Stephanie Grond

Human commensals producing a novel antibiotic impair pathogen colonization

The vast majority of systemic bacterial infections are caused by facultative, often antibiotic-resistant, pathogens colonizing human body surfaces. Nasal carriage of Staphylococcus aureus predisposes to invasive infection, but the mechanisms that permit or interfere with pathogen colonization are largely unknown. Whereas soil microbes are known to compete by production of antibiotics, such processes have rarely been reported for human microbiota. We show that nasal Staphylococcus lugdunensis strains produce lugdunin, a novel thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus, and a rare example of a non-ribosomally synthesized bioactive compound from human-associated bacteria. Lugdunin is bactericidal against major pathogens, effective in animal models, and not prone to causing development of resistance in S. aureus. Notably, human nasal colonization by S. lugdunensis was associated with a significantly reduced S. aureus carriage rate, suggesting that lugdunin or lugdunin-producing commensal bacteria could be valuable for preventing staphylococcal infections. Moreover, human microbiota should be considered as a source for new antibiotics.

Piriformospora indica affects plant growth by auxin production

Piriformospora indica has been shown to improve the growth of many plant species including Arabidopsis thaliana, but the mechanism by which this is achieved is still unclear. Arabidopsis root colonization by P. indica was examined in sterile culture on the medium of Murashige and Skoog. P. indica formed intracellular structures in Arabidopsis root epidermal cells and caused changes in root growth, leading to stunted and highly branched root systems. This effect was because of a diffusible factor and could be mimicked by IAA. In addition, P. indica was shown to produce IAA in liquid culture. We suggest that auxin production affecting root growth is responsible for, or at least contributes to, the beneficial effect of P. indica on its host plants.

Metagenome-Derived Clones Encoding Two Novel Lactonase Family Proteins Involved in Biofilm Inhibition in Pseudomonas aeruginosa†

ABSTRACT Here we report the isolation and characterization of three metagenome-derived clones that interfere with bacterial quorum sensing and degrade N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL). By using a traI-lacZ gene fusion, the metagenome-derived clones were identified from a soil DNA library and analyzed. The open reading frames linked to the 3-oxo-C8-HSL-degrading activities were designated bpiB01, bpiB04, and bpiB07. While the BpiB07 protein was similar to a known lactonase, no significant similarities were observed for the BpiB01 and BpiB04 proteins or the deduced amino acid sequences. High-performance liquid chromatography-mass spectrometry analyses confirmed that the identified genes encode novel lactone-hydrolyzing enzymes. The original metagenome-derived clones were expressed in Pseudomonas aeruginosa and employed in motility and biofilm assays. All clones were able to reproducibly inhibit motility in P. aeruginosa. Furthermore, these genes clearly inhibited biofilm formation in P. aeruginosa when expressed in P. aeruginosa PAO1. Thus, this is the first study in which metagenome-derived proteins have been expressed in P. aeruginosa to successfully inhibit biofilm formation.

Molecular Analysis of the Kirromycin Biosynthetic Gene Cluster Revealed β-Alanine as Precursor of the Pyridone Moiety

Kirromycin is a complex linear polyketide that acts as a protein biosynthesis inhibitor by binding to the bacterial elongation factor Tu. The kirromycin biosynthetic gene cluster was isolated from the producer, Streptomyces collinus Tü 365, and confirmed by targeted disruption of essential biosynthesis genes. Kirromycin is synthesized by a large hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) encoded by the genes kirAI-kirAVI. This complex involves some very unusual features, including the absence of internal acyltransferase (AT) domains in KirAI-KirAV, multiple split-ups of PKS modules on separate genes, and swapping in the domain organization. Interestingly, one PKS enzyme, KirAVI, contains internal AT domains. Based on in silico analysis, a route to pyridone formation involving PKS and NRPS steps was postulated. This hypothesis was experimentally proven by feeding studies with [U-13C3(15)N]beta-alanine and NMR and MS analyses of the isolated pure kirromycin.

A Novel Metagenomic Short-Chain Dehydrogenase/ Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies.

The COP9 signalosome mediates transcriptional and metabolic response to hormones, oxidative stress protection and cell wall rearrangement during fungal development.

The COP9 signalosome complex (CSN) is a crucial regulator of ubiquitin ligases. Defects in CSN result in embryonic impairment and death in higher eukaryotes, whereas the filamentous fungus Aspergillus nidulans survives without CSN, but is unable to complete sexual development. We investigated overall impact of CSN activity on A. nidulans cells by combined transcriptome, proteome and metabolome analysis. Absence of csn5/csnE affects transcription of at least 15% of genes during development, including numerous oxidoreductases. csnE deletion leads to changes in the fungal proteome indicating impaired redox regulation and hypersensitivity to oxidative stress. CSN promotes the formation of asexual spores by regulating developmental hormones produced by PpoA and PpoC dioxygenases. We identify more than 100 metabolites, including orsellinic acid derivatives, accumulating preferentially in the csnE mutant. We also show that CSN is required to activate glucanases and other cell wall recycling enzymes during development. These findings suggest a dual role for CSN during development: it is required early for protection against oxidative stress and hormone regulation and is later essential for control of the secondary metabolism and cell wall rearrangement.

