-di-GMP, a reduced cupA fimbriae expression and a switch to wild-type colony morphology.     

Cloning and expression in Escherichia coli of Serratia marcescens genes encoding prodigiosin biosynthesis

Prodigiosin, the bright red pigment produced by many strains of Serratia marcescens, is synthesized by a bifurcated pathway that terminates in the enzymatic condensation of the two final products, a monopyrrole and a bipyrrole . Sau3A fragments of S. marcescens ( Nima ) DNA were introduced into a strain of Escherichia coli K-12 by use of the cosmid vector pHC79 , and transformed clones were selected based on resistance to ampicillin. Among 879 transformants screened, 2 could be induced to synthesize prodigiosin when supplied with either one or both terminal products of the bifurcated pathway. Data are presented to support the idea that production of prodigiosin is not usually mediated by a plasmid.     

茎瘤固氮根瘤菌ORS571 GGDEF和EAL结构域蛋白的预测及功能研究

[目的]环二鸟苷酸c-di-GMP是细菌中广泛存在的第二信使,能够调控多种细胞功能。c-di-GMP的合成与水解分别由含有GGDEF结构域和EAL结构域的蛋白催化。本研究针对茎瘤固氮根瘤菌ORS571的GGDEF和EAL结构域相关蛋白进行基因组学分析,并对三个同时含有GGDEF和EAL结构域的蛋白（AZC_3085、AZC_3226和AZC_4658）进行功能研究。[方法]利用SMART数据库对含有GGDEF和EAL结构域的蛋白进行结构域预测。利用CLUSTALW程序对蛋白序列进行比较分析。通过同源重组的方法构建突变株,并对突变株的细胞运动能力、胞外多糖合成、生物膜形成及与豆科宿主的结瘤等表型进行测定。[结果]茎瘤固氮根瘤菌ORS571中一共存在37个GGDEF和EAL结构域蛋白。突变株△4658的运动能力较野生型有下降,但是其胞外多糖合成能力、生物膜形成能力和竞争性结瘤能力较野生型有提高。此外,实验结果表明突变株△4658的胞内c-di-GMP水平高于野生型。突变株△3085和△3226的各种表型与野生型相比没有明显差异。[结论]茎瘤固氮根瘤菌ORS571编码如此大数量的GGDEF和EAL结构域蛋白,表明c-di-GMP可能在其信号转导过程中起到非常重要的作用。同时具有GGDEF和EAL结构域的蛋白AZC_4658对茎瘤固氮根瘤菌ORS571的运动能力、胞外多糖合成、生物膜形成及与宿主的结瘤起到一定的调节作用。     

Role of the GGDEF protein family in Salmonella cellulose biosynthesis and biofilm formation

Salmonella enterica serovar Typhimurium is capable of producing cellulose as the main exopolysaccharide compound of the biofilm matrix. It has been shown for Gluconacetobacter xylinum that cellulose biosynthesis is allosterically regulated by bis-(3',5') cyclic diguanylic acid, whose synthesis/degradation depends on diguanylate cyclase/phosphodiesterase enzymatic activities. A protein domain, named GGDEF, is present in all diguanylate cyclase/phosphodiesterase enzymes that have been studied to date. In this study, we analysed the molecular mechanisms responsible for the failure of Salmonella typhimurium strain SL1344 to form biofilms under different environmental conditions. Using a complementation assay, we were able to identify two genes, which can restore the biofilm defect of SL1344 when expressed from the plasmid pBR328. Based on the observation that one of the genes, STM1987, contains a GGDEF domain, and the other, mlrA, indirectly controls the expression of another GGDEF protein, AdrA, we proceeded on a mutational analysis of the additional GG[DE]EF motif containing proteins of S. typhimurium. Our results demonstrated that MlrA, and thus AdrA, is required for cellulose production and biofilm formation in LB complex medium whereas STM1987 (GGDEF domain containing protein A, gcpA) is critical for biofilm formation in the nutrient-deficient medium, ATM. Insertional inactivation of the other six members of the GGDEF family (gcpB-G) showed that only deletion of yciR (gcpE) affected cellulose production and biofilm formation. However, when provided on plasmid pBR328, most of the members of the GGDEF family showed a strong dominant phenotype able to bypass the need for AdrA and GcpA respectively. Altogether, these results indicate that most GGDEF proteins of S. typhimurium are functionally related, probably by controlling the levels of the same final product (cyclic di-GMP), which include among its regulatory targets the cellulose production and biofilm formation of S. typhimurium.     

