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bioRxiv2020

Estimation of Full-Length TprK Diversity in Treponema pallidum subspecies pallidum

Immune evasion and disease progression of Treponema pallidum subspecies pallidum are associated with sequence diversity in the hypervariable, putative outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the proteins 7 variable regions, or were conducted on strains passed through rabbits. As a consequence, a complete profiling of tprK during infection in the human host is still lacking. Furthermore, prior studies examining how T. pallidum uses its repertoire of genomic donor sites to generate diversity within the V regions of the tprK also yielded a partial understanding of this process, due to the limited number of tprK alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length tprK alleles from T. pallidum collected from early lesions of patients attending two STD clinics in Italy. Our data, combined with recent data available on Chinese T. pallidum strains, show the near complete absence of overlap in TprK sequences among the 41 strains profiled to date. Moreover, our data allowed us to redefine the boundaries of tprK V regions, identify 55 donor sites, and estimate the total number of TprK variants that T. pallidum can potentially generate. Altogether, our results support how T. pallidum TprK antigenic variation system is an unsurmountable obstacle for the human immune system to naturally achieve infection eradication, and reiterate the importance of this mechanism for pathogen persistence in the host. ImportanceSyphilis continues to be a significant public health issue in both low- and high-income nations, including the United States, where the number of infectious syphilis cases has increased dramatically over the past five years. T. pallidum, the causative agent of syphilis, encodes an outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed deep TprK profiling to understand full-length TprK diversity in T. pallidum-positive clinical specimens and compared these to all samples for which TprK deep sequencing is available. We found almost no overlap in TprK sequences between different patients. We further estimate that the total baseline junctional diversity of full-length TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows T. pallidum to establish lifelong infection.

microbiology↗

bioRxiv2020

Analysis of cell-associated DENV RNA by oligo(dT) primed 5' capture scRNAseq

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5 capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5 to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5 end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5 capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naive B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5 capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells. IMPORTANCEDengue is one of the most widespread vector-borne viral diseases in the world. However, it is still unclear which cells harbor virus during DENV infection, and how viral reservoirs in serum and infected cells are related. These results demonstrate for the first time that intracellular DENV RNA can be identified and infected cells quantified by 5 capture scRNAseq. This strategy provides a significantly greater throughput and higher sensitivity than previously published methods and has the potential to provide additional information on the genomic heterogeneity of intracellular DENV RNA.

microbiology↗

bioRxiv2020

Plasmid diversity among genetically related Klebsiella pneumoniae blaKPC-2 and blaKPC-3 isolates collected in the Dutch national surveillance

Carbapenemase-producing Klebsiella pneumoniae emerged over the past decades as an important pathogen causing morbidity and mortality in hospitalized patients. For infection prevention and control, it is important to track the spread of bacterial strains in humans including the plasmids they contain. However, little is known concerning the plasmid repertoire among K. pneumoniae strains. Therefore, the major aim was to recapitulate the size, contents and diversity of the plasmids of genetically related K. pneumoniae strains harboring the beta-lactamase gene blaKPC-2 or blaKPC-3 to determine their dissemination in the Netherlands and the former Dutch Caribbean islands from 2014-2019. Next-generation sequencing was combined with long-read third-generation sequencing to reconstruct 18 plasmids of K. pneumoniae. wgMLST revealed five genetic clusters (termed KpnClusters) comprised of K. pneumoniae blaKPC-2 isolates and four clusters consisted of blaKPC-3 isolates. Each cluster was characterized by a distinct resistome and plasmidome. KpnCluster-019 blaKPC-2 isolates were found both in the Netherlands and the Caribbean islands. K. pneumoniae blaKPC-3 isolates were found in the collection of the Netherlands. The 18 plasmids were mostly unrelated and varied between K. pneumoniae blaKPC-2 and blaKPC-3 clusters. However, the large and medium sized plasmids contained a variety of antibiotic resistance genes, transposons, insertion sequence elements, conjugal transfer systems, cation transport systems, toxin/antitoxin systems, and prophage-related sequence elements. The small plasmids carried genes implicated in virulence. Thus, implementing long-read plasmid sequencing analysis for K. pneumoniae surveillance provided important insights in the success and understanding of transmission of a KpnCluster-019 blaKPC-2 strain between the Netherlands and the Caribbean. ImportanceCarbapenemase-producing Klebsiella pneumoniae has spread globally and is of great concern for debilitated patients. K. pneumoniae is notorious for spreading antimicrobial resistance genes by plasmids among Enterobacterales. Combining short and long read sequencing enables reconstruction of plasmids containing antibiotic resistance genes, conjugation machinery, transposons, toxins and/or virulence determinants and thereby enhancing international pathogen surveillance.

