Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species

Background

Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology.

Results

Draft genome sequences of Nocardia asteroides NBRC 15531^T, Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402^T, and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4–11, 7–13, and 1–6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text.

Conclusion

We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied, and 4) different N. brasiliensis strains have some different gene clusters of PKS-I/NRPS, although the rest of the clusters are common within the N. brasiliensis strains. Genome sequencing suggested that Nocardia strains are highly promising resources in the search of novel secondary metabolites.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-323) contains supplementary material, which is available to authorized users.     

Characterization of clinical isolates of pathogenic <Emphasis Type="Italic">Nocardia</Emphasis> strains and related actinomycetes in Thailand from 1996 to 2003

In Thailand from 1996 to 2003, 171 strains of pathogenic aerobic actinomycetes from clinical specimens were isolated. Of those strains, 134 were mycolic acid containing actinomycetes, including 96 strains of Nocardia species. Others included 10 strains of Gordonia, 14 strains of Rhodococcus, and 22 strains of Mycobacterium. One strain each of the genera Tsukamurella and Corynebacterium were also isolated. Also identified were 27 strains of non-mycolic acid containing actinomycetes. Our identification studies of 96 strains of Nocardia species showed that significant pathogens in Thailand were N. beijingensis (18 strains), N. cyriacigeorgica (13 strains), and N. farcinica (34 strains); the most prevalent species was N. farcinica (35.4%). We also isolated four strains of N. asiatica, five strains of N. asteroides sensu stricto, four strains of N. nova, seven strains of N. otitidiscaviarum, eight strains of N. transvalensis, and two strains of N. pseudobrasiliensis.     

The experimental pathogenicity in Swiss mice of various strains of Nocardia isolated in soil of Tucumán (Argentina)

The isolation of Nocardia pathogenic for man in soil of Tucumán revealed the convenience of undertaking a study of their virulence, to evaluate any potential danger it may imply.Thirty three strains were studied: 28 N. brasiliensis, 3 N. asteroides and 2 N. caviae by inoculating albino Swiss mice into the footpads and intraperitoneally.The results obtained revealed the existence of Nocardia of variable aggresiveness in the soil of Tucumán.     

Natural occurrence of Nocardia in soil of Tucumán: Physiological characteristics

This is the first study initiated in Argentina to establish the presence of species of Nocardia from soil samples. These samples were gathered in different areas of Tucumán.Thirty three pathogenic strains of Nocardia were isolated by the paraffin bait method. Out of them, 28 were N. brasiliensis, 3 N. asteroides and 2 N. caviae. N. brasiliensis was widely distributed in the soil of the areas tested.It is proved that N. caviae, so rarely found in other regions of the world, occurs in Tucumán.A detailed study of the morphological and physiological characteristics for identification is discussed.     

Pathogenicity of Nocardia caviae,N. asteroides and N. brasiliensis

The pathogenicity ofNocardia caviae, N. asteroides andN. brasiliensis has been tested for white mice, guinea pigs and rabbits and chorio-allantoic membrane of the developing chick embryo. Altogether, 14 strains belonging to the 3Nocardia species originating from soil, human and animal sources in India or abroad were tested. All of them proved pathogenic though the degree of virulence varied from strain to strain. Incorporation of hog gastric mucin in the inoculum enhanced the virulence of all the 3Nocardia species for white mice.N. caviae strains were uniformly more virulent than those ofN. asteroides andN. brasiliensis.In the white mice inoculated intraperitoneally, a greater dissemination of the disease was apparent withN. caviae than withN. asteroides. Of the 6 strains ofN. caviae tested, 5 disseminated to the lung, 3 to the heart and 2 to the brain. InN. asteroides dissemination of the disease to the brain was observed with 2 of its 3 strains.N. brasiliensis showed no dissemination.N. caviae was found to be equally virulent for white mice, guinea pigs and rabbits. On the other hand,N. asteroides andN. brasiliensis were more virulent for white mice than for guinea pigs and rabbits. The lesions caused byN. caviae in mice, guinea pigs and rabbits persisted up to 4 weeks. In strong contrast to this the lesions due toN. asteroides andN. brasiliensis found in the guinea pigs and rabbits showed a strong tendency towards spontaneous clearance.Histologically, the lesions caused byN. caviae, N. asteroides andN. brasiliensis in mice, guinea pigs and rabbits were in the form of abscesses which showed an acute or chronic reaction. In the case ofN. caviae these abscesses showed both granules and freely dispersed cocco-bacillary bodies or filaments. As forN. asteroides it occurred in the form of cocco-bacillary bodies or filaments whereasN. brasiliensis consistently produced granules in the lesions.The lesions caused by the 3Nocardia species on the chorio-allantoic membrane of the developing chick embryo were in the form of abscesses which contained cocco-bacillary bodies and branching filaments but no granules.This forms a part of the thesis submitted by P.V.K. for Ph. D. degree, of the University of Delhi.     

