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Fulltext Plant Growth Promoting Potentials of Beneficial Endophytic Escherichia coli USML2 in Association with Rice Seedlings

Tharek M, Khairuddin D, Najimudin N, Ghazali AH

Trop Life Sci Res, 2021 Mar;32(1):119-143.
PMID: 33936555 DOI: 10.21315/tlsr2021.32.1.8

An endophytic Escherichia coli USML2 originally isolated from the inner part of an oil palm (Elaeis guineensis Jacq.) leaf tissue was inoculated to rice seedlings to investigate its ability in colonising plant inner tissues and promoting growth. Infection of E. coli USML2 was initiated by colonisation on the root surface, invasion of the interior root system followed by endophytic spreading. Inoculation of E. coli USML2 in the rice rhizosphere zone resulted in a significant increase in leaf numbers (33.3%), chlorophyll content (33.3%), shoot height (34.8%) and plant dry weight (90.4%) of 42 days old rice seedlings as compared to the control. These findings also demonstrated the ability of E. coli USML2 to spread endophytically which serves as a beneficial strategy for the bacterium to colonise the host plant and gain protection against adverse soil conditions. The genome of E. coli USML2 had also revealed predicted genes essential for endophytic bacterial colonisation and plant growth promotion which further proven potentials of E. coli USML2 as Plant Growth Promoting Endophyte (PGPE).
Low electric field DNA separation and in-channel amperometric detection by microchip capillary electrophoresis

Ghanim MH, Najimudin N, Ibrahim K, Abdullah MZ

IET Nanobiotechnol, 2014 Jun;8(2):77-82.
PMID: 25014078 DOI: 10.1049/iet-nbt.2012.0044

Miniaturisation of microchip capillary electrophoresis (MCE) is becoming an increasingly important research topic, particularly in areas related to micro total analysis systems or lab on a chip. One of the important features associated with the miniaturised MCE system is the portable power supply unit. In this work, a very low electric field MCE utilising an amperometric detection scheme was designed for use in DNA separation. The device was fabricated from a glass/polydimethylsiloxane hybrid engraved microchannel with platinum electrodes sputtered onto a glass substrate. Measurement was based on a three-electrode arrangement, and separation was achieved using a very low electric field of 12 V/cm and sample volume of 1.5 µl. The device was tested using two commercial DNA markers of different base pair sizes. The results are in agreement with conventional electrophoresis, but with improved resolution. The sensitivity consistently higher than 100 nA, and the separation time approximately 45 min, making this microchip an ideal tool for DNA analysis.
Sequence analysis and structure prediction of type II Pseudomonas sp. USM 4-55 PHA synthase and an insight into its catalytic mechanism

Wahab HA, Ahmad Khairudin NB, Samian MR, Najimudin N

BMC Struct Biol, 2006;6:23.
PMID: 17076907

Polyhydroxyalkanoates (PHA), are biodegradable polyesters derived from many microorganisms such as the pseudomonads. These polyesters are in great demand especially in the packaging industries, the medical line as well as the paint industries. The enzyme responsible in catalyzing the formation of PHA is PHA synthase. Due to the limited structural information, its functional properties including catalysis are lacking. Therefore, this study seeks to investigate the structural properties as well as its catalytic mechanism by predicting the three-dimensional (3D) model of the Type II Pseudomonas sp. USM 4-55 PHA synthase 1 (PhaC1P.sp USM 4-55).
Crystal structure of truncated haemoglobin from an extremely thermophilic and acidophilic bacterium