microbiology↗

bioRxiv2020

Swine ANP32A supports avian influenza virus polymerase

Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as mixing vessels, being susceptible to both avian and human origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports some, albeit limited, activity of avian origin influenza virus polymerases. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the super pro-viral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the LRR and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the mixing vessel trait of swine and further our understanding of the evolution and ecology of viruses in this host.

microbiology↗

bioRxiv2020

Evolution and maintenance of microbe-mediated protection under occasional pathogen attack

Every host is colonized by a variety of microbes, some of which can protect their hosts from pathogen infection. However, pathogen presence naturally varies over time in nature, such as in the case of seasonal epidemics. We experimentally coevolved populations of Caenorhabditis elegans worm hosts with bacteria possessing protective traits (Enterococcus faecalis), in treatments varying the infection frequency with pathogenic Staphylococcus aureus every host generation, alternating host generations, every fifth host generation or never. We additionally investigated the effect of initial pathogen presence at the formation of the defensive symbiosis. Our results show that enhanced microbe-mediated protection evolved during host-protective microbe coevolution when faced with rare infections by a pathogen. Initial pathogen presence had no effect on the evolutionary outcome of microbe-mediated protection. We also found that protection was only effective at preventing mortality during the time of pathogen infection. Overall, our results suggest that resident microbes can be a form of transgenerational immunity against rare pathogen infection.

microbiology↗

bioRxiv2020

Isolation and characterization of bovine herpes virus 5 (BHV5) from cattle in India

Bovine herpesvirus 1 (BHV1) and 5 (BHV5) are genetically and antigenically related alphaherpesviruses. Infection with one virus induces protective immunity against the other. However, disease associated with BHV1 and BHV5 varies significantly; whereas BHV1 infection is usually associated with rhinotracheitis and abortion, BHV5 causes encephalitis in cattle. BHV5 outbreaks are sporadic and mainly restricted to the South American countries. We report BHV5 infection for the first time from aborted cattle in India. Based on the characteristic cytopathic effects in MDBK cells, amplification of the viral genome in PCR, differential PCR for BHV1/BHV5, nucleotide sequencing and restriction endonuclease patterns, identity of the virus was confirmed as BHV5 subtype A. Serum samples from the aborted cattle strongly neutralized both BHV1 and BHV5 suggesting an active viral infection in the herd. Upon UL27, UL44 and UL54 gene-based sequence and phylogenetic analysis, the isolated virus clustered with BHV5 strains and showed highest similarity with the Brazilian BHV5 strains. Author SummaryBHV5 causes fatal meningoencephalitis that may result in a mortality rate of up to 100% in cattle. BHV5 is rarely associated with abortion and its distribution is restricted to South American countries. Only a few cases of this disease have been reported from other countries. For the first time, we provide a strong evidence of BHV5 infection from aborted cattle in India. The finding may necessitate inclusion of BHV5 test protocol in testing of semen for sexually transmitted diseases. Also, the isolated virus would be useful for developing diagnostic, prophylactic and therapeutic agents to combat BHV5 disease in the country.

microbiology↗

bioRxiv2020

Host-derived Circular RNAs Display Proviral Activities in Hepatitis C Virus - Infected Cells

