Marie-Joelle Virolle

The polyphosphate kinase plays a negative role in the control of antibiotic production in Streptomyces lividans

The polyphosphate kinase gene (ppk) from Streptomyces lividans, which encodes a 774‐amino‐acid protein (86.4 kDa) showing extensive homology to other bacterial polyphosphate kinases, was cloned by polymerase chain reaction (PCR) using oligonucleotides derived from the putative ppk gene from the closely related species, Streptomyces coelicolor. In vitro, the purified Ppk was shown to be able to synthesize the polyphosphate [poly(P)] from ATP (forward reaction) as well as to regenerate ATP from the poly(P) in the presence of an excess of ADP (reverse reaction). In conditions of poly(P) synthesis, a phosphoenzyme intermediate was detected, indicating an autophosphorylation of the enzyme in the presence of ATP. The ppk gene was shown to be transcribed as a monocistronic mRNA from a unique promoter. Its transcription was only detectable during the late stages of growth in liquid minimal medium. A mutant strain interrupted for ppk was characterized by increased production of the antibiotic actinorhodin on rich R2YE solid medium (0.37 mM KH2PO4 added). This production was enhanced on the same medium with no KH2PO4 added but was completely abolished by the addition of 1.48 mM KH2PO4. In the ppk mutant strain, this increased production correlated with enhanced transcription of actII‐ORF4 encoding the specific activator of the actinorhodin pathway. In that strain, the transcription of redD and cdaR, encoding the specific activators of the undecylprodigiosin and calcium‐dependent antibiotic biosynthetic pathways, respectively, was also increased but to a lesser extent. The enhanced expression of these regulators did not seem to be related to increased relA‐dependent ppGpp synthesis, as no obvious increase in relA expression was observed in the ppk mutant strain. These results suggested that the negative regulatory effect exerted by Ppk on antibiotic biosynthesis was most probably caused by the repression exerted by the endogenous Pi, resulting from the hydrolysis of the poly(P) synthesized by Ppk, on the expression of the specific activators of the antibiotic biosynthetic pathways.

Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the gamma phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

Sequences involved in growth-phase-dependent expression and glucose repression of a Streptomyces α-amylase gene

In glycerol-grown, but not in glucose-grown cultures, expression in Streptomyces lividans TK24 of the cloned alpha-amylase gene (aml) of Streptomyces limosus is switched on toward the end of exponential growth. During this period, aml expression is further inducible by maltotriose. We showed that a 378 bp fragment, extending from position -204 through to +174 (relative to the transcriptional start site), included cis-acting sequences involved in aml regulation. When this fragment was present on a multicopy plasmid, the growth-phase-dependent aml expression conferred by a DNA fragment cloned on a compatible low-copy-number plasmid was greatly enhanced, as if negative regulators were being titrated. A study of the regulation of aml expression in variants with deletions in the aml promoter region indicated that a direct repeat (DR) between positions -124 and -106 (relative to the transcriptional start site) and an inverted repeat (IR) between positions +9 and +24 were good candidates for secondary and primary operator sites, respectively. Deletion of a 29 bp fragment containing the IR rendered aml expression partly growth-phase-independent, resistant to glucose repression, and insensitive to maltotriose induction.

Repression of Antibiotic Production and Sporulation in Streptomyces coelicolor by Overexpression of a TetR Family Transcriptional Regulator

ABSTRACT The overexpression of a regulatory gene of the TetR family (SCO3201) originating either from Streptomyces lividans or from Streptomyces coelicolor was shown to strongly repress antibiotic production (calcium-dependent antibiotic [CDA], undecylprodigiosin [RED], and actinorhodin [ACT]) of S. coelicolor and of the ppk mutant strain of S. lividans. Curiously, the overexpression of this gene also had a strong inhibitory effect on the sporulation process of S. coelicolor but not on that of S. lividans. SCO3201 was shown to negatively regulate its own transcription, and its DNA binding motif was found to overlap its −35 promoter sequence. The interruption of this gene in S. lividans or S. coelicolor did not lead to any obvious phenotypes, indicating that when overexpressed SCO3201 likely controls the expression of target genes of other TetR regulators involved in the regulation of the metabolic and morphological differentiation process in S. coelicolor. The direct and functional interaction of SCO3201 with the promoter region of scbA, a gene under the positive control of the TetR-like regulator, ScbR, was indeed demonstrated by in vitro as well as in vivo approaches.

Comparative Proteomic Analysis of Streptomyces lividans Wild-Type and ppk Mutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

ABSTRACT Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.

Extracellular sugar phosphates are assimilated by Streptomyces in a PhoP-dependent manner

Filamentous microorganisms of the bacterial genus Streptomyces have a complex life cycle that includes physiological and morphological differentiations. It is now fairly well accepted that lysis of Streptomyces vegetative mycelium induced by programmed cell death (PCD) provides the required nutritive sources for the bacterium to erect spore-forming aerial hyphae. However, little is known regarding cellular compounds released during PCD and the contribution of these molecules to the feeding of surviving cells in order to allow them to reach the late stages of the developmental program. In this work we assessed the effect of extracellular sugar phosphates (that are likely to be released in the environment upon cell lysis) on the differentiation processes. We demonstrated that the supply of phosphorylated sugars, under inorganic phosphate limitation, delays the occurrence of the second round of PCD, blocks streptomycetes life cycle at the vegetative state and inhibits antibiotic production. The mechanism by which sugar phosphates affect development was shown to involve genes of the Pho regulon that are under the positive control of the two component system PhoR/PhoP. Indeed, the inactivation of the response regulator phoP of Streptomyces lividans prevented the ‘sugar phosphate effect’ whereas the S. lividansppk (polyphosphate kinase) deletion mutant, known to overexpress the Pho regulon, presented an enhanced response to phosphorylated sugars.

