Melanin and novel melanin precursors from Aeromonas media

Many bacteria produce reddish brown to black pigments and some of these have been characterised. This report describes the isolation and characterisation of a diffusible brown melanin-like pigment from the bacterium Aeromonas media. Physico-chemical testing suggested that the pigment is a true melanin. New butanol-soluble yellow, red and brown pigments were isolated from the A. media strain under reducing conditions during melanogenesis and these pigments were shown to be unstable precursors of the polymeric brown melanin product.     

Multiple-Locus Sequence Typing Analysis of Bacillus cereus and Bacillus thuringiensis Reveals Separate Clustering and a Distinct Population Structure of Psychrotrophic Strains

We used multilocus sequence typing (MLST) to characterize phylogenetic relationships for a collection of Bacillus cereus group strains isolated from forest soil in the Paris area during a mild winter. This collection contains multiple strains isolated from the same soil sample and strains isolated from samples from different sites. We characterized 115 strains of this collection and 19 other strains based on the sequences of the clpC, dinB, gdpD, panC, purF, and yhfL loci. The number of alleles ranged from 36 to 53, and a total of 93 allelic profiles or sequence types were distinguished. We identified three major strain clusters—C, T, and W—based on the comparison of individual gene sequences or concatenated sequences. Some less representative clusters and subclusters were also distinguished. Analysis of the MLST data using the concept of clonal complexes led to the identification of two, five, and three such groups in clusters C, T, and W, respectively. Some of the forest isolates were closely related to independently isolated psychrotrophic strains. Systematic testing of the strains of this collection showed that almost all the strains that were able to grow at a low temperature (6°C) belonged to cluster W. Most of these strains, including three independently isolated strains, belong to two clonal complexes and are therefore very closely related genetically. These clonal complexes represent strains corresponding to the previously identified species Bacillus weihenstephanensis. Most of the other strains of our collection, including some from the W cluster, are not psychrotrophic. B. weihenstephanensis (cluster W) strains appear to comprise an effectively sexual population, whereas Bacillus thuringiensis (cluster T) and B. cereus (cluster C) have clonal population structures.     

Loss of Homogentisate 1,2-Dioxygenase Activity in Bacillus anthracis Results in Accumulation of Protective Pigment

Melanin production is important to the pathogenicity and survival of some bacterial pathogens. In Bacillus anthracis, loss of hmgA, encoding homogentisate 1,2-dioxygenase, results in accumulation of a melanin-like pigment called pyomelanin. Pyomelanin is produced in the mutant as a byproduct of disrupted catabolism of L-tyrosine and L-phenylalanine. Accumulation of pyomelanin protects B. anthracis cells from UV damage but not from oxidative damage. Neither loss of hmgA nor accumulation of pyomelanin alter virulence gene expression, sporulation or germination. This is the first investigation of homogentisate 1,2-dioxygenase activity in the Gram-positive bacteria, and these results provide insight into a conserved aspect of bacterial physiology.     

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.     

Enhanced Transformation Efficiency of Recalcitrant Bacillus cereus and Bacillus weihenstephanensis Isolates upon In Vitro Methylation of Plasmid DNA

Digestion patterns of chromosomal DNAs of Bacillus cereus and Bacillus weihenstephanensis strains suggest that Sau3AI-type restriction modification systems are widely present among the isolates tested. In vitro methylation of plasmid DNA was used to enhance poor plasmid transfer upon electroporation to recalcitrant strains that carry Sau3AI restriction barriers.     