First human case of nocardiosis caused by Nocardia pseudobrasiliensis in Japan

Nocardia sp. IFM 0896, an actinomycete with biochemical characteristics that differed from Nocardia brasiliensis, was isolated from a 71-year-old Japanese man with a history of tuberculosis and cancer. Although the isolate was tentatively identified as N. brasiliensis, the morphological and physiological characteristics of strain IFM 0896 were different from those of N. brasiliensis IFM 0236^T. The results of 16S rRNA gene sequence phylogenies and PCR-RFLP analysis of a heat shock protein revealed that Nocardia sp. IFM 0896 belongs to the species N. pseudobrasiliensis. This is the first clinical isolation report of N. pseudobrasiliensis in Japan.This revised version was published online in October 2005 with corrections to the Cover Date.     

Nocardia aciditolerans sp. nov., isolated from a spruce forest soil

Actinomycetes growing on acidified starch-casein agar seeded with suspensions of litter and mineral soil from a spruce forest were provisionally assigned to the genus Nocardia based upon colonial properties. Representative isolates were found to grow optimally at pH 5.5, have chemotaxonomic and morphological features consistent with their assignment to the genus Nocardia and formed two closely related subclades in the Nocardia 16S rRNA gene tree. DNA:DNA relatedness assays showed that representatives of the subclades belong to a single genomic species. The isolates were distantly associated with their nearest phylogenetic neighbour, the type strain of Nocardia kruczakiae, and were distinguished readily from the latter based on phenotypic properties. On the basis of these data it is proposed that the isolates merit recognition as a new species, Nocardia aciditolerans sp. nov. The type strain is isolate CSCA68^T (=KACC 17155^T = NCIMB 14829^T = DSM 45801^T).     

Temperature resistant Nocardia biovarieties in Tucumán,Argentina?

Eighty-six regional strains of the pathogenic Nocardia species isolated from soil and human mycetoma were tested for their response to different incubation temperatures and for their tolerance to different temperatures. The aim was to assess whether growth temperature and tolerance to elevated temperatures are valuable criteria for the differentiation of pathogenic species of local strains based on the results obtained from a large number of strains. The results showed that 75.34% of all N. brasiliensis isolates from both sources grew at a temperature higher than 37 °C. 20% of the mycetoma strains and 11.32% of those from soil grew at 45 °C. 98.1% of N. brasiliensis from soil and 55.0% of the mycetoma strains tolerated 50 °C for 8 h and many isolates from both sources endured this temperature for an even longer time and tolerated yet higher temperatures. Both properties (growth temperature and temperature tolerance) are used to identify N. asteroides complex (N. farcinica) and N. otitidiscaviarum, and according to our results they are not suitable to differentiate regional strains of this species. The N. asteroides strains assayed showed an ability to grow at and tolerate elevated temperatures superior to those belonging to the other species. Although adaptation of local N. asteroides and N. otitidiscaviarum strains to temperature is important, it is more significant for N. brasiliensis, because this species is predominant in the Tucumán soil and responsible for the major number of diseases in the area.This revised version was published online in October 2005 with corrections to the Cover Date.     

Pathogenicity of Nocardia transvalensis for laboratory mice

Nocardia transvalensis was found to be virulent for laboratory mice both by intraperitoneal and intravenous routes of inoculation, the latter route producing a more progressive and disseminating infection. Cortisone administration was found to enhance the susceptibility of mice, the LD₅₀ for the cortisone treated mice being five times less than that for the untreated animals. In the tissues of cortisone treated mice, N. transvalensis grew as dispersed filaments with coccobacillary forms or sometimes their loose aggregates, whereas in normal animals granule formation was a conspicuous feature. The pathogenicity of N. transvalensis is compared with that of N. asteroides, N. brasiliensis and N. caviae from published reports.     

