characterization and description of novel actinomycete...

Chapter 3

Characterization and Description

of Novel Actinomycete Taxa

Characterization and Description of Novel Actinomycete Taxa

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3.1. INTRODUCTION

Of the 10 potentially novel species of actinobacteria recoverd from Hundung

limestone site, three (3) were characterized by polyphasic taxonomy. Polyphasic

taxonomy - a term coined by Colwell (1970) - incorporates all genotypic, phenotypic,

and phylogenetic information. Genotypic information is derived from nucleic acids

present in the cell, whereas phenotypic information is derived from proteins and their

functions, different chemotaxonomic markers, and a wide range of other expressed

factors. These are used for the delineation of taxa at all levels (Murray et al. 1990).

3.2. METHODOLOGY

3.2.1. Phenotypic characterization

The classical phenotypic characteristics of bacteria comprise morphological,

physiological, and biochemical features. Morphological features of a bacterium

include both cellular and colonial characteristics. The biochemical features include

data on presence or absence of various enzymes, diversity of carbon or energy sources

used and metabolic versatility whereas chemotaxonomic features include information

on the various chemical constituents of the cell.

3.2.1.1. Morphological characterization

Microscopy

The axenic actinomycete strain was cultivated aerobically on GM1 (28 °C) for 5

weeks for strain MBRL 34 and SCNA (28 °C) for 2 weeks for strains MBRL 201 and


86

MBRL 251. Morphology of the spores and mycelia was observed by light microscopy

(Olympus BH2) and scanning electron microscopy (Quanta 200, FEI). Bacterial cells

producing spore-bearing mycelia were cultured by cover-slip techniques. The fully

grown mycelia on the cover-slip were then observed under the light microscope. For

electron microscopy, the mycelia were sputter coated with gold and observed under

scanning electron microscope (SEM) (Williams and Davies 1967).

Growth pattern on various culture media

The actinomycete strain was grown on International Streptomyces Project (ISP) media

(Shirling and Gottlieb, 1966), tryptic soy agar (TSA, Difco), SCNA, Czapek’s Dox

agar (CDA) and Nutrient agar (NA) at 28 °C for 7 d. The colour of aerial and

substrate mycelia and production of soluble pigments was then observed. The colony

colour was determined in accordance with the Inter-Society Colour Council –

National Bureau of Standards (ISCC-NBS) colour charts (Kelly 1964).

3.2.1.2. Biochemical characterization

Aesculin decomposition (Gordon et al. 1974)

Actinomycete cultures were inoculated into aesculin broth, and observed for growth

and blackening of the medium after 2 weeks.

Casein hydrolysis (Leboffe and Pierce 2008)

Actinomycete cultures were spot-inoculated on Skim Milk Agar and incubated

aerobically for 1 d. Formation of halo zones around the growth indicates positive

result.


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Catalase activity (Leboffe and Pierce 2008)

A loopful of actinomycete culture was transferred to a microscope slide. One or two

drops of H2O2 (3 %, v/v) was then aseptically placed directly onto the bacteria and

observed for the formation of bubbles.

Citrate utilization (Leboffe and Pierce 2008)

Using an inoculating needle and light inoculum, the Simmons Citrate tube was

stabbed with the test actinomycete and incubated for 4 d. A change in colour of the

medium from green to deep blue indicates a positive reaction.

Gelatin liquefaction (Leboffe and Pierce 2008; Collins et al. 2004)

Nutrient Gelatin medium was stab-inoculated with heavy inoculum of the

actinomycete culture, and was kept incubated for 1 week. The tube was then kept at 4

°C for 1 h and checked for liquefaction.

Indole production (Cappucino and Sherman 2004)

Actinomycete culture was inoculated in Nutrient broth medium and incubated for 2 d.

Formation of red ring on addition of 0.6 ml of Kovac’s reagent indicates production

of indole from tryptophan in the culture medium.

Methyl Red-Voges Proskaeur test (Leboffe and Pierce 2008)

Actinomycete cultures were inoculated into MR-VP broth and incubated at 37 °C for

2-3 d. For Methyl Red test, 3 drops of Methyl Red reagent was added and checked for

red colour formation (positive reaction). For Voges Proskaeur test, 0.6 ml of VP


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Reagent A and 0.2 ml of VP Reagent B were added and kept for 10 min to 1 h.

Formation of red colour indicates positive result.

Nitrate reduction (Leboffe and Pierce 2008, Lanyi 1987)

Actinomycete cultures were inoculated into Nitrate Broth. After 2 d of incubation,

eight drops each of Reagent A and Reagent B were added to each tube, and allowed to

stand undisturbed for 10 min. Formation of red colour indicates positive result. A

small amount of zinc powder was added to cultures not producing a colour change.

The development of red colour confirmed negative result for nitrate reduction.

Oxidase activity (Kovacs 1956)

Sterile discs (HiMedia) were impregnated with tetramethyl-p-phenylenediamine

solution (1 %, w/v) and air dried. To the disc, a single colony of the actinomycete

culture was smeared and observed for change into violet colour within 20 s.

Production of acids from carbohydrates (Leboffe and Pierce 2008)

The Phenol red broth is used to test for acid production from carbohydrates (fructose,

glucose, lactose, maltose, mannitol and sucrose). 5 ml each of the medium were

distributed in screw-capped test tubes and autoclaved. The medium was impregnated

with a carbohydrate disc (HiMedia) using a sterilized forcep. The medium was then

inoculated with actinomycete mycelial suspension and incubated for 4 d. The medium

was then checked for change of colour from red to yellow.


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Starch hydrolysis (Leboffe and Pierce 2008)

Actinomycete cultures were spot-inoculated on the starch agar plate and incubated

aerobically for 2 d. The cultures were covered with Gram’s iodine and observed for

clearing zones surrounding the cultures. Presence of halo zones indicate positive

result.

Tween hydrolysis (Sierra 1957)

To Tween hydrolysis medium, the actinomycete strain was cross-streaked and

inoculated for 1-2 weeks. The plates were then observed for formation of fuzzy halos

around the actinomycete lawns.

Urea hydrolysis (Leboffe and Pierce 2008)

A heavy inoculum of the actinomycete culture was inoculated on the sterilized

Rustigian and Stuart’s Urea Broth. The tube was inoculated for 4 d, and examined for

change of colour from red to pink. Colour change indicates positive result.

Utilization of sole C source (Shirling and Gottlieb 1966)

Actinomycete cell (or mycelium) suspension was inoculated in the basal medium

containing the carbon source (0.5 %, w/v). The carbon sources used for the test were

adonitol, arabinose, cellobiose, dulcitol, fructose, galactose, inositol, inulin, lactose,

maltose, mannitol, mannose, melibiose, raffinose, rhamnose, ribose, salicin, sodium

malate, sorbose, sorbitol, succinic acid, trehalose, xylitol and xylose. Appearance of

growth was observed after 2 weeks.


