Search bioRxivSearch

SEARCH · Search bioRxiv

Results for “Microbiology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources

Characteristics of a Block to Entry via an HIV-2 Envelope, MCR

Successful viral infection depends not only on targeting the correct cell type, but also on the route taken into the cell. After entry, a retrovirus must reverse transcribe its genome and access the nucleus. Blocks to infection can arise at many different stages of the cycle; if they occur only in some cell types it can reveal hitherto unknown aspects of viral or cellular biology. A block to infection of a primary isolate of HIV-2 was previously identified in specific cell types. In this study, parameters of route of entry of the envelope protein from this primary isolate, MCR, were investigated. A critical component of the block acts at a pre-reverse transcription stage, as virions pseudotyped with MCR envelope did not undergo fusion and entry rates commensurate with productive infection. Furthermore, expression of p56lck, which regulates CD4 surface expression, partially rescued infection of MCR envelope-pseudotyped virus in restrictive cell types. Based on these findings, we propose that a part of this block results from poor cell-surface expression of CD4.

microbiology

Replication of early and recent Zika virus isolates throughout mouse brain development

Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus associated injury. Therefore, a critical aspect of ZIKV induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and mid-gestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly-elongated basal processes of the radial glial progenitor cells, and impairment of postmitotic neuronal migration were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.\n\nSignificanceZika virus infection has been associated with multiple pathologies of the central nervous system (CNS) including microcephaly, Guillain-Barre syndrome, lissencephaly, the loss of white and grey matter volume and acute myelitis. Using organotypic brain slice cultures, we determined that ZIKV replicates across different embryonic developmental stages, and viral infection can disrupt proper brain development leading to congenital CNS complications. These data illustrate that all lineages of ZIKV tested are neurotropic, and that infection may disrupt neuronal migration during brain development. The results expand our understanding of neuropathologies associated with congenital Zika virus syndrome.

microbiology

Translated Blast of L Polymerase as a Hit for Novel Arenaviruses Species

Many pathogenic viruses can transmit between human and animals as zoonotic viruses and cause dangerous diseases with obvious clinical signs globally. However, the world deals seriously with these viruses when the viruses infected either human or animals especially if the infection were confirmed that classified as zoonotic (Lal et al. 2005). There are many viruses distribution in many countries around the world including Ebolavirus, Marburgvirus, SARS and MERS coronaviruses, Hendra, Nipah and arenavirus haemorrhagic fever viruses were categorized as zoonotic RNA viruses that cause epidemic in some regions such as African countries (Fichet-Calvet & Rogers 2009) (Ehichioya et al. 2010). Consequently, structural bioinformatics of virus protein like L polymerase of arenaviruses was used for monitor the future outbreak that could be happens by new species of viruses. At this research, significant similarities with hemorrhagic fever viruses including arenaviruses were found on GenBank database. Translated blast (tBLASTn) available on https://blast.ncbi.nlm.nih.gov/Blast.cgi was used for searching translated nucleotide databases using a protein query of arenavirus L polymerase (McGinnis & Madden 2004). At this research, the new and archival metazoan transcriptome sequence data of the new TSA species that available on NCBI was used for identification with arenaviruses genes. Therefore, structure bioinformatics was utilized for better understanding and predication the evolution and natural history of the pools of uncharacterized virus on Genbank database that have led to emerging haemorrhagic fever in near future around the world.

microbiology

Metagenomic sequencing to replace semi-quantitative urine culture for detection of urinary tract infections: a proof of concept

