Chapter 4 DISCUSSIONshodhganga.inflibnet.ac.in/bitstream/10603/4391/14/14_chapter 4.pdf · habitats of India, including a few habitats and regions hitherto unexplored by the earlier

V. Venkata Ramana, Ph. D. Thesis, 2011 Discussion

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Chapter – 4

DISCUSSION


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According to the International Union for Conservation of Nature

and Natural Resources, biodiversity encompasses all life forms,

ecosystems, and ecological processes, and acknowledges the hierarchy at

genetic, taxon, and ecosystem levels (McNeely et al., 1990). Importance of

biodiversity in this era of global climate change is recognized and hence

United Nations declared 2010 as the International year of biodiversity.

India is recognized as one of the 12 mega diversity regions of the world

for its richness in overall species diversity and its contribution to the

global biodiversity is around 8% (Kapur and Jain 2004). Diversity

inventorisation is the first step towards conservation. Present thesis

focuses on inventorisation of a special group of bacteria, Anoxygenic

phototrophic Bacteria (APB) with particular reference to purple bacteria

in the classes Alphaproteobacteria, Betaproteobacteria (purple nonsulfur

bacteria) and Gammaproteobacteria (purple sulfur bacteria) from diverse

habitats of India, including a few habitats and regions hitherto

unexplored by the earlier workers. Apart from satisfying the intellectual

curiosity, studies on cultured diversity can provide raw material for

possible exploitation for human welfare and it is a source of innovation

in biotechnology (Bull et al., 1992).

Most of the new species (and other taxa) of bacteria described from

India in the past ten years belongs to the three phyla, Proteobacteria,

Actinobacteria and Firmicutes. Though new ptoteobacterial species were

extensively described, they were all restricted to chemotrophs. There

were no descriptions of new species of phototrophic Proteobacteria till

2004, though sporadic reports of their distribution in India are available.


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Purple non sulfur bacteria are reported from industrial effluents near

Hyderabad (Renuka et al., 1987; Sasikala et al., 1995), paddy soils of

south India (Sasikala et al., 2002), and reverine ecosystems (Sarkar and

Banerjee, 1979), while purple sulfur bacteria were isolated from tropical

waters of Pichavaram mangroves near Port Novo and other coastal

ecosystems (Loka Bharathi and Chandramohan, 1986; Krishnamurthy et

al., 1986). Though description of novel species of Anoxygenic

Phototrophic Bacteria from India started relatively recently (Ramana et

al., 2005), there are 40 species names and 2 genera names validly

described till July 2011 (http://www.bacterio.cict.fr).

Sample collection was random and arbitrary and, it was one time

sampling at a particular site (Fig. 3.1). Diverse habitats of different

regions of sampling are given in the methodology (section, 2.9). Apart

from samples such as soil, sediment, water, microbial mats and natural

purple blooms (Fig. 4.1) which were earlier analyzed for APB diversity

from India (Arunasri, 2005; Srinivas, 2008; Anil Kumar, 2008; Kalyan,

2011), dead marine invertebrate samples (jellyfish with pink, brown and

red coloured spots) (Fig. 4.1d) were also utilized for the isolation of purple

bacteria. Most of the other samples studied were from the Indian

Himalayas (20) and coast of south India (30). During sampling,

pink/purple/brown coloured ponds (Fig. 4.1a and 4.1b) and sands (Fig.

4.1c) with blooms of purple phototrophic bacteria were observed and

sampled. Apart from visual observation, habitats rich in purple bacteria

can be located (even when blooms are not present) by remote sensing

(Gitelson et al., 1999) using bacteriochlorophylls as biomarkers.

http://www.bacterio.cict.fr/


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For isolation of bacteria from environmental samples, two

strategies can be used: direct isolation and enrichment isolation. Since

normally purple bacteria occur in relatively low numbers in natural

habitats, they require enrichment prior to isolation. However, from

natural blooms, which are rather quite rare, they can be isolated by

direct plating or streaking on agar. Since microbial population of would

have acclimatized to respective environmental conditions of the sample,

Fig. 4.1 Few natural purple blooms and dead marine

specimen (sampling sites). (a) Purple pond near seashore, (b)

Purple bloom in the pond, (c) Purple sand near seashore at

Pamban Bridge and (d) Dead Jelly fish at seashore


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mineral salts such as calcium chloride; sodium chloride and magnesium

sulfate were adjusted according to the nature of the sample

(marine/fresh water) in the enrichment media.

In the present study, different types of bacteria were enriched in

the same mineral media by varying carbon source/e- donor, nitrogen

source, salt concentration, sulfur source and pH. For example, Rba.

maris JA276T (Fig. 3.2) was enriched from moist sediment of seashore at

Cochin in a mineral medium containing 0.04% (w/v) NaCl and pyruvate

as carbon source/e- donor, whereas Mch. gracile sp. JA260 (Table 3.1)

(purple sulfur bacteria) was isolated from the same sample by replacing

pyruvate with bicarbonate as inorganic carbon source and sodium

sulfide as e- donor with 2% (w/v) NaCl. Similarly, strain JA225 of the

genus Rhodobacter could be enriched from microbial mat of hot sulfur

spring of Manikaran (Table 3.1) by using pyruvate as carbon source,

whereas Rps. faecalis JA227 (Table 3.1) was enriched from the same

sample by replacing pyruvate with sodium bicarbonate as inorganic

carbon source and hydrogen as electron donor. Few strains, those

belonging to the genera Rhodothalssium, Blastochloris and

Rhodomicrobium were isolated for the first time from India by using

different physico-chemical parameters for enrichments such as high salt

concentration (5-8% w/v)/glutarate and maltose as carbon

source/succinate as carbon source at low pH (5.5) respectively.

Incubation conditions such as light and temperature were also

varied which could allow enrichment of a wide variety of purple bacteria.

For example, strains JA349 and JA350 with two completely different


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carotenoid series (spirilloxanthin and okenone respectively) of the same

genus Marichromatium were enriched from the pink coloured moist sand

at seashore at Pamban Bridge, by using fluorescent light and

incandescent light respectively (Table 3.1). Hence, it is suggested to use

multiple enrichment media that mainly vary in medium components like

carbon source, nitrogen source, NaCl concentration and pH; and

physical parameters like temperature and light (intensity and quality). In

contrast, maintaining laboratory conditions that are similar to that of the

environment may enable the growth of dominant bacteria of respective

environments.

It is suggested to isolate purple bacterial strains from the

enrichments as early as possible since algal succession was observed in

certain enrichment cultures upon prolonged incubation. However, the

incubation of the enrichment media for prolonged time (if not dominated

by undesired organisms) enables detection of slow growing organisms or

organisms that develop only after modification of the environment by

other strains.

The isolation and purification methods used here are streaking on

agar slants and plates/ agar shake cultures. Since most of the target

isolates from the study are obligate anaerobes, they were isolated by the

method of streaking on agar slants, in which anaerobic condition is

maintained by sealing with butyl rubber stoppers and flushing with inert

gas argon to replace the air in it. Isolation of facultative anaerobes of

purple nonsulfur bacteria can also be done by streaking on agar plates.

However, the same could not be followed for purple sulfur bacteria, as

they require reduced sulfur compounds like sulfide as electron donor in


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their medium, which could be oxidized on exposure to air. One more

method, paraffin wax overlay (Archana et al., 2004) was also suggested

for the isolation of these bacteria which however is applicable only for

those strains which can withstand exposure to melting temperature of

wax (45-50OC).

All the 59 isolated purple bacterial strains (Table 3.2) could not be

subjected to the laborious polyphasic characterization, which includes

large numbers (about 150) of tests. Hence, for detailed polyphasic

characterization, 9 strains (JA194T, JA276T, JA296T, JA248T, JA430T,

JA310T, JA531T, JA349T, JA553T) which showed unique characters

(Table 3.2) were selected by rapid typing based on characteristics such as

habitat, morphological characters, bacteriochlorophylls and 16S RNA

gene sequence similarity (< 97%) (Stackebrandt and Goebel, 1994).

Polyphasic taxonomy includes the study of multiple parameters

with respect to the delineating taxa. As there is no single golden standard

to classify and identify the bacterial taxa, polyphasic characterization is

well accepted approach in the present bacterial taxonomic studies. The

standards and number of methods and/or parameters of polyphasic

taxonomy are not final and are subject to constant changes. Two

publications that were consulted for the description of novel taxa of

anoxygenic phototrophic bacteria are ―Recommended standards for the

description of new species of the anoxygenic phototrophic bacteria‖

(Imhoff and Caumette, 2004) and ―Notes on the characterization of

prokaryote strains for taxonomic purposes‖ (Tindall et al 2010) (Table

4.1).


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Until 2010, purple bacterial taxa description has been carried out

based on recommended standards for polyphasic taxonomic study by

Imhoff and Caumette 2004 (Rdv. marinum [Srinivas et al., 2006];

Rubrivivax bezoatilyticus [Ramana et al., 2006]; Rba. vinaykumarii

[Srinivas et al., 2007]; Rba. maris [Venkata Ramana et al., 2008];

Thiohalocapsa marina [Anil Kummar et al., 2009]) with no need of

optional characters (chemotaxonomy). Since then, chemotaxonomic

characters such as FAME, polar lipids, quinones and genetic

characterization such as phylogenetic analysis based on housekeeping

genes, Multi Locus Sequence Anlaysis (MLSA) (Richter et al., 2006;

Delétoile et al., 2010) became essential (Table 4.1) and, are being used to

delineate the taxa. However, information of chemotaxonomic data are

not available for all type strains and there is an urgent need to create

libraries or databases of cellular fatty acids, quinones and polar lipids to

enable comparative analysis in taxonomic studies which may also lead to

discovery of possible biotechnological potentials. For polyphasic

characterization, the target strain has to be comparatively studied with

all nearest type strains of the respective higher taxa in all aspects at

author‘s laboratory (Tindall et al., 2010). This requirement is becoming a

bottleneck in description of novel taxa, since frequently no type strains

are available and sometimes legitimacy also is questionable (Okamura et

al., 2009).