Combining infrared and mode synthesizing atomic force microscopy: Application to the study of lipid vesicles inside Streptomyces bacteria

We propose a new analytical approach combining vibrational spectroscopy and acoustic tomography for the detection and characterization of vesicles inside Streptomyces bacteria. Using atomic force microscopy and infrared spectroscopy (AFM-IR), we detect the presence of triglyceride vesicles. Their sizes in depth are measured with high accuracy using mode synthesizing atomic force microscopy (MS-AFM). We conducted a comparative study of AFM-IR and MS-AFM, and highlighted the advantages of the coupling of these techniques in having a full characterization (chemical, topographical, and volumetric) of a biological sample. With these complementary techniques, a complete access to the vesicle size distribution has been achieved with an accuracy of less than 50 nm. A 3D reconstruction of bacteria showing the in-depth distribution of vesicles is given to underline the great potential of the acoustic method.

Community-level physiological profiling analyses show potential to identify the copiotrophic bacteria present in soil environments

Community-level physiological profiling (CLPP) analyses from very diverse environments are frequently used with the aim of characterizing the metabolic versatility of whole environmental bacterial communities. While the limitations of the methodology for the characterization of whole communities are well known, we propose that CLPP combined with high-throughput sequencing and qPCR can be utilized to identify the copiotrophic, fast-growing fraction of the bacterial community of soil environments, where oligotrophic taxa are usually dominant. In the present work we have used this approach to analyze samples of litter and soil from a coniferous forest in the Czech Republic using BIOLOG GN2 plates. Monosaccharides and amino acids were utilized significantly faster than other C substrates, such as organic acids, in both litter and soil samples. Bacterial biodiversity in CLPP wells was significantly lower than in the original community, independently of the carbon source. Bacterial communities became highly enriched in taxa that typically showed low abundance in the original soil, belonging mostly to the Gammaproteobacteria and the genus Pseudomonas, indicating that the copiotrophic strains, favoured by the high nutrient content, are rare in forest litter and soil. In contrast, taxa abundant in the original samples were rarely found to grow at sufficient rates under the CLPP conditions. Our results show that CLPP is useful to detect copiotrophic bacteria from the soil environments and that bacterial growth is substrate specific.

Disruption of sblA in Streptomyces lividans permits expression of a heterologous alpha-amylase gene in the presence of glucose.

In a transposition mutant of Streptomyces lividans TK24, the usually glucose-repressible expression of a heterologous alpha-amylase gene (aml) became resistant to glucose repression. The transposon had inserted into an ORF called sblA which encodes a 274 aa product sharing significant sequence similarities with various phosphatases that act on small phosphorylated substrates. sblA was transcribed as a monocistronic mRNA and its transcription was enhanced at the transition phase. Because its transcriptional and putative translational start points coincide, sblA is likely to be translated in the absence of a conventional RBS. The sblA-disrupted mutant is characterized by early growth arrest in glucose-grown cultures and by partial relief of glucose repression of aml expression.In a transposition mutant of Streptomyces lividans TK24, the usually glucose-repressible expression of a heterologous α-amylase gene (aml) became resistant to glucose repression. The transposon had inserted into an ORF called sblA which encodes a 274 aa product sharing significant sequence similarities with various phosphatases that act on small phosphorylated substrates. sblA was transcribed as a monocistronic mRNA and its transcription was enhanced at the transition phase. Because its transcriptional and putative translational start points coincide, sblA is likely to be translated in the absence of a conventional RBS. The sblA-disrupted mutant is characterized by early growth arrest in glucose-grown cultures and by partial relief of glucose repression of aml expression.

Pathway-Consensus Approach to Metabolic Network Reconstruction for Pseudomonas putida KT2440 by Systematic Comparison of Published Models

Over 100 genome-scale metabolic networks (GSMNs) have been published in recent years and widely used for phenotype prediction and pathway design. However, GSMNs for a specific organism reconstructed by different research groups usually produce inconsistent simulation results, which makes it difficult to use the GSMNs for precise optimal pathway design. Therefore, it is necessary to compare and identify the discrepancies among networks and build a consensus metabolic network for an organism. Here we proposed a process for systematic comparison of metabolic networks at pathway level. We compared four published GSMNs of Pseudomonas putida KT2440 and identified the discrepancies leading to inconsistent pathway calculation results. The mistakes in the models were corrected based on information from literature so that all the calculated synthesis and uptake pathways were the same. Subsequently we built a pathway-consensus model and then further updated it with the latest genome annotation information to obtain modelPpuQY1140 for P. putida KT2440, which includes 1140 genes, 1171 reactions and 1104 metabolites. We found that even small errors in a GSMN could have great impacts on the calculated optimal pathways and thus may lead to incorrect pathway design strategies. Careful investigation of the calculated pathways during the metabolic network reconstruction process is essential for building proper GSMNs for pathway design.