الثلاثاء، 5 مايو، 2009

Actinomycetes

Actinomycetes


Actinomycetes are best known for their ability to produce antibiotics
and are gram positive bacteria which comprise a group of
branching unicellular microorganisms. They produce branching
mycelium which may be of two kinds viz., substrate mycelium and
aerial mycelium. Among actinomycetes, the streptomycetes are the
dominant. The non‐streptomycetes are called rare actinomycetes,
comprising approximately 100 genera.
Members of the actinomycetes, which live in marine
environment, are poorly understood and only few reports are available
pertaining to actinomycetes from mangroves (Siva Kumar, 2001;
Vikineswari et al. 1997; Rathana Kala & Chandrika, 1993;
Lakshmanaperumalsamy, 1978).
Isolation of Actinomycetes from Sediments For isolation actinomycetes, the samples can be collected by inserting a polyvinyl corer (10cm dia.) (previously sterilized with alcohol) into the sediments.
The corer is sterilized with alcohol before sampling at each station. The
central portion of the top 2 cm sediment sample can be taken out with
the help of a sterile spatula. This sample can be transferred to a sterile
polythene bag and transported immediately to the laboratory. The sediment samples thus collected are air‐dried aseptically. After a week, the sediment samples are to be incubated at 550 C for 5 min (Balagurunathan, 1992). Then, 10‐fold serial dilutions of the sediment samples should be prepared, using filtered and sterilized 50% seawater.
One ml of the serially diluted samples should be plated in the Kuster’s
Agar (Siva Kumar, 2001) in triplicate petriplates. To minimize fungal
contamination, all agar plates should be supplemented with 50 ug/ml of nystatin. The actinomycete colonies that appear on the petriplates can be
counted from 5th day onwards, upto 28th day. All the colonies that are growing on the petriplates can be separately streaked in petriplates, subcultured, ensured for their axenicity and maintained in slants.



Identification of Actinomycets.
Various approaches for the identification of actinomycets are given briefly below:
a) Molecular Approach
The most powerful approaches to taxonomy are through the study of
nucleic acids. Because these are either direct gene products or the genes
themselves and comparisons of nucleic aids yield considerable
information about true relatedness.
Molecular systematics, which includes both classification and
identification, has its origin in the early nucleic acid hybridization
studies, but has achieved a new status following the introduction of
nucleic acid sequencing techniques (O’Donnell et al., 1993). Significance
of phylogenetic studies based on 16S rDNA sequences is increasing in
the systematics of bacteria and actinomycetes (Yokota, 1997). Sequences
of 16S ribosomal DNA have provided actinomycetologists with a
phylogenetic tree that allows the investigation of evolution of
actinomycetes and also provides the basis for identification.
Analysis of the 16S rDNA begins by isolating DNA (Hapwood,
1985) and amplifying the gene coding for 16S rRNA using the
polymerase chain reaction (e.g. Siva Kumar, 2001). The purified DNA
fragments are directly sequenced. The sequencing reactions are performed
using DNA sequencer in order to determine the order in which the bases
are arranged within the length of sample (Xu Li‐Hua, et al., 1999) and a
computer is then used for studying the sequence for identification using
phylogenetic analysis procedures. However, analysis of 16S rDNA
generally allows us to identify the organisms upto the genus level only.
b) Chemotaxonomical Approach
Chemotaxonomy is the study of chemical variation in organisms and the
use of chemical characters in the classification and identification. It is
one of the valuable methods to identify the genera of actinomycetes.
Studies of Cummins and Harris (1956) established that
actinomycetes have a cell wall composition akin to that of gram‐positive
bacteria, and also indicated that the chemical composition of the cell
wall might furnish practical methods of differentiating various types of
actinomycetes. This is because of the fact that chemical components of
the organisms that satisfy the following conditions, have significant
meaning in systematics.
K. Sivakumar 199
i. They should be distributed universally among the
microorganisms studied; and,
ii. The components should be homologous among the strains
within a taxon, while significant differences exist between the
taxa to be differentiated.
Presence of Diaminopimelic Acid (DAP) isomers is one of the
most important cell‐wall properties of gram‐positive bacteria and
actinomycetes. Most bacteria have a characteristic wall envelope,
composed of peptidoglycan. The 2, 6‐ Diaminopimelic Acid (DAP) is
widely distributed as a key aminoacid and it has optical isomers. The
systematic significance lies mostly in the key aminoacid with two amino
bases, and determination of the key aminoacid is usually sufficient for
characterisation. If DAP is present, bacteria generally contain one of the
isomers, the LL‐form or the meso‐form, mostly located in the
peptidoglycan. Major constituents of cell wall of actinomycetes (Lechevalier and
Lechevalier, 1970) are as follows:

I + +
II + +**
III +
** hydroxy DAP (may also be present)
The sugar composition often provides valuable information on
the classification and identification of actinomycetes. Actinomycete cells
contain some kinds of sugars, in addition to the glucosamine and
muramic acid of peptidoglycan. The sugar pattern plays a key role in the
identification of sporulating actinomycetes which have meso‐DAP in
their cell walls. However, the actinomycetes which have LL‐DAP along
with glycine (wall chemo type‐I) have no characteristic pattern of sugars
(Lechevalier and Lechevalier, 1970) and hence the whole cell sugar test
has not received much attention here.
c) Classical Approach
Classical approaches for classification make use of morphological,
physiological, and biochemical characters. The classical method
200 Actinomycetes
described in the identification key by Nonomura (1974) and Bergey’s
Manual of Determinative Bacteriology (Buchanan & Gibbons, 1974) is
very much useful in the identification of streptomycetes. These
characteristics have been commonly employed in taxonomy of
streptomycetes for many years. They are quite useful in routine
identification. They are as follows.
- Aerial Mass Colour
The colour of the mature sporulating aerial mycelium is recorded in a
simple way (White, grey, red, green, blue and violet). When the aerial
mass colour falls between two colour series, both the colours are
recorded. If the aerial mass colour of a strain to be studied shows
intermediate tints, then also, both the colour series are noted.
- Melanoid Pigments
The grouping is made on the production of melanoid pigments (i.e.
greenish brown, brownish black or distinct brown, pigment modified by
other colours) on the medium. The strains are grouped as melanoid
pigment produced (+) and not produced (‐).
- Reverse Side Pigments
The strains were divided into two groups, according to their ability to
produce characteristic pigments on the reverse side of the colony,
namely, distinctive (+) and not distinctive or none (‐). In case, a colour
with low chroma such as pale yellow, olive or yellowish brown occurs, it
is included in the latter group (‐).
. Soluble Pigments
The strains are divided into two groups by their ability to produce
soluble pigments other than melanin: namely, produced (+) and not
produced (‐). The colour is recorded (red, orange, green, yellow, blue
and violet).
Spore Chain Morphology
With regard to spore chains, the strains can be grouped into ‘sections’.
The species belonging to the genus Streptomyces are divided into three
sections (Shirling & Gottlieb, 1966), namely rectiflexibiles (RF),
retinaculiaperti (RA) and Spirales (S). When a strain forms two types of
spore chains, both are noted (e.g. SRA).
K. Sivakumar 201
Characteristics of the spore‐bearing hyphae and spore chains
should be determined by using direct microscopic examination of the
culture surface. Adequate magnification (400x) could be used to
establish the presence or absence of spore chains and to observe the
nature of sporophores.
Spore morphological characters of the strains can be studied by
inoculating a loopful of one week old cultures into 1.5% agar medium
contained in test tubes at 370C. The actinomycete should be suspended
and thoroughly mixed in the semisolid agar medium and 1 or 2 drops of
the medium could be aseptically pipetted on to a sterile glass slide. A
drop of agar should be spread well on the slide and allowed to solidify
into a thin film so as to facilitate direct observation under microscope.
The cultures should be incubated at 28 + 20C and examined periodically
for the formation of aerial mycelium, sporophore structure and spore
morphology.
Spore Surface
Spore morphology and its surface features should be observed under the
scanning electron microscope. The cross hatched cultures prepared for
observation under the light microscope can be used for this purpose.
RF Spore chains (400X)
RA Spore chains (400X)
Spiral Spore chains (400X)
202 Actinomycetes
The electron grid should be cleaned and adhesive tape should be placed
on the surface of the grid. The mature spores of the strain should be
carefully placed on the surface of the adhesive tape and gold coating
should be applied for half an hour and the specimen can be examined
under the electron microscope at different magnifications. The spore
silhouettes can be characterized as smooth, spiny, hairy and warty.
Assimilation of Carbon Source
The ability of different actinomycete strains in utilizing various carbon
compounds as source of energy should be studied following the method
recommended by International Streptomyces Project (Shirling and
Gottlieb, 1966).
Chemically, pure carbon sources, certified to be free of
admixture with other carbohydrates or contaminating materials, should
be used for this purpose. Carbon sources for this test could be arabinose,
xylose, inositol, mannitol, fructose, rhamnose, sucrose and raffinose.
These carbon sources should be sterilized by ether sterilization without
heating.
Comparing the properties of the isolated strain with the
representative species found in the key of Nonomura (1974) and
Bergey’s Manual of Determinative Bacteriology (1974, 1989, 1994 & 2005) can help in the species level identification. If the isolated strain
could not be assigned to any of the valid representatives listed in the key
of Nonomura (1974) and Bergey’s Manual of Determinative Bacteriology
(Buchanan & Gibbons, 1974), then it can be identified based on the
numerical taxonomic studies.
d) Numerical Taxonomic Approach
Numerical taxonomy involves examining many strains for a large
number of characters prior to assigning the test organism to a cluster
based on shared features. The numerically defined taxa are polythetic;
so, no single property is either indispensable or sufficient to entitle an
organism for membership of a group. Once classification has been
achieved, cluster‐specific or predictive characters can be selected for
identification (Williams et al, 1983).
Numerical taxonomy was first applied to Streptomyces by
Silvestri et al. (1962). The numerical taxonomic study of the genus
Streptomyces by Williams et al. (1983) involves determination of 139 unit
characters for 394 type cultures of Streptomyces; clusters were defined at
77.5% or 81% Ssm and 63% Sj similarity levels, and the former co‐effieient
K. Sivakumar 203
is being used to define the clusters. His study includes 23 major, 20
minor and 25 single member clusters.
The numerical classification of the genus Streptomyces by
Kampfer et al. (1991) involves determination of 329 physiological tests.
His study includes 15 major clusters, 34 minor clusters and 40 single
member clusters which are defined at 81.5% similarity level Ssm using
the simple matching coefficient (Sokal and Michener, 1958) and 59.6 to
64.6% similarity level Sj using Jaccard coefficient (Sneath, 1957). Thus,
numerical taxonomy provides us with an invaluable framework for
Streptomyces taxonomy, including identification of species.
Preservation
The preservation methods are similar to that of bacteria such as subculturing, freezing especially in liquid nitrogen, freeze‐drying and
maintenance of strains in mineral oil.
Conclusion Studies on actinomycetes are very limited and the actinomycetes have been mentioned incidentally, on the microbial community of marine habitats. Further, only little information is available on the actinomycetes of the mangrove environment (which is one among the most productive coastal ecosystems) with regard to their occurrence and distribution (Vikineswari et al., 1997; Rathna Kala and Chandrika, 1993; Lakshmanaperumalsamy, 1978).