Jamil F, Teh AH, Schadich E, Saito JA, Najimudin N, Alam M

J. Biochem., 2014 Aug;156(2):97-106.
PMID: 24733432 DOI: 10.1093/jb/mvu023

A truncated haemoglobin (tHb) has been identified in an acidophilic and thermophilic methanotroph Methylacidiphilium infernorum. Hell's Gate Globin IV (HGbIV) and its related tHbs differ from all other bacterial tHbs due to their distinctively large sequence and polar distal haem pocket residues. Here we report the crystal structure of HGbIV determined at 1.96 Å resolution. The HGbIV structure has the distinctive 2/2 α-helical structure with extensions at both termini. It has a large distal site cavity in the haem pocket surrounded by four polar residues: His70(B9), His71(B10), Ser97(E11) and Trp137(G8). This cavity can bind bulky ligands such as a phosphate ion. Conformational shifts of His71(B10), Leu90(E4) and Leu93(E7) can also provide more space to accommodate larger ligands than the phosphate ion. The entrance/exit of such bulky ligands might be facilitated by positional flexibility in the CD1 loop, E helix and haem-propionate A. Therefore, the large cavity in HGbIV with polar His70(B9) and His71(B10), in contrast to the distal sites of other bacterial tHbs surrounded by non-polar residues, suggests its distinct physiological functions.
Construction and characterization of a Burkholderia pseudomallei wzm deletion mutant

Yuen CW, Ong EB, Mohamad S, Manaf UA, Najimudin N

J Microbiol Biotechnol, 2012 Oct;22(10):1336-42.
PMID: 23075783

In Burkholderia pseudomallei, the pathogen that causes melioidosis, the gene cluster encoding the capsular polysaccharide, is located on chromosome 1. Among the 19 capsular genes in this cluster, wzm has not been thoroughly studied. To study the function of wzm, we generated a deletion mutant and compared it with the wild-type strain. The mutant produced less biofilm in minimal media and was more sensitive to desiccation and oxidative stress compared with the wild-type strain, indicating that wzm is involved in biofilm formation and membrane integrity. Scanning electron microscopy showed that the bacterial cells of the mutant strain have more defined surfaces with indentations, whereas cells of the wild-type strain do not.
Fulltext Complete Genome Sequence of Salmonella enterica subsp. enterica Serovar Typhi Isolate B/SF/13/03/195 Associated with a Typhoid Carrier in Pasir Mas, Kelantan, Malaysia

Muhamad Harish S, Sim KS, Mohd Nor F, Mat Hussin H, Hamzah WM, Najimudin N, et al.

Genome Announc, 2015;3(6).
PMID: 26564035 DOI: 10.1128/genomeA.01285-15

We report here the complete genome sequence of Salmonella enterica subsp. enterica serovar Typhi B/SF/13/03/195 obtained from a typhoid carrier, who is a food handler in Pasir Mas, Kelantan.
Fulltext Genome Sequence of a Gram-Positive Diazotroph, Paenibacillus durus Type Strain ATCC 35681

Halim MA, Rahman AY, Sim KS, Yam HC, Rahim AA, Ghazali AH, et al.

Genome Announc, 2016;4(1).
PMID: 26893411 DOI: 10.1128/genomeA.00005-16

Here, we report the complete genome sequence of Paenibacillus durus type strain ATCC 35681, which can fix atmospheric nitrogen even in the presence of nitrate.
Production of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Erwinia sp. USMI-20

Majid MI, Akmal DH, Few LL, Agustien A, Toh MS, Samian MR, et al.

Int J Biol Macromol, 1999 Jun-Jul;25(1-3):95-104.
PMID: 10416655

A locally isolated soil microorganism identified as Erwinia sp. USMI-20 has been found to produce poly(3-hydroxybutyrate), P(3HB), from either palm oil or glucose and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), from a combination of palm oil and a second carbon source of either one of the following compounds: propionic acid, n-propanol, valeric acid and n-pentanol. It was found that Erwinia sp. USMI-20 could produce P(3HB) up to 69 wt.% polymer content with a dry cell weight of 4.4 g/l from an initial amount of 14.5 g/l of glucose followed by a feeding rate of glucose at 0.48 g/h glucose. On the other hand, the bacteria can achieve 46 wt.% of P(3HB) and a dry cell weight of 3.6 g/l from a batch fermentation in a 10-l fermentor from an initial concentration of 4.6 g/l of palm oil. Further characterisation of the polymer production was also carried out by using different types of palm oil. Among the different palm oils that were used, crude palm oil was the best lipid source for P(3HB) production as compared to palm olein and palm kernel oil. In the production of the copolymer, P(3HB-co-3HV), the highest mole fraction of 3-HV units could be as high as 47 mol% from a single feeding of valeric acid upon initial growth on palm oil.
Validation of a Burkholderia pseudomallei hypothetical protein and determination of its translational start codon using chromosomal integration of His-Tag coding sequence

Yam H, Abdul Rahim A, Gim Luan O, Samian R, Abdul Manaf U, Mohamad S, et al.

