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PubMed2006

Marine sponge Craniella austrialiensis-associated bacterial diversity revelation based on 16S rDNA library and biologically active Actinomycetes screening, phylogenetic analysis.

AIMS: The aim of this study was to investigate the bacterial diversity associated with the sponge Craniella australiensis using a molecular strategy and isolating Actinomycetes with antimicrobial potentials. METHODS AND RESULTS: The bacterial diversity associated with South China Sea sponge C. austrialiensis was assessed using a 16S rDNA clone library alongside restriction fragment length polymorphism and phylogenetic analysis. It was found that the C. austrialiensis-associated bacterial community consisted of alpha, beta and gamma-Proteobacteria, Firmicutes, Bacteroidetes as well as Actinobacterium. Actinomycetes were isolated successfully using seawater medium with sponge extracts. According to the BLAST and phylogenetic analysis based on about 600-bp 16S rDNA sequences, 11 of the representative 23 isolates closely matched the Streptomyces sp. while the remaining 12 matched the Actinomycetales. Twenty Actinomycetes have antimicrobial potentials, of which 15 are found to possess broad-spectrum antimicrobial potentials. CONCLUSIONS: The sponge C. austrialiensis-associated bacterial community is very abundant including Proteobacteria, Firmicutes, Bacteroidetes and Actinobacterium while Actinomycetes is not predominant. Artificial seawater medium with sponge extracts is suitable for Actinomycetes isolation. Most of the isolated C. austrialiensis-associated Actinomycetes have a broad spectrum of antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed the diversity of the bacterial community and the isolated Actinomycetes with antimicrobial potentials associated with sponge C. australiensis.

Actinobacteria↗

PubMed2000

Polyphasic microbial community analysis of petroleum hydrocarbon-contaminated soils from two northern Canadian communities.

The cold-adapted bacterial communities in petroleum hydrocarbon-contaminated and non-impacted soils from two northern Canadian environments, Kuujjuaq, Que., and Alert, Nunavut, were analyzed using a polyphasic approach. Denaturing gradient gel electrophoresis (DGGE) separation of 16S rDNA PCR fragments from soil total community DNA revealed a high level of bacterial diversity, as estimated by the total number of bands visualized. Dendrogram analysis clustered the sample sites on the basis of geographical location. Comparison of the overall microbial molecular diversity suggested that in the Kuujjuaq sites, contamination negatively impacted diversity whereas in the Alert samples, diversity was maintained or increased as compared to uncontaminated controls. Extraction and sequencing analysis of selected 16S rDNA bands demonstrated a range of similarity of 86-100% to reference organisms, with 63.6% of the bands representing high G+C Gram-positive organisms in the order Actinomycetales and 36.4% in the class Proteobacteria. Community level physiological profiles generated using Biolog GN plates were analyzed by cluster analysis. Based on substrate oxidation rates, the samples clustered into groups similar to those of the DGGE dendrograms, i.e. separation based upon geographic origin. The coinciding results reached using culture-independent and -dependent analyses reinforces the conclusion that geographical origin of the samples, rather than petroleum contamination level, was more important in determining species diversity within these cold-adapted bacterial communities.

Journal Article↗

PubMed2007

Bacterial community structure associated with the Antarctic soft coral, Alcyonium antarcticum.

The structure and composition of microbial communities inhabiting the soft coral Alcyonium antarcticum were investigated across three differentially contaminated sites within McMurdo Sound, Antarctica. Diverse microbial communities were revealed at all sites using culture-based analysis, denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone-library analysis, and FISH. Phylogenetic analysis of isolates and retrieved sequences demonstrated close affiliation with known psychrophiles from the Antarctic environment and high similarity to Gammaproteobacteria clades of sponge-associated microorganisms. The majority of bacteria detected with all techniques reside within the Gammaproteobacteria, although other phylogenetic groups including Alpha- and Betaproteobacteria, Bacteroidetes, Firmicutes, Actinomycetales, Planctomycetes, and Chlorobi and bacteria from the functional group of sulfate-reducing bacteria were also present. Multivariate (nMDS) analysis of DGGE banding patterns and principal component analysis of quantitative FISH data revealed no distinct differences in community composition between differentially contaminated sites. Rather, conserved coral-associated bacterial groups were observed within and between sites, providing evidence to support specific coral-microbial interactions. This is the first investigation of microbial communities associated with Antarctic soft corals, and the results suggest that spatially stable microbial associations exist across an environmental impact gradient.

