7659546
The PhoBR two-component system regulates antibiotic biosynthesis in Serratia in response to phosphate
BackgroundSecondary metabolism in Serratia sp. ATCC 39006 (Serratia 39006) is controlled via a complex network of regulators, including a LuxIR-type (SmaIR) quorum sensing (QS) system. Here we investigate the molecular mechanism by which phosphate limitation controls biosynthesis of two antibiotic secondary metabolites, prodigiosin and carbapenem, in Serratia 39006.ResultsWe demonstrate that a mutation in the high affinity phosphate transporter pstSCAB-phoU, believed to mimic low phosphate conditions, causes upregulation of secondary metabolism and QS in Serratia 39006, via the PhoBR two-component system. Phosphate limitation also activated secondary metabolism and QS in Serratia 39006. In addition, a pstS mutation resulted in upregulation of rap. Rap, a putative SlyA/MarR-family transcriptional regulator, shares similarity with the global regulator RovA (regulator of virulence) from Yersina spp. and is an activator of secondary metabolism in Serratia 39006. We demonstrate that expression of rap, pigA-O (encoding the prodigiosin biosynthetic operon) and smaI are controlled via PhoBR in Serratia 39006.ConclusionPhosphate limitation regulates secondary metabolism in Serratia 39006 via multiple inter-linked pathways, incorporating transcriptional control mediated by three important global regulators, PhoB, SmaR and Rap.
25765508
Characterization of PitA and PitB fromEscherichia coli
ABSTRACT Escherichia coli contains two major systems for transporting inorganic phosphate (Pi). The low-affinity Pi transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external Pi concentrations by the phoregulon and is an ABC transporter. We isolated a third putative Pi transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional Pi transporter that may be repressed at low Pi levels by the pho regulon. While apitB+ cosmid clone allowed growth on medium containing 500 μM Pi, E. coli with wild-type genomic pitB (pitAΔpstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitBΔpstC345 triple mutant. The mutation ΔpstC345 constitutively activates thepho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting thephoB-phoR operon allowed thepitB+pitAΔpstC345 strain to utilize Pi, with Pi uptake rates significantly higher than background levels. In addition, the apparent Kmof PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitAmutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.
35122942
Two systems for the uptake of phosphate in Escherichia coli
Mutants of Escherichia coli K-12 were constructed such that each possessed one single major system for phosphate transport. A comparison of these strains showed that one of the systems (PIT) was fully constitutive, required no binding protein, and operated in spheroplasts. It permitted the complete exchange of intracellular phosphate with extracellular phosphate (or arsenate) and was completely inhibited by uncouplers. The other system, PST, was repressible by phosphate concentrations above 1 mM, required the phosphate-binding protein for full activity, and did not operate in spheroplasts. It catalyzed very little exchange between internal and external phosphate and was resistant to uncouplers. The maximal velocities attained by the two systems were approximately the same, but the affinity for phosphate in the PST system was greater by two orders of magnitude. In strains in which both systems were fully operative, the initial rates of uptake was nearly additive, and the systems appeared to interact with a common intracellular phosphate pool.
2971172
Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli.
The pstA(=phoT), pstB and phoU genes are situated at 84 minutes on the Escherichia coli genetic map. All of them are involved in the negative regulation of the phosphate regulon, and all of them except for phoU are required for the binding-protein-mediated, highly specific phosphate transport. We have determined the DNA sequence of about 4 X 10(3) bases of chromosomal segment containing these genes. Four translational reading frames (TRFs) were detected in the region. We attempted to assign the TRFs to the mutant alleles. Plasmids were constructed so that each contained only one of the TRFs, downstream from the lac promoter, to be used for the complementation tests. By this test, TRF-2, TRF-3 and TRF-4 were identified with the pstA(=phoT), pstB and phoU genes, respectively. Alkaline phosphatase-constitutive mutations of the two strains in our collection were complemented by the plasmid with the TRF-1 region. Therefore, we propose to designate the allele phoW. The order of the genes in this region has been established to be phoS-phoW-pstA(=phoT)-pstB-phoU counterclockwise on the E. coli genetic map.
20550418
Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships
The DNA nucleotide sequence of four genes for the phosphate-specific transport system of Escherichia coli is reported. Along with the DNA sequence for the phoS gene reported previously (Surin et al., J. Bacteriol. 157:772-778, 1984; Magota et al., J. Bacteriol. 157:909-917, 1984), this study completes the nucleotide sequence of the phosphate-specific transport region. The complete sequence (including phoS) contains five open reading frames oriented in the same direction, each preceded by a putative ribosome-binding site near the presumed translation initiation codon ATG. The complete sequence is transcribed counterclockwise, in the order phoS pstC pstA pstB phoU. Genetic complementation shows that of the four open reading frames in the new sequence, three correspond to known mutant alleles; the fourth, which was designated pstC, has not been described before and could not be related to any known mutant allele. We have confirmed that pstA was allelic to phoT32. The pstC, pstB, and phoU gene products were identified as peripheral membrane proteins. The pstA gene product appears to be an integral membrane protein.
8163403
Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices.
The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.
42866546
Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli
Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems. Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg [dry weight]-1min-1) and will grow in the presence of arsenate in the medium. However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg [dry weight]-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium. Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide. Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells. Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity. In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system).
