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A MALDI TOF MS-based minisequencing method for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains of Enterobacteriaceae

Ikryannikova LN, Shitikov EA, Zhivankova DG, Il'ina EN, Edelstein MV, Govorun VM

J Microbiol Methods, 2008 Dec;75(3):385-91.
PMID: 18694787 DOI: 10.1016/j.mimet.2008.07.005

A minisequencing method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was developed for rapid identification of single nucleotide polymorphisms at bla(TEM) gene codons 104, 164 and 238 associated with extended-spectrum activity on TEM-type beta-lactamases. The method was validated by testing the Escherichia coli and Klebsiella pneumoniae strains possessing the known bla(TEM) gene sequences.

Matched MeSH terms: Bacterial Proteins/genetics*
A genome level survey of Burkholderia pseudomallei immunome expressed during human infection

Su YC, Wan KL, Mohamed R, Nathan S

Microbes Infect., 2008 Oct;10(12-13):1335-45.
PMID: 18761419 DOI: 10.1016/j.micinf.2008.07.034

Burkholderia pseudomallei is the etiological agent of melioidosis, a severe infectious disease of humans and animals. The role of the bacterium's proteins expressed in vivo during human melioidosis continues to remain an enigma. This study's aim was to identify B. pseudomallei target proteins that elicit the humoral immune response in infected humans. A small insert genomic expression library was constructed and immunoscreened to identify peptides that reacted exclusively with melioidosis patients' sera. Sero-positive clones expressing immunogenic peptides were sequenced and annotated, and shown to represent 109 proteins involved in bacterial cell envelope biogenesis, cell motility and secretion, transcription, amino acid, ion and protein metabolism, energy production, DNA repair and unknown hypothetical proteins. Western blot analysis of three randomly selected full-length immunogenic polypeptides with patients' sera verified the findings of the immunome screening. The patients' humoral immune response to the 109 proteins suggests the induction or significant upregulation of these proteins in vivo during human infection and thus may play a role in the pathogenesis of B. pseudomallei. Identification of B. pseudomallei immunogens has shed new light on the elucidation of the bacterium's pathogenesis mechanism and disease severity. These immunogens can be further evaluated as prophylactic and serodiagnostic candidates as well as drug targets.

Matched MeSH terms: Bacterial Proteins/genetics
A mutation in anti-sigma factor MAB_3542c may be responsible for tigecycline resistance in Mycobacterium abscessus

Ng HF, Tan JL, Zin T, Yap SF, Ngeow YF

J Med Microbiol, 2018 Dec;67(12):1676-1681.
PMID: 30351265 DOI: 10.1099/jmm.0.000857

In this study, we characterized 7C, a spontaneous mutant selected from tigecycline-susceptible Mycobacterium abscessus ATCC 19977. Whole-genome sequencing (WGS) was used to identify possible resistance determinants in this mutant. Compared to the wild-type, 7C demonstrated resistance to tigecycline as well as cross-resistance to imipenem, and had a slightly retarded growth rate. WGS and subsequent biological verifications showed that these phenotypes were caused by a point mutation in MAB_3542c, which encodes an RshA-like protein. In Mycobacterium tuberculosis, RshA is an anti-sigma factor that negatively regulates the heat/oxidative stress response mechanisms. The MAB_3542c mutation may represent a novel determinant of tigecycline resistance. We hypothesize that this mutation may dysregulate the stress-response pathways which have been shown to be linked to antibiotic resistance in previous studies.

Matched MeSH terms: Bacterial Proteins/genetics
A new cold-adapted, organic solvent stable lipase from mesophilic Staphylococcus epidermidis AT2

Kamarudin NH, Rahman RN, Ali MS, Leow TC, Basri M, Salleh AB

Protein J, 2014 Jun;33(3):296-307.
PMID: 24777627 DOI: 10.1007/s10930-014-9560-3

The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.

