Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.
Comparative whole-genome sequencing enables the identification of specific mutations during adaptation of bacteria to new environments and allelic replacement can establish their causality. However, the mechanisms of action are hard to decipher and little has been achieved for epistatic mutations, especially at the metabolic level. Here we show that a strain of Escherichia coli carrying mutations in the rpoC and glpK genes, derived from adaptation in glycerol, uses two distinct metabolic strategies to gain growth advantage. A 27-bp deletion in the rpoC gene first increases metabolic efficiency. Then, a point mutation in the glpK gene promotes growth by improving glycerol utilization but results in increased carbon wasting as overflow metabolism. In a strain carrying both mutations, these contrasting carbon/energy saving and wasting mechanisms work together to give an 89% increase in growth rate. This study provides insight into metabolic reprogramming during adaptive laboratory evolution for fast cellular growth.
Human enterovirus 71 (EV-71) is genotyped for molecular epidemiological investigation mainly using the two structural genes, VP1 and VP4. Based on these, EV-71 is divided into three genotypes, A, B and C, and within the genotypes B and C, there are further subgenotypes, B1-B5 and C1-C5. Classification using these genes is useful but gives incomplete phylogenetic information. In the present study, the phylogenetic relationships amongst all the known EV-71 and human enterovirus A (HEV-A) isolates with complete genome sequences were examined. A different tree topology involving EV-71 isolates of subgenotypes, C4 and B5 was obtained in comparison to that drawn using VP1. The nucleotide sequence divergence of the C4 isolates was 18.11% (17-20%) when compared to other isolates of subgenotype C. However, this positions the C4 isolates within the cut-off divergence value of 17-22% used to designate the virus genotypes. Hence, it is proposed here that C4 should be designated as a new genotype D. In addition, the subgenotype B5 isolates had an average nucleotide divergence of only 6.14% (4-8%) when compared to other subgenotype B4 isolates. This places the B5 isolates within the subgenotype B4. It is proposed here that the B5 isolates to be redesignated as B4. With the newly proposed genotype D and inclusion of subgenotype B5 within B4, the average nucleotide divergence between genotypes was 18.99% (17-22%). Inter- and intra-subgenotype average divergences were 12.02% (10-14%) and 3.92% (1-10%), respectively. A phylogenetic tree built using the full genome sequences is robust as it takes into consideration changes in the sequences of both the structural and non-structural genes. Similar nucleotide similarities, however, were obtained if only VP1 and 3D RNA polymerase genes were used. Furthermore, addition of 3D RNA polymerase sequences will also show recombination events. Hence, in the absence of full genome sequences, it is proposed here that a combination of VP1 and 3D RNA polymerase gene sequences be used for initial genotyping of EV-71 isolates.
Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions--exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.
In 1998, Nipah virus (NV) emerged in peninsular Malaysia, causing fatal encephalitis in humans and a respiratory disease in swine. NV is most closely related to Hendra virus (HV), a paramyxovirus that was identified in Australia in 1994, and it has been proposed that HV and NV represent a new genus within the family Paramyxoviridae. This report describes the analysis of the sequences of the polymerase gene (L) and genomic termini of NV as well as a comparison of the full-length, genomic sequences of HV and NV. The L gene of NV is predicted to be 2244 amino acids in size and contains the six domains found within the L proteins of all nonsegmented, negative-stranded (NNS) RNA viruses. However, the GDNQ motif found in most NNS RNA viruses was replaced by GDNE in both NV and HV. The 3' and 5' termini of the NV genome are nearly identical to the genomic termini of HV and share sequence homology with the genomic termini of other members of the subfamily Paramyxovirinae. At 18,246 nucleotides, the genome of NV is 12 nucleotides longer than the genome of HV and they have the largest genomes within the family Paramyxoviridae. The comparison of the structures of the genomes of HV and NV is now complete and this information will help to establish the taxonomic position of these novel viruses within the family Paramyxoviridae.
In this study we analysed three bacterial strains coded L10.10T, A4R1.5 and A4R1.12, isolated in the course of a study of quorum-quenching bacteria occurring in Antarctic soil. The 16S rRNA gene sequence was identical in the three strains and showed 99.7% pairwise similarity with respect to the closest related species Pseudomonas weihenstephanensis WS4993T. Therefore, the three strains were classified within the genus Pseudomonas. Analysis of housekeeping genes (rpoB, rpoD and gyrB) sequences showed similarities of 84-95% with respect to the closest related species of Pseudomonas, confirming its phylogenetic affiliation. The ANI values were less than 86% to the closest related species type strains. The respiratory quinone is Q9. The major fatty acids are C16:0, C16:1 ω7c/ C16:1 ω6c in summed feature 3 and C18:1 ω7c / C18:1 ω6c in summed feature 8. The strains are oxidase- and catalase-positive. Growth occurs at 4-30°C, and at pH 4.0-10. The DNA G+C content is 58.2-58.3mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains L10.10T, A4R1.5 and A4R1.12 into a novel species of Pseudomonas, for which the name P. versuta sp. nov. is proposed. The type strain is L10.10T (LMG 29628T, DSM 101070T).