Multilocus sequence analysis of Bacillus thuringiensis serovars navarrensis, bolivia and vazensis and Bacillus weihenstephanensis reveals a common phylogeny

The Bacillus cereus group sensu lato includes six closely-related bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. B. thuringiensis is distinguished from the other species mainly by the appearance of an inclusion body upon sporulation. B. weihenstephanensis is distinguished based on its psychrotolerance and the presence of specific signature sequences in the 16S rRNA gene and cspA genes. A total of seven housekeeping genes (glpF, gmK, ilvD, pta, purH, pycA and tpi) from different B. thuringiensis serovars and B. weihenstephanensis strains were amplified and their nucleotide sequences determined. A maximum likelihood phylogenetic tree was inferred from comparisons of the concatenated sequences. B. thuringiensis serovars navarrensis, bolivia and vazensis clustered not with the other B. thuringiensis serovars but rather with the B. weihenstephanensis strains, indicative of a common phylogeny. In addition, specific signature sequences and single nucleotide polymorphisms common to B. thuringiensis serovars navarrensis, bolivia and vazensis and the B. weihenstephanensis strains, and absent in the other B. thuringiensis serovars, were identified.     

Discrimination Between Mesophilic and Psychrotolerant Strains in the Bacillus cereus Group Based on the PstI Digestion of the pycA Gene

A simple and rapid assay for the detection of Bacillus weihenstephanensis isolates and other psychrotolerant strains in the Bacillus cereus group was developed. It is based on the presence of a nucleotide substitution at position 795 on the housekeeping pycA gene in all B. weihenstephanensis strains. This mutation creates a PstI recognition site. It is absent in mesophilic strains in the B. cereus group. The pycA gene is amplified by PCR and the amplicons submitted to PstI digestions. In mesophilic strains, a single band of 1,718 bp in length is visualised on an agarose gel. In B. weihenstephanensis strains and in all other psychrotolerant strains from the B. cereus group, the amplicons are cleaved and two bands of 1,175 and 543 bp, respectively, are visualised. This method could be used for the screening of B. cereus collections and for the identification of psychrotolerant and mesophilic isolates from different environments.     

Occurrence and pathogenic potential of Bacillus cereus group bacteria in a sandy loam

The major part (94%) of the Bacillus cereus-like isolates from a Danish sandy loam are psychrotolerant Bacillus weihenstephanensis according to their ability to grow at temperatures below 7 °C and/or two PCR-based methods, while the remaining 6% are B. cereus. The Bacillus mycoides-like isolates could also be␣divided into psychrotolerant and mesophilic isolates. The psychrotolerant isolates of B. mycoides could␣be discriminated from the mesophilic by the two PCR-based methods used to characterize B.␣weihenstephanensis. It is likely that the mesophilic B. mycoides strains are synonymous with Bacillus pseudomycoides, while psychrotolerant B. weihenstephanensis, like B. mycoides, are B. mycoides senso stricto. B. cereus is known to produce a number of factors, which are involved in its ability to cause gastrointestinal and somatic diseases. All the B. cereus-like and B. mycoides like isolates from the sandy loam were investigated by PCR for the presence of 12 genes encoding toxins. Genes for the enterotoxins (hemolysin BL and nonhemolytic enterotoxin) and the two of the enzymes (cereolysin AB) were present in the major part of the isolates, while genes for phospolipase C and hemolysin III were present in fewer isolates, especially among B. mycoides like isolates. Genes for cytotoxin K and the hemolysin II were only present in isolates affiliated to B. cereus. Most of the mesophilic B. mycoides isolates did not possess the genes for the nonhemolytic enterotoxin and the cereolysin AB. The presence of multiple genes coding for virulence factors in all the isolates from the B. cereus group suggests that all the isolates from the sandy loam are potential pathogens.     

The Hemolytic Enterotoxin HBL Is Broadly Distributed among Species of the Bacillus cereus Group

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L₁, L₂, and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.     