23672862
Regulation of the phosphate regulon of Escherichia coli: analysis of mutant phoB and phoR genes causing different phenotypes
The phoB gene product of Escherichia coli is the transcriptional activator for the genes in the phosphate regulon as well as for phoB itself, all of which are induced by phosphate starvation. The phoR gene product modulates PhoB function in response to the phosphate concentrations in the medium. We quantitatively compared the levels of expression of the phoA, phoB, phoE, and pstS genes in several phoB mutants with different phenotypes by constructing operon fusions of these genes with the gene for chloramphenicol acetyltransferase. Although all the phoB mutants examined had little activator function for phoA, three among the four mutants showed various levels of the activator function for phoB, pstS, and phoE. To study the functional motifs of the PhoB and PhoR proteins, we cloned and sequenced the four classical phoB and six phoR mutant genes. All of the phoB mutations and one of the phoR mutations were missense mutations, and most of the altered amino acids were in the highly conserved amino acids among the regulatory proteins homologous to PhoB or PhoR protein, such as the OmpR, SfrA, and VirG proteins or the EnvZ, CpxA, and VirA proteins. The other five phoR mutations were nonsense mutations.
6545909
Regulation of the phosphate regulon of Escherichia coli: Characterization of the promoter of the pstS gene
SummaryThe pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the-10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the-10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gelmobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.
22264169
Regulation of the phosphate regulon of Escherichia coli. Activation of pstS transcription by PhoB protein in vitro.
Expression of the genes in the phosphate regulon, including the pstS (phoS) and phoB genes, is positively regulated by PhoB protein when phosphate is limited. We purified PhoB protein from overproducing cells and studied its interaction with the pstS gene. It binds specifically to the DNA fragment containing the promoter region of pstS. The transcription initiation site of the gene in vivo was identified by S1 nuclease mapping and primer-extension experiments. In-vitro transcription of pstS was activated by the PhoB protein, and the initiation site of transcription agreed with the in-vivo initiation site. Activation of in-vitro transcription by PhoB protein required both the normal sigma factor (sigma 70) and core RNA polymerase. PhoB protein binding sites on the promoter regions of pstS and phoB were determined by footprinting experiments with DNase I and a methylating agent. In both cases the protein binds to the pho box, the concensus sequence shared by regulatory regions of genes in the phosphate regulon. Our findings indicate that PhoB protein recognizes and binds to the pho box and activates transcription of the genes in the phosphate regulon.
39135059
Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12.
phoB encodes a positive regulator for a number of the genes belonging to the phosphate regulon of Escherichia coli, including phoA, phoS, phoE and ugpAB. We have determined the nucleotide sequence of the chromosomal segment containing the promoter and the coding region of the phoB gene. The sequence data combined with the known amino-terminal amino acid sequence of a PhoB-LacZ hybrid protein suggest that the PhoB protein consists of 228 amino acid residues with a Mr of 26,302. In the regulatory region of the gene, a consensus nucleotide sequence shared by the regulatory regions of the phoA, phoS and phoE genes, which we name the "phosphate box", was found. Since these genes are positively regulated by the product of phoB, this suggests that transcription of the phoB gene is also regulated positively by its own product. Extensive homology was found in the amino acid sequences of the products of phoB, ompR and dye, all of which are positive regulatory genes for a number of genes coding for envelope proteins. This implies that these genes were originally duplications of a protogene that evolved to have divergent but related functions.
25642553
The signal‐transduction network for Pho regulation in Bacillus subtilis
Depletion of nutrients, including phosphate, is a stress often encountered by a bacterial cell, and results in slowed growth, marking the cessation of exponential growth. Genes that are transcriptionally activated during phosphate starvation have been used to examine the signal‐transduction mechanisms governing the Pho regulon in Bacillus subtilis. Alkaline phosphatase, the traditional reporter protein for Pho regulation in prokaryotes, is encoded by a multigene family in B. subtilis. Characterization of the alkaline phosphatase family was a breakthrough in the study of regulation of the Pho regulon, especially the discovery of promoter elements exclusively responsive to phosphate‐starvation regulation. Current data suggest that at least three two‐component signal‐transduction systems interact, forming a regulatory network that controls the phosphate‐deficiency response in B. subtilis. The interconnected pathways involve the PhoP–PhoR system, whose primary role is to mediate the phosphate‐deficiency response; the Spo0 phosphorelay required for the initiation of sporulation; and a newly discovered signal‐transduction system, ResD–ResE, which also has a role in respiratory regulation during late growth. Parallel pathways positively regulate the Pho response via PhoP–PhoR. One pathway includes the ResD–ResE system, while the other involves a transition‐state regulator, AbrB. The Spo0 system represses the Pho response by negatively regulating both pathways. This review will discuss how the characterization of the APase multigene family made possible studies which show that the Pho regulon in B. subtilis is regulated by the integrated action of the Res, Pho and Spo signal‐transduction systems.
2663252
Target genes and structure of the direct repeats in the DNA-binding sequences of the response regulator PhoP in Streptomyces coelicolor
Expression of genes belonging to the pho regulon in Streptomyces coelicolor is positively regulated (as shown by comparing the wild-type and a ΔphoP mutant) by binding of the response regulator PhoP to 11-nt direct repeats (DRus). These sequences have been found in over 100 genes of Streptomyces coelicolor; 20 of them were cloned and the binding of PhoPDBD to most of their promoters has been shown by electrophoretic mobility shift assays. Deletion experiments showed that at least two DRus are required for proper binding of PhoPDBD. Deletion of 1 nt leaving a 10-nt direct repeat reduced drastically binding of PhoPDBD. Three different types of operators have been identified. Complex operators (class III) contain up to six DRus, some of them with poor conservation of the 11-nt consensus sequence, which however were protected by PhoPDBD in footprinting analyses. A cooperative binding of PhoPDBD molecules initiated at conserved core DRus appears to be the mechanism involved in binding of several PhoPDBD monomers to those complex operators. The information theory-based model that incorporates the positive or negative contribution to the binding of PhoPDBD of adjacent sequences has been used to deduce the structure of PHO boxes and the relevance of each DRu.