Animals↗

PubMed2001

Phylogenetic diversity of thermophilic actinomycetes and Thermoactinomyces spp. isolated from mushroom composts in Korea based on 16S rRNA gene sequence analysis.

Forty one strains isolated from 21 samples of various mushroom composts in Korea were analyzed by 16S rRNA gene sequencing to investigate the phylogenetic diversity of thermophilic actinomycetes. The 25 strains of thermophilic actinomycete isolates were related to the five genera, Pseudonocardia, Saccharomonospora, Saccharopolyspora, Streptomyces, and Thermobifida, within the order Actinomycetales, and 16 strains were classified into the genus Thermoactinomyces within the family Bacillaceae. Most of 41 isolates were encompassed by two genera, Streptomyces and Thermoactinomyces, that were isolated mainly in composts prepared from waste cotton and hay, respectively. Among them, M104 and M109 were placed in distinct taxonomic positions although these strains formed phylogenetic lineages related to the genus Streptomyces and to the family Streptosporangiaceae, respectively. Therefore, a phenetic and genetic characterization of these strains will be needed to pinpoint their taxonomic position.

Agaricales↗

PubMed1993

Penicillin-binding proteins of Rhodococcus equi: potential role in resistance to imipenem.

Rhodococcus equi is a gram-positive coccobacillus which, like other members of the order Actinomycetales, is increasingly reported as an opportunistic pathogen in patients with AIDS. The use of combinations of antibiotics that include imipenem (IMP) has been suggested for the treatment of patients infected with R. equi. An antagonism between IMP, meropenem, cefoxitin, ceftriaxone, moxalactam, and oxacillin and other beta-lactams, such as penicillin, amoxicillin, cephalothin, and ticarcillin, was detected in vitro both on Mueller-Hinton agar and in broth for all 10 IMP-susceptible R. equi strains examined. To study the mechanism of the antagonism between beta-lactams, a mutant with decreased susceptibility to IMP (isolate IpR) was selected in vitro from a susceptible clinical isolate of R. equi (isolate IpS). IpR exhibited decreased susceptibility to IMP, meropenem, cefoxitin, ceftriaxone, moxalactam, and oxacillin but not to penicillin, amoxicillin, cephalothin, or ticarcillin. No beta-lactamase was found in IpS, IpS cultured with antagonistic beta-lactams, or IpR strains. Labeling of penicillin-binding proteins (PBPs) revealed four PBPs with molecular masses of ca. 59, 56, 43, and 26 kDa in IpS. In IpR, PBP 3 disappeared and was replaced by PBP 3a of 40 kDa. The 50% saturation of PBP 3 and PBP 3a by the carbapenems correlated with the MICs of these antibiotics, respectively, for IpS and IpR strains. However, PBP 3a was not detected in IpS when IpS was cultured in the presence of beta-lactams, with which antagonism was observed. The present work describes the PBPs of R. equi and reports that IMP resistance in R. equi is related to an altered PBP pattern.

Anti-Bacterial Agents↗

PubMed2006

Microbial community structure and dynamics of dark fire-cured tobacco fermentation.

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.

Bacteria↗

PubMed1990

Effectiveness of Different Frankia Cell Types as Inocula for the Actinorhizal Plant Casuarina.