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Property Comments

©Morphology:

Cell shapea*

Cell size (diameter, length)a

Motilitya and flagellation b

Colony morphology

Type of cell division b

Cell differentiation and life cycle

Formation of typical cellular

aggregates a

eg. Net or platelet

formation, capsule and

slime formation

Visible internal or external

structures b

eg. spores, gas vesicles

Gram-stain a Optional in the case of

established Proteobacteria

Ultra structure c eg. internal membranes,

chlorosomes and others

Chemical composition:

Color of cell suspensions

In-vivo absorbtion spectra

Type of bacteriochlorophylls

Carotenoides / Pigments

Quinones

®Fatty acids

®Polar lipids Two dimendional

Cell wall analysis Optional

Reserve materials eg. PHB, polyglucose, So

DNA base ratio i.e. G+C content ®Phylogenetic analysis:

Sequence Use complete and good

quality sequence.

16S rRNA gene sequence,

phylogenetic tree position

Alignment databases Multiple alignment using

databases ARB, RDP, SILVA

and LTP

Alignment softwares CLUSTAL_X, CLUSTAL W,

CLUSTAL X2, CLUSTAL W2,

MEGA, T-COFFEE,

MUSCLE), should be used

followed by manual editing.

Alignment file Should be submitted as

supplementary data. Never


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truncate an aligned file.

Pair wise similarity calculation To use ARB, PHYDIT and

jPHYDIT programs

Phylogenetic tree To use distance matrix,

maximum-parsimony,

maximum-likelihood

methods.

Genetic analyses:

DNA-DNA hybridization If applicable

®MLSA, MLST If applicable

®Sub species and strain level AFLP-PCR, PFGE, RAPD,

rep-PCR, REP-PCR, ERIC-

PCR, BOX-PCR, (GTG)5 -

PCR and ribotyping.

Physiology:

Growth medium

Temperature range and optimum

pH range and optimum

NaCl range and optimum

Vitamin requirements

Carbon, nitrogen and sulfur

Sources

Relations to sulphide Tolerance, use, oxidation

products

Relation to oxygen Anaerobic, aerobic,

microaerobic growth,

tolerance

Light responses Minimum requirement,

light sensitivity: optional

Modes of energy generation:

Phototrophy,

Potential chemotrophy,

Electron donors,

Electron acceptors

Ecology:

Natural habitat Physico-chemical

properties, pH, salinity,

temperature

Source of isolates Geographical location, GPS

positioning

Mineral salts composition of source

Abundance at habitat

Culture deposition: Minimum of 2 culture

collection certificates

Sequence deposition: To be accessible to public


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EMBL Accession number

Comparitive study

Reference strains

Should be characterized at

author‘s laboratory

Table 4.1 Revised table of characteristics / parameters required for the description of new species of anoxygenic phototrophic bacteria. (Imhoff and Caumette 2004; modification after recommendations of ®Tindall et al., 2010). Those marked as optional provide possibly important properties of the bacterium but are not regarded as essential for the species description. *To be demonstrated by: a) a light micrograph; b) a light or electron micrograph; c) an electron micrograph.

STRAINS OF THE GENUS Rhodobacter (Rba)

The family Rhodobacteraceae of Alphaproteobacteria consists of 5

phototrophic genera; Rhodobacter, Rhodobaca, Rhodovulum,

Rubribacterium and Rhodothalassium. Rhodobacter and Rhodovulum are

the two genera each having relatively large number of species (12 each)

among APB. Rhodobacter is the type genus of the family

Rhodobacteraceae, order Rhodobacteriales, class Alphaproteobacteria.

(http://www.bacterio.cict.fr/classifgenerafamilies.html#Rhodobacteracea

e).

The genus Rhodobacter is differentiated from other genera with few

characteristic properties such as ovoid to rod shaped cell morphology,

the presence of vesicular internal membranes (Except in Rba. balsticus)

and carotenoides of spheroidene series (Imhoff, 2005). Although

Rhodobacter spp. and Rhodovulum spp. rather differ in NaCl requirement

and they share common chemotaxonomic characteristics such as large

type cytochrome C2 (Amber et al., 1979), sole quinone component Q-10

(Imhoff, 1984a), lipopolysaccharides with gulucosamine as sole amino

sugar in their lipid A moieties, phosphate and amide-linked 3-OH-14:0


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and/or 3-oxo-14:0 and ester linked 3-OH-10:0 (Weckesser et al., 1995),

those enable their distinction from other genera. Rhodobacter spp are

differentiated from each other based on morphology, slime production,

sulfate assimilation, denitrification, vitamin requirement, utilization of

carbon source and fatty acid composition, 16S rRNA gene sequence

comparison and DNA-DNA hybridization (de Bont et al., 1981; Ivanova et

al., 1988; Hiraishi et al., 1996).

In enrichment cultures set up for purple nonsulfur bacteria,

members of this genus grown faster and outcompete other purple

nonsulfur bacteria. In the present study, 11 different strains of the genus

Rhodobacter; JA194T, JA247, JA260, JA276T, JA296T, JA312, JA313,

JA431, JA533, JA542 and JA555 were isolated from diverse habitats

such as soil, estuarine water, fresh water pond, water and sediment of

seashore, microbial mat, soil below the ice (high attitude) and hot sulfur

springs, whereas the existing type strains are only from fresh water,

sewage ponds and eutrophic lakes

(http://www.bacterio.cict.fr/qr/rhodobacter.html), which reflects the

identification of novel habitats of Rhodobacter strains.

Among the above 11 strains, 3 strains JA194T, JA276T,

JA296T exhibited unique characters in the rapid typing and were

subjected to detailed polyphasic study. Strains isolated from similar

ecological niche showed close phylogenetic relationship which was

observed by the phylogenetic tree of Rhodobacter spp. from the present

study (Fig. 4.2) In the phylogenetic tree, strains JA276T, JA296T, JA431T,

JA533, JA542 and JA555 which were isolated from marine and estuarine

http://www.bacterio.cict.fr/qr/rhodobacter.html


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habitats clustered together along with Rba. vinaykumarii which was also

of marine origin representing the group of marine cluster (Fig. 4.2).

Similarly, strains JA225, JA194T, JA247, JA312, and JA313, isolated

from fresh water habitats clustered with type strains of fresh water origin

and branched separately from strains of marine cluster.

Fig. 4.2 Dendrogram depicting the phylogenetic relationship of Rhodobacter strains (bold) isolated from diverse habitats of India.


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Strains JA194T and JA247

Strain JA194T is completely oval, similar to Rba. ovatus, whereas

remaining all strains are oval to rods except Rba. sphaeroides which is

spherical to rods. The strain JA194T able to grow from 5-400C (Fig. 3.9),

Strain JA194T lacked an ester peak (1735 cm-1) and differed in other

cellular components, including proteins (C–N stretch amines 1230 cm-1),

Table 4.2b. Carbon sources and electron donors used by species of

the genus Rhodobacter. Taxa: 1, Rba. johrii JA192T (Girija et al., 2010); 2, JA194T ; 3, Rba. sphaeroides (Imhoff, 2005); 4, JA276T; 5, Rba. veldkampii (Hansen & Imhoff, 1985; Imhoff, 2005); 6, Rba. vinaykumarii (Srinivas et al., 2007); 7, Rba. ovatus (Srinivas et al., 2008); 8, Rba. blasticus (Imhoff, 2005); 9, Rba. azotoformans (Hiraishi et al., 1996); 10, JA296T; 11, Rba. capsulatus (Imhoff, 2005); 12, Rba. changlensis (Anil Kumar et al., 2007a). For all taxa, organic substrate utilization was tested during photo-organoheterotrophic growth. All taxa utilized pyruvate, but not benzoate or arginine. +, substrate utilized; _, substrate not utilized or absent; ±, variable in different strains; (+), weak growth; NR, not reported.

Table 4.2 (a) Differentiating characteristics of species of the genus

Rhodobacter. Taxa: 1, Rba. johrii JA192T; 2, JA194T; 3, Rba. sphaeroides DSM 158T; 4, JA276T; 5, Rba. veldkampii; 6, Rba. vinaykumarii JA123T; 7, Rba. ovatus JA234T; 8, Rba. blasticus; 9, Rba. azotoformans KA25T; 10, JA296T; 11, Rba. capsulatus; 12, Rba. changlensis JA139T. Data represent a comparative analysis done in the authors‘ laboratory unless indicated. Motile species exhibit polar flagella. +, Present; 2, absent; ±, variable in different strains; NR, not reported; *C, Chains; O, ovoid; R, rod-shaped; S, spherical; †GB, Greenish brown; OB, orange–brown; YB, yellowish brown. ‡DMSE, Demethylspheroidene; SE, spheroidene; SO, spheroidenone; OH-SE, hydroxyspheroidene; OH-SO, hydroxyspheroidenone; Me-SE, methoxyneurosporene; Polar, polar carotenoid; tr, trace; §Polar carotenoid with nine conjugated double bonds; ||L, Lamellar; V, vesicular; ¶NaCl not required, but optimal growth occurs at 3% NaCl; #Optimal growth in the absence of NaCl but growth occurs at 3% NaCl; **Optimal growth in the absence of NaCl but growth occurs at 5% NaCl; ††Data from Hiraishi et al. (1996); ‡‡b, Biotin; B12, vitamin B12; n, niacin; p-ABA, p-aminobenzoic acid; t, thiamine; (b, n), a few strains require biotin and/or niacin; §Ranges represent values for more

than one strain, including the type strain; ||||-, Not detected/not reported.