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Utilization of sole N source (Shirling and Gottlieb 1966)

The basal medium containing Nitrogen source (0.5 %, w/v) was inoculated with

actinomycete suspension and growth was observed after 2 weeks. The nitrogen

sources used in the study were L-alanine, L-arginine hydrochloride, L-asparagine, L-

aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, glycine, L-

histidine hydrochloride, L-isoleucine, L-leucine, L-methionine, L-ornithine,

potassium nitrate, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-

tyrosine and L-valine.

3.2.1.3. Chemotaxonomy

Whole cell amino acid and sugar analyses (Staneck and Roberts 1974; Tang et al.

2009)

Purified cell wall of the actinomycete culture was prepared by hydrolysis of

approximately 3 mg dry cell mass in a sealed ampoule with 0.2 ml HCl (6 M) at 120

°C for 12 h. For determination of the type of diamino pimelic (DAP) acid, 5 l of the

whole cell hydrolysate was spotted at the base of the cellulose TLC sheet (Merck

2330); 1 l meso-DAP (10 mM) was applied separately on the side as the standard

reference. Ascending TLC was performed with the solvent system, Methanol-distilled

water-HCl (6 M)-Pyridine (80:26:4:10 v/v/v/v) for approximately 3.5 h. The

chromatogram was then air-dried and spots were visualized by spraying with

ninhydrin reagent (0.2 % in acetone). For whole cell sugars, the hydrolysis was done

with HCl (0.2 M) at 120 °C for 2 h. 80 l each of the whole cell hydrolysates, 1-

phenyl-3-methyl-5-pyrazolone solution (0.25 M in methanol) and NaOH solution (0.2


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M) were mixed and incubated for 30 min at 70 °C. The mixture was then cooled to

room temperature and neutralized with HCl (0.2 M) to pH 7.0, and extracted with

isoamyl acetate. The organic phase was discarded. The extraction process was

continued for three more cycles (except that chloroform was used instead of isoamyl

acetate in the last cycle). The aqueous phase was collected and analyzed in HPLC.

Determination of menaquinones (Collins et al. 1977; Tamaoko et al. 1983)

Approximately 100 mg freeze-dried actinomycete culture was mixed with 20 mL

chloroform-methanol (2:1, v/v) and the suspension was kept incubated under shaking

condition overnight. The biomass was then removed by filtration and the extract

evaporated to dryness under reduced pressure at low temperature (37 °C). Analytical

TLC of quinones was performed in silica gel TLC plate GF254 (Merck) for 20 min

using toluene as the developing solvent. Usually menaquinones have an Rf value of ~

0.7 and was detected after brief irradiation with short-wave ultraviolet light (254 nm).

The menaquinone fraction was then extracted from the gel, dissolved in methanol and

concentrated under reduced pressure. The final extract was then analyzed in HPLC.

Polar lipids analysis (Lechevalier et al. 1977; Minnikin et al. 1984)

In a 50 ml centrifuge tube containing approximately 1 g cell mass, 15 ml methanol

was added and kept in boiling water bath (100 °C) for 5 min. The mixture was cooled

to room temperature, followed by addition of 10 ml chloroform and equal volume of

NaCl solution (2 %, w/v). The tube was vigorously shaken (10 min) and then

centrifuged (8000 rpm, 10 min). The organic phase was dried on a rotary evaporator.

The lipid extract was then dissolved in 200 l chloroform-methanol (2:1, v/v) and


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used for chromatographic analysis of the phospholipids. 10 l of the whole cell lipid

extract was applied on one corner of silica gel 60 thin-layer chromatographic plates

(10x10 cm) and developed in two dimensions using the following solvent systems:

Chloroform-methanol-water (65:24:4, v/v/v) in the first direction, and chloroform-

acetic acid-methanol-water (80:18:12:5, v/v/v/v) in the second direction. The

chromatogram was visualized by spraying with molybdatophosphoric acid and

charring at 150 °C for 2 min.

Determination of cellular fatty acids (Sasser 1990)

The five steps involved in the preparation of cells for fatty acid composition analysis

by Gas chromatography is mentioned below:

i. Harvesting – About 40 mg cell mass from a quadrant streaked plate was

harvested using a 4 mm loop and placed in a clean 13x100 culture tube.

ii. Saponification – 1 ml of Reagent 1 was added to the tube. The tube was

securely sealed with teflon lined cap, vortexed briefly and heated in a boiling

water bath for 5 min, and then vigorously vortexed for 5-10 s and returned to

the water bath to complete the 30 min heating.

iii. Methylation – The cooled tube was then uncapped and 2 ml of Reagent 2 was

added. The tube was capped again and briefly vortexed. After vortexing, the

tube was heated for 10 min at 80 °C.

iv. Extraction – 1.25 ml of Reagent 3 was added to the cooled tube followed by

recapping and gentle tumbling on a clinical rotator for about 10 min. The tubes

were uncapped and the aqueous (lower) phase was pipetted out and discarded.


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v. Base wash – About 3 ml of Reagent 4 was added to the remaining organic

phase in the tube. The tube was recapped and tumbled for 5 min. Following

uncapping, about 2/3 of the organic phase was pipetted into a GC vial and

capped.

The fatty acid methyl esters (FAMEs) obtained was then analyzed by GC (Agilent

Technologies 7890A GC System) by using the Microbial Identification Software

Package (Sherlock Version 6.1; MIDI database: TSBA6, MIDI Inc).

3.2.1.4. Physiological characterization (Goodfellow 1986)

Growth temperature

Actinomycete cultures were inoculated on TSA and incubated at different

temperatures (5, 15, 28, 37, 42, 50 and 60 °C). Growth was observed at regular

intervals upto the 7th

day.

Growth at different concentration of NaCl

TSA was prepared with different NaCl concentrations (0, 2, 5, 7 and 10 % w/v). The

actinomycete cultures were inoculated on the plates and growth observed at regular

intervals upto the 7th

day.

pH tolerance

pH of TSA was adjusted with different buffer solutions to give a pH range of 4 – 10.

The petri dishes containing the media were inoculated and incubated at 28 °C.

Actinomycete cultures were examined for growth at regular intervals upto the 10th

day.


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3.2.2. Genotypic characterization

3.2.2.1. Genomic DNA isolation and PCR amplification of 16S rRNA gene (Li et al.

2007)

The genomic DNA isolation and PCR amplification of 16S rRNA gene were

performed as described in Chapter 2.

3.2.2.2. Cloning of amplified 16S rRNA gene

Purification of amplified 16S rRNA gene

Purification of the amplified 16S rRNA gene product was done using the EasyPure

Quick Gel Extraction Kit (TransGen) as per the instruction manual.

Transformation

The purified PCR products were cloned using the TransGen TA kit (TransGen

Biotech) following the user’s instructions.

Analysis of recombinant clones by colony PCR

The cells were checked for transformation by PCR using the following mixture: 5 l

Buffer for Taq pol (with Mg2+

, 10X), 4 l dNTPs (2.5 mM), 1 l of each M13 +/-

primers (2.5 mM), 0.3 l Taq polymerase (2.5U/l), 37.7 l H2O and 1 l DNA

template.

