Antimicrobial properties of cultivable bacteria associated ... with classical biochemical identification ... 80 predominant role in the establishment of cross relationships between

Draft

Antimicrobial properties of cultivable bacteria associated

with seaweeds in Gulf of Mannar of South East Coast of

India

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2015-0769.R2

Manuscript Type: Article

Date Submitted by the Author: 31-Mar-2016

Complete List of Authors: Thilakan, Bini; Central Marine Fisheries Research Institute, Marine

Biotechnology Division Chakraborty, Kajal; Central Marine Fisheries Research Institute, Marine Biotechnology Division Chakraborty, RekhaDevi; Central Marine Fisheries Research Institute

Keyword: seaweed, nonribosomal peptide synthetase, polyketide synthetase, marine bacteria, antibacterial activity

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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Antimicrobial properties of cultivable bacteria associated with seaweeds in Gulf of Mannar of 1

South East Coast of India 2

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B. Thilakan*, K. Chakraborty1,*, and R. D. Chakraborty 4

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Running head: Seaweed associated bacteria from Gulf of Mannar 9

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B. Thilakan, and K. Chakraborty.1 Marine Biotechnology Division, Central Marine Fisheries Research Institute, 16

Ernakulam North P.O., P.B. No. 1603, Cochin-682018, Kerala, India 17

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R. D. Chakraborty. Crustacean Fisheries Division, Central Marine Fisheries Research Institute, Ernakulam North 19

P.O., P.B. No. 1603, Cochin-682018, Kerala, India 20

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1 Corresponding author (e-mail: [email protected]). 22

* Equal contribution 23

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Abstract: In this study, 234 bacterial strains were isolated from 7 seaweed species in the Gulf-of-Mannar South-24

East coast of India. The strains having consistent antimicrobial activity were chosen for further studies, and this 25

constituted about 9.8% of the active strains isolated. Phylogenetic analysis using 16S rDNA sequencing, assisted 26

with classical biochemical identification indicated the existence of two major phyla, Firmicutes and Proteobacteria. 27

Antimicrobial activity analysis combined with the results of amplifying genes encoding for polyketide synthetase 28

and nonribosomal peptide synthetase showed that seaweed-associated bacteria had broad-spectrum antimicrobial 29

activity. These epibionts might be beneficial to seaweeds by limiting or preventing the development of competing or 30

fouling bacteria. Phylogenetic analysis of ketosynthase regions with respect to the diverse range of ketosynthase 31

(KS) domains showed that the KS domains from the candidate isolates were of Type I. The bacterial cultures 32

retained their antimicrobial activities after plasmid curing, which further suggested that the antimicrobial activity of 33

these isolates was not encoded by plasmid, and the genes encoding the antimicrobial product might be present 34

within the genome. Seaweed-associated bacteria with potential antimicrobial activity suggested the seaweed species 35

as an ideal ecological niche harboring specific bacterial diversity representing a largely underexplored source of 36

antimicrobial secondary metabolites. 37

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Key words: seaweed, nonribosomal peptide synthetase, polyketide synthetase, marine bacteria, antibacterial activity. 39

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Résumé: Dans cette étude, 234 souches bactériennes ont été isolées à partir de 7 espèces d'algues dans la côte du 41

Golfe-du-Mannar Sud-Est de l'Inde. Les souches ayant une activité antimicrobienne cohérente ont été choisis pour 42

des études ultérieures, et ce qui constituait environ 9.8% des souches actives isolées. L'analyse phylogénétique 43

utilisant 16S séquençage de l'ADNr, aidé à l'identification biochimique classique indiqué l'existence de deux 44

embranchements majeur, Firmicutes et Proteobacteria. Analyse de l'activité antimicrobienne combinée avec les 45

résultats de l' amplification des gènes codant pour des polycétide synthétase et peptides non ribosomiques synthétase 46

ont montré que les bactéries d'algues associées aux algues ont un large spectre d' activité anti-microbienne. Ces 47

épibiontes pourrait être bénéfique pour les algues en limitant ou en empêchant le développement de la compétition 48

ou l'encrassement des bactéries. L'analyse phylogénétique des régions de cétosynthase par rapport à la grande 49

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diversité de cétosynthase (KS) des domaines a montré que les domaines KS provenant des isolats candidats étaient 50

de type I. Les cultures bactériennes ont conservé leurs activités antimicrobiennes après plasmide durcissement, ce 51

qui suggère en outre que l'activité antimicrobienne de ces isolats a pas codés par le plasmide, et les gènes codant 52

pour les produits antimicrobiens peuvent être présents au sein du génome. Bactéries d'algues associée avec une 53

activité antimicrobienne potentiel suggéré les espèces d'algues comme une niche écologique idéal abritant la 54

diversité bactérienne spécifique représentant une source largement sous de métabolites secondaires antimicrobiens. 55

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Mots-clés: algues, peptides non ribosomiques synthétase, polycétide synthétase, bactéries marines, une activité 57

antibactérienne. 58

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Introduction 72

The emergence of antibiotic-resistant bacteria and the need for novel, antimicrobial compounds led to the 73

investigation of new habitats to screen the production of bioactive substances (Meklat et al. 2011; Gram et al. 2010). 74

Every surface immersed in the sea, including those of seaweeds and sponges, provide an organic material-rich 75

habitat for the microorganisms to thrive. They are regularly exposed to the high concentrations of potentially 76

harmful microbes, even though they suffer remarkably low levels of microbial infection, despite lacking the cell-77

based immune systems (Gram et al. 2010; Kubaneck et al. 2003). Antimicrobial defenses of marine organisms are 78

largely uncharacterized, although from a small number of studies it appeared that chemical defenses might play a 79

predominant role in the establishment of cross relationships between the marine surface-associated microorganisms 80

and their eukaryotic host (Imhoff et al. 2011; Kubaneck et al. 2003). Marine microbial symbionts are possibly the 81

true producers or take part in the biosynthesis of bioactive marine natural products isolated from the eukaryotic hosts 82

as reported in similar studies (Li 2009; Kubanek et al. 2003; Zhang et al. 2009). Studies on the sponges and their 83

associated microbiota validated this hypothesis (Quevrain et al. 2014). 84

A greater percentage of epiphytic isolates possessing antimicrobial activities also highlighted the 85

biotechnological potential for targeted isolation of marine eukaryote-associated bacteria (Zeng et al. 2005; 86

Kanagasabhapathy et al. 2006; Penesyan et al. 2009; Ali et al. 2012). A few interesting studies have been presented 87

on associates of macroalgae, but a detailed knowledge of the interaction of algae with their associated microbes, and 88

among the microbes on algal surfaces and tissues is still lacking (Kubaneck et al. 2003; Goecke et al. 2010; Wiese et 89

al. 2009, Ali et al. 2012). Investigation of the pharmaceutical metabolites may reveal the biosynthesis mechanisms 90

of related natural products, and solve the current problem of supply limitation in marine drug development (Li 91