Protein J, 2012 Mar;31(3):246-9.
PMID: 22354666 DOI: 10.1007/s10930-012-9398-5

In this post genomic era, there are a great number of in silico annotated hypothetical genes. However, experimental validation of the functionality of these genes remains tentative. Two of the major challenges faced by researcher are whether these hypothetical genes are protein-coding genes and whether their corresponding predicted translational start codons are correct. In this report, we demonstrate a convenient procedure to validate the presence of a hypothetical gene product of BPSS1356 from Burkholderia pseudomallei as well as its start codon. It was done by integration of a His-Tag coding sequence into C-terminal end of BPSS1356 gene via homologous recombination. We then purified the native protein using affinity chromatography. The genuine start codon of BPSS1356 was then determined by protein N-terminal sequencing.
Fulltext Draft Genome Sequence of Flavobacterium sp. Strain PL002, Isolated from Antarctic Porphyra Algae

Teoh CP, Lavin P, Najimudin N, Lee PC, Iancu L, Purcarea C, et al.

Microbiol Resour Announc, 2021 Feb 25;10(8).
PMID: 33632855 DOI: 10.1128/MRA.00063-21

Here, we report the draft genome sequence of Flavobacterium sp. strain PL002, isolated from Antarctic Porphyra algae. The 4,299,965-bp genome sequence is assembled into 170 contigs, has 32.92% GC content, and 3,734 predicted genes.
Salmonella enterica serovar Typhi cutF is upregulated during environmental stress

Lau KL, Ong EB, Zainudin ZF, Samian MR, Ismail A, Najimudin N

J Gen Appl Microbiol, 2013;59(3):239-44.
PMID: 23863294
Fulltext Draft Genome Sequence of Plant Growth-Promoting Rhizobacterium Burkholderia sp. Strain USMB20, Isolated from Nodules of Mucuna bracteata

Aziz NA, Shaffie S, Rahman AYA, Hokchai Y, Najimudin N, Ghazali AHA

Microbiol Resour Announc, 2021 Mar 18;10(11).
PMID: 33737365 DOI: 10.1128/MRA.01051-20

Burkholderia sp. strain USMB20 is a plant growth-promoting rhizobacterium that was isolated from nodules of the leguminous cover crop Mucuna bracteata. The draft genome sequence of Burkholderia sp. strain USMB20 has an assembly size of 7.7 Mbp in 26 contigs with a GC content of 66.88%.
Transcriptional analysis of nitrogen fixation in Paenibacillus durus during growth in nitrogen-enriched medium

Halim MA, Choo QC, Ghazali AHA, Wajidi MFF, Najimudin N

Lett Appl Microbiol, 2021 May;72(5):610-618.
PMID: 33525052 DOI: 10.1111/lam.13455

Paenibacillus durus strain ATCC 35681T is a Gram-positive diazotroph that displayed capability of fixing nitrogen even in the presence of nitrate or ammonium. However, the nitrogen fixation activity was detected only at day 1 of growth when cultured in liquid nitrogen-enriched medium. The transcripts of all the nifH homologues were present throughout the 9-day study. When grown in nitrogen-depleted medium, nitrogenase activities occurred from day 1 until day 6 and the nifH transcripts were also present during the course of the study albeit at different levels. In both studies, the absence of nitrogen fixation activity regardless of the presence of the nifH transcripts raised the possibility of a post-transcriptional or post-translational regulation of the system. A putative SigA box sequence was found upstream of the transcription start site of nifB1, the first gene in the major nitrogen fixation cluster. The upstream region of nifB2 showed a promoter recognizable by SigE, a sigma factor normally involved in sporulation.
Cloning and characterization of poly(3-hydroxybutyrate) biosynthesis genes from Pseudomonas sp. USM 4-55

Tan Y, Neo PC, Najimudin N, Sudesh K, Muhammad TS, Othman AS, et al.