Matched MeSH terms: Bacterial Proteins/genetics
A new organic solvent tolerant protease from Bacillus pumilus 115b

Rahman RN, Mahamad S, Salleh AB, Basri M

J Ind Microbiol Biotechnol, 2007 Jul;34(7):509-17.
PMID: 17492323

Five out of the nine benzene-toulene-ethylbenzene-xylene (BTEX) tolerant bacteria that demonstrated high protease activity on skim milk agar were isolated. Among them, isolate 115b identified as Bacillus pumilus exhibited the highest protease production. The protease produced was stable in 25% (v/v) benzene and toluene and it was activated 1.7 and 2.5- fold by n-dodecane and n-tetradecane, respectively. The gene encoding the organic solvent tolerant protease was cloned and its nucleotide sequence determined. Sequence analysis revealed an open reading frame (ORF) of 1,149 bp that encoded a polypeptide of 383 amino acid residues. The polypeptide composed of 29 residues of signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids with a calculated molecular mass of 27,846 Da. This is the only report available to date on organic solvent tolerant protease from B. pumilus.

Matched MeSH terms: Bacterial Proteins/genetics
A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene

Sulong MR, Abdul Rahman RN, Salleh AB, Basri M

Protein Expr. Purif., 2006 Oct;49(2):190-5.
PMID: 16769222

An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity.

Matched MeSH terms: Bacterial Proteins/genetics
Fulltext A persistent antimicrobial resistance pattern and limited methicillin-resistance-associated genotype in a short-term Staphylococcus aureus carriage isolated from a student population

Mat Azis N, Pung HP, Abdul Rachman AR, Amin Nordin S, Sarchio SNE, Suhaili Z, et al.

J Infect Public Health, 2017 Mar-Apr;10(2):156-164.
PMID: 27033676 DOI: 10.1016/j.jiph.2016.02.013

The aim of the present study was to assess and compare the antimicrobial susceptibility pattern against a panel of antibiotics and molecular and methicillin resistance-associated genotypes of 120 carriage S. aureus isolates previously isolated from a student population at two isolation events within a one-month interval. The antibiotic susceptibility of isolates was determined using the Kirby-Bauer disc-diffusion method (cefoxitin by Etest). The MRSA was screened using polymerase chain reaction for the presence of the mecA gene. The mecA-positive isolates were subjected to staphylococcal cassette chromosome (SCC) mec typing, multilocus sequence typing (MLST) and eBURST analysis. All isolates were characterized for the presence of the Panton-Valentine leukocidin (PVL) gene, an enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) pattern and the spa type. For the two occasions where S. aureus was isolated, the highest frequency of resistance was observed for penicillin (70% and 65%, respectively), with a lower rate against erythromycin and tetracycline (<12%). All isolates were susceptible to ciprofloxacin and gentamycin. As for methicillin resistance, eight isolates had minimum inhibitory concentrations (MIC) of resistant categories, but 10 isolates (8.33%) were positive for the mecA gene. The mecA-positive isolates belonged to SCCmec types I (n=9) and V (n=1). MLST was resolved for only three MRSAs, ST508 (n=1), ST88 (n=1) and ST96 (n=1). The results of the eBURST analysis showed that the MRSA isolates analyzed in the present study were potentially related to MRSA identified in other countries. Approximately half of the persistent S. aureus carriers harbored S. aureus of a similar spa type in the respective individuals during both isolation events. A persistent antimicrobial pattern and limited distinct MRSAs were observed over the short study period. The latter frequently exhibited SCCmec type I, commonly associated with hospital-acquired (HA) characteristics, but further delineation is needed to justify the origins of these bacteria.

Matched MeSH terms: Bacterial Proteins/genetics
Fulltext A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity

Ooi JD, Jiang JH, Eggenhuizen PJ, Chua LL, van Timmeren M, Loh KL, et al.

Nat Commun, 2019 07 29;10(1):3392.
PMID: 31358739 DOI: 10.1038/s41467-019-11255-0

Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.

