Thiocyanate (SCN-) is a contaminant requiring remediation in gold mine tailings and wastewaters globally. Seepage of SCN--contaminated waters into aquifers can occur from unlined or structurally compromised mine tailings storage facilities. A wide variety of microorganisms are known to be capable of biodegrading SCN-; however, little is known regarding the potential of native microbes for in situ SCN- biodegradation, a remediation option that is less costly than engineered approaches. Here we experimentally characterize the principal biogeochemical barrier to SCN- biodegradation for an autotrophic microbial consortium enriched from mine tailings, to arrive at an environmentally realistic assessment of in situ SCN- biodegradation potential. Upon amendment with phosphate, the consortium completely degraded up to ∼10 mM SCN- to ammonium and sulfate, with some evidence of nitrification of the ammonium to nitrate. Although similarly enriched in known SCN--degrading strains of thiobacilli, this consortium differed in its source (mine tailings) and metabolism (autotrophy) from those of previous studies. Our results provide a proof of concept that phosphate limitation may be the principal barrier to in situ SCN- biodegradation in mine tailing waters and also yield new insights into the microbial ecology of in situ SCN- bioremediation involving autotrophic sulfur-oxidizing bacteria.
Aureobasidium pullulans AY4 is an opportunistic pathogen that was isolated from the skin of an immunocompromised patient. We present here the draft genome of strain AY4, which reveals an abundance of genes relevant to bioindustrial applications, including biocontrol and biodegradation. Putative genes responsible for the pathogenicity of strain AY4 were also identified.
A draft genome sequence of Pichia kudriavzevii M12 is presented here. The genome reveals the presence of genes encoding enzymes involved in xylose utilization and the pentose phosphate pathway for bioethanol production. Strain M12 is also a potential producer of phytases, enzymes useful in food processing and agriculture.
Background: The indigenous people of Peninsular Malaysia, also known as Orang Asli, have gradually been urbanized. A shift towards non-communicable diseases commonly associated with sedentary lifestyles have been reported in many tribes. This study engaged with a semi-urbanized Temiar tribe from Kampong Pos Piah, Perak, who are experiencing an epidemiological transition. Methods: Weight, height, waist circumference, blood pressure, HbA1C and lipid levels were measured as indicators of cardio-metabolic health. DNA was extracted from saliva using salting-out method followed by PCR amplification of the V3-V4 region of the 16S rRNA gene and sequencing on Illumina MiSeq. Microbiome analysis was conducted on Qiime v1.9. Statistical analysis was conducted using Qiime v1.9 and R. Results: The study revealed that 60.4% of the Temiar community were overweight/obese, with a higher prevalence among women. HbA1C levels showed that 45% of Temiar had pre-diabetes. Insulin resistance was identified in 21% of Temiar by using a surrogate marker, TG/HDL. In total, 56.5% of Temiar were pre-hypertensive, and the condition was prevalent across all age-groups. The saliva microbiome profiles of Temiar revealed significant differences by gender, BMI, abdominal obesity as well as smoking status. The relative abundance of the genus Bifidobacterium was increased in men whereas the genera Prevotella, Capnocytophaga, Leptotrichia, Neisseria and Streptococcus were increased in women. Proteobacteria was significantly depleted in smokers. Conclusions: Temiar from Pos Piah had a high prevalence of cardio-metabolic risks, including general and abdominal obesity, pre-diabetes, prehypertension and hypertension. This phenomenon has not been previously reported in this tribe. The saliva microbiome profiles were significantly different for individuals of different gender, BMI, abdominal obesity and smoking status.