J Basic Microbiol, 2010 Apr;50(2):179-89.
PMID: 20082371 DOI: 10.1002/jobm.200900138

Pseudomonas sp. USM 4-55 is a locally isolated bacterium that possesses the ability to produce polyhydroxyalkanoates (PHA) consisting of both poly(3-hydroxybutyrate) [P(3HB)] homopolymer and medium-chain length (mcl) monomers (6 to 14 carbon atoms) when sugars or fatty acids are utilized as the sole carbon source. In this study, the P(3HB) biosynthesis operon carrying the phbC(Ps) P(3HB) synthase was successfully cloned and sequenced using a homologous probe. Three open reading frames encoding NADPH-dependent acetoacetyl-coenzyme A reductase (PhbB(Ps)), beta-ketothiolase (PhbA(Ps)) and P(3HB) synthase (PhbC(Ps)) were found in the phb operon. The genetic organization of phb operon showed a putative promoter region, followed by phbB(Ps)-phbA(Ps)-phbC(Ps). phbR(Ps)which encoded a putative transcriptional activator was located in the opposite orientation, upstream of phbBAC(Ps). Heterologous expression of pGEM''ABex harboring phbC(Ps) in Escherichia coli JM109 resulted in P(3HB) accumulation of up to 40% of dry cell weight (DCW).
Crystal structure of a compact α-amylase from Geobacillus thermoleovorans

Mok SC, Teh AH, Saito JA, Najimudin N, Alam M

Enzyme Microb Technol, 2013 Jun 10;53(1):46-54.
PMID: 23683704 DOI: 10.1016/j.enzmictec.2013.03.009

A truncated form of an α-amylase, GTA, from thermophilic Geobacillus thermoleovorans CCB_US3_UF5 was biochemically and structurally characterized. The recombinant GTA, which lacked both the N- and C-terminal transmembrane regions, functioned optimally at 70°C and pH 6.0. While enzyme activity was not enhanced by the addition of CaCl2, GTA's thermostability was significantly improved in the presence of CaCl2. The structure, in complex with an acarbose-derived pseudo-hexasaccharide, consists of the typical three domains and binds one Ca(2+) ion. This Ca(2+) ion was strongly bound and not chelated by EDTA. A predicted second Ca(2+)-binding site, however, was disordered. With limited subsites, two novel substrate-binding residues, Y147 and Y182, may help increase substrate affinity. No distinct starch-binding domain is present, although two regions rich in aromatic residues have been observed. GTA, with a smaller domain B and several shorter loops compared to other α-amylases, has one of the most compact α-amylase folds that may contribute greatly to its tight Ca(2+) binding and thermostability.
Fulltext Hypothetical Protein BPSL3393 of Burkholderia pseudomallei is Involved in Ethanolamine Catabolism

Luan OG, Yam H, Samian R, Wajidi MFF, Mahadi NM, Mohamad S, et al.

Trop Life Sci Res, 2017 Jul;28(2):57-74.
PMID: 28890761 MyJurnal DOI: 10.21315/tlsr2017.28.2.5

Burkholderia pseudomallei is a soil-dwelling bacterium that causes a globally emerging disease called melioidosis. Approximately one third of the in silico annotated genes in its genome are classified as hypothetical genes. This group of genes is difficult to be functionally characterised partly due to the absence of noticeable phenotypes under conventional laboratory settings. A bioinformatic survey of hypothetical genes revealed a gene designated as BPSL3393 that putatively encodes a small protein of 11 kDA with a CoA binding domain. BPSL3393 is conserved in all the B. pseudomallei genomes as well as various in other species within the genus Burkholderia. Taking into consideration that CoA plays a ubiquitous metabolic role in all life forms, characterisation of BPSL3393 may uncover a previously over-looked metabolic feature of B. pseudomallei. The gene was deleted from the genome using a double homologous recombination approach yielding a null mutant. The BPSL3393 mutant showed no difference in growth rate with the wild type under rich and minimal growth conditions. An extensive metabolic phenotyping test was performed involving 95 metabolic substrates. The deletion mutant of BPSL3393 was severely impaired in its ethanolamine metabolism. The growth rate of the mutant was attenuated when ethanolamine was used as the sole carbon source. A transcriptional analysis of the ethanolamine metabolism genes showed that they were down-regulated in the BPSL3393 mutant. This seemed to suggest that BPSL3393 functions as a positive regulator for ethanolamine metabolism.
Ethyl caffeate ameliorated amyloid-beta42 protein-associated toxicity in PC12 cells and Drosophila melanogaster

