scholarly journals Evolutionarily stable gene clusters shed light on the common grounds of pathogenicity in the Acinetobacter calcoaceticus-baumanniicomplex

2022 ◽  
Author(s):  
Bardya Djahanschiri ◽  
Gisela Di Venanzio ◽  
Jesus S. Distel ◽  
Jennifer Breisch ◽  
Marius Alfred Dieckmann ◽  
...  
Keyword(s):  
Phylogenetic Signal ◽  
Functional Integration ◽  
Acinetobacter Calcoaceticus ◽  
Gene Clusters ◽  
Resistant Bacteria ◽  
Stable Gene ◽  
Last Common Ancestor ◽  
Human Host ◽  
Evolutionarily Stable ◽  
Shed Light

Nosocomial pathogens of the Acinetobacter calcoaceticus-baumannii (ACB) complex are a cautionary example for the world-wide spread of multi- and pan-drug resistant bacteria. Aiding the urgent demand for novel therapeutic targets, comparative genomics studies between pathogens and their apathogenic relatives shed light on the genetic basis of human-pathogen interaction. Yet, existing studies are limited in taxonomic scope, sensing of the phylogenetic signal, and resolution by largely analyzing genes isolated from their functional contexts. Here, we explored more than 3,000 Acinetobacter genomes in a phylogenomic framework integrating orthology-based phylogenetic profiling and micro-synteny conservation analyses. This allowed to delineate gene clusters in the type strain A. baumannii ATCC 19606 whose evolutionary conservation indicates a functional integration of the subsumed genes. These evolutionarily stable gene clusters (ESGCs) reveal metabolic pathways, transcriptional regulators residing next to their targets but also tie together sub-clusters with distinct functions to form higher-order functional modules. We shortlisted 150 ESGCs that either co-emerged with, or are found preferentially in, the pathogenic ACB clade. They unveil, at an unprecedented resolution, the genetic makeup that coincides with the manifestation of the pathogenic phenotype in the last common ancestor of the ACB clade. Key innovations are the remodeling of the regulatory-effector cascade connecting LuxR/LuxI quorum sensing via an intermediate messenger to biofilm formation, the extension of micronutrient scavenging systems, and the increase of metabolic flexibility by exploiting carbon sources that are provided by the human host. Specifically, we could show that only members of the ACB clade use kynurenine as a sole carbon and energy source, a substance produced by humans to fine-tune the antimicrobial innate immune response. In summary, this study provides a rich and unbiased set of novel testable hypotheses on how pathogenic Acinetobacter interact with and ultimately infect their human host. They disclose promising routes for future therapeutic strategies.

scholarly journals Identification of a New Antimicrobial, Desertomycin H, Utilizing a Modified Crowded Plate Technique

Marine Drugs ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. 424
Author(s):  
Osama G. Mohamed ◽  
Sadaf Dorandish ◽  
Rebecca Lindow ◽  
Megan Steltz ◽  
Ifrah Shoukat ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Gene Clusters ◽  
Multidrug Resistant ◽  
Microbial Interactions ◽  
Mass Spectrometry Data ◽  
Metagenomic Data ◽  
Resistant Bacteria ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Plate Technique

The antibiotic-resistant bacteria-associated infections are a major global healthcare threat. New classes of antimicrobial compounds are urgently needed as the frequency of infections caused by multidrug-resistant microbes continues to rise. Recent metagenomic data have demonstrated that there is still biosynthetic potential encoded in but transcriptionally silent in cultivatable bacterial genomes. However, the culture conditions required to identify and express silent biosynthetic gene clusters that yield natural products with antimicrobial activity are largely unknown. Here, we describe a new antibiotic discovery scheme, dubbed the modified crowded plate technique (mCPT), that utilizes complex microbial interactions to elicit antimicrobial production from otherwise silent biosynthetic gene clusters. Using the mCPT as part of the antibiotic crowdsourcing educational program Tiny Earth®, we isolated over 1400 antibiotic-producing microbes, including 62, showing activity against multidrug-resistant pathogens. The natural product extracts generated from six microbial isolates showed potent activity against vancomycin-intermediate resistant Staphylococcus aureus. We utilized a targeted approach that coupled mass spectrometry data with bioactivity, yielding a new macrolactone class of metabolite, desertomycin H. In this study, we successfully demonstrate a concept that significantly increased our ability to quickly and efficiently identify microbes capable of the silent antibiotic production.