Disruption of the copper efflux pump (CopA) of Serratia marcescens ATCC 274 pleiotropically affects copper sensitivity and production of the tripyrrole secondary metabolite, prodigiosin

The prodigiosin biosynthetic gene cluster (pig cluster) of Serratia marcescens ATCC 274 (Sma 274) is flanked by cueR/copA homologues. Inactivation of the copA homologue resulted in an increased sensitivity to copper, confirming that CopA is involved in copper homeostasis in Sma 274. The effect of copper on the biosynthesis of prodigiosin in Sma 274 and the copA mutant strain was investigated. Increased levels of copper were found to reduce prodigiosin production in the wild type Sma 274, but increase production in the copA mutant strain. The physiological implications for CopA mediated prodigiosin production are discussed. We also demonstrate that the gene products of pigB–pigE of Sma 274 are sufficient for the biosynthesis of 2-methyl-3-n-amyl-pyrrole and condensation with 4-methoxy-2,2′-bipyrrole-5-carboxyaldehyde to form prodigiosin, as we have shown for Serratia sp. ATCC 39006.     

Inverse relationship between the flagella formation and prodigiosin synthesis in Serratia marcescens

Treatment by polymyxin B sulfate and ethylenediaminetetraacetate separated a 40 kilodalton (kDa) protein from the nonpigmented Serratia marcescens and even from the nonpigmented bacteria of the pigmented strains, whereas the same treatment separated the 100 kDa protein associated with the pigment formation from the pigmented bacteria. Lysozyme treatment separated the 100 kDa and/or 40 kDa proteins correlated with the pigmented level. The 40 kDa protein was not an outer membrane protein but a flagellin. These results suggest that the flagella formation was inversely related with the pigment formation.     

Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces

The biosynthetic pathway of the red-pigmented antibiotic, prodigiosin, produced by Serratia sp. is known to involve separate pathways for the production of the monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP) and the bipyrrole, 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) which are then coupled in the final condensation step. We have previously reported the cloning, sequencing and heterologous expression of the pig cluster responsible for prodigiosin biosynthesis in two Serratia sp. In this article we report the creation of in-frame deletions or insertions in every biosynthetic gene in the cluster from Serratia sp. ATCC 39006. The biosynthetic intermediates accumulating in each mutant have been analysed by LC-MS, cross-feeding and genetic complementation studies. Based on these results we assign specific roles in the biosynthesis of MBC to the following Pig proteins: PigI, PigG, PigA, PigJ, PigH, PigM, PigF and PigN. We report a novel pathway for the biosynthesis of MAP, involving PigD, PigE and PigB. We also report a new chemical synthesis of MAP and one of its precursors, 3-acetyloctanal. Finally, we identify the condensing enzyme as PigC. We reassess the existing literature and discuss the significance of the results for the biosynthesis of undecylprodigiosin by the Red cluster in Streptomyces coelicolor A3(2).     

Seed traits are pleiotropically regulated by the flowering time gene PERPETUAL FLOWERING 1 (PEP1) in the perennial Arabis alpina

The life cycles of plants are characterized by two major life history transitions—germination and the initiation of flowering—the timing of which are important determinants of fitness. Unlike annuals, which make the transition from the vegetative to reproductive phase only once, perennials iterate reproduction in successive years. The floral repressor PERPETUAL FLOWERING 1 (PEP1), an ortholog of FLOWERING LOCUS C, in the alpine perennial Arabis alpina ensures the continuation of vegetative growth after flowering and thereby restricts the duration of the flowering episode. We performed greenhouse and garden experiments to compare flowering phenology, fecundity and seed traits between A. alpina accessions that have a functional PEP1 allele and flower seasonally and pep1 mutants and accessions that carry lesions in PEP1 and flower perpetually. In the garden, perpetual genotypes flower asynchronously and show higher winter mortality than seasonal ones. PEP1 also pleiotropically regulates seed dormancy and longevity in a way that is functionally divergent from FLC. Seeds from perpetual genotypes have shallow dormancy and reduced longevity regardless of whether they after‐ripened in plants grown in the greenhouse or in the experimental garden. These results suggest that perpetual genotypes have higher mortality during winter but compensate by showing higher seedling establishment. Differences in seed traits between seasonal and perpetual genotypes are also coupled with differences in hormone sensitivity and expression of genes involved in hormonal pathways. Our study highlights the existence of pleiotropic regulation of seed traits by hub developmental regulators such as PEP1, suggesting that seed and flowering traits in perennial plants might be optimized in a coordinated fashion.