Genes involved in the 4-aminobenzenesulfonate (4-ABS) degradation pathway of Hydrogenophaga sp. PBC were identified using transposon mutagenesis. The screening of 10,000 mutants for incomplete 4-ABS biotransformation identified four mutants with single transposon insertion. Genes with insertions that impaired the ability to utilize 4-ABS for growth included (1) 4-sulfocatechol 1,2-dioxygenase β-subunit (pcaH2) and 3-sulfomuconate cycloisomerase involved in the modified β-ketoadipate pathway; (2) 4-aminobenzenesulfonate 3,4-dioxygenase component (sadA) involved in aromatic ring hydroxylation; and (3) transposase gene homolog with a putative cis-diol dehydrogenase gene located downstream. The pcaH2 mutant strain accumulated brown metabolite during growth on 4-ABS which was identified as 4-sulfocatechol through thin layer chromatography and HPLC analyses. Supplementation of wild-type sadA gene in trans restored the 4-ABS degradation ability of the sadA mutant, thus supporting the annotation of its disrupted gene.
The Malaysian and global shrimp aquaculture production has been significantly impacted by acute hepatopancreatic necrosis disease (AHPND) typically caused by Vibrio parahaemolyticus harboring the pVA plasmid containing the pirAVp and pirBVp genes, which code for Photorhabdus insect-related (Pir) toxin. The limited genomic resource for V. parahaemolyticus strains from Malaysian aquaculture farms precludes an in-depth understanding of their diversity and evolutionary relationships. In this study, we isolated shrimp-associated and environmental (rearing water) V. parahaemolyticus from three aquaculture farms located in Northern and Central Malaysia followed by whole-genome sequencing of 40 randomly selected isolates on the Illumina MiSeq. Phylogenomic analysis and multilocus sequence typing (MLST) reveal distinct lineages of V. parahaemolyticus that harbor the pirABVp genes. The recovery of pVA plasmid backbone devoid of pirAVp or pirABVp in some V. parahaemolyticus isolates suggests that the toxin genes are prone to deletion. The new insight gained from phylogenomic analysis of Asian V. parahaemolyticus, in addition to the observed genomic instability of pVa plasmid, will have implications for improvements in aquaculture practices to diagnose, treat or limit the impacts of this disease.
Here we report the draft genomes and annotation of four N-acyl homoserine lactone (AHL)-producing members from the family Sphingomonadaceae. Comparative genomic analyses of 62 Sphingomonadaceae genomes were performed to gain insights into the distribution of the canonical luxI/R-type quorum sensing (QS) network within this family. Forty genomes contained at least one luxR homolog while the genome of Sphingobium yanoikuyae B1 contained seven Open Reading Frames (ORFs) that have significant homology to that of luxR. Thirty-three genomes contained at least one luxI homolog while the genomes of Sphingobium sp. SYK6, Sphingobium japonicum, and Sphingobium lactosutens contained four luxI. Using phylogenetic analysis, the sphingomonad LuxR homologs formed five distinct clades with two minor clades located near the plant associated bacteria (PAB) LuxR solo clade. This work for the first time shows that 13 Sphingobium and one Sphingomonas genome(s) contain three convergently oriented genes composed of two tandem luxR genes proximal to one luxI (luxR-luxR-luxI). Interestingly, luxI solos were identified in two Sphingobium species and may represent species that contribute to AHL-based QS system by contributing AHL molecules but are unable to perceive AHLs as signals. This work provides the most comprehensive description of the luxI/R circuitry and genome-based taxonomical description of the available sphingomonad genomes to date indicating that the presence of luxR solos and luxI solos are not an uncommon feature in members of the Sphingomonadaceae family.
Cherax destructor, the yabby, is an iconic Australian freshwater crayfish species, which, similar to other major invertebrate groups, is grossly under-represented in genomic databases. The yabby is also the principal commercial freshwater crustacean species in Australia subject to explotation via inland fisheries and aquaculture. To address the genomics knowledge gap for this species and explore cost effective and efficient methods for genome assembly, we generated 106.8 gb of Nanopore reads and performed a long-read only assembly of the Cherax destructor genome. On a mini-server configured with an ultra-fast swap space, the de novo assembly took 131 h (∼5.5 days). Genome polishing with 126.3 gb of PCR-Free Illumina reads generated an assembled genome size of 3.3 gb (74.6% BUSCO completeness) with a contig N50 of 80,900 bp, making it the most contiguous for freshwater crayfish genome assemblies. We found an unusually large number of cellulase genes within the yabby genome which is relevant to understanding the nutritional biology, commercial feed development, and ecological role of this species and crayfish more generally. These resources will be useful for genomic research on freshwater crayfish and our methods for rapid and super-efficient genome assembly will have wide application.
