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1.
Nat Rev Genet ; 21(9): 526-540, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32533119

RESUMO

It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome-wide transposon mutagenesis with high-throughput sequencing to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome. Four TIS variations were published in 2009: transposon sequencing (Tn-Seq), transposon-directed insertion site sequencing (TraDIS), insertion sequencing (INSeq) and high-throughput insertion tracking by deep sequencing (HITS). TIS has since become an important tool for molecular microbiologists, being one of the few genome-wide techniques that directly links phenotype to genotype and ultimately can assign gene function. In this Review, we discuss the recent applications of TIS to answer overarching biological questions. We explore emerging and multidisciplinary methods that build on TIS, with an eye towards future applications.


Assuntos
Elementos de DNA Transponíveis/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Animais , Humanos
2.
Nucleic Acids Res ; 51(12): 6101-6119, 2023 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-37158230

RESUMO

Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.


Assuntos
Acinetobacter baumannii , Proteínas de Escherichia coli , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Acinetobacter baumannii/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Fator sigma/genética , Fator sigma/metabolismo , Regulação Bacteriana da Expressão Gênica
3.
Drug Resist Updat ; 66: 100911, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36592567

RESUMO

AIMS: This study examined the origins and evolution of the AdeABC, AdeFGH and AdeIJK efflux pumps in the Acinetobacter genus, including human and animal pathogens and species from non-clinical environments. METHODS: Comparative genome analyses were performed using the reference sequences for 70 Acinetobacter species to identify putative orthologs of AdeABC, AdeFGH and AdeIJK and their regulators. Sequence similarities and the genomic locations of coding sequences were correlated with phylogeny to infer modes of evolution. Intraspecies variation was assessed in species of interest using up to 236 complete genome sequences. Mutants overproducing adeIJK in A. baylyi were examined to identify regulators of this system in a non A. baumannii species. RESULTS: The results indicate that adeIJK has been a stable part of Acinetobacter genomes since the genesis of this genus, whereas adeABC and adeFGH were carried by less than half of the species, but showed some lineage specificity. The organisation and local genetic contexts of adeABC loci were particularly variable to the sub-species level, suggesting frequent recombination. Cognate regulatory systems were almost always found in the genomes of species encoding pumps. Mutations in adeN, which encodes a repressor of adeIJK, were selected by antibiotic exposure in A. baylyi, similar to previous findings in pathogenic lineages. CONCLUSIONS: The multidrug efflux capacity of clinical Acinetobacter strains stems from accessory and core genetic features. AdeIJK is likely to have ancient core function(s) that have promoted its maintenance, whereas recent antibiotic use may be driving the evolution of the AdeABC pump.


Assuntos
Acinetobacter baumannii , Proteínas de Membrana Transportadoras , Animais , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Acinetobacter baumannii/genética , Farmacorresistência Bacteriana Múltipla/genética , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana
4.
Neurobiol Dis ; 179: 106051, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36822548

RESUMO

Emerging evidence suggests the presence of bidirectional interactions between the central nervous system and gut microbiota that may contribute to the pathogenesis of neurodegenerative diseases. However, the potential role of gut microbes in forms of spinocerebellar ataxia, such as the fatal neurodegenerative disease Machado Joseph disease (MJD), remains unexplored. Here, we examined whether gut microbiota alterations may be an early disease phenotype of MJD. We profiled the gut microbiota of male and female transgenic MJD mice (CMVMJD135) expressing human ATXN3 with expanded CAG repeats (133-143 CAG) at pre-symptomatic, symptomatic and well-established stages of the disease (7, 11 and 15 weeks of age, respectively). We compared these profiles with the gut microbiota of male and female wild-type (WT) littermate control mice at same ages. Correlation network analyses were employed to explore the relevance of microbiota changes to disease progression. The results demontrated distinct sex-dependent effects in disease development whereby male MJD mice displayed earlier motor impairments than female MJD mice. The gut microbiota community structure and composition also demonstrated sex-specific differences between MJD and WT mice. In both male and female MJD mice, the shifts in the microbiota were present by 7 weeks, before the onset of any symptoms. These pre-symptomatic microbial changes correlated with the severity of neurological impairments present at later stages of the disease. Previous efforts towards developing treatments for MJD have failed to yield meaningful outcomes. Our study reports a novel relationship between the gut microbiota and MJD development and severity. Elucidating how gut microbes are involved in MJD pathogenesis may offer new and efficacious treatment strategies for this currently untreatable disease.