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polysaccharides (1183, 1131 and 1100 cm-1) and aromatic compounds

(980 cm-1), compared with Rba. sphaeroides DSM 158T. A peak in strain

JA194T at 3150 cm-1 in the lipid FT-IR fingerprint ( Fig. 3.10) was due to

N–H stretching of peptide linkages of the polypeptide chain of

lipoproteins (Lam et al., 2004) which is not observed in the Rba.

sphaeroides DSM 158T. Furthermore, the high intensities of peaks

observed in strain JA194T indicate the presence of longer acyl chains of

fatty acids (Ami et al., 2006).

Strain JA194T vary from speices of the genus Rhodobacter in

utilizing organic carbon sources/e- donors (Table 4.2b), for their

phototrophic growth. Though the strain JA194T has distinct phenotypic,

physiological and chemotaxonomic properties, its 16S RNA gene

sequence is 99% similar to the nearest type strain Rba. sphaeroides.

Furthur, genomic relatedness (67% DNA-DNA homology) and metabolite

fingerprinting of strain JA194T supported its distant phylogenetic

relatedness with the nearest type strain Rba. sphaeroides DSM 158T and

necessitated the description of strain JA194T as a novel species of the

genus Rhodobacter named as Rhodobacter megalophilus JA194T. Strain

JA247 has all characteristics similar to strain JA194T and proposed as

an additional strain of Rhodobacter megalophilus JA194T.

Description of Rhodobacter megalophilus JA194T sp. nov.

Rhodobacter megalophilus [me.ga.lo.phi‘lus. Gr. adj. megas wide; Gr. adj.

philos loving; N.L. masc. adj. megalophilus wide (temperature)-loving].

Cells are Gram-negative and oval-shaped (1.2–1.5 X 1.5– 2.0 mm).

Multiplication occurs by binary fission. Cells have vesicular


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intracytoplasmic membranes and lack flagella. Phototaxis occurs. Whole-

cell absorption maxima are found at 374, 407, 446, 476, 509, 590, 800

and 854 nm. Growth occurs photo-organoheterotrophically with various

sources, including malate, fumarate, acetate, propionate, butyrate,

valerate, caproate, glutamate, aspartate, glucose, tartrate, ethanol,

mannitol, sorbitol and glycerol. Photolithoautotrophic growth is possible

in the presence of thiosulfate as electron donor and NaHCO3 as carbon

source. Nitrogen sources providing good growth are ammonium chloride

and glutamate. Nitrate and molecular nitrogen also support growth as

nitrogen sources. Thiamine is required for growth. Growth occurs from 5

to 400C. The DNA G+C content of the type strain is 66.67 mol% (by

HPLC). The type strain, JA194T (=KCTC 5602T =JCM 14598T), and an

additional strain of the species, JA247, were isolated from soils of the

Indian Himalayas.

Strain JA276T

Chain formation is observed in strain JA276T like in JA296T, Rba.

capsulatus (Molisch, 1907; Imhoff, 2005) and Rba. changlesis (Anil

Kumar et al., 2007a). Unlike, Rba. vinaykumarii (Sriniva et al., 2007)

isolated from marine water, which had obligate requirement for NaCl,

strain JA276T do not require NaCl though isolated from similar habitat,

but could tolerate upto 3% NaCl (w/v). In the genus Rhodobacter, the

fatty acids C19:1ω7c / ω 6c / 19 cyclo were observed only in strain

JA276T (Table 4.2a). Strain JA276T also varies in utilizing organic carbon

sources/e- donors (Table 4.2b) during phototrophic growth and its 16S

rRNA gene sequence is 96.2% similar to Rba. capsulatus. Morphological,


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physiological and genomic (16S rRNA gene sequence) differences of strain

JA276T (Table 4.2a and 4.2b) from other species of the genus

Rhodobacter, enable strain JA276T to be described as novel species

named Rhodobacter maris JA276T.

Description of Rhodobacter maris JA276T sp. nov.

Rhodobacter maris (ma'ris. L. gen. n. maris, of the sea, pertaining to the

habitat from where the type strain was isolated).

Cells are ovoid to rods, 0.6-1.0 m wide and 1.0-1.5 m long, non-

motile, divide by binary fission and can form chains of 4-6 cells. Growth

occurs under anaerobic conditions in the light (photoorganoheterotrophy)

or under aerobic conditions in the dark (chemoorganoheterotrophy).

Internal photosynthetic membranes are of the vesicular type. The color of

phototrophic cultures is yellowish brown. The in vivo absorption

spectrum of intact cells in sucrose exhibits maxima at 377, 476, 509,

590, 803, and 860 nm confirming the presence of bacteriochlorophyll a.

Carotenoids include spheroidene and spheroidenone. Substrates that

were utilized by strain JA276T as carbon sources and electron donors

under photoorganoheterotrophic conditions include; acetate, fumarate,

pyruvate, malate, glycerol, valerate, lactate, caproate, propionate and

butyrate. Strain JA276T could not utilize formate, caprylate, gluconate,

succinate, thiosulfate, aspartate, ascorbate, benzoate, glutamate, sulfur,

proponol, glucose, fructose, mannitol, peptone, sucrose, casaminoacids,

sorbitol, ethanol, tartrate, sulfite, citrate, oxaloacetate, 2-ketoglutarate,

lactose, maltose, starch, sulfide, bicarbonate, pelarganate, arginine,

yeast extract and oleic acid. Ammonium chloride, glutamate, glutamine


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and molecular nitrogen were utilized as nitrogen sources, while urea,

aspartate, nitrate and nitrite did not support growth. Magnesium sulfate,

thiosulfate, sulfite, thioglycolate and sulfide were utilized as sulfur

sources, while elemental sulfur, methionine and cysteine did not support

growth. NaCl was not obligatory for growth of strain JA276T; but

tolerates up to 3% (w/v) NaCl. Strain JA276T grew at pH 6.0–8.0

(optimum at 6.5-7.0) and at 25–35C (optimum at 30C). Thiamine is

required as growth factor. Autotrophic and fermentative growth is not

possible. The type strain, JA276T (=JCM 14794T =ATCC BAA 1549T

=CCUG 55129T) was isolated from a sediment samples collected from sea

shore of Cochin, Kerala State, India. G+C content of the type strain is

62.85 mol% (by HPLC).

Strain JA296T

The sample yielding strain JA296T was a coloured microbial mat of

brakish water, which is different from the samples that yielded other

strains of the genus Rhodobacter. Strain JA296T also has unique

characters among Rhodobacter spp such as chain formation, marine

origin, lack of NaCl requirement (with tolerance up to 2% NaCl w/v) and

different organic substrate utilization. Apart from the above, strain

JA296T is 95.5% similar to Rba. capsulatus by 16S rRNA gene sequence.

Carotenoid demethylspheroidene (DMSE) was observed only in the strain

JA296T. Therefore, based on morphological, physiological,

chemotaxonomical (Table 4.2a and 4.2b) and genetic differences, strain

JA296T was described as novel species named Rhodobacter aestuarii.


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Description of Rhodobacter aestuarii JA296T sp. nov.

Rhodobacter aestuarii (aes.tu.a‘ rii. L. gen. n. aestuarii of the estuary, the

habitat from which the type strain was isolated).

Cells are ovoid to rod-shaped, 0.7-1.0 m wide and 1.5-2.0 m

long, motile, divide by binary fission and form chains of 4-8 cells. Growth

occurs under anaerobic conditions in the light (photo-

organoheterotrophy) or under aerobic conditions in the dark (chemo-

organoheterotrophy). Internal photosynthetic membranes are of the

vesicular type. The color of phototrophic cultures is yellowish green.

Bacteriochlorophyll a and the carotenoids, sphaeroidene and

spheroidenone are present. Substrates that are utilized by strain JA296T

as carbon sources and electron donors under photoorganoheterotrophic

conditions include; pyruvate and lactate. NaCl was not obligatory for the

growth of strain JA296T; but tolerates up to 2% (w/v) NaCl. Strain

JA296T grew at pH 6.0–8.5 (optimum at 7.0) and at 25–35 C (optimum

at 30 C). Thiamine is required as growth factor. Photolithoautotrophic,

chemolithoautotrophic and fermentative growth could not be

demonstrated. The DNA G + C content of the type strain is 65.1 mol% (by

HPLC).

The type strain, JA296T (=ATCC BAA-1550T = CCUG 55130T) was

isolated from a brown colored microbial mat of brackish water from the

Bhitarkanika mangrove forest at Dangmal, Orissa, India.


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Objectionable reclassification of Rhodobacter changlensis to the

new genus Catellibacterium

Rba. changlensis (Anil Kumar et al., 2007a) is now reclassified into the

new genus Catellibacterium (Zheng et al., 2011), based on phylogenetic

dendrograms constructed using 16S rRNA gene sequences. Since,

taxonomy of phototrophic bacteria is interspersed with chemotrophs, it

might be possible in clustering Rba. changlensis with the members of the

genus Catellibacterium. However, phototrophy is a genus specific

character of Rhodobacter, Rba. changlesis should not be reclassified into

a genus Catellibacteriuim (non phototrophic members) as none of the

species of the genus Catellibacterium have phototrophy. Therefore,

Rhodobacter changlensis to be further studied carefully to delineate as

novel phototrophic genera.

STRAINS OF THE GENUS Rhodopseudomonas (Rps)

Rhodopseudomonas and Rhodoblastus are the only phototrophic

genera of the family Bradyrhizobiaceae of order Rhizobiales. Genus

Rhodopseudomonas differentiated from other genera of the family

principally based on 16S rRNA gene sequence and phototrophy. Ever

since the description of the genus Rhodopseudomonas (Czurda &

Maresch 1937), the members of this genus were subject to significant

changes including reclassification of species (Imhoff et al., 1984b;

Hiraishi & Ueda, 1994; Hiraishi, 1997) and currently, there are only

three species names remaining with this

genus(http://www.bacterio.cict.fr/qr/Rhodopseudomonas.html);Rhodpse


Results

175

udomonas palustris, Rps. rhenobacensis and Rps. faecalis. Since, all the

three type strains are differentiated among themselves in the species

level based on DNA-DNA hybridization, they are called as

Genomospecies.

