Endophytic Bacteria Enterobacter hormaechei Fabricated Silver Nanoparticles and Their Antimicrobial Activity.

Antimicrobial resistance (AMR), one of the greatest issues for humankind, draws special attention to the scientists formulating new drugs to prevent it. Great emphasis on the biological synthesis of silver nanoparticles (AgNPs) for utilization in single or combinatorial therapy will open up new avenues to the discovery of new antimicrobial drugs. The purpose of this study was to synthesize AgNPs following a green approach by using an endophytic bacterial strain, Enterobacter hormaechei, and to assess their antimicrobial potential against five pathogenic and four multidrug-resistant (MDR) microbes. UV-Vis spectroscopy, fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), and zeta potential (ζ) were used to characterize the synthesized AgNPs. Endophytic E. hormaechei-mediated AgNPs (Eh-AgNPs) were represented by a strong UV-Vis absorbance peak at 418 nm within 5 min, forming spherical and polydispersed nanoparticles in the size range of 9.91 nm to 92.54 nm. The Eh-AgNPs were moderately stable with a mean ζ value of -19.73 ± 3.94 mV. The presence of amine, amide, and hydroxyl functional groups was observed from FTIR analysis. In comparison to conventional antibiotics, the Eh-AgNPs were more effective against Bacillus cereus (ATCC 10876) and Candida albicans (ATCC 10231), exhibiting 9.14 ± 0.05 mm and 8.24 ± 0.05 mm zones of inhibition (ZOIs), respectively, while displaying effective inhibitory activity with ZOIs ranging from 10.98 ± 0.08 to 13.20 ± 0.07 mm against the MDR bacteria. Eh-AgNP synthesis was rapid and eco-friendly. The results showed that Eh-AgNPs are promising antimicrobial agents that can be used in the development and formulation of new drugs to curb the menace of antimicrobial resistance in pathogenic and MDR microbes.

Secondary Metabolites Profiling of Acinetobacter baumannii Associated with Chili (Capsicum annuum L.) Leaves and Concentration Dependent Antioxidant and Prooxidant Properties.

Secondary bioactive compounds of endophytes are inevitable biomolecules of therapeutical importance. In the present study, secondary metabolites profiling of an endophytic bacterial strain, Acinetobacter baumannii, were explored using GC-MS study. Presence of antioxidant substances and antioxidant properties in chloroform (CHL), diethyl ether (DEE), and ethyl acetate (EA) crude extracts of the endophytic bacteria were studied. Total phenolic content (TPC), total flavonoid content (TFC), total antioxidant capacity (TAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and ferrous ion chelating assay were evaluated. A total of 74 compounds were identified from the GC-MS analysis of the EA extract representing mostly alkane compounds followed by phenols, carboxylic acids, aromatic heterocyclic compounds, ketones, aromatic esters, aromatic benzenes, and alkenes. Among the two phenolic compounds, namely, phenol, 2,4-bis(1,1-dimethylethyl)- and phenol, 3,5-bis(1,1-dimethylethyl)-, the former was found in abundance (11.56%) while the latter was found in smaller quantity (0.14%). Moreover, the endophytic bacteria was found to possess a number of metal ions including Fe(II) and Cu(II) as 1307.13 ± 2.35 ppb and 42.38 ± 0.352 ppb, respectively. The extracts exhibited concentration dependent antioxidant and prooxidant properties at high and low concentrations, respectively. The presence of phenolic compounds and metal ions was believed to play an important role in the antioxidant and prooxidant potentials of the extracts. Further studies are suggested for exploring the untapped resource of endophytic bacteria for the development of novel therapeutic agents.

Silver Nanoparticles Synthesized by Using the Endophytic Bacterium Pantoea ananatis are Promising Antimicrobial Agents against Multidrug Resistant Bacteria.