ABSTRACTSemi-quantitative bacterial culture is the standard method to diagnose urinary tract infections (UTI), but bacterial growth rate limits diagnostic speed and it is unreliable when patients have been pre-treated with antibiotics. Metagenomics could increase diagnostic speed and accuracy by sequencing the microbiome and resistome directly from urine samples, bypassing culture. However, a semi-quantitative approach - as needed for diagnosing UTIs - has not been established.\n\nMetagenomics was deployed to identify and semi-quantify bacterial presence indicative of UTI, predict antimicrobial susceptibility (AMR), and results were compared to semi-quantitative culture. Whole genome sequencing of the corresponding uropathogens was done for comparison. Analysis time and cost were tracked.\n\nForty-one consecutive urine samples underwent metagenomic analysis. All culture positive samples contained >200ng of DNA, suggestive of a threshold below which UTI could be ruled out solely based on DNA quantity. A semi-quantitative Diagnostic Index (DI) was created by multiplying the total DNA quantity by the relative abundance of uropathogens per urine sample. The DI allowed discrimination of UTI from non-UTI samples in all but 1 case. Metagenomic detection of AMR determinants correctly predicted the phenotype of uropathogens in 20 of 32 cases. The metagenomic work-flow was 31h and cost {euro}116 per sample, but could be reduced to 4.5h and {euro}5 for low-DNA-yield non-UTI samples.\n\nThe genomic determinants of AMR and their distribution across uropathogens need to be better understood for prediction of AMR phenotypes by metagenomics. The introduction of the DI demonstrates the potential of semi-quantitative metagenomics to replace culture as rapid diagnostic method for UTI.

microbiology

Labeling of prokaryotic mRNA in live cells using fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB)

High-throughput sequencing and cellular imaging have expanded our knowledge of microbial diversity and expression of cellular activity. However, it remains challenging to characterize low-abundance, slow-growing microorganisms that play key roles in biogeochemical cycling. With the goal of isolating transcriptionally active cells of these microorganisms from environmental samples, we developed fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB) to label living prokaryotic cells. FISH-TAMB utilizes polyarginine cell-penetrating peptides to deliver molecular beacons across cell walls and membranes. Target cells are fluorescently labeled via hybridization between molecular beacons and messenger RNA of targeted functional genes. FISH-TAMBs target specificity and deliverance into both bacterial and archaeal cells were demonstrated by labeling intracellular methyl-coenzyme M reductase A (mcrA) transcripts expressed by Escherichia coli mcrA+, Methanosarcina barkeri, and a methanogenic enrichment of deep continental fracture fluid. Growth curve analysis supported sustained cellular viability following FISH-TAMB treatment. Flow cytometry and confocal microscopy detected labeled single cells and single cells in aggregates with unlabeled cells. As FISH-TAMB is amenable to target any functional gene of interest, when coupled with cell sorting, imaging, and sequencing techniques, FISH-TAMB will enable characterization of key uncharacterized rare biosphere microorganisms and of the syntrophically activated metabolic pathways between physically associated microorganisms.

microbiology

Identification and kinetics characterization of a wax ester hydrolase from a feather-degrading actinomycete

Streptomyces fradiae var. k11 is a Gram-positive soil microorganism capable of degrading chicken feathers. Apart from being mostly protein, chicken feathers have a considerable level of lipids, with wax esters being the largest lipid class. The waxes may pose a challenge while rendering the feathers into coproducts, such as feather meal, and so the identification of a wax-ester hydrolase is warranted. A draft genome sequence of S. fradiae var. k11 was used to identify 14 gene sequences of potential lipid-degrading enzymes. The genes were expressed in E. coli BL21(DE3) cells on a pET vector and screened for activity. Four of the 14 enzymes had detectable activity, with two of the enzymes, SFK3309 and SFK3087, active against p-nitrophenyl palmitate, a representative water-insoluble substrate. A modified enzymatic assay was designed to measure activity against three model wax substrates: jojoba oil, beeswax, and cetyl-palmitate. SFK3309 was characterized to hydrolyze all three wax substrates. Kinetic experiments for SFK3309 were performed with cetyl-palmitate at 37{degrees}C, pH 8.0. The Km was determined to be 850 {micro}M and the Kcat was 11.63 s-1. Through the characterization of SFK3309 as a wax-ester hydrolase, biotechnological implications of wax ester hydrolases in the rendering of many industrial wastes can be substantiated for further studies.