Matched MeSH terms: Bacterial Proteins/genetics
Fulltext A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species

Liew PS, Teh CS, Lau YL, Thong KL

Trop Biomed, 2014 Dec;31(4):709-20.
PMID: 25776596 MyJurnal

Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

Matched MeSH terms: Bacterial Proteins/genetics
A single-gene approach for the subspecies classification of Mycobacteroides abscessus

Ng HF, Ngeow YF

Pathog Dis, 2020 11 11;78(8).
PMID: 32945880 DOI: 10.1093/femspd/ftaa055

The subspecies classification of Mycobacteroides abscessus complex into M. abscessus, M. massiliense and M. bolletii requires the amplification and sequencing of multiple genes. The objective of this study was to evaluate the possibility of subspecies classification using a single PCR target. An in silico study was performed to classify 1613 strains deposited in a public database using 9 genes (partial gene sequences of hsp65, rpoB, sodA, argH, cya, glpK, gnd, and murC, and the full gene sequence of MAB_3542c). We found the housekeeping gene gnd to be able to classify the M. abscessus subspecies with high accuracy (99.94%). A single-gene PCR approach based on gnd would be a suitable replacement for the more expensive, labor-intensive and time-consuming multi-gene PCR analysis currently in use for the subspecies identification of M. abscessus.

Matched MeSH terms: Bacterial Proteins/genetics
A stop-gain mutation in sigma factor SigH (MAB_3543c) may be associated with tigecycline resistance in Mycobacteroides abscessus

Lee CL, Ng HF, Ngeow YF, Thaw Z

J Med Microbiol, 2021 Jul;70(7).
PMID: 34236301 DOI: 10.1099/jmm.0.001378

Introduction. Tigecycline is currently acknowledged to be one of the most effective antibiotics against infections caused by Mycobacteroides abscessus.Gap statement. The genetic determinants of tigecycline resistance in M. abscessus are not well understood.Aim. In this study, we characterized a tigecycline-resistant M. abscessus mutant, designated CL7, to identify the potential resistance mechanism.Methodology. CL7 was characterized using antimicrobial susceptibility testing, whole-genome sequencing, PCR and RT-qPCR. For biological verification, gene overexpression assays were carried out.Results. Whole-genome sequencing and the subsequent gene overexpression assays showed that CL7 harboured a stop-gain mutation in MAB_3543 c, which may be responsible for the tigecycline resistance phenotype. This gene encodes an orthologue of SigH, which is involved in the positive regulation of physiological stress response and is negatively regulated by the RshA anti-sigma factor in Mycobacterium tuberculosis. We hypothesized that the MAB_3543 c mutation may disrupt the interaction between SigH and RshA (MAB_3542 c). RT-qPCR analyses revealed the upregulation of MAB_3543 c and other key stress response genes, which has previously been shown to be a hallmark of SigH-RshA bond disruption and tigecycline resistance.Conclusion. The MAB_3543c mutation may represent a novel determinant of tigecycline resistance in M. abscessus. The findings of this study will hopefully contribute to our knowledge of potential tigecycline resistance mechanisms in M. abscessus, which may lead to better diagnostics and treatment modalities in the future.

Matched MeSH terms: Bacterial Proteins/genetics*
Fulltext Acinetobacter baumannii in suspected bacterial infections: Association between multidrug resistance, virulence genes, & biofilm production

Gautam D, Dolma KG, Khandelwal B, Goyal RK, Mitsuwan W, Pereira MLG, et al.

Indian J Med Res, 2023 Oct 01;158(4):439-446.
PMID: 38006347 DOI: 10.4103/ijmr.ijmr_3470_21