Antibiotic resistance is one of the most important global problems currently confronting the world. Different biomedical applications of silver nanoparticles (AgNPs) have indicated them to be promising antimicrobial agents. In the present study, extracellular extract of an endophytic bacterium, Pantoea ananatis, was used for synthesis of AgNPs. The synthesized AgNPs were characterized by UV⁻Vis spectroscopy, FTIR, transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), and Zeta potential. The antimicrobial potential of the AgNPs against pathogenic Staphylococcus aureus subsp. aureus (ATCC 11632), Bacillus cereus (ATCC 10876), Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 10145) and Candida albicans (ATCC 10231), and multidrug resistant (MDR) Streptococcus pneumoniae (ATCC 700677), Enterococcus faecium (ATCC 700221) Staphylococcus aureus (ATCC 33592) Escherichia coli (NCTC 13351) was investigated. The synthesized spherical-shaped AgNPs with a size range of 8.06 nm to 91.32 nm exhibited significant antimicrobial activity at 6 µg/disc concentration against Bacillus cereus (ATCC 10876) and Candida albicans (ATCC 10231) which were found to be resistant to conventional antibiotics. The synthesized AgNPs showed promising antibacterial efficiency at 10 µg/disc concentration against the MDR strains. The present study suggests that AgNPs synthesized by using the endophytic bacterium P. ananatis are promising antimicrobial agent.

Nucleotide sequence of Phaseolus vulgaris L. alcohol dehydrogenase encoding cDNA and three-dimensional structure prediction of the deduced protein.

BACKGROUND: Common beans (Phaseolus vulgaris L.) are widely consumed as a source of proteins and natural products. However, its yield needs to be increased. In line with the agenda of Phaseomics (an international consortium), work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated. Alcohol dehydrogenase (AD) encoding gene cDNA was a noticeable transcript among the generated ESTs. This AD is an important enzyme; therefore, to understand more about it this study was undertaken. OBJECTIVE: The objective of this study was to elucidate P. vulgaris L. AD (PvAD) gene cDNA sequence and to predict the three-dimensional (3D) structure of deduced protein. MATERIALS AND METHODS: positive and negative strands of the PvAD cDNA clone were sequenced using M13 forward and M13 reverse primers to elucidate the nucleotide sequence. Deduced PvAD cDNA and protein sequence was analyzed for their basic features using online bioinformatics tools. Sequence comparison was carried out using bl2seq program, and tree-view program was used to construct a phylogenetic tree. The secondary structures and 3D structure of PvAD protein were predicted by using the PHYRE automatic fold recognition server. RESULTS: The sequencing results analysis showed that PvAD cDNA is 1294 bp in length. It's open reading frame encodes for a protein that contains 371 amino acids. Deduced protein sequence analysis showed the presence of putative substrate binding, catalytic Zn binding, and NAD binding sites. Results indicate that the predicted 3D structure of PvAD protein is analogous to the experimentally determined crystal structure of s-nitrosoglutathione reductase from an Arabidopsis species. CONCLUSIONS: The 1294 bp long PvAD cDNA encodes for 371 amino acid long protein that contains conserved domains required for biological functions of AD. The predicted deduced PvAD protein's 3D structure reflects the analogy with the crystal structure of Arabidopsis thaliana s-nitrosoglutathione reductase. Further study is required to validate the predicted structure.

Analysis of common bean (Phaseolus vulgaris L., genotype BAT93) calmodulin cDNA using computational tools.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs) is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM) gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it. OBJECTIVE: The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM) gene cDNA and its deduced protein (amino acids) sequence. MATERIALS AND METHODS: Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D) structure of PvCaM protein were predicted using the Phyre automatic fold recognition server. RESULTS: Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM. CONCLUSIONS: This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is essential.

Insights from computational analysis of full-length ß-ketoacyl-[ACP] synthase-II cDNA isolated from American and African oil palms.

