Beauveria is a globally distributed genus of soil-borne entomopathogenic hyphomycetes of interest as a model system for the study of entomopathogenesis and the biological control of pest insects. Species recognition in Beauveria is difficult due to a lack of taxonomically informative morphology. This has impeded assessment of species diversity in this genus and investigation of their natural history. A gene-genealogical approach was used to investigate molecular phylogenetic diversity of Beauveria and several presumptively related Cordyceps species. Analyses were based on nuclear ribosomal internal transcribed spacer (ITS) and elongation factor 1-alpha (EF1-alpha) sequences for 86 exemplar isolates from diverse geographic origins, habitats and insect hosts. Phylogenetic trees were inferred using maximum parsimony and Bayesian likelihood methods. Six well supported clades within Beauveria, provisionally designated A-F, were resolved in the EF1-alpha and combined gene phylogenies. Beauveria bassiana, a ubiquitous species that is characterized morphologically by globose to subglobose conidia, was determined to be non-monophyletic and consists of two unrelated lineages, clades A and C. Clade A is globally distributed and includes the Asian teleomorph Cordyceps staphylinidaecola and its probable synonym C. bassiana. All isolates contained in Clade C are anamorphic and originate from Europe and North America. Clade B includes isolates of B. brongniartii, a Eurasian species complex characterized by ellipsoidal conidia. Clade D includes B. caledonica and B. vermiconia, which produce cylindrical and comma-shaped conidia, respectively. Clade E, from Asia, includes Beauveria anamorphs and a Cordyceps teleomorph that both produce ellipsoidal conidia. Clade F, the basal branch in the Beauveria phylogeny includes the South American species B. amorpha, which produces cylindrical conidia. Lineage diversity detected within clades A, B and C suggests that prevailing morphological species concepts underestimate species diversity within these groups. Continental endemism of lineages in B. bassiana s.l. (clades A and C) indicates that isolation by distance has been an important factor in the evolutionary diversification of these clades. Permutation tests indicate that host association is essentially random in both B. bassiana s.l. clades A and C, supporting past assumptions that this species is not host specific. In contrast, isolates in clades B and D occurred primarily on coleopteran hosts, although sampling in these clades was insufficient to assess host affliation at lower taxonomic ranks. The phylogenetic placement of Cordyceps staphylinidaecola/bassiana, and C. scarabaeicola within Beauveria corroborates prior reports of these anamorph-teleomorph connections. These results establish a phylogenetic framework for further taxonomic, phylogenetic and comparative biological investigations of Beauveria and their corresponding Cordyceps teleomorphs.

Beauveria is a globally distributed genus of soil-borne entomopathogenic hyphomycetes of interest as a model system for the study of entomopathogenesis and the biological control of pest insects. Species recognition in Beauveria is difficult due to a lack of taxonomically informative morphology. This has impeded assessment of species diversity in this genus and investigation of their natural history. A gene-genealogical approach was used to investigate molecular phylogenetic diversity of Beauveria and several presumptively related Cordyceps species. Analyses were based on nuclear ribosomal internal transcribed spacer (ITS) and elongation factor 1-alpha (EF1-alpha) sequences for 86 exemplar isolates from diverse geographic origins, habitats and insect hosts. Phylogenetic trees were inferred using maximum parsimony and Bayesian likelihood methods. Six well supported clades within Beauveria, provisionally designated A-F, were resolved in the EF1-alpha and combined gene phylogenies. Beauveria bassiana, a ubiquitous species that is characterized morphologically by globose to subglobose conidia, was determined to be non-monophyletic and consists of two unrelated lineages, clades A and C. Clade A is globally distributed and includes the Asian teleomorph Cordyceps staphylinidaecola and its probable synonym C. bassiana. All isolates contained in Clade C are anamorphic and originate from Europe and North America. Clade B includes isolates of B. brongniartii, a Eurasian species complex characterized by ellipsoidal conidia. Clade D includes B. caledonica and B. vermiconia, which produce cylindrical and comma-shaped conidia, respectively. Clade E, from Asia, includes Beauveria anamorphs and a Cordyceps teleomorph that both produce ellipsoidal conidia. Clade F, the basal branch in the Beauveria phylogeny includes the South American species B. amorpha, which produces cylindrical conidia. Lineage diversity detected within clades A, B and C suggests that prevailing morphological species concepts underestimate species diversity within these groups. Continental endemism of lineages in B. bassiana s.l. (clades A and C) indicates that isolation by distance has been an important factor in the evolutionary diversification of these clades. Permutation tests indicate that host association is essentially random in both B. bassiana s.l. clades A and C, supporting past assumptions that this species is not host specific. In contrast, isolates in clades B and D occurred primarily on coleopteran hosts, although sampling in these clades was insufficient to assess host affliation at lower taxonomic ranks. The phylogenetic placement of Cordyceps staphylinidaecola/bassiana, and C. scarabaeicola within Beauveria corroborates prior reports of these anamorph-teleomorph connections. These results establish a phylogenetic framework for further taxonomic, phylogenetic and comparative biological investigations of Beauveria and their corresponding Cordyceps teleomorphs.