Asexual Propagation of a Virulent Clone Complex in a Human and Feline Outbreak of Sporotrichosis

Sporotrichosis is one of the most frequent subcutaneous fungal infections in humans and animals caused by members of the plant-associated, dimorphic genus Sporothrix. Three of the four medically important Sporothrix species found in Brazil have been considered asexual as no sexual stage has ever been reported in Sporothrix schenckii, Sporothrix brasiliensis, or Sporothrix globosa. We have identified the mating type (MAT) loci in the S. schenckii (strain 1099-18/ATCC MYA-4821) and S. brasiliensis (strain 5110/ATCC MYA-4823) genomes by using comparative genomic approaches to determine the mating type ratio in these pathogen populations. Our analysis revealed the presence of a MAT1-1 locus in S. schenckii while a MAT1-2 locus was found in S. brasiliensis representing genomic synteny to other Sordariomycetes. Furthermore, the components of the mitogen-activated protein kinase (MAPK)-pheromone pathway, pheromone processing enzymes, and meiotic regulators have also been identified in the two pathogens, suggesting the potential for sexual reproduction. The ratio of MAT1-1 to MAT1-2 was not significantly different from 1:1 for all three Sporothrix species, but the population of S. brasiliensis in the outbreaks originated from a single mating type. We also explored the population genetic structure of these pathogens using sequence data of two loci to improve our knowledge of the pattern of geographic distribution, genetic variation, and virulence phenotypes. Population genetics data showed significant population differentiation and clonality with a low level of haplotype diversity in S. brasiliensis isolates from different regions of sporotrichosis outbreaks in Brazil. In contrast, S. schenckii isolates demonstrated a high degree of genetic variability without significant geographic differentiation, indicating the presence of recombination. This study demonstrated that two species causing the same disease have contrasting reproductive strategies and genetic variability patterns.     

Contribution to deterioration of polymeric materials by a slow-growing bacterium Nocardia corynebacterioides

Bacteria capable of degrading polymeric products were isolated from several sources including tap water, sediment, and a deteriorated polymeric product. Alcaligenes xylosoxidans T2, Pseudomonas aeruginosa GP10, and Nocardia corynebacterioides S3 were able to utilize rubber products as a sole source of carbon and energy. These microorganisms showed different growth patterns in mineral salt media (MSM) supplemented with rubber strips or with the rubber extract. A. xylosoxidans T2, P. aeruginosa GP10 and N. corynebacterioides S3 reduced the weight of the rubber product by approximately 2.0, 4.0 and 5.3%, respectively, after 70 days of incubation with the rubber product in MSM. On average, 0.45 mg (water-soluble carbon) g^?1 of the rubber product was released into solution phase after 7 days of incubation. Growth of N. corynebacterioides S3 was initially slower but exceeded the other two bacteria upon colonization of the polymer products. N. corynebacterioides S3 formed a dense biofilm on surfaces of the rubber product as observed under scanning electron microscopy. This study suggests that indigenous microorganisms in a range of environments are capable of degrading polymeric products through utilization of chemicals released from the polymer matrix, but the slow-growing microorganisms on material surfaces have a much more pronounced role in the materials deterioration.     

Differences in Cell Morphometry,Cell Wall Topography and Gp70 Expression Correlate with the Virulence of Sporothrix brasiliensis Clinical Isolates

Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.     

A Population Genetics-Based and Phylogenetic Approach to Understanding the Evolution of Virulence in the Genus Listeria