BACKGROUND: Palm oil derived from fruits (mesocarp) of African oil palm (Elaeis guineensis Jacq. Tenera) and American oil palm (E. oleifera) is important for food industry. Due to high yield, Elaeis guineensis (Tenera) is cultivated on commercial scale, though its oil contains high (~54%) level of saturated fatty acids. The rate-limiting activity of beta-ketoacyl-[ACP] synthase-II (KAS-II) is considered mainly responsible for the high (44%) level of palmitic acid (C16:0) in the oil obtained from E. guineensis. OBJECTIVE: The objective of this study was to annotate KAS-II cDNA isolated from American and African oil palms. MATERIALS AND METHODS: The full-length E. oleifera KAS-II (EoKAS-II) cDNA clone was isolated using random method of gene isolation. Whereas, the E. guineensis KAS-II (EgTKAS-II) cDNA was isolated using reverse transcriptase polymerase chain reaction (RT-PCR) technique; and missing ends were obtained by employing 5'and 3' RACE technique. RESULTS: The results show that EoKAS-II and EgTKAS-II open reading frames (ORFs) are of 1689 and 1721 bp in length, respectively. Further analysis of the both EoKAS-II and EgTKAS-II predicted protein illustrates that they contains conserved domains for 'KAS-I and II', 'elongating' condensing enzymes, 'condensing enzymes super-family', and '3-oxoacyl-[ACP] synthase II'. The predicted protein sequences shows 95% similarity with each other. Consecutively, the three active sites (Cys, His, and His) were identified in both proteins. However, difference in positions of two active Histidine (His) residues was noticed. CONCLUSION: These insights may serve as the foundation in understanding the variable activity of KAS-II in American and African oil palms; and cDNA clones could be useful in the genetic engineering of oil palms.

Diversity of endophytic bacteria in medicinally important Nepenthes species.

BACKGROUND: Nepenthes species are used in traditional medicines to treat various health ailments. However, we do not know which types of endophytic bacteria (EB) are associated with Nepenthes spp. OBJECTIVE: The objective of this study was to isolate and to identify EB associated with Nepenthes spp. MATERIALS AND METHODS: Surface-sterilized leaf and stem tissues from nine Nepenthes spp. collected from Peninsular Malaysia were used to isolate EB. Isolates were identified using the polymerase chain reaction-amplified 16S ribosomal DNA (rDNA) sequence similarity based method. RESULTS: Cultivable, 96 isolates were analyzed; and the 16S rDNA sequences analysis suggest that diverse bacterial species are associated with Nepenthes spp. Majority (55.2%) of the isolates were from Bacillus genus, and Bacillus cereus was the most dominant (14.6%) among isolates. CONCLUSION: Nepenthes spp. do harbor a wide array of cultivable endophytic bacteria.

A summary report of the workshop on the 'academic leadership training in the AIMST University, Malaysia'.

In Malaysia, there are 81 (as on February 15, 2013) higher education institutions including satellite branches of the foreign universities. In northern part of the Peninsular Malaysia, AIMST University is the first private not-for-profit university and aims to become a premier private university in the country and the region. The workshop described in this article was designed to develop and enhance the capacity of academic staff-in-leadership-role for the University. This type of workshops may be a good method to enhance the leadership qualities of the head of each unit, department, school and faculty in each university.

Endophytic bacteria as a source of novel antibiotics: An overview.

World human population is increasing with an alarming rate; and a variety of new types of health issues are popping up. For instance, increase in number of drug-resistant bacteria is a cause of concern. Research on antibiotics and other microbial natural products is pivotal in the global fight against the growing problem of antibiotic resistance. It is necessary to find new antibiotics to tackle this problem. The use of therapeutic plant species in traditional medicine is as old as mankind; and currently, it is strongly believed that all types of plant species across the plant kingdom do harbour endophytic bacteria (EB). The natural therapeutic compounds produced by EB do have several potential applications in pharmaceutical industry. The EB derived natural products such as Ecomycins, Pseudomycins, Munumbicins and Xiamycins are antibacterial, antimycotic and antiplasmodial. Some of these natural products have been reported to possess even antiviral (including Human Immunodeficiency Virus (HIV)) properties. Therefore, to deal with increasing number of drug-resistant pathogens EB could serve as a potential source of novel antibiotics.