scholarly journals Recombinant transfer in the basic genome ofEscherichia coli

2015 ◽  
Vol 112 (29) ◽  
pp. 9070-9075 ◽  
Author(s):  
Purushottam D. Dixit ◽  
Tin Yau Pang ◽  
F. William Studier ◽  
Sergei Maslov
Keyword(s):  
Common Ancestor ◽  
Recipient Cell ◽  
Gene Clusters ◽  
Selective Advantage ◽  
Last Common Ancestor ◽  
Genome Sequences ◽  
Bacterial Genomes ◽  
Basic Genome ◽  
Single Base Pair ◽  
Generalized Transduction

An approximation to the ∼4-Mbp basic genome shared by 32 strains ofEscherichia colirepresenting six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly between genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome pairs have one or two recombinant transfers of length ∼40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kilobase pairs. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. Most recombinant transfers seem likely to be due to generalized transduction by coevolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.

scholarly journals Refactoring the formicamycin biosynthetic gene cluster to make high-level producing strains and new molecules

2020 ◽  
Author(s):  
Rebecca Devine ◽  
Hannah McDonald ◽  
Zhiwei Qin ◽  
Corinne Arnold ◽  
Katie Noble ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Large Scale ◽  
Liquid Culture ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistant Bacteria ◽  
Drug Resistant ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes

AbstractThe formicamycins are promising antibiotics with potent activity against Gram-positive pathogens including VRE and MRSA and display a high barrier to selection of resistant isolates. They were first identified in Streptomyces formicae KY5, which produces the formicamycins at low levels on solid agar but not in liquid culture, thus hindering further investigation of these promising antibacterial compounds. We hypothesised that by understanding the organisation and regulation of the for biosynthetic gene cluster, we could rationally refactor the cluster to increase production levels. Here we report that the for biosynthetic gene cluster consists of 24 genes expressed on nine transcripts. Seven of these transcripts, including those containing all the major biosynthetic genes, are repressed by the MarR-regulator ForJ which also controls the expression of the ForGF two-component system that initiates biosynthesis. A third cluster-situated regulator, ForZ, autoregulates and controls production of the putative MFS transporter ForAA. Consistent with these findings, deletion of forJ increased formicamycin biosynthesis 5-fold, while over-expression of forGF in the ΔforJ background increased production 10-fold compared to the wild-type. De-repression by deleting forJ also switched on biosynthesis in liquid-culture and induced the production of two novel formicamycin congeners. By combining mutations in regulatory and biosynthetic genes, six new biosynthetic precursors with antibacterial activity were also isolated. This work demonstrates the power of synthetic biology for the rational redesign of antibiotic biosynthetic gene clusters both to engineer strains suitable for fermentation in large scale bioreactors and to generate new molecules.ImportanceAntimicrobial resistance is a growing threat as existing antibiotics become increasingly ineffective against drug resistant pathogens. Here we determine the transcriptional organisation and regulation of the gene cluster encoding biosynthesis of the formicamycins, promising new antibiotics with activity against drug resistant bacteria. By exploiting this knowledge, we construct stable mutant strains which over-produce these molecules in both liquid and solid culture whilst also making some new compound variants. This will facilitate large scale purification of these molecules for further study including in vivo experiments and the elucidation of their mechanism of action. Our work demonstrates that understanding the regulation of natural product biosynthetic pathways can enable rational improvement of the producing strains.