microbiology

Acute Hepatitis E Virus infection in two geographical regions of Nigeria

Hepatitis E virus (HEV) remains a major public health concern in resource limited regions of the world. Yet data reporting is suboptimal and surveillance system inadequate. In Nigeria, there is dearth of information on prevalence of acute HEV infection. This study was therefore designed to describe acute HEV infection among antenatal clinic attendees and asymptomatic community dwellers from two geographical regions in Nigeria.\n\nIn this study 750 plasma samples were tested for HEV IgM by Enzyme Linked lmmunosorbent Assay (ELISA) technique. The tested samples were randomly selected from a pool of 1,115 samples previously collected from selected populations (pregnant women - 272, Oyo community dwellers - 438, Anambra community dwellers - 405) for viral hepatitis studies between September 2012 and August 2013.\n\nOne (0.4%) pregnant woman in her 3rd trimester had detectable HEV IgM, while community dwellers from the two study locations had zero prevalence rates of HEV IgM.\n\nDetection of HEV IgM in a pregnant woman, especially in her 3rd trimester is of clinical and epidemiological significance. The need therefore exists for establishment of a robust HEV surveillance system in Nigeria, and especially amidst the pregnant population in a bid to improve maternal and child health.

microbiology

Global transcriptome analysis of Aedes aegypti mosquitoes in response to Zika virus infection

Zika virus (ZIKV) of the Flaviviridae family is a recently emerged mosquito-borne virus that has been implicated in the surge of the number of microcephaly instances in South America. The recent rapid spread of the virus led to its declaration as a global health emergency by the World Health Organization. The virus is transmitted mainly by the mosquito Aedes aegypti that also vectors dengue virus, however little is known about the interactions of the virus with the mosquito vector. In this study, we investigated the transcriptome profiles of whole Ae. aegypti mosquitoes in response to ZIKV infection at 2, 7, and 14 days post-infection using RNA-Seq. Results showed changes in the abundance of a large number of transcripts at each time point following infection, with 18 transcripts commonly changed among the three time points. Gene ontology analysis revealed that most of the altered genes are involved in metabolic process, cellular process and proteolysis. In addition, 486 long intergenic non-coding RNAs were identified that were altered upon ZIKV infection. Further, we found correlational changes of a number of potential mRNA target genes with that of altered host microRNAs. The outcomes provide a basic understanding of Ae. aegypti responses to ZIKV and helps to determine host factors involved in replication or mosquito host anti-viral response against the virus.\n\nImportanceVector-borne viruses pose great risks on human health. Zika virus has recently emerged as a global threat, rapidly expanding its distribution. Understanding the interactions of the virus with mosquito vectors at the molecular level is vital for devising new approaches in inhibiting virus transmission. In this study, we embarked on analyzing the transcriptional response of Aedes aegypti mosquitoes to Zika virus infection. Results showed large changes both in coding and long non-coding RNAs. Analysis of these genes showed similarities with other flaviviruses, including dengue virus, which is transmitted by the same mosquito vector. The outcomes provide a global picture of changes in the mosquito vector in response to Zika virus infection.

microbiology

Genomic and structural features of the Yellow Fever virus from the 2016-2017 Brazilian outbreak

Brazil has been suffering a severe sylvatic epidemic of yellow fever virus (YFV) since late 2016. Analysis of full-length YFV genomes from all hosts involved in the Brazilian 2017 outbreak reveals that they belong to sub-lineage 1E within modern-lineage, but display several unique amino acid substitutions in highly conserved positions at NS3 and NS5 viral proteins. Evolutionary analyses indicate that YFV carrying that set of amino acid substitution circulates in the Southern Brazilian region for several months before being detected in December 2016. Structural and selection analyses support that some of these substitutions were under positive selection and could impact enzyme structure and function. Altogether, this evidence demonstrated that the current Brazilian YFV carries unique amino acid signatures in the non-structural proteins and support the hypothesis that those substitutions may be affecting the viral fitness and transmissibility.

microbiology

Comparison of algorithms for the detection of enteroviruses in stool specimens from children diagnosed with Acute Flaccid Paralysis