3.2.2.3. Sequencing of cloned 16S rRNA gene

Sequencing was done by Sangon Sequencing Company, Shanghai, China. Sequences

obtained were then assembled using the SeqMan II (DNASTAR).The almost


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complete 16S rRNA gene sequence of the actinomycete strain was submitted to the

EzTaxon-e server database (Kim et al. 2012; http://eztaxon-e.ezbiocloud.net/) and

aligned with the 16S rRNA gene sequences of other closely related species using

CLUSTAL X version 2.1 (Larkin et al., 2007). Phylogenetic analyses were performed

using the software package MEGA version 5.2 (Tamura et al. 2011). Distances (using

distance options according to Kimura’s two-parameter model; Kimura 1983) were

calculated and clustering was performed with the neighbour-joining (NJ) (Saitou and

Nei 1987), Maximum Likelihood (ML) (Felsenstein 1981) and Maximum Parsimony

(MP) (Kluge and Farris 1969) methods. To determine the support of each clade,

bootstrap analysis was performed with 1000 resamplings (Felsenstein 1985).

3.2.2.4. Purification of Genomic DNA for G+C content analysis and DNA-DNA

hybridization studies

Isolation of Genomic DNA

Actinomycete strains were cultured in TSB shake flasks (200 rpm, 37 °C, 7 d). Cell

biomass was collected by centrifugation (5,000 rpm) and washed twice with distilled

water. Approximately 1-2 g cell mass was taken in a 50 ml oak ridge tube, mixed with

7 ml TE buffer (1X) and 0.7 ml EDTA (0.5 M); 0.7 ml lysozyme (50 mg/ml) was then

added and the tube was kept incubated overnight under shaking conditions (200 rpm,

37 °C). This was followed by addition of 0.7 ml SDS (20 %, w/v) and 70 l

proteinase K (5 mg/ml) and the tube was then kept on water bath (55 °C, 2 h) after

thorough mixing. Equal volume of Phenol-Chloroform (1:1, v/v) was then added,

mixed and centrifuged (12,000 rpm, 10 min). The aqueous phase was transferred to a

fresh 50 ml oak ridge tube. The extraction process was repeated 2 more times. The

http://eztaxon-e.ezbiocloud.net/


96

final aqueous extract was mixed with equal volume of isopropanol and 1/10 volume

of sodium acetate (3 M), and mixed carefully. The precipitated DNA was spooled out

in a fresh eppendorf tube, washed with ethanol (70 %) and dried until no smell of

alcohol was detected. Finally the DNA was dissolved in a minimum volume of TE

buffer (1X).

Purification of Genomic DNA

The DNA obtained by the above method is likely to contain significant amount of

RNA. RNAs were, therefore, degraded using RNA-degrading enzyme, RNase. The

volume of the DNA solution was made up to 500 l with TE (1X) and 15 l RNase A

(400 U/ml) and 150 l RNase T1 (400U/ml) were added to it, mixed and kept

incubated in a shaker (37 °C, 30 min). Extraction was done by adding equal volume

of Phenol-Chloroform-Isoamyl alcohol (25:24:1, v/v/v) followed by slow mixing and

centrifugation at 12,000 rpm. The aqueous phase was carefully transferred to a fresh

eppendorf tube, and the extraction was repeated for another round. The final

extraction was done using chloroform only. To the aqueous extract, an equal volume

of isopropanol was added to precipitate the DNA. The mixture was allowed to stand

for 10 min at room temperature followed by centrifugation. The supernatant was

carefully decanted, and the tube was allowed to dry until no smell of alcohol was

detected. The final purified DNA was then dissolved in 30 l sterile milliQ water.


97

Determination of purity and concentration of DNA

The purity and concentration of DNA was checked by monitoring the absorbances at

260 and 280 nm. Pure DNA has A260/ A280 between 1.7 and 1.9. For DNA with A260/

A280 ratio below 1.7 or above 1.9, DNA purification was repeated.

DNA concentration in the purified sample was calculated using the relation:

DNA concentration = 50 g/ml x A260 x dilution factor

where, 50 g/ml is the concentration of DNA

corresponding to A260 = 1

3.2.2.5. Determination of G+C content of genomic DNA

DNA enzymolysis

10 l purified DNA was heated for 10 min at 100 °C, followed by immediately

cooling on ice for 5 min. To the denatured DNA, 10 l RNase P1 (63 U/ml) was

added and incubated in water bath (50 °C, 1 h). It was incubated for another 1 h at 37

°C after addition of 10 l alkaline phosphatase (70 U/ml). The final mixture was used

for analysis of G+C content.

Measurement of G+C content (Mesbah et al., 1989)

The hydrolyzed DNA was analyzed in HPLC. The G+C content was calculated

according to the peak area of the deoxynucleotides in the sample chromatogram using

the following formula:

𝐆 + 𝐂 𝐜𝐨𝐧𝐭𝐞𝐧𝐭 (𝐦𝐨𝐥%) = 𝐆+𝐂

𝐆+𝐂+𝐓+𝐀 × 𝟏𝟎𝟎%


98

3.2.2.6. DNA-DNA hybridization (DDH) studies

Digestion of Genomic DNA

The purified DNA sample was fragmented to ~ 300 bp fragment in a sonicator (5 s

sonication followed by 5 s interval, 3 rounds). The DNA fragment was analyzed by

agarose gel electrophoresis (1 %, w/v) using DNA ladder (100 bp) as the reference.

Hybridization reaction by thermal renaturation method (DeLey et al. 1970)

The study was done using Lambda 35 UV/Vis Spectrophotometer (Perkin Elmer)

equipped with 6+6 Peltier Temperature Programmer (Perkin Elmer) and BG-Chiller

E15 (Baygene Biotech). DNA-DNA relatedness value below 70% is considered the

delineating limit for species determination (Wayne et al. 1987).

3.3. RESULTS

Based on the polyphasic characterization, the strains MBRL 34, MBRL 201 and

MBRL 251 were characterized as Micromonospora kangleipakensis sp. nov.,

Streptomyces manipurensis sp. nov. and Streptomyces hundungensis sp. nov.

respectively. The detailed characterization results of the three novel species are

discussed below.


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3.3.1. Micromonospora kangleipakensis sp. nov.

3.3.1.1. Morphological characteristics

Strain MBRL 34T was isolated as a green colour forming colony from the GM1 plate.

The strain forms single spores (~0.87m) on short sporophores in the substrate

mycelium. Figure 3.1 shows its growth morphology on GM1 plates and as observed

under SEM.

Fig. 3.1. Growth morphology of MBRL 34T in GM1 and as observed under SEM after 35 d of

incubation, Bar 5 m.

The strain grew with colours ranging from orange to green (Table 3.1) in most of the

media tested, and no growth (or poor growth) is observed on ISP6, ISP7, NA and

TSA.