2009). 92

Polyketide synthetases (PKS) and non-ribosomal peptide synthetases (NRPS) are multifunctional enzymes 93

catalyzing the biosynthesis of structurally diverse bioactive natural products (Hutchinson 2003), which has been 94

commonly employed for designing molecular tools to assess the metabolically active bacterial groups (Ayuso-95

Sacido and Genilloud 2005; Zhang et al. 2009; Kennedy et al. 2009). There are several reports regarding the 96

screening of the polyketide synthetase (pks) and nonribosomal peptide synthetase (nrps) genes from sponge-97

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associated and soil-borne microrganisms, such as bacteria and fungi (Meklat et al. 2011; Zhou et al. 2011). Despite 98

being very important marker gene systems, little is known about the presence of nrps and pks-I in the diverse 99

seaweed-associated microbiota. 100

In this study, we have adopted a culture-dependent method to assess the cultivable antimicrobial 101

heterotrophic bacterial communities associated with seven species of intertidal seaweeds collected from the Gulf of 102

Mannar on the south-east coast of India, and to explore them as a source for potentially useful antimicrobial 103

substances. The potential of the seaweed-associated microbiota to produce secondary metabolites was analyzed by 104

polymerase chain reaction employing degenerate primers of polyketide synthetase (pks-I) and nonribosomal peptide 105

synthetase (nrps) genes exploiting their conserved nature. 106

107

Materials and methods 108

Specimen collection and processing to isolate seaweed-associated bacteria 109

Intertidal seaweeds belonging to Phaeophyceae and Rhodophyceae were collected by scuba diving from the 110

intertidal zone of Mandapam situated at 9° 17' 0" North, 79° 7' 0" East, Gulf of Mannar region in the south-east 111

coast of India. The brown seaweeds collected were Anthophycus longifolium, Sargassum myriocystum, Padina 112

gymnospora, Turbinaria ornata and Dictyota dichotoma, whereas red seaweeds were Hypnea valentiae and 113

Laurencia papillosa. The seaweed samples were placed in a sterile polythene bag filled with seawater. The samples 114

were kept in the dark at 4°C until further processing. The specimens of seaweed samples were thoroughly washed 115

with sterile seawater to remove the loosely attached bacteria from the surface. For isolation of seaweed-associated 116

bacteria, the specimen samples (10 g) were suspended in sterile seawater (10 mL), and aseptically homogenized by 117

using a pestle and mortar in a laminar air flow hood. The suspension was serially diluted in sterile seawater (9 mL), 118

and different dilutions were plated on the aseptically prepared isolation media. The isolation media used in this 119

study was nutrient agar supplemented with sodium chloride (NA, NaCl, 1% w/v), Zobell marine agar (ZMA), 120

seawater agar (SWA) and nutrient agar (half strength). Incubation was performed in the dark at 30°C for a period of 121

7 days. The pure cultures were obtained by respective isolation and purification steps on nutrient agar medium 122

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supplemented with sodium chloride (NA, NaCl, 1% w/v) (Wiese et al. 2009; Wilson et al.2010; Kanagasabhapathy 123

et al. 2006; Lemos et al. 1985; Ali et al. 2012; Boyd et al. 2009). 124

Antimicrobial assay 125

The pathogenic organisms used in the study were Vibrio harveyi, Aeromonas hydrophilla, Vibrio 126

alginolyticus, Vibrio angullarum, Vibrio parahaemolyticus MTCC451, Vibrio vulnificus MTCC1145, and Vibrio 127

parahaemolyticus ATCC17802. For preliminary characterization of antimicrobial bacteria, an inhibition test on the 128

solid media was carried out by a spot over lawn assay (Chakraborty et al. 2014). The pathogens were grown to the 129

log phase in nutrient broth supplemented with 1 % sodium chloride for 18–24 h (108 CFU/mL). They were thereafter 130

swabbed on the Mueller–Hinton agar (Himedia, India) with 1 % sodium chloride/50 % seawater over which the 131

purified isolates (5 per dish) were spotted (3 mm diameter) using sterile toothpicks. The plates were incubated at 132

25°C for 24–72 h. The clearing zones in the turbid growth of pathogen were scored as antibacterial activity. A 133

measure of antimicrobial activity was recorded as the diameter of inhibition zones determined as a distance of ≥1 134

mm between the circular area (=lawn of the isolate) and the end of the clear zone bounded by the lawn of the test 135

strain (Gram et al. 2010; Lemos et al. 1985). The live cells were stained with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-136

diphenyltetrazolium bromide (MTT) to visualize the growth inhibition around the cultured isolates. 137

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Biochemical identification and 16S rRNA-based phylogeny 139

Bacteria with antimicrobial properties were identified using the biochemical methods. The colony 140

morphology was analyzed on agar plates and gram staining was performed. The strains were identified by carrying 141

out biochemical and physiological tests as described in the Bergey’s Manual of Determinative Bacteriology (Krieg 142

and Holt 1984). Conventional tests, such as motility, gas from glucose, starch hydrolysis, indole, nitrate reduction, 143

Vogues-proskauer, citrate utilization, gelatin hydrolysis, esculin hydrolysis, growth at various temperatures and 144

NaCl concentrations etc. were used. The results were further confirmed by 16S rRNA gene sequence-based 145

phylogeny. Total genomic DNA was extracted from the bacterial cultures grown in nutrient broth by using phenol–146

chloroform extraction method (Sambrook and Russell 2001), and quantified using a Biophotometer (Eppendorf, 147

Germany). The molecular characterization of the cultured bacterial strains was performed by 16s rRNA gene 148

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sequencing assisted with BLAST similarity search. The primer sequences used for the PCR reaction were presented 149

in Table 1. PCR was performed in a total volume of 25 µL containing 1x reaction buffer with MgCl2 (Sigma), 0.25 150

mM of each dNTP (Fermentas), 0.5 mM of each primer (Sigma), 1 ng DNA, and 0.3 U Taq DNA polymerase 151

(Sigma). The following cycling conditions were used: initial denaturation at 94°C for 5 min, followed by 30 cycles 152

of 95°C for 1 min, 58°C for 1 min, and 72°C for 2 min, with a final extension at 72°C for 5 min (Biorad, USA). The 153

molecular sizes of the amplified fragments were estimated by comparing with a 1 Kb ladder on a 1.5% (w/v) 154

agarose gel using 1X TAE buffer at 80V. The fragments of the expected size were gel-purified using GelEluteTM

gel 155

extraction kit (Sigma) following the manufacturer’s protocol and sequenced. The sequence data were deposited in 156

GenBank, and compared with the existing sequences using blastn search program. Sequences were aligned against 157

the reference sequences with CLUSTALW software of Bioedit program, and the aligned dataset was used as input 158

for phylogenetic analysis program. The evolutionary history was inferred by using the Maximum Likelihood method 159

based on the Kimura 2-parameter model (Kimura et al. 1980), and the bootstrap analysis with 1000 replications. 160

Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011). 161

Antagonism assays among the isolated marine bacteria and their auto inhibition 162

Marine bacteria were grown in broth for a period of 18 h, and a swab of the organism has been employed as 163

test cultures (108

CFU/mL). Marine bacterial cells were scraped off with a loop from a pre-inoculated plate, and 164

antibacterial activities of the test organisms were determined following established method (Lemos et al. 1985). The 165

plates were incubated overnight to analyze inhibitory zone. All the experiments were carried out in triplicates. 166

Plasmid profiling 167

The plasmid of the strain was extracted by the alkaline method, and identified by agarose gel 168

electrophoresis. Briefly, the cells were grown overnight in the Luria Bertane broth containing sodium chloride (LBS, 169

NaCl, 2% w/v) and incubated at 37oC in a shaker incubator (120 rpm) for 16-18 h. The culture (1.5 mL) was used 170

for plasmid preparation following the method of alkaline lysis (Sambrook et al. 1989; Devi et al. 2009). The nucleic 171

acid was re-dissolved in Tris-EDTA buffer (50 µL,10 mM Tris-HCl, 1 mM Na2EDTA, pH 8.0) containing DNAase 172

free RNAase (20 mg/mL), vortexed briefly, before being stored at -20°C for further use. Electrophoresis was 173

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performed using 0.8% agarose gel system in the Tris-borate buffer. The gels were stained with ethidium bromide 174

(0.5 µg/mL). The resolved bands were visualized on a UV transilluminator at a wavelength of 360 nm and 175

photographed using a UV gel documentation system (Biorad, USA). 176

Plasmid curing 177

Plasmid curing was carried out to determine whether the antibacterial substance was plasmid-encoded. For 178

the curing experiment, a chemical agent, sodium dodecyl sulfate (SDS) was used. Each bacterial isolate was 179

inoculated into the LBS broth and incubated at 37°C under shaking in an incubator. Thereafter, the cultured strain 180

(50 µL, 10%) was added into 5 mL of fresh LB medium containing 0.005% SDS with three consecutive transfers for 181

every 24 h. Every day a portion of each culture was withdrawn and checked for the presence of plasmid in the 182

agarose gel (0.8% w/v) electrophoresis. The antimicrobial activities of the colonies were determined after plasmid 183

curing by the established method (Hu et al. 2010). 184

Screening of polyketide synthetase I (pks-I) and nonribosomal peptide synthetase (nrps) genes 185

The highly conserved sequences of β-ketoacyl synthase (KS) domains are shared among all PKSs, and 186

therefore, the KS domains are useful in the screening for PKS genes in bacteria. Similarly, the most conserved 187

Adenylation (A) domain can be used for the design of the PCR primer, and also to survey the NRPS gene diversity. 188

Different sets of degenerate primers targeting genes encoding pks-I and nrps were used to screen the biosynthetic 189

potential of the bacterial isolates to elicit bioactive polyketide compounds characterized by the repetitive occurrence 190

of ketide (-CH2-CO-) moieties and non-ribosomal peptides. The primers have been listed in the Table 1. PCR was 191

performed in a total volume of 25 µL containing 1X reaction buffer with MgCl2 (Sigma), 0.25 mM of each dNTP 192

(Fermentas), 0.5 mM of each primer (Sigma), 1 ng DNA, and 0.3 U Taq DNA polymerase (Sigma). The PCR 193

process was set as initial denaturation time of 5 min at 94°C followed by 35 cycles of 1 min at 95°C, 1 min at 45°C, 194

1 min at 72°C, and a final extension of 5 min at 72°C. The amplified products were examined by 1.5% agarose gel 195

electrophoresis, and bands of 700 to 800 bp and 1000 to 1,400 bp were classified as products of pks-I and nrps 196

genes, respectively. The polymerase chain reaction amplicons were separated by agarose gel electrophoresis. The 197

bands of the expected size (pks-I 700-800 bp and nrps 1000-1400 bp) were purified using Gel EluteTM

Gel 198

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Extraction Kit (Sigma) before being cloned into the pGET-Blunt M13 vector (Fermentas) following the 199

manufacturer’s instructions and transformed into chemocompetent E. coli cells. The positive recombinants were 200

screened by ampicillin resistant recombinant selection method assisted by colony PCR with M13 specific primers. 201

The positive clones were used for plasmid isolation using GenJETTM

Plasmid isolation kit (Fermentas) and 202

sequenced using the M13 F and M13 R primers. 203

Table 1 204

Sequence analysis 205

Forward and reverse sequences of the PKS gene amplified product were assembled using Dna Baser v2.exe 206

and the vector sequences were removed using Vescreen (NCBI). The sequence data were subjected to GenBank 207

searches with blastx algorithm. The nucleotide sequences were translated into peptide sequences EMBOSS Transeq 208

(EMBL-EBI), and blastp searches of deduced amino acid sequences were also performed. Multiple alignments of 209

amino acid sequences with reference sequences of the GenBank were carried out by CLUSTALW software of 210

Bioedit program, and then the aligned data set was used as input for the phylogenetic analysis program (Zhu et al. 211

2009). The evolutionary history was inferred by using the Maximum Likelihood method based on the Whelan And 212

Goldman model (Whelan and Goldman 2001) and the bootstrap analysis with 1000 replications in MEGA5 213

(Tamura et al. 2011). Multiple alignments of active sites of type I KS domains with reference sequences was also 214

performed to verify the conserved sequence motifs. 215

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Accession numbers 217

Partial 16S rRNA gene sequences were deposited under the Accession numbers KC559432-KC559434; 218

KC510279-KC510286; JX203227-JX203230, and the partial PKS sequences were deposited under the Accession 219

numbers KC589396- KC589400; KC607821- KC607823. 220

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Results 222

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Isolation and antimicrobial screening 223

Among the total number of 234 isolates screened during isolation, 53, i.e. about 22% were found to be 224

active against at least one test pathogen used in the preliminary screening (Figure 1). From the above stated 53 225

isolates, only 23 were succeeded in overcoming further laboratory subculturing strategies, and could withstand the 226

activity. The bactericidal activity was ensured by spraying MTT, which stained the live cells as blue (Figure 1.4). 227

The antimicrobial patterns of these isolates against the test pathogens used in the study have been summarized in 228

Table 2. 229

Figure 1 230

Among different seaweed species considered in the present study, L. papillosa contributed a share of 27% 231

of the active bacterial isolates, followed by A. longifolium (22% of the total seaweed-associated bacterial isolates). 232