Species of the genus Rhodopseudomonas belong to the class

Alphaproteobacteria and share common features such as Gram-negative

rod-shaped cells, motile cells by means of polar or subpolar flagella,

polar growth, budding and asymmetric cell division. Primary

characteristic feature is the formation of rosette like aggregates.

Photosynthetic pigments are bacteriochlorophyll a and carotenoides of

the spirilloxanthin series. Preferred mode of growth is photoheterotrophic

under anoxic conditions in the light.

During the study, 11 strains of genus Rhodopseudomonas JA226,

JA227, JA228, JA229, JA251, JA253, JA310T, JA311, JA531T, JA640

and JA641 were isolated from diverse habitats (Table 3.1) of different

geographical regions of India. All the above strains share genus specific

characteristics of Rhodopseudomonas such as Gram-negative rod-shaped

cells, cells are motile by means of polar or subpolar flagella, budding,

asymmetric cell division, formation of rosette like aggregates of the genus

Rhodopseudomonas.

Based on 16S rRNA gene sequence similarity, 9 among 11 isolated

strains belongs to Rps. faecalis (Table 3.2) probably indicating their

predominance. The percentage of relatedness between strains obtained

by 16S rRNA gene sequence alignment tool did not concur with that of

phylogenetic tree. This may probably be because all three type strains


Results

176

being Genomospecies, not much variation in the 16S rRNA gene

sequences was observed among them. Though most of the strains JA226,

JA228, JA229, JA227, JA251, JA640, JA641, JA310T and JA311 showed

highest 16S rRNA gene sequence similarity with Rps.faecalis, they have

clustered together and formed a single clade with Rps. palustris. Strains

JA253 and JA531T have separately claded from the node of two clusters

of Rps. faecalis and Rps. rhenobacensis (Fig. 4.3).

Strains yielding samples include fresh water of lake and stagnant

water pool, sediment of saltpan, sewage water, soil, microbial mats and

hot sulfur spring waters, whereas type strains of the genus

Rhodopseudomonas were isolated from soil/sludge/pond water (Molisch

1907), sediment of fresh water lake (Hougardy et al., 2000) and chicken

feces (Zhang et al., 2002).

Like strains of the genus Rhodobacter (Table 3.1), strains of genus

Rhodopseudomonas are also frequently isolated from most of the

samples. A mineral medium prescribed by Biebl and Pfennig 1981 for the

isolation of PNSB or the members of Rhodospirillaceae, can be applied for

isolation and cultivation of Rhodopseudomonas species (Imhoff, 1998a).

In the present study, strains of the genus Rhodopseudomonas were

widely isolated from the enrichment media (Table 2.1) formulated with

pyruvate as a carbon source with different NaCl concentrations. Based

on rapid typing, 2 strains JA310T and JA531T were selected for detailed

polyphasic characterization.


Results

177

Fig. 4.3 Dendrogram depicting the phylogenetic relationships strains

JA531T, JA253, DSM 123T, JA251, JA229, JA640, JA641, JA311, JA226, JA228, JA310T and JA227 with the members of the genus Rhodopseudomonas along with interspersing chemotrophs as determined using 16S rRNA gene sequence. Tree was constructed by the neibour joining method using the MEGA4 software. Bar; two nucleotide substitutions per 100 nucleotides.


Results

178

Strain JA531T

Strain JA531T is the only strain of Rhodopseudomonas so far

isolated from the hyper saline habitat (saltpan 10% NaCl w/v). However,

this strain has no obligate requirement for NaCl but can tolerate up to

4% NaCl. Strain JA531T could grow under a wide range of pH (5-9) (Table

4.3), which was not observed in any of the other species of the genus

Rhodopseudomonas. Utilization of benzoate (up to 30 mM) was observed

by strain JA531T, which is a characteristic feature of Rps. palustris.

These unique characters made us to go for polyphasic characterization to

ascertain if it represents a new species. Strain JA531T has interspecies

variability through their habitat, cell size, type of budding, colour of the

culture, temperature, pH range, growth factor requirement, polar lipid

composition, fatty acid composition, carbon source utilization, DNA-DNA

relatedness and G+C content (Table 4.3). Since 16S rRNA gene sequence

similarity of strain JA531T is 97-98% (i.e, >97%) with Rps. palustris, Rps.

faecalis and Rps. rhenobacensis, DNA-DNA relatedness studies were

carried out. A genome relatedness of 48-62% observed further confirmed

that strain JA531T represents a novel species which was named

Rhodopseudomonas harwoodiae JA531T.


179

Characteristic §1 2 3 4 5 6

Isolation source Saltpan sediment

Soil Soil/pond water

Soil/Sludge/pond water

Chicken faces

Eutrophic pond

Cell shape Rods Rods Rods Rods with rounded ends

Vibriod Rods

Cell size ( width X length in μm)

0.8-1 X 2.0-3.5

0.8-1.2 X 2.0-4.0

0.5-1.0 X 2.0-6.0

0.4-1.0 X 1.5-3.0 0.6-0.8 X 1.0 – 2.0

0.4 – 0.6 X 1.5-2.0

Type of Budding Tube Sessile Sessile Tube Sessile Sessile

Color of cultures R RB RB RB R R

Optimal pH (range) 6-7.5 (5-9) 6.5-7 (6- 9) 6-8 (7) 6.5-8.5 (6-9) 7 (6.5-7) 5.5 (5-7)

Temperatureoptimum (range; oC

) 30 (25-40) 30 (15-35) 30 (20-35) 30 (25-40) 30 (25-35) 25-35 (30)

Aerobic dark growth + + + + - +

Photoautotrophic growth with

- - - H2, Thiosulfate, Sulfide

H2, Sodium bicarbonate

-

Growth factors PABA

Thiamine, PABA

PABA ¥PABA Thiamin PABA

Major Polar lipids DPG, PG, PE, PC,

AL1 and 3, GL1-3, L1-3 and 7,8

DPG, PG, PE, PC,

AL1 and 3, GL2, L1-3

DPG, PG, PE, PC, OL, AL1-3, GL1-3, L1-

3 and 7

DPG, PG, PE, PC, OL, AL1-3,GL1-2,

L1-7

DPG, PG, PE, PC, OL, AL1-3, GL1, L1-3

DPG, PG, PE, PC, OL, AL1-3, GL1-2, L1-3 and

7

Fatty acid composition (mol%)

C12:0 - 1.1 2.4 1.6 1.7 0.2

C14:0 - - - 1.8 - -

C14:0 2OH - - 1.0 - - 0.1

C14:0 3OH 0.7 0.6 1.7 - - -

C16:0 7.6 17.8 18.2 20.8 14.9 13.4 &C16:1ω7c alcohol 1.0 1.1 1.5 0.3 1.7 0.5


180

C16:1 isoI - 0.5 0.8 - - -

C16:1ω7c/6c 2.8 2.5 3.3 10.2 6.2 7.8

C18:0 6.9 5.3 5.0 4.0 2.8 3.5

C18:1 ω5c - 0.7 - 0.1 1.1 0.5

C18:1 ω7c 74.2 53.2 56.5 51 63 69.6

C18:1 ω7c11methyl 3.2 7.5 4.0 4.3 5.8 1.8

C19:0 iso - - 4.2 0.3 - 0.2

C20:0 - 1.3 2.4 0.1 - 0.1

C20:1 ω7c - 1.2 - 0.2 - 0.2

Organic substrates supporting growth ψBenzoate + - + + - -

Butyrate + - + + + +

Caproate + - + (+) (+) -

Caprylate - - + - - -

Ethanol - - +/- + - +

Formate - - + - - +

Fumarate (+) + + + (+) +

D- Glucose - (+) +/- + +/- -

Glutamate - - + - - -

Glycerol (+)/- + + + - (+)

Glycolate - - + - -

Malate - + + + + +

Malonate (+)/- - + + - -

Propanol - - + - -

Propionate (+) - + + (+) -

Sorbitol - + + - -

Succinate (+) + + + + +

Tartarate - - - - - +

Valerate + - + - -


181

Molecular features $DDH(%) relatedness 1 100

2 33.1 + 5.2 100 3 45 + 9.2 48 + 4.2 100 4 47.5 + 4.4 45.3 + 2.8 32.8 + 3.8

[25] 100

5 28.4 + 2.9 32.1 + 2.2 41 + 8.3 [25] 40.7 + 2 [18] 100 6 27 + 4.6 24.1 + 3.8 32.5 + 14.5

[20] 38.6 + 4.4 [29] 43.5 + 4.2

[25] 100

@16S rRNA gene sequence similarity (%)

1 100

2 96.1 100

3 96.5 98.8 100

4 98.4 96.7 97.1 100

5 97.0 97.8 98.1 97.4 100

6 96.8 97.7 98.0 97.2 99.0 100 €DNA G+C content (mol%) 62.4 63.8 65.8 64.9 64.0 65.4

Table 4.3 *Differentiating characteristics between species of the genus Rhodopseudomonas. *Sources: Data pertains to comparative analysis done at author‘s laboratory. Taxa: 1, Rhodopseudomonas harwoodiae sp. nov. JA531T; 2, Rhodopseudomonas parapalustris sp. nov. JA310T; 3, Rhodopseudomonas pseudopalustris sp. nov. DSM 123T; 4, Rhodpseudomonas palustris ATCC 17001T; 5, Rhodopseudomonas faecalis JCM 11668T; 6, Rhodopseudomonas rhenobacensis DSM 12706T. For all taxa organic substrate utilization was tested during photoheterotrophic growth. Acetate, lactate and pyruvate are utilized by all the strains. All strains could not utilize: citrate, fructose, mannitol, methanol and aspertate. Intracellular photosynthetic membranes are of lamellae for all strains. All strains have bacteriochlorophyll-a, show tryptophanase –ve, have no salt requirement, assimilate sulfate, do not ferment glucose/fructose and have Q10 as major quinone. +, substrate utilized/present; -, substrate not utilized/absent; (+), weak growth; R, Red; RB, Red Brown; PABA, para-aminobenzoic acid; PE, phosphatidyl ethanolamine; PC, phosphatidyl choline; DPG, diphosphatidyl glycerol; PG, phosphatidyl glycerol; OL, ornithine lipid; AL1-3 unknown aminolipids; GL1-3, unknown glycolipids; L1-8, unknown lipids. Numbering of polar lipids is according to the convention adopted by Kampfer et al., 2006. ψTested at 0.5 and 1 mM. ¥Few strains require biotin; €HPLC method, &Alcohol (not a fatty acid); §Data of JA531T includes data of additional strains, JA447 and JA490. $Values given in the square brackets ([]) is taken from Okamura et al. (2009) and data given are the mean and standard deviation values of 3 independent experiments performed in triplicates for each sample (control: reversal of strains was used for binding and labeling); @Sequence similarity was calculated from the dendrogram (Fig 3.35).