The soil bacterium Frankia of the Actinomycetales, capable of forming N(2)-fixing symbiotic root nodules on a diverse array of actinorhizal plants, has several morphological forms when grown in pure culture. Fresh hydrated preparations of whole cells, hyphae, and spores were all infective on seedlings of Casuarina at different dilutions. Desiccated hyphae showed no infection capacity, while desiccated spores remained infective, although at a reduced level. On the basis of most-probable-number statistics, spore suspensions were 3 orders of magnitude more infective than hyphae.

Journal Article↗

PubMed1997

Bacterial diversity in Adirondack mountain lakes as revealed by 16S rRNA gene sequences.

Bacterial communities of seven lakes in the Adirondack Mountains of New York State were characterized by amplification and sequencing of 16S ribosomal DNA. Analysis of over 100 partial sequences revealed a diverse collection of lineages, largely of the class Proteobacteria (19% alpha subdivision, 31% beta subdivision, and 9% gamma subdivision), the phylum Cytophaga-Flavobacteria-Bacteroides (15%), and the order Actinomycetales (18%). Additionally, a number of the sequences were similar to those of the order Verrucomicrobiales. However, few of the sequence types are closely related to those of characterized species. The relative contributions of the groups of sequences differed among the lakes, suggesting that bacterial population structure varies and that it may be possible to relate aquatic bacterial community structure to water chemistry.

Bacteria↗

PubMed2002

Effects of T4 lysozyme release from transgenic potato roots on bacterial rhizosphere communities are negligible relative to natural factors.

Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for Bacteria, Actinomycetales, or alpha- or beta-Proteobacteria. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.

Bacteria↗

PubMed2004

Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria.

In organisms other than higher plants, family 19 chitinase was first discovered in Streptomyces griseus HUT6037, and later, the general occurrence of this enzyme in Streptomyces species was demonstrated. In the present study, the distribution of family 19 chitinases in the class Actinobacteria and the phylogenetic relationship of Actinobacteria family 19 chitinases with family 19 chitinases of other organisms were investigated. Forty-nine strains were chosen to cover almost all the suborders of the class Actinobacteria, and chitinase production was examined. Of the 49 strains, 22 formed cleared zones on agar plates containing colloidal chitin and thus appeared to produce chitinases. These 22 chitinase-positive strains were subjected to Southern hybridization analysis by using a labeled DNA fragment corresponding to the catalytic domain of ChiC, and the presence of genes similar to chiC of S. griseus HUT6037 in at least 13 strains was suggested by the results. PCR amplification and sequencing of the DNA fragments corresponding to the major part of the catalytic domains of the family 19 chitinase genes confirmed the presence of family 19 chitinase genes in these 13 strains. The strains possessing family 19 chitinase genes belong to 6 of the 10 suborders in the order Actinomycetales, which account for the greatest part of the Actinobacteria: Phylogenetic analysis suggested that there is a close evolutionary relationship between family 19 chitinases found in Actinobacteria and plant class IV chitinases. The general occurrence of family 19 chitinase genes in Streptomycineae and the high sequence similarity among the genes found in Actinobacteria suggest that the family 19 chitinase gene was first acquired by an ancestor of the Streptomycineae and spread among the Actinobacteria through horizontal gene transfer.

Actinobacteria↗

PubMed2005

Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora.

Salinispora is the first obligate marine genus within the order Actinomycetales and a productive source of biologically active secondary metabolites. Despite a worldwide, tropical or subtropical distribution in marine sediments, only two Salinispora species have thus far been cultivated, suggesting limited species-level diversity. To further explore Salinispora diversity and distributions, the phylogenetic diversity of more than 350 strains isolated from sediments collected around the Bahamas was examined, including strains cultured using new enrichment methods. A culture-independent method, using a Salinispora-specific seminested PCR technique, was used to detect Salinispora from environmental DNA and estimate diversity. Overall, the 16S rRNA gene sequence diversity of cultured strains agreed well with that detected in the environmental clone libraries. Despite extensive effort, no new species level diversity was detected, and 97% of the 105 strains examined by restriction fragment length polymorphism belonged to one phylotype (S. arenicola). New intraspecific diversity was detected in the libraries, including an abundant new phylotype that has yet to be cultured, and a new depth record of 1,100 m was established for the genus. PCR-introduced error, primarily from Taq polymerase, significantly increased clone library sequence diversity and, if not masked from the analyses, would have led to an overestimation of total diversity. An environmental DNA extraction method specific for vegetative cells provided evidence for active actinomycete growth in marine sediments while indicating that a majority of sediment samples contained predominantly Salinispora spores at concentrations that could not be detected in environmental clone libraries. Challenges involved with the direct sequence-based detection of spore-forming microorganisms in environmental samples are discussed.