Actinomycetes from Sediments in the Trondheim Fjord, Norway. Diversity and Biological Activity
Abstract: The marine environment represents a largely untapped source for isolation of new microorganisms with potential to produce biologically active secondary metabolites. Among such microorganisms, Gram-positive actinomycete bacteria are of special interest, since they are known to produce chemically diverse compounds with a wide range of biological activities.
We have set out to isolate and characterize actinomycete bacteria from the sediments in one of the largest Norwegian fjords, the Trondheim fjord, with respect to diversity and antibiotic-producing potential. Approximately 3,200 actinomycete bacteria were isolated using four different agar media from the sediment samples collected at different locations and depths (4.5 to 450 m). Grouping of the isolates first according to the morphology followed by characterization of isolates chosen as group representatives by molecular taxonomy revealed that Micromonospora was the dominating actinomycete genus isolated from the sediments.
The deep water sediments contained a higher relative amount of Micromonospora compared to the shallow water samples. Nine percent of the isolates clearly required sea water for normal growth, suggesting that these strains represent obligate marine organisms. Extensive screening of the extracts from all collected isolates for antibacterial and antifungal activities revealed strong antibiotic-producing potential among them. The latter implies that actinomycetes from marine sediments in Norwegian fjords can be potential sources for the discovery of novel anti-infective agents.
Keywords: Actinomycete bacteria, fjord sediments, molecular taxonomy, antimicrobial activities
The demand for new antibiotics continues to grow due to the rapid spread of antibiotic-resistant pathogens causing life-threatening infections. Although considerable progress is being made within the fields of chemical synthesis and engineered biosynthesis of antimicrobial compounds, nature still remains the richest and the most versatile source for new antibiotics [1,2,3].
Bacteria belonging to the family Actinomycetaceae are well known for their ability to produce secondary metabolites, many of which are active against pathogenic microorganisms. Traditionally, these bacteria have been isolated from terrestrial sources although the first report of mycelium-forming actinomycetes being recovered from marine sediments appeared several decades ago [4]. It is only more recently that marine-derived actinomycetes have become recognized as a source of novel antibiotics and anti-cancer agents with unusual structures and properties [5].
Many microbiologists believe that free-living bacteria are cosmopolitan due to their easy dispersal [6]. However, chemical and physical factors contribute to selection of species and strains that are best adapted to that particular environment. Due to the broad bacterial species definition one may find members of one species in two very different environments [7,8]. However, comprehensive analysis of the recent studies strongly suggests that free-living microbial taxa exhibit biogeographic patterns [7,9]. Some of the unusual structures and properties of compounds isolated from marine sources and the fact that 58 % of the isolated actinomycetes from sediments collected around Guam in the Pacific ocean required sea water for growth [5] implies that one may find microorganisms adapted to the marine environment and producing compounds not found among microorganisms adapted to the terrestrial sources.
Fjords are narrow inlets of the sea, which have been formed as a result of marine inundation of glaciated valleys. Typical characteristics of a fjord include a relatively narrow inlet, significantly eroded bottom and communication with the open sea. The ecology of the microorganisms, especially bacteria, inhabiting the fjords is poorly studied. The Trondheim fjord (135 km long) differs from many other fjords by a large fresh water supplement from six major river systems. In one year these rivers bring fresh water to the fjord corresponding to 6.5 % of it total water volume [10]. From May until September the fjord contains a 5-25m thick layer of brackish surface water, depending on time, weather and location. In the brackish water layer the salt content varies from approximately 18 to 32 practical salinity units (PSU). The dissolved and particulate organic matter in the sediments from the fjord has both marine origin from phytoplankton and macroalgae as well as terrestrial from soil due to fresh water run-off, highly influenced by snow melting in April-May.
The Norwegian marine environments are largely unexplored, and may provide a rich source of the microorganisms producing novel and efficient anti-infective compounds. The purpose of this study was to investigate if fjord sediments from temperate areas could be suitable sources for isolation of mycelium-forming actinomycetes producing antimicrobial compounds.
Mar. Drugs 2008, 6(1) 14
Collection and analysis of the sediment samples
Sediment samples from two different locations and five depths were collected and processed (see Table 1 for description and ID). The first site (B-site) was close to the shore and here samples were collected at depths of 4.5, 6, 27 and 28 m. The 4.5 m and 6 m sampling sites were situated in the kelp forest belt and the 6 m sampling site had a brown layer of sedimented micro algae on the surface.
At 27 and 28 m depth, the sediment surface was dominated by sand/silt. The second site (T-site) was located in the middle of the fjord and here samples were taken at 450 m depth, where the sediment was dominated by clay particles. Visual inspection indicated a higher content of fine organic matter than in the other sediments. Similar to the B2 sample the sediments from this location also had a layer of dead micro algae on the surface.
Table 1. Description of sediments and sampling sites.