Viruses subvert macromolecular pathways in infected host cells to aid in viral gene amplification or to counteract innate immune responses. Roles for host-encoded RNAs, including microRNAs, have been found to provide pro- and anti-viral functions. Recently, circular RNAs (circRNAs), that are generated by a nuclear back-splicing mechanism of pre-mRNAs, have been implicated to have roles in DNA virus-infected cells. This study examines the circular RNA landscape in uninfected and hepatitis C virus (HCV)-infected liver cells. Results showed that the abundances of distinct classes of circRNAs were up-regulated or down-regulated in infected cells. Identified circRNAs displayed pro-viral effects. One particular up-regulated circRNA, circPSD3, displayed a very pronounced effect on viral RNA abundances in both hepatitis C virus- and Dengue virus-infected cells. Surprisingly, circPSD3 also inhibited the cellular nonsense-mediated decay (NMD) pathway in liver cells. Thus, enhanced abundance of circPSD3 in virus-infected cells aids in viral replication and likely contributes to the known inhibition of NMD in HCV-infected cells. Findings from the global analyses of the circular RNA landscape argue pro-, and likely, anti-viral functions are executed by circRNAs that modulate both viral gene expression as well as host pathways. Because of their long half-lives, circRNAs likely play hitherto unknown, important roles in viral pathogenesis. Author SummaryUsually, cells are infected by one or a few virus particles that carry genomes with limited expression capacity. Thus, the expression of viral genomes has to compete with a sea of cellular components that aid in viral translation, replication and virion production. Depending on their lifestyle, viruses have evolved to avoid or to subvert cellular pathways, especially those that display anti-viral functions. Host-derived circular RNA molecules have recently been discovered in the cytoplasm of cells, although, as-of yet, few functions have been assigned to them. Here, we describe alterations in the circular RNA landscape in hepatitis C virus-infected liver cells. Up-regulated and down-regulated circular RNAs were identified, and three of the upregulated RNAs were shown to promote HCV infection. One of them, circPSD3, inhibited the cellular nonsense-mediated RNA decay that is a powerful antiviral response in infected cells. Because circular RNAs are more stable than linear RNAs, they may have important functions during viral infection, dictating the outcomes of innate immune responses and viral pathogenesis.

microbiology↗

bioRxiv2020

Community sequencing on a natural experiment reveals little influence of host species and timing but a strong influence of compartment on the composition of root endophytes in three annual Brassicaceae

The plant family Brassicaceae includes some of the most studied hosts of plant microbiomes, targeting microbial diversity, community assembly rules, and effects on host performance. Compared to bacteria, eukaryotes in the brassicaceous microbiome remain understudied, especially under natural settings. Here, we assessed the impact of host identity and age on the assembly of fungal and oomycete root communities, using DNA metabarcoding of roots and associated soil of three annual co-habiting Brassicaceae collected at two time points. Our results showed that fungal communities are more diverse and structured than those of oomycetes. In both cases, plant identity and sampling time had little influence on community variation, whereas root/soil compartment had a strong effect by exerting control on the entry of soil microorganisms into the roots. The enrichment in roots of specific fungi suggests a specialization towards the asymptomatic colonization of plant tissues, which could be relevant to hosts fitness and health.

microbiology↗

bioRxiv2020

Remodeling of the core leads HIV-1 pre-integration complex in the nucleus of human lymphocytes.

Retroviral replication proceeds through obligate integration of the viral DNA into the host genome. To enter the nucleus, the viral DNA must be led through the nuclear pore complex (NPC). During HIV-1 cytoplasmic journey, the viral core acts like a shell to protect the viral genetic material from antiviral sensors and ensure an adequate environment for the reverse transcription. However, the relatively narrow size of the nuclear pore channel requires that the HIV-1 core reshapes into a structure that fits the pore. On the other hand, the organization of the viral CA proteins that remain associated to the pre-integration complex (PIC) during and after nuclear translocation, in particular, in human lymphocytes, the main target cells of HIV-1, is still enigmatic. In this study, we analysed the progressive organizational changes of viral CA proteins within the cytoplasm and the nucleus by immuno-gold labelling. Furthermore, we set up a novel technology, HIV-1 ANCHOR, which enables specific detection of the retrotranscribed DNA by fluorescence microscopy, thereby uncovering the architecture of the potential HIV-1 PIC. Thus, we revealed DNA- and CA-positive complexes by correlated light- and electron microscopy (CLEM). During and after nuclear translocation, HIV-1 appears as a complex of viral DNA decorated by multiple viral CA proteins remodelled in a "pearl necklace" shape. Thus, we observed how CA proteins reshape around the viral DNA to permit the entrance of the HIV-1 in the nucleus. This particular CA protein complex composed by the integrase and the retrotranscribed DNA leads HIV-1 genome inside the host nucleus to potentially replicate. Our findings contribute to the understanding of the early steps of HIV-1 infection and provide new insights into the organization of HIV-1 CA proteins during and after viral nuclear entry. ImportanceHow the reverse transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. HIV-1 core has a size of [~]100 nm, largely exceeding that of the NPC channel ([~]39 nm). Thus, a rearrangement of the viral CA proteins organization is required to achieve effective nuclear translocation. The mechanistic of this process remains undefined due to the lack of a technology capable to visualize potential CA sub-complexes in association with the viral DNA in the nucleus of HIV-1-infected cells. By the means of state-of-the-art technologies (HIV-1 ANCHOR system combined with CLEM), our study shows that remodeled viral complexes retain multiple CA proteins but not intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed in the outer and inner side of the NE, and they represent potential PIC. Thus, our study shed light on critical early steps characterizing HIV-1 infection, thereby revealing novel, therapeutically exploitable points of intervention. Furthermore, we developed and provided a powerful tool enabling direct, specific and high-resolution visualization of intracellular and intranuclear HIV-1 subviral structures.