Bacteriological Techniques↗

PubMed1968

Quantitative analysis of Actinomyces cell walls.

Quantitative data on the amino acid composition of cell walls of five species of Actinomyces were obtained by using a Beckman-Spinco amino acid analyzer. The major amino acids in A. israelii, A. naeslundii, A. eriksonii, and A. bovis species included alanine, glutamic acid, lysine, aspartic acid, and ornithine, as reported by previous workers, whereas A. propionicus contained diaminopimelic acid. Other amino acids, including glycine, valine, leucine, proline, isoleucine, and threonine, were present in at least some of the walls in quantities equal to or slightly less than that of lysine. This raised the question of whether these may represent cross-links in the peptidoglycan or other cell wall structural components or whether the wall preparations contained nonpeptidoglycan material despite the use of electron microscopy as a standard of purity; further work is required to supply the answer. The quantitative data furnish relative molar concentrations of amino acids, which can provide definitive identification of some of the species and differentiation of Actinomyces from other members of the Actinomycetales and from morphologically similar genera such as Corynebacterium and Propionibacterium.

Actinomyces↗

PubMed1973

Biosynthesis of prodigiosin, a secondary metabolite of Serratia marcescens.

Prodigiosenes (prodigiosin and prodigiosin-like pigments) are known to be synthesized by only one genus of Eubacteriales and by two genera of Actinomycetales. Biosynthesis by Serratia marcescens occurs over a relatively narrow range of temperatures, although the bacteria grow over a broad range. When cultures of S. marcescens were incubated at 27 C in 1.0% casein hydrolysate, viable count and protein attained maximal values within 24 to 48 h, whereas prodigiosin did not reach a maximum until 96 h. The greatest amount of pigment was synthesized when cultures were in the senescent phase of growth. Suspensions of nonproliferating bacteria incubated at 27 C in only L-alanine also synthesized prodigiosin, although at a slower rate than growing cultures. Kinetics of growth for the wild-type, red S. marcescens and a white mutant were identical when incubated at 27 C, but the wild type produced abundant pigment. These results plus other data obtained from the literature suggest that prodigiosin is a secondary metabolite. The importance of this proposal to understanding the function of prodigiosin in S. marcescens is discussed.

Aerobiosis↗

PubMed2006

Type III pilus of corynebacteria: Pilus length is determined by the level of its major pilin subunit.

Multiple pilus gene clusters have been identified in several gram-positive bacterial genomes sequenced to date, including the Actinomycetales, clostridia, streptococci, and corynebacteria. The genome of Corynebacterium diphtheriae contains three pilus gene clusters, two of which have been previously characterized. Here, we report the characterization of the third pilus encoded by the spaHIG cluster. By using electron microscopy and biochemical analysis, we demonstrate that SpaH forms the pilus shaft, while SpaI decorates the structure and SpaG is largely located at the pilus tip. The assembly of the SpaHIG pilus requires a specific sortase located within the spaHIG pilus gene cluster. Deletion of genes specific for the synthesis and polymerization of the other two pilus types does not affect the SpaHIG pilus. Moreover, SpaH but not SpaI or SpaG is essential for the formation of the filament. When expressed under the control of an inducible promoter, the amount of the SpaH pilin regulates pilus length; no pili are assembled from an SpaH precursor that has an alanine in place of the conserved lysine of the SpaH pilin motif. Thus, the spaHIG pilus gene cluster encodes a pilus structure that is independently assembled and antigenically distinct from other pili of C. diphtheriae. We incorporate these findings in a model of sortase-mediated pilus assembly that may be applicable to many gram-positive pathogens.