Isolation and identification of bacterial endophytes from pharmaceutical agarwood-producing Aquilaria species.

BACKGROUND: Resins and gums are used in traditional medicine and do have potential applications in pharmacy and medicine. Agarwood is the fragrant resinous wood, which is an important commodity from Aquilaria species and has been used as a sedative, analgesic, and digestive in traditional medicine. Endophytic bacteria are potentially important in producing pharmaceutical compounds found in the plants. Hence, it was important to understand which types of endophytic bacteria are associated with pharmaceutical agarwood-producing Aquilaria species. OBJECTIVE: This study was undertaken to isolate and identify endophytic bacteria associated with agarwood-producing seven (7) Aquilaria species from Malaysia. MATERIALS AND METHODS: Botanical samples of seven Aquilaria species were collected, and endophytic bacteria were isolated from surface-sterilized-tissue samples. The 16S rRNA gene fragments were amplified using PCR method, and endophytic bacterial isolates (EBIs) were identified based on 16S rRNA gene sequence similarity based method. RESULTS: Culturable, 77 EBIs were analyzed, and results of 16S rRNA gene sequences analysis suggest that 18 different types of endophytic bacteria are associated with (seven) Aquilaria species. From 77 EBIs, majority (36.4%) of the isolates were of Bacillus pumilus. CONCLUSION: These findings indicate that agarwood-producing Aquilaria species are harboring 18 different types of culturable endophytic bacteria.

Identification of bacterial endophytes associated with traditional medicinal plant Tridax procumbens Linn.

BACKGROUND: In traditional medicine, Tridax procumbens Linn. is used in the treatment of injuries and wounds. The bacterial endophytes (BEs) of medicinal plants could produce medicinally important metabolites found in their hosts; and hence, the involvement of BEs in conferring wound healing properties to T. Procumbens cannot be ruled out. But, we do not know which types of BEs are associated with T. Procumbens. OBJECTIVE: The objective of this study was to investigate the fast growing and cultivable BEs associated with T. procumbens. MATERIALS AND METHODS: Leaves and stems of healthy T. Procumbens plants were collected and cultivable BEs were isolated from surface-sterilized leaf and stem tissue samples using Luria-Bertani (LB) agar (medium) at standard conditions. A polymerase chain reaction was employed to amplify 16S rRNA coding gene fragments from the isolates. Cultivable endophytic bacterial isolates (EBIs) were identified using 16S rRNA gene nucleotide sequence similarity based method of bacterial identification. RESULTS: Altogether, 50 culturable EBIs were isolated. 16S rRNA gene nucleotide sequences analysis using the Basic Local Alignment Search Tool (BLAST) revealed identities of the EBIs. Analysis reveals that cultivable Bacillus spp., Cronobacter sakazakii, Enterobacter spp., Lysinibacillus sphaericus, Pantoea spp., Pseudomonas spp. and Terribacillus saccharophilus are associated with T. Procumbens. CONCLUSION: Based on the results, we conclude that 24 different types of culturable BEs are associated with traditionally used medicinal plant, T. Procumbens, and require further study.

Insights from the GC content analysis of 76genome survey sequences (GSS) from Elaeisoleifera.