Despite the urgent need for sustained development of novel antibacterial compounds to combat the drastic rise in antibiotic resistant and emerging bacterial infections, only a few clinically relevant antibacterial drugs have been recently developed. One of the bottlenecks impeding the development of novel antibacterial compounds is the identification of new enzymatic targets. The nutritionally essential amino acid anabolic pathways, for example lysine biosynthesis, provide an opportunity to explore the development of antibacterial compounds, since human genomes do not possess the genes necessary to synthesize these amino acids de novo. The diaminopimelate (DAP)/lysine (lys) anabolic pathways are attractive targets for antibacterial development since the penultimate lys precursor meso-DAP (m-DAP) is a cross-linking amino acid in the peptidoglycan (PG) cell wall of most Gram-negative bacteria and lys plays a similar role in the PG of most Gram-positive bacteria, in addition to its role as one of the 20 proteogenic amino acids. The L,L-diaminopimelate aminotransferase (DapL) pathway was recently identified as a novel variant of the DAP/lys anabolic pathways. The DapL pathway has been identified in the pathogenic bacteria belonging to the genus; Chlamydia, Leptospira, and Treponema. The dapL gene has been identified in the genomes of 381 or approximately 13% of the 2771 bacteria that have been sequenced, annotated and reposited in the NCBI database, as of May 23, 2014. The narrow distribution of the DapL pathway in the bacterial domain provides an opportunity for the development and or discovery of narrow spectrum antibacterial compounds.
The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/C Vs ) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44-46°C. Its apparent K m values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum.
Typhoid fever, caused by Salmonella enterica serovar Typhi, remains an important public health burden in Southeast Asia and other endemic countries. Various genotyping methods have been applied to study the genetic variations of this human-restricted pathogen. Multilocus sequence typing (MLST) is one of the widely accepted methods, and recently, there is a growing interest in the re-application of MLST in the post-genomic era. In this study, we provide the global MLST distribution of S. Typhi utilizing both publicly available 1,826 S. Typhi genome sequences in addition to performing conventional MLST on S. Typhi strains isolated from various endemic regions spanning over a century. Our global MLST analysis confirms the predominance of two sequence types (ST1 and ST2) co-existing in the endemic regions. Interestingly, S. Typhi strains with ST8 are currently confined within the African continent. Comparative genomic analyses of ST8 and other rare STs with genomes of ST1/ST2 revealed unique mutations in important virulence genes such as flhB, sipC, and tviD that may explain the variations that differentiate between seemingly successful (widespread) and unsuccessful (poor dissemination) S. Typhi populations. Large scale whole-genome phylogeny demonstrated evidence of phylogeographical structuring and showed that ST8 may have diverged from the earlier ancestral population of ST1 and ST2, which later lost some of its fitness advantages, leading to poor worldwide dissemination. In response to the unprecedented increase in genomic data, this study demonstrates and highlights the utility of large-scale genome-based MLST as a quick and effective approach to narrow the scope of in-depth comparative genomic analysis and consequently provide new insights into the fine scale of pathogen evolution and population structure.
The genus Pseudomonas has one of the largest diversity of species within the Bacteria kingdom. To date, its taxonomy is still being revised and updated. Due to the non-standardized procedure and ambiguous thresholds at species level, largely based on 16S rRNA gene or conventional biochemical assay, species identification of publicly available Pseudomonas genomes remains questionable. In this study, we performed a large-scale analysis of all Pseudomonas genomes with species designation (excluding the well-defined P. aeruginosa) and re-evaluated their taxonomic assignment via in silico genome-genome hybridization and/or genetic comparison with valid type species. Three-hundred and seventy-three pseudomonad genomes were analyzed and subsequently clustered into 145 distinct genospecies. We detected 207 erroneous labels and corrected 43 to the proper species based on Average Nucleotide Identity Multilocus Sequence Typing (MLST) sequence similarity to the type strain. Surprisingly, more than half of the genomes initially designated as Pseudomonas syringae and Pseudomonas fluorescens should be classified either to a previously described species or to a new genospecies. Notably, high pairwise average nucleotide identity (>95%) indicating species-level similarity was observed between P. synxantha-P. libanensis, P. psychrotolerans-P. oryzihabitans, and P. kilonensis- P. brassicacearum, that were previously differentiated based on conventional biochemical tests and/or genome-genome hybridization techniques.