P. gymnospora and H. valentiae contributed equally (17% each) towards the total number of bacterial isolates with 233

potentially greater antibacterial properties against the pathogens. The cumulative share of the seaweeds S. 234

myriocystum, D. dichotoma, and T. ornata towards the total number of active isolates was found to be significantly 235

lesser (17% of the total seaweed-associated bacterial isolates). The contribution of active isolates from the different 236

seaweed host species was presented as a pie diagram (Figure 2A). It is intriguing to note that the representatives of 237

the γ-Proteobacteria were most abundant (40% of the active isolates) as seaweed association, the majority of which 238

were affiliated with the Shewanellae algae (Figure 2B). The predominant bacterial groups belonging to Firmicutes 239

was found to be Bacillus subtilis followed by Bacillus amyloliquefaciens (31 and 17% of the active isolates, 240

respectively). Bacillus cereus, Pseudomonas putida, and Vibrio alginolyticus contributed the individual share of 4% 241

of the aggregate number of seaweed-associated active isolates (Figure 2B). 242

Figure 2 243

Table 2 244

Biochemical identification and 16S rRNA-based phylogeny 245

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The bacteria with antimicrobial properties were characterized using different biochemical tests 246

(Chakraborty et al. 2014). Among the selected bacterial strains, a total of 12 strains were classified as Gram-247

positive, and the remaining 11 belonged to Gram negative as determined by Gram staining and KOH screening 248

experiments. Biochemical and molecular analysis had clustered the promising bacterial isolates into two major phyla 249

Firmicutes and Proteobacteria. The 16S rRNAs of the isolated strains were compared with the closest relatives in 250

the GenBank, and a phylogenetic tree was constructed by comparing the sequences of the 23 isolates with their 251

closest relatives (Figure 3). The isolates were identified as B. subtilis, B. amyloliquefaciens, V. alginolyticus, S. 252

algae and P. putida. 253

Figure 3 254

Antagonism assays among the isolated marine bacteria and their autoinhibition 255

The autoinhibition and mutual inhibition patterns of the antimicrobial isolates associated with seaweed 256

revealed that some of the isolated strains with antibacterial activity did not exhibit autoinhibition, although a few 257

bacterial strains showed inhibition towards other antibacterial isolates considered in the present study. The 258

autoinhibition and mutual inhibition patterns of antimicrobial isolates used in the study have been summarized in 259

Table 3. 260

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Table 3 262

Plasmid profiling, curing and antibacterial activity 263

The Shewanellae sp were found to possess a plasmid above 10 kb, whereas V. alginolyticus showed two 264

plasmids sized greater than 10 kb and 1 kb, respectively. Among the Bacillus strains, SWI1 (~ >10 kb) and SWI8 265

showed the presence of three plasmids namely, 6 kb, 8 kb and above 10 kb. However, no bands appeared after 266

plasmid curing (Figure 4), and the cultures retained their antimicrobial activity even after the process of plasmid 267

curing. 268

Figure 4 269

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Analysis of pks and nrps sequences 270

The PKS specific primers were successful in obtaining the PCR amplicons showing significant homology 271

to the sequences deposited in the GenBank. No correct products were detected when the NRPS primers were used 272

for PCR. The negative results from the initial NRPS and PKS PCRs were also confirmed through subsequent 273

annealing-temperature gradient NRPS and PKS PCRs. The primers used in the study were shown in the Table 1. 274

Among the 23 candidate antimicrobial isolates, 8 were found to be PKS positive with an amplicon of PKS gene (~ 275

0.7 kb). All the PKS positive isolates were found to be the genus Bacillus. The PKS primers could not amplify the 276

Shewanella isolates. To verify the amplified product, the sequenced results obtained through cloning were subjected 277

to blastx analysis in the GenBank. Further, the DNA sequences were translated, and the deduced amino acid 278

sequences were also analyzed through blastp program of NCBI. From the blast analysis report, it was found that 4 of 279

the pks-I positive isolates had sequence similarity with B. subtilis (99%) PKS, and the remaining 4 sequences were 280

similar to the gene sequences reported from B. amyloliquefaciens (99%). All of the 8 bacterial isolates with an 281

amplified pks-I gene product exhibited antibacterial activities against multiple aquaculture pathogens, which 282

demonstrated their broad spectra of antimicrobial potential (Table 2). 283

Phylogeny of KS domain sequences 284

On the basis of the architecture and mode of action of the enzyme assembly lines, the PKSs have been 285

classified into Type I, Type II and Type III. The Type I PKSs refer to linearly arranged and covalently fused 286

catalytic domains within the large multifunctional enzymes, whereas the term Type II indicates a dissociable 287

complex of discrete and usually monofunctional enzymes. Furthermore, the third group of multifunctional enzymes 288

of the chalcone synthase type is denoted as Type III PKSs (Hertweck 2009). The phylogeny of the sequenced KS 289

domains characterized the deduced amino acid sequences as Type I bacterial PKSs (Figure 5). The analysis of 290

deduced amino acid sequences demonstrated that the KS domain shared the conserved motif TACSSSLVA (Figure 291

6). 292

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Figure 5 294

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Figure 6 295

296

Discussion 297

Antimicrobial assay 298

Seaweeds were proposed to have chemical defense strategies against targeted microorganisms (Kubanek et 299

al. 2003), and the high proportion of antimicrobial isolates from seaweeds further validated this hypothesis (Lemos 300

et al. 1985; Burgess et al. 1999; Wiese et al. 2009). In the present study, 22 percent of cultured bacterial isolates 301

associated with seaweeds were found to have antimicrobial activity during the preliminary screening. About 9 302

percent of the isolates showed consistent results on further screenings, and these results were found to be similar to 303

those observed by other researchers (Burgess et al. 1999; Lemos et al. 1985). Earlier reports have demonstrated that 304

the proportion of bacteria with inhibitory activity associated with seaweeds and invertebrates was greater (11%) than 305

that of seawater and sediments (Zeng et al. 2005). Bacteria associated with live or inert surfaces were more likely to 306

display antibacterial properties (Gram et al. 2010). There were several reports demonstrating that compounds of the 307

associated bacterium help the host in certain ways to deter the pathogenic microbial flora, and that the bioactive 308

compounds isolated from host have structural similarities to the compounds of the microbial origin (Kubanek et al. 309

2003; Zhang et al. 2009). Bacterial origin of sponge-derived metabolites has been validated in earlier studies 310

(Schneemann et al. 2010; Quevrain et al. 2014). Epimanzamine D, which was originally isolated from a Palaun 311

sponge, and was produced by Streptomyces fulvorobeus strain HB113, and coproporphyrin, produced by the strain 312

HB100, was previously described as products of sponges (Schneemann et al. 2010). Similarly, the bacteria affiliated 313

to the genus Pseudoalteromonas were found to possess the similar spectrum of activity as that of C. clathrus that 314

was reported to produce the compounds monopalmitin and monostearin, which were previously isolated from the 315

whole sponge (Quevrain et al. 2014). The bacterial isolates with antibiotic activity in the present study were 316

pigmented, which further corroborate the observation of the earlier study that antibiotic-producing bacteria were 317

pigmented (Lemos et al. 1985). 318

319

Biochemical identification and 16S rRNA gene-based phylogeny 320

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The results obtained by biochemical identification were further confirmed by 16S rRNA gene phylogeny. 321