25236528
Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon
The phosphate regulon is negatively regulated by the PstSCAB transporter and PhoU protein by a mechanism that may involve protein-protein interaction(s) between them and the Pi sensor protein, PhoR. In order to study such presumed interaction(s), mutants with defined deletions of the pstSCAB-phoU operon were made. This was done by construction of M13 recombinant phage carrying these mutations and by recombination of them onto the chromosome by using a rep host (which cannot replicate M13) for allele replacement. These mutants were used to show that delta (pstSCAB-phoU) and delta (pstB-phoU) mutations abolished Pi uptake by the PstSCAB transporter, as expected, and that delta phoU mutations had no effect on uptake. Unexpectedly, delta phoU mutations had a severe growth defect, and this growth defect was (largely) alleviated by a compensatory mutation in the pstSCAB genes or in the phoBR operon, whose gene products positively regulate expression of the pstSCAB-phoU operon. Because delta phoU mutants that synthesize a functional PstSCAB transporter constitutively grew extremely poorly, the PhoU protein must have a new role, in addition to its role as a negative regulator. A role for the PhoU protein in intracellular Pi metabolism is proposed. Further, our results contradict those of M. Muda, N. N. Rao, and A. Torriani (J. Bacteriol. 174:8057-8064, 1992), who reported that the PhoU protein was required for Pi uptake.
43107410
Fine tuning the specificity of the periplasmic phosphate transport receptor. Site-directed mutagenesis, ligand binding, and crystallographic studies.
Phosphorous, primarily in the form of phosphate, is a critical nutrient for the life of a cell. We have previously determined the 1.7-A resolution structure of the phosphate-binding protein, an initial receptor for the high-affinity phosphate active transport system or permease in Escherichia coli (Luecke, H., and Quiocho, F.A. (1990) Nature 347, 402-406). This structure is the first to reveal the key role of hydrogen bonding interactions in conferring the high specificity of the permease, a specificity also shared by other phosphate transport systems. Both monobasic and dibasic phosphates are recognized by the phosphate-binding protein with Asp56 playing a key role. Here we report site-directed mutagenesis, ligand binding, and crystallographic studies of the binding protein which show that introduction of one additional Asp by mutagenesis of the Thr141 in the ligand-binding site restricts binding to only the monobasic phosphate.
22188351
The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation.
The prodigiosin biosynthesis gene cluster (pig cluster) from two strains of Serratia (S. marcescens ATCC 274 and Serratia sp. ATCC 39006) has been cloned, sequenced and expressed in heterologous hosts. Sequence analysis of the respective pig clusters revealed 14 ORFs in S. marcescens ATCC 274 and 15 ORFs in Serratia sp. ATCC 39006. In each Serratia species, predicted gene products showed similarity to polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs) and the Red proteins of Streptomyces coelicolor A3(2). Comparisons between the two Serratia pig clusters and the red cluster from Str. coelicolor A3(2) revealed some important differences. A modified scheme for the biosynthesis of prodigiosin, based on the pathway recently suggested for the synthesis of undecylprodigiosin, is proposed. The distribution of the pig cluster within several Serratia sp. isolates is demonstrated and the presence of cryptic clusters in some strains shown. The pig cluster of Serratia marcescens ATCC 274 is flanked by cueR and copA homologues and this configuration is demonstrated in several S. marcescens strains, whilst these genes are contiguous in strains lacking the pig cluster.
24846876
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.
6330005
Synthesis, proton-affinity and anti-cancer properties of the prodigiosin-group natural products.
The prodigiosin-group natural products are a family of tripyrrole red-pigments that are produced by microorganisms such as Streptomyces and Serratia and contain a common 4-methoxy-2,2'-bipyrrole ring system. They were first isolated in 1929 and studied as antibiotic and cytotoxic agents in the 1960s, but were not developed clinically due to their high systemic toxicity. However, during the past decade some prodigiosins have shown potentially useful immunosuppressive activity when administered at doses that are not toxic. They have also been found to exhibit selective cytotoxicity against melanoma and liver cancer cells. These results have fueled various studies on the biological mechanisms of the prodigiosins and it has now been established that they inhibit phosphorylation and activation of JAK-3, a cytoplasmic tyrosine kinase associated with the cell surface receptor component called common gamma-chain. They also uncouple lysosomal vacuolar-type ATPases through promotion of H(+)/Cl(-) symport and facilitate oxidative double-strand DNA cleavage in the presence of copper. A simple and elegant synthesis of the prodigiosins has also been developed, which has allowed a number of the natural prodigiosins and synthetic analogues to be prepared. These studies have served to renew interest in the prodigiosin-group natural products. In this review the recent advances on the synthesis, proton-affinity and biological activities of the prodigiosins are discussed. With regard to their anti-cancer properties, particular attention is given to their ability to facilitate oxidative DNA damage, which provides a rationale for the cytotoxic properties of the prodigiosin-group natural products.
5688610
The prodigiosins, proapoptotic drugs with anticancer properties.
The family of natural red pigments, called prodigiosins (PGs), characterised by a common pyrrolylpyrromethene skeleton, are produced by various bacteria. Some members have immunosuppressive properties and apoptotic effects in vitro and they have also displayed antitumour activity in vivo. Understanding the mechanism of action of PGs is essential for drug development and will require the identification and characterisation of their still unidentified cell target. Four possible mechanisms of action have been suggested for these molecules: (i) PGs as pH modulators; (ii) PGs as cell cycle inhibitors; (iii) PGs as DNA cleavage agents; (iv) PGs as mitogen-activated protein kinase regulators. Here, we review the pharmacological activity of PG and related compounds, including novel synthetic PG derivatives with lower toxicity and discuss the mechanisms of action and the molecular targets of those molecules. The results reported in this review suggest that PGs are a new class of anticancer drugs, which hold out considerable promise for the Pharmacological Industry.
22936030
Anticancer and immunosuppressive properties of bacterial prodiginines.
