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Fulltext Quorum sensing activity in Pandoraea pnomenusa RB38

Ee R, Lim YL, Kin LX, Yin WF, Chan KG

Sensors (Basel), 2014;14(6):10177-86.
PMID: 24919016 DOI: 10.3390/s140610177

Strain RB38 was recovered from a former dumping area in Malaysia. MALDI-TOF mass spectrometry and genomic analysis identified strain RB-38 as Pandoraea pnomenusa. Various biosensors confirmed its quorum sensing properties. High resolution triple quadrupole liquid chromatography-mass spectrometry analysis was subsequently used to characterize the N-acyl homoserine lactone production profile of P. pnomenusa strain RB38, which validated that this isolate produced N-octanoyl homoserine lactone as a quorum sensing molecule. This is the first report of the production of N-octanoyl homoserine lactone by P. pnomenusa strain RB38.
Chania multitudinisentens gen. nov., sp. nov., an N-acyl-homoserine-lactone-producing bacterium in the family Enterobacteriaceae isolated from landfill site soil

Ee R, Madhaiyan M, Ji L, Lim YL, Nor NM, Tee KK, et al.

Int J Syst Evol Microbiol, 2016 Jun;66(6):2297-2304.
PMID: 26978486 DOI: 10.1099/ijsem.0.001025

Phylogenetic and taxonomic characterization was performed for bacterium RB-25T, which was isolated from a soil sample collected in a former municipal landfill site in Puchong, Malaysia. Growth occurred at 20-37 °C at pH 5-8 but not in the presence of 9 % (w/v) NaCl or higher. The principal fatty acids were C16:0, C18:1ω7c and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). Ubiquinone-8 was the only isoprenoid quinone detected. Polar lipid analysis revealed the presence of phospholipid, phosphoaminolipid, phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminolipid. DNA G+C content was 50.9 mol% phylogenetic analysis based on 16S rRNA gene sequence showed that strain RB-25T formed a distinct lineage within the family Enterobacteriaceae of the class Gammaproteobacteria. It exhibited a low level of 16S rRNA gene sequence similarity with its phylogenetic neighbours Pantoea rwandensis LMG 26275T (96.6 %), Rahnella aquatilis CIP 78.65T (96.5 %), Pectobacterium betavasculorum ATCC 43762T (96.4 %), Pantoea rodasii LMG 26273T (96.3 %), Gibbsiella dentisursi NUM 1720T (96.3 %) and Serratia glossinae C1T (96.2 %). Multilocus sequence analyses based on fusA, pyrG, rplB, rpoB and sucA sequences showed a clear distinction of strain RB-25T from the most closely related genera. Isolate RB-25T could also be distinguished from members of these genera by a combination of the DNA G+C content, respiratory quinone system, fatty acid profile, polar lipid composition and other phenotypic features. Strain RB-25T represents a novel species of a new genus, for which the name Chaniamultitudinisentens gen. nov., sp. nov. is proposed. The type strain is RB-25T (=DSM 28811T=LMG 28304T).
Complete genome sequence of oxalate-degrading bacterium Pandoraea vervacti DSM 23571(T)

Ee R, Yong D, Lim YL, Yin WF, Chan KG

J Biotechnol, 2015 Jun 20;204:5-6.
PMID: 25848988 DOI: 10.1016/j.jbiotec.2015.03.020

Pandoraea vervacti DSM 23571(T) is an oxalate metabolizing bacterium isolated from an uncultivated field soil in Mugla, Turkey. Here, we present the first complete genome sequence of P. vervacti DSM 23571(T). A complete pathway for degradation of oxalate was revealed from the genome analysis. These data are important to path new opportunities for genetic engineering in the field of biotechnology.
Fulltext Novel Methyltransferase Recognition Motif Identified in Chania multitudinisentens RB-25(T) gen. nov., sp. nov

Ee R, Lim YL, Yin WF, See-Too WS, Roberts RJ, Chan KG

Front Microbiol, 2016;7:1362.
PMID: 27630623 DOI: 10.3389/fmicb.2016.01362
Fulltext Quorum sensing activity of Serratia fonticola strain RB-25 isolated from an ex-landfill site

Ee R, Lim YL, Tee KK, Yin WF, Chan KG

Sensors (Basel), 2014 Mar 12;14(3):5136-46.
PMID: 24625739 DOI: 10.3390/s140305136