182

Description of Rhodopseudomonas harwoodiae JA531T sp. nov.

Rhodopseudomonas harwoodiae (har.wo.o.di'a.e. N.L. fem. gen. n.

harwoodiae of Harwood, named after Professor Dr. Caroline S. Harwood,

a microbiologist who has significantly contributed to the aromatic

hydrocarbon metabolism of Rps. palustris)

Cells are rod shaped, 0.8-1m wide and 2.0-3.5 m long, budding

division, motile with lamellar ICM structures. Phototrophic cultures are

red. In vivo absorption spectra of intact cells exhibit maxima at 497, 590,

605, 863 nm. Bacteriochlorophyll-a and carotenoids of spirilloxanthin

series are present. No NaCl requirement (tolerates up to 4% [w/v]) The

type strain is mesophilic (range; 25-40 oC) with pH optimum at 6.0-7.5

(range; 5-9 pH). The preferred mode of growth is

photoorganoheterotrophy with a few organic compounds. Good growth

occurs on benzoate, butyrate, caproate, and valerate. Feeble growth

occurs with fumarate, glycerol, malonate, propionate and succinate as

carbon sources. Photoautotrophic and fermentative growth is absent.

Para-aminobenzoic acid is required as growth factor. C18:1 ω7c is the

major fatty acid with sufficient amount of C16:0, C18:0, C18:1ω7c11methyl,

C16:1ω7c/ω6c. Ubiquinone-10 is the major quinone. Cardiolipin,

phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine,

two unidentified aminophospholipids and an unidentified aminolipid are

the major polar lipids. The DNA G+C content of the type strain is 62.4

mol% (by HPLC). Natural habitats are salterns of coastal ecosystem. The

type strain, JA531T (=NBRC 107575T =KCTC 5841T), is isolated from a

sediment of a saltpan at Kalambakam, Tamil Nadu, India.


183

Strain JA310T

The enrichment and growth of strain JA310T occurred between 15-

350C with an optimum of 300C. Though the strain JA310T is

phylogenetically close to Rps. palustris, did not share the property of

benzoate utilization, which is a major characteristic feature of Rps.

palustris. Presence of small amounts of C14:03OH and C16:1 iso are

observed only in strains JA310T and Rps. palustris.

Though, strain JA310T showing >97% 16S rRNA gene sequence

similarity with nearest type strains (Imhoff and Caumette, 2004;

Stackebrandt and Ebers, 2006), its description as novel species is

supported by distant DNA-DNA homology (32.1 + 2.2) and polar lipids

composition (apart from phenotypic differences) (Table 4.3).

Polar lipid analysis revealed the presence of diphosphatidyl glycerol

(DPG), phosphatidyl glycerol (PG), phosphatidyl ethanolamine (PE),

phosphatidyl choline (PC) and an unidentified aminolipid (AL1) as the

major polar lipids in all the Rhodopseudomonas strains tested (Fig 4.3).

The major polar lipids composition observed in this study is in

concurrence with the earlier reports for the members of the genus

Rhodopseudomonas-Bradyrhizobium-213 Nitrobacter-Afipia (Hougary et

al., 2000; Imhoff et al., 1982). Major differences in the polar lipids among

the strains of Rhodopseudomonas were observed with respect to the

minor polar lipids. Glycolipid (GL3) was observed only with strains DSM

123T and JA531T which distinguish from other members of

Rhodopseudomonas, while presence of aminolipid (AL2) in strain DSM

123Tdelineates it from strain JA531T. Absence of a glycolipid (GL1) and


184

an aminolipid (AL2) differentiates strain JA310T with other members. A

few unidentified polar lipids (L4-L6) are unique to stain ATCC17001T (R.

palustris) and absence of a glycolipid (GL2) in stain JCM11668T (R.

faecalis) differentiates from the rest.

Like strain JA531T, strain JA310T also share genus specific

characters and showed interspecies variability (Table 4.3), which

necessitated the proposal of strain JA310T as a novel species named as

Rhodopseudomonas parapalustris sp, nov., (for sharing most of the

characteristics of Rps. palustris with few exception including separate

clustering with Rps. palustris).

Description of Rhodopseudomonas parapalustris JA310T sp. nov.

Rhodopseudomonas parapalustris (pa.ra.pa.lus'tris. Gr. prep. para,

alongside of, resembling; L. fem. adj. palustris, marshy, and also a

bacterial epithet; N.L. fem. adj. parapalustris, resembling to

Rhodopseudomonas palustris).

Cells are rod shaped, 0.8-1.2 m wide and 2.0-4.0 m long. Cells

are motile by means of polar flagella and multiply by budding. Cells of

old culture form rosettes. Internal photosynthetic membranes are

present as lamellar stacks parallel to the cytoplasmic membrane.

Phototrophically grown cultures are reddish brown. The in vivo

absorption spectrum of intact cells in sucrose exhibits maxima at 497,

590, 800 and 863. Bacteriochlorophyll a and carotenoids of the

spirilloxanthin series are present. The type strain is mesophilic (range;

15-35C). No NaCl requirement (tolerates up to 0.5% [w/v]) and growth

pH optima is from 6.5-7.0 (range, 6.0-9.0). The preferred mode of growth


185

is photoorganoheterotrophy with a few organic compounds. Malate,

succinate, pyruvate and sorbitol are good carbon sources. Phototrophic

growth also occurs on D-glucose. Benzoate, butyrate, caproate,

caprylate, ethanol, formate, glutamate, glycolate, malonate, proponol,

propionate, tartarate and valerate are not utilized as carbon sources.

Photoautotrophic and chemoautotrophic growth with thiosulfate / H2 /

sulfide / sulfur / sulfite / thioglycolate as the electron donor and

NaHCO3 as the carbon source is not observed. Fermentative growth does

not occur in the presence of glucose, fructose or pyruvate. Nitrate and

aspargine are used as nitrogen sources.

Thiamine and para-aminobenzoic acid are required as growth

factors. C18:1 ω7c is the major fatty acid with sufficient amounts of C12:0,

C16:0, C18:1 ω7c 11methyl, C18:0 and C16:1 ω7c/ω6c. Ubiquinone-10 is the

major Quinone. Cardiolipin, phosphatidylethanolamine,

phosphatidylglycerol and two unidentified aminophospholipids are the

major polar lipids. The G+C of genomic DNA is 63.8 mol% (by HPLC).

Natural habitats are surface soils near lake at high altitudes. The type

strain JA310T (=NBRC 106083T =KCTC 5839T) was isolated from a

sediment soil sample near Pangong Lake at Leh, Jammu and Kashmir,

India.

Strain DSM 123T

According to Okamura et al., (2009), ATCC17001T and DSM 123T

which are known to be type strain Rps. palustris are not the same.

Hence, both these strains were obtained from respective culture

collections and subjected to polyphasic characterization. The differences


186

exhibited by the strain DSM 123T with all other types strains of the

genus Rhodopseudomonas including DNA homology data (Table 4.3)

supported the statement made by Okamura et al., 2009 and necessitated

the description of DSM 123T as a novel species and named as

Rhodopseudomonas pseudopalustris (Venkata Ramana et al., MS under

revision).

Description of Rhodopseudomonas pseudopalustris DSM 123T sp.

nov.

Rhodopseudomonas pseudopalustris (pseudês, Gr. adj. false; L. fem. adj.

palustris, marshy, and also a bacterial epithet; N.L. fem. adj.

pseudopalustris, the false [Rhodopseudomonas] palustris)

Cells are rod shaped, 0.8-1μm wide and 2.0-6.0 μm long, budding

division, motile and have lamellar ICM structures. Phototrophic cultures

are reddish brown. In vivo absorption spectra of intact cells exhibit

maxima at 497, 590, 605, 865 nm. Bacteriochlorophyll-a and

carotenoids of spirilloxanthin series are present. No NaCl requirement

(tolerates up to 0.5% [w/v]) The type strain is mesophilic (range; 20-35

oC) with pH optimum at 7.0 (range; 6-8 pH). The preferred mode of

growth is photoorganoheterotrophy with a few organic compounds. Good

growth occurs on benzoate, butyrate, caproate, caprylate, fumarate,

glutamate, glycerol, glycolate, malate, malonate, proponal, propionate,

sorbitol, succinate and valerate. Feeble growth occurs with ethanol and

D-glucose. Photoautotrophic and fermentative growth is absent. Para-

aminobenzoic acid is required as growth factor. C18:1ω7c is the major

fatty acid 328 with sufficient amount of C16:0, C18:0,


187

C18:1ω7c11methyl and C16:1ω7c/ω6c. Ubiquinone-10 is the major

quinone. Diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl

ethanolamine, phosphatidyl choline, ornithine lipid and an unidentified

aminolipid (AL1) are the major polar lipids. The DNA G+C content of the

type strain is 65.8 mol% (by HPLC). The type strain DSM123T

(=NBRC100419T; confirmed by Okamura et al., 2009) was isolated and

deposited at DSMZ by van Niel (1944).

