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MBio May 2015Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require...
UNLABELLED
Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling.
IMPORTANCE
A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.
Topics: Base Sequence; Chromosome Mapping; DNA Barcoding, Taxonomic; DNA Transposable Elements; Escherichia coli; Gene Library; Genetic Fitness; High-Throughput Nucleotide Sequencing; Mutagenesis, Insertional; Mutation; Phenotype; Pseudomonas; Reproducibility of Results; Rhodobacteraceae; Shewanella
PubMed: 25968644
DOI: 10.1128/mBio.00306-15 -
MSphere Apr 2022Mobile genetic elements (MGEs) drive bacterial evolution, alter gene availability within microbial communities, and facilitate adaptation to ecological niches. In...
Mobile genetic elements (MGEs) drive bacterial evolution, alter gene availability within microbial communities, and facilitate adaptation to ecological niches. In natural systems, bacteria simultaneously possess or encounter multiple MGEs, yet their combined influences on microbial communities are poorly understood. Here, we investigate interactions among MGEs in the marine bacterium Sulfitobacter pontiacus. Two related strains, CB-D and CB-A, each harbor a single prophage. These prophages share high sequence identity with one another and an integration site within the host genome, yet these strains exhibit differences in "spontaneous" prophage induction (SPI) and consequent fitness. To better understand mechanisms underlying variation in SPI between these lysogens, we closed their genomes, which revealed that in addition to harboring different prophage genotypes, CB-A lacks two of the four large, low-copy-number plasmids possessed by CB-D. To assess the relative roles of plasmid content versus prophage genotype on host physiology, a panel of derivative strains varying in MGE content were generated. Characterization of these derivatives revealed a robust link between plasmid content and SPI, regardless of prophage genotype. Strains possessing all four plasmids had undetectable phage in cell-free lysates, while strains lacking either one plasmid (pSpoCB-1) or a combination of two plasmids (pSpoCB-2 and pSpoCB-4) produced high (>10 PFU/mL) phage titers. Homologous plasmid sequences were identified in related bacteria, and plasmid and phage genes were found to be widespread in Oceans metagenomic data sets. This suggests that plasmid-dependent stabilization of prophages may be commonplace throughout the oceans. The consequences of prophage induction on the physiology of microbial populations are varied and include enhanced biofilm formation, conferral of virulence, and increased opportunity for horizontal gene transfer. These traits lead to competitive advantages for lysogenized bacteria and influence bacterial lifestyles in a variety of niches. However, biological controls of "spontaneous" prophage induction, the initiation of phage replication and phage-mediated cell lysis without an overt stressor, are not well understood. In this study, we observed a novel interaction between plasmids and prophages in the marine bacterium Sulfitobacter pontiacus. We found that loss of one or more distinct plasmids-which we show carry genes ubiquitous in the world's oceans-resulted in a marked increase in prophage induction within lysogenized strains. These results demonstrate cross talk between different mobile genetic elements and have implications for our understanding of the lysogenic-lytic switches of prophages found not only in marine environments, but throughout all ecosystems.
Topics: Bacteriophages; Ecosystem; Plasmids; Prophages; Rhodobacteraceae
PubMed: 35311569
DOI: 10.1128/msphere.00930-21 -
Applied and Environmental Microbiology Apr 2018Members of the genus have been isolated worldwide from a great variety of marine sources as both free-living and host-associated bacteria. So far, the available data... (Review)
Review
Members of the genus have been isolated worldwide from a great variety of marine sources as both free-living and host-associated bacteria. So far, the available data depict a group of alphaproteobacteria characterized by a versatile metabolism, which allows them to use a variety of substrates to meet their carbon, nitrogen, sulfur, and phosphorous requirements. Additionally, -related bacteria have been shown to proliferate under extreme oligotrophic conditions, tolerate high heavy-metal concentrations, and metabolize potentially toxic compounds. Considering this versatility, it is not surprising that they have been detected from temperate to tropical regions and are often the most abundant isolates obtained from marine invertebrates. Such an association is particularly recurrent with marine sponges and corals, animals that play a key role in benthic marine systems. The data so far available indicate that these bacteria are mainly beneficial to the host, and besides being involved in major nutrient cycles, they could provide the host with both vitamins/cofactors and protection from potential pathogens via the synthesis of antimicrobial secondary metabolites. In fact, the biosynthetic abilities of spp. have been emerging in recent years, and both genomic and analytic studies have underlined how these organisms promise novel natural products of biotechnological value.