Pigment Production on L-Tryptophan Medium by Cryptococcus gattii and Cryptococcus neoformans

In recent years strains previously grouped within Cryptococcus neoformans have been divided into two species C. neoformans and C. gattii, with Cryptococcus neoformans comprising serotypes A, D, and AD and C. gattii comprising serotypes B and C. Cryptococcus neoformans have also been subdivided into two varieties C. neoformans var. grubii, serotype A, and C. neoformans var. neoformans, serotype D. We analyzed the growth and pigment production characteristics of 139 strains of Cryptococcus spp. in L-tryptophan containing media. Nearly all strains of Cryptococcus, including each variety and serotype tested produced a pink water-soluble pigment (molecular weight of 535.2 Da) from L-tryptophan. Consequently, the partial separation of the species was based on whether the pink pigment was secreted into the medium (extracellular) or retained as an intracellular pigment. On L-tryptophan medium C. neoformans var. grubii and serotype AD produced a pink extracellular pigment. In contrast, for C. gattii, the pink pigment was localized intracellularly and masked by heavy production of brown pigments. Pigment production by C. neoformans var. neoformans was variable with some strains producing the pink extracellular pigment and others retained the pink pigment intracellularly. The pink intracellular pigment produced by strains of C. neoformans var. neoformans was masked by production of brown pigments. Cryptococcus laccase mutants failed to produce pigments from L-tryptophan. This is the first report that the enzyme laccase is involved in tryptophan metabolism. Prior to this report Cryptococcus laccase produced melanin or melanin like-pigments from heterocyclic compounds that contained ortho or para diphenols, diaminobenzenes and aminophenol compounds. The pigments produced from L-tryptophan were not melanin.     

Construction of a new cloning vector utilizing a cryptic plasmid and the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus

Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin pigment and harbors a cryptic 7.4-kb plasmid, pHY6202. We constructed a Streptomyces cloning vector, pSY10CMM, consisting of the HUT6202 rep gene, the thiostrepton resistance gene and an operon encoding the synthesis of melanin pigment, abbreviated mel, from S. castaneoglobisporus. The vector, which has SphI and BamHI sites as cloning sites with insertional inactivation of the mel, is a more convenient cloning vector than commonly used pIJ702, because of its broad host range for antibiotic-producing Streptomyces strains and its much greater production of diffusible melanin pigment.     

Germination and proliferation of emetic Bacillus cereus sensu lato strains in milk

The Bacillus cereus sensu lato group includes potentially pathogenic bacteria that are ubiquitous in the environment and, importantly, could also be present in food products. This study focuses on emetic isolates which presumably could cause acute food poisoning and emetic syndrome. Here, we evaluate the ability of psychrotolerant Bacillus weihenstephanensis MC118 (isolated from soil) and mesophilic B. cereus BOD3/9 isolated from milk to germinate and multiply at 7 and 30 °C. Whereas the rates of germination at 30 °C in milk and nutrient broth of MC118 and BOD3/9 were similar, MC118, but not BOD3/9, proliferated to achieve relatively high numbers (～10⁶?colony-forming units/g) within 7 days of incubation at 7 °C. Mesophilic BOD3/9 showed a slight decrease of cell concentration in similar studies at 7 °C. Genotyping with repetitive extragenic palindromic sequence-based PCR and pulsed field gel electrophoresis revealed significant similarities between BOD3/9 and emetic reference B. cereus F4810/72 strain, while the B. weihenstephanensis MC118 isolate was more similar to the B. weihenstephanensis non-emetic reference DSMZ11821 strain. Our data suggest that emetic isolates that are also psychrotolerant, such as MC118, could constitute a hazard in the dairy industry, where milk could be a suitable medium for germination and growth.     