GENUS Blastochloris (Blc)

Blastochloris (Blc) is a phototrophic genus of the family

Hypomicrobiaceae, order Rhizobiales and class Alphaprotebacteria.

Members of the genus Blastochloris have unique identity due to the

presence of BChl b, thus help in identification till genus level and

differentiating from rest of the purple non-sulfur bacteria which have

BChl a alone (Hiraishi, 1997). Strains of the genus Blastochloris also

found to have a little amount of BChl a (peak at 812 nm), since it is the

reaction centre chlorophyll even in those bacteria containing other

bacteriochlorophylls as predominant components. The genus

Blastochloris currently comprises of two validly published species names,

Blc. viridis and Blc. sulfoviridis. The two species of Blastochloris were

previously assigned to the genus Rhodopseudomonas (Drews and

Giesbrecht, 1966; Keppen and Gorlenko, 1975) based on significant

similarities in cell morphology and cell division to Rps. palustris.

Subsequently, significant differences in 16S rRNA gene sequences with

respect to other Purple nonsulfur bacteria was the reason for


188

reconsideration of the taxonomic position of these bacteria and

reevaluation of phenotypic differences from the type species of

Rhodopseudomonas, leading to their reassignment to the new genus

Blastochloris as Blc. viridis and Blc. sulfoviridis (Kawasaki et al., 1993b;

Hiraishi, 1997).

The sequence similarity between the 16S rRNA genes of Blc. viridis

and Blc. sulfoviridis was 98.7%, which suggest that the two species are

phylogenetically closely related organisms. However, their separation and

delineation as different species was confirmed based on the supportive

results of DNA-DNA hybridization values of 38% between the two species

(Hiraishi, 1997).

Their green to olive green coloration, together with the

characteristic long wavelength absorption maxima between 1015-1035

nm, which is due to bacteriochlorophylls b, clearly separates Blstochloris

species from other phototrophic Alphaproteobacteria.

The interspecies differentiating characteristics of the genus

Blastochloris include type of budding, colour of the culture, optimum pH,

sulfate assimilation, phototrophic growth modes, growth factor

requirements, utilization of carbon source, G+C content, major quinones,

cellular fatty acid composition and DNA-DNA relatedness.

Strain JA248T

In the present study, strain JA248T which was isolated from the epilithic

biofilm (Fig. 3.22) shares genus specific characters of Blastochloris such

as rod-shaped cells, asymmetric mode of growth and cell division,

formation of rosette like aggregates, presence of bacteriochlorophylls b


189

and the lamellar structure of internal photosynthetic membranes and,

also phylogenetically close to the members of the genus Blastochloris.

The same strain JA248T differentiate with Blc. sulfoviridis and Blc. viridis

regarding habitat of isolation, colour of the culture, range and optimum

of temperature for growth, presence of aerobic dark growth, quinone

composition, carotenoid composition, utilization of carbon source/e-

donor, cellular fatty acid composition, 16S rRNA gene sequence

dissimilarity (Fig. 3.27), G+C content, MLSA and Fatty acids (Table 4.4).

Particularly, the strain JA248T grows well at temperature range of 15-

400C with an optimum of 250C, whereas the other two species cannot

grow below 250C. The size of the cells of strain JA248T is twice the size of

other two species of Blastochloris and the colour is yellowish green

whereas the other two are green to olive green. Few characteristics of

strain JA248T were found to coincide with Blc. viridis such as sulfate

assimilation, presence of carotenoid lycopene, absence of

photolithoautotrophy and presence or absence of few minor cellular fatty

acids. Some characters of strain JA248T were found to coincide with Blc.

sulfoviridis, are sessile type of budding, maximum temperature tolerance,

vitamin requirement, quinones, presence of carotenoid 7, 8, 11, 12- tetra

hydro lycopene and lack of few minor cellular fatty acids.

Strain JA248T showed closest 16S rRNA gene sequence similarity

with the type strain Blastochloris sulfoviridis DSM 729T (98.5% sequence)

and Blastochloris viridis DSM 133T (98.1%) (Table 3.2). Though strain

JA248T is showing close 16S rRNA gene sequence similarity with Blc.

sulfoviridis, it clustered with Blc. viridis in the phylogenetic tree (Fig.


190

3.27) and does not require any reduced sulfur source which is obligate

for the growth of Blc. sulfoviridis. The genome of strain JA248T, however,

shares only 46.5 and 32.5% relatedness (based on DNA–DNA

hybridization) with Blc. viridis DSM 133T and Blc. sulfoviridis DSM 729T,

respectively, which is within the recommended standards to delineate a

bacterial species (Stackebrandt & Goebel, 1994; Stackebrandt & Ebers,

2006). Sequence similarity based on concatenated sequences of five

house keeping genes (16S rRNA, ITS, pufM, recA and dnaK) ranged from

95 to 90% and is well within the recommended limits (<97.5% similarity)

to delineate phototrophic species (Serrano et al., 2010). The molecular

analysis, strongly supported by differences in phenotypic, physiological

and chemotaxonomic characteristics (Table 4.4), suggest that strain

JA248T represents a novel species in the genus Blastochloris, for which

the name Blastochloris gulmargensis sp. nov. is proposed. ―gumargensis‖

is derived from the place ―Gulmarg‖ from where the strain had been

isolated.

Characteristic 1 2 3

Cell size (μm) 1.0-1.5 x 3-5 0.6-0.9 x 1.2-2.0

0.5-1.0 x 1.0-1.8

Color of cell Suspensions

Yellow green Green to Olive green

Olive green

Type of budding Sessile Tube Sessile

Optimal pH for growth 6.5-7.5 6.5-7.0 6.5-7.0

Optimal temperature for growth (range)

25 (15-40) 30 (25-35) 30 (25-40)

Vitamin requirement PABA, biotin PABA, Biotin, Pyridoxal-P

PABA, Biotin

Sulfate assimilation + + -

Aerobic dark growth + (+) (+)

Photolithoautotrophy - - Thisulfate, sulfide

Major Quinones Q-8, MK-8 Q-9, MK-9 Q-10, MK-8


191

Carotenoid composition (mol%)

Neurosporene 10 9 4

1,2-Dihydroneurosporene 57 71 70

Lycopene 2 3 -

1,2-Dihydrolycopene 5 12 1

1,2-Dihydro-3,4-didehydrolycopene

9 5 2

7,8,11,12-Tetrahydrolycopene

17 - 23

$Carbon sources/electron donors utilized for growth

Acetate - + -

Glucose - (+) +

Sulfide - - +

Thiosulfate - - +

Fumarate + + (+)

α-keto glutarate + + -

Malate + (+) +

Fructose - - +

Glucose - (+) +

Ethanol - (+) (+)

Glutamate - (+) (+)

Maltose (+) - +

Sucrose - (+) +

Glycerol - - +

Proponol - - +

Acetate - + -

Fatty acid composition (%)

C16:0

13.8 9.4 10.0

C16:1

ω7cOH 1.0 1.0 1.0

C16:1

ω7c/C16:1

ω6c 8.0 10.5 6.8

C16:0

3OH 1.0 0.5 -

C17:1

ω8c - 1.9 0.6

C18:0

1.2 1.9 1.3

C18:0

3OH - - 1.0

C18:1

ω5c - 1.8 0.9

C18:1

ω7c 67.7 65.8 70.4

C18:1

ω7c 11-methyl - - 1.0

C20:1

ω7c 3.0 3.6 3.0

⊕G+C content of genomic

DNA (mol%)

63.8 61.8 65.5

Table 4.4…


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Table 4.4 *Differentiating characteristics of species of the genus Blastochloris. *Sources: Data pertains to comparative analysis done at authors‘ laboratory. 1,

strain JA248T; 2, Blc. viridis DSM133T; 3, Blc. sulfoviridis DSM729T. $All the

results are analysis after 3 subcultures with the respective substrates and compared against control (without the test substrate). Substrates that were utilized by all the strains include- pyruvate and succinate; those that could not be utilized include- tartrate, citrate, formate and benzoate. Other similarities of the three strains include rod shape, cell division by budding, presence of a single polar flagellum, rosette formation, lamellar internal membrane and presence of BChl b. Type strains do not have NaCl requirement for growth. &The reassociation values are the mean + 1 SD of two independent

determinations. Symbols: +, substrate utilized; -, substrate not utilized; (+), poorly utilized; PABA = para-aminobenzoic acid; NT, not tested, ND, not

determined. ⊕HPLC analysis.

Description of Blastocholris gulmargensis sp. nov.

Blastochloris gulmargensis (gul.mar.gen'sis. N.L. fem. adj. gulmargensis

pertaining to Gulmarg, a hilly resort in Kashmir, India, from where the

type strain was isolated).

Cells are rod shaped, 1.0-1.5 μm wide and 3.0-5.0 μm long,

budding division and motile. Growth occurs well under anaerobic

conditions in the light. Lamellar intracytoplasmic membrane structures.

Phototrophic cultures are yellow green. The in vivo absorption spectra of

intact cells exhibit maxima at 404, 455, 485, 605, 812, 1022 nm.

Bacteriochlorophyll b and carotenoids of spirilloxanthin series are

present. The type strain is mesophilic (25oC) and grows well at a pH

optimum of 6.5-7.5. The preferred mode of growth is


occurs on fumarate, pyruvate, succinate, α-ketoglutarate and malate.