Quorum sensing is a unique bacterial communication system which permits bacteria to synchronize their behaviour in accordance with the population density. The operation of this communication network involves the use of diffusible autoinducer molecules, termed N-acylhomoserine lactones (AHLs). Serratia spp. are well known for their use of quorum sensing to regulate the expression of various genes. In this study, we aimed to characterized the AHL production of a bacterium designated as strain RB-25 isolated from a former domestic waste landfill site. It was identified as Serratia fonticola using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis and this was confirmed by 16S ribosomal DNA sequencing. High resolution triple quadrupole liquid chromatography-mass spectrometry analysis of S. fonticola strain RB-25 spent culture supernatant indicated the existence of three AHLs namely: N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl) homoserine-lactone (3-oxo-C6 HSL). This is the first report of the production of these AHLs in S. fonticola.
Fulltext Lineage-Specific Methyltransferases Define the Methylome of the Globally Disseminated Escherichia coli ST131 Clone

Forde BM, Phan MD, Gawthorne JA, Ashcroft MM, Stanton-Cook M, Sarkar S, et al.

mBio, 2015 Nov 17;6(6):e01602-15.
PMID: 26578678 DOI: 10.1128/mBio.01602-15

Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.
IMPORTANCE: DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone.
Fulltext Population dynamics of an Escherichia coli ST131 lineage during recurrent urinary tract infection

Forde BM, Roberts LW, Phan MD, Peters KM, Fleming BA, Russell CW, et al.

Nat Commun, 2019 08 13;10(1):3643.
PMID: 31409795 DOI: 10.1038/s41467-019-11571-5

Recurrent urinary tract infections (rUTIs) are extremely common, with ~ 25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.
Fulltext Labrenzia sp. BM1: a quorum quenching bacterium that degrades N-acyl homoserine lactones via lactonase activity

Ghani NA, Norizan SN, Chan XY, Yin WF, Chan KG

Sensors (Basel), 2014;14(7):11760-9.
PMID: 24995373 DOI: 10.3390/s140711760

We report the degradation of quorum sensing N-acylhomoserine lactone molecules by a bacterium isolated from a Malaysian marine water sample. MALDI-TOF and phylogenetic analysis indicated this isolate BM1 clustered closely to Labrenzia sp. The quorum quenching activity of this isolate was confirmed by using a series of bioassays and rapid resolution liquid chromatography analysis. Labrenzia sp. degraded a wide range of N-acylhomoserine lactones namely N-(3-hexanoyl)-L-homoserine lactone (C6-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). Re-lactonisation bioassays confirmed Labrenzia sp. BM1 degraded these signalling molecules efficiently via lactonase activity. To the best of our knowledge, this is the first documentation of a Labrenzia sp. capable of degrading N-acylhomoserine lactones and confirmation of its lactonase-based mechanism of action.
Fulltext Rhodotorula mucilaginosa, a quorum quenching yeast exhibiting lactonase activity isolated from a tropical shoreline

Ghani NA, Sulaiman J, Ismail Z, Chan XY, Yin WF, Chan KG

Sensors (Basel), 2014;14(4):6463-73.
PMID: 24721765 DOI: 10.3390/s140406463

Two microbial isolates from a Malaysian shoreline were found to be capable of degrading N-acylhomoserine lactones. Both Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry and 18S rDNA phylogenetic analyses confirmed that these isolates are Rhodotorula mucilaginosa. Quorum quenching activities were detected by a series of bioassays and rapid resolution liquid chromatography analysis. The isolates were able to degrade various quorum sensing molecules namely N-hexanoyl-L-homoserine lactone (C6-HSL), N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). Using a relactonisation assay to verify the quorum quenching mechanism, it is confirmed that Rh. mucilaginosa degrades the quorum sensing molecules via lactonase activity. To the best of our knowledge, this is the first documentation of the fact that Rh. mucilaginosa has activity against a broad range of AHLs namely C6-HSL, 3-oxo-C6-HSL and 3-hydroxy-C6-HSL.
Fulltext Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic Escherichia coli

Goh KGK, Phan MD, Forde BM, Chong TM, Yin WF, Chan KG, et al.

mBio, 2017 10 24;8(5).
PMID: 29066548 DOI: 10.1128/mBio.01558-17

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.
Fulltext Unusual multiple production of N-acylhomoserine lactones a by Burkholderia sp. strain C10B isolated from dentine caries

Goh SY, Tan WS, Khan SA, Chew HP, Abu Kasim NH, Yin WF, et al.