Bacterial prodiginines are a family of red-pigmented, tripyrrolic compounds that display numerous biological activities, including antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive and anticancer properties. Recently, significant progress has been made in understanding the biosynthesis and regulation of bacterial prodiginines. An understanding of the biosynthesis of prodiginines will allow engineering of bacterial strains capable of synthesizing novel prodiginines through rational design and mutasynthesis experiments. Bacterial prodiginines and synthetic derivatives are effective proapoptotic agents with multiple cellular targets, and they are active against numerous cancer cell lines, including multidrug-resistant cells, with little or no toxicity towards normal cell lines. A synthetic derivative, GX15-070 (Obatoclax), developed through structure-activity relationship studies of the pyrrolic ring A of GX15, is in multiple Phase I and II clinical trials in both single and dual-agent studies to treat different types of cancer. Therefore, prodiginines have real therapeutic potential in the clinic.
24720158
The biosynthetic implications of acetate and glutamate incorporation into (3R,5R)-carbapenam-3-carboxylic acid and (5R)-carbapen-2-em-3-carboxylic acid by Serratia sp.
Two new beta-lactams have been isolated from strains of Serratia and Erwinia sp. and identified as (3R,5R)- and (3S,5R)-carbapenam-3-carboxylic acid. These novel carbapenams lack antibacterial activity, are resistant to both beta-lactamases I and II from Bacillus cereus and are not detected by the lactamase induction assay. Radiolabelled and stable isotope experiments have established that both metabolites together with the antibiotic 5R-carbapenem-3-carboxylic acid are glutamate and acetate derived. A number of possible pathways for the biosynthesis of these compounds as well as their relationship to the more complex members of the carbapenem family of beta-lactam antibiotics are discussed.
32820780
SQ 27,860, a simple carbapenem produced by species of Serratia and Erwinia.
In our continued search for the production of β-lactam-containing molecules from bacteria, we report the isolation and structure determination of a simple carbapenem SQ 27, 860, produced by species of Serratia and Erwinia. The antibiotic is highly unstable and isolation was achieved through the p-nitrobenzyl ester.
24442087
Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control
Serratia sp. ATCC 39006 produces the carbapenem antibiotic, carbapen‐2‐em‐3‐carboxylic acid and the red pigment, prodigiosin. We have previously reported the characterization of a gene, carR, controlling production of carbapenem in this strain. We now describe further characterization of the carR locus to locate the genes encoding carbapenem biosynthetic and resistance functions. A novel family of diverse proteins showing sequence similarity to the C‐terminal domain of CarF (required for carbapenem resistance) is described. We also report the isolation of the locus involved in the biosynthesis of the red pigment, prodigiosin. A cosmid containing ≈ 35 kb of the Serratia chromosome encodes synthesis of the pigment in the heterologous host, Erwinia carotovora, demonstrating, for the first time, that the complete prodigiosin biosynthetic gene cluster had been cloned and functionally expressed. We report the isolation of a third locus in Serratia, containing convergently transcribed genes, smaI and smaR, encoding LuxI and LuxR homologues respectively. SmaI directs the synthesis of N‐acyl homoserine lactones involved in the quorum sensing process. We demonstrate that biosynthesis of the two secondary metabolites, carbapenem antibiotic and prodigiosin pigment, is under pheromone‐mediated transcriptional regulation in this bacterium. Finally, we describe a new prodigiosin‐based bioassay for detection of some N‐acyl homoserine lactones.
24989838
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).
11649828
The biosynthesis and regulation of bacterial prodiginines
The red-pigmented prodiginines are bioactive secondary metabolites produced by both Gram-negative and Gram-positive bacteria. Recently, these tripyrrole molecules have received renewed attention owing to reported immunosuppressive and anticancer properties. The enzymes involved in the biosynthetic pathways for the production of two of these molecules, prodigiosin and undecylprodigiosin, are now known. However, the biochemistry of some of the reactions is still poorly understood. The physiology and regulation of prodiginine production in Serratia and Streptomyces are now well understood, although the biological role of these pigments in the producer organisms remains unclear. However, research into the biology of pigment production will stimulate interest in the bioengineering of strains to synthesize useful prodiginine derivatives.
25706497
Biosynthesis of tripyrrole and β‐lactam secondary metabolites in Serratia: integration of quorum sensing with multiple new regulatory components in the control of prodigiosin and carbapenem antibiotic production
Serratia sp. ATCC 39006 (39006) uses a complex hierarchical regulatory network allowing multiple inputs to be assessed before genes involved in secondary metabolite biosynthesis are expressed. This taxonomically ill‐defined Serratia sp. produces a carbapenem antibiotic (Car; a β‐lactam) and a red pigmented antibiotic, prodigiosin (Pig; a tripyrrole), which are controlled by the smaIR quorum sensing (QS) locus. SmaR is a repressor of Pig and Car when levels of N‐acyl‐ l‐homoserine lactones, produced by SmaI, are low. In this study, we demonstrate direct DNA binding of purified SmaR to the promoter of the Car biosynthetic genes and abolition of this binding by the QS ligand. We have also identified multiple new secondary metabolite regulators. QS controls production of secondary metabolites, at least in part, by modulating transcription of three genes encoding regulatory proteins, including a putative response regulator of the GacAS two‐component signalling system family, a novel putative adenylate cyclase and Rap (regulator of antibiotic and pigment). Mutations in another gene encoding a novel predicted global regulator, pigP, are highly pleiotropic; PigP has a significant ‘master’ regulatory role in 39006 where it controls the transcription of six other regulators. The PigP protein and its homologues define a new family of regulators and are predicted to bind DNA via a helix‐turn‐helix domain. There are regulatory overlaps between the QS and PigP regulons that enable the information from different physiological cues to be funnelled into the control of secondary metabolite production.