We improved upon the previously reported draft genome of Hydrogenophaga intermedia strain PBC, a 4-aminobenzenesulfonate-degrading bacterium, by supplementing the assembly with Nanopore long reads which enabled the reconstruction of the genome as a single contig. From the complete genome, major genes responsible for the catabolism of 4-aminobenzenesulfonate in strain PBC are clustered in two distinct genomic regions. Although the catabolic genes for 4-sulfocatechol, the deaminated product of 4-aminobenzenesulfonate, are only found in H. intermedia, the sad operon responsible for the first deamination step of 4-aminobenzenesulfonate is conserved in various Hydrogenophaga strains. The absence of pabB gene in the complete genome of H. intermedia PBC is consistent with its p-aminobenzoic acid (pABA) auxotrophy but surprisingly comparative genomics analysis of 14 Hydrogenophaga genomes indicate that pABA auxotrophy is not an uncommon feature among members of this genus. Of even more interest, several Hydrogenophaga strains do not possess the genomic potential for hydrogen oxidation, calling for a revision to the taxonomic description of Hydrogenophaga as "hydrogen eating bacteria."
Crown gall (CG) is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of CG is Agrobacterium or Allorhizobium strains that harbor a tumor-inducing plasmid (pTi). The microbial community within the CG tumor has not been widely elucidated and it is not known if certain members of this microbial community promote or inhibit CG. This study investigated the microbiotas of grapevine CG tumor tissues from seven infected vineyards located in Hungary, Japan, Tunisia, and the United States. Heavy co-amplification of grapevine chloroplast and mitochondrial ribosomal RNA genes was observed with the widely used Illumina V3-V4 16S rRNA gene primers, requiring the design of a new reverse primer to enrich for bacterial 16S rRNA from CG tumors. The operational taxonomic unit (OTU) clustering approach is not suitable for CG microbiota analysis as it collapsed several ecologically distinct Agrobacterium species into a single OTU due to low interspecies genetic divergence. The CG microbial community assemblages were significantly different across sampling sites (ANOSIM global R = 0.63, p-value = 0.001) with evidence of site-specific differentially abundant ASVs. The presence of Allorhizobium vitis in the CG microbiota is almost always accompanied by Xanthomonas and Novosphingobium, the latter may promote the spread of pTi plasmid by way of acyl-homoserine lactone signal production, whereas the former may take advantage of the presence of substrates associated with plant cell wall growth and repair. The technical and biological insights gained from this study will contribute to the understanding of complex interaction between the grapevine and its microbial community and may facilitate better management of CG disease in the future.
The quality of traditional Chinese soy sauce is determined by microbial communities and their inter-related metabolic roles in the fermentation tank. In this study, traditional Chinese soy sauce brine samples were obtained periodically to monitor the transitions of the microbial population and functional properties during the 6 months of fermentation process. Whole genome shotgun method revealed that the fermentation brine was dominated by the bacterial genus Weissella and later dominated by the fungal genus Candida. Metabolic reconstruction of the metagenome sequences demonstrated a characteristic profile of heterotrophic fermentation of proteins and carbohydrates. This was supported by the detection of ethanol with stable decrease of pH values. To the best of our knowledge, this is the first study that explores the temporal changes in microbial successions over a period of 6 months, through metagenome shotgun sequencing in traditional Chinese soy sauce fermentation and the biological processes therein.