100

Table 3.1. Cultural characteristics of MBRL 34T on various ISP and other

selective media as observed using ISCC-NBS Color Chart

Medium Colour of mycelium

Aerial (Spore mass) Substrate

ISP2

ISP3

ISP4

ISP5

ISP6

ISP7

NA

SCNA

CDA

TSA

GM 1

Orange

Orange

Orange

Orange

NG*

PG#

PG#

Orange

Olive Green

PG#

Olive Green

Orange Yellow

Orange Yellow

Orange

Orange Yellow

-

-

-

Orange Yellow

Deep Green

-

Deep Green

* NG, No Growth; #PG, Poor Growth

3.3.1.2. Biochemical characteristics

The strain hydrolyzed casein, but not aesculin, gelatin, Tween 20, tyrosine and urea. It

was positive for catalase but negative for methyl red, indole production, nitrate

reduction, and citrate utilization tests. The strain utilized inulin and mannose as sole

carbon sources and L-alanine, L-glutamine, L-isoleucine, L-leucine, L-lysine

hydrochloride, L-methionine, L-serine, L-tyrosine and L-valine as sole nitrogen

sources. It could not utilize adonitol, arabinose, cellobiose, dulcitol, fructose,

galactose, inositol, lactose, maltose, mannitol, melibiose, raffinose, rhamnose, salicin,

sorbitol, sucrose, trehalose, xylose, L-arginine hydrochloride, L-asparagine, L-

cysteine, L-cystine, L-glycine, L-histidine, L-phenylalanine, L-proline, L-threonine

and L-tryptophan as either sole carbon or nitrogen sources. Table 3.2 lists the

biochemical characteristics for strain MBRL 34T.


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Table 3.2. Biochemical characteristics of strain MBRL 34T

Characteristics Result Characteristics Result

Casein hydrolysis

Catalase activity

Citrate utilization

Gelatin liquefaction

Utilization of sole C sources

Adonitol

Arabinose

Cellobiose

Dulcitol

Fructose

Galactose

Inositol

Inulin

Lactose

Maltose

Mannitol

Mannose

Melibiose

Raffinose

Rhamnose

Salicin

Sorbitol

Trehalose

Xylose

+

+

-

-

-

-

-

-

-

-

-

+

-

-

-

+

-

-

-

-

-

-

-

Indole production

Methyl Red test

Nitrate reduction

Starch hydrolysis

Tween 20 hydrolysis

Urea hydrolysis

Utilization of sole N sources

L-Alanine

L-Arginine hydrochloride

L-Asparagine

L-Cysteine

L-Cystine

L-Glutamine

Glycine

L-Histidine hydrochloride

L-Isoleucine

L-Leucine

L-Methionine

L-Phenylalanine

L-Proline

L-Serine

L-Threonine

L-Tryptophan

L-Valine

-

-

-

-

-

-

+

+

+

-

-

+

-

-

+

+

+

-

-

+

+

-

+

Note: + positive; - negative

3.3.1.3. Chemotaxonomical characteristics

Strain MBRL 34T had meso-DAP as the diagnostic cell wall diamino acid. Galactose

(53.5 %) and glucose (23.1 %) were the main sugars detected in the whole cell

hydrolysates along with small amounts of mannose, xylose, rhamnose and ribose.

MK-10(H6) (36.6 %), MK-10(H2) (24.1 %), MK-11(H4) (16.4 %) and MK-10(H4)

(11.1 %) were the predominant menaquinones detected. The major polar lipids


102

detected were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE),

phosphatidylglycerol (PG), phosphatidylinositol (PI) and

phosphatidylinositolmannoside (PIM), with other unknown lipids (UL) (Figure 3.2).

The FAME profile (>1%) contained iso-C16:0 (36.2 %), iso-C15:0 (24.4 %), iso-C17:0

(8.1 %), C17:0 (5.2 %), Summed Feature 9 containing C17:1c and/or 10-methyl C16:0

(4.2%), 10-methyl C17:0 (3.4%), anteiso-C17:0 (2.8%), iso-C18:0 (2.2%), 2-OH C16:1

(2.0%), anteiso-C15:0 (1.9%), C18:0 (1.8%), iso-C14:0 (1.6%) and iso-H C16:1 (1.0%).

Fig. 3.2 Two-dimensional thin-layer chromatogram of polar lipids of strain MBRL 34T

(1, PIM; 2, PI; 3, PG; 4, PE; 5, DPG; 6, UL)

3.3.1.4. Physiological characteristics

The strain grows at the temperature range of 15-37 °C with optimum growth at 28 °C.

It grows over the pH range of 5-7 with optimum at pH 6. It has low tolerance of NaCl

and doesn’t grow at ≥ 2 % NaCl.

1

2

3 4

5

6


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3.3.1.5. Genotypic characteristics

The GenBank accession number for the 16S rRNA gene sequence of strain MBRL

34T is JN560152. Comparison of 16S rRNA gene sequences of strain MBRL 34

T and

other members of the genus Micromonospora showed sequence similarities ranging

from 96.36 % to 98.39 % with 43 strains showing similarities above 97%, of which

six had similarities above 98% (M. echinaurantica DSM 43904T, 98.39%; M. eburnea

LK2-10T, 98.37%; M. coerulea DSM 43143

T, 98.25%; M. viridifaciens DSM 43909

T,

98.18%; M. echinofusca DSM 43913T, 98.11% and M. chaiyaphumensis MC5-1

T,

98.02%). In the phylogenetic tree (Figure 3.3) based on the NJ method, supported by

the ML and the MP methods, strain MBRL 34T forms a monophyletic clade with

Micromonospora coerulea DSM 43143T.

The Genomic DNA G+C content for strain MBRL 34T was found to be 73.46 mol%.

The DNA-DNA relatedness value between strain MBRL 34T and Micromonospora

coerulea DSM 43143T was calculated as 53.14% which is below the 70% delineating

limit for species determination.

3.3.1.6. Differentiating characteristics between strain MBRL 34T and DSM 43143

T

Apart from low DNA-DNA reassociation value, the strain MBRL 34T could be

differentiated from the type strain M. coerulea DSM 43143T by several phenotypic

characteristics as shown in Tables 3.3 and 3.4.


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Table 3.3. Differential physiological and biochemical characteristics between

strain MBRL 34T and M. coerulea DSM 43143T

Characteristics MBRL 34T DSM 43143T

pH range for growth

Optimum pH for growth

Growth temperature range

Nitrate reduction


Adonitol

Cellobiose

Dulcitol

Fructose

Galactose

Inositol

Lactose

Maltose

Mannitol

Melibiose

Raffinose


L-Arginine

L-Asparagine

Glycine

L-Histidine

L-Isoleucine

L-Methionine

L-Phenylalanine

L-Proline

L-Threonine

L-Valine

G+C mol %

5-7

6

15-37

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

+

-

-

-

+

73.5

6-8

7

28-37

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

-

+

+

+

-

72.1


105

Fig. 3.3. Neighbour-joining tree, based on 16S rRNA gene sequences, showing the

relationships between strain MBRL 34T and all the reported type strains of Micromonospora

species. Catellatospora citrea NBRC 14495T is used as the outgroup. Asterisks indicate branches that

were also recovered using the maximum parsimony tree. Numbers at nodes are levels of bootstrap

support (%) for branch points (1000 resamplings). Bar, 0.005 substitutions per nucleotide position.