The genus Listeria includes (i) the opportunistic pathogens L. monocytogenes and L. ivanovii, (ii) the saprotrophs L. innocua, L. marthii, and L. welshimeri, and (iii) L. seeligeri, an apparent saprotroph that nevertheless typically contains the prfA virulence gene cluster. A novel 10-loci multilocus sequence typing scheme was developed and used to characterize 67 isolates representing six Listeria spp. (excluding L. grayi) in order to (i) provide an improved understanding of the phylogeny and evolution of the genus Listeria and (ii) use Listeria as a model to study the evolution of pathogenicity in opportunistic environmental pathogens. Phylogenetic analyses identified six well-supported Listeria species that group into two main subdivisions, with each subdivision containing strains with and without the prfA virulence gene cluster. Stochastic character mapping and phylogenetic analysis of hly, a gene in the prfA cluster, suggest that the common ancestor of the genus Listeria contained the prfA virulence gene cluster and that this cluster was lost at least five times during the evolution of Listeria, yielding multiple distinct saprotrophic clades. L. welshimeri, which appears to represent the most ancient clade that arose from an ancestor with a prfA cluster deletion, shows a considerably lower average sequence divergence than other Listeria species, suggesting a population bottleneck and a putatively different ecology than other saprotrophic Listeria species. Overall, our data suggest that, for some pathogens, loss of virulence genes may represent a selective advantage, possibly by facilitating adaptation to a specific ecological niche.Population genetics-based and phylogenetic studies have greatly contributed to the understanding of the evolutionary history and ecology of bacterial pathogens. In particular, multilocus sequence analyses (MLSA) and single-nucleotide polymorphism (SNP)-based population genetics research have revealed the microevolutionary patterns of species complexes like the Bacillus cereus complex (12) or the microevolution of well-known pathogens like Yersinia pestis (2), Salmonella enterica serovar Typhi (57), and Mycobacterium tuberculosis (18). One of the common findings of these studies is that obligate pathogens generally have a genetically clonal population structure as inferred by MLSA (1), while the population structure of free-living facultative pathogenic bacteria is characterized by relatively high genetic variability (12, 70). It has been hypothesized that these differences in population structure are related to the fact that some obligate pathogens represent epidemic clones (38), i.e., clonal lineages whose members have an epidemiological advantage compared to other lineages and are therefore able to quickly spread within the population. Because this dispersal of the members of an epidemic clone occurs rapidly, there is not enough time to accumulate mutations.In this paper we present a phylogenetic and population genetics study of the genus Listeria. This genus consists of six closely related pathogenic (L. monocytogenes and L. ivanovii) and nonpathogenic (L. innocua, L. welshimeri, L. seeligeri, and a newly described species, L. marthii) species as well as a distantly related species, L. grayi (22). Another new species, L. rocourtiae, has been recently reported (33), but isolates were not available for inclusion in the study reported here. Because of the distant phylogenetic relatedness of L. grayi to the other Listeria species, it has been suggested that this species should be put in a separate genus, Murraya (63); L. grayi was thus not included in our study reported here. L. monocytogenes and L. ivanovii are facultative pathogens of warm-blooded animals and are the causative agents of a severe infectious disease, listeriosis (67). While L. monocytogenes has a wide host range, including humans, the host range of L. ivanovii seems to be largely restricted to ruminants, in particular sheep (13), even though some human listeriosis cases caused by L. ivanovii have been reported (34).Key virulence genes in Listeria include (i) six genes (prfA, plcA, hly, mpl, actA, and plcB) clustered in a genomic element, designated the prfA virulence cluster or the Listeria pathogenicity island (LiPI), and (ii) members of the internalin family (61). Genes in the prfA cluster encode functions that that are necessary for inter- and intracellular motility and intracellular survival in the host cell. While some internalin genes encode proteins essential for host cell invasion (e.g., inlA and inlB) (3), inlC has recently been shown to encode a protein critical for cell-to-cell spread (52), and the functions of a number of other internalin proteins still remain to be elucidated (40). A number of internalin genes are also organized in clusters, including the inlAB operon, the inlGHE operon (which can also be present as an inlGC2DE or as an inlC2DE operon), which is found in L. monocytogenes and an L. ivanovii species-specific pathogenicity island encoding sphingomyelinase and numerous internalins (13). Importantly, the presence or absence of the prfA cluster and virulence characteristics can also be used to classify Listeria species and clades into three groups, including (i) species that do contain the prfA virulence cluster and are known pathogens, like L. monocytogenes and L. ivanovii, (ii) species that lack the prfA virulence cluster and are nonpathogenic (L. marthii and L. welshimeri), and (iii) species in which the presence of the prfA virulence cluster varies by strain. The last group contains L. seeligeri, which is nonpathogenic, although the majority of strains in the population contain the prfA virulence cluster (69), and L. innocua, which is also nonpathogenic, and although most strains lack the prfA virulence cluster, a small proportion of strains do carry this cluster (31, 68). The facts that the genus Listeria contains closely related nonpathogenic and pathogenic species and that strains with and without the prfA cluster within the same species make this genus an interesting model system for studies on the evolution of pathogenicity in opportunistic environmental pathogens. In addition, an improved understanding of the phylogeny and evolution of pathogenic and nonpathogenic Listeria spp. will also help in the development of appropriate assays for the specific detection and identification of human and animal pathogenic Listeria strains as well as regulations and intervention strategies that specifically target pathogenic species and strains.     

