Interactions between bacteria and fungi cover a wide range of incentives, mechanisms and outcomes. The genus Collimonas consists of soil bacteria that are known for their antifungal activity and ability to grow at the expense of living fungi. In non-contact confrontation assays with the fungus Aspergillus niger, Collimonas fungivorans showed accumulation of biomass concomitant with inhibition of hyphal spread. Through microarray analysis of bacterial and fungal mRNA from the confrontation arena, we gained new insights into the mechanisms underlying the fungistatic effect and mycophagous phenotype of collimonads. Collimonas responded to the fungus by activating genes for the utilization of fungal-derived compounds and for production of a putative antifungal compound. In A. niger, differentially expressed genes included those involved in lipid and cell wall metabolism and cell defense, which correlated well with the hyphal deformations that were observed microscopically. Transcriptional profiles revealed distress in both partners: downregulation of ribosomal proteins and upregulation of mobile genetic elements in the bacteria and expression of endoplasmic reticulum stress and conidia-related genes in the fungus. Both partners experienced nitrogen shortage in each other's presence. Overall, our results indicate that the Collimonas/ Aspergillus interaction is a complex interplay between trophism, antibiosis and competition for nutrients.

Two formate dehydrogenases (CO 2 -reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionateoxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H 2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO 2 -reduction rates, and low K m values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 UAEmg )1 and a K m for formate of 0.04 mM were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 UAEmg )1 and a K m of 0.01 mM for formate with benzyl viologen as electron acceptor. The specific CO 2 -reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 UAEmg )1 and 89 UAEmg )1 , respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 ± 3, 56 ± 2 and 19 ± 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 ± 3 kDa and a 33 ± 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected.

Summary The antifungal activity of bacteria from the genus Collimonas has been well documented, but the chemistry and gene functions that underlie this phenotype are still poorly understood. Screening of a random plasposon insertion library of Collimonas fungivorans Ter331 for loss‐of‐function mutants revealed the importance of gene cluster K, which is annotated to code for the biosynthesis of a secondary metabolite and which features genes for fatty acid desaturases and polyketide synthases. Mutants in gene cluster K had lost the ability to inhibit hyphal growth of the fungus Aspergillus niger and were no longer able to produce and secrete several metabolites that after extraction and partial purification from wildtype strain Ter331 were shown to share a putative ene‐triyne moiety. Some but not all of these metabolites were able to inhibit growth of A. niger, indicating functional variation within this group of Collimonas‐produced polyyne‐like ‘collimomycins’. Polymerase chain reaction analysis of isolates representing different Collimonas species indicated that the possession of cluster K genes correlated positively with antifungal ability, further strengthening the notion that this cluster is involved in collimomycin production. We discuss our findings in the context of other bacterially produced polyynes and the potential use of collimomycins for the control of harmful fungi.

Plasmid pTer331 from the bacterium Collimonas fungivorans Ter331 is a new member of the pIPO2/pSB102 family of environmental plasmids. The 40 457-bp sequence of pTer331 codes for 44 putative ORFs, most of which represent genes involved in replication, partitioning and transfer of the plasmid. We confirmed that pTer331 is stably maintained in its native host. Deletion analysis identified a mini-replicon capable of replicating autonomously in Escherichia coli and Pseudomonas putida. Furthermore, plasmid pTer331 was able to mobilize and retromobilize IncQ plasmid pSM1890 at typical rates of 10(-4) and 10(-8), respectively. Analysis of the 91% DNA sequence identity between pTer331 and pIPO2 revealed functional conservation of coding sequences, the deletion of DNA fragments flanked by short direct repeats (DR), and sequence preservation of long DRs. In addition, we experimentally established that pTer331 has no obvious contribution in several of the phenotypes that are characteristic of its host C. fungivorans Ter331, including the ability to efficiently colonize plant roots. Based on our findings, we hypothesize that cryptic plasmids such as pTer331 and pIPO2 might not confer an individual advantage to bacteria, but, due to their broad-host-range and ability to retromobilize, benefit bacterial populations by accelerating the intracommunal dissemination of the mobile gene pool.

Summary Collimonas is a genus of soil bacteria comprising three recognized species: C. fungivorans, C. pratensis and C. arenae. Collimonads share the ability to degrade chitin (chitinolysis), feed on living fungal hyphae (mycophagy), and dissolve minerals (weathering), but vary in their inhibition of fungi (fungistasis). To better understand this phenotypic variability, we analysed the genomic content of four strains representing three Collimonas species (Ter14, Ter6, Ter91 and Ter10) by hybridization to a microarray based on reference strain C. fungivorans Ter331. The analysis revealed genes unique to strain Ter331 (e.g. those on the extrachromosomal element pTer331) and genes present in some but not all of the tested strains. Among the latter were several candidates that may contribute to fungistasis, including genes for the production and secretion of antifungals. We hypothesize that differential possession of these genes underlies the specialization of Collimonas strains towards different fungal hosts. We identified a set of 136 genes that were common in all tested Collimonas strains, but absent from the genomes of three other members of the family Oxalobacteraceae. Predicted products of these ‘Collimonas core’ genes include lytic, secreted enzymes such as chitinases, peptidases, nucleases and phosphatases with a putative role in mycophagy and weathering.

Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions. Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate. Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and A1-pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0.93 nkat (mg protein)-l and 4 3 nkat (mg protein)'l, respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified t o homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF2OO. The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28.9 kDa as calculated from the sequence. Apparent Km values for PCA and NADH were 019 mM and 0025 mM, respectively. The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline pH values above 8 and higher substrate concentrations (apparent Km values: 1-55 mM for proline and 10-5 mM for NAD at pH 10-0). Studies with growing cells of C. sticklandii and [15N]proline revealed that proline is not oxidized in vivo because 15N was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction. The proC gene encoding PCA reductase of C. sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli. The enzyme exhibited high homologies to PCA reductases from different sources. Thus, C. sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.

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