BACKGROUND OBJECTIVES: Acinetobacter baumannii has emerged as a nosocomial pathogen with a tendency of high antibiotic resistance and biofilm production. This study aimed to determine the occurrence of A. baumannii from different clinical specimens of suspected bacterial infections and furthermore to see the association of biofilm production with multidrug resistance and expression of virulence factor genes in A. baumannii.
METHODS: A. baumannii was confirmed in clinical specimens by the detection of the blaOXA-51-like gene. Biofilm production was tested by microtitre plate assay and virulence genes were detected by real-time PCR.
RESULTS: A. baumannii was isolated from a total of 307 clinical specimens. The isolate which showed the highest number of A. baumannii was an endotracheal tube specimen (44.95%), then sputum (19.54%), followed by pus (17.26%), urine (7.49%) and blood (5.86%), and <2 per cent from body fluids, catheter-tips and urogenital specimens. A resistance rate of 70-81.43 per cent against all antibiotics tested, except colistin and tigecycline, was noted, and 242 (78.82%) isolates were multidrug-resistant (MDR). Biofilm was detected in 205 (66.78%) with a distribution of 54.1 per cent weak, 10.42 per cent medium and 2.28 per cent strong biofilms. 71.07 per cent of MDR isolates produce biofilm (P<0.05). Amongst virulence factor genes, 281 (91.53%) outer membrane protein A (OmpA) and 98 (31.92%) biofilm-associated protein (Bap) were detected. Amongst 100 carbapenem-resistant A. baumannii, the blaOXA-23-like gene was predominant (96%), the blaOXA-58-like gene (6%) and none harboured the blaOXA-24-like gene. The metallo-β-lactamase genes blaIMP-1 (4%) and blaVIM-1(8%) were detected, and 76 per cent showed the insertion sequence ISAba1.
INTERPRETATION CONCLUSIONS: The majority of isolates studied were from lower respiratory tract specimens. The high MDR rate and its positive association with biofilm formation indicate the nosocomial distribution of A. baumannii. The biofilm formation and the presence of Bap were not interrelated, indicating that biofilm formation was not regulated by a single factor. The MDR rate and the presence of OmpA and Bap showed a positive association (P<0.05). The isolates co-harbouring different carbapenem resistance genes were the predominant biofilm producers, which will seriously limit the therapeutic options suggesting the need for strict antimicrobial stewardship and molecular surveillance in hospitals.

Matched MeSH terms: Bacterial Proteins/genetics
Activated ADI pathway: the initiator of intermediate vancomycin resistance in Staphylococcus aureus

Tan XE, Neoh HM, Looi ML, Chin SF, Cui L, Hiramatsu K, et al.

Can J Microbiol, 2017 Mar;63(3):260-264.
PMID: 28059579 DOI: 10.1139/cjm-2016-0439

Comparative proteomic profiling between 2 vancomycin-intermediate Staphylococcus aureus (VISA) strains, Mu50Ω-vraSm and Mu50Ω-vraSm-graRm, and vancomycin-susceptible S. aureus (VSSA) strain Mu50Ω revealed upregulated levels of catabolic ornithine carbamoyltransferase (ArcB) of the arginine catabolism pathway in VISA strains. Subsequent analyses showed that the VISA strains have higher levels of cellular ATP and ammonia, which are by-products of arginine catabolism, and displayed thicker cell walls. We postulate that elevated cytoplasmic ammonia and ATP molecules, resulting from activated arginine catabolism upon acquisition of vraS and graR mutations, are important requirements facilitating cell wall biosynthesis, thereby contributing to thickened cell wall and consequently reduced vancomycin susceptibility in VISA strains.

Matched MeSH terms: Bacterial Proteins/genetics
Fulltext AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp

See-Too WS, Ee R, Lim YL, Convey P, Pearce DA, Yin WF, et al.

Sci Rep, 2017 02 22;7:42968.
PMID: 28225085 DOI: 10.1038/srep42968

Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15T, isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions.

Matched MeSH terms: Bacterial Proteins/genetics*
Alcanivorax hongdengensis sp. nov., an alkane-degrading bacterium isolated from surface seawater of the straits of Malacca and Singapore, producing a lipopeptide as its biosurfactant

Wu Y, Lai Q, Zhou Z, Qiao N, Liu C, Shao Z

Int J Syst Evol Microbiol, 2009 Jun;59(Pt 6):1474-9.
PMID: 19502338 DOI: 10.1099/ijs.0.001552-0