The 16S rRNA gene sequence-based homology searches showed that most of the isolates of the present study were 322

closely related to each other (>99 percent similarity). However, every individual isolate was included in the study as 323

separate as they were found to be different in their inhibition and growth patterns. In the present survey of 324

antimicrobial bacteria associated with seaweeds, the representatives from two bacterial phyla, Firmicutes and 325

Proteobacteria were found. These results found harmony with earlier findings (Wiese et al. 2009; Kennedy et al. 326

2009). The bacteria isolated from brown seaweed Laminaria saccharina were found to be affiliated to the bacterial 327

domain of the Gram-positive Firmicutes, the Gram-negative Proteobacteria and Bacteroidetes. Representatives of 328

the Proteobacteria were found to be most abundant, the majority of which were affiliated with the γ-subgroup 329

(Wiese et al. 2009). The γ-proteobacterial phylum was demonstrated to be the most dominant cultivable group in a 330

recent study of sponge-associated bacteria from Haliclona simulans collected from the Irish waters (Kennedy et al. 331

2009). The predominant bacterial group found in H. simulans (Kennedy et al. 2008) and on the surface of J. rubens 332

(Ali et al. 2012) (about 73%) was Proteobacteria as determined by the total 16S rRNA gene library (77 percent of 333

clones). The γ-proteobacterial S. algae, however, would be less likely to be enriched by the selection process, and 334

are probably the dominant group of cultivable bacteria from the seaweeds. The entire set of isolates of Firmicutes 335

belonged to the genus Bacillus. A large number of Bacillus isolates found in this study might be due to the 336

selectivity of the media used in the present study as also supported by published literature (Zhang et al. 2009; 337

Kanagasabhapathy et al. 2006). Within the Firmicutes, especially strains belonging to the genus Bacillus are 338

common producers of antimicrobial compounds. Approximately 800 bacterial metabolites with antibiotic activity 339

have been isolated from Bacillus spp (Wiese et al. 2009). The Bacillus clade in the present study had representatives 340

of the species B. cereus, B. subtilis and B. amyloliquefaciens. The DNA similarity searches of the partial 16S rRNA 341

gene sequences of the bacterial strains isolated in the study with the GenBank database had shown that the strains 342

that were phylogenetically associated with B. amyloliquefaciens and B. subtilis have been clustered as one, and 343

therefore, difficult to distinguish. B. amyloliquefaciens and B. subtilis were reported to harbor several rRNA gene 344

clusters in which 16S rDNA sequence variation was found to exist (Hu et al. 2010). However, we could not 345

differentiate them with the sequence results of the present study, possibly because the regions amplified with our 346

primers do not belong to those clusters with sequence variation. 347

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Antagonism assays among the isolated marine bacteria and their autoinhibition 348

None of the cells were found to be autoinhibitory. The absence of inhibitory activity of the producer strains 349

against other epiphytic producers indicated that the production of inhibitors could be of great importance in 350

microhabitats, such as an algal surface, where competition for an attachment site is surely a frequent event. In 351

general, a few producer strains inhibited the growth of the other producers, while the activity among the majority of 352

the strains was non-inhibitory. Only one strain, SWI 1 was found to be inhibited by all the producers tested. 353

Interestingly, on the other hand, in contradiction to the Lemos’s (1985) observation, a greater number of 354

antimicrobial relationships between producer strains isolated from the algae were not observed. Most of the bacterial 355

strains considered under the present study were not inhibitory to each other except SWI1. Detailed autoinhibition 356

and mutual inhibition patterns were shown in the Table 3. These results suggested that these beneficial populations 357

coexisted in the seaweed biofilm, and might significantly contribute in protecting the host seaweeds from the 358

deleterious pathogenic microbial populations or other colonizers. 359

360

Plasmid profiling, curing and antibacterial activity 361

The Shewanellae sp were found to possess a plasmid of molecular size greater than 10 kb, and among 362

different Bacillus strains, SWI1 (~>10 kb) and SWI8 (~>10 kb, ~>8 kb, ~>1 kb) showed the presence of plasmids. 363

V. alginolyticus isolated in the present study manifested the occurrence of two plasmids. However, no bands 364

appeared on the electrophorized gel after plasmid curing. The cultures retained their antimicrobial activities even 365

after plasmid curing, suggesting that the antimicrobial activities of the bacterial isolates, which have been considered 366

in the present study, was not encoded by plasmid, and the genes encoding the antimicrobial product might be present 367

within the genome. The antibiotic whose biosynthesis was determined by the SCPI plasmid of Streptomyces 368

coelicolor had been characterized as methylenomycin A (2-methylene-cyclopentan-3-one-4, 5-epoxy-4, 5-dimethyl-369

carboxylic acid) (Wright 1975). Plasmid linkage of bacteriocin activity was reported in an earlier literature 370

(Schillinger 1989). In contrast, chromosomally encoded class II bacteriocin LCI protein of Bacillus 371

amyloliquefaciens Bg-C31 was also reported (Hu et al. 2010). It is of note that several gene clusters aid in 372

biosynthesizing the bioactive peptides and polyketides by enzymes, for example, the non-ribosomal peptide 373

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synthetase and polyketide synthetase. In addition to this, bacteriocins are ribosomally synthesized proteins that elicit 374

bactericidal activity. The genetic determinants for bacteriocins are either chromosomal or plasmid encoded as 375

demonstrated previously (Hu et al. 2010). The results obtained in the present study confirmed that the antibacterial 376

activities of the bacterial strains were not due to a plasmid-encoded bacteriocin. 377

378

Analysis of pks-I and nrps gene sequences 379

Polyketides and nonribosomal peptides became immensely important over the past few decades, and the 380

numbers of various novel polyketide and non-ribosomal peptide compounds have been found from marine-derived 381

microbes, most of which showed different biological activities and ecological functions (Zhou et al. 2011). 382

Polyketides, nonribosomal peptides, and PKS/NRPS hybrid compounds are important classes of natural products 383

and include many important drugs. Phycochemical studies showed the ability of algae to produce and store 384

polyketides as polycyclic ether macrolides and open chain polyketides. Although macrolides produced by the 385

terrestrial microorganisms have been used for long in human therapeutics, microlides from marine algae is a recent 386

addition (Cardozo et al. 2007). Compounds of polyketide origin, with potential bioactivities, have been isolated from 387

seaweeds, and were reported to have structural similarity to the known compounds of terrestrial cyanobacteria. It is 388

apparent that the seaweeds use targeted antimicrobial chemical defense strategies, and that the secondary 389

metabolites important in the ecological interactions between marine macroorganisms and microorganisms could be a 390

promising source of novel bioactive compounds, but this hypothesis has rarely been tested (Kubanek et al. 2003). In 391

support, it was found that the deduced amino acid sequence of Type III PKS (SbPKS) from a brown seaweed, 392