Assuntos
Microbioma Gastrointestinal , Doença de Machado-Joseph , Ataxias Espinocerebelares , Masculino , Humanos , Feminino , Camundongos , Animais , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/patologia , Camundongos Transgênicos , Fenótipo , Ataxina-3/genética
5.
Microbiology (Reading) ; 169(5)2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37252766

RESUMO

Genes encoding a novel multidrug efflux pump, AadT, from the Drug:H+ antiporter 2 family, were discovered in Acinetobacter multidrug resistance plasmids. Here, we profiled the antimicrobial resistance potential, and examined the distribution of these genes. aadT homologs were found in many Acinetobacter and other Gram-negative species and were typically adjacent to novel variants of adeAB(C), which encodes a major tripartite efflux pump in Acinetobacter. The AadT pump decreased bacterial susceptibility to at least eight diverse antimicrobials, including antibiotics (erythromycin and tetracycline), biocides (chlorhexidine), and dyes (ethidium bromide and DAPI) and was able to mediate ethidium transport. These results show that AadT is a multidrug efflux pump in the Acinetobacter resistance arsenal and may cooperate with variants of AdeAB(C).


Assuntos
Acinetobacter baumannii , Anti-Infecciosos , Antibacterianos/farmacologia , Proteínas de Membrana Transportadoras/genética , Farmacorresistência Bacteriana Múltipla/genética , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana
6.
BMC Microbiol ; 23(1): 137, 2023 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-37202723

RESUMO

BACKGROUND: Acinetobacter baumannii is an opportunistic human pathogen that causes a variety of infections in immunosuppressed individuals and patients in intensive care units. The success of this pathogen in nosocomial settings can be directly attributed to its persistent nature and its ability to rapidly acquire multidrug resistance. It is now considered to be one of the top priority pathogens for development of novel therapeutic approaches. Several high-throughput techniques have been utilised to identify the genetic determinants contributing to the success of A. baumannii as a global pathogen. However, targeted gene-function studies remain challenging due to the lack of appropriate genetic tools. RESULTS: Here, we have constructed a series of all-synthetic allelic exchange vectors - pALFI1, pALFI2 and pALFI3 - with suitable selection markers for targeted genetic studies in highly drug resistant A. baumannii isolates. The vectors follow the Standard European Vector Architecture (SEVA) framework for easy replacement of components. This method allows for rapid plasmid construction with the mutant allele, efficient conjugational transfer using a diaminopimelic acid-dependent Escherichia coli donor strain, efficient positive selection using the suitable selection markers and finally, sucrose-dependent counter-selection to obtain double-crossovers. CONCLUSIONS: We have used this method to create scar-less deletion mutants in three different strains of A. baumannii, which resulted in up to 75% deletion frequency of the targeted gene. We believe this method can be effectively used to perform genetic manipulation studies in multidrug resistant Gram-negative bacterial strains.


Assuntos
Acinetobacter baumannii , Antibacterianos , Humanos , Antibacterianos/farmacologia , Alelos , Plasmídeos/genética , Farmacorresistência Bacteriana Múltipla/genética , Mutagênese , Testes de Sensibilidade Microbiana
7.
Chem Rev ; 121(9): 5417-5478, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33761243

RESUMO

Bacterial multidrug efflux pumps have come to prominence in human and veterinary pathogenesis because they help bacteria protect themselves against the antimicrobials used to overcome their infections. However, it is increasingly realized that many, probably most, such pumps have physiological roles that are distinct from protection of bacteria against antimicrobials administered by humans. Here we undertake a broad survey of the proteins involved, allied to detailed examples of their evolution, energetics, structures, chemical recognition, and molecular mechanisms, together with the experimental strategies that enable rapid and economical progress in understanding their true physiological roles. Once these roles are established, the knowledge can be harnessed to design more effective drugs, improve existing microbial production of drugs for clinical practice and of feedstocks for commercial exploitation, and even develop more sustainable biological processes that avoid, for example, utilization of petroleum.