microbiology↗

bioRxiv2020

Structural insights into the insecticidal Vip3A toxin of Bacillus thuringiensis

The vegetative insecticidal proteins (Vips) secreted by Bacillus thuringiensis are regarded as the new generation of insecticidal toxins because they have different insecticidal properties compared with commonly applied insecticidal crystal proteins (Cry toxins). Vip3A toxin, representing the vast majority of Vips, has been used commercially in transgenic crops and bio-insecticides. However, the lack of both structural information of Vip3A and a clear understanding of its insecticidal mechanism at the molecular level, limits its further development and broader application. Here we present the first crystal structure of the Vip3A toxin in an activated form. Since all members of this insecticidal protein family are highly conserved, the structure of Vip3A provides unique insight into the general domain architecture and protein fold of the Vip3 family of insecticidal toxins. Our structural analysis reveals a four-domain organization, featuring a potential membrane insertion region, a receptor binding domain, and two glycan binding domains of activated Vip3A. We further identify the specific glycan moieties recognized by Vip3A through a glycan array screen. Taken together, these findings provide insights into the mode of action of Vip3 family of insecticidal toxins, and will boost the development of Vip3 into more efficient bio-insecticides.

microbiology↗

bioRxiv2020

Competition sensing alters antibiotic production in Streptomyces

One of the most important ways that bacteria compete for resources and space is by producing antibiotics that inhibit competitors. Because antibiotic production is costly, the biosynthetic gene clusters coordinating their synthesis are under strict regulatory control and often require "elicitors" to induce expression, including cues from competing strains. Although these cues are common, they are not produced by all competitors and so the phenotypes causing induction remain unknown. By studying interactions between 24 antibiotic-producing streptomycetes we show that inhibition between competitors is common and occurs more frequently if strains are closely related. Next, we show that antibiotic production is more likely to be induced by cues from strains that are closely related or that share secondary metabolite biosynthetic gene clusters (BGCs). Unexpectedly, antibiotic production is less likely to be induced by competitors that inhibit the growth of a focal strain, indicating that cell damage is not a general cue for induction. In addition to induction, antibiotic production often decreased in the presence of a competitor, although this response was not associated with genetic relatedness or overlap in BGCs. Finally, we show that resource limitation increases the chance that antibiotic production declines during competion. Our results reveal the importance of social cues and resource availability in the dynamics of interference competition in streptomycetes. SIGNIFICANCE STATEMENTBacteria secrete antibiotics to inhibit their competitors, but the presence of competitors can determine whether these toxins are produced. Here, we study the role of the competitive and resource environment on antibiotic production in Streptomyces, bacteria renowned for their production of antibiotics. We show that Streptomyces are more likely to produce antibiotics when grown with closely related competitors or that share biosynthetic pathways for secondary metabolites, but not when they are threatened by competitors toxins, in contrast to predictions of the competition sensing hypothesis. Streptomyces also often reduce their output of antibiotics when grown with competitors, especially under nutrient limitation. Our findings highlight that interactions between the social and resource environments strongly regulate antibiotic production in these medicinally important bacteria.

microbiology↗

bioRxiv2020

Proteomic changes in bacteria caused by exposure to environmental conditions can be detected by Matrix-Assisted Laser Desorption/Ionization - Time of Flight (MALDI-ToF) Mass Spectrometry

In the past decade, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) has become a timely and cost-effective alternative to bacterial identification. The MALDI-ToF MS technique analyzes the total protein of culturable microorganisms at the species level and produces a mass spectra based on peptides which is compared to a database of identified profiles. Consequently, unique signatures of each microorganism are produced allowing identification at the species and, more importantly, strain level. Our present study proposes that the MALDI-ToF MS can be further used to screen functional and metabolic differences. While other studies applied the MALDI-ToF technique to identify subgroups within species, we investigated how various environmental factors could alter the unique bacterial signatures. We found that genetic and phenotypic differences between microorganisms belonging to the same species can be reflected in peptide-mass fingerprints generated by MALDI-ToF MS. These results suggest that MALDI-ToF MS can screen intra-species phenotypic differences of several microorganisms.

microbiology↗

bioRxiv2020

From SARS-CoV to Wuhan 2019-nCoV: Will History Repeat Itself?