Photoautotrophic and fermentative growth are absent. PABA and biotin


193

are required as growth factors. C18:1

ω7c, C16:0

are the dominant fatty

acids; sufficient amounts of C16:1 ω7c/C16:1 ω6c and C20:1 ω7c are also

found. Major quinones are Q-8 and MK-8. The DNA G + C content of the

type strain is 63.8 mol% (by HPLC). Natural habitats are cold sulfur

springs. The type strain, JA248T (=JCM 14795

=DSM 19786), was

isolated from an epilithic biofilm sample from a cold sulfur spring at

Gulmarg, Jammu and Kashmir, India.

GENUS Ectothiorhodospira (Ect)

Ectothiorhodospira is a phototrophic genus of purple sulfur

bacteria classified in the family Ectothiorhodispiriaceae, order

Chromatiales, class Gammaproteobacteria. Members of the family

Ectothiorhodospiraceae form a distinct lineage from Chromatiaceae on the

basis of 16S rRNA gene sequence analysis (Imhoff and Süling, 1996).

Among the 13 genera recognized in the family Ectothiorhodospiraceae,

only 4 genera (Ectothiorhodosinus, Ectothiorhodospira, Halorhodospira

and Thiorhodospira) are represented by members of true phototrophic

species(http://www.bacterio.cict.fr/classifgenerafamilies.html#Ectothiorh

odospiraceae)

All the phototrophic members of the family Ectothiorhodospiraceae

accumulate sulfur granules outside the cell and thus phenotypically

distinct from the members of the Chromatiaceae (Imhoff, 2006b). After

reclassification due to revision of species delineation, the genus

Ectothiorhodospira was left with only 4 species (Ect. mobilis, Ect.

shaposhnikovii, Ect. marina and Ect. Haloalkaliphila. E. vacuolata and E.


194

marismortui are considered as heterotypic synonyms of Ect.

shaposhnikovii and Ect. mobilis respectively based on DNA-DNA

reassociation and ribotyping (Ventura et al., 2000). With the

subsequently described Ect. variabilis (Gorlenko et al. 2009), the number

of species became five.

The species of the genus Ectothiorhodospira represent by few

characteristic features such as rod-shaped or vibrioid or true spiral

shaped cells, gram-negative, motile by polar tuft of flagella, multiply by

binary fission and many contain gas vesicles. Internal photosynthetic

membranes are lamellar type and photosynthetic pigments are

bacteriochlorophyll-a and carotenoides. Sulfide oxidized to sulfate, with

S0 as an intermediate, which is deposited outside the cells. Most species

live in marine and saline environments that contain sulfide and have

slight to extreme alkaline pH and occasionally found in soil.

Strain JA430T

Strain JA430T was isolated from a solar saltern, a novel habitat,

though Ectothiorhodospira spp have been reported from other marine and

halophilic environments (Imhoff, 2006). Strain JA430T was enriched in

the medium supplemented with pyruvate / bicarbonate as carbon source

and sulfide as electron donor. Since, strain JA430T did not have

photolithoautotrophy, it must have used the pyruvate as carbon source

and reduced sulfide as electron donor, in contrast to all other species of

Ectothiorhodospira which are having photolithoautotrophic growth. By

not supplementing the medium with an organic carbon source, the strain

could have been missed. This may explain why all the species described


195

till now are photoautotrophic. Thus, it becomes clear that restricting to

one medium composition will restrict the discovery of novel species.

A 16S rRNA gene sequence similarity of < 97% with the species of

the genus Ectothiorhodospira (Table 3.2) has clearly indicated the strain

JA430T representing a novel species, which necessitated detailed

polyphasic characterization. Though phylogenetic tree constructed by

16S rRNA gene sequence of strain JA430T (Fig. 3.21) show closest

similarity with the type strain Ect. variablis WN22T (96.1% sequence

similarity), it clustered with type strain Ect. mobilis DSM 237T.

Polyphasic characterization of strain JA430T has revealed its

differentiating characteristics with existing type strains of the genus

Ectothiorhodospira such as size of cells, colour of the cell suspension and

type of carbon source utilization. In addition, requirement of vitamins p-

aminobenzoic acid, pantothente and pyridoxal phosphate was observed

for the growth of strain JA430T, whereas none of the other strains of the

same genus require vitamin source. Sulfate assimilation was not

observed in any of the strains except JA430T and Ect. variablis. In

contrast to the property of having diazotrophy in species of the genus

Ectothiorhodospira, strain JA430T did not have the same.


196


197

Apart from phenotypic and physiological differences, 16S rRNA

gene sequence similarity (96.1%) of strain JA430T is also within the

recommended standards to delineate a bacterial species (Stackebrandt

and Goebel, 1994; Stackebrandt and Ebers, 2006). The above differences

of strain JA430T from other Ectothiorhodospira spp. (Table 4.5) enable

description of a novel species of the genus Ectothiorhodospira as

Ectothiorhodospira salini.

Description of Ectothiorhodospira salini sp. nov.

Ectothiorhodospira salini (sa.li'ni. L. gen. n. salini, of a salt-cellar).

Cells are vibrioid to spiral shaped, 1.0–1.5 m wide and 2.0–3.5 m long.

Cells are motile by means of polar flagella and divide by binary fission.

Growth occurs under anaerobic conditions in the light. Internal

photosynthetic membranes are of lamellar type arranged parallel to the

cytoplasmic membrane. Phototrophic cultures are reddish brown. The in

vivo absorption spectra of intact cells exhibit maxima at 311, 377, 512-

518, 590-593, 797 and 860 nm. Bacteriochlorophyll a and carotenoids;

spirilloxanthin, rhodopin, anhydrorhodovibrin, tetrahydrolycopene and

rhodovibrin are present. The type strain is mesophilic (300C) growing at a

pH optimum at 7.5 (range, pH 7.0-10.0) and requires 5% NaCl for

optimal growth (NaCl range, 0.5-12.0%). The preferred mode of growth is


occurs on acetate, malate and pyruvate. Photoautotrophic and

fermentative growth is absent. PABA, pantothenate and pyridoxal

phosphate are required as growth factors. C18:1ω7c is the dominant fatty

acid; sufficient amounts of C16:0, C19:0cyclo8c and C16:1 ω7c/C16:16c are

http://sa.li/


198

also found. Major quinones are MK-7 and Q-7. The DNA G+C content of

the type strain is 63 mol% (by HPLC). Natural habitats are solar salterns.

The type strain JA430T (=NBRC105915 =KCTC 5805) was isolated from

sediment sample from a solar saltern at kanyakumari, Tamil Nadu,

India.

STRAINS OF THE GENUS Marichromatium (Mcr)

Marichromatium (Mcr) is a phototrophic genus of purple sulfur

bacteria, classified in the family Chromaticaea, order Chromatiales, class

Gammaproteobactreia. Marichromatium species are distinct from other

members of the family Chromatiaceae by their specific salt requirement

and the very high mol% G+C content of their DNA. Species of this genus

have previously been classified as belonging to the genus Chromatium

(Pfennig and Truper, 1974; Pfennig, 1989b). However because of

significant phenotypic and genetic differences to true Chromatium spp.

and other members of the family Chromatiaceae, they have been

transferred to the new genus Marichromatium (Imhoff et al., 1998b).

Till date, 5 Marichromatium species have been validly published

(http://www.bacterio.cict.fr/m/marichromatium.html). Mcr. gracile

biotype thermosulfidiphilum (Serrano et al., 2009) is a newly described

biotype, the only one among anoxygenic phototrophic bacteria. The

common property among all the members of the genus Marichromatium

is that they represent the true marine species with wide growth

capabilities. Marichromatium species had been distinguished based on

the differences in colour of cell suspension, substrate utilization, cell

http://www.bacterio.cict.fr/m/marichromatium.html


199

size, salt responses, carotenoides group, growth modes and whole

genome DNA-DNA hybridization.

In the present study, the highest number of strains of purple

sulfur bacteria (JA550, JA548, JA349, JA546, JA350, JA387, JA547,

JA536, JA637, JA552, JA537, JA551, JA540, JA638, JA553 and JA260)

isolated belong to the genus Marichromatium (16 strains out of 24 = 67%

of PSB) and all were from the marine habitats. Most of the strains (8 out

of 16) among the above showed closest 16S rRNA gene sequence

similarity (Table 3.2) to Marichromatium gracile revealing their

predominance.

Though the species of the genus Marichromatium (except Mcr.

bheemlicum and Mcr. fluminis) share more than 99% 16S rRNA gene

sequence similarity, they exhibit differences in ribotyping (Acinas et al.,

2004). The DNA-DNA hybridization of the Marichromatium species

(Serrano et al., 2009) revealed that Mcr. gracile and Mcr. purpuratum are

71% similar which are to be considered as heterotypic synonyms.

However, this conflict is resolved by the use of MLSA approach where

they have used concatenated sequences of 6 protein-coding genes (gyrB,

recA, fusA, dnaK, pufM and soxB) together with 16S rRNA gene and the

internal transcriber spacer ITS region for comparison (a minimum use of

7 genes is recommended for MLSA [Gevers et al., 2005; Maiden et al.,

1998]) and have concluded that the established taxonomy of the genus

Marichromatium with four validly described species (the recently

described species, Marichromatium fluminis (Sucharita et al., 2010a) is

not included in their analysis) is acceptable.


200

A noteworthy observation in the present study is that use of

glutamate as carbon source in the media enhanced the enrichment of

most strains of Mcr. purpuratum, whereas pyruvate as carbon source

enriched strains of other species of the genus Marichromatium. Based on

16S rRNA gene sequence similarity (Table 3.2), strain JA387 is close to

Mcr. indicum (Arunasri et al., 2005), while JA536 and JA637 are close to

Mcr. gracile DSM 203T (Table 3.2). But all these three strains closely

clustered with Mcr. purpuratum in the phylogenetic tree (Fig. 4.4).