Assuntos
Antibacterianos/metabolismo , Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Resistência Microbiana a Medicamentos , Humanos , Proteínas de Membrana Transportadoras/química
8.
Environ Microbiol ; 24(12): 6071-6085, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36054310

RESUMO

Osmotic stress, caused by high or fluctuating salt concentrations, is a crucial abiotic factor affecting microbial growth in aquatic habitats. Many organisms utilize common responses to osmotic stress, generally requiring active extrusion of toxic inorganic ions and accumulation of compatible solutes to protect cellular machinery. We heterologously expressed and purified predicted osmoprotectant, proline/glycine betaine-binding proteins (ProX) from two phylogenetically distinct Synechococcus spp. MITS9220 and WH8102. Homologues of this protein are conserved only among Prochlorococcus LLIV and Synechococcus clade I, III and CRD1 strains. Our biophysical characterization show Synechococcus ProX exists as a dimer, with specificity solely for glycine betaine but not to other osmoprotectants tested. We discovered that MITS9220_ProX has a 10-fold higher affinity to glycine betaine than WH8102_ProX, which is further elevated (24-fold) in high salt conditions. The stronger affinity and effect of ionic strength on MITS9220_ProX glycine betaine binding but not on WH8102_ProX alludes to a novel regulatory mechanism, providing critical functional insights into the phylogenetic divergence of picocyanobacterial ProX proteins that may be necessary for their ecological success.


Assuntos
Betaína , Synechococcus , Betaína/metabolismo , Synechococcus/genética , Synechococcus/metabolismo , Ecótipo , Filogenia , Glicina/metabolismo
9.
Environ Microbiol ; 24(9): 4065-4078, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35437913

RESUMO

The production of methane as an end-product of organic matter degradation in the absence of other terminal electron acceptors is common, and has often been studied in environments such as animal guts, soils and wetlands due to its potency as a greenhouse gas. To date, however, the study of the biogeographic distribution of methanogens across coal seam environments has been minimal. Here, we show that coal seams are host to a diverse range of methanogens, which are distinctive to each geological basin. Based on comparisons to close relatives from other methanogenic environments, the dominant methanogenic pathway in these basins is hydrogenotrophic, with acetoclastic being a second major pathway in the Surat Basin. Finally, mcrA and 16S rRNA gene primer biases were predominantly seen to affect the detection of Methanocellales, Methanomicrobiales and Methanosarcinales taxa in this study. Subsurface coal methanogenic community distributions and pathways presented here provide insights into important metabolites and bacterial partners for in situ coal biodegradation.


Assuntos
Euryarchaeota , Gases de Efeito Estufa , Animais , Archaea/metabolismo , Carvão Mineral/microbiologia , Euryarchaeota/genética , Gases de Efeito Estufa/metabolismo , Metano/metabolismo , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Solo
10.
Microb Cell Fact ; 21(1): 190, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104783

RESUMO

BACKGROUND: Eukaryotic algae have recently emerged as hosts for metabolic engineering efforts to generate heterologous isoprenoids. Isoprenoid metabolic architectures, flux, subcellular localization, and transport dynamics have not yet been fully elucidated in algal hosts. RESULTS: In this study, we investigated the accessibility of different isoprenoid precursor pools for C15 sesquiterpenoid generation in the cytoplasm and chloroplast of Chlamydomonas reinhardtii using the Abies grandis bisabolene synthase (AgBS) as a reporter. The abundance of the C15 sesquiterpene precursor farnesyl pyrophosphate (FPP) was not increased in the cytosol by co-expression and fusion of AgBS with different FPP synthases (FPPSs), indicating limited C5 precursor availability in the cytoplasm. However, FPP was shown to be available in the plastid stroma, where bisabolene titers could be improved several-fold by FPPSs. Sesquiterpene production was greatest when AgBS-FPPS fusions were directed to the plastid and could further be improved by increasing the gene dosage. During scale-up cultivation with different carbon sources and light regimes, specific sesquiterpene productivities from the plastid were highest with CO2 as the only carbon source and light:dark illumination cycles. Potential prenyl unit transporters are proposed based on bioinformatic analyses, which may be in part responsible for our observations. CONCLUSIONS: Our findings indicate that the algal chloroplast can be harnessed in addition to the cytosol to exploit the full potential of algae as green cell factories for non-native sesquiterpenoid generation. Identification of a prenyl transporter may be leveraged for further extending this capacity.