Although intensively investigated for biological control of insect pests, little is known about the ecology of the fungal entomopathogenic genus Beauveria in natural or agricultural habitats. In this study, we used molecular phylogenetic and genotypic information to infer species diversity, reproductive potential and genetic structure of Beauveria occurring within a single arable field and bordering hedgerow in Denmark. Isolates were sampled from cultivated field and hedgerow soils, from insects harbouring latent fungal infections, and from the phylloplanes of three plant species common in the hedgerow flora. A nuclear phylogeny of this local Beauveria assemblage resolved seven phylogenetic species, including (i) five phylogenetic species within Beauveria bassiana sensu stricto; (ii) Clade C, a taxonomically uncharacterized species that is morphologically indistinguishable but phylogenetically distant from B. bassiana s.s.; and (iii) Beauveria brongniartii. All seven species were present throughout the hedgerow habitat, including as infections in insects. Significantly, only B. bassiana s.s. phylogenetic species Eu_1 was isolated from tilled soils. Mating type polymerase chain reaction assays demonstrated that all five B. bassiana s.s. phylogenetic species possess bipolar outcrossing mating systems. Of these, only the Eu_1 population contained two mating types; however, a 31:2 skew in MAT1:MAT2 mating types suggests a low frequency of sexual reproduction in this population. The four remaining B. bassiana s.s. phylogenetic species were fixed for single mating types and these populations are evidently clonal. Multilocus microsatellite genotyping revealed polymorphism in all five phylogenetic species of B. bassiana s.s.; however, all show evidence of clonal genetic structure.

The glpK genes of Enterococcus casseliflavus and Enterococcus faecalis, encoding glycerol kinase, the key enzyme of glycerol uptake and metabolism in bacteria, have been cloned and sequenced. The translated amino acid sequences exhibit strong homology to the amino acid sequences of other bacterial glycerol kinases. After expression of the enterococcal glpK genes in Escherichia coli, both glycerol kinases were purified and were found to be phosphorylated by enzyme I and the histidine-containing protein of the phosphoenolpyruvate: glycose phosphotransferase system. Phosphoenolpyruvate-dependent phosphorylation caused a 9-fold increase in enzyme activity. The site of phosphorylation in glycerol kinase of E. casseliflavus was determined as His-232. Site-specific mutagenesis was used to replace His-232 in glycerol kinase of E. casseliflavus with an alanyl, glutamate, or arginyl residue. The mutant proteins could no longer be phosphorylated confirming that His-232 of E. casseliflavus glycerol kinase represents the site of phosphorylation. The His 232 3 Arg glycerol kinase exhibited an about 3-fold elevated activity compared with wild-type glycerol kinase. Fructose 1,6-bisphosphate was found to inhibit E. casseliflavus glycerol kinase activity. However, neither EIIA Glc from E. coli nor the EIIA Glc domain of Bacillus subtilis had an inhibitory effect on glycerol kinase of E. casseliflavus.Glycerol uptake in Gram-negative and Gram-positive bacteria is mediated by the glycerol diffusion facilitator, an integral membrane protein of 30 kDa, catalyzing the rapid equilibration of concentration gradients of glycerol across the cytoplasmic membrane (1, 2). Intracellular glycerol is converted to glycerol-3-P by the enzyme glycerol kinase that uses ATP as phosphoryl donor (3, 4). Glycerol-3-P is not a substrate of the glycerol diffusion facilitator (5) and hence remains entrapped in the cell, where it is further metabolized. Phosphorylation of glycerol by glycerol kinase provides the driving force for the uptake of glycerol, as it creates a constant imbalance of the glycerol concentrations inside and outside the cell. It was therefore not surprising that glycerol kinase is the target of several regulation mechanisms.