Hijacking of Rho GTPases during bacterial infection

Highly pathogenic bacteria, including Yersinia, Salmonella, E. coli and Clostridia, produce an amazing array of virulence factors that target Rho proteins. These pathogens exploit and/or impair many aspects of Rho protein activities by activating or inhibiting these key molecular switches. Here, we describe examples illustrating how modulation of Rho protein activity is the underlying molecular mechanism used by pathogens to disrupt host epithelial/endothelial barriers, paralyze immune cell migration and phagocytic functions, invade epithelial cells, replicate, and form reservoirs or disseminate in epithelia. Remarkably, emerging evidence points to the capacity of target cells to not only perceive the imbalance of Rho activity induced by virulence factors but also to respond by stimulating the production of anti-microbial responses that alert the host to the pathogenic threat. Furthermore, toxins that activate Rho proteins have been extremely useful in revealing the exquisite cellular regulations of these GTPases, notably by the ubiquitin and proteasome system. Finally, a number of studies indicate that toxins targeting Rho proteins have great potential in the development of new therapeutic tools.     

Effects of sterols,rat hematin and coproporphyrin on the free-living stages of Nematospiroides dubius and Nippostrongylus brasiliensis

Nematospiroides dubius, unlike the closely related nematode Nippostrongylus brasiliensis, lacks a sterol requirement for the completion of its development to the L₃ stage in an aqueous suspension of formalin-killed Escherichia coli. These larvae were justascapable of completing their development to adults in mice as those grown on charcoal-feces cultures. In addition, sterols had a toxic effect on the larvae of N. dubius which was removed by substituting a Krebs-Ringer solution for distilled water.In order to interpret the absence of a sterol requirement for the development of N. dubius larvae, an analysis of the egg lipids for both species was made. N. brasiliensis had about twice the sterol concentration of N. dubius eggs. A comparison of the development for both species showed, however, that N. brasiliensis grows more than twice as much as N. dubius to reach the L₃ larval stage. Therefore, the amount of endogenous sterol is insufficient for N. brasiliensis to develop from the egg to the L₃ stage.N. dubius and N. brasiliensis also differ in their porphyrin requirements. Unlike N. brasiliensis, coproporphyrin did not increase the size of N. dubius larvae so that they were comparable to those grown on charcoal-feces cultures. Coproporphyrin did result in larvae that were more robust in appearance than those grown without a supplement.     

Resistance to Nocardia brasiliensis infection in mice immunized with either Nocardia or BCG

Different vaccination procedures to increase the mecha nisms of host resistance to Nocardia brasiliensis were studied in mice. When mice were challenged in the footpad, 2×10⁸ N. brasiliensis 20 days after footpad inoculation with either viable or killed N. brasiliensis, the mice demonstrated significant resistance to infection when compared with noninfected and nonimmunized mice. The degree of resistance seems to be correlated with the delayed-type hypersensitivity response in the vaccinated animals. Vaccination with another acid-fast bacilli, BCG, afforded both a mild protection and low DTH reactivity. Antibody levels to Nocardia were similar in either Nocardia- or BCG- treated groups indicating that they do not play an important role in resistance to infection by N. brasiliensis.     

Comparative Genomic Analysis Identifies Divergent Genomic Features of Pathogenic Enterococcus cecorum Including a Type IC CRISPR-Cas System,a Capsule Locus,an epa-Like Locus,and Putative Host Tissue Binding Proteins