The contents of organic material in the different sediment samples were measured as total carbon (C) and nitrogen (N), in order to test possible correlation with actinomycete microbiota and the ability to produce antimicrobial compounds. The highest content of organic carbon (1.8 %) and nitrogen (0.15 %) was found in the T1 sediment (Table 1). The contents of organic carbon and nitrogen in the B-site sediments varied from 0.6 to 1.1 % and 0.04 to 0.06 %, respectively, where the B2 sample was clearly the one with highest content of the organic matter. Higher total N over C ratio may indicate a higher content of more rapidly degradable organic material [11]. The highest N/C ratio was found for the B3 and T1 sediments, suggesting the presence of more easily degradable organic matter in these samples.
Isolation of actinomycete bacteria
Settled suspensions of the sediments were plated onto four different selective media adding up to a total of 320 primary isolation plates (diameter 14 cm) of which 248 (78%) yielded myceliumforming actinomycete colonies. The total number of actinomycetes observed on the primary isolation plates was 7874. An average of 24.6 mycelium-forming actinomycete colonies per plate were observed with numbers increasing to 31.8 when only considering those plates that yielded actinomycetes.
3200 colonies were picked based on actinomycete-like morphology, of which around 900 formed powdery colonies with well-developed aerial hyphae fragmented into spore chains. Thes isolates were tentatively termed as Streptomyces-like actinomycetes. The main part of the remaining isolates formed orange to red pigmented colonies with solid colony texture, non fragmenting substrat Mar. Drugs 2008, 6(1) 15 mycelium that lacked aerial hyphae and often turned purple, brown or black upon sporulation, an was tentatively termed as MNSA (mycelium-forming non-streptomycete actinomycetes). The tota CFU (colony forming units) for Streptomyces-like and MNSA isolates were registered (Table 2). Th highest total CFU was found on soil agar from the B1 sample and the highest number of MNS colonies was found in the same sediment when plated onto chitin agar (IM7b). Highest number o Streptomyces-like colonies was found when Biologen 6 m sediments wer plated on to IM6 (modifie Kusters) agar Table 2. Viable counts of bacteria and actinomycetes in sediments from different depths in the Trondheim fjord after plating onto selective agar media. The number are mean values of CFU (colony forming units) per mL wet sediment









The T1 sample contained the highest relative number of MNSA colonies (93 %) and the lowest number of Streptomyces-like colonies (0.7 %) (Table 2). However the total CFU on selective media from this sediment was approximately one tenth of that from the B-site sediments. For the latter sediments the highest relative numbers of MNSA colonies varied from 17 to 30 % when plated on to IM7b agar. In general, the IM7b (colloid chitin) gave the highest numbers of MNSA colonies with the exception of the T1 sample where IM6 was clearly the best in this respect. IM5 (humic acid sea water Mar. Drugs 2008, 6(1) 16 agar) was the isolation medium that gave the lowest number of mycelium-forming actinomycetes from fjord sediments.
Different types of selective pre-treatments were applied in order to increase the number of mycelium-forming actinomycetes relative to the non-actinomycetal heterotrophic microbial flora.
These treatments included dry heat, phenol treatment, dry heat followed by phenol treatment, dry heat followed by benzethonium chloride treatment, and pollen baiting. For the B-site sediment samples (Figure 1) it was possible to obtain increased relative numbers of actinomycetes on the agar plates with all the different types of pretreatments, except for the pollen baiting, which did not yield any isolates. With some of the pretreatments (dry heat followed by phenol treatment), it was possible to obtain isolation plates only containing actinomycetes with morphologies typical for the genera Micromonospora.
Figure 1. The effect of different types of pre-treatments, applied to the sediments samples, on the relative numbers of actinomycetes appearing on the isolation plates




However, for the T1 (450 m) samples these pretreatments were detrimental, and apparently eliminated most of the actinomycete microbiota. We were not able to isolate any mycelium-forming actinomycetes from the sediments with the pollen baiting technique, suggesting that actinomycetes producing zoospores are rare in the fjord sediments we have investigated.
Sea water requirement
All original media for isolation of actinomycetes contained sea water, and we decided to test whether the presence of this media component can be crucial for growth of isolated actinomycetes.
Therefore, the isolates were transferred to the respective media with or without sea water, and their Mar. Drugs 2008, 6(1) 17 growth was monitored over a period of 8 weeks. Growth of approximately 8 % percent of the isolates from T1 sample was found to be completely dependent on the presence of sea water, while the respective average figure for the B-site samples was 9 %. The individual percentages for the B-site samples were: 4.5 m; 8 %; 6 m; 10 %, 27 m; 5 % and 28 m; 8 %. In addition, around 20 % of the isolates grew considerably faster on agar media containing sea water. The major part of the isolates (50 %) did not show any clear preference for media with or without sea water, while around 20 % of the isolates grew better in the absence of sea water.
Figure 2. The percentage of the mycelium-forming actinomycete isolates
displaying antibacterial activities against Micrococcus luteus and antifungal activities against Candida albicans in agar diffusion assays. A. Non-streptomycete isolates. B. Streptomyces-like isolates.









Biological activities

The 3,200 isolated actinomycete bacteria were transferred to three different solid (agar) production media. After an incubation period of one to six weeks depending on growth rate of the isolates, the media and the cells were dried and extracted with DMSO. The extracts were tested for antibacterial and antifungal activity against the Gram-positive bacterium Micrococcus luteus and the yeast Candida albicans using traditional agar diffusion assays. The results of this analysis are presented in Figure 2.
Somewhat surprisingly, the MNSA isolates from the T1 sediments showed the highest frequency of activity against Gram-positive bacterium M. luteus (58 %) and yeast C. albicans (39 %). For the B-site MNSA isolates the frequency of the corresponding activities varied between 25-32%and 13-21%, respectively.
The frequency of activities against M. luteus and C. albicans among the Streptomyces-like strains varied between 34-47 % and 23-42 %, respectively. Within this group, the highest frequency of antibacterial activities was found among the isolates from the B2 sample and the highest antifungal activity among the B1 sample.