Sensors (Basel), 2014;14(5):8940-9.
PMID: 24854358 DOI: 10.3390/s140508940

Bacteria realize the ability to communicate by production of quorum sensing (QS) molecules called autoinducers, which regulate the physiological activities in their ecological niches. The oral cavity could be a potential area for the presence of QS bacteria. In this study, we report the isolation of a QS bacterial isolate C10B from dentine caries. Preliminary screening using Chromobacterium violaceum CV026 biosensor showed that isolate C10B was able to produce N-acylhomoserine lactones (AHLs). This bacterium was further identified as a member of Burkholderia, an opportunistic pathogen. The isolated Burkholderia sp. was confirmed to produce N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL), N-decanoyl-L-homoserine lactone (C10-HSL) and N-dodecanoyl-L-homoserine lactone (C12-HSL).
Fulltext Quorum sensing activity of Citrobacter amalonaticus L8A, a bacterium isolated from dental plaque

Goh SY, Khan SA, Tee KK, Abu Kasim NH, Yin WF, Chan KG

Sci Rep, 2016;6:20702.
PMID: 26860259 DOI: 10.1038/srep20702

Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased cell density. In this study, a bacterium (L8A) isolated from dental plaque biofilm was identified as Citrobacter amalonaticus by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Its N-acylhomoserine-lactone (AHL) production was screened by using two types of AHL biosensors namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Citrobacter amalonaticus strain L8A was identified and confirmed producing numerous types of AHL namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL). We performed the whole genome sequence analysis of this oral isolate where its genome sequence reveals the presence of QS signal synthase gene and our work will pave the ways to study the function of the related QS genes in this bacterium.
Fulltext Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme

Hancock SJ, Phan MD, Peters KM, Forde BM, Chong TM, Yin WF, et al.

Antimicrob Agents Chemother, 2017 02;61(2).
PMID: 27872077 DOI: 10.1128/AAC.01740-16

Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies.
Fulltext Quorum quenching revisited--from signal decays to signalling confusion

Hong KW, Koh CL, Sam CK, Yin WF, Chan KG

Sensors (Basel), 2012;12(4):4661-96.
PMID: 22666051 DOI: 10.3390/s120404661

In a polymicrobial community, while some bacteria are communicating with neighboring cells (quorum sensing), others are interrupting the communication (quorum quenching), thus creating a constant arms race between intercellular communication. In the past decade, numerous quorum quenching enzymes have been found and initially thought to inactivate the signalling molecules. Though this is widely accepted, the actual roles of these quorum quenching enzymes are now being uncovered. Recent evidence extends the role of quorum quenching to detoxification or metabolism of signalling molecules as food and energy source; this includes "signalling confusion", a term coined in this paper to refer to the phenomenon of non-destructive modification of signalling molecules. While quorum quenching has been explored as a novel anti-infective therapy targeting, quorum sensing evidence begins to show the development of resistance against quorum quenching.
Fulltext Methylome Characterization of Burkholderia pseudomallei Strain 982 at Single-Base Resolution

Hong KW, Tee KK, Yin WF, Roberts RJ, Chan KG

Microbiol Resour Announc, 2019 Oct 24;8(43).
PMID: 31649075 DOI: 10.1128/MRA.00898-19

Burkholderia pseudomallei is the etiological agent of melioidosis, which has been studied by transcriptome and secretome analyses. However, little is known about the methylome of this pathogen. Here, we present the complete genome and methylome of melioidosis-causing B. pseudomallei strain 982.
Fulltext Draft genome sequence of Pantoea sp. strain A4, a Rafflesia-associated bacterium that produces N-acylhomoserine lactones as quorum-sensing molecules

Hong KW, Gan HM, Low SM, Lee PK, Chong YM, Yin WF, et al.