Topics: Animals; Anthozoa; Biotechnology; Environment; Porifera; Rhodobacteraceae; Symbiosis
PubMed: 29453252
DOI: 10.1128/AEM.02516-17 -
Genome Biology and Evolution Aug 2019In Bacteria, chromosome replication starts at a single origin of replication and proceeds on both replichores. Due to its asymmetric nature, replication influences...
In Bacteria, chromosome replication starts at a single origin of replication and proceeds on both replichores. Due to its asymmetric nature, replication influences chromosome structure and gene organization, mutation rate, and expression. To date, little is known about the distribution of highly conserved genes over the bacterial chromosome. Here, we used a set of 101 fully sequenced Rhodobacteraceae representatives to analyze the relationship between conservation of genes within this family and their distance from the origin of replication. Twenty-two of the analyzed species had core genes clustered significantly closer to the origin of replication with representatives of the genus Celeribacter being the most apparent example. Interestingly, there were also eight species with the opposite organization. In particular, Rhodobaca barguzinensis and Loktanella vestfoldensis showed a significant increase of core genes with distance from the origin of replication. The uneven distribution of low-conserved regions is in particular pronounced for genomes in which the halves of one replichore differ in their conserved gene content. Phage integration and horizontal gene transfer partially explain the scattered nature of Rhodobacteraceae genomes. Our findings lay the foundation for a better understanding of bacterial genome evolution and the role of replication therein.
Topics: Bacterial Proteins; Chromosomes, Bacterial; DNA Replication; Gene Expression Regulation, Bacterial; Genome, Bacterial; Phylogeny; Rhodobacteraceae
PubMed: 31273387
DOI: 10.1093/gbe/evz138 -
The ISME Journal Nov 2021Coral mucus, tissue, and skeleton harbor compositionally different microbiota, but how these coral compartments shape the microbial evolution remains unexplored. Here,...
Coral mucus, tissue, and skeleton harbor compositionally different microbiota, but how these coral compartments shape the microbial evolution remains unexplored. Here, we sampled bacteria inhabiting a prevalent coral species Platygyra acuta and sequenced genomes of 234 isolates comprising two populations in Rhodobacteraceae, an alphaproteobacterial lineage representing a significant but variable proportion (5-50%) of the coral microbiota. The Ruegeria population (20 genomes) contains three clades represented by eight, six, and six isolates predominantly sampled from the skeleton (outgroup), mucus (clade-M), and skeleton (clade-S), respectively. The clade-M possesses functions involved in the utilization of coral osmolytes abundant in the mucus (e.g., methylamines, DMSP, taurine, and L-proline), whereas the clade-S uniquely harbors traits that may promote adaptation to the low-energy and diurnally anoxic skeleton (e.g., sulfur oxidation and swimming motility). These between-clade genetic differences were largely supported by physiological assays. Expanded analyses by including genomes of 24 related isolates (including seven new genomes) from other marine environments suggest that clade-M and clade-S may have diversified in non-coral habitats, but they also consolidated a key role of distinct coral compartments in diversifying many of the above-mentioned traits. The unassigned Rhodobacteraceae population (214 genomes) varies only at a few dozen nucleotide sites across the whole genomes, but the number of between-compartment migration events predicted by the Slatkin-Maddison test supported that dispersal limitation between coral compartments is another key mechanism diversifying microbial populations. Collectively, our results suggest that different coral compartments represent ecologically distinct and microgeographically separate habitats that drive the evolution of the coral microbiota.
Topics: Animals; Anthozoa; Microbiota; Rhodobacteraceae
PubMed: 34017056
DOI: 10.1038/s41396-021-01009-6 -
Phylogenomics of Rhodobacteraceae reveals evolutionary adaptation to marine and non-marine habitats.The ISME Journal Jun 2017Marine Rhodobacteraceae (Alphaproteobacteria) are key players of biogeochemical cycling, comprise up to 30% of bacterial communities in pelagic environments and are...