Assuntos
Chlamydomonas reinhardtii , Microalgas , Sesquiterpenos , Carbono/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Microalgas/metabolismo , Neopreno , Plantas , Fosfatos de Poli-Isoprenil , Sesquiterpenos/metabolismo , Terpenos/metabolismo
11.
Microb Ecol ; 84(3): 780-793, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34686899

RESUMO

The global trend of transiting to more renewable energy sources requires transition fuels, such as coal seam gas, to supplement and secure energy needs. In order to optimise strategies and technologies for enhancing gas production, an understanding of the fundamental microbial processes and interactions would be advantageous. Models have recently begun mapping the microbial roles and interactions in coal seam environments, from direct coal degradation to methanogenesis. This study seeks to expand those models by observing community compositional shifts in the presence of differing organic matter by conducting 16S rRNA microbial surveys using formation water from the Surat and Sydney Basins grown on varying types of organic matter (black and brown coal, oil shale, humic acid, and lignin). A total of 135 microbes were observed to become enriched in the presence of added organic matter in comparison to carbon-free treatments. These surveys allowed detailed analysis of microbial compositions in order to extrapolate which taxa favour growth in the presence of differing organic matter. This study has experimentally demonstrated shifts in the microbial community composition due to differing carbon sources and, for the first time, generated a conceptual model to map putative degradation pathways regarding subsurface microbial consortia.


Assuntos
Carvão Mineral , Microbiota , RNA Ribossômico 16S/genética , Metano/metabolismo , Consórcios Microbianos/genética
12.
Proc Natl Acad Sci U S A ; 116(36): 18015-18020, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31416917

RESUMO

Acinetobacter baumannii has rapidly emerged as a major cause of gram-negative hospital infections worldwide. A. baumannii encodes for the transport protein AceI, which confers resistance to chlorhexidine, a widely used antiseptic. AceI is also the prototype for the recently discovered proteobacterial antimicrobial compound efflux (PACE) family of transport proteins that confer resistance to a range of antibiotics and antiseptics in many gram-negative bacteria, including pathogens. The gene encoding AceI is conserved in the core genome of A. baumannii, suggesting that it has an important primordial function. This is incongruous with the sole characterized substrate of AceI, chlorhexidine, an entirely synthetic biocide produced only during the last century. Here we investigated a potential primordial function of AceI and other members of the PACE family in the transport of naturally occurring polyamines. Polyamines are abundant in living cells, where they have physiologically important functions and play multifaceted roles in bacterial infection. Gene expression studies revealed that the aceI gene is induced in A. baumannii by the short-chain diamines cadaverine and putrescine. Membrane transport experiments conducted in whole cells of A. baumannii and Escherichia coli and also in proteoliposomes showed that AceI mediates the efflux of these short-chain diamines when energized by an electrochemical gradient. Assays conducted using 8 additional diverse PACE family proteins identified 3 that also catalyze cadaverine transport. Taken together, these results demonstrate that short-chain diamines are common substrates for the PACE family of transport proteins, adding to their broad significance as a novel family of efflux pumps.


Assuntos
Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Diaminas , Farmacorresistência Bacteriana , Proteínas de Membrana Transportadoras , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clorexidina/farmacologia , Diaminas/química , Diaminas/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo
13.
J Bacteriol ; 203(7)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33257523

RESUMO

Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacterium that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used for this species. In this study, we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses, and transport were underrepresented. The majority of Pf-5 essential genes were part of the P. protegens core genome. Comparison of the essential gene set of Pf-5 with those of two plant-associated pseudomonads, P. simiae and P. syringae, and the well-studied opportunistic human pathogen P. aeruginosa PA14 showed that the four species share a large number of essential genes, but each species also had uniquely essential genes. Comparison of the Pf-5 in silico-predicted and in vitro-determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles may improve our understanding of the biological processes important for bacterial survival and growth.IMPORTANCE Essential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analyses of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of three other pseudomonads highlights how gene essentiality varies between closely related species.