Sargassum binderi, shared a higher sequence similarity with bacterial PKSs (38% identity) than plant PKSs 393

(Baharum et al. 2011). This further strengthens the hypothesis of ecological interactions between the seaweed host 394

and their associated bacterial flora. 395

In the present report, 23 antimicrobial isolates with broad spectrum activities against aquaculture pathogens 396

were screened for the presence of secondary metabolite genes, and among those only 8 were able to amplify the 397

desired genes. These results provided us with the basic information on the presence of metabolite genes, but the 398

correlation to specific metabolites was found to be limited due to the lengths of the PCR fragments (Schneemann et 399

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al. 2010). The possible absence of the screened amplicons might be due to the chosen primer system, albeit 400

favorable for the great majority of known metabolite genes, are not the working polyketides with unusual molecular 401

constructions (Schirmer et al. 2005; Schneemann et al. 2010) or the coding genes for the antimicrobial products of 402

the screened strains might be different from that of the pks or nrps genes. Our results ratified the statement that there 403

were examples of strains possessing the functional genes with no inhibitory activity and vice versa (Zhao et al. 404

2011). Further, the existence of cultured isolates with the potential to synthesize bioactive compounds, and without a 405

metabolite gene amplified product showed that culturing remains a powerful tool for exploring the bioactive 406

metabolites of bacterial origin (Penesyan et al. 2009). Even though the cultivation-based studies possess some 407

limitations, it remains essential as it provides opportunities to study and understand the microbial ecology, 408

physiology, and to design antibiotic screening assays (Ali et al. 2012). Further, the absence of screened metabolite 409

gene products in the tested active strains demonstrated the possibility of other biosynthetic genes. We, therefore, 410

suggest that the biosynthetic gene-guided screening of bioactive bacterial population also needs to consider the 411

conserved gene sequences of other biosynthetic pathways (Zhu et al. 2009). 412

A phylogeny based on the KS domain sequences from other well-described organisms can be employed to 413

determine the structural similarity of the obtained KS domain sequences (Zhu et al. 2009). The deduced amino acid 414

sequences obtained in the study were aligned with the relative sequences of the GenBank (Figure 3). The 415

phylogenetic study showed that amplified gene products of the present study were of bacterial Type I PKSs. Earlier 416

studies in sponge-associated bacteria too found that the bacterial strains harbor Type I bacterial PKSs (Zhu et al. 417

2009; Zhang et al. 2009). In the present study, KS domain sequence-based phylogeny further clustered four 418

sequences with B. subtilis, and the remaining four with B. amyloliquefaciens. Hence, the KS domain sequences 419

enabled us to differentiate B. subtilis from the B. amyloliquefaciens strains, which we could not do through 16S 420

rRNA gene-based phylogenetic approach. B. cereus strain isolated in the present study was unable to amplify the 421

metabolite gene product. Multiple sequence alignment of the sequenced data with the known sequences from the 422

GenBank further enabled to identify the conserved sequence motif TACSSSLVA. The KS domain conserved 423

residues from the sponge, Hymeniacidon perleve associated bacteria was reported as VDTACSSSLVA (Zhu et al. 424

2009). In our study, this sequence motif amino acids showed some variations at certain specific locations. In B. 425

subtilis, the acidic amino acid, asparte, located at the third position from the cysteine active site in the N-terminal 426

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has been replaced with glutamate. Likewise, in B. amyloliquefaciens the amino acid, valine, located at the fourth

427

position from the cysteine active site in the N-terminal was replaced by isoleucine. However, due to the structural 428

similarities of acidic amino acids, aspartate (2-aminosuccinic acid) with glutamate (2-aminopentanedioic acid), and 429

isoleucine (2-amino-3-methylpentanoic acid) with valine (2-amino-3-methylbutanoic acid), it is apparent that the KS 430

domains of the bioactive Bacillus strains in the present study shared a common catalytic mode of action. Due to their 431

versatile assemblage mechanism, polyketides exhibit remarkable diversity both in terms of structure and biological 432

activities. To date, it is estimated that only a small fraction of the antimicrobial molecules potentially produced by 433

the Gram-positive bacteria has been identified. The recent advances in genome sequencing highlighted the genus 434

Bacillus as a potentially important source of antibiotic-like compounds (Fickers 2010). 435

In summary, the seaweeds possess greater potential as potential sources for screening bioactive bacterial 436

isolates that facilitate novel natural product discovery from the marine environment. These epibionts might be 437

beneficial to the seaweeds by limiting or preventing the development of competing, pathogenic and fouling bacteria. 438

Our results further suggest that the antimicrobial activity cannot be solely assessed by metagenomic studies as some 439

strains may escape the amplification of the desired genes. In that case, the antimicrobial potential may only be 440

assessed by screening the inhibition potential of the desired indicator organisms. Further, we recommend metabolite 441

gene-based screening of bioactive organisms should also exploit biosynthetic gene clusters of biosynthetic pathways 442

other than PKSs and NRPSs. 443

Acknowledgements 444

The Authors thank the Ministry of Earth Science, New Delhi for funding under the project “Drugs from the 445

sea” (grant number MoES-2/DS/6/2007 PC-IV) and Indian Council of Agricultural Research, New Delhi for 446

providing the necessary facilities. The authors thank the Director, Central Marine Fisheries Research Institute for his 447

guidance and support. Thanks are due to the Head, Marine Biotechnology Division, Central Marine Fisheries 448

Research Institute for facilitating the research activity. B.T. acknowledges ICAR Outreach Activity-3 for a 449

fellowship. 450

451

Conflict of interest: The authors declare that there is no conflict of interest. 452

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453

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568

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Table 1. Polymerase chain reaction (PCR) primersa used in this study 569

Primer Targetb Sequence (5’-3’) References

fD1 16SrRNA AGAGTTT GATCCTGGCTCAG Weisburg et al, 1991

rP2 16SrRNA ACGGCTACCTTGTTACGACTT Weisburg et al, 1991

GBF PKS RTRGAYCCNCAGCAICG Zang et al, 2009

GBR PKS VGTNCCNGTGCCRTG Zang et al, 2009

GCF PKS GCSATGGAYCCSCARCARCGSVT Schirmer et al, 2005

GCR PKS GTSCCSGTSCRTGSSCYTCSAC Schirmer et al, 2005

KSDPOOF PKS MGNGARGARGCNNWNSMNATGGAYCCNCARCANMG Zang et al, 2009

KSHGTGr PKS GGRTCNCCNARNSWNGTNCCNGTNCCRTG Zang et al, 2009

KS11f PKS GCIATGGAYCCICARCARMGIVT Schirmer et al, 2005

KS12r PKS GTICCIGTICCRTGISCYTCIAC Schirmer et al, 2005

MTF NRPS GCNGGYGGYGCNTAYGTNCC Zhao et al, 2008

MTR NRPS CCNCGDATYTTNACYTG Zhao et al, 2008

570

a The primer sequences used for the PCR reaction have been represented. PCR was performed in a total volume of 25 571