Bacterial Proteins↗

PubMed2006

The crystal structure of Rv0813c from Mycobacterium tuberculosis reveals a new family of fatty acid-binding protein-like proteins in bacteria.

The gene Rv0813c from Mycobacterium tuberculosis, which codes for a hypothetical protein of unknown function, is conserved within the order Actinomycetales but absent elsewhere. The crystal structure of Rv0813c reveals a new family of proteins that resemble the fatty acid-binding proteins (FABPs) found in eukaryotes. Rv0813c adopts the 10-stranded beta-barrel fold typical of FABPs but lacks the double-helix insert that covers the entry to the binding site in the eukaryotic proteins. The barrel encloses a deep cavity, at the bottom of which a small cyclic ligand was found to bind to the hydroxyl group of Tyr192. This residue is part of a conserved Arg-X-Tyr motif much like the triad that binds the carboxylate group of fatty acids in FABPs. Most of the residues forming the internal surface of the cavity are conserved in homologous protein sequences found in CG-rich prokaryotes, strongly suggesting that Rv0813c is a member of a new family of bacterial FABP-like proteins that may have roles in the recognition, transport, and/or storage of small molecules in the bacterial cytosol.

Amino Acid Sequence↗

PubMed2003

The cell wall of the pathogenic bacterium Rhodococcus equi contains two channel-forming proteins with different properties.

We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA(Req) (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA(Req) has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB(Req) [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.

Animals↗

PubMed2006

Assembly of distinct pilus structures on the surface of Corynebacterium diphtheriae.

Different surface organelles contribute to specific interactions of a pathogen with host tissues or infectious partners. Multiple pilus gene clusters potentially encoding different surface structures have been identified in several gram-positive bacterial genomes sequenced to date, including actinomycetales, clostridia, corynebacteria, and streptococci. Corynebacterium diphtheriae has been shown to assemble a pilus structure, with sortase SrtA essential for the assembly of a major subunit SpaA and two minor proteins, SpaB and SpaC. We report here the characterization of a second pilus consisting of SpaD, SpaE, and SpaF, of which SpaD and SpaE form the pilus shaft and SpaF may be located at the pilus tip. The structure of the SpaDEF pilus contains no SpaABC pilins as detected by immunoelectron microscopy. Neither deletion of spaA nor sortase srtA abolishes SpaDEF pilus formation. The assembly of the SpaDEF pilus requires specific sortases located within the SpaDEF pilus gene cluster. Although either sortase SrtB or SrtC is sufficient to polymerize SpaDF, the incorporation of SpaE into the SpaD pili requires sortase SrtB. In addition, an alanine in place of the lysine of the SpaD pilin motif abrogates pilus polymerization. Thus, SpaD, SpaE, and SpaF constitute a different pilus structure that is independently assembled and morphologically distinct from the SpaABC pili and possibly other pili of C. diphtheriae.

ATP-Binding Cassette Transporters↗

PubMed1990

DNA probes for the identification of Nocardia asteroides.

DNA probes for the rapid identification of Nocardia asteroides were obtained by constructing a genomic library of strain GUH-2 in the lambda cloning vector EMBL3. Of 50 recombinant clones tested, 2 were identified that hybridized with 31% of the N. asteroides strains in a reference collection without cross-hybridization with related members of the Actinomycetales. Additional libraries were then generated from selected strains of N. asteroides that had failed to hybridize with any of the GUH-2 clones. Four additional clones were obtained from these strains which, when pooled, provided DNA probes specific for all of the N. asteroides strains tested.

DNA Probes↗