Partial gene sequences of phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) were evaluated for species delineation and detection of recombination among enterococci populations recovered from a bathing beach impacted by low tide river flow. At inter-species level, a maximum similarity of 86.5% and 94.8% was observed among the enterococci pheS and rpoA sequence, respectively. A superimposed plot of delimited pairwise similarity values obtained for 266 pair-wise observations revealed that while there was a harmony between species identity obtained from both genes, pheS was more discriminatory than rpoA. The difference was more pronounced for inter-species comparison. A number of putative recombination events between indigenous and non-indigenous strains was detected based on a library of aligned sequences. Virulence genes cyl, esp, gelE and asa were detected in 7, 22, 100 and 63%, respectively among river isolates but at lower proportion of 0, 20, 67 and 42%, respectively among beach water isolates. Random amplified polymorphic DNA profiling presented evidence suggesting low tide river as a source of fecal enterococci entering the recreation beach water. Multilocus sequence typing analysis of a number of Enterococcus faecalis isolates presented four sequence types, ST59, 117, 181 and 474. The presence of genetically diverse fecal enterococci with associated virulence traits and a background of recombination events in surface recreational water could present a potential public health risk.
This study describes our investigation on the prevalence and molecular identification of bartonellae from Rattus diardii and R. norvegicus in the urban areas of Malaysia. Of 95 rats investigated, Bartonella tribocorum, B. rattimassiliensis, B. coopersplainsensis, B. elizabethae, and B. queenslandensis were isolated from kidney and spleen homogenates of four rats. Bartonellae DNA was amplified from the rat organ tissues by using primers specific for the bartonellae RNA polymerase beta subunit (rpoB) gene in nine other rats. Sequence analysis of the rpoB gene fragments shows the identification of B. queenslandensis in five rats, B. elizabethae in three rats, and B. tribocorum in one rat. Combining the results of isolation and molecular detection of bartonellae, we found that the prevalence of Bartonella infection in the Rattus spp. investigated in this study was 13.7%. Implementation of effective rat control program in the urban areas is necessary to prevent the spillover of bartonellosis from rats to humans.
Antimicrobial resistance in Acinetobacter baumannii is a growing public health concern and an important pathogen in nosocomial infections. We investigated the genes involved in resistance to carbapenems and cephalosporins in clinical A. baumannii isolates from a tertiary medical centre in Malaysia. A. baumannii was isolated from 167 clinical specimens and identified by sequencing of the 16S rRNA and rpoB genes. The MIC for imipenem, meropenem, ceftazidime and cefepime were determined by the E-test method. The presence of carbapenemase and cephalosporinase genes was investigated by PCR. The isolates were predominantly nonsusceptible to carbapenems and cephalosporins (>70 %) with high MIC values. ISAba1 was detected in all carbapenem-nonsusceptible A. baumannii harbouring the blaOXA-23-like gene. The presence of blaOXA-51-like and ISAba1 upstream of blaOXA-51 was not associated with nonsusceptibility to carbapenems. A. baumannii isolates harbouring ISAba1-blaADC (85.8 %) were significantly associated with nonsusceptibility to cephalosporins (P<0.0001). However, ISAba1-blaADC was not detected in a minority (<10 %) of the isolates which were nonsusceptible to cephalosporins. The acquired OXA-23 enzymes were responsible for nonsusceptibility to carbapenems in our clinical A. baumannii isolates and warrant continuous surveillance to prevent further dissemination of this antibiotic resistance gene. The presence of ISAba1 upstream of the blaADC was a determinant for cephalosporin resistance. However, the absence of this ISAba1-blaADC in some of the isolates may suggest other resistance mechanisms and need further investigation.
In this study, PCR-RFLP analysis (PRA) targeting hsp65 and rpoB gene regions was evaluated for the identification of mycobacterial species isolated from Malaysian patients. Overall, the hsp65 PRA identified 92.2 % of 90 isolates compared to 85.6 % by the rpoB PRA. With 47 rapidly growing species, the hsp65 PRA identified fewer (89.4 %) species than the rpoB PRA (95.7 %), but with 23 slow-growing species the reverse was true (91.3 % identification by the hsp65 PRA but only 52.5 % by the rpoB PRA). There were 16 isolates with discordant PRA results, which were resolved by 16S rRNA and hsp65 gene sequence analysis. The findings in this study suggest that the hsp65 PRA is more useful than the rpoB PRA for the identification of Mycobacterium species, particularly with the slow-growing members of the genus. In addition, this study reports 5 and 12 novel restriction patterns for inclusion in the hsp65 and rpoB PRA algorithms, respectively.