Molecular taxonomy
Examination of the colony morphology of the actinomycete isolates suggested that they may tentatively belong to the genera Streptomyces and Micromonospora. In order to confirm preliminary classification, we selected 30 isolates from the T1 sample having different “representative” morphologies.
These isolates were morphologically more homogeneous than the B-site isolates. Fragments of the 16S rRNA genes from these isolates were PCR-amplified, sequenced, and a phylogenetic tree was constructed, allowing the sorting of the sequences into seven different clusters (Figure 3). Cluster 1 consisted of 12 isolates showing 99 % to 100 % homology to Micromonospora matsumotoense. All displayed moderate antibacterial activity, except MP38-F9 and MP38-F5 which displayed strong and no activity respectively. They all also produced a weak antifungal activity, except MP38-F5. Cluster 2 contained four isolates showing 99 to 100 % homology to Micromonospora sp. e24. None of these isolates showed antibacterial or antifungal activity. The six isolates in Cluster 3 showed 99 to 100 % identity to M. chokoriensis. Of these isolates only MP38-A6 showed antimicrobial activity which was moderate antibacterial and weak antifungal. Cluster 4 consisted of one isolate with its partial 16S rDNA sequence showing 99 % identity to M. aurantiaca. This isolate showed no antibacterial or antifungal activity. Cluster 5 contained three isolates that showed 99 % homology to M. marina. Two of the isolates MP38-E11 and MP38-D12 displayed moderate antibacterial activity. Cluster 6 consisted of one isolate showing 100 % homology to Verrucosispora gifhornensis. No antimicrobial activity was detected for this strain. Cluster 7 contained three isolates which showed 99 % homology to M.
chersina, all of them showing antibacterial activity. Despite clear division of the isolates to different clusters according to the partial 16S rDNA sequences, there was just as much morphological variation among the isolates within the clusters as between the different clusters. For example, among 12 Micromonospora isolates from Cluster 1, only three have shown differences in the partial 16S rDNA sequences. However, all isolates in this cluster exhibited differences in morphology, and some of them were clearly different in terms of biological activity profiles.
Mar. Drugs 2008, 6(1) 19
Figure 3. Phylogenetic relationship of partial 16S rDNA sequences generated in
this study, rooted using the 16S rDNA sequence of Streptomyces coelicolor. See
Material and methods for tree description. Numbers at tree nodes represent the
number of times the topology to the right of the node was recovered in 1000
bootstrap re-samplings; values lower than 50 are not shown. Accession numbers
for the sequences are in parentheses. Scale bar represents the number of changes per base position.