UNLABELLED: South American oil-palm (Elaeis oleifera) is not cultivated in tropical countries like Malaysia on large scale due to low yield of palm oil derived from its fruit mesocarp. However, its fruit mesocarp oil contains about 68.6 % oleic acid (C(18:1)) which is more than double in comparison to commercially cultivated oilpalm, E. guineensis Jacq Tenera (hybrid of Dura (♀) x Pisifera (♂)). It is also known that E. oleifera is a good source of tocotrienols and carotenoids. Therefore, it is of interest to know the genome sequence of E. oleifera. The objective of this study is to generate genome survey sequences (GSS) to get GC content insight in the E. oleifera genome. The nuclear genomic DNA isolated from young leaf-tissues was digested with EcoRI and NdeI/DraI restriction enzymes; and three genomic DNA libraries were constructed using Lambda ZAP-II, pGEM®-T Easy, and pDONR 222™ as cloning vectors. Generated 76 GSSs were analyzed by using Bioinformatics tools. The analysis result indicates that the adenine, cytosine, guanine and thymine content in generated GSSs are 30%, 20%, 20%, and 30% respectively. In conclusion, based on the precise GC content analysis of the randomly isolated 76 GSSs by using Bioinformatics tools we hypothesize that GC content in E. oleifera genome is 40%. The hypothesized 40% GC content in E. oleifera genome is expected to remain close to the GC content based on the whole genome analysis.(ψ)The nucleotide sequence data reported in this paper have been submitted to dbGSS division of the international DNA database (GenBank/DDBJ/EMBL) under accession numbers: DX575945- DX575972 and EI798032-EI798079. ABBREVIATIONS: gDNA - Nuclear genomic DNA, GSSs - Genome survey sequences K12, SAOP - South American oil-palm Db1.

Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library.

UNLABELLED: It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (Chyb) enzyme encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected Chyb amino acid sequences from other plant species and algal members with E. oleifera Chyb using ClustalW and its phylogenetic analysis suggest that Chyb from monocotyledonous plant species, Lilium hubrid, Crocus sativus and Zea mays are the most evolutionary related with E. oleifera Chyb. This study reports the annotation of E. oleifera Chyb. ABBREVIATIONS: ESTs - expressed sequence tags, EoChyb - Elaeis oleifera beta-carotene hydroxylase, MC - main cluster.

Screening of endophytic bacteria isolated from leaves of Sambung Nyawa [Gynura procumbens (Lour.) Merr.] for cytokinin-like compounds.

UNLABELLED: Endophytic bacteria are harmless in most plant species; and known to boost the growth and development of the host plants probably by secreting growth hormones. The isolation, identification and screening of endophytic bacteria for the plant growth regulators like cytokinin are needed to get the leads for their applications in agriculture sector. We describe the isolation and identification of the bacterial endophytes from the leaves of Sambung Nyawa [Gynura procumbens (Lour.) Merr.] and their screening for cytokinin-like compounds. We isolated three endophytic bacteria from the leaves of G. procumbens collected from the forest research institute of Malaysia (FRIM). They were further identified using amplified 16S rRNA gene sequence based method of bacterial identification. The ethyl acetate extracts of the isolates-broth were analyzed using cucumber cotyledon greening bioassay (CCGB) to determine the presence of cytokinin-like compounds. Consequently, the bacterial putative endophytes were identified as Psuedomonas resinovorans, Paenibacillus polymaxa, and Acenitobacter calcoaceticus. Broth-extracts from two (Psuedomonas resinovorans and Paenibacillus polymaxa) of the three putative bacterial endophytes show the positive results in their screening for cytokinin-like compounds using CCGB. Thus, we hypothesize that the bacterial putative endophytes of G. procumbens that produce cytokinin-like compounds might have a role in the growth and development of G. procumbens. ABBREVIATIONS: CCGB - Cucumber cotyledon greening bioassay, rDNA - Ribosomal DNA, K12, BAP - 6-Benzylaminopurine, Db1, MSA - Multiple sequence alignment. 8081.

Micropropagation of Elaeis guineensis Jacq. 'Dura': Comparison of three basal media for efficient regeneration.

Three basal plant tissue culture media, namely, N6, MS, and modified Y3, were compared to optimize micropropagation protocol for E. guineensis. Full strength media were used separately to regenerate plantlets directly using immature zygotic embryos (IZEs), and through somatic embryogenesis of calli obtained from IZEs. The plantlets regenerated by direct regeneration on three media were examined for shoot length and rooting percentage. For the induction of callus, somatic embryogenesis, and rooting modified Y3 medium was the most effective. In conclusion, the results indicate that modified Y3 medium is the most suitable for direct regeneration, callus induction and somatic embryogenesis in E. guineensis.