Tan FHP, Hadri NAB, Najimudin N, Watanabe N, Azzam G

Geriatr Gerontol Int, 2021 Dec;21(12):1125-1130.
PMID: 34699118 DOI: 10.1111/ggi.14296

AIM: Alzheimer's disease (AD) is the most pervasive neurodegenerative disorder in societies globally. Till now, the mechanism behind this disease is still equivocal. Amyloid-beta42 protein (Aβ42), the most toxic and aggressive Aβ species, is the main focus of this study. The naturally occurring ethyl caffeate (EC) is associated with various medicinal properties. Here, EC was tested for its protective properties against Aβ42's toxic effects.
METHODS: As treatment of Aβ42 has been shown to cause neuronal cell death, EC was first screened with Aβ42-incubated PC12 neuronal cells. Next, the compound was tested on the Drosophila melanogaster AD model using the rough eye phenotype assay, lifespan assay and negative geotaxis assay.
RESULTS: EC ameliorated PC12 cells from cell death linked to Aβ42 exposure. Using Drosophila expressing human Aβ42, feeding of EC was able to partially rescue the rough eye phenotype, lengthen the lifespan of AD Drosophila and enhanced the mobility of middle-aged AD Drosophila.
CONCLUSION: Overall, the results of this study showed that EC might possess therapeutic properties for AD. Geriatr Gerontol Int 2021; 21: 1125-1130.
Alleviatory effects of Danshen, Salvianolic acid A and Salvianolic acid B on PC12 neuronal cells and Drosophila melanogaster model of Alzheimer's disease

Tan FHP, Ting ACJ, Leow BG, Najimudin N, Watanabe N, Azzam G

J Ethnopharmacol, 2021 Oct 28;279:114389.
PMID: 34217797 DOI: 10.1016/j.jep.2021.114389

ETHNOPHARMACOLOGICAL RELEVANCE: Danshen water extract (DWE), obtained from the Salvia miltiorrhiza Bunge (Family Lamiaceae) root, is usually employed in Chinese traditional medicine as treatment to cardiovascular ailments and cerebrovascular diseases. Intriguingly, the extract was also found to contain vast beneficial properties in Alzheimer's disease (AD) treatment.
AIM OF THE STUDY: Alzheimer's disease is the most significant type of neurodegenerative disorder plaguing societies globally. Its pathogenesis encompasses the hallmark aggregation of amyloid-beta (Aβ). Of all the Aβ oligomers formed in the brain, Aβ42 is the most toxic and aggressive. Despite this, the mechanism behind this disease remains elusive. In this study, DWE, and its major components, Salvianolic acid A (SalA) and Salvianolic acid B (SalB) were tested for their abilities to attenuate Aβ42's toxic effects.
METHODS: The composition of DWE was determined via Ultra-Performance Liquid Chromatography (UPLC). DWE, SalA and SalB were first verified for their capability to diminish Aβ42 fibrillation using an in vitro activity assay. Since Aβ42 aggregation results in neuronal degeneration, the potential Aβ42 inhibitors were next evaluated on Aβ42-exposed PC12 neuronal cells. The Drosophila melanogaster AD model was then employed to determine the effects of DWE, SalA and SalB.
RESULTS: DWE, SalA and SalB were shown to be able to reduce fibrillation of Aβ42. When tested on PC12 neuronal cells, DWE, SalA and SalB ameliorated cells from cell death associated with Aβ42 exposure. Next, DWE and its components were tested on the Drosophila melanogaster AD model and their rescue effects were further characterized. The UPLC analysis showed that SalA and SalB were present in the brains and bodies of Drosophila after DWE feeding. When human Aβ42 was expressed, the AD Drosophila exhibited degenerated eye structures known as the rough eye phenotype (REP), reduced lifespan and deteriorated locomotor ability. Administration of DWE, SalA and SalB partially reverted the REP, increased the age of AD Drosophila and improved most of the mobility of AD Drosophila.
CONCLUSION: Collectively, DWE and its components may have therapeutic potential for AD patients and possibly other forms of brain diseases.
Characterization of P(3HB) from untreated raw palm oil mill effluent using Azotobacter vinelandii ΔAvin_16040 lacking S-layer protein