scholarly journals A Distinct Contractile Injection System Gene Cluster Found in a Majority of Healthy Adult Human Microbiomes

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Maria I. Rojas ◽  
Giselle S. Cavalcanti ◽  
Katelyn McNair ◽  
Sean Benler ◽  
Amanda T. Alker ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Gene Cluster ◽  
Bowel Disease ◽  
Human Microbiome ◽  
Gene Clusters ◽  
The United States ◽  
Injection System ◽  
Human Gut ◽  
Human Host ◽  
Inflammatory Bowel

ABSTRACT Many commensal bacteria antagonize each other or their host by producing syringe-like secretion systems called contractile injection systems (CIS). Members of the Bacteroidales family have been shown to produce only one type of CIS—a contact-dependent type 6 secretion system that mediates bacterium-bacterium interactions. Here, we show that a second distinct cluster of genes from Bacteroidales bacteria from the human microbiome may encode yet-uncharacterized injection systems that we term Bacteroidales injection systems (BIS). We found that BIS genes are present in the gut microbiomes of 99% of individuals from the United States and Europe and that BIS genes are more prevalent in the gut microbiomes of healthy individuals than in those individuals suffering from inflammatory bowel disease. Gene clusters similar to that of the BIS mediate interactions between bacteria and diverse eukaryotes, like amoeba, insects, and tubeworms. Our findings highlight the ubiquity of the BIS gene cluster in the human gut and emphasize the relevance of the gut microbiome to the human host. These results warrant investigations into the structure and function of the BIS and how they might mediate interactions between Bacteroidales bacteria and the human host or microbiome. IMPORTANCE To engage with host cells, diverse pathogenic bacteria produce syringe-like structures called contractile injection systems (CIS). CIS are evolutionarily related to the contractile tails of bacteriophages and are specialized to puncture membranes, often delivering effectors to target cells. Although CIS are key for pathogens to cause disease, paradoxically, similar injection systems have been identified within healthy human microbiome bacteria. Here, we show that gene clusters encoding a predicted CIS, which we term Bacteroidales injection systems (BIS), are present in the microbiomes of nearly all adult humans tested from Western countries. BIS genes are enriched within human gut microbiomes and are expressed both in vitro and in vivo. Further, a greater abundance of BIS genes is present within healthy gut microbiomes than in those humans with with inflammatory bowel disease (IBD). Our discovery provides a potentially distinct means by which our microbiome interacts with the human host or its microbiome.

scholarly journals The final steps of [FeFe]-hydrogenase maturation

2019 ◽  
Vol 116 (32) ◽  
pp. 15802-15810 ◽  
Author(s):  
Oliver Lampret ◽  
Julian Esselborn ◽  
Rieke Haas ◽  
Andreas Rutz ◽  
Rosalind L. Booth ◽  
...  
Keyword(s):  
Functional Integration ◽  
Structural Features ◽  
Site Directed Mutagenesis ◽  
Structural Reorganization ◽  
Biologically Inspired ◽  
Hydrogenase Maturation ◽  
Flexible Loop ◽  
Loop Region ◽  
Shed Light ◽  
Secondary Ligand

The active site (H-cluster) of [FeFe]-hydrogenases is a blueprint for the design of a biologically inspired H2-producing catalyst. The maturation process describes the preassembly and uptake of the unique [2FeH] cluster into apo-hydrogenase, which is to date not fully understood. In this study, we targeted individual amino acids by site-directed mutagenesis in the [FeFe]-hydrogenase CpI of Clostridium pasteurianum to reveal the final steps of H-cluster maturation occurring within apo-hydrogenase. We identified putative key positions for cofactor uptake and the subsequent structural reorganization that stabilizes the [2FeH] cofactor in its functional coordination sphere. Our results suggest that functional integration of the negatively charged [2FeH] precursor requires the positive charges and individual structural features of the 2 basic residues of arginine 449 and lysine 358, which mark the entrance and terminus of the maturation channel, respectively. The results obtained for 5 glycine-to-histidine exchange variants within a flexible loop region provide compelling evidence that the glycine residues function as hinge positions in the refolding process, which closes the secondary ligand sphere of the [2FeH] cofactor and the maturation channel. The conserved structural motifs investigated here shed light on the interplay between the secondary ligand sphere and catalytic cofactor.