With poliovirus eradication within reach, the WHO has included in its recommendations a cell-culture independent algorithm for enterovirus surveillance. This study was designed to compare both the cell culture dependent and independent algorithms and assess how either might impact our perception of the diversity of enterovirus types present in a sample.\n\nSixteen paired samples (16 isolates from RD cell culture and their corresponding stool suspension. i.e. 32 samples) from AFP cases in Nigeria were analyzed in this study. One of these 16 sample pairs (the control) was previously identified and confirmed as poliovirus 2 (PV-2). All the samples were subjected to RNA extraction, cDNA synthesis, RT-snPCR (the WHO recommended cell-culture independent algorithm) and its modifications for co-infection detection and resolution. Amplicons were sequenced and strains identified using the enterovirus genotyping tool and phylogenetic analysis.\n\nThe enterovirus diversity was shown to be the same between RD cell culture isolates and fecal suspension for the control and five (7, 10, 11, 12 & 14) of the samples analyzed. It was however, different for the remaining 10 (62.5%) samples analyzed. Fourteen different enterovirus types were identified in this study. To be precise, 9 (CV-B4, E6, E7, E13, E14, E19, E29, EV-B75 and EV-B77) and 5 (CV-A1, CV-A11, CV-A13, EV-C99 and PV2) EV-B and EV-C types, respectively where detected in this study. It is crucial to mention that E19 and EV-B75were only recovered from RD cell culture isolates while E14, EV-B77, CV-A11 and CV-A13 were only recovered from fecal suspension.\n\nThe results of this study show that both the cell culture dependent and independent protocols recommended by the WHO for enterovirus detection unavoidably bias our perception of the diversity of enterovirus types present in a sample. Hence, rather than jettison one for the other, effort should be directed at harmonizing both for increased sensitivity.

microbiology

Sugar metabolism of the first thermophilic Planctomycetes Thermogutta terrifontis: comparative genomic and transcriptomic approache

Xanthan gum, a complex polysaccharide comprising glucose, mannose and glucuronic acid residues, is involved in numerous biotechnological applications in cosmetics, agriculture, pharmaceuticals, food and petroleum industries. Additionally, its oligosaccharides were shown to possess antimicrobial, antioxidant and few other properties. Yet, despite its extensive usage, little is known about xanthan gum degradation pathways and mechanisms.\n\nThermogutta terrifontis R1 was described as the first thermophilic representative of the Planctomycetes phylum. As other cultivated planctomycetes, it grows well on various carbohydrates including oligo- and polysaccharides, however, its capability of anaerobic growth with or without electron acceptors was a novel finding among the representatives of this phylum.\n\nThe aim of this work is to examine T. terrifontis catabolic pathways with a special focus on the xanthan gum degradation pathway using genomic and transriptomic sequencing. Genomic analysis revealed more than a hundred glycosidases, polysaccharide lyases and other CAZymes, involved in oligo- and polysaccharide degradation by T. terrifontis, proteins of central carbohydrate metabolism and aerobic and anaerobic respiration. Furthermore, the combination of genomic and transcriptomic approaches revealed a putative novel xanthan gum degradation pathway involving unusual catalytic steps and enzymes: novel glycosidase(s) of DUF1080 family, hydrolyzing xanthan gum beta-glucosidic backbone linkages and beta-mannosidases instead of xanthan lyases for degradation of terminal beta-mannosidic linkages. Surprisingly, the genes coding DUF1080 proteins were found in high number in T. terrifontis and in many other Planctomycetes genomes, which, together with our observation that xanthan gum being a selective substrate for many planctomycetes, supports the important role of DUF1080 in xanthan gum degradation. Our findings shed light on the metabolism of the first thermophilic planctomycete, capable to degrade a number of polysaccharides, either aerobically or anaerobically, including the biotechnologically important bacterial polysaccharide xanthan gum. The results serve as good foundation for future exploration of T. terrifontis and its enzymes in biotechnological applications.