Liew PWY, Jong BC, Sudesh K, Najimudin N, Mok PS

World J Microbiol Biotechnol, 2023 Jan 06;39(3):68.
PMID: 36607449 DOI: 10.1007/s11274-022-03503-1

The production of poly(3-hydroxybutyrate) [P(3HB)] from untreated raw palm oil mill effluent (urPOME), the first wastewater discharge from crude palm oil extraction, is discussed. The mutant strain Azotobacter vinelandii ΔAvin_16040, which lacks the S-layer protein but has a better P(3HB) synthesis capability than the wild type strain ATCC 12,837, was chosen for this study. UrPOME substrate, with high biological oxygen demand (BOD), chemical oxygen demand (COD) and suspended solids, was used without pre-treatment. DSMZ-Azotobacter medium which was devoid of laboratory sugar(s) was used as the basal medium (BaM). Initially, Azotobacter vinelandii ΔAvin_16040 generated 325.5, 1496.3, and 1465.7 mg L-1 of P(3HB) from BaM with 20% urPOME, 2BaM with 20% urPOME and 20 g L-1 sucrose, and 2BaM with 20% urPOME and 2 mL L-1 glycerol, respectively. P(3HB) generation was enhanced by nearly tenfold using statistical optimization, resulting in 13.9 g L-1. Moreover, the optimization reduced the compositions of mineral salts and sugar in the medium by 48 and 97%, respectively. The urPOME-based P(3HB) product developed a yellow coloration most possibly attributed to the aromatic phenolics content in urPOME. Despite the fact that both were synthesised by ΔAvin_16040, thin films of urPOME-based P(3HB) had superior crystallinity and tensile strength than P(3HB) produced only on sucrose. When treated with 10 and 50 kGy of electron beam irradiation, these P(3HB) scissioned to half and one-tenth of their original molecular weights, respectively, and these cleavaged products could serve as useful base units for specific polymer structure construction.
3-[[(3S)-1,2,3,4-Tetrahydroisoquinoline-3-Carbonyl]Amino]Propanoic Acid (THICAPA) Is Protective Against Aβ42-Induced Toxicity In Vitro and in an Alzheimer's Disease Drosophila

Tan FHP, Ting ACJ, Najimudin N, Watanabe N, Shamsuddin S, Zainuddin A, et al.

J Gerontol A Biol Sci Med Sci, 2023 Oct 28;78(11):1944-1952.
PMID: 37453137 DOI: 10.1093/gerona/glad169

Alzheimer's disease (AD) is the most prevalent type of dementia globally. The accumulation of amyloid-beta (Aβ) extracellular senile plaques in the brain is one of the hallmark mechanisms found in AD. Aβ42 is the most damaging and aggressively aggregating Aβ isomer produced in the brain. Although Aβ42 has been extensively researched as a crucial peptide connected to the development of the characteristic amyloid fibrils in AD, the specifics of its pathophysiology are still unknown. Therefore, the main objective was to identify novel compounds that could potentially mitigate the negative effects of Aβ42. 3-[[(3S)-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl]amino]propanoic acid (THICAPA) was identified as a ligand for Aβ42 and for reducing fibrillary Aβ42 aggregation. THICAPA also improved cell viability when administered to PC12 neuronal cells that were exposed to Aβ42. Additionally, this compound diminished Aβ42 toxicity in the current AD Drosophila model by rescuing the rough eye phenotype, prolonging the life span, and enhancing motor functions. Through next-generation RNA-sequencing, immune response pathways were downregulated in response to THICAPA treatment. Thus, this study suggests THICAPA as a possible disease-modifying treatment for AD.

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