Assuntos
Proteínas de Bactérias/genética , Genes Essenciais , Pseudomonas/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , Biblioteca Gênica , Genoma Bacteriano , Mutagênese Insercional , Plantas/microbiologia , Pseudomonas/metabolismo
14.
Ecol Lett ; 24(7): 1487-1504, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33896087

RESUMO

Bacteria and archaea have very different ecology compared to plants. One similarity, though, is that much discussion of their ecological strategies has invoked concepts such as oligotrophy or stress tolerance. For plants, so-called 'trait ecology'-strategy description reframed along measurable trait dimensions-has made global syntheses possible. Among widely measured trait dimensions for bacteria and archaea three main axes are evident. Maximum growth rate in association with rRNA operon copy number expresses a rate-yield trade-off that is analogous to the acquisitive-conservative spectrum in plants, though underpinned by different trade-offs. Genome size in association with signal transduction expresses versatility. Cell size has influence on diffusive uptake and on relative wall costs. These trait dimensions, and potentially others, offer promise for interpreting ecology. At the same time, there are very substantial differences from plant trait ecology. Traits and their underpinning trade-offs are different. Also, bacteria and archaea use a variety of different substrates. Bacterial strategies can be viewed both through the facet of substrate-use pathways, and also through the facet of quantitative traits such as maximum growth rate. Preliminary evidence shows the quantitative traits vary widely within substrate-use pathways. This indicates they convey information complementary to substrate use.


Assuntos
Archaea , Ecologia , Archaea/genética , Bactérias/genética , Fenótipo , Plantas
15.
Artigo em Inglês | MEDLINE | ID: mdl-33820764

RESUMO

Fluoroquinolones are one of the most prescribed broad-spectrum antibiotics. However, their effectiveness is being compromised by high rates of resistance in clinically important organisms, including Acinetobacter baumannii We sought to investigate the transcriptomic and proteomic responses of the clinical A. baumannii strain AB5075-UW upon exposure to subinhibitory concentrations of ciprofloxacin. Our transcriptomics and proteomics analyses found that the most highly expressed genes and proteins were components of the intact prophage phiOXA. The next most highly expressed gene (and its protein product) under ciprofloxacin stress was a hypothetical gene, ABUW_0098, named here the Acinetobacterciprofloxacin tolerance (aciT) gene. Disruption of this gene resulted in higher susceptibility to ciprofloxacin, and complementation of the mutant with a cloned aciT gene restored ciprofloxacin tolerance to parental strain levels. Microscopy studies revealed that aciT is essential for filamentation during ciprofloxacin stress in A. baumannii Sequence analysis of aciT indicates the encoded protein is likely to be localized to the cell membrane. Orthologs of aciT are found widely in the genomes of species from the Moraxellaceae family and are well conserved in Acinetobacter species, suggesting an important role. With these findings taken together, this study has identified a new gene conferring tolerance to ciprofloxacin, likely by enabling filamentation in response to the antibiotic.


Assuntos
Acinetobacter baumannii , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Testes de Sensibilidade Microbiana , Proteômica
16.
Biochem Soc Trans ; 49(6): 2465-2481, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34882230

RESUMO

Marine cyanobacteria are key primary producers, contributing significantly to the microbial food web and biogeochemical cycles by releasing and importing many essential nutrients cycled through the environment. A subgroup of these, the picocyanobacteria (Synechococcus and Prochlorococcus), have colonised almost all marine ecosystems, covering a range of distinct light and temperature conditions, and nutrient profiles. The intra-clade diversities displayed by this monophyletic branch of cyanobacteria is indicative of their success across a broad range of environments. Part of this diversity is due to nutrient acquisition mechanisms, such as the use of high-affinity ATP-binding cassette (ABC) transporters to competitively acquire nutrients, particularly in oligotrophic (nutrient scarce) marine environments. The specificity of nutrient uptake in ABC transporters is primarily determined by the peripheral substrate-binding protein (SBP), a receptor protein that mediates ligand recognition and initiates translocation into the cell. The recent availability of large numbers of sequenced picocyanobacterial genomes indicates both Synechococcus and Prochlorococcus apportion >50% of their transport capacity to ABC transport systems. However, the low degree of sequence homology among the SBP family limits the reliability of functional assignments using sequence annotation and prediction tools. This review highlights the use of known SBP structural representatives for the uptake of key nutrient classes by cyanobacteria to compare with predicted SBP functionalities within sequenced marine picocyanobacteria genomes. This review shows the broad range of conserved biochemical functions of picocyanobacteria and the range of novel and hypothetical ABC transport systems that require further functional characterisation.