Discussion
The Trondheim fjord is approximately 135 km long and is characterized by the large water supply from six rivers entering the fjord. The dissolved and particulate organic matter in the sediments from the fjord originates from both marine phytoplankton and macro algae, and terrestrial material from the Mar. Drugs 2008, 6(1) 20 run-off caused by snow melting in the spring brought to the fjord by the rivers. An average of 9 % of the actinomycete isolates in this investigation required sea water for growth, most of them originating from the deep-water sediment sample. The latter can probably be explained by a better adaptation of the bacteria in this sediment to the sea water environment, since fresh water supply at such depth shall be minimal. Jensen et al. [5] reported that as much as 58 % of the actinomycetes, isolated from sediment samples collected around the island of Guam required sea water for growth. While the Trondheim fjord is a sea inlet into the terrestrial environment, Guam is positioned far out into the Pacific Ocean. These facts suggests that although there is a clear evidence of metabolically active marine actinomycetes in the fjord sediments, there is also a considerable number of actinomycetes ofterrestrial origin present as well.
Micromonospora and Streptomyces-like actinomycetes were the dominating actinomycetes isolated from the near shore shallow water sediments, where the numbers of streptomycetes decreased with depth and distance from the shore, as have also been reported by others [12]. However, a considerable number of isolates displayed morphologies that did not conform with these two genera. Their relative phylogenetic positions are under investigation and will be reported elsewhere.
The use of different types of selective treatments originally designed for selective isolation of actinomycetes from soil increased the relative numbers of actinomycetes on the agar plates inoculated from the shallow water (4.5 to 28 m) near shore samples. Although these techniques have previously been applied with success for isolation of groups of actinomycetes from soil [13-15], no reports exist to our knowledge on effectiveness of such techniques applied to the marine sediments from temperate areas. For the deep-water sediment samples (450 m) these treatments were clearly detrimental, mainly resulting in agar plates free for actinomycetes. This suggests that the selective pre treatments designed to isolate actinomycetes from soil are not optimal for marine samples and more effort is required in order to establish methods allowing specific enrichment of marine actinomycetes. The dominating actinomycete genus isolated from the deep sediments under the conditions tested was Micromonospora.
Some of the selective treatments as for example dry heat at 120 ◦C for 60 min and exposure to 1.5 % phenol is indeed designed for the selective isolation of Micromonospora. However, very few Micromonospora isolates from the deep water samples survived these treatments. This could suggest that although their relative phylogenetic position determined by 16S rDNA is identical or close to known terrestrial organisms, they still have signs of adaptation to their marine environment. Micromonospora species growing at 450 m experience an environment with relatively low and constant ambient temperature (8 ◦C), stable and high salinity, and are not exposed to desiccation and may therefore differ significantly from their terrestrial counterparts. Without the use of selective pre-treatments the relative numbers of actinomycetes (mainly Micromonospora) could account for as much as 90 % of the colonies on some of the selective agar media. At the moment, however, we can not exclude the possibility that the dominance of Micromonospora on our isolation plates was caused by the chosen isolation procedures, and that other actinomycetes are also present in considerable numbers in these sediments.
Although we have analyzed too few samples to draw any solid conclusions, the trend in our results was that the percentage of isolates displaying activity against M. luteus among the MNSA isolates was roughly proportional to the carbon content in the different sediments. That is, the percentage of mycelium-forming actinomycetes with this activity increased with the content of organic carbon Mar. Drugs 2008, 6(1) 21 in the sediments (Table 1 and Figure 2). No correlation could be seen between sampling depth and antimicrobial activity among MNSA isolates. With the exception of the T1 sample (collected from 450 m depth), there was a decrease in the percentage of Streptomyces-like isolates displaying activity against C. albicans with increasing sampling depth. At the same time, we are aware of the fact that our test organism, Candida albicans, is of terrestrial origin, and thus we might have missed antibiotic activities directed against marine fungi.
Despite the fact that the T1 sample had the highest content of organic carbon and nitrogen, it gave the lowest CFU number on our selective media. It also contained less morphological diversity among the actinomycete isolates than what was found for the shallow water B-site samples. The reason for this is most probably our failure to cultivate many of the actinomycetes present in the sample due to their need for special cultivation conditions. This is supported by previous reports where high actinobacterial diversity was found in marine sediments by constructing actinobacteriumspecific 16S rDNA clone libraries. Furthermore, the information from the cultivation-independent techniques could be used to improve the recovery of novel actinobacteria [16-18]. Even though, the diversity and biological activities of actinomycetes from the Trondheim fjord sediments unraveled so far suggests that they might be a rich source for discovery of new anti-infective agents.
Experimental Section
Sample collection and isolation of bacteria
Sediments from 4.5, 6.0, 27 and 28 m depths were collected by scuba divers, while sediments from 450 m depth were sampled with a box-corer. The upper 5 cm of the sediments were collected in zip-lock bags (scuba) or with a sterile spade (box –corer) and transferred to 1 liter sterile plastic containers. Approximately 10 % of the container volume was filled with 60 % sediment and 40 % sea water from the sampling site. This was done in order to ensure aerobic conditions under storage upon processing. Samples were processed the same day or the day after sampling. All storage was done in the dark at 4 oC. Sediments were diluted 1:10 v/v with sterile sea water and vigorously shaken with glass beads for 30 sec. Settled (5 min) sediment suspensions were plated on to different selective agar media and incubated at 20 ◦C for two to six weeks. Selective treatments were performed on dried sediments (Speedvac 30 ◦C, 16 h) and included dry heat (120 ◦C, 60 min), phenol (1.5 %, 30 min at 30 ◦C), dry heat and phenol, dry heat and benzethonium chloride (0.02 %, 30 min at 30 ◦C), as well as pollen baiting [19,20].
Carbon and nitrogen content analysis
Sediment samples were dried and ground with a pester and mortar, before the total carbon (C) and nitrogen (N) content in the sediment samples were measured using a NA 1500 Nitrogen/Carbon/Sulphur analyzer from Carlo Erba Instruments. To ensure good average values each sediment sample was analyzed in five parallels.
Mar. Drugs 2008, 6(1) 22
Isolation and production media
Isolation media consisted of the following: IM5 (humic acid agar [21], with sea water), humic acid (1 g), K2HPO4 (0.5 g), FeSO4•7H2O (1 mg), agar (20 g), vitamin B solution (1 mL), natural sea water (0.5 L) and distilled water (0.5 L); IM6 glycerol (0.5 g), starch (0.5 g), sodium propionate (0.5 g), KNO3 (0.1 g), asparagine (0.1 g), casein (0.3 g), K2HPO4 (0.5 g), FeSO4•7H2O (1 mg), agar (20 g), vitamin B solution (1 mL), natural sea water (0.5 L) and distilled water(0.5 L); IM7 (chitin agar [9], with sea water) chitin (Sigma), K2HPO4 (0.5 g), FeSO4•7H2O (1 mg), agar (20 g), vitamin B solution (1 mL), natural sea water (0.7 L) and distilled water (0.3 L); IM8, malt extract (1 g), glycerol (1 g), glucose (1 g), peptone (1 g), yeast extract (1 g), agar
(20 g), natural sea water (0.5 L) and distilled water (0.5 L). The pH of the isolation
media was adjusted to pH 8.2. Vitamin B solution consisted of the following:
thiamine-HCl (50 mg), riboflavin (50 mg), niacin (50 mg), pyridoxine-HCl (50 mg),
inositol (50 mg), Ca-pantothenate (50 mg), p-aminobenzoic acid (50 mg), biotin
(25 mg) and distilled water (100 mL). All isolation media were amended with filtered (0.2-µm pore size) cycloheximide (50 µg/mL), nystatin (75 µg/mL) and nalidixic acid (30 µg/mL).
Production media: PM2, mannitol (5.0 g), soya bean flour (5.0 g), Clerol (antifoam, 0.1 g), dry yeast (0.9 g), agarose (10.0 g), tap water (1 L); PM3, oatmeal (20 g), glycerol (2.5 g), FeSO4•7H2O (0.1 mg), MnCl2•4H2O (0.1 mg), ZnSO4•7H2O (0.1 mg), agarose (10 g), tap water (1 L); PM4, glucose (0.5 g), glycerol (2.5 g), oatmeal (5.0 g), soybean meal (5.0 g), yeast extract (0.5 g), casaminoacids (2.0 g), CaCO3 (2.0 g), Clerol (0.1 g), agarose (10 g) and tap water (1 L).
Media for nucleic acid extraction: organic agar Gause 2 (modified), glucose (10 g), trypton (3 g), peptone (5 g), agar (20 g), tap water (0.5 L) and sea water (0.5 L).
Plates with isolation and production media were incubated at 20 oC for periods of 2 to 6 weeks.
Cultivation, extraction and bioactivity testing
The 3200 isolated mycelium-forming actinomycetes were transferred to three different solid (agar) production media PM2, PM3 and PM4 in 96-well plates. After an incubation at 20 ◦C for one to six weeks, depending on growth rate of the isolates, the media and the cells were dried directly in the plates, and extracted with dimethylsulfoxide (DMSO, Sigma, 200 µL). The extracts were tested for antibacterial and antifungal activity against the Gram-positive bacterium Micrococcus luteus ATCC 9341 and the yeast Candida albicans ATCC 10231 using traditional agar diffusion assays. Each DMSO extract (1 µL) was applied onto the surface of the agar inoculated with the test organism and activity registered as inhibition zones after 16 hours of incubation at 34 ◦C.
Nucleic acid extraction,
16S rDNA amplification, sequencing and analysis Fresh colonies grown on Gause 2 organic agar [22] were macerated and transferred to sterile distilled water (100 µL) and heated to 98 ◦C for 10 min. The suspensions were centrifuged (5000 x g, 1 min) and DNA from the clear supernatant precipitated with three volumes of ethanol, centrifuged (12000 rpm, 15 min) and pellets dissolved in distilled water to the original volume.
16S ribosomal DNA (rDNA) sequencing templates were amplified from genomic
Mar. Drugs 2008, 6(1) 23 DNA by PCR using previously described [18] actinomycete specific primers
S-C-Act-235-S-20 (5’-CGCGGCCTATCAGCTTGTTG-3’) and S-C-Act-878-A-19
(5’-CCGTACTCCCCAGGCGGGG-3’). Reaction mixture (50 µL) contained genomic DNA extract (1 µL), Thermopol Buffer (New England Biolabs), DMSO (2 µL), Bovine serum albumin (2 µL), deoxynucleoside triphosphate mixture (2.5 pmol), each primer (20 pmol), and Taq DNA polymerase (2.5 U). All sequencing reactions were carried out with an ABI PRISM 3100 genetic analyzer at the Department of Biology, The Norwegian University of Science and Technology. DNA sequences were deposited to GenBank under accession numbers DQ645597 to DQ645626. The 16S rDNA sequences (500-625 bp) were used to search the GeneBank database with the BlastN algorithm to reveal closest matches to the 16S rDNA sequences for known species. Sequences were aligned with representative actinomycete 16S rDNA sequences and a phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 [23].












































