scholarly journals Reconstructing the ancestral phenotypes of great apes and humans (Homininae) using subspecies-level phylogenies

2019 ◽  
Author(s):  
Keaghan J Yaxley ◽  
Robert A Foley
Keyword(s):  
Dna Sequences ◽  
Phenotypic Variation ◽  
Common Ancestor ◽  
Phylogenetic Signal ◽  
Great Apes ◽  
Last Common Ancestor ◽  
Ancestral State ◽  
Great Ape ◽  
Subspecies Level ◽  
African Great Apes

Abstract Owing to their close affinity, the African great apes are of interest in the study of human evolution. Although numerous researchers have described the ancestors we share with these species with reference to extant great apes, few have done so with phylogenetic comparative methods. One obstacle to the application of these techniques is the within-species phenotypic variation found in this group. Here, we leverage this variation, modelling common ancestors using ancestral state reconstructions (ASRs) with reference to subspecies-level trait data. A subspecies-level phylogeny of the African great apes and humans was estimated from full-genome mitochondrial DNA sequences and used to implement ASRs for 14 continuous traits known to vary between great ape subspecies. Although the inclusion of within-species phenotypic variation increased the phylogenetic signal for our traits and improved the performance of our ASRs, whether this was done through the inclusion of subspecies phylogeny or through the use of existing methods made little difference. Our ASRs corroborate previous findings that the last common ancestor of humans, chimpanzees and bonobos was a chimp-like animal, but also suggest that the last common ancestor of humans, chimpanzees, bonobos and gorillas was an animal unlike any extant African great ape.

scholarly journals Reservoir water in Singapore contains ESBL-producing and carbapenem-resistant bacteria with conjugatable conserved gene cluster transfer between different species.

2021 ◽  
Author(s):  
Yang Zhong ◽  
Siyao Guo ◽  
Joergen Schlundt
Keyword(s):  
One Health ◽  
Gene Clusters ◽  
Resistant Bacteria ◽  
Klebsiella Pneumonia ◽  
Sequencing Analysis ◽  
Metagenomic Sequencing ◽  
Beta Lactamase ◽  
Reservoir Water ◽  
The One ◽  
Conserved Gene

As the role of the aquatic environment in the One-Health approach has called increasing attention, the studies of Antimicrobial resistance (AMR) spreading in the water bodies have been reported worldwide. However, there are still limited studies on the AMR carrier in the reservoir water in Singapore. Since 2018, our group has collect water samples from six reservoirs in Singapore and isolated the beta-lactam-resistant bacteria from them. We then characterized the isolates with Whole-genome sequencing (WGS) and successfully identified ESBL-producing bacteria from three sampling reservoirs, and confirmed their resistance with both phenotypic and sequencing methods. To better understand the AMR spreading locally, we compared our isolates with isolates from other WGS studies in Singapore covered humans, food, and the enviroment. From there, we noticed the same sequence type (ST) as ST10, ST23, and ST38 has been shared among the environment, food, and humans, as well as the same beta-lactamase genes, are widely distributed among multiple sources. Further genetic environment comparison of beta-lactamase has suggested their spreading as conserved gene clusters among different species and sources. And this hypothesis has been supported by the successful conjugation of blaCTX-M-15 from Klebsiella pneumonia to Escherichia coli (E .coli). We also applied the shotgun metagenomic sequencing to understand the community of bacteria in reservoir water and detect the AMR genes. The composition of bacteria has shown different diversity among different samples. Besides, different beta-lactamase genes have been identified compared to culture depended methods. Here, we suggest that sequencing analysis has great potential in understanding AMR spreading in the One-Health approach. A genetic-based AMR risk assessment is in urgent need in Singapore.

scholarly journals Production of the antimicrobial compound tetrabromopyrrole and the Pseudomonas quinolone system precursor, 2-heptyl-4-quinolone, by a novel marine species Pseudoalteromonas galatheae sp. nov.