Assuntos
Proteínas de Transporte/metabolismo , Cianobactérias/metabolismo , Nutrientes/metabolismo , Água do Mar/microbiologia , Proteínas de Transporte/química , Metais/metabolismo , Nitrogênio/metabolismo , Fósforo/metabolismo , Conformação Proteica , Oligoelementos/metabolismo
17.
Appl Environ Microbiol ; 87(22): e0171821, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34495707

RESUMO

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, in soil, and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Metal ion homeostasis in the hospital pathogen Acinetobacter baumannii contributes to pathogenesis; however, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. We also show that the CzcCBA heavy metal efflux system contributes to cadmium efflux. Collectively, these systems provide A. baumannii with a comprehensive cadmium translocation pathway from the cytoplasm to the periplasm and subsequently the extracellular space. Furthermore, analysis of the A. baumannii metallome under cadmium stress showed zinc depletion, as well as copper enrichment, both of which are likely to influence cellular fitness. Overall, this work provides new knowledge on the role of a broad arsenal of membrane transporters in A. baumannii metal ion homeostasis. IMPORTANCE Cadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, including Acinetobacter baumannii, which is notorious for persisting in harsh environments. Here, we show that A. baumannii utilizes a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be a primary means by which cadmium exerts toxicity upon the bacterium.


Assuntos
Acinetobacter baumannii , Cádmio , Farmacorresistência Bacteriana/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Transporte Biológico , Cádmio/toxicidade , Proteínas de Membrana Transportadoras/genética , Zinco/metabolismo
18.
Biotechnol Bioeng ; 118(2): 647-658, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33022743

RESUMO

Yeast has been engineered for cost-effective organic acid production through metabolic engineering and synthetic biology techniques. However, cell growth assays in these processes were performed in bulk at the population level, thus obscuring the dynamics of rare single cells exhibiting beneficial traits. Here, we introduce the use of monodisperse picolitre droplets as bioreactors to cultivate yeast at the single-cell level. We investigated the effect of acid stress on growth and the effect of potassium ions on propionic acid tolerance for single yeast cells of different species, genotypes, and phenotypes. The results showed that the average growth of single yeast cells in microdroplets experiences the same trend to those of yeast populations grown in bulk, and microdroplet compartments do not significantly affect cell viability. This approach offers the prospect of detecting cell-to-cell variations in growth and physiology and is expected to be applied for the engineering of yeast to produce value-added bioproducts.


Assuntos
Saccharomyces cerevisiae/crescimento & desenvolvimento , Engenharia Metabólica
19.
Trends Genet ; 38(2): 209-210, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34711424

Assuntos
Clorófitas
20.
Microb Ecol ; 80(1): 34-46, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31828390

RESUMO

Diverse microbial communities living in subsurface coal seams are responsible for important geochemical processes including the movement of carbon between the geosphere, biosphere and atmosphere. Microbial conversion of the organic matter in coal to methane involves a complex assemblage of bacteria and archaea working in syntrophic relationships. Despite the importance and value of this microbial process, very few of the microbial taxa have defined metabolic or ecological roles in these environments. Additionally, the genomic features mediating life in this chemically reduced, energy poor, deep subsurface environment are not well characterised. Here we describe the isolation and genomic and catabolic characterisation of three alphaproteobacterial Stappia indica species from three coal basins across Australia. S. indica genomes from coal seams were compared with those from closely related S. indica isolated from diverse surface waters, revealing a coal seam-specific suite of genes associated with life in the subsurface. These genes are linked to processes including viral defence, secondary metabolite production, polyamine metabolism, polypeptide uptake membrane transporters and putative energy neutral pressure-dependent CO2 fixation. This indicates that subsurface Stappia have diverse metabolisms for biomass recycling and pressure-dependent CO2 fixation and require a suite of defensive and competitive strategies relative to their surface-dwelling relatives.


Assuntos
Ciclo do Carbono , Água Doce/microbiologia , Genoma Bacteriano , Sedimentos Geológicos/microbiologia , Rhodobacteraceae/fisiologia , Biomassa , Carvão Mineral , Mineração , New South Wales , Queensland , Rhodobacteraceae/genética , Rhodobacteraceae/isolamento & purificação , Especificidade da Espécie
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