This manuscript has been withdrawn as it was submitted without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

microbiology↗

bioRxiv2020

Human stem cell derived sensory neurons are positioned to support varicella zoster virus latency

The neuropathogenesis of varicella-zoster virus (VZV) has been challenging to study due to the strict human tropism of the virus and the resultant difficulties in establishing tractable experimental models. In vivo, sensory neurons of the dorsal root ganglia and trigeminal ganglia serve as cellular niches that support viral latency, and VZV can subsequently reactivate from these cells to cause disease. Whether sensory neurons possess intrinsic properties that position them to serve as a reservoir of viral latency remains unknown. Here, we utilize a robust human sensory neuron system to investigate lytic infection and viral latency. We find that sensory neurons exhibit resistance to lytic infection by VZV. On the other hand, latent infection in sensory neurons is associated with an episomal-like configuration of viral DNA and expression of the VZV latency-associated transcript (VLT), thus closely mirroring the in vivo state. Moreover, despite the relative restriction in lytic infection, we demonstrate that viral reactivation is possible from latently infected sensory neurons. Taken together, our data suggest that human sensory neurons possess intrinsic properties that serve to facilitate their role as a latent reservoir of VZV. IMPORTANCEVaricella-zoster virus (VZV) has infected over 90% of people worldwide. Following primary infection, the virus can remain dormant in the nervous system and may reactivate later in life, with potentially severe consequences. Here, we develop a model of VZV infection in human sensory neurons in order to determine whether these cells are intrinsically positioned to support latency and reactivation. We find that human sensory neurons are relatively resistant to lytic infection, but can support latency and reactivation. Moreover, during in vitro latency human sensory neurons, but not other neurons, express the newly discovered VZV latency-associated transcript (VLT), thus closely mirroring the in vivo latent state. Taken together, these data indicate that human sensory neurons are uniquely positioned to support latency. We anticipate that this human sensory neuron model will serve to facilitate further understanding of the mechanisms of VZV latency and reactivation.

microbiology↗

bioRxiv2020

Restricted sequence variation in Streptococcus pyogenes penicillin binding proteins

A recent clinical report has linked Streptococcus pyogenes {beta}-lactam antibiotic resistance to mutations in the Penicillin Binding Protein PBP2x. To determine whether this is an isolated case or reflects a broader prevalence of mutations that might confer reduced {beta}-lactam susceptibility, we investigated the relative frequency of penicillin binding protein (PBP) sequence variation within a global database of 9,667 S. pyogenes isolates. We found that mutations in S. pyogenes PBPs (PBP2x, PBP1a, PBP1b and PBP2a) occur infrequently across this global database with less than 3 amino acid changes differing between >99% of the global population. Only 4 of the 9,667 strains contained mutations near transpeptidase active sites. The reported PBP2x T553K substitution was not identified. These findings are in contrast to those of 2,520 S. pneumococcus sequences where PBP mutations are relatively frequent and are often located in key {beta}-lactam binding pockets. These data, combined with the general lack of penicillin resistance reported in S. pyogenes worldwide, suggests that extensive, unknown, constraints restrict S. pyogenes PBP sequence plasticity. These findings imply that while heavy antibiotic pressure may select for mutations in the PBPs, there is currently no evidence of such mutations becoming fixed in the S. pyogenes population nor that mutations are being sequentially acquired in the PBPs. ImportancePenicillin is the first line therapeutic option for Streptococcus pyogenes infections. Despite the global high prevalence of S. pyogenes infections and widespread use of penicillin, reports of resistance to penicillin have been incredibly rare. Recently, penicillin resistance was detected in two clinical S. pyogenes isolates with accompanying mutations in the active site of the penicillin binding protein PBP2x, raising concerns that penicillin resistance may become more widespread. We screened a global database of S. pyogenes genome sequences to investigate the frequency of penicillin binding protein (PBP) mutations, identifying that PBP mutations are uncommon relative to Streptococcus pneumoniae. These findings support clinical observations that penicillin resistance is rare in S. pyogenes, and suggest that there are considerable constraints on S. pyogenes PBP sequence variation.