M. chokoriensis 2-19/6T (AB241454) M. saelicesensis Lupac 09T (AJ783993)

M. lupini lupac 14NT (AJ783996)

M. mirobrigensis WA201T (AJ626950)

M. siamensis TT2-4T (AB193565)

M. matsumotoense IMSNU 22003T (AF152109)

M. rifamycinica AM105T (AY561829)

M. krabiensis MA-2T (AB196716)

M. carbonacea DSM 43815T (X92606)

M. purpureochromogenes DSM 43821T (X92611) M. coxensis 2-30-b/28T (AB241455)

M. halophytica DSM 43026T (X92601)

M. humi P0402T (GU459068)

M. echinospora ATCC 15837T (U58532)

M. tulbaghiae TVU1T (EU196562)

M. chalcea DSM 43026T (X92594)

M. aurantiaca ATCC 27029T (CP002162)

M. maritima D10-9-5T (HQ704071)

M. marina JSM1-1T (AB196712)

M. sediminicola SH2-13T (AB609325)

M. eburnea LK2-10T (AB107231)

M. narathiwatensis BTG4-1T (AB193559)

M. nigra DSM 43818T (X92609)

M. sagamiensis DSM 43912T (X92624)

M. inyonensis DSM 46123T (X92629)

M. pallida DSM 43817T (X92608)

M. inositola DSM 43819T (X92610)

M. fulviviridis DSM 43906T (X92620)

M. rosaria DSM 803T (X92631)

M. chersina DSM 44151T (X92628)

M. endolithica DSM 44398T (AJ560635)

M. cremea CR30T (FN658654)

M. coriariae NAR01T (AJ784008)

M. echinofusca DSM 43913T (X92625)

M. peucetia DSM 43363T (X92603)

M. citrea DSM 43903T (X92617)

M. chaiyaphumensis MC5-1T (AB196710)

M. auratinigra TT1-11T (AB159779)

M. yangpuensis FXJ6.011T (GU002071)

M. echinaurantiaca DSM 43904T (X92618)

Micromonospora manipurensis MBRL 34T (JN560152)

Micromonospora coerulea DSM 43143T (X92598)

M. viridifaciens DSM 43909T (X92623)

M. equina Y22T (JF912511)

M. pattaloongensis TJ2-2T (AB275607)

M. pisi GUI 15T (AM944497)

M. olivasterospora DSM 43868T (X92613)

M. rhizosphaerae 211018T (FJ261956)

Catellatospora citrea NBRC 14495T (D85477)

97*

91

94*

77*

74

92*

62*

60

96*

50

76*

*

52*

*

*

52

*

*

*

0.005


106

Table 3.4. Cellular fatty acid compositions of MBRL 34T and M. coerulea DSM 43143T (%)

Fatty acid MBRL 34T DSM 43143T

Straight-chain

C17:0

C18:0

Branched

iso-C14:0

iso-C15:0

anteiso-C15:0

iso-C16:1 H#

iso-C16:0

iso-C17:0

anteiso-C17:0

2-OH C16:1

10-methyl C17:0

iso-C18:0

Summed features*

9

5.2

1.8

1.6

24.4

1.9

1.0

36.2

8.1

2.8

2.0

3.4

2.2

4.2

2.9

1.9

1.1

28.0

1.0

-

27.6

13.7

3.7

3.3

3.0

1.3

6.0

* Summed features represent two or three fatty acids that cannot be separated by the Microbial

Identification System. Summed feature 9 consisted of iso-C17:19c and/or 10-methyl C16:0.

# Indicates that the position and configuration of the double bond is not known.

The morphological, 16S rRNA gene sequence and chemotaxonomical characteristics

indicate the affiliation of the strain MBRL 34T to the genus Micromonospora. The

differences between the strain MBRL 34T and the closest phylogenetic neighbour M.

coerulea DSM 43143T obtained from the results of DDH experiment and differential

phenotypic characteristics indicate that the strain MBRL 34T could be placed as a

novel taxon among the genus Micromonospora, for which the name Micromonospora

kangleikpakensis sp. nov. has been proposed.


107

3.3.1.7. Etymology of the strain MBRL 34T

Micromonospora kangleipakensis (kang.lei.pak.en´sis. N.L. masc. adj.

kangleipakensis pertaining to Kangleipak, the old name of Manipur, a state in North

East India, the source of the soil from where the type strain was isolated).

The type strain, MBRL 34T has been deposited in the Deutsche Sammlung von

Mikroorganismen und Zellkulturen, Germany (DSM 45612T) and Japan Collection of

Microorganisms, Japan (JCM 17696T).

3.3.2. Streptomyces manipurensis sp. nov.


Strain MBRL 201T formed extensive substrate and aerial mycelia bearing long spore

chains (~ 70 spores per chain). At maturity, the strain formed rectiflexible spore

chains. Growth morphology on SCNA and the scanning electron micrograph of the

aerial mycelia for strain MBRL 201T are shown in Figure 3.4. The strain grew well in

all the media tested (Table 3.5).


108

Fig. 3.4. Growth morphology of MBRL 201T in SCNA medium and Scanning Electron

Micrograph of spore chains after 2 weeks of incubation, Bar 5 m

Table 3.5. Cultural characteristics of MBRL 201T on various ISP and other

selective media as observed using ISCC-NBS Color Chart



ISP2

ISP3

ISP4

ISP5

ISP6

ISP7

NA

CDA

SCNA

TSA

Grey Brown

Grey Brown

White

Yellow Brown

Grey

Yellow Pink

Grey

Yellow Pink

Reddish Brown

Yellow Brown

Brown

Grey

Yellow White

Yellow Brown

N.D.#

Brown

Orange Yellow

Orange Yellow

Yellow Brown

Yellow Brown

#The result was not determined due to deep colour in the medium


109


The strain could hydrolyze tyrosine, starch (weakly) and Tweens 20, 40, 60 and 80

but not casein and gelatin. The strain was positive for catalase and indole production

tests but negative for methyl red, Voges-Proskauer, citrate utilization and nitrate

reduction tests. It could not produce acid from fructose, glucose, lactose, maltose,

mannitol or sucrose. The strain utilized arabinose, cellobiose, fructose, galactose,

maltose, mannose, raffinose, ribose, sodium malate and succinic acid as sole carbon

sources and L-alanine, L-arginine, L-arginine, L-asparagine, glycine, L-histidine, L-

hydroxyproline, L-leucine, potassium nitrate, proline, L-serine, L-tryptophan and L-

tyrosine as sole nitrogen sources. It could not utilize dulcitol, meso-inositol, lactose,

mannitol, rhamnose, sorbose, trehalose, xylitol, xylose, L-aspartic acid, glutamic acid,

L-methionine, L-ornithine, L-phenylalanine or L-valine as either sole carbon or

nitrogen sources. The biochemical characteristics of strain MBRL 201T are

summarized in Table 3.6.