Percentage of 16S rRNA gene sequences similarity of remaining 9 strains

of Marichromatium coincides with the percentage of distance in

phylogenetic tree (Fig. 4.4). This kind of unconcurrent result

(phylogenetic distances caliculated by dendrogram and by 16S rRNA

Blast) is observed with genomospecies, because they are genotypically

very close to each other.


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Fig. 4.4 Dendrogram showing phylogenetic relationship of strains ((JA550, JA548, JA349, JA546, JA350, JA387, JA547, JA536, JA637, JA552, JA537, JA551, JA540, JA638, JA553 and JA260) with phototrophic members of Gammaproteobacteria. (Tree was constructed using NJ method of MEGA4 software. The gene bank accession numbers for 16S rRNA gene sequences are shown in parentheses. Bar, one nucleotide substitution for 100 nucleotides. Bootstrap values below 50 have been removed from

the dendrogram)


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STRAINS OF THE GENUS Rhodovulum (Rdv)

Till date, the genus Rhodovulum (Rdv) comprises twelve validly

published species: Rdv. sulfidophilum, Rdv. adriaticum, Rdv.

euryhalinum, Rdv. strictum, Rdv. iodosum, Rdv. robiginosum, Rdv.

steppense, Rdv. lacipunicei, Rdv. kholense, Rdv. imhoffii, Rdv.

visakhapatnamense and Rdv. marinum

(http://www.bacterio.cict.fr/qr/rhodovulum.html). The major

differentiating characterstic of the genus Rhodovulum with other genera

is 16S rRNA gene sequence dissimilarity. Species of this genus (Rdv.

sulfidophilum, Rdv. adriaticum) have formerly been included in the genus

Rhodopseudomonas. The recognition of morphologically and

chemotaxonomically distinct characteristics and phylogenetic analysis

led to their separation from Rhodopseudomonas and classification in the

genus Rhodobacter (Imhoff et al., 1984b). Later, the data of 16S rRNA

gene sequences phylogenetically separated fresh water and marine forms

of the genus Rhodobacter. Hence, Rhodobacter sulfidophilum and

Rhodobacter adriaticum were transferred into the new genus Rhodovulum

(Hiraishi and Ueda, 1994).

In the present study, four strains of genus Rhodovulum, JA255,

JA390, JA545 and A595 were isolated (Fig. 3.2). Based on rapid typing

(Table 3.2), strain JA595 was sorted and subjected for detailed

polyphasic characterization. Strian JA595 showed distinct physiological

and 16S rRNA gene sequence similarities with the species of the genus

Rhodovulum, but exhibited all similar characteristics and close 16S rRNA

gene sequence similarity (99.5%) with strain JA580T (which is recently

http://www.bacterio.cict.fr/qr/rhodovulum.html


203

proposed as a novel species named Rhodovulum phaeolacus [Lakshmi et

al., 2011]) (Fig. 4.5). Therefore, strain JA595 is propsed as an additional

strain of Rhodovulum phaeolacus.

Fig. 4.5 Phylogenetic tree based on almost-complete 16S rRNA gene

sequences showing the relationship of strain JA595T within the class

Alphaproteobacteria. The tree was constructed by the neighbor joining method using the MEGA4 software and rooted by using Rhodospirillum rubrum as out group as the out-group. Numbers at nodes represent bootstrap values (based on 100 resamplings). The GenBank accession numbers for 16S rRNA gene sequences are shown in parentheses. Bar, 1 nucleotide substitutions per 100 nucleotides. Other three strains JA255, JA390 and JA545 are not included in the dendrogram for having insufficient length and quality of 16S rRNA gene sequence.


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STRAINS OF THE LESS PREDOMINANT GENERA: Rhodothalassium,

Rubrivivax, Roseospira, Rhodomicrobium, Rhodoplanes AND

Rhodospirillum.

The obligate requirement for salt, cell morphology and internal

membrane structure clearly distinguish Rhodothalassium from other

spiral shaped phototrophic Alphaproteobacteria. Strain JA389 and JA473

of genus Incertae Sedis Rhodothalassium, were isolated from halophilic

(saltpan) habitats, like that of existing type strain Rhodothalassium

salexigens DSM 2132T, which prooved their wide occurence in

hypersaline environments. Based on rapid typing (Table 3.2), strains

JA389 and JA473 are similar to Rhodothalassium salexigens (Table 4.6).

Two strains of the genus Rubrivivax, 2 of Roseospira and one each

of Rhodomicrobium, Rhodoplanes and Rhodospirillum were found to be

very similar to their respective closest type strains (Table 3.2), based on

colour of the culture, pigments and carotenoides, microscopic

observation and 16S rRNA gene sequence similarity. Hence, they were

considered as strains of their respective closest type strains. Now the

genus Rhodomicrobium consists of only one validly published species

Rhodomicrobium vanielli. Isolation of strain of Rhodomicrobium vannielli

(present study) is the first report from India.


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STRAINS OF THE GENERA Thiophaeococcus, Allochromatium

Thiohalocapsa.

Genera Thiophaecoccus, Allochromatium and Thiohalocapsa are belong to

the purple sulfur bacteria classified under the family Chromatiales, order

Rhodocyclales of the class Betaproteobacteria distributed under the class

Fig. 4.6 Phylogenetic tree based 16S rRNA gene sequences showing the relationship of strains JA389 and JA473 within the class Alphaproteobacteria. The tree was constructed by the neighbor joining method within the MEGA4 software and rooted by using Escherichia coli as the out-group. Numbers at nodes represent bootstrap values (based on 100 resamplings). The GenBank accession numbers for 16S rRNA gene sequences are shown in parentheses. Bar, 2

nucleotide substitutions per 100 nucleotides.


206

Betaproteobacteria. Thiophaeococcus is a recently described (Anil Kumar

et al., 2008b) genus isolated from brackish water of mangrove forest

(Bhitarkanika) from India on the east coast, whereas the strains JA535,

JA541 and JA592 of Thiophaeococcus (of present study), were isolated

from microbial mat and mud of saltpan. All strains of Thiophaeococcus

have obligate requirement for NaCl. 16S rRNA gene sequence similarity of

strains JA541, JA592 and JA535 (from the present study) with

Thiophaeococcus mangrovi is 95.5%, 95.6% and 95.7% respectively (

Table 3.2 and Fig. 4.7). All these three strains were isolated from Tamil

Nadu coast. The similarity between each of the three strains is 99%.

These three strains are yet to be characterized completely to reveal the

differentiating characteristics at species level.

Strains JA543 and JA549 have similar morphological and

microscopic characteristics with Allochromatium phaeobacterium JA144T,

Allochromatium vinosum DSM 180T respectively. 99.4% similarity of 16S

rRNA gene sequence of strain JA549 with Allochromatium vinosum (Table

3.2 and Fig. 4.7) also supports close relatedness with type strain.

One strain JA534 of the genus Thiohalocapsa was isolated which is

from seashore, Akkarpettai,Tamil Nadu and its 16S rRNA gene sequence

similarity is 98.9% with Thiohalocapsa marina JA142T (Table 3.2).

Thiohalocapsa spp. abound in purple ponds imparting bright purple

colour to the water body (Fig. 4.1).


207

Fig. 4.7 Dendrogram showing phylogenetic relationship of strains of the genera Thiophaeococcus, Allochromatium and Thiohalocapsa with phototrophic members of class Gammaproteobacteria. Tree was constructed using NJ method of MEGA4 software. The gene bank accession numbers for 16S rRNA gene sequences are shown in parentheses. Bar, two nucleotide substitutions for 100 nucleotides. Bootstrap values below 50 have been removed from the dendrogram.


208

From the results it was found that all purple sulfur bacteria (with

the exception of Allochromatium) could be isolated from marine habitats,

whereas most of the purple nonsulfur strains were from fresh water

habitats (Table 3.1 and 3.2). Among purple sulfur bacteria isolated, most

of strains belong to the genus Marichromatium. Among purple non sulfur

bacteria, most of the strains/species belong to the genera Rhodobacter

and Rhodopseudomonas.

Most of the strains (47%) isolated in the present study are from

coastal habitats of south India, followed by strains isolated from

Himalayan region of north India (24%). Remaining percentage of samples

include all other habitats (section, 2.9). From the present study it was

observed that Purple sulfur bacteria (mostly Genus Marichromatium) are

predominant in marine and coastal habitats and purple nonsulfur

bacteria (mostly Rhodobacter and Rhodopseudomonas) are predominant

in fresh water habitats.

As a whole, among 59 strains, it possible to describe 7 novel

species and 3 additional strains of type strains, which had been isolated

from different regions of India. Six among 7 novel species belongs to

purple nonsulfur bacteria and one is of purple sulfur bacteria.

Until now, a total number of 137 (75 PNSB + 62 PSB) purple

bacterial species were described and validly published from 13 different

countries. Among 137 novel purple bacterial species, 38 species are

from India, which contributes 27.7% to total novel purple bacterial

species of the world (Fig. 4.8).


209

Based on the earlier and present study, it came to know that the

species of the genera Rhodobacter and Rhodovulum of purple nonsulfur

bacteria and Marichromatium of purple sulfur bacteria were predominant

in the natural habitats in the environment and has high rate of

evolutionary divergence in India.

Fig. 4.8 Present status of purple bacterial species discovery

by different countries.

Data taken from Bergey‘s Manual of Systematic Bacteriology

(2005), List of prokaryotic names with standing in nomenclature (LPSN)(http://www.bacterio.cict.fr/) and unpublished data

(Manuscprits under revision from our lab)

http://www.bacterio.cict.fr/

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