36525626
Phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum‐sensing‐dependent and ‐independent pathways
Serratia sp. ATCC 39006 produces two secondary metabolite antibiotics, 1‐carbapen‐2‐em‐3‐carboxylic acid (Car) and the red pigment, prodigiosin (Pig). We have previously reported that production of Pig and Car is controlled by N‐acyl homoserine lactone (N‐AHL) quorum sensing, with synthesis of N‐AHLs directed by the LuxI homologue SmaI, and is also regulated by Rap, a member of the SlyA family. We now describe further characterization of the SmaI quorum‐sensing system and its connection with other regulatory mechanisms. We show that the genes responsible for biosynthesis of Pig, pigA–O, are transcribed as a single polycistronic message in an N‐AHL‐dependent manner. The smaR gene, transcribed convergently with smaI and predicted to encode the LuxR homologue partner of SmaI, was shown to possess a negative regulatory function, which is uncommon among the LuxR‐type transcriptional regulators. SmaR represses transcription of both the pig and car gene clusters in the absence of N‐AHLs. Specifically, we show that SmaIR exerts its effect on car gene expression via transcriptional control of carR, encoding a pheromone‐independent LuxR homologue. Transcriptional activation of the pig and car gene clusters also requires a functional Rap protein, but Rap dependency can be bypassed by secondary mutations. Transduction of these suppressor mutations into wild‐type backgrounds confers a hyper‐Pig phenotype. Multiple mutations cluster in a region upstream of the pigA gene, suggesting this region may represent a repressor target site. Two mutations mapped to genes encoding pstS and pstA homologues, which are parts of a high‐affinity phosphate transport system (Pst) in Escherichia coli. Disruption of pstS mimicked phosphate limitation and caused concomitant hyper‐production of Pig and Car, which was mediated, in part, through increased transcription of the smaI gene. The Pst and SmaIR systems define distinct, yet overlapping, regulatory circuits which form part of a complex regulatory network controlling the production of secondary metabolites in Serratia ATCC 39006.
17609826
Quorum sensing in Serratia.
Many bacteria use cell-cell communication to monitor their population density, synchronize their behaviour and socially interact. This communication results in a coordinated gene regulation and is generally called quorum sensing. In gram-negative bacteria, the most common quorum signal molecules are acylated homoserine lactones (AHLs), although other low-molecular-mass signalling molecules have been described such as Autoinducer-2 (AI-2). The phenotypes that are regulated in Serratia species by means of AHLs are remarkably diverse and of profound biological and ecological significance, and often interconnected with other global regulators. Furthermore, AHL- and AI-2-mediated systems (less profoundly studied) are continuously being discovered and explored in Serratia spp., many having interesting twists on the basic theme. Therefore, this review will highlight the current known quorum sensing systems in Serratia spp., including the important nosocomial pathogen Serratia marcescens.
22889999
The Rap and Hor proteins of Erwinia, Serratia and Yersinia: a novel subgroup in a growing superfamily of proteins regulating diverse physiological processes in bacterial pathogens
The enteric bacterium Serratia marcescens is an opportunistic human pathogen. The strain ATCC39006 makes the red pigment, prodigiosin (Pig), and the β‐lactam antibiotic carbapenem (Car). Mutants were isolated that were concomitantly defective for Pig and Car production. These mutants were found to have a mutation in the rap gene (regulation of antibiotic and pigment). Sequence analysis of the rap gene revealed a predicted protein product showing strong homology to SlyA, originally thought to be a haemolytic virulence determinant in Salmonella typhimurium. Homologues of rap were detected in several bacterial genera, including Salmonella, Yersinia, Enterobacter, and species of the plant pathogen, Erwinia. The Erwinia horEr (homologue of rap) and the Yersinia horYe genes were also found to be very similar to rap and slyA. Marker exchange mutagenesis of horEr revealed that it encoded a regulatory protein controlling the production of antibiotic and exoenzyme virulence determinants in the phytopathogen, Erwinia carotovora subspecies carotovora. We have shown that these new homologues of SlyA form a highly conserved subgroup of a growing superfamily of bacterial regulatory proteins controlling diverse physiological processes in human, animal and plant pathogens.
11145219
RovA, a global regulator of Yersinia pestis, specifically required for bubonic plague
The pathogenic species of Yersinia contain the transcriptional regulator RovA. In Yersinia pseudotuberculosis and Yersinia enterocolitica, RovA regulates expression of the invasion factor invasin (inv), which mediates translocation across the intestinal epithelium. A Y. enterocolitica rovA mutant has a significant decrease in virulence by LD50 analysis and an altered rate of dissemination compared with either wild type or an inv mutant, suggesting that RovA regulates multiple virulence factors. Here, we show the involvement of RovA in the virulence of Yersinia pestis, which naturally lacks a functional inv gene. A Y. pestis ΔrovA mutant is attenuated ≈80-fold by LD50 and is defective in dissemination/colonization of spleens and lungs after s.c. inoculation. However, the ΔrovA mutant is only slightly attenuated when given via an intranasal or i.p. route, indicating a more important role for RovA in bubonic plague than pneumonic plague or systemic infection. Microarray analysis was used to define the RovA regulon. The psa locus was among the most highly down-regulated loci in the ΔrovA mutant. A ΔpsaA mutant had a significant dissemination defect after s.c. infection but only slight attenuation by the pneumonic-disease model, closely mimicking the virulence defect seen with the ΔrovA mutant. DNA-binding studies revealed that RovA specifically interacts with the psaE and psaA promoter regions, indicating a direct role for RovA in regulating this locus. Thus, RovA appears to be a global transcription factor in Y. pestis and, through its regulatory influence on genes such as psaEFABC, contributes to the virulence of Y. pestis.
39408334
Invasin and beyond: regulation of Yersinia virulence by RovA.
RovA, a member of the MarR/SlyA family of winged-helix transcription factors, regulates expression of invasin, the major adhesion and invasion factor in Yersinia enterocolitica and Yersinia pseudotuberculosis. Disruption of rovA increases the LD(50) of the organism when inoculated using the oral route. However, when administered by intraperitoneal injection only a slight difference in LD(50) between mutant and wild-type organisms is apparent. The study of RovA and the genes it regulates provides a unique opportunity to gain insight into the initial stages of a Yersinia infection.