microbiology

The use of hyperimmune chicken reference sera is not appropriate for the validation of influenza pseudotype neutralization assays

Pseudotype particle neutralization (pp-NT) is a next-generation serological assay employed for the sensitive study of influenza antibody responses, especially haemagglutinin stalk-directed antibodies. However, to date a validation of this assay has not been performed, and this limits its use to primarily research laboratories. To identify possible serological standards to be used in optimization and validation of the pp-NT, we have evaluated the cross-reactivity of hyperimmune chicken reference antisera in this assay. Our findings show that the cross-reactivity detected by the pp-NT assay is only in part explained by phylogenetic relationships and protein homology between the HA subtypes analysed; further studies are necessary to understand the origin of the cross-reactivity detected, and reference standards with higher specificity should be evaluated or generated de novo for future use in pp-NT.

microbiology

Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV)

Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF), causing immunosuppression and morbidity in young chickens. In addition to strains that cause classical Gumboro disease, the so-called very virulent (vv) strain, also in circulation, causes more severe disease and increased mortality. IBDV has traditionally been controlled through the use of live attenuated vaccines, with attenuation resulting from serial passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated phenotypes are poorly understood. In order to address this, we aimed to investigate host cell-IBDV interactions using a recently described chicken primary B cell model, where chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken CD40L. We demonstrated that these cells could support the replication of IBDV when infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by microarray. We found that key genes involved in B cell activation and signaling (TNFSF13B, CD72 and GRAP) were down-regulated following infection relative to mock, which we speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the induction was far less pronounced upon infection with UK661, which we speculate could contribute to its virulence.

microbiology

Metabolic Model-Based Analysis of the Emergence of Bacterial Cross-Feeding through Extensive Gene Loss

Metabolic dependencies between microbial species are common and have a significant impact on the assembly and resilience of microbial communities. However, the origins of such metabolic dependencies, the evolutionary forces that drive metabolic cross-feeding, and the impact of metabolic and genomic architecture on their emergence are not clear. To address these questions, we developed a novel simulation-based framework coupling a model of reductive evolution with a multi-species genome-scale model of microbial metabolism. We used this framework to model the evolution of a two-species microbial community, simulating thousands of independent evolutionary trajectories and investigating the link between genome reductive evolution and the emergence of metabolic interactions. Surprisingly, even though our model does not impose explicit selection for cooperation, metabolic dependencies emerged in nearly half of all evolutionary runs. Evolved dependencies involved cross-feeding of a diverse set of metabolites at varying frequencies, reflecting various constraints imposed by the metabolic network architecture. We additionally found metabolic missed opportunities, wherein species failed to capitalize on metabolites made available by their partners. When cross-feeding did evolve, it generally emerged immediately after a metabolite became available, but a complete dependence on such cross-fed metabolites often evolved relatively slowly. Examining the genes deleted and retained in each evolutionary trajectory and the timing of gene deletion events along these trajectories, we were further able to identify both genome-wide properties and specific gene retentions that were associated with metabolic phenotypes. Our findings provide insight into the evolution of cooperative metabolic interaction among microbial species, offering a unique view into the way such relationships could emerge in natural settings.

microbiology

Prey range and genome evolution of Halobacteriovorax marinus predatory bacteria from an estuary