Actinomycetes

CHARACTERISTICS OF SOIL ACTINOMYCETES
FROM ANTARCTICA

Forty-seven actinomyces strains were isolated from Antarctic soils – nineteen of them showed antagonistic activity against Gram-positive and Gram-negative bacteria.
Six of the strains possessed a broad spectrum of antibacterial activity. Results obtained from the physiological and biochemical analyses including determination of 39 characteristics proved that two of the strains (23 and 29) were similar whereas all the rest differed among each other. Morphological studies indicated that the strains belonged to the genera Streptomyces, Actinomadura and Kitasatosporia.
Antibacterial activity of three actinomycetes strains (designed as 29, 30 and 47) was confirmed in batch culture. They were active against clinical isolates from the species Staphylococcus aureus and Streptococcus pneumoniae. The three strains also showed antibacterial activity against the phytopathogenic bacteria Xanthomonas axonopodis pv. glycines, X. vesicatoria, X. axonopodis pv. phaseoli, Pseudomonas syringae pv. tomato and Clavibacter michiganensis, for which no biological means for control, had been developed yet. The broadest spectrum of antibacterial action had the strain 29. The antibacterial compounds produced by these strains probably possessed non-polar structure and consisted of several active components.


ACTINOMYCETES ISOLATED FROM SOIL SAMPLES FROM THE CROCKER RANGE SABAH




A diversity of actinomycetes was isolated from various sites of top soils throughout the Crocker Range in Sabah. The soils were mainly collected during the expedition (15—25 October 1999) together with 2 soil samples collected on 28 November 1999 under Rafflesia keithii in the Rafflesia Reserve Forest, Gunung Mas. A total of 78 strains of actinomycetes, probably mostly Streptomyces, were obtained from different sites. Amongst these strains 20 have been aerobically grown in shaking liquid cultures. Acetone extracts of these cultures were screened for MAPK Kinase and MAP Kinase Phosphatase in a yeast system in the preliminary screening of novel cancer drugs. This screening system is based on the fact that the MAP kinase pathway is homologues from yeast to human. However, no such inhibitors were found. A few strains with pigmentation were collected from specific locations.

INTRODUCTION

Actinomycetes are gram positive bacteria frequently filamentous and sporulating with DNA rich in G+C from 57—75%. Some of their secondary metabolites have employed as useful microbial compounds (Prescott, Harley & Klein, 1993). Examples include streptomycin from Streptomyces griseus for treatment of tuberculosis caused by Mycobacterium tuberculosis and the immunosuppress drug, tacrolimus (FK506) produced by S. tsukubaensis. Actinomycetes of about 100 genera exist in soils (Yokota, 1997). In their natural habitat, such as forests, the actinomycetes interact in various ways with the higher plants.
The fallen tress, barks and flowers first provide nutrients both to the microbes and plants through microbial degradation of carbohydrates, lipids and proteins to sugars, fatty acids, glycerol and amino acids and ultimately to mineralisation. Besides providing these nutrients, plant secondary metabolites (such as dipterocarp resins) that are generally toxic to microorganisms, will need to be degraded or detoxified by certain microbes. These degraders (microbes) are selectively pressured and ultimately evolve to produce novel secondary metabolites of their possibly to counteract the toxic plant secondary metabolites (Park et al, 1999 and Ho et al, 2000).
In this study, soil samples were collected in different habitats in the Crocker Range National Park to investigate the diversity of actinomycetes. Actinomycetes were then isolated on selective medium humic-acid + modified B vitamins and extracts were screened for biological activities of the secondary metabolites.