Strain MBRL 201T had LL-DAP as the diagnostic cell wall diamino acid. Glucose

was the main sugar detected in the whole cell hydrolysates along with small amounts

of galactose, mannose, rhamnose, ribose and xylose. MK-9(H6) (58.7 %) and MK-

9(H8) (41.3 %) were the predominant menaquinones detected. The major polar lipids

detected were DPG, phosphatidylmethylethanolamine (PME), PG, PE, PI and PIM,

with other ULs and unknown phospholipids (PLs) (Figure 3.5). The FAME profile

(>1%) contained anteiso-C15:0 (38.9 %), iso-C15:0 (19.9 %), anteiso-C17:0 (14.7 %),


110

C16:0 (7.7 %), iso-C17:0 (6.6 %), iso-C16:0 (6.5 %), iso-C14:0 (1.1 %), and Summed

Feature 9 containing C17:1c and/or 10-methyl C16:0 (1.0 %).

Fig. 3.5. Two-dimensional thin-layer chromatogram of polar lipids of strain MBRL 201T

(1, PIM; 2, PI; 3, PG; 4, PE; 5, PME; 6, DPG; 7-12, ULs; 13-15, PLs)


The strain grows between 15-37 °C and pH 6-10, with optimum growth at 28 °C and

pH 8. It can grow in presence of up to 2 % NaCl.


111



Acid production from carbohydrate

Fructose

Glucose

Lactose

Maltose

Mannitol

Sucrose

Catalase activity

Citrate utilization


Arabinose

Cellobiose

Dulcitol

Fructose

Galactose

Inositol

Lactose

Maltose

Mannitol

Mannose

Raffinose

Rhamnose

Ribose

Salicin

Sodium malate

Sorbose

Succinic acid

Trehalose

Xylitol

Xylose

-

-

-

-

-

-

+

+

+

+

-

+

+

-

-

+

-

+

+

-

+

-

+

-

+

-

-

-

Hydrolysis of

Casein

Tween 20

Tween 40

Tween 60

Tween 80

Starch


Indole production

Nitrate reduction

Methyl Red test

Voges Proskaeur test


L-Alanine


L-Asparagine

L-Aspartic acid

L-Glutamic acid

Glycine


L-Leucine

L-Methionine

L-Ornithine

Potassium nitrate

L-Phenylalanine

L-Proline

L-Serine

L-Tryptophan

L-Tyrosine

L-Valine

-

+

+

+

+

w

-

-

-

-

-

+

+

+

-

-

+

+

+

-

-

+

-

+

+

+

+

-

Note: w weakly positive; + positive; - negative,


112



201T is JN560156. EzTaxon-e sequence similarity results showed that the strain

MBRL 201T

has 16S rRNA gene sequence homologies (>97 %) with 209

Streptomyces type strains. Analysis of EzTaxon-e and phylogenetic NJ tree results

(Figure 3.6) indicated that strain MBRL 201T is closely related to S. virginiae NBRC

12827T and S. cinnamonensis NBRC 15873

T (16S rRNA gene sequence similarities of

99.66% and 99.66%, respectively) and this phylogenetic relationship was also

supported in the tree generated with the MP algorithm.

The Genomic DNA G+C content was found to be 72.9 mol%. Strain MBRL 201T

exhibited low DNA-DNA reassociation values with S. virginiae NBRC 12827T

(44.5±3.1 %) and S. cinnamonensis NBRC 15873T (35.6±2.2 %), thereby indicating

that the whole genomic DNA relatedness values are well below the delineating 70%

cut-off point for species identification. The new species fall in the cluster 39 of the

family Streptomycetaceae as identified by Labeda et al. (2012).

3.3.2.6. Differentiating characteristics among strains MBRL 201T, NBRC 12827

T and

NBRC 15873T

The genotypic and phenotypic features described above suggest that strain MBRL

201T

could be clearly distinguished from its closest phylogenetic relatives. Besides

low DNA-DNA relatedness with the closest phylogenetic neighbours, the strain is

also distinguished from them by several phenotypic properties as listed in Table 3.7.

Therefore, the Hundung strain MBRL 201T is considered to represent a new species


113

of the genus Streptomyces, for which the name Streptomyces manipurensis sp. nov.

has been proposed.

Fig. 3.6. Neighbour-joining tree, based on 16S rRNA gene sequences, showing the relationship

between strain MBRL 201T and type strains of Streptomyces species. Asterisks indicate

branches that were also recovered using the maximum parsimony tree. Numbers at nodes are

levels of bootstrap support (%) for branch points (1000 resamplings). Bar, 0.001 substitutions

per nucleotide position.

S. xanthophaeus NBRC 12829T (AB184177)

S. spororaveus LMG 20313T (AJ781370)

S. nojiriensis LMG 20094T (AJ781355)

S. cirratus NRRL B-3250T (AY999794)

S. vinaceus NBRC 13425T (AB184394)

S. subrutilus DSM 40445T (X80825)

S. avidinii NBRC 13429T (AB184395)

S. lavendulae subsp. lavendulae NBRC 12789T (AB184146)

S. colombiensis NRRL B-1990T (DQ026646)

S. goshikiensis NBRC 12868T (AB184204)

S. sporoverrucosus NBRC 15458T (AB184684)

S. virginiae NBRC 12827T (AB184175)

S. cinnamonensis NBRC 15873T (AB184707)

Streptomyces manipurensis MBRL 201T (JN560156)

S. yokosukanensis NRRL B-3353T (DQ026652)

S. katrae NBRC 13447T (AB184409)

S. polychromogenes NBRC 13072T (AB184292)

S. racemochromogenes NRRL B-5430T (DQ026656)

S. flavotricini NBRC 12770T (AB184132)

S. globosus LMG 19896T (AJ781330)

S. toxytricini NBRC 12823T (AB184173) 99*

94

99

100

95*

88*

68*

91 56

57

53

0.001

*


114

Table 3.7. Differential characteristics among strains MBRL 201T, S. virginiae NBRC 12827T and

S. cinnamonensis NBRC 15873T

Characteristics S. manipurensis

MBRL 201T

S. virginiae

NBRC 12827T

S. cinnamonensis

NBRC 15873T

pH range


Hydrolysis of

Casein

Starch

Tyrosine

Nitrate reduction


Arabinose

Cellobiose

Fructose

Galactose

Mannose

Raffinose

Sodium malate

Succinic acid

Trehalose


L-Arginine

DL-Methionine

L-Ornithine

L-Phenylalanine

L-Valine

Major fatty acids (>5%)

iso-C15:0

anteiso-C15:0

iso-H C16:1

iso-C16:0

C16:0

Sum in Feature 9

anteiso-C17:19c

iso-C17:0

anteiso-C17:0

G+C mol %

6-10

8

-

w

+

-

+

+

+

+

+

+

+

+

-

+

-

-

-

-

19.9

38.9

-

6.5

7.7

-

-

6.6

14.7

72.9

6-8

7

+

-

-

+

-

+

-

+

+

-

-

-

-

-

-

+

+

+

13.0

27.7

-

24.5

6.2

-

-

-

10.1

72.0

6-9

7

+

+

-

+

-

-

+

-

-

+

-

-

+

+

+

-

+

+

15.9

22.1

5.1

17.1

5.6

6.3

5.4

-

7.8

72.4

Note: +, Positive; - negative; w weakly positive; all the data were from this study.


115


Streptomyces manipurensis (ma.ni.pur.en´sis. N.L. masc. adj. manipurensis

pertaining to Manipur, a state in North East India, the source of the soil from which

the type strain was isolated).