23697651
Environmental control of invasin expression in Yersinia pseudotuberculosis is mediated by regulation of RovA, a transcriptional activator of the SlyA/Hor family
Invasin is the primary invasive factor of Yersinia pseudotuberculosis that allows efficient internalization into eukaryotic cells. We investigated invasin expression and found that the inv gene is regulated in response to a variety of environmental signals, such as temperature, growth phase, nutrients, osmolarity and pH, and requires the product of rovA, a member of the SlyA/Hor transcriptional activator family. The rovA gene was found by a genetic complementation strategy that restores temperature regulation of an unexpressed inv–phoA fusion in Escherichia coli K‐12. RovA plays a role in the invasion of Y. pseudotuberculosis into mammalian cells and mediates the regulation of invasin in response to all environmental signals analysed. Deletion analysis of the inv promoter region revealed a DNA segment extending 207 bp upstream of the transcriptional start site, which is required for maximal RovA‐induced inv transcription. Gel retardation assays showed that RovA interacts preferentially with this promoter fragment and suggested two potential RovA binding sites. Studies with chromosomal gene fusions also demonstrated that rovA follows the same pattern of regulation as invasin, indicating that environmental control of inv expression is mainly mediated by the control of RovA synthesis. Furthermore, we showed that a rovA–lacZ fusion is only slightly expressed in a rovA mutant strain, indicating that a positive autoregulatory mechanism is also involved in rovA expression.
25143552
Virulence and Prodigiosin Antibiotic Biosynthesis in Serratia Are Regulated Pleiotropically by the GGDEF/EAL Domain Protein, PigX
ABSTRACT Gram-negative bacteria of the genus Serratia are opportunistic human, plant, and insect pathogens. Serratia sp. strain ATCC 39006 secretes pectinases and cellulases and produces the secondary metabolites carbapenem and prodigiosin. Mutation of a gene (pigX) resulted in an extremely pleiotropic phenotype: prodigiosin antibiotic biosynthesis, plant virulence, and pectinase production were all elevated. PigX controlled secondary metabolism by repressing the transcription of the target prodigiosin biosynthetic operon (pigA-pigO). The transcriptional start site of pigX was determined, and pigX expression occurred in parallel with Pig production. Detailed quantitative intracellular proteome analyses enabled the identification of numerous downstream targets of PigX, including OpgG, mutation of which reduced the production of the plant cell wall-degrading enzymes and virulence. The highly pleiotropic PigX regulator contains GGDEF and EAL domains with noncanonical motifs and is predicted to be membrane associated. Genetic evidence suggests that PigX might function as a cyclic dimeric GMP phosphodiesterase. This is the first characterization of a GGDEF and EAL domain protein in Serratia and the first example of the regulation of antibiotic production by a GGDEF/EAL domain protein.
24915960
PigZ, a TetR/AcrR family repressor, modulates secondary metabolism via the expression of a putative four‐component resistance‐nodulation‐cell‐division efflux pump, ZrpADBC, in Serratia sp. ATCC 39006
The Gram‐negative enterobacterium, Serratia sp. ATCC 39006 synthesizes several secondary metabolites, including prodigiosin (Pig) and a carbapenem antibiotic (Car). A complex hierarchical network of regulatory proteins control Pig and Car production. In this study we characterize a TetR family regulator, PigZ, which represses transcription of a divergently transcribed putative resistance‐nodulation‐cell‐division (RND) efflux pump, encoded by zrp (PigZ repressed pump) ADBC, via direct binding to the zrpA‐pigZ intergenic region. Unusually, this putative RND pump contains two predicted membrane fusion proteins (MFPs), ZrpA and ZrpD. A mutation in pigZ resulted in multiple phenotypic changes, including exoenzyme production, motility and differential regulation of Pig and Car production. A polar suppressor mutation, within zrpA, restored all tested phenotypes to parental strain levels, indicating that the changes observed are due to the increase in expression of ZrpADBC in the absence of the repressor, PigZ. Genomic deletions of zrpA and zrpD indicate that the MFP ZrpD, but not ZrpA, is essential for activity of the putative pump. Bioinformatic analysis revealed that putative RND efflux pumps encoding two MFP components are not uncommon, particularly among plant‐associated, Gram‐negative bacteria. In addition, based on phylogenetic analysis, we propose that these pairs of MFPs consist of two distinct subtypes.
24020369
Substrate analysis and molecular cloning of the extracellular alkaline phosphatase of Streptomyces griseus.
Streptomyces species secrete large amounts of alkaline phosphatase (AP) enzymes that have not been characterized so far. An AP has been purified to homogeneity from cultures of Streptomyces griseus IMRU 3570. The enzyme has a monomer size of 62 kDa and is processed in the culture to a 33 kDa protein as shown by immunoblotting. The enzyme was purified by ammonium sulfate precipitation, CM-Sephadex cationic exchange, chromatofocusing and HPLC Sphaerogel 3000SW filtration. The pure enzyme uses a variety of organic phosphorylated compounds as substrates. The N-terminal end of the mature protein was found to be RLREDPFTLGVASGDPHP. The gene phoA has been cloned using as probe an oligomer based on the N-terminal sequence of the S. griseus AP. phoA encodes a protein of 62678 Da with low homology to the AP of Escherichia coli. The phoA gene was found to be homologous to three alkaline-phosphatase-encoding genes previously identified in the Streptomyces coelicolor genome. On the basis of the optimal pH, substrate specificity and differences in amino acid sequence of motifs defining the active centre of APs, the S. griseus AP uses a wide range of organic phosphate substrates and is different from the phosphatases of Gram-negative bacteria.