Wet markets in low-and middle-income countries are often reported to have inadequate sanitation resulting in fecal contamination of sold produce. Consumption of contaminated wet market-sourced foods has been linked to individual illness and disease outbreaks. This pilot study, conducted in two major wet markets in Dhaka city, Bangladesh during a 4-month period in 2021 aimed to assess the occurrence and characteristics of Escherichia coli and non-typhoidal Salmonella spp. (NTS) from tilapia (Oreochromis niloticus) and shrimp (Penaeus monodon). Fifty-four individuals of each species were collected. The identity of the bacterial isolates was confirmed by PCR and their susceptibility toward 15 antimicrobials was tested by disk diffusion. The whole genome of 15 E. coli and nine Salmonella spp. were sequenced using Oxford Nanopore Technology. E. coli was present in 60-74% of tilapia muscle tissue and 41-44% of shrimp muscle tissue. Salmonella spp. was found in skin (29%) and gills (26%) of tilapia, and occasionally in muscle and intestinal samples of shrimp. The E. coli had several Multilocus sequence typing and serotypes and limited antimicrobial resistance (AMR) determinants, such as point mutations on glpT and pmrB. One E. coli (BD17) from tilapia carried resistance genes for beta-lactams, quinolones, and tetracycline. All the E. coli belonged to commensal phylogroups B1 and A and showed no Shiga-toxin and other virulence genes, confirming their commensal non-pathogenic status. Among the Salmonella isolates, five belonged to Kentucky serovar and had similar AMR genes and phenotypic resistance patterns. Three strains of this serovar were ST198, often associated with human disease, carried the same resistance genes, and were genetically related to strains from the region. The two undetermined sequence types of S. Kentucky were distantly related and positioned in a separate phylogenetic clade. Two Brunei serovar isolates, one Augustenborg isolate, and one Hartford isolate showed different resistance profiles. This study revealed high fecal contamination levels in tilapia and shrimp sold at two main wet markets in Dhaka. Together with the occurrence of Salmonella spp., including S. Kentucky ST198, a well-known human pathogen, these results stress the need to improve hygienic practices and sanitation standards at markets to improve food safety and protect consumer health.
The true mahseer (Tor spp.) is one of the highest valued fish in the world due to its high nutritional value and great unique taste. Nevertheless, its morphological characterization and single mitochondrial gene phylogeny in the past had yet to resolve the ambiguity in its taxonomical classification. In this study, we sequenced and assembled 11 complete mahseer mitogenomes collected from Java of Indonesia, Pahang and Terengganu of Peninsular Malaysia as well as Sarawak of East Malaysia. The mitogenome evolutionary relationships among closely related Tor spp. samples were investigated based on maximum likelihood phylogenetic tree construction. Compared to the commonly used COX1 gene fragment, the complete COX1, Cytb, ND2, ND4 and ND5 genes appear to be better phylogenetic markers for genetic differentiation at the population level. In addition, a total of six population-specific mitolineage haplotypes were identified among the mahseer samples analyzed, which this offers hints towards its taxonomical landscape.
Mammal diversity assessments based on DNA derived from invertebrates have been suggested as alternatives to assessments based on traditional methods; however, no study has field-tested both approaches simultaneously. In Peninsular Malaysia, we calibrated the performance of mammal DNA derived from blowflies (Diptera: Calliphoridae) against traditional methods used to detect species. We first compared five methods (cage trapping, mist netting, hair trapping, scat collection, and blowfly-derived DNA) in a forest reserve with no recent reports of megafauna. Blowfly-derived DNA and mist netting detected the joint highest number of species (n = 6). Only one species was detected by multiple methods. Compared to the other methods, blowfly-derived DNA detected both volant and non-volant species. In another forest reserve, rich in megafauna, we calibrated blowfly-derived DNA against camera traps. Blowfly-derived DNA detected more species (n = 11) than camera traps (n = 9), with only one species detected by both methods. The rarefaction curve indicated that blowfly-derived DNA would continue to detect more species with greater sampling effort. With further calibration, blowfly-derived DNA may join the list of traditional field methods. Areas for further investigation include blowfly feeding and dispersal biology, primer biases, and the assembly of a comprehensive and taxonomically-consistent DNA barcode reference library.