The type strain, MBRL 201T, has been deposited in the Deutsche Sammlung von

Mikroorganismen und Zellkulturen, Germany (DSM 42029T) and Japan Collection of

Microorganisms, Japan (JCM 17351T).

3.3.3. Streptomyces hundungensis sp. nov.


Strain MBRL 251T formed extensive substrate and aerial mycelia bearing long spore

chains (approximately 50 spores per chain). At maturity, the strain formed

rectiflexibile spore chains. Growth morphology of MBRL 251T in the SCNA medium

and scanning electron micrograph showing spore chains in the aerial mycelia are

shown in Figure 3.7. The strain grew well in all the media tested (Table 3.8).


116

Fig. 3.7. Growth morphology of MBRL 251T in SCNA medium and Scanning Electron

Micrograph of spore chains after 2 weeks of incubation, Bar 5 m.

Table 3.8. Cultural characteristics of MBRL 251T on various ISP and other selective

media as observed using ISCC-NBS Color Chart



ISP2

ISP3

ISP4

ISP5

ISP6

ISP7

NA

CDA

SCNA

TSA

Grey Brown

Grey Brown

White

Yellow Brown

Grey

Yellow Pink

Grey

Yellow Pink

Reddish Brown

Yellow Brown

Brown

Grey

Yellow White

Grey Yellow Brown

N.D.*

Brown

Orange Yellow

Grey Yellow Brown

Orange Yellow

Yellow Brown

*The result was not determined due to deep colour in the medium.


117


Strain MBRL 251T could produce acid from fructose and glucose, but not from

lactose, maltose, mannitol or sucrose. The strain was found to hydrolyze tyrosine,

gelatin and Tweens 20, 40, 60 and 80 but not casein and starch. The strain was

positive for catalase, methyl red and indole production and nitrate reduction tests but

negative for Voges-Proskauer and citrate utilization tests. It could utilize arabinose,

cellobiose, fructose, galactose, maltose, mannose, sodium malate, succinic acid and

xylose as sole carbon sources; and L-alanine, L-arginine, glycine, L-histidine, L-

hydroxyproline, DL-methionine, L-ornithine, L-phenylalanine, proline, L-serine and

L-valine as sole nitrogen sources. It could not utilize dulcitol, meso-inositol, lactose,

mannitol, raffinose, rhamnose, sorbose, trehalose, or potassium nitrate as either sole

carbon or nitrogen sources. The biochemical characteristics of strain MBRL 251T are

summarized in Table 3.9.


Strain MBRL 251T had LL-DAP as the diagnostic cell wall diamino acid. Glucose

(32.9 %) and xylose (32.1 %) were the main sugars detected in the whole cell

hydrolysates along with small amounts of galactose, mannose, rhamnose and ribose.

MK-9(H6) (72.4 %) and MK-9(H8) (27.6 %) were the predominant menaquinones

detected. The major polar lipids detected were DPG, PE, PG, PI and PIM, with other

unknown ULs, PL, aminophospholipid (APL) (Figure 3.8). The FAME profile (>1%)

contained anteiso-C15:0 (35.1 %), iso-C16:0 (21.1 %), anteiso-C17:0 (13.2 %), iso-C14:0

(6.5 %), C16:0 (6.1 %), iso-C15:0 (3.8 %), iso-C17:0 (2.9 %), cyclo-C17:0 (2.8 %), and iso-

C16:1 H (1.7 %).


118

Fig. 3.8. Two-dimensional thin-layer chromatogram of polar lipids of strain MBRL 251T

1, PIM; 2, PI; 3, PG; 4, PE; 5, DPG; 6-7, ULs; 8, PL; 9, APL.


The strain MBRL 251T grows at 15-37 °C and between pH 5-10, with optimum

growth at 28 °C and pH 8. It can grow in presence of up to 7 % NaCl.



251T is JN560156. EzTaxon-e analysis showed that the strain MBRL 251

T shared

close 16S rRNA gene sequence homologies (>97%) with 205 Streptomyces type

strains. Analysis of EzTaxon-e and NJ tree results (Figure 3.9) indicated that strain

MBRL 251T is closely related to S. xanthochromogenes NRRL B-5410

T (99.66 %)

and S. michiganensis NBRC 12797T (99.66 %). The strain MBRL 251

T falls in the

cluster 29 of the family Streptomycetaceae as suggested by Labeda et al. (2012).

1 2

3 4

5

8

6 7

9


119



Acid production from carbohydrate

Fructose

Glucose

Lactose

Maltose

Mannitol

Sucrose

Catalase activity


Arabinose

Cellobiose

Dulcitol

Fructose

Galactose

Inositol

Lactose

Maltose

Mannitol

Mannose

Raffinose

Rhamnose

Sodium malate

Sorbose

Succinic acid

Trehalose

Xylose

+

+

-

-

-

-

+

+

+

-

+

+

-

-

+

-

+

-

-

+

-

+

-

+

Hydrolysis of

Casein

Starch

Tween 20

Tween 40

Tween 60

Tween 80

Tyrosine

Citrate utilization


Indole production

Nitrate reduction

Methyl Red test

Voges Proskaeur test


L-Alanine


Glycine


DL-Methionine

L-Ornithine

Potassium nitrate

L-Phenylalanine

L-Proline

L-Serine

L-Valine

-

-

+

+

+

+

+

-

+

+

+

+

-

+

+

+

+

+

+

-

+

+

+

+

Note: + positive; - negative


120

Fig. 3.9. Neighbour-joining tree, based on 16S rRNA gene sequences, showing the

relationships between strain MBRL 251T and other type strains of Streptomyces species.

Asterisks indicate branches that were also recovered using the maximum parsimony tree.

Numbers at nodes are levels of bootstrap support (%) for branch points (1000 resamplings).

Bar, 0.002 substitutions per nucleotide position.

The G+C content of the genomic DNA was determined to be 72.3 mol%. DDH

studies for strain MBRL 251T was done with the two closest relatives, S.

xanthochromogenes NBRC 12828T and S. michiganensis NBRC 12797

T. The results

indicated that strain MBRL 251T had low DNA-DNA reassociation values with S.

xanthochromogenes NBRC 12828T (46.6±8.9 %) and S. michiganensis NBRC 12797

T

(40.7±3.7 %), thereby indicating that the whole genomic DNA relatedness values are

well below the delineating 70% cut-off point for species identification.

S. badius NRRL B-2567T (AY999783)

S. globisporus subsp. globisporus NBRC 12867T (AB184203)

S. sindenensis NBRC 3399T (AB184759)

S. parvus NBRC 3388T (AB184756)

S. fimicarius ISP 5322T (AY999784)

S. anulatus NRRL B-2000T (DQ026637)

S. griseorubiginosus NBRC 13047T (AB184276)

S. microflavus NBRC 13062T (AB184284)

S. mauvecolor LMG 20100T (AJ781358)

S. violascens NBRC 12920T (AB184246)

Streptomyces hundungensis MBRL 251T (JN560157)

S. xanthochromogenes NRRL B-5410T (DQ442559)

S. michiganensis NBRC 12797T (AB184153)

S. melanogenes NBRC 12890T (AB184222)

S. noboritoensis NBRC 13065T (AB184287)

S. gobitricini NBRC 15419T (AB184666)

100*

98* 82*

93*

77

60

88*

*

59*

99

68

0.002


121

3.3.3.6. Differentiating characteristics among strains MBRL 251T, NBRC 12828

T and

NBRC 12797T

The genotypic and phenotypic features described above suggest that strain MBRL

251T

could be clearly distinguished from its closest phylogenetic relatives. Besides

low DNA-DNA relatedness with the closest phylogenetic neighbours, the strain is

also distinguished from them by several phenotypic properties as listed in Table 3.10.