26972298
The Acid-Inducible asr Gene inEscherichia coli: Transcriptional Control by thephoBR Operon
ABSTRACT Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, ∼450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. Theasr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encodedasr::lacZ fusion under a nativeasr promoter was reduced ∼15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of aphoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Δ(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoRregulatory system to sense an external acidity and regulate transcription of the asr gene.
7121534
The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis.
Bacterial pathogens regulate virulence factor gene expression coordinately in response to environmental stimuli, including nutrient starvation. The phosphate (Pho) regulon plays a key role in phosphate homeostasis. It is controlled by the PhoR/PhoB two-component regulatory system. PhoR is an integral membrane signaling histidine kinase that, through an interaction with the ABC-type phosphate-specific transport (Pst) system and a protein called PhoU, somehow senses environmental inorganic phosphate (P(i)) levels. Under conditions of P(i) limitation (or in the absence of a Pst component or PhoU), PhoR activates its partner response regulator PhoB by phosphorylation, which, in turn, up- or down-regulates target genes. Single-cell profiling of PhoB activation has shown recently that Pho regulon gene expression exhibits a stochastic, "all-or-none" behavior. Recent studies have also shown that the Pho regulon plays a role in the virulence of several bacteria. Here, we present a comprehensive overview of the role of the Pho regulon in bacterial virulence. The Pho regulon is clearly not a simple regulatory circuit for controlling phosphate homeostasis; it is part of a complex network important for both bacterial virulence and stress response.
30475776
Phosphate Control of the Biosynthesis of Antibiotics and Other Secondary Metabolites Is Mediated by the PhoR-PhoP System: an Unfinished Story
The biosynthesis of many different types of antibiotics and other secondary metabolites is regulated by phosphate. Production of these valuable compounds occurs only under phosphate-limiting nutritional conditions. In a few cases, there is evidence showing that the negative phosphate control is exerted at the transcriptional level. Recently, it was shown that phosphate control of antibiotic biosynthesis in Streptomyces lividans and Streptomyces coelicolor is mediated by the two-component PhoR-PhoP system that also controls the alkaline phosphatase gene (phoA). The PhoR protein is a standard membrane sensor kinase, whereas PhoP is a member of the DNA-binding response regulators. In Escherichia coli and Bacillus subtilis, the phosphorylated PhoP protein (PhoP∼P) activates, in response to phosphate starvation, expression of the pho regulon genes by binding to consensus phosphate boxes in the promoter regions (PHO boxes). Expression of phoA in S. lividans is induced by PhoP∼P, and mutants lacking phoP (or phoR and phoP) do not form PhoA. These mutants overproduce large amounts of actinorhodin and undecylprodigiosin. No consensus PHO boxes occur in the upstream region of phosphate-regulated secondary metabolism genes. However, pathway-specific activator proteins (ActII-open reading frame 4 [ORF4], RedD, CcaR, and DnrI) are known to bind to these regions. In S. coelicolor, actII-orf4 is positively regulated by the AfsS protein, which, in turn, is induced by the phosphorylated AfsR protein. It is likely that the PhoR-PhoP system exerts its action on actinorhodin and undecylprodigiosin by a cascade mechanism mediated by AfsR and AfsS. Directed phoR-phoP gene disruption will be very useful for the construction of tailored phosphate-deregulated strains overproducing valuable secondary metabolites.
24376715
The two-component PhoR-PhoP system controls both primary metabolism and secondary metabolite biosynthesis in Streptomyces lividans
The biosynthesis of most secondary metabolites in different bacteria is strongly depressed by inorganic phosphate. The two-component phoR-phoP system of Streptomyces lividans has been cloned and characterized. PhoR showed all of the characteristics of the membrane-bound sensor proteins, whereas PhoP is a member of the DNA-binding OmpR family. Deletion mutants lacking phoP or phoR-phoP, were unable to grow in minimal medium at low phosphate concentration (10 μM). Growth was fully restored by complementation with the phoR-phoP genes. Both S. lividans ΔphoP and ΔphoR-phoP deletion mutants were unable to synthesize extracellular alkaline phosphatase (AP) as shown by immunodetection with anti-AP antibodies and by enzymatic analysis, suggesting that the PhoR-PhoP system is required for expression of the AP gene (phoA). Synthesis of AP was restored by complementation of the deletion mutants with phoR-phoP. The biosynthesis of two secondary metabolites, actinorhodin and undecylprodigiosin, was significantly increased in both solid and liquid medium in the ΔphoP or ΔphoR-phoP deletion mutants. Negative phosphate control of both secondary metabolites was restored by complementation with the phoR-phoP cluster. These results prove that expression of both phoA and genes implicated in the biosynthesis of secondary metabolites in S. lividans is regulated by a mechanism involving the two-component PhoR-PhoP system.
24273695
The evolutionary role of secondary metabolites--a review.
It is argued that organisms have evolved the ability to biosynthesise secondary metabolites ('natural products') due to the selectional advantages they obtain as a result of the functions of the compounds. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes in Streptomyces, is interpreted in terms of the defense roles of antibiotics. The clustering together of antibiotic biosynthesis, regulation, and resistance genes, and in particular the staggering complexity shown in the case of the gene cluster for erythromycin A biosynthesis, implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature.
34118919
Secondary metabolism, inventive evolution and biochemical diversity--a review.
Evidence now being obtained through nucleotide (nt) sequence analysis supports the concept that secondary metabolism has arisen by modification of existing primary metabolic reactions. Although amino acid sequence identity deduced from nt sequences of genes encoding proteins from related primary and secondary metabolic pathways is sufficient to indicate a common ancestry, the match is often better when genes in different rather than in the same species are compared. The information so far available suggests that gene transfer between organisms has been an important factor in the evolution of secondary metabolism. Many secondary pathways may be of relatively ancient origin and they may have arisen only infrequently. Much subsequent elaboration of the pathways has probably taken place after their acquisition by other species and so has been influenced by a variety of selective conditions. The characteristic diversity of secondary metabolites and their functions can be accounted for by the random manner in which the pathways initially evolved and have subsequently been exploited.