A taxonomic study was carried out on strain A-11-3(T), which was isolated from an oil-enriched consortia from the surface seawater of Hong-Deng dock in the Straits of Malacca and Singapore. Cells were aerobic, Gram-negative, non-spore-forming irregular rods. The strain was catalase- and oxidase-negative. It grew on a restricted spectrum of organic compounds, including some organic acids and alkanes. 16S rRNA gene sequence comparisons showed that strain A-11-3(T) was most closely related to the type strains of Alcanivorax jadensis (96.8 % sequence similarity), Alcanivorax borkumensis (96.8 %), Alcanivorax dieselolei (94.8 %), Alcanivorax venustensis (94.2 %) and Alcanivorax balearicus (94.0 %). The predominant fatty acids were C(16 : 0) (31.2 %), C(18 : 1)omega7c (24.8 %), C(18 : 0) (9.6 %), C(12 : 0) (8.3 %), C(16 : 1)omega7c (8.3 %) and C(16 : 0) 3-OH (5.1 %). The G+C content of the genomic DNA was 54.7 mol%. Moreover, the strain produced lipopeptides as its surface-active compounds. According to physiological and biochemical tests, DNA-DNA hybridization results and sequence comparisons of the 16S-23S internal transcribed spacer, the gyrB gene and the alkane hydroxylase gene alkB1, strain A-11-3(T) was affiliated with the genus Alcanivorax but could be readily distinguished from recognized Alcanivorax species. Therefore strain A-11-3(T) represents a novel species of the genus Alcanivorax for which the name Alcanivorax hongdengensis sp. nov. is proposed. The type strain is A-11-3(T) (=CGMCC 1.7084(T)=LMG 24624(T)=MCCC 1A01496(T)).

Matched MeSH terms: Bacterial Proteins/genetics
Alteration in apyrase enzyme attenuated virulence of Shigella flexneri

BangaSingh KK, Nisha M, Lau HY, Ravichandran M, Salleh MZ

Microb Pathog, 2016 Feb;91:123-8.
PMID: 26706344 DOI: 10.1016/j.micpath.2015.12.004

Virulence of Shigella is attributed to the genes presence in chromosome or in the megaplasmid. The apy gene which is located in the megaplasmid of Shigella species encodes for apyrase enzyme, a pathogenesis-associated enzyme causing mitochondrial damage and host cell death. In this study we constructed an apy mutant of Shigella flexneri by insertional activation using a kanamycin resistant gene cassette. The wild type apy gene of S. flexneri 2a was PCR amplified, cloned and mutated with insertion of kanamycin resistant gene cassette (aphA). The mutated construct (apy: aphA) was subcloned into a conjugative suicidal vector (pWM91) at the unique Sma1 and Sac1 sites. The mutation of the wild apy gene in the construct was confirmed by DNA sequencing. The mutated construct was introduced into wild type S. flexneri 2a by conjugation with Escherichia coli. After undergoing homologous recombination, the wild apy gene was deleted from the construct using the sucrose selection method. Non-functional activity of the apyrase enzyme in the constructed strain by colorimetric test indicated the successful mutation of the apyrase enzyme. This strain with mutated apy gene was evaluated for its protective efficacy using the guinea pig keratoconjunctivitis model. The strain was Sereny negative and it elicited a significant protection following challenge with wild S. flexneri strain. This apy mutant strain will form a base for the development of a vaccine target for shigellosis.

Matched MeSH terms: Bacterial Proteins/genetics
Fulltext Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Masomian M, Rahman RN, Salleh AB, Basri M

PLoS One, 2016;11(3):e0149851.
PMID: 26934700 DOI: 10.1371/journal.pone.0149851

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca(2+)-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65 °C and retained ≥ 97% activity after incubation at 50 °C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.

Matched MeSH terms: Bacterial Proteins/genetics*
Fulltext Analysis of anoxybacillus genomes from the aspects of lifestyle adaptations, prophage diversity, and carbohydrate metabolism

Goh KM, Gan HM, Chan KG, Chan GF, Shahar S, Chong CS, et al.