Therefore, the Hundung strain MBRL 251T is considered to represent a new species

of the genus Streptomyces, for which the name Streptomyces hundungensis sp. nov.

has been proposed.


Streptomyces hundungensis (hun.dung.en´sis. N.L. masc. adj. hundungensis

belonging to Hundung in Ukhrul, a hill district in Manipur, India, the source of the

type strain).

The type strain, MBRL 251T has been deposited in Japan Collection of

Microorganism, Japan (JCM 17577T) and Korean Collection for Type Cultures, Korea

(KCTC 29125T).


122

Table 3.10. Differential characteristics among strains MBRL 251T, S. xanthochromogenes NBRC

12828T and S. michiganensis NBRC 12797T

Characteristics S. hundungensis

MBRL 251T

S. xanthochromogenes

NBRC 12828T

S. michiganensis

NBRC 12797T

pH range


NaCl tolerance (%, w/v)

Hydrolysis of

Casein

Gelatin

Tyrosine

Catalase

MR

Indole production

Utilization of sole C-sources

Arabinose

Cellobiose

Maltose

Mannitol

Raffinose

Sodium malate

Sorbose

Trehalose

Utilization of sole N-sources

L-Arginine

Potassium nitrate

Major fatty acids (>5%)

iso-C14:0

iso-C15:0

anteiso-C15:0

iso-C16:0

C16:0

anteiso-C17:19c

anteiso-C17:1

G+C mol %

5-10

8

7

-

+

+

w

+

+

+

+

+

-

-

+

-

-

+

-

6.5

-

35.1

21.1

6.1

-

13.2

72.3

6-9

7

2

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

+

-

5.7

44.2

12.9

-

5.1

12.9

72.3

6-9

7

2

+

+

-

+

+

+

+

+

+

+

+

-

+

+

+

+

-

5.2

40.6

13.2

8.1

-

19.2

71.9

Note: + Positive; w weakly positive; - negative


123

3.4. DISCUSSION

Limited studies have been done on the microbiology of limestone habitats. Most of

these studies have been focussed on the aspects of microbial biogeography (Groth et

al. 1999; Stomeo et al. 2008; Yamac et al. 2011; Niyomvong et al. 2012; Rule and

Cheeptham 2013). The rich diversity of actinomycetes in limestone habitats has been

demonstrated earlier by the discovery of two novel genera Knoellia and Hoyosella

(Groth et al. 2002; Jurado et al. 2009) from such less explored habitats.

Caves, another limestone ecosystem, with large calcium carbonate deposits, are

considered to be hostile environments for life (Howarth 1993) and are often severely

resource-limited due to absence of light that precludes primary production of organic

materials (Poulson and Lavoie 2000). Microbes, especially the mineral oxidizing

bacteria, play a major role in the dissolution of limestones and other calcareous rocks

for the formation of caves. Microbially influenced dissolution or corrosion of mineral

surfaces can occur through secretion of exoenzymes, organic acids e.g. carbonic acid

(Gillieson 1996; Schwabe et al. 2001) and mineral acids e.g. sulphuric acid (Hill

1987, 1990, 1995, 1996, 2000; Jagnow et al. 2000), and a variety of other mechanisms

(Sand 1997; Jones 2001; Bullen et al. 2008). In addition, bacteria isolated from caves

are shown to be effective in extracellular precipitation of calcium carbonate through a

variety of processes that include ammonification, denitrification, sulphate reduction,

and anaerobic sulphide oxidation (Danielli and Edington 1983; Simkiss and Wilbur

1989; Ehrlich 1996; Castanier et al. 1999; Riding 2000; Engel et al. 2001).

Microorganisms are commonly found fossilized within carbonate speleotherms (Jones

and Motyka 1987; Polyak and Cokendolpher 1992; Jones and Kahle 1995; Melim et


124

al. 2001). Carbonate pool fingers in Lechuguilla Cave in New Mexico, USA were first

identified by Davis (2000) as potentially biogenic in orgin. Similar results were

found by Baskar et al. (2006) from microcrystalline calcites within stalactites.

Considering the ununsual nature of the limestone habitats and the interaction of

microorganisms in cave formation, it is not surprising to identify novel species from

such habitats. Barton and Jurado (2007) studied the diversity of microbes in

Lechuguilla Cave. They found that 40 out of 83 isolates were possible representatives

of previously uncultivated species. Some of the novel species of actinobacteria

reported from limestone rocks and related habitats are Amycolatopsits halotolerans

sp. nov. (Lee 2006a), Amycolatopsis jejuensis sp. nov. (Lee 2006a), Amycolatopsis

saalfeldensis sp. nov. (Carlsohn et al. 2007a), Catellatospora koreensis sp. nov. (Lee

et al. 2000), Hoyosella altamirensis gen. nov., sp. nov. (Jurado et al. 2009), Knoellia

sinensis gen. nov., sp.nov. (Groth et al. 2002), Knoellia subterranea sp.nov. (Groth et

al. 2002), Kribbella aluminosa sp. nov. (Carlsohn et al. 2007b), Microlunatus

cavernae sp. nov. (Cheng et al. 2013a), Nocardia jejuensis sp. nov. (Lee 2006b) and

Saccharopolyspora cavernae sp. nov. (Cheng et al. 2013b).

This chapter reports the isolation of three novel species from limestone samples. The

isolation of a moderately acidophilic actinomycete Micromonospora kangleipakensis

sp. nov. (Nimaichand et al. 2013a) from the alkaline environment is an interesting

finding. Presence of acidophilic bacteria have also been reported from limestone

caves of the Frasassi Gorge, Italy (Vlasceanu et al. 2000; Macalady et al. 2006). The

formation of weak organic acids from biogenic carbon dioxide or strong mineral acids

during the oxidation of minerals might result in creation of acidic microhabitats


125

within the alkaline limestone environments. Ruamps et al. (2011) studied microbial

biogeography in different layers of soil and found different microhabitats providing

diverse conditions for microbial growth, activity and survival at the various layers.

This concept of micro-environments within a large environment could be the reason

behind isolation of the strain Micromonospora kangleipakensis strain MBRL 34T. The

isolation of Streptomyces manipurensis sp. nov. (Nimaichand et al. 2012) and

Streptomyces hundungensis sp. nov. (Nimaichand et al. 2013b), and 7 other

potentially novel strains of actinomycetes (among the representative phylotypic

strains) indicate that the Hundung ecosystem (underexplored limestone habitat) could

be a rich source for novel actinomycetes.

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