8406430
Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria
Abstract. A highly efficient method of transposon mutagenesis was developed for genetic analysis of Xanthobacter autotrophicus Py2. The method makes use of a transposon delivery vector that encodes a hyperactive Tn5 transposase that is 1,000-fold more active than the wild-type transposase. In this construct, the transposase is expressed from the promoter of the tetA gene of plasmid RP4, which is functional in a wide variety of organisms. The transposon itself contains a kanamycin resistance gene as a selectable marker and the origin of replication from plasmid R6K to facilitate subsequent cloning of the resulting insertion site. To test the effectiveness of this method, mutants unable to produce the characteristic yellow pigment (zeaxanthin dirhamnoside) of X. autotrophicus Py2 were isolated and analyzed. Transposon insertions were obtained at high frequency: approximately 1×10–3 per recipient cell. Among these, pigment mutants were observed at a frequency of approximately 10–3. Such mutants were found to have transposon insertions in genes homologous to known carotenoid biosynthetic genes previously characterized in other pigmented bacteria. Mutants were also isolated in Pseudomonasstutzeri and in an Alcaligenesfaecalis, demonstrating the effectiveness of the method in diverse Proteobacteria. Preliminary results from other laboratories have confirmed the effectiveness of this method in additional phylogenetically diverse species.
30677138
Transport of basic amino acids by the dinitrogen-fixing cyanobacterium Anabaena PCC 7120.
Two transport systems for L-arginine were evident in Anabaena sp. strain PCC 7120: a high-affinity one (Km, 1.7 microM) that accumulated arginine within the cells through an energy-requiring process and another one that exhibited low affinity for L-arginine (Km, 0.75 mM) and was unable to accumulate the substrate. Both systems were inhibited by L-canavanine, L-lysine, and L-ornithine. Two systems were also evident for L-lysine uptake (Km, 1.9 and 110 microM, respectively). After selection for resistance to canavanine or hydroxylysine, independent mutants were isolated which were impaired in the high-affinity uptake of arginine and lysine. A common permease appears, therefore, to be involved in the high-affinity transport of these basic amino acids. Both the high- and the low-affinity systems can contribute to the growth of Anabaena sp. on L-arginine. However, arginine did not effectively repress either nitrogenase or nitrate reductase.
36623406
N-(3-oxohexanoyl)-L-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora.
Erwinia carotovora A.T.C.C. 39048 produces the antibiotic 1-carbapen-2-em-3-carboxylic acid. A number of mutants with a carbapenem-non-producing phenotype were selected as part of an investigation into the molecular and genetic basis of carbapenem biosynthesis. Cross-feeding studies revealed that the mutants fell into two discrete groups. Group 1 mutants were found to secrete a diffusible low-molecular-mass compound which restored carbapenem production in group 2 mutants. This compound was isolated from the spent culture supernatant of a group 1 mutant using solvent extraction, hydrophobic-interaction chromatography and silica-gel chromatography, and finally purified by reverse-phase semipreparative h.p.l.c. M.s. and n.m.r. spectroscopy revealed that the compound was N-(3-oxohexanoyl)homoserine lactone. Both D- and L-isomers were synthesized, and subsequent analysis by c.d. established that the natural product has the L-configuration. Although carbapenem production was restored by both isomers, dose-response curves indicated that the L-isomer has greater activity, with an induction threshold of about 0.5 micrograms/ml. N-(3-Oxohexanoyl)-L-homoserine lactone is, therefore, an autoregulator of carbapenem biosynthesis rather than a biosynthetic intermediate. This compound is already known for its role in autoinduction of bioluminescence in the marine bacterium Vibrio fischeri. It is also structurally-related to the A- and I-factors which are known to regulate production of antibiotics in some Streptomyces species. Its association in this work with the regulation of carbapenem biosynthesis implies a broader role for autoregulator-controlled gene expression in prokaryotes.
11876379
Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria
A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.
9456596
A GntR family transcriptional regulator (PigT) controls gluconate-mediated repression and defines a new, independent pathway for regulation of the tripyrrole antibiotic, prodigiosin, in Serratia.
Biosynthesis of the red, tripyrrole antibiotic prodigiosin (Pig) by Serratia sp. ATCC 39006 (39006) is controlled by a complex regulatory network involving an N-acyl homoserine lactone (N-AHL) quorum-sensing system, at least two separate two-component signal transduction systems and a multitude of other regulators. In this study, a new transcriptional activator, PigT, and a physiological cue (gluconate), which are involved in an independent pathway controlling Pig biosynthesis, have been characterized. PigT, a GntR homologue, activates transcription of the pigA-O biosynthetic operon in the absence of gluconate. However, addition of gluconate to the growth medium of 39006 repressed transcription of pigA-O, via a PigT-dependent mechanism, resulting in a decrease in Pig production. Finally, expression of the pigT transcript was shown to be maximal in exponential phase, preceding the onset of Pig production. This work expands our understanding of both the physiological and genetic factors that impinge on the biosynthesis of the secondary metabolite Pig in 39006.
85658872
Molecular Cloning: A Laboratory Manual
Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
41779374
Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages.
Abstract φ80 transducing phages for the proC † , phoA and phoB genes of Escherichia coli have been obtained. Two mutants have been isolated, in which the brnQ, phoA, proC, phoB (and possibly phoR ) genes have been deleted. Derivatives of a phoA, phoB deletion strain which are lysogenic for a φ80 phoA transducing phage make only very low levels of alkaline phosphatase activity. These results are in agreement with a positive control mechanism for the regulation of alkaline phosphatase synthesis.