Enterococcus cecorum (EC) is the dominant enteric commensal of adult chickens and contributes to the gut consortia of many avian and mammalian species. While EC infection is an uncommon zoonosis, like other enterococcal species it can cause life-threating nosocomial infection in people. In contrast to other enterococci which are considered opportunistic pathogens, emerging pathogenic strains of EC cause outbreaks of musculoskeletal disease in broiler chickens. Typical morbidity and mortality is comparable to other important infectious diseases of poultry. In molecular epidemiologic studies, pathogenic EC strains were found to be genetically clonal. These findings suggested acquisition of specific virulence determinants by pathogenic EC. To identify divergent genomic features and acquired virulence determinants in pathogenic EC; comparative genomic analysis was performed on genomes of 3 pathogenic and 3 commensal strains of EC. Pathogenic isolates had smaller genomes with a higher GC content, and they demonstrated large regions of synteny compared to commensal isolates. A molecular phylogenetic analysis demonstrated sequence divergence in pathogenic EC genomes. At a threshold of 98% identity, 414 predicted proteins were identified that were highly conserved in pathogenic EC but not in commensal EC. Among these, divergent CRISPR-cas defense loci were observed. In commensal EC, the type IIA arrangement typical for enterococci was present; however, pathogenic EC had a type IC locus, which is novel in enterococci but commonly observed in streptococci. Potential mediators of virulence identified in this analysis included a polysaccharide capsular locus similar to that recently described for E. faecium, an epa-like locus, and cell wall associated proteins which may bind host extracellular matrix. This analysis identified specific genomic regions, coding sequences, and predicted proteins which may be related to the divergent evolution and increased virulence of emerging pathogenic strains of EC.     

Regulation of Natural Rubber Biosynthesis by Proteins Associated with Rubber Particles

Natural rubber, cis-1,4-polyisoprene, is an essential raw material used in thousands of products, many of which are absolutely necessary for medical purposes. Natural rubber is obtained from latex, an aqueous emulsion present in the laticiferous vessels of the natural rubber-producing plants. Hevea brasiliensis (the Brazilian rubber tree) currently is the only commercially important source of natural rubber. H. brasiliensis crops have very little genetic variability, leaving rubber plantations at risk of serious pathogenic attacks. In addition, repeated exposure to residual proteins in latex products derived from H. brasiliensis have led to serious and widespread allergic (type I) hypersensitivity. Therefore, identification of alternative sources of natural rubber is a very important biotechnological task. Potentially, Russian dandelion (Taraxacum kok-saghyz) may be such an alternative because significant amounts of natural rubber are produced in its root system. However, H. brasiliensis is a more efficient producer of natural rubber than T. kok-saghyz. Thus, improvement of rubber biosynthesis in plants is a first-priority problem of modern biotechnology. In this review, we describe proteins that may increase the concentration of natural rubber in laticiferous vessels of T. kok-saghyz and its close relative Taraxacum brevicorniculatum, when overexpressed in the plants. These proteins, cis-prenyltransferases, rubber transferase activator, and small rubber particle proteins, are directly involved in synthesis of the polyisoprene chain. We also analyze the effects of their expression levels on the production of natural rubber in vivo.     

Interspecies competition triggers virulence and mutability in Candida albicans–Pseudomonas aeruginosa mixed biofilms

Inter-kingdom and interspecies interactions are ubiquitous in nature and are important for the survival of species and ecological balance. The investigation of microbe-microbe interactions is essential for understanding the in vivo activities of commensal and pathogenic microorganisms. Candida albicans, a polymorphic fungus, and Pseudomonas aeruginosa, a Gram-negative bacterium, are two opportunistic pathogens that interact in various polymicrobial infections in humans. To determine how P. aeruginosa affects the physiology of C. albicans and vice versa, we compared the proteomes of each species in mixed biofilms versus single-species biofilms. In addition, extracellular proteins were analyzed. We observed that, in mixed biofilms, both species showed differential expression of virulence proteins, multidrug resistance-associated proteins, proteases and cell defense, stress and iron-regulated proteins. Furthermore, in mixed biofilms, both species displayed an increase in mutability compared with monospecific biofilms. This characteristic was correlated with the downregulation of enzymes conferring protection against DNA oxidation. In mixed biofilms, P. aeruginosa regulates its production of various molecules involved in quorum sensing and induces the production of virulence factors (pyoverdine, rhamnolipids and pyocyanin), which are major contributors to the ability of this bacterium to cause disease. Overall, our results indicate that interspecies competition between these opportunistic pathogens enhances the production of virulence factors and increases mutability and thus can alter the course of host-pathogen interactions in polymicrobial infections.     

Structure and function of a mating-type gene from the homothallic species Neurospora africana

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.