Melanin pigmentation gives rise to black spots on the wings of the silkworm Bombyx mori

Several mutants of the silkworm Bombyx mori show body color variation at the larval and adult stages. The Wild wing spot (Ws) mutant exhibits a phenotype in which the moth has a spot on the apex of the forewing. In this study, we investigated this trait to elucidate the molecular mechanism underlying the color pattern. Microscopy of the black spot of Ws mutants showed that the pigment emerges in the scales of the wing, and accumulation of the pigment becomes strong just before eclosion. We next examined the relationship between the black spot of the Ws mutant and melanin. The spectrophotometry using alkaline extracts from the black spot in the wing showed the highest absorption intensity at 405 nm, which is the absorbance wavelength of melanin. Moreover, inhibition assays for enzymes implicated in melanin synthesis using 3-iodo-l-tyrosine (a tyrosine hydroxylase inhibitor) and L-α-methyl-DOPA (a dopa decarboxylase inhibitor) revealed that treatment with each inhibitor disrupted the pigmentation of the wing of the Ws mutant. On the basis of these results, we analyzed the expression pattern of five genes involved in melanin formation, and found that the expression levels of yellow and laccase2 were increased just before pigmentation, whereas those of DDC, tan, and TH were increased when the apex of the wing turned black. These results showed that melanin pigmentation gives rise to the black spot on the wing.     

The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur

Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb) labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization.     

Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea

Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.     

Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “Bacillus cereus Group”

Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the “B. cereus group,” advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus.     

Isolation of Bartonella henselae and Two New Bartonella Subspecies,Bartonella koehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats

Domestic cats are the natural reservoir of Bartonella henselae, B. clarridgeiae and B. koehlerae. To determine the role of wild felids in the epidemiology of Bartonella infections, blood was collected from 14 free-ranging California mountain lions (Puma concolor) and 19 bobcats (Lynx rufus). Bartonella spp. were isolated from four (29%) mountain lions and seven (37%) bobcats. These isolates were characterized using growth characteristics, biochemical reactions, molecular techniques, including PCR-RFLP of selected genes or interspacer region, pulsed-field gel electrophoresis (PFGE), partial sequencing of several genes, and DNA-DNA hybridization. Two isolates were identical to B. henselae genotype II. All other isolates were distinguished from B. henselae and B. koehlerae by PCR-RFLP of the gltA gene using endonucleases HhaI, TaqI and AciI, with the latter two discriminating between the mountain lion and the bobcat isolates. These two novel isolates displayed specific PFGE profiles distinct from B. henselae, B. koehlerae and B. clarridgeiae. Sequences of amplified gene fragments from the three mountain lion and six bobcat isolates were closely related to, but distinct from, B. henselae and B. koehlerae. Finally, DNA-DNA hybridization studies demonstrated that the mountain lion and bobcat strains are most closely related to B. koehlerae. We propose naming the mountain lion isolates B. koehlerae subsp. boulouisii subsp. nov. (type strain: L-42-94), and the bobcat isolates B. koehlerae subsp. bothieri subsp. nov. (type strain: L-17-96), and to emend B. koehlerae as B. koehlerae subsp. koehlerae. The mode of transmission and the zoonotic potential of these new Bartonella subspecies remain to be determined.     

Efficacy of entomopathogenic bacteria for control of Musca domestica

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.     

Genetic instability of the short-living ascomycetous fungus <Emphasis Type="Italic">Podospora anserina</Emphasis> induced by prolonged submerged cultivation

Investigation of genetic variability of the short-living filamentous fungus Podospora anserina during its adaptation to conditions of prolonged submerged cultivation has been carried out for the first time. Cultivation of P. anserina under aeration (on a shaker) provides pronounced selective pressure, which makes it possible to obtain isolates with specific features, which are well adapted to cultivation in liquid media and have a life span several times exceeding that of the original strain. Static cultivation did not prevent the ageing of P. anserina. Repeated transfers in the shaker culture resulted in formation of mycelium deprived of the dark pigment melanin and actively producing carotenoids under illumination. The qualitative composition of P. anserina carotenoids was the same as in the closely-related species Neurospora crassa. The features obtained during the shaker cultivation (including changes in the colony morphology and decreased capacity for melanin synthesis) are inherited by their hybrids with the wild type strains, i.e., they resulted from the intragenomic rearrangements occurring during submerged cultivation of the fungus.