Marine Rhodobacteraceae (Alphaproteobacteria) are key players of biogeochemical cycling, comprise up to 30% of bacterial communities in pelagic environments and are often mutualists of eukaryotes. As 'Roseobacter clade', these 'roseobacters' are assumed to be monophyletic, but non-marine Rhodobacteraceae have not yet been included in phylogenomic analyses. Therefore, we analysed 106 genome sequences, particularly emphasizing gene sampling and its effect on phylogenetic stability, and investigated relationships between marine versus non-marine habitat, evolutionary origin and genomic adaptations. Our analyses, providing no unequivocal evidence for the monophyly of roseobacters, indicate several shifts between marine and non-marine habitats that occurred independently and were accompanied by characteristic changes in genomic content of orthologs, enzymes and metabolic pathways. Non-marine Rhodobacteraceae gained high-affinity transporters to cope with much lower sulphate concentrations and lost genes related to the reduced sodium chloride and organohalogen concentrations in their habitats. Marine Rhodobacteraceae gained genes required for fucoidan desulphonation and synthesis of the plant hormone indole 3-acetic acid and the compatible solutes ectoin and carnitin. However, neither plasmid composition, even though typical for the family, nor the degree of oligotrophy shows a systematic difference between marine and non-marine Rhodobacteraceae. We suggest the operational term 'Roseobacter group' for the marine Rhodobacteraceae strains.
Topics: Acclimatization; Base Sequence; DNA, Bacterial; Ecosystem; Evolution, Molecular; Molecular Sequence Data; Phylogeny; Rhodobacteraceae
PubMed: 28106881
DOI: 10.1038/ismej.2016.198 -
MBio Mar 2020Dimethylsulfoniopropionate (DMSP) is abundant in marine environments and an important source of reduced carbon and sulfur for marine bacteria. While both and possessed...
Dimethylsulfoniopropionate (DMSP) is abundant in marine environments and an important source of reduced carbon and sulfur for marine bacteria. While both and possessed genes encoding the DMSP demethylation and cleavage pathways, their responses to DMSP differed. A glucose-fed, chemostat culture of consumed 99% of the DMSP even when fed a high concentration of 5 mM. At the same time, cultures released 19% and 7.1% of the DMSP as dimethylsulfide (DMS) and methanethiol, respectively. Under the same conditions, consumed only 28% of the DMSP and formed one-third of the amount of gases. To examine the pathways of sulfur and methyl C assimilation, glucose-fed chemostats of both species were fed 100 μM mixtures of unlabeled and doubly labeled [-C, S]DMSP. Both species derived nearly all of their sulfur from DMSP despite high sulfate availability. In addition, only 33% and 50% of the methionine was biosynthesized from the direct capture of methanethiol in and , respectively. The remaining methionine was biosynthesized by the random assembly of free sulfide and methyl-tetrahydrofolate derived from DMSP. Thus, although the two species possessed similar genes encoding DMSP metabolism, their growth responses were very different. Dimethylsulfoniopropionate (DMSP) is abundant in marine environments and an important source of reduced carbon and sulfur for marine bacteria. DMSP is the precursor for the majority of atmospheric dimethylsulfide (DMS), a climatically active gas that connects the marine and terrestrial sulfur cycles. Although research into the assimilation of DMSP has been conducted for over 20 years, the fate of DMSP in microbial biomass is not well understood. In particular, the biosynthesis of methionine from DMSP has been a focal point, and it has been widely believed that most methionine was synthesized via the direct capture of methanethiol. Using an isotopic labeling strategy, we have demonstrated that the direct capture of methanethiol is not the primary pathway used for methionine biosynthesis in two species, a genus comprised primarily of globally abundant marine bacteria. Furthermore, although the catabolism of DMSP by these species varied greatly, the anabolic pathways were highly conserved.