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sara Skøtt Paulsen ◽  
Thomas Isbrandt ◽  
Markus Kirkegaard ◽  
Yannick Buijs ◽  
Mikael Lenz Strube ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Marine Species ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Halogenated Compound ◽  
Genome Wide ◽  
A Genome ◽  
Rapid Spread ◽  
Bioassay Guided Fractionation ◽  
Type Strains

AbstractNovel antimicrobials are urgently needed due to the rapid spread of antibiotic resistant bacteria. In a genome-wide analysis of Pseudoalteromonas strains, one strain (S4498) was noticed due to its potent antibiotic activity. It did not produce the yellow antimicrobial pigment bromoalterochromide, which was produced by several related type strains with which it shared less than 95% average nucleotide identity. Also, it produced a sweet-smelling volatile not observed from other strains. Mining the genome of strain S4498 using the secondary metabolite prediction tool antiSMASH led to eight biosynthetic gene clusters with no homology to known compounds, and synteny analyses revealed that the yellow pigment bromoalterochromide was likely lost during evolution. Metabolome profiling of strain S4498 using HPLC-HRMS analyses revealed marked differences to the type strains. In particular, a series of quinolones known as pseudanes were identified and verified by NMR. The characteristic odor of the strain was linked to the pseudanes. The highly halogenated compound tetrabromopyrrole was detected as the major antibacterial component by bioassay-guided fractionation. Taken together, the polyphasic analysis demonstrates that strain S4498 belongs to a novel species within the genus Pseudoalteromonas, and we propose the name Pseudoalteromonas galatheae sp. nov. (type strain S4498T = NCIMB 15250T = LMG 31599T).

scholarly journals Design, Overproduction and Purification of the Chimeric Phage Lysin MLTphg Fighting against Staphylococcus aureus

Processes ◽  
2020 ◽  
Vol 8 (12) ◽  
pp. 1587
Author(s):  
Feng Wang ◽  
Xiaohang Liu ◽  
Zhengyu Deng ◽  
Yao Zhang ◽  
Xinyu Ji ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Infections ◽  
Antimicrobial Agents ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Conventional Antibiotics ◽  
Phage Lysin ◽  
Shed Light

With the increasing spread of multidrug-resistant bacterial pathogens, it is of great importance to develop alternatives to conventional antibiotics. Here, we report the generation of a chimeric phage lysin, MLTphg, which was assembled by joining the lysins derived from Meiothermus bacteriophage MMP7 and Thermus bacteriophage TSP4 with a flexible linker via chimeolysin engineering. As a potential antimicrobial agent, MLTphg can be obtained by overproduction in Escherichia coli BL21(DE3) cells and the following Ni-affinity chromatography. Finally, we recovered about 40 ± 1.9 mg of MLTphg from 1 L of the host E. coli BL21(DE3) culture. The purified MLTphg showed peak activity against Staphylococcus aureus ATCC6538 between 35 and 40 °C, and maintained approximately 44.5 ± 2.1% activity at room temperature (25 °C). Moreover, as a produced chimera, it exhibited considerably improved bactericidal activity against Staphylococcus aureus (2.9 ± 0.1 log10 reduction was observed upon 40 nM MLTphg treatment at 37 °C for 30 min) and also a group of antibiotic-resistant bacteria compared to its parental lysins, TSPphg and MMPphg. In the current age of growing antibiotic resistance, our results provide an engineering basis for developing phage lysins as novel antimicrobial agents and shed light on bacteriophage-based strategies to tackle bacterial infections.

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