µL containing 1x reaction buffer with MgCl2 (Sigma), 0.25 mM of each dNTP (Fermentas), 0.5 mM of each primers 572

(Sigma), 1 ng DNA and 0.3 U Taq DNA polymerase (Sigma). 573

574

b Phylogenetic analysis was carried out using 16S rRNA sequencing. Different sets of degenerate primers targeting 575

genes encoding pks-I and nrps were used as described in the text. All of the amplification products were examined by 576

agarose gel electrophoresis, and bands of 700 to 800 bp and 1000 to 1,400 bp were classified as products of pks-I and 577

nrps genes, respectively. 578

579

580

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Table 2. Antimicrobial activity of the seaweed-associated bacteria against pathogenic organisms and their screening for secondary metabolite genes 1

(pks and nrps) responsible for bioactivity 2

Strains Seaweed host Seaweed-associated

strains

Antimicrobial activity (mm) against test pathogens Presence of

indicated

gene#

V.

alg

ino

lyti

cus

V.p

ara

ha

emo

lyti

cus

17

802

V.p

ara

hem

oly

ticu

s

45

1

V.v

uln

ific

us

MT

CC

11

45

V.

an

gu

lla

rum

A.

hyd

roph

illa

V.

ha

rvey

i

pks nrps

SWI 1 Anthophycus longifolium B. cereus ++ ++ - ++ - +++ - N N

SWI 2 Anthophycus longifolium B. subtilis +++ +++ ++ +++ ++ +++ ++ Pa

N

SWI 3 Padina gymnospora B. subtilis +++ +++ +++ +++ ++ +++ ++ Pb

N

SWI 4A Laurencia papillosa B. subtilis ++ ++ +++ +++ ++ +++ ++ N N

SWI 4B Laurencia papillosa B. amyloliquefacens - ++ ++ +++ - ++ ++ Pc

N

SWI 5 Turbinaria ornata B. amyloliquefacens +++ +++ +++ +++ ++ +++ +++ Pd

N

SWI 6 Hypnea valentiae B. amyloliquefacens ++ ++ +++ ++ ++ +++ ++ Pe

N

SWI 7 Padina gymnospora B. amyloliquefacens ++ +++ +++ +++ ++ +++ ++ Pf

N

SWI 8 Laurencia papillosa B. subtilis - - ++ ++ - - - N N

SWI 9 Hypnea valentiae S. algae - +++ ++ - - - +++ N N

SWI 10 Laurencia papillosa S. algae - +++ ++ - - - - N N

SWI 11 Padina gymnospora S. algae - +++ ++ - - - +++ N N

SWI 12 Hypnea valentiae S. algae - +++ - - - - ++ N N

SWI 12B Hypnea valentiae V. alginolyticus - - - - - - ++ N N

SWI 13 Padina gymnospora S. algae - ++ + - - - +++ N N

SWI 14 Anthophycus longifolium

S. algae - +++ + - - - +++ N N

SWI 16A Anthophycus longifolium B. subtilis - ++ - ++ - - - N N

SWI 16B Anthophycus longifolium B. subtilis ++ +++ ++ +++ ++ +++ ++ Pg

N

SWI 17 Dictyota dichotoma S. algae - +++ + +++ - - +++ N N

SWI 18 Dictyota dichotoma S. algae - +++ + +++ - - - N N

SWI19 Sargassum myriocystum B. subtilis + +++ ++ - ++ +++ +++ Ph

N

SWI20 Laurencia papillosa S. algae - +++ - - - - - N N

SWI21 Laurencia papillosa P. putida - - - +++ - - ++ N N

# A total of 23 antimicrobial isolates with broad spectrum activities against aquaculture pathogens were screened for the presence of secondary 3

metabolite genes, and among those only 8 were able to amplify the desired genes. 4

GenBank No for the sequenced amplicons a KC589397;

b KC607823;

c KC607821;

d KC589396;

e KC607822;

f KC589396;

g KC589400;

h KC589398 5

(-) signifies no inhibition; (+) signifies the inhibition zone as less than 10 mm; (++) inhibition zone 10-15 mm; (+++) inhibition zone >15mm 6

(P) signifies positive and (N) as negative. 7

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Table 3. Autoinhibition and mutual inhibition pattern (expressed in mm) of antimicrobial isolates associated with the 1

seaweeds 2

SWI1 SWI2 SWI3 SWI4A SWI4B SWI5 SWI6 SWI7 SWI8

SWI1 ND 10.3 (1.5) 10.3 (1.53) 12.7 (0.58) ND 9.0 (1.00) 9.33 (1.15) 10.0 (1.0) ND

SWI2 3.0 (2.0) ND ND ND ND ND ND ND ND

SWI3 3.7 (1.5) ND ND ND ND ND ND ND ND

SWI4A 2.3 (0.6) ND ND ND ND ND ND ND ND

SWI4B ND ND ND ND ND ND ND ND ND

SWI5 ND ND ND ND ND ND ND ND ND


SWI7 ND ND ND ND ND ND ND ND 3.33 (0.58)

SWI8 0.7 (0.6) 2.67 (0.51) 10.7 (1.15) 9.67 (0.58) 11.3 (1.15) 10.3 (0.58) 10.7 (1.15) 2.7 (0.60) ND





SWI12B ND ND ND ND ND ND ND 9.3 (1.20) ND

SWI13 1.0 (1.7) ND 9.33 (0.58) 11.3 (1.15) 14.0 (1.0) 11 (1.0) ND 2.0 (0.00) ND

SWI14 0.7 (1.2) 2.33 (0.19) 1.33 (0.58) 1.67 (0.58) ND ND ND 9.7 (0.60) ND

SWI16A 0.7 (1.2) 9.33 (0.19) 11.7 (1.53) 11.0 (1.00) 13.0 (1.0) 11.7 (0.58) ND 12.0 (0.60) 2.67 (0.58)

SWI16B ND ND ND ND ND ND ND ND ND


SWI18 10 (1.5) 12.0 (1.00) 14.3 (0.58) 12 (1.0) 11.7 (1.53) 17.7 (0.58) 15 (1.0) ND ND


SWI20 ND ND 1.67 (0.58) 2.33 (0.58) 1.33 (0.58) ND ND ND ND


3

ND: No autoinhibition recorded 4

Figures in parentheses indicate the mutual inhibition pattern 5

6

7

8

9

10

11

12

13

14

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Figure captions 15

Figure 1 Indicative photograph showing the inhibitory zones exhibited by the seaweed-associated 16

antimicrobial bacterial flora during antibacterial screening against the listed pathogens. (1) 17