Soil samples: Soil samples were collected by sterile method from various locations visited throughout this scientific expedition to Crocker Range Park (Figure 1), from an area of mist forest (1400—1500m from sea level), submontane rain forest (Mahua), hill forest (uphill of Mensalog River, Ulu Senagang) to cultivated areas (of introduced Theobroma cacao and Tectonia grandis). Soil samples were air-dried under room temperature for about 30 days before isolation (Table 1). A second set (Table 2) used air-dried soils stored at room temperature over a long period (9-11 months).
Isolation of actinomycetes: 0.5g of soil samples was suspended in 9.5m1 of sterile distilled water and was 1000-fold diluted. 0. lml of the dilutions was spread on humic acid + modified B vitamins agar (HV) medium, pH 7.2, supplemented with cycloheximide. The plates were incubated at 280C for 2 weeks.
Classification of actinomycetes: Isolated strains were transferred from HV medium onto oatmeal agar medium, pH 7.2 and incubated at 280C for 14 days. Colouration of aerial mycelium (on the surface of agar), substrate mycelium (underside of plate) and diffusible pigment were observed.
Extraction of secondary metabolites: Submerged fermentation of purified cultures were carried out in liquid medium of 2% mannitol, 2% peptone and 1% glucose, pH 7.2, for 5 days at 280C, 220rpm. Resultant broths were added with equal volume of acetone to extract secondary metabolites (final concentration of extract in 50% acetone).
Screening:The acetone extracts were tested for inhibitory activity against MAPK kinase and MAP kinase phosphatase inhibitors into yeast strains Saccharomyces cerevisiae MKKlP386 and S. cerevisiae MKK1P386— MSG5 respectively.

RESULTS AND DISCUSSION:

A total of 78 isolates of actinomycetes were isolated from 22 soil samples (Table 1 & 2) while 16 other soil samples without any isolate (Table 3). Some strains were isolated from soil under Theobroma cacao, Rhododendron sp. and particularly Rafflesia kethii (Figure 2) and R. pricei (Figure 3). Most of the isolates were presumed to be of the genera Streptomyces as they showed good sporulation with compact, chalk-like dry colonies of different colours. A few pigmented strains, unique to individual sites were observed.





• All of the isolates were recovered from humic acid + B vitamins* agar plates (pH 7.2) which had been incubated for 14-30 days at 280C.
*B vitamins: thiamine-HC1, pyridoxin-HC1 and inositol.
• The isolates from E1-El1 were recovered from humic acid + B vitamins** agar plates (pH7.2) which had been incubated for 14-30 days at 280C.
*B vitamins: thiamine-HC1, pyridoxin-HC1, ribiflavin, niacin, inositol, Ca-pantothenate & p-aminobenzoic acid.
• Isolated by ‘L’-Lo, C.W; ‘C’- Cheah, H-Y; ‘W’-Wong, N.K. ‘E’- Lai, N.S. Eric






• All of the isolates were recovered from humic acid + B vitamins** agar plates (pH 7.2)
which had been incubated for 14-30 days at 280C. Isolated by Lo.C.W.
** B vitamins: thiamine-HC1, pyridoxin-HC1, ribiflavin, niacin, inositol, Capantothenate, paminobenzoic acid and biotin



All the isolates were grouped into 3 colour groups (white series, grey series and brown series) based on the colour of aerial mycelium on oatmeal agar, after 14 days incubation at 280C (Figure 4). Majority of the strains were of the grey series, followed by white series and brown the least

(Table 4). The grey series include pale grey, light grey, medium grey and dark grey; white colour group includes yellowish white, milky white and orange white while brown colour group includes greyish orange, brownish orange and greyish brown. As description of colour is quite subjective, a colour chart from Nippon 9000(1997) was used for standardization. A few strains varied according to sites are as follow: L-28 exhibited red pigmentation all over the agar medium, while L-27 exhibited orange colour extracellular pigment. Both strains were isolated from soil samples obtained under Theobroma cacao and Tectonia grandis respectively (Table 5). In the MAP kinase screening, 20 extracts were screened but none was found to be inhibitory.







The search for novel metabolites especially from actinomycetes requires a large number of isolates (over thousands) in order to discover a novel compound of pharmaceutical interest. The search will be more promising if diverse actinomycetes are sampled and screened. For this reason, soils were specifically collected under identified trees. This is based on the hypothesis that actinomycetes diversity may be influenced by the diversity of plant species as these bacteria grow profusely in the humus and leaf litter layer. Furthermore, different plants produce different type of secondary metabolites and some of these chemical compounds are toxic to soil microorganisms including actinomycetes. However, adaptation has in turn lead the actinomycetes to produce their own secondary metabolites.
Although the collection sites have mainly been limited to fairly disturbed forests in the fringes of Crocker Range, yet they possess many actinomycetes in the leaf-litter humus layer. The conservation of this park will ensure the survival of these commercially important industrial microbes of biotechnological and pharmaceutical importance together with striking Rafflesia, orchids and Rhododendron.

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