J Bacteriol, 2012 Dec;194(23):6610.
PMID: 23144374 DOI: 10.1128/JB.01619-12

Pantoea sp. strain A4 is a Gram-negative bacterium isolated from the Rafflesia flower. We present here, for the first time, the genome sequence of Rafflesia-associated Pantoea sp. strain A4, which exhibited quorum-sensing activity.
Fulltext Whole-genome sequence of Cupriavidus sp. strain BIS7, a heavy-metal-resistant bacterium

Hong KW, Thinagaran Da, Gan HM, Yin WF, Chan KG

J Bacteriol, 2012 Nov;194(22):6324.
PMID: 23115161 DOI: 10.1128/JB.01608-12

Cupriavidus sp. strain BIS7 is a Malaysian tropical soil bacterium that exhibits broad heavy-metal resistance [Co(II), Zn(II), Ni(II), Se(IV), Cu(II), chromate, Co(III), Fe(II), and Fe(III)]. It is particularly resistant to Fe(II), Fe(III), and Zn(II). Here we present the assembly and annotation of its genome.
Fulltext Whole-genome sequence of N-acylhomoserine lactone-synthesizing and -degrading Acinetobacter sp. strain GG2

Hong KW, Koh CL, Sam CK, Yin WF, Chan KG

J Bacteriol, 2012 Nov;194(22):6318.
PMID: 23105061 DOI: 10.1128/JB.01579-12

Acinetobacter sp. strain GG2 is a quorum-sensing and quorum-quenching bacterium isolated from the ginger rhizosphere. It degrades a broad range of N-acylhomoserine lactone molecules via lactonase. The genome sequence of strain GG2 may provide insights on the regulation of quorum-sensing and quorum-quenching mechanisms in this bacterium.
Fulltext Complete genome sequence of Burkholderia sp. Strain GG4, a betaproteobacterium that reduces 3-oxo-N-acylhomoserine lactones and produces different N-acylhomoserine lactones

Hong KW, Koh CL, Sam CK, Yin WF, Chan KG

J Bacteriol, 2012 Nov;194(22):6317.
PMID: 23105060 DOI: 10.1128/JB.01578-12

Burkholderia sp. strain GG4, isolated from the ginger rhizosphere, possesses a unique N-acylhomoserine lactone (AHL)-modifying activity that reduces 3-oxo-AHLs to 3-hydroxy-AHLs. To the best of our knowledge, this is the first sequenced genome from a bacterium of the genus Burkholderia that shows both quorum-sensing and signaling confusion activities.
Comparative genomic and phylogenetic analysis of a toxigenic clinical isolate of Corynebacterium diphtheriae strain B-D-16-78 from Malaysia

Hong KW, Asmah Hani AW, Nurul Aina Murni CA, Pusparani RR, Chong CK, Verasahib K, et al.

Infect Genet Evol, 2017 Oct;54:263-270.
PMID: 28711373 DOI: 10.1016/j.meegid.2017.07.015

In this study, we report the comparative genomics and phylogenetic analysis of Corynebacterium diphtheriae strain B-D-16-78 that was isolated from a clinical specimen in 2016. The complete genome of C. diphtheriae strain B-D-16-78 was sequenced using PacBio Single Molecule, Real-Time sequencing technology and consists of a 2,474,151-bp circular chromosome with an average GC content of 53.56%. The core genome of C. diphtheriae was also deduced from a total of 74 strains with complete or draft genome sequences and the core genome-based phylogenetic analysis revealed close genetic relationship among strains that shared the same MLST allelic profile. In the context of CRISPR-Cas system, which confers adaptive immunity against re-invading DNA, 73 out of 86 spacer sequences were found to be unique to Malaysian strains which harboured only type-II-C and/or type-I-E-a systems. A total of 48 tox genes which code for the diphtheria toxin were retrieved from the 74 genomes and with the exception of one truncated gene, only nucleotide substitutions were detected when compared to the tox gene sequence of PW8. More than half were synonymous substitution and only two were nonsynonymous substitutions whereby H24Y was predicted to have a damaging effect on the protein function whilst T262V was predicted to be tolerated. Both toxigenic and non-toxigenic toxin-gene bearing strains have been isolated in Malaysia but the repeated isolation of toxigenic strains with the same MLST profile suggests the possibility of some of these strains may be circulating in the population. Hence, efforts to increase herd immunity should be continued and supported by an effective monitoring and surveillance system to track, manage and control outbreak of cases.

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