PLoS One, 2014;9(6):e90549.
PMID: 24603481 DOI: 10.1371/journal.pone.0090549

Species of Anoxybacillus are widespread in geothermal springs, manure, and milk-processing plants. The genus is composed of 22 species and two subspecies, but the relationship between its lifestyle and genome is little understood. In this study, two high-quality draft genomes were generated from Anoxybacillus spp. SK3-4 and DT3-1, isolated from Malaysian hot springs. De novo assembly and annotation were performed, followed by comparative genome analysis with the complete genome of Anoxybacillus flavithermus WK1 and two additional draft genomes, of A. flavithermus TNO-09.006 and A. kamchatkensis G10. The genomes of Anoxybacillus spp. are among the smaller of the family Bacillaceae. Despite having smaller genomes, their essential genes related to lifestyle adaptations at elevated temperature, extreme pH, and protection against ultraviolet are complete. Due to the presence of various competence proteins, Anoxybacillus spp. SK3-4 and DT3-1 are able to take up foreign DNA fragments, and some of these transferred genes are important for the survival of the cells. The analysis of intact putative prophage genomes shows that they are highly diversified. Based on the genome analysis using SEED, many of the annotated sequences are involved in carbohydrate metabolism. The presence of glycosyl hydrolases among the Anoxybacillus spp. was compared, and the potential applications of these unexplored enzymes are suggested here. This is the first study that compares Anoxybacillus genomes from the aspect of lifestyle adaptations, the capacity for horizontal gene transfer, and carbohydrate metabolism.

Matched MeSH terms: Bacterial Proteins/genetics
Analysis of differentially expressed proteins in late-stationary growth phase of Mycobacterium tuberculosis H37Rv

Ang KC, Ibrahim P, Gam LH

Biotechnol Appl Biochem, 2014 Mar-Apr;61(2):153-64.
PMID: 23826872 DOI: 10.1002/bab.1137

Mycobacterium tuberculosis is a causative agent of tuberculosis (TB). The ability of M. tuberculosis to be quiescent in the cell has caused the emergence of latent infection. A comprehensive proteomic analysis of M. tuberculosis H37Rv over three growth phases, namely mid-log (14-day culture), early stationary (28-day culture), and late stationary (50-day culture), was performed in order to study the change in proteome from the mid-log phase to late-stationary phase. Combination methods of two-dimensional electrophoresis (2-DE) and tandem mass spectrometry were used to generate proteome maps of M. tuberculosis at different growth phases. Ten proteins were detected differentially expressed in the late-stationary phase compared with the other two phases. These proteins were SucD, TrpD, and Rv2161c, which belong to metabolic pathway proteins; FadE5, AccD5, DesA1, and Rv1139c are proteins involved in cell wall or lipid biosynthesis, whereas TB21.7 and Rv3224 are conserved hypothetical proteins with unknown function. A surface antigen protein, DesA1, was not detectable in the late-stationary phase, although present in both log and early-stationary phases. The changes in the expression levels of these proteins were in line with the growth environment changes of the bacteria from mid-log phase to late-stationary phase. The information gathered may be valuable in the intervention against latent TB infection.

Matched MeSH terms: Bacterial Proteins/genetics
Analysis of lolB gene sequence and its use in the development of a PCR assay for the detection of Vibrio cholerae

Lalitha P, Siti Suraiya MN, Lim KL, Lee SY, Nur Haslindawaty AR, Chan YY, et al.

J Microbiol Methods, 2008 Sep;75(1):142-4.
PMID: 18579241 DOI: 10.1016/j.mimet.2008.05.001

A PCR assay has been developed based on a lolB (hemM) gene, which was found to be highly conserved among the Vibrio cholerae species but non-conserved among the other enteric bacteria. The lolB PCR detected all O1, O139 and non-O1/non-O139 serogroup and biotypes of V. cholerae. The analytical specificity of this assay was 100% while the analytical sensitivity was 10 pg/microL and 10(3) CFU/mL at DNA and bacterial level respectively. The diagnostic sensitivity and specificity was 98.5% and 100% respectively.

Matched MeSH terms: Bacterial Proteins/genetics*

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