microbiology↗

bioRxiv2020

Profiling the microbial community structure and functional diversity of a dam-regulated river undergoing gravel bar restoration

BackgroundRiver restoration efforts are expected to influence and change the diversity and functions of microbial communities following the recovery of habitat characteristics in the river ecosystem. The recreation or restoration of gravel bars in the Trinity River in California aims to rehabilitate the environmental heterogeneity downstream of the dam impounded channel. Here, we profiled the community composition, estimated diversity, and annotated putative metabolic functions of the sediment microbial communities to assess whether the construction and restoration of gravel bars in the Trinity River in California enhanced environmental heterogeneity, with the increase in the microbial beta diversity of these in-channel structures against the free-flowing reach of the main channel with comparison to its undisturbed tributaries. ResultsMicrobial community composition of the free-flowing (i.e., no gravel bars) communities were relatively closer regardless of dam influence, whereas the Trinity River gravel bar and tributaries gravel bar communities were highly dissimilar. Proteobacteria, Bacteroidetes, and Acidobacteria were the highly abundant sediment microbial phyla on most sites, specifically in the Trinity River gravel bar communities. Putative functional annotation of microbial taxa revealed that chemoheterotrophy and aerobic chemoheterotrophy were the most prevalent microbial processes, with the Trinity River gravel bars having relatively higher representations. The considerably large abundance of heterotrophic taxa implies that gravel bars provide suitable areas for heterotrophic microorganisms with metabolic functions contributing to the net respiration in the river. ConclusionsOur results provide supporting evidence on the positive impact of habitat restoration being conducted in the Trinity River with the non-dam influenced, undisturbed tributaries as the basis of comparison. Gravel bar recreation and restoration contributed to the increased microbial biodiversity through the restoration of environmental heterogeneity at the river scale. We provided valuable insights into the potential microbial processes in the sediment that might be contributing to the biogeochemical processes carried out by the microbial communities in the Trinity River. The significant positive correlation between the functional diversity of the identified microbial taxa and beta diversity suggests that differences in the detected metabolic functions were closely related to dissimilarities in community composition.

microbiology↗

bioRxiv2020

Origin time and epidemic dynamics of the 2019 novel coronavirus

The 2019 novel coronavirus (2019-nCoV) have emerged from Wuhan, China. Studying the epidemic dynamics is crucial for further surveillance and control of the outbreak. We employed a Bayesian framework to infer the time-calibrated phylogeny and the epidemic dynamics represented by the effective reproductive number (Re) changing over time from 33 genomic sequences available from GISAID. The time of the most recent common ancestor (MRCA) was December 17, 2019 (95% HPD: December 7, 2019 - December 23, 2019). The median estimate of Re shifted from 1.6 to 1.1 on around January 1, 2020. This study provides an early insight of the 2019-nCoV epidemic. However, due to limited amount of data, one should be cautious when interpreting the results at this stage.

microbiology↗

bioRxiv2020

Recombinant protein production and purification of SiiD, SiiE and SiiF - components of the SPI4-encoded type I secretion system from Salmonella Typhimurium

In humans, Salmonella enterica infections are responsible for a pleiotropy of medical conditions. These include intestinal inflammation and typhoid fever. The initial contact between Salmonella and polarized epithelial cells is established by the SPI4-encoded type I secretion system (T1SS), which secrets SiiE, a giant non-fimbrial adhesin. We have recombinantly produced various domains of this T1SS from Salmonella enterica serovar Typhimurium in Escherichia coli for further experimental characterization. We purified three variants of SiiD, the periplasmic adapter protein spanning the space between the inner and outer membrane, two variants of the SiiE N-terminal region and the N-terminal domain of the SiiF ATP-binding cassette (ABC) transporter. In all three proteins, at least one variant yielded high amounts of pure soluble protein. Proper folding and cooperative unfolding were investigated by circular dichroism (CD) spectroscopy. Secondary structure content estimations from CD spectra were in good agreement with the values derived from SiiD and SiiF homology models or, in case of the SiiE N-terminal region, a secondary structure prediction. For one SiiD variant, protein crystals could be obtained that diffracted X-rays to approximately 4 [A] resolution.

microbiology↗