Regulation of production of the blue pigment indigoidine by the pseudo γ-butyrolactone receptor FarR2 in Streptomyces lavendulae FRI-5.

The γ-butyrolactone autoregulator signaling cascade is widely distributed among Streptomyces species as an important regulatory system of secondary metabolism. In Streptomyces lavendulae FRI-5, a γ-butyrolactone autoregulator IM-2 and the IM-2 specific receptor FarA control production of the blue pigment indigoidine together with two types of antibiotics: d-cycloserine and the nucleoside antibiotics. Here, we demonstrated by in silico analysis that farR2 (a farA homologue), which is located in a cluster of regulatory genes including farA, belongs to the family of pseudoreceptor regulator genes, and that the expression of farR2 is controlled by the IM-2/FarA regulatory system. Disruption of farR2 resulted in delayed production of indigoidine and in transcriptional derepression of the clustered far regulatory genes. Moreover, FarR2 bound to the FarA-binding sequences in the promoter regions of the regulatory genes that were downregulated by FarR2.

Identification and characterization of target genes of the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius.

The GinI/GinR quorum-sensing system represses oxidative fermentation, including acetic acid and gluconic acid fermentation, as well as antifoam activity in Gluconacetobacter intermedius NCI1051. An 89 aa protein, GinA, whose production is induced by the quorum-sensing system, represses both oxidative fermentation and antifoam activity via a still unknown mechanism, although an OmpA family protein, GmpA, as a target of the GinI/GinR quorum-sensing system via GinA, has been found to repress oxidative fermentation. In this study, four novel GinA-inducible genes (gltA, pdeA, pdeB and nagA) were identified and their involvement in oxidative fermentation and antifoam activity was examined by gene disruption. Disruption of nagA (which encodes a putative N-acetylglucosamine-6-phosphate deacetylase) decreased the growth rate in the exponential growth phase, indicating that nagA was required for the rapid growth of the strain. This unexpected finding revealed a new aspect of the GinI/GinR quorum-sensing system: it accelerates exponential growth by induction of nagA. In contrast, gltA (a putative glycosyltransferase) and pdeA (a putative cyclic-di-GMP phosphodiesterase) were shown to repress oxidative fermentation, including acetic acid and gluconic acid fermentation. gltA was also shown to repress antifoam activity. Disruption of pdeB (a putative phosphodiesterase/diguanylate cyclase) caused no phenotypic changes. Taking our previous results into consideration, these results showed an apparently complex mechanism for repressing oxidative fermentation by the quorum-sensing system; at least three GinA-inducible genes, gltA, pdeA and gmpA, were involved in the repression of oxidative fermentation by the GinI/GinR quorum-sensing system, the most characteristic feature of the acetic acid bacteria.

Identification of genes involved in the butyrolactone autoregulator cascade that modulates secondary metabolism in Streptomyces lavendulae FRI-5.

The gamma-butyrolactone-autoregulator signalling system is widely distributed across many Streptomyces species and it controls secondary metabolism and/or morphological differentiation. IM-2 [(2R,3R,1'R)-2-1'-hydroxybutyl-3-hydroxymethyl-gamma-butanolide] is a gamma-butyrolactone autoregulator which, in Streptomyces lavendulae FRI-5, switches off the production of D-cycloserine, but switches on the production of several nucleoside antibiotics and blue pigment. In the IM-2 system, an IM-2 specific receptor (FarA) plays a critical role in the biosynthetic regulation of these metabolites, including IM-2 itself. Here, we identified five additional regulatory genes in the farA-flanking region and demonstrated that, in addition to farA, at least two more genes (farR1 and farR2) are involved in the IM-2/FarA system as the direct transcriptional target of FarA. The gel-shift assay revealed that FarA was bound to the upstream region of the four genes (including farR1 and farR2) in an IM-2-dependent manner. The FarA-binding sites were localized by DNase I footprinting to 27- to 33-bp palindromic structures, suggesting that FarA-binding sequences consist of two conserved hexamers separated by six nucleotides. Both farR1 and farR2 were transcribed in a growth-dependent manner, and marked expression was induced in the presence of IM-2, whereas transcripts of other two genes were not detected under the cultivation conditions used. The FarA-binding sites of farR1 and far2 overlap the promoter regions, suggesting that FarA represses the transcription of these two genes in the absence of IM-2 by inhibiting RNA polymerase access.

An OmpA family protein, a target of the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius, controls acetic acid fermentation.

Via N-acylhomoserine lactones, the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius NCI1051, a gram-negative acetic acid bacterium, represses acetic acid and gluconic acid fermentation. Two-dimensional polyacrylamide gel electrophoretic analysis of protein profiles of strain NCI1051 and ginI and ginR mutants identified a protein that was produced in response to the GinI/GinR regulatory system. Cloning and nucleotide sequencing of the gene encoding this protein revealed that it encoded an OmpA family protein, named GmpA. gmpA was a member of the gene cluster containing three adjacent homologous genes, gmpA to gmpC, the organization of which appeared to be unique to vinegar producers, including "Gluconacetobacter polyoxogenes." In addition, GmpA was unique among the OmpA family proteins in that its N-terminal membrane domain forming eight antiparallel transmembrane beta-strands contained an extra sequence in one of the surface-exposed loops. Transcriptional analysis showed that only gmpA of the three adjacent gmp genes was activated by the GinI/GinR quorum-sensing system. However, gmpA was not controlled directly by GinR but was controlled by an 89-amino-acid protein, GinA, a target of this quorum-sensing system. A gmpA mutant grew more rapidly in the presence of 2% (vol/vol) ethanol and accumulated acetic acid and gluconic acid in greater final yields than strain NCI1051. Thus, GmpA plays a role in repressing oxidative fermentation, including acetic acid fermentation, which is unique to acetic acid bacteria and allows ATP synthesis via ethanol oxidation. Consistent with the involvement of gmpA in oxidative fermentation, its transcription was also enhanced by ethanol and acetic acid.

Control of acetic acid fermentation by quorum sensing via N-acylhomoserine lactones in Gluconacetobacter intermedius.

A number of gram-negative bacteria regulate gene expression in a cell density-dependent manner by quorum sensing via N-acylhomoserine lactones (AHLs). Gluconacetobacter intermedius NCI1051, a gram-negative acetic acid bacterium, produces three different AHLs, N-decanoyl-l-homoserine lactone, N-dodecanoyl-L-homoserine lactone, and an N-dodecanoyl-L-homoserine lactone with a single unsaturated bond in its acyl chain, as determined by liquid chromatography-tandem mass spectrometry. Two genes encoding an AHL synthase and a cognate regulator were cloned from strain NCI1051 and designated ginI and ginR, respectively. Disruption of ginI or ginR abolished AHL production, indicating that NCI1051 contains a single set of quorum-sensing genes. Transcriptional analysis showed that ginI is activated by GinR, which is consistent with the finding that there is an inverted repeat whose nucleotide sequence is similar to the sequence bound by members of the LuxR family at position -45 with respect to the transcriptional start site of ginI. A single gene, designated ginA, located just downstream of ginI is transcribed by read-through from the GinR-inducible ginI promoter. A ginA mutant, as well as the ginI and ginR mutants, grew more rapidly in medium containing 2% (vol/vol) ethanol and accumulated acetic acid at a higher rate with a greater final yield than parental strain NCI1051. In addition, these mutants produced larger amounts of gluconic acid than the parental strain. These data demonstrate that the GinI/GinR quorum-sensing system in G. intermedius controls the expression of ginA, which in turn represses oxidative fermentation, including acetic acid and gluconic acid fermentation.