BackgroundHalobacteriovorax are saltwater-adapted predatory bacteria that attack Gram-negative bacteria and therefore may play an important role in shaping microbial communities. To understand the impact of Halobacteriovorax on ecosystems and develop them as biocontrol agents, it is important to characterize variation in predation phenotypes such as prey range and investigate the forces impacting Halobacteriovorax genome evolution across different phylogenetic distances.\n\nResultsWe isolated H. marinus BE01 from an estuary in Rhode Island using Vibrio from the same site as prey. Small, fast-moving attack phase BE01 cells attach to and invade prey cells, consistent with the intraperiplasmic predation strategy of H. marinus type strain SJ. BE01 is a prey generalist, forming plaques on Vibrio strains from the estuary as well as Pseudomonas from soil and E. coli. Genome analysis revealed that BE01 is very closely related to SJ, with extremely high conservation of gene order and amino acid sequences. Despite this similarity, we identified two regions of gene content difference that likely resulted from horizontal gene transfer. Analysis of modal codon usage frequencies supports the hypothesis that these regions were acquired from bacteria with different codon usage biases compared to Halobacteriovorax. In BE01, one of these regions includes genes associated with mobile genetic elements, such as a transposase not found in SJ and degraded remnants of an integrase occurring as a full-length gene in SJ. The corresponding region in SJ included unique mobile genetic element genes, such as a site-specific recombinase and bacteriophage-related genes not found in BE01. Acquired functions in BE01 include the dnd operon, which encodes a pathway for DNA modification that may protect DNA from nucleases, and a suite of genes involved in membrane synthesis and regulation of gene expression that was likely acquired from another Halobacteriovorax lineage.\n\nConclusionsOur results support previous observations that Halobacteriovorax prey on a broad range of Gram-negative bacteria. Genome analysis suggests strong selective pressure to maintain the genome in the H. marinus lineage represented by BE01 and SJ, although our results also provide further evidence that horizontal gene transfer plays an important role in genome evolution in predatory bacteria.

microbiology

The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A Carbohydrate in Streptococcus pyogenes

In many Lactobacillales species (i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes, synthesizes a key antigenic surface polymer--the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N-acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphorylundecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltranferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis. In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.

microbiology

Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1

Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate (NO3-) to nitrite (NO2-), which accumulates in the medium. These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (NarGHJI). M. nitratireducenticrescens strain JAM1 also contains gene clusters encoding two putative nitric oxide reductase (NO) reductases and one putative nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. In this study, we show that strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory copper-(NirK) or cytochrome cd1-type (NirS) NO2- reductase, NO3--amended strain JAM1 cultures produce N2O, representing up to 6% of the N-input. NO2- was shown to be the key intermediate of this production process. In NO3-amended cultures, we analyzed denitrification genes in succession of net N2O-production and -consumption phases at the gene expression level. These phases were found to correlate with changes in the expression levels of the NO reductase gene cnorB1 and nnrS, which indicated NO production in the cultures.\n\nImportanceBy showing that all the three denitrification reductases are active, this demonstrates that Methylophaga nitratireducenticrescens JAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of a denitrification reactor from which it originates. The small production of N2O via NO in strain JAM1 did not involve Nar contrary to what was demonstrated in Escherichia coli. M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using NO3- and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities.

microbiology

Super-Resolution Microscopy Reveals Structural Mechanisms Driving the Nanoarchitecture of a Viral Chromatin Tether

By tethering their circular genomes (episomes) to host chromatin, DNA tumor viruses ensure retention and segregation of their genetic material during cell divisions. Despite functional genetic and crystallographic studies, there is little information addressing the three-dimensional structure of these tethers in cells, issues critical for understanding persistent infection by these viruses. Here, we have applied direct stochastic optical reconstruction microscopy (dSTORM) to establish the nanoarchitecture of tethers within cells latently infected with the oncogenic human pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV). Each KSHV tether comprises a series of homodimers of the latency-associated nuclear antigen (LANA) that bind with their C-termini to the tandem array of episomal terminal repeats (TRs) and with their N-termini to host chromatin. Super-resolution imaging revealed that individual KSHV tethers possess similar overall dimensions and, in aggregate, fold to occupy the volume of a prolate ellipsoid. Using plasmids with increasing numbers of TRs, we found that tethers display polymer power-law scaling behavior with a scaling exponent characteristic of active chromatin. For plasmids containing a two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corresponding to a single TR. From these data, we have generated a three-dimensional model of the episomal half of the tether that integrates and extends previously established findings from epi-fluorescent, crystallographic, and epigenetic approaches. Our findings also validate the use of dSTORM in establishing novel structural insights into the physical basis of molecular connections linking host and pathogen genomes.

microbiology