Involvement of Multiple Loci in Quorum Quenching of Autoinducer I Molecules in the Nitrogen-Fixing Symbiont Rhizobium (Sinorhizobium) sp. Strain NGR234

ABSTRACT Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. Since we have previously described the complete genome sequence of NGR234, we now report on a genome-wide functional analysis of the genes and enzymes involved in autoinducer I hydrolysis in this microbe. Altogether we identified five cosmid clones that repeatedly gave a positive result in our function-based approach for the detection of autoinducer I hydrolase genes. Of these five cosmid clones, two were located on pNGR234b and three were on cNGR234. Subcloning and in vitro mutagenesis in combination with BLAST analyses identified the corresponding open reading frames (ORFs) of all cosmid clones: dlhR, qsdR1, qsdR2, aldR, and hitR-hydR. Analyses of recombinant DlhR and QsdR1 proteins by using high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrate that these enzymes function as acyl homoserine lactone (AHL) lactonases. Furthermore, we showed that these enzymes inhibited biofilm formation and other quorum-sensing-dependent processes in Pseudomonas aeruginosa, Chromobacterium violaceum, and Agrobacterium tumefaciens. Finally, our experimental data suggest that competitive colonization of roots in the rhizospheres of cowpea plants is affected by DlhR and QsdR1.

Highly active ansamitocin derivatives: mutasynthesis using an AHBA-blocked mutant.

Despite the fact that natural products represent a very important source of drugs in several therapeutic fields such as antiinfective agents and cancer therapy, the development of natural product based drugs is often hampered by their structural complexity. This fact precludes facile total synthetic access to analogues or the development of natural product libraries. Therefore, semisynthetic and biotechnological approaches are commonly pursued in pharmaceutical research and development. A very interesting strategy combines chemical semisynthesis with biosynthesis using genetically engineered microorganisms, a technique termed mutational biosynthesis or mutasynthesis (Figure 1). Recently, mutasynthesis has expe-

Archazolid A Binds to the Equatorial Region of the c-Ring of the Vacuolar H+-ATPase

The macrolactone archazolid is a novel, highly specific V-ATPase inhibitor with an IC50 value in the low nanomolar range. The binding site of archazolid is presumed to overlap with the binding site of the established plecomacrolide V-ATPase inhibitors bafilomycin and concanamycin in subunit c of the membrane-integral VO complex. Using a semi-synthetic derivative of archazolid for photoaffinity labeling of the V1VO holoenzyme we confirmed binding of archazolid to the VO subunit c. For the plecomacrolide binding site a model has been published based on mutagenesis studies of the c subunit of Neurospora crassa, revealing 11 amino acids that are part of the binding pocket at the interface of two adjacent c subunits (Bowman, B. J., McCall, M. E., Baertsch, R., and Bowman, E. J. (2006) J. Biol. Chem. 281, 31885–31893). To investigate the contribution of these amino acids to the binding of archazolid, we established in Saccharomyces cerevisiae mutations that in N. crassa had changed the IC50 value for bafilomycin 10-fold or more and showed that out of the amino acids forming the plecomacrolide binding pocket only one amino acid (tyrosine 142) contributes to the binding of archazolid. Using a fluorescent derivative of N,N′-dicyclohexylcarbodiimide, we found that the binding site for archazolid comprises the essential glutamate within helix 4 of subunit c. In conclusion the archazolid binding site resides within the equatorial region of the VO rotor subunit c. This hypothesis was supported by an additional subset of mutations within helix 4 that revealed that leucine 144 plays a role in archazolid binding.

BpiB05, a novel metagenome-derived hydrolase acting on N-acylhomoserine lactones

The N-acyl-homoserine lactones (N-AHLs) play an important role in bacterial cell-cell signaling. Up to date, however, only a few different experimentally proven classes of N-AHL ring-cleaving enzymes are known. Here we report on the isolation and biochemical characterization of a novel hydrolase derived from the soil metagenome and acting on N-AHLs. The identified protein designated BpiB05 is weakly similar to hypothetical proteins from Bacteroides fragilis, the draft genomes of two Burkholderia species as well as a marine metagenomic ORF but is otherwise not similar to any known protein. BpiB05 was overexpressed in Escherichia coli as a 10× His-tagged fusion protein. The recombinant protein revealed a molecular weight of about 70kDa and was tested for its quorum quenching (QQ) activities using a lacZ-bioassay. Additional HPLC-MS analyses confirmed the lactonolytic activity of the purified protein in the presence of Ca²⁺. Further tests suggested that BpiB05 strongly reduces motility in Pseudomonas aeruginosa, pyocyanin synthesis and biofilm formation in this microbe. Because BpiB05 is not distantly related to any of the currently known hydrolases it forms probably a novel group within the growing number of proteins acting on N-AHLs.