Seaweed material A. longifolium used for isolation, (2) Antibacterial activities of seaweed-18

associated bacterium SWI 2 against V. vulnificus MTCC1145, (3) Gram staining 19

photomicrograph of SWI2 (B. subtilis), (4) Antibacterial activities of seaweed-associated 20

bacterium SWI 14 against V. parahaemolyticus 17802, whereas the bactericidal zones were 21

yellow, whereas live cells were blue in color, (5) Gram staining photomicrograph of SWI 14 (S. 22

algae). The clearance zones realized by the isolates signify the antibacterial activity. 23

Antimicrobial activity was recorded as the diameter of inhibition zones determined as a 24

distance of ≥1 mm between the circular area (=lawn of the isolate) and the end of the clear zone 25

bounded by the lawn of the test strain. 26

Figure 2 Pie diagrams showing the (A) distribution of seaweed-associated active isolates contributed by the 27

representative seaweed hosts screened under the study, (B) contribution (as percent share towards 28

the total number of bacterial isolates with potentially greater antibacterial properties against the 29

pathogens) of the individual representative bacterium as seaweed association. 30

Figure 3 Phylogenetic tree derived from nearly complete 16S rRNA gene sequences, showing relationships 31

between the antimicrobial bacterial isolates associated with seaweeds and their phylogenetic 32

neighbors. The evolutionary history was inferred by using the Maximum Likelihood method based 33

on the Kimura 2-parameter model. Evolutionary analyses were conducted in MEGA5. 34

Figure 4 Plasmid profiles of the antimicrobial isolates before and after curing. (A) Plasmid profiles of Gram 35

negative isolates of the study 4th

lane (down) V. alginolyticus, M (Molecular marker) (B) Plasmid 36

profiles of Bacillus strain Lane 1 (SWI1), 12th

Lane (SWI8), M (Molecular marker) (C) Plasmid 37

profiles of the strains after curing, M (Molecular marker). Molecular marker used is Gene 38

RulerTM

1kb DNA Ladder (Thermo scientific, 250bp-10,000bp). 39

Figure 5 Molecular phylogeny analysis of ketosynthase regions with respect to the diverse range of 40

ketosynthase domains including. Type I, II and III. The evolutionary history was inferred by using 41

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28

the Maximum Likelihood method based on the Whelan and Goldman model. A discrete gamma 42

distribution was used to model evolutionary rate differences among sites. The tree with the highest 43

log likelihood is shown. The sequences in the experiment are preceded by circles. 44

Figure 6 Amino acid sequence alignments of active sites of Type I KS domains. The characteristic 45

conserved motifs of Type I KS domains were predicted from the multiple alignments of active 46

sites. The cysteine active site and the conserved amino acid sequence motif are marked by asterisk 47

and frame, respectively. The sequences in this study are from seaweed-associated bacteria and are 48

SWI02, SWI19, SWI03, SWI16B, SWI05, SWI07, SWI4B and SWI06. Other reference sequences 49

aligned sequences in the GenBank were derived from pks (B. subtilis, ABR19774.1), NidKS2 50

(Streptomyces aelestis, AF016585), EryA (Saccharopolyspora erythraea, CAA44448), PimS0 51

(Streptomyces natalensis, AJ278573), EpoA (Sorangium cellulosum AF217189), McyD 52

(Microcystis aeroginosa, AF183408.1), NJ6-3-2 (Pseudoalteromonas sp, DQ666948), and BaeN 53

(B. amyloliquefaciens 154686134). 54

55

56

57

58

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Figure 1 Indicative photograph showing the inhibitory zones exhibited by the seaweed-associated antimicrobial bacterial flora during antibacterial screening against the listed pathogens. (1) Seaweed

material A. longifolium used for isolation, (2) Antibacterial activities of seaweed-associated bacterium SWI 2

against V. vulnificus MTCC1145, (3) Gram staining photomicrograph of SWI2 (B. subtilis), (4) Antibacterial activities of seaweed-associated bacterium SWI 14 against V. parahaemolyticus 17802, whereas the

bactericidal zones were yellow, whereas live cells were blue in color, (5) Gram staining photomicrograph of SWI 14 (S. algae). The clearance zones realized by the isolates signify the antibacterial activity.

Antimicrobial activity was recorded as the diameter of inhibition zones determined as a distance of ≥1 mm between the circular area (=lawn of the isolate) and the end of the clear zone bounded by the lawn of the

test strain. 308x231mm (300 x 300 DPI)

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Figure 2 Pie diagrams showing the (A) distribution of seaweed-associated active isolates contributed by the representative seaweed hosts screened under the study, (B) contribution (as percent share towards the total

number of bacterial isolates with potentially greater antibacterial properties against the pathogens) of the individual representative bacterium as seaweed association.

32x33mm (600 x 600 DPI)

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Figure 3 Phylogenetic tree derived from nearly complete 16S rRNA gene sequences, showing relationships between the antimicrobial bacterial isolates associated with seaweeds and their phylogenetic neighbors. The

evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-

parameter model. Evolutionary analyses were conducted in MEGA5. 274x243mm (96 x 96 DPI)

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Figure 4 Plasmid profiles of the antimicrobial isolates before and after curing. (A) Plasmid profiles of Gram negative isolates of the study 4th lane (down) V. alginolyticus, M(Molecular marker) (B) Plasmid profiles of Bacillus strain Lane 1 (SWI1), 12th Lane (SWI8), M (Molecular marker) (C) Plasmid profiles of the strains

after curing, M (Molecular marker). Molecular marker used is Gene RulerTM1kb DNA Ladder (Thermo scientific, 250bp-10,000bp). 20x15mm (600 x 600 DPI)

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Figure 5 Molecular phylogeny analysis of ketosynthase regions with respect to the diverse range of ketosynthase domains including. Type I, II and III. The evolutionary history was inferred by using the

Maximum Likelihood method based on the Whelan and Goldman model. A discrete gamma distribution was used to model evolutionary rate differences among sites. The tree with the highest log likelihood is shown.

The sequences in the experiment are preceded by circles. 325x241mm (72 x 72 DPI)

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Figure 6 Amino acid sequence alignments of active sites of Type I KS domains. The characteristic conserved motifs of Type I KS domains were predicted from the multiple alignments of active sites. The cysteine active

site and the conserved amino acid sequence motif are marked by asterisk and frame, respectively. The

sequences in this study are from seaweed-associated bacteria and are SWI02, SWI19, SWI03, SWI16B, SWI05, SWI07, SWI4B and SWI06. Other reference sequences aligned sequences in the GenBank were

derived from pks (B. subtilis, ABR19774.1), NidKS2 (Streptomyces aelestis, AF016585), EryA (Saccharopolyspora erythraea, CAA44448), PimS0 (Streptomyces natalensis, AJ278573), EpoA (Sorangium

cellulosum AF217189), McyD (Microcystis aeroginosa, AF183408.1), NJ6-3-2 (Pseudoalteromonas sp, DQ666948), and BaeN (B. amyloliquefaciens 154686134).

210x118mm (96 x 96 DPI)

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Antimicrobial properties of cultivable bacteria associated ... with classical biochemical identification ... 80 predominant role in the establishment of cross relationships between