Topics: Carbon; Glucose; Methionine; Rhodobacteraceae; Sulfhydryl Compounds; Sulfonium Compounds; Sulfur
PubMed: 32209679
DOI: 10.1128/mBio.00329-20 -
Chemical Reviews Mar 2018This review focuses on the type A cytochrome c oxidases (C cO), which are found in all mitochondria and also in several aerobic bacteria. C cO catalyzes the respiratory... (Review)
Review
This review focuses on the type A cytochrome c oxidases (C cO), which are found in all mitochondria and also in several aerobic bacteria. C cO catalyzes the respiratory reduction of dioxygen (O) to water by an intriguing mechanism, the details of which are fairly well understood today as a result of research for over four decades. Perhaps even more intriguingly, the membrane-bound C cO couples the O reduction chemistry to translocation of protons across the membrane, thus contributing to generation of the electrochemical proton gradient that is used to drive the synthesis of ATP as catalyzed by the rotary ATP synthase in the same membrane. After reviewing the structure of the core subunits of C cO, the active site, and the transfer paths of electrons, protons, oxygen, and water, we describe the states of the catalytic cycle and point out the few remaining uncertainties. Finally, we discuss the mechanism of proton translocation and the controversies in that area that still prevail.
Topics: Adenosine Triphosphate; Biocatalysis; Electron Transport Complex IV; Electrons; Oxidation-Reduction; Oxygen; Protein Subunits; Protons; Rhodobacteraceae; Water
PubMed: 29350917
DOI: 10.1021/acs.chemrev.7b00664 -
Applied and Environmental Microbiology Apr 2019LZB033 (), which produces dimethylsulfoniopropionate (DMSP) and reduces nitrate to nitrogen, was isolated from seawater of the East China Sea. Its genome encodes a...
LZB033 (), which produces dimethylsulfoniopropionate (DMSP) and reduces nitrate to nitrogen, was isolated from seawater of the East China Sea. Its genome encodes a large number of transcriptional regulators which may be important for its adaptation to diverse marine environments. The alternative σ factor (RpoN) is a central regulator of many bacteria, regulating the transcription of multiple genes and controlling important cellular functions. However, the exact role of RpoN in spp. is unknown. In this study, an in-frame deletion mutant was constructed in LZB033, and the function of RpoN was determined. To systematically identify RpoN-controlled genes, we performed a detailed analysis of gene expression differences between the wild-type strain and the Δ mutant using RNA sequencing. The expression of 175 genes was shown to be controlled by RpoN. Subsequent phenotypic assays showed that the Δ mutant was attenuated in flagellar biosynthesis and swimming motility, utilized up to 13 carbon substrates differently, lacked the ability to assimilate malic acid, and displayed markedly decreased biofilm formation. In addition, stress response assays showed that the Δ mutant was impaired in the ability to survive under different challenge conditions, including osmotic stress, oxidative stress, temperature changes, and acid stress. Moreover, both the DMSP synthesis and catabolism rates of LZB033 decreased after was knocked out. Our work provides essential insight into the regulatory function of RpoN, revealing that RpoN is a key determinant for LZB033 flagellar formation, motility, biofilm formation, and environmental fitness, as well as DMSP production and degradation. This study established an in-frame gene deletion method in the alphaproteobacterium LZB033 and generated an gene mutant. A comparison of the transcriptomes and phenotypic characteristics between the mutant and wild-type strains confirmed the role of RpoN in LZB033 flagellar formation, motility, biofilm formation, and carbon usage. Most importantly, RpoN is a key factor for survival under different environmental challenge conditions. Furthermore, the ability to synthesize and metabolize dimethylsulfoniopropionate (DMSP) was related to RpoN. These features revealed RpoN to be an important regulator of stress resistance and survival for LZB033 in marine environments.
Topics: Adaptation, Physiological; Bacterial Proteins; Biofilms; China; Flagella; Gene Expression Regulation, Bacterial; Gene Knockout Techniques; Osmotic Pressure; Oxidative Stress; RNA Polymerase Sigma 54; RNA, Bacterial; Rhodobacteraceae; Sequence Analysis, RNA; Sulfonium Compounds; Temperature; Transcriptome
PubMed: 30709822
DOI: 10.1128/AEM.02844-18 -
ELife Dec 2021Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (P) ATPases...
Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (P) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the P-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here, we present structures and complementary functional analyses of an archetypal P-ATPase, sCoaT from sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy-metal-binding domains (HMBDs), and provide fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turnover of P-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in for example drug discovery, since P-ATPases function as virulence factors in many pathogens.
Topics: Binding Sites; Biological Transport; Cation Transport Proteins; Ions; Metals, Heavy; Models, Molecular; P-type ATPases; Protein Conformation; Rhodobacteraceae; Zinc
PubMed